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1. Flavor Research: Principles and Techniques, R. Teranishi, I. Hornstein, P. Issenberg, and E. L. Wick
2. Principles of Enzymology for the Food Sciences, John R. Whitaker
3. Low-Temperature Preservation of Foods and Living Matter, Owen R. Fennema, William D. Powrie, and Elmer H. Marth
4. Principles of Food Science
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5. Food Emulsions, edited by Stig E. Friberg
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7. Flavor Research: Recent Advances, edited by R. Teranishi, Robert A. Flath,
and Hiroshi Sugisawa
8. Computer-Aided Techniques in Food Technology, edited by Israel Saguy
9. Handbook of Tropical Foods, edited by Harvey T. Chan
10. Antimicrobials in Foods, edited by Alfred Larry Branen and P. Michael
Davidson
11. Food Constituents and Food Residues: Their Chromatographic Determination,
edited by James F. Lawrence
49. Food Extrusion Science and Technology, edited by Jozef L. Kokini, Chi-Tang
Ho, and Mukund V. Karwe
50. Surimi Technology, edited by Tyre C. Lanier and Chong M. Lee
51. Handbook of Food Engineering, edited by Dennis R. Heldman and Daryl B.
Lund
52. Food Analysis by HPLC, edited by Leo M. L. Nollet
53. Fatty Acids in Foods and Their Health Implications, edited by Ching Kuang
Chow
54. Clostridium botulinum: Ecology and Control in Foods, edited by Andreas H. W.
Hauschild and Karen L. Dodds
55. Cereals in Breadmaking: A Molecular Colloidal Approach, Ann-Charlotte Eliasson and Kre Larsson
56. Low-Calorie Foods Handbook, edited by Aaron M. Altschul
57. Antimicrobials in Foods: Second Edition, Revised and Expanded, edited by P.
Michael Davidson and Alfred Larry Branen
58. Lactic Acid Bacteria, edited by Seppo Salminen and Atte von Wright
59. Rice Science and Technology, edited by Wayne E. Marshall and James I.
Wadsworth
60. Food Biosensor Analysis, edited by Gabriele Wagner and George G. Guilbault
61. Principles of Enzymology for the Food Sciences: Second Edition, John R.
Whitaker
62. Carbohydrate Polyesters as Fat Substitutes, edited by Casimir C. Akoh and
Barry G. Swanson
63. Engineering Properties of Foods: Second Edition, Revised and Expanded, edited by M. A. Rao and S. S. H. Rizvi
64. Handbook of Brewing, edited by William A. Hardwick
65. Analyzing Food for Nutrition Labeling and Hazardous Contaminants, edited by
Ike J. Jeon and William G. Ikins
66. Ingredient Interactions: Effects on Food Quality, edited by Anilkumar G.
Gaonkar
67. Food Polysaccharides and Their Applications, edited by Alistair M. Stephen
68. Safety of Irradiated Foods: Second Edition, Revised and Expanded, J. F. Diehl
69. Nutrition Labeling Handbook, edited by Ralph Shapiro
70. Handbook of Fruit Science and Technology: Production, Composition, Storage,
and Processing, edited by D. K. Salunkhe and S. S. Kadam
71. Food Antioxidants: Technological, Toxicological, and Health Perspectives,
edited by D. L. Madhavi, S. S. Deshpande, and D. K. Salunkhe
72. Freezing Effects on Food Quality, edited by Lester E. Jeremiah
73. Handbook of Indigenous Fermented Foods: Second Edition, Revised and Expanded, edited by Keith H. Steinkraus
74. Carbohydrates in Food, edited by Ann-Charlotte Eliasson
75. Baked Goods Freshness: Technology, Evaluation, and Inhibition of Staling,
edited by Ronald E. Hebeda and Henry F. Zobel
76. Food Chemistry: Third Edition, edited by Owen R. Fennema
77. Handbook of Food Analysis: Volumes 1 and 2, edited by Leo M. L. Nollet
78. Computerized Control Systems in the Food Industry, edited by Gauri S. Mittal
79. Techniques for Analyzing Food Aroma, edited by Ray Marsili
80. Food Proteins and Their Applications, edited by Srinivasan Damodaran and
Alain Paraf
81. Food Emulsions: Third Edition, Revised and Expanded, edited by Stig E. Friberg and Kre Larsson
82. Nonthermal Preservation of Foods, Gustavo V. Barbosa-Cnovas, Usha R.
Pothakamury, Enrique Palou, and Barry G. Swanson
83. Milk and Dairy Product Technology, Edgar Spreer
84. Applied Dairy Microbiology, edited by Elmer H. Marth and James L. Steele
85. Lactic Acid Bacteria: Microbiology and Functional Aspects, Second Edition,
Revised and Expanded, edited by Seppo Salminen and Atte von Wright
86. Handbook of Vegetable Science and Technology: Production, Composition,
Storage, and Processing, edited by D. K. Salunkhe and S. S. Kadam
87. Polysaccharide Association Structures in Food, edited by Reginald H. Walter
88. Food Lipids: Chemistry, Nutrition, and Biotechnology, edited by Casimir C.
Akoh and David B. Min
89. Spice Science and Technology, Kenji Hirasa and Mitsuo Takemasa
90. Dairy Technology: Principles of Milk Properties and Processes, P. Walstra, T.
J. Geurts, A. Noomen, A. Jellema, and M. A. J. S. van Boekel
91. Coloring of Food, Drugs, and Cosmetics, Gisbert Ottersttter
92. Listeria, Listeriosis, and Food Safety: Second Edition, Revised and Expanded,
edited by Elliot T. Ryser and Elmer H. Marth
93. Complex Carbohydrates in Foods, edited by Susan Sungsoo Cho, Leon
Prosky, and Mark Dreher
94. Handbook of Food Preservation, edited by M. Shafiur Rahman
95. International Food Safety Handbook: Science, International Regulation, and
Control, edited by Kees van der Heijden, Maged Younes, Lawrence Fishbein,
and Sanford Miller
96. Fatty Acids in Foods and Their Health Implications: Second Edition, Revised
and Expanded, edited by Ching Kuang Chow
97. Seafood Enzymes: Utilization and Influence on Postharvest Seafood Quality,
edited by Norman F. Haard and Benjamin K. Simpson
98. Safe Handling of Foods, edited by Jeffrey M. Farber and Ewen C. D. Todd
99. Handbook of Cereal Science and Technology: Second Edition, Revised and
Expanded, edited by Karel Kulp and Joseph G. Ponte, Jr.
100. Food Analysis by HPLC: Second Edition, Revised and Expanded, edited by
Leo M. L. Nollet
101. Surimi and Surimi Seafood, edited by Jae W. Park
102. Drug Residues in Foods: Pharmacology, Food Safety, and Analysis, Nickos A.
Botsoglou and Dimitrios J. Fletouris
103. Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection,
edited by Luis M. Botana
104. Handbook of Nutrition and Diet, Babasaheb B. Desai
105. Nondestructive Food Evaluation: Techniques to Analyze Properties and
Quality, edited by Sundaram Gunasekaran
106. Green Tea: Health Benefits and Applications, Yukihiko Hara
107. Food Processing Operations Modeling: Design and Analysis, edited by Joseph
Irudayaraj
108. Wine Microbiology: Science and Technology, Claudio Delfini and Joseph V.
Formica
109. Handbook of Microwave Technology for Food Applications, edited by Ashim K.
Datta and Ramaswamy C. Anantheswaran
110. Applied Dairy Microbiology: Second Edition, Revised and Expanded, edited by
Elmer H. Marth and James L. Steele
111. Transport Properties of Foods, George D. Saravacos and Zacharias B.
Maroulis
112. Alternative Sweeteners: Third Edition, Revised and Expanded, edited by Lyn
OBrien Nabors
113. Handbook of Dietary Fiber, edited by Susan Sungsoo Cho and Mark L. Dreher
114. Control of Foodborne Microorganisms, edited by Vijay K. Juneja and John N.
Sofos
115. Flavor, Fragrance, and Odor Analysis, edited by Ray Marsili
116. Food Additives: Second Edition, Revised and Expanded, edited by A. Larry
Branen, P. Michael Davidson, Seppo Salminen, and John H. Thorngate, III
117. Food Lipids: Chemistry, Nutrition, and Biotechnology: Second Edition, Revised
and Expanded, edited by Casimir C. Akoh and David B. Min
118. Food Protein Analysis: Quantitative Effects on Processing, R. K. OwusuApenten
119. Handbook of Food Toxicology, S. S. Deshpande
120. Food Plant Sanitation, edited by Y. H. Hui, Bernard L. Bruinsma, J. Richard
Gorham, Wai-Kit Nip, Phillip S. Tong, and Phil Ventresca
121. Physical Chemistry of Foods, Pieter Walstra
122. Handbook of Food Enzymology, edited by John R. Whitaker, Alphons G. J.
Voragen, and Dominic W. S. Wong
123. Postharvest Physiology and Pathology of Vegetables: Second Edition, Revised
and Expanded, edited by Jerry A. Bartz and Jeffrey K. Brecht
124. Characterization of Cereals and Flours: Properties, Analysis, and Applications,
edited by Gnl Kaletun and Kenneth J. Breslauer
125. International Handbook of Foodborne Pathogens, edited by Marianne D.
Miliotis and Jeffrey W. Bier
Preface
There is no book dealing with food protein analysis exclusively, that is, with
the analysis of proteins in the food system. This books attempts to ll this
niche. Protein analysis comes in two forms: 1) Quantitative analysis, and 2)
fractionation and characterization. The rst activity is described here. This
publication provides a reference for planning, performing and interpreting
assays for food proteins. Many approved methods derive from the late-19th
century, but they have undergone rigorous testing and modernization. This
book does not focus on reviewing the latest research methods for protein
analysis. With the exceptions of Chapters 6 and 7, each of the 14 selfcontained chapters describes one protein assayprinciples, practices, and
expected results.
This book describes the effect of food processing on protein assay
results with the emphasis on how to analyze proteins in real foods. A
number of ``Methods'' sections provide instructions for specic tests.
Sample pretreatment and clean-up procedures are described. General
pretreatment strategies help in the avoidance of interference. More specic
clean-up methods apply to particular protein assays and are described along
with these. Example results, performance characteristics, case reports, and
practical problems and solutions related to a wide range of foods are
detailed in numerous gures, tables, and references.
v
vi
Preface
Contents
Preface
Part 1.
Chapter 1.
Part 2.
Chapter 2.
v
Fundamental Techniques
Kjeldahl Method, Quantitative Amino Acid Analysis and
Combustion Analysis
1. Introduction to Food Protein Analyses
2. Kjeldahl Analysis
3. Colorimetric Analysis of Kjeldahl Nitrogen
4. Quantitative Amino Acid Analysis
5. Combustion Nitrogen Analyzers
References
1
1
7
18
25
29
38
47
47
48
50
53
vii
viii
Chapter 3.
Chapter 4.
Part 3.
Chapter 5.
Chapter 6.
Contents
5.
6.
7.
55
56
69
69
70
73
77
80
57
64
86
87
93
99
99
103
105
109
110
112
113
116
121
125
125
127
131
133
147
169
169
147
160
Contents
ix
2.
3.
Part 4.
Chapter 8.
Chapter 9.
Bradford AssayApplications
1. Introduction
2. Coomassie Brilliant Blue Dye-Binding Assays
3. Performance Characteristics of CBBG Dye-Binding
Assays
4. Applications to Food Protein Analysis
References
171
183
184
184
185
186
191
195
195
195
201
204
218
221
221
225
230
241
247
247
252
255
257
260
265
268
270
271
274
Contents
Chapter 10.
Chapter 11.
Part 5.
Chapter 12.
285
289
289
292
292
297
297
301
305
312
329
281
281
281
341
341
346
348
354
366
374
Chapter 13.
381
381
381
384
386
393
398
401
402
Chapter 14.
411
411
411
414
416
Contents
xi
5.
447
1
Kjeldahl Method, Quantitative Amino
Acid Analysis and Combustion
Analysis
Chapter 1
Technique
Dumasa
Nessler's reagenta
Biuret method
Alkali-phenol reagent or Bethelot's methoda
Kjeldahla
Folin-Ciocalteau
Dye bindinga
Lowry
Direct alkaline distillation
Near-infrared reectance (NIR)a
Modied Berthelot reaction
Modied Lowry method (Peterson)
Bradford method (Coomassie Blue binding method)
Bicinchoninic acid (BCA) method
Techniques for which semiautomated or fully automated apparatus has been manufactured.
high sample throughput, simplicity, and low capital costs. Some of the most
signicant methods (Dumas, Kjeldahl, and biuret assays) date from the
late 1800s (Table 1). Techniques for food protein analysis are described
in this book. I will focus on the techniques that feature most often in
the food science literature. Infrared analysis of food proteins is not discussed
here.
1.1.
Techniques for food protein analysis need to be robust. This means one of
several things. Foremost is compatibility with fresh produce (cereals, fruits,
vegetables, meat, milk) and processed foods. Samples in various physical
states (powders, slurries, dilute liquids, emulsions, gels, pastes) should be
analyzable. A robust assay will also deal effectively with foods from either
animal or plant sources. Such techniques are unaffected by the presence of
dyes or pigments that absorb infrared, visible, or ultraviolet light. A robust
protein assay needs mimimal sample pretreatment, which increases error
and decrease analytical precision. Sample cleanup also increases the time per
analysis (reduces sample throughput) and adds to costs. In the worst-case
scenario, pretreatment can be too invasive, thereby invalidating results. In
summary, a robust protein assay is simple, quick, sensitive, and reliable. It is
also compatible with a diverse range of foods. The economic imperative
Kjeldahl Method
Accuracy
Precision, repeatability, or
reproducibility
Specicity
Reliability
Lower limit of detection (LLD)
Sample throughput (time per
analysis)
Explanation
Range over which signal is proportional to
analyte concentration
Slope of the calibration graph; analytical
response per unit change in protein
concentration. cf. parameters a, a0 in Eqs.
(1)(4)
Degree of agreement of results with a true
value
Agreement between repeated measurements
taken with a single sample or with
different paired samples
Ability to discriminate between protein and
interfering substance. Ratio of sensitivity
for the analyte and interference
A composite parameter combining
specicity, accuracy, precision, and
sensitivity
Minimal protein concentration detectable
above background noise
Numbers of samples analyzed per unit time,
speed of analysis
Chapter 1
1.2.
The two common forms of calibration are (a) method calibration and (b)
sample calibrations. With method calibration a set of food samples are
analyzed using a new test method and a reference method that has been
validated by a committee of the Association of Ofcial Analytical Chemists
(AOAC). A calibration graph is then drawn by plotting results from the
reference method (% Kjeldahl protein) on the Y-axis and the test results on
the X-axis. The Xi and Yi observations are usually related by an equation for
a straight line:
Yi aXi b
where a is the gradient and b is the intercept for the calibration graph. For
each Xi result we can determine the calculated % Kjeldahl protein value
(Ycalc) via Eq. (2).
Ycalc aXi b
Values for Yi and Ycalc can be compared in order to evaluate the test method
(see later). Some investigators choose to plot the Kjeldahl results on the Xaxis. Therefore, rather than Eq. (1) we get
Yi* a0 X * b0
where Xi* is % Kjeldahl protein and Yi* is the test result. To compare Eq.
(1) and Eq. (3), notice that a0 1/a and b0 Yi (Xi / a).
For sample calibration, the assay technique is assumed to be valid. We
analyze a set of (standard) samples containing known amounts of protein.
In Eq. (1), Xi now represents a range of known protein concentrations and
Yi are the corresponding instrument responses. Calibration factors (a, b,
etc.) can be determined from simple algebra or statistical analysis of paired
(Xi, Yi) results. From the principles of least-squares analysis,
P
Xi Xm = Yi Ym
4
a
P
X i X m 2
Kjeldahl Method
and
b Ym
aXm
where Xm and Ym are the mean values for all Xi and Yi observations.
Agreement between the reference and test results is measured by the
correlation coefcient (R); R&1 shows excellent agreement. When Yi and
Ycalc observations are poorly correlated, R & 0. The squared correlation
coefcient (R2) can be calculated from Eq. (6). Most handheld calculators
can perform this operation automatically.
"
#
Yi Ycalc 2
2
6
R 1
P
Yi Ym 2
Precision is another measure of the (dis)agreement between Yi and
Ycalc values. This can be expressed as the standard deviation (SD) or
coefcient of variation (CV). High-precision methods produce low values
for the SD and CV.
P
Yi Ycalc 2
2
7
SD
n 2
CV SD=Ym 6100
We can also measure precision (commonly called error) from n-replicate (Yi)
measurements on a single test sample. Thereafter, the numerator in Eq. (7)
becomes (Yi Ym)2, which is the square of the differences between
individual observations and the mean for all observations. A low CV
implies good agreement between successive test results.
1.3.
Assay Performance
Chapter 1
Yo 2:326SDo
a
Usually Yo and b are both set to zero when the analyst sets the instrument
baseline response to zero. Consequently, Eq. (9) becomes
LLD 2:326SDo =a
10
This relation shows that LLD decreases with increasing assay sensitivity and
with increasing baseline quality (see decrease in the value for SDo). In order
to ensure high sensitivity, it is important to obtain a stable instrumental
baseline.
1.4.
The Kjeldahl method is used for calibrating other protein assays. Duda and
Szot (6) evaluated six methods for analyzing porcine plasma protein during
its manufacture. The techniques are simple and therefore of wider interest
(Table 3). The protein content of porcine plasma was 5.58% (w/v). All
techniques showed a good correlation with Kjeldahl results (R 0.905
0.952). The precision for density and Kjeldahl assays was the same
(CV 10.8%). The sensitivity of the former method was better. With
appropriate calibration, density or viscosity measurements could be suitable
for the routine analysis during the manufacture of plasma proteins.
Instrument
Standard picnometer
Laboratory refractometer
Laboratory refractometer
UV spectrophotometer
UV spectrophotometer
UV spectrophotometer
Kjeldahl Method
Chapter 1
Test method
Analysis time
(min)
Biuret
10
Dye bindingc
Infrared
15
0.51.0
Alkaline
distillation
90
Regression line
and correlation
coefcienta
Y 0.857 cP
1.942
R 0.972
Y 1.060 cP
1.03 R 0.96
Y 1.070 cP
0.670
Standard error of
analysisb
0.336 and 0.2336
0.8381.980
2.383 and 1.540
cP, crude protein determined by Kjeldhal method (N 6 6.25); Y, response from the test
method.
P
b
Assay standard error calculated from Ycalc Yi 2 =n.
c
No information given.
Source: Ref. 8.
Kjeldahl Method
11
Digestion is complete when the mixture turns clear (light green color),
usually after 2030 minutes of heating. A further (after-boiling) period of
heating is necessary to ensure quantitative recovery of nitrogen. Data from
McKenzie and Wallace (cited in Ref. 14) show that adding X (mg) of
potassium sulfate per mL of sulfuric acid increases its boiling point
according to the relation Y (8C) 55.8X 331.2. A maximum boiling point
elevation of 1308C is achivable by adding 2 mg (potassium sulfate) per mL
acid. A high boiling point reduces the sample digestion time. Sample
digestion can also be facilitated by using a catalyst; the order of effectiveness
for metal oxide catalysts is Hg > Se > Te > Ti > Mo > Fe > Cu > V >W > Ag
(16). A proprietary brand of Kjeldahl catalyst (Kjeltabs from Foss
Electric Ltd.) comes as tablets. Each tablet contains 0.25 g of mercuric
oxide and 5 g of potassium sulfate. A working selenium catalyst can be
formulated with potassium sulfate (32 g), mercuric sulfate (5 g), and
selenium powder (1 g). Chemical oxidants (hydrogen peroxide, perchloric
acid, or chromic acid) can be added to the sulfuric acid to speed up sample
digestion.
Ammonium sulfate is rst neutralized with alkali to form ammonia.
This is then distilled and trapped using 4% boric acid. Ammonium borate is
then titrated with standard acid in the presence of a suitable indicator. Lowcost Quick-t glassware is readily available for distillation and titration.
Sophisticated semiautomatic distillation systems are also available. The
10
Chapter 1
12
13
14
Suitable titration indicators include methyl orange, methyl red, Congo red,
and Tashiro indicator (a 1:1 mixture of 0.2% methyl red solution and 0.1%
methylene blue).
2.1.
15
The units for SN are g-N 100 g 1 (g-nitrogen released per 100 g of sample).
The Fk (g-protein g 1 N) is the amount of protein that produces a gram of
nitrogen. Fk is also called the nitrogen-to-protein conversion factor. AOACrecommended values for FK for meat and other food are summarized by
Benedict (17). Frequently, FK is given a default value of 6.25 or 5.7 for
animal and plant proteins, which are assumed to have an average N content
of 16% and 17.5%, respectively. In fact, most proteins deviate signicantly
from these averages (18). FK is also affected by the presence of NPN (e.g.,
adenine, ammonia, choline, betaine, guanidine, nucleic acid, urea, free
amino acids). Soya beans have 310% NPN, which increases to about 30%
for immature seeds. The amount of NPN also changes with growth
conditions as well as with geographic factors. There is generally no
correlation between NPN and protein content (19). No single FK value
applies to all food types. Ideally, FK should be determined for each
individual food type (Table 5).
FK can be calculated from amino acid data (18,2024). Table 5 lists
the 20 naturally occurring amino acids along with their formula weight,
number of nitrogen atoms, percent nitrogen, and the value for FK. For
arginine, FK is 3.11 100=32:15. An idealized protein having all 20 amino
acids in equal numbers has a nitrogen content of 14.73%. The FK value is
therefore 6.79 (100 g/14.73). Evaluating FK from amino acid data (for
32.15
27.06
21.20
19.16
19.15
18.64
15.71
13.71
13.32
12.16
11.96
11.75
11.56
10.67
10.67
10.52
9.52
9.38
8.47
7.73
14.73
6.79
Average
FK(1)
4
%N
4
3
2
2
2
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
N atoms
FK
3.11
3.70
4.72
5.22
5.22
5.36
6.36
7.29
7.51
8.22
8.36
8.51
8.65
9.37
9.37
9.51
10.51
10.66
11.80
12.94
Sum
5
Amino
Acid FK
6.01
234.0
188.0
292
783
487.0
128.0
219.0
90.0
338.0
571.0
428.0
278.0
47.0
290.0
600.0
214.0
574.0
148.0
341.0
297.0
5669.7
6
AA composition
(mg/g N)
75.2
50.9
61.9
150.1
93.3
23.9
34.4
12.3
45.0
69.5
51.2
32.7
5.4
30.9
64.0
22.5
54.6
13.9
28.9
22.9
943.5
7
AA-N
(mg/g N)
0.001343
0.001211
0.00221
0.005359
0.003331
0.001704
0.002458
0.000441
0.003216
0.004961
0.003655
0.002334
0.000388
0.00221
0.004573
0.001608
0.003902
0.000992
0.002064
0.001639
0.04960
8
AA
(moles/g N)
0.027082
0.024422
0.044564
0.108048
0.067157
0.034362
0.049553
0.008886
0.064837
0.100016
0.073687
0.047059
0.007825
0.044563
0.092199
0.032415
0.078669
0.019999
0.041615
0.033045
1.0
9
Mole fraction
(Xi)
4.71761
3.79022
5.88694
15.7859
9.81828
2.58058
4.4152
1.81447
6.81433
11.5118
8.6288
5.60469
0.94756
5.84662
12.0964
4.3144
11.5723
2.98379
6.87481
5.98774
132.0
biX
10
a
FK(1) is determined from the average nitrogen content for all amino acids, i.e., 14.73%. FK(2) value of whole protein or sum of amino acid nitrogen
divided by sum of weight of nitrogen. FK 5669.7/943.5. Columns 810 contain data for calculating total protein content from quantitative amino
acid analysis (Sec. 4).
174.2
155.2
132.1
146.1
146.2
75.1
89.1
204.2
105.1
115.1
117.1
119.1
121.1
131.2
131.2
133.1
147.1
149.2
165.2
181.2
2
Formula
weight (bi)
Determination of the Kjeldahl Factor for Milk Protein Using Amino Acid Composition Dataa
Arginine
Histidine
Asparagine
Glutamine
Lysine
Glycine
Alanine
Tryptophan
Serine
Proline
Valine
Threonine
Cystine
Isoleucine
Leucine
Aspartic acid
Glutamic acid
Methionine
Phenylalanine
Tyrosine
1
Amino
Acid
TABLE 5
Kjeldahl Method
11
12
Chapter 1
skimmed milk) can be achieved in the following steps: (a) express each
amino acid as mg per gram of total nitrogen (see column 6 of Table 5) and
(b) calculate the mass of nitrogen derived from each amino acid (column 7 in
Table 5). FK is the weight of amino acids divided by the weight of amino
acid nitrogen (AA-N).
FK
total weight of AA
5696:7
6:01
total weight of AA N
943:5
16
Some typical values for FK are listed in Table 6 for a range of foods. The use
of FK values for quantitative amino acid analysis is discussed in Sec. 4.
Fk
6.15
6.02
6.13
5.73
5.96
5.72
5.82
5.82
5.14
5.30
5.715.75
5.615.64
5.72
5.93
5.40
5.44
5.69
Food product
Roots and tuber
Carrot
Beet
Potato
Potato protein
Fk
5.80
5.27
5.18
5.94
Fruit
Tomato
Banana
Apple
Microbial and fungal
Yeast
Mushrooms
5.78
5.61
Buckwheat
Oats
Millet
Mustard seed
Rapeseed meal
Sunower meal
Flax meal
5.53
5.50
5.68
5.40
5.53
5.36
5.41
6.26
5.32
5.72
Kjeldahl Method
2.2.
13
A.
Kaul and Sharma (25) analyzed a range of legume and cereal grains by
micro-Kjeldahl analysis. About 200 mg of each sample was weighed into
several 75-mL Kjeldahl digestion tubes. Concentrated sulfuric acid (3 mL),
hydrogen peroxide (1.5 mL), and one Kjeltab tablet were added. The tubes
were heated using a Tectator digestion block at 3748C for exactly 25 minutes
and then allowed to cool. The contents of each tube were diluted to 75 mL
and any ammonia produced quantied by colorimetric analysis (Sec. 3).
B.
Potatoes
C.
14
D.
Chapter 1
Beer Protein
17
Commenta
Kjel-Tec 1030 (Auto), Kjel-Tec 1007/ 1002*;
Kjel-Foss 16120y
0:92+0:23 g (barley or malt) range 0.51.5 g, 14+8:4
mL (beer) range 525 mL
K2SO4 CuSO4 TiO2; 3.515.8 g
16+4:3 mL, range 1025 mL
411148C, range 3804308C
75 min or 9.6 min
Mainly colorimetric
Kjeldahl Method
2.3.
15
Kjeldahl analysis has undergone three forms of automation. The KjelFoss1 instrument mechanizes the entire micro-Kjeldahl procedure (digestion, neutralization, distillation, and titration). The Kjel-Tec1 technique
uses a digestion block in conjunction with apparatus for automated
distillation and titrimetric analysis. The nal form of automation is the
Technicon AutoAnalyzer Instrument1, which uses continuous ow analysis
(CFA).
A.
16
Chapter 1
FIGURE 2 Calibration graph for the Kjel-Foss automated method for protein
determinations. (Data from Table 1 of Ref. 34.)
The Technicon AutoAnalyzer has two reaction modules (36). The rst
module digests water-dispersible samples. The digest is then pumped to a
second module (AutoAnalyzer Sampler II). Colorigenic reagents are added
in quick succession before the ow stream passes to a delay coil to allow
color formation. Ammonia is detected using Berthelot's reaction or the
ninhydrin assay (Sec. 3). The AutoAnalyzer was applied for protein
determinations in plant material (37), feedstuffs (38), grain our (39),
instant breakfasts, meat analogues (40), meat products (41), and over 40
assorted canned and processed foods (42,43). In general AutoAnalyzer
results agreed with micro-Kjeldahl analysis.
The AutoAnalyzer digestion unit is heated in two stages at 3804008C
and 3003208C. To achieve efcient digestion, the ratio of acid to sample is
Kjeldahl Method
17
Kjel-Foss (% protein)
Kjeldahl (% protein)
14.64
14.42
16.76
16.64
16.01
14.54
14.43
16.60
16.74
16.02
14.40
14.77
17.42
14.09
14.52
14.80
17.33
14.21
14.33
18.87
14.41
18.86
higher than normal for batch digestion. A superheated layer of acid forms,
which facilitates sample digestion (44). Later tests showed that the recovery
of nitrogen from refractory materials (arginine, creatine, or nicotinic acid)
was only 70%. Davidson, et al. (45) concluded that the AutoAnalyzer
digestion module was not reliable if an accuracy of 1% was desired for
Kjeldahl analysis. Over 70 different animal feeds (corn grain, wheat, barley,
rice, alfalfa, mixed feeds, feed concentrates) were analyzed using the
AutoAnalyzer digestion module. The recovery of nitrogen was 8890% (46).
In contrast, using a Technicon block digestor followed by AutoAnalyzer
Sampler II led to 100% recovery of nitrogen from cattle supplement, swine
ration, pig starter, and poultry ration (47). Suitable catalysts include copper
sulfate and oxides of mercury, selenium, or titanium. Ammonia was
detected using alkaline phenol reagent (Sec. 3.1). Quantitative recovery of
nitrogen was also demonstrated by Kaul and Sharma (25), who used a
Tectator1 heating block to digest 23 assorted strains of rice and 15 other
cereal-legume mixtures. The electrically heated block digests 40 samples per
hour under controlled temperature conditions. Samples were then transferred to the AutoAnalyzer Sampler II for ammonia detection using the
alkaline phenol reagent.
18
3.
Chapter 1
The alkali-phenol reagent is frequently used for the Technicon AutoAnalyzer. Under alkaline conditions, ammonia, sodium hypochlorite, and
phenol react to form a blue product. Berthelot rst reported this reaction in
1859. The principles of the APR assay have been reviewed (4851) although
the underlying reactions remain uncertain. Ammonia probably reacts with
hypochlorite to form chloramine (NH2Cl). This reacts with phenol to form
N-chloro-p-hydroxybenzoquinone monoimine or quinochloroamine (I).
NH3 OCl ?NH2 Cl
PhO NH2 Cl?I
PhO I?II
Alternatively, ammonia may rst react with phenol to form paminophenol, which is then oxidized by hypochlorite to form (I).
Irrespective of how (I) forms, it reacts with 1 mole of phenol to form
indophenol (II). The yellow compound ionizes under strongly alkaline
conditions (pKa 13.4) giving a blue anion De 1 26104 L mol 1 cm 1 .
Substituted phenols (o-chlorophenol, m-cresol, and guaiacol) undergo the
indophenol reaction. However, para-substituted phenols and some metaderivatives do not react. Color intensity and the rate of color formation
increase in the presence of manganese (2) ions or acetone. Sodium
nitroprusside (1040 mg L 1) is another catalyst.
A simple APR assay suitable for detecting 3 ppm ammonia is
described in Ref. 48 (Fig. 3). Indophenol formation is pH and temperature
dependent. The linear dynamic range for ammonia was 0.33 ppm with a
sensitivity of 0.3284 (absorbance units/1 ppm NH3). The assay precision (for
1 ppm NH3) was +3%. Although performed with boric acid as the
background medium, the simple APR assay is probably not suitable for
Kjeldahl Method
FIGURE 3
19
Kjeldahl-N determination. Copper, zinc, and iron salts were found to act as
interferences.
Tetlow and Wilson (49) added ethylenediaminetetraacetic acid
(EDTA) to APR to reduce metal ion interference. Temperature control
was also improved using a thermostated water bath. An outline protocol is
described below.
Method 1
Analysis of ammonia using the APR assay (48,49).
Reagents
1. Phenol (crystalline, 85% pure)
2. Sodium hydroxide solution (5 N)
3. Sodium hypochlorite solution (or commercial bleach)
4. Ammonium chloride solid
5. EDTA (6% w/v)
6. Acetone
20
Chapter 1
Procedure
Preparation of alkali-phenol reagent. Place 62.5 g of solid phenol in a
500-mL beaker and add 135 mL of sodium hydroxide (5 N) slowly
with stirring. Caution: Use an ice bath to avoid excessive heat
buildup. Add 12 mL of acetone and make up the volume to 500 mL
with deionized water.
Sodium hypochlorite (1% w/v available chloride). Prepare by diluting
commercially available bleach.
Ammonium chloride standards (1000 ppm NH3 and 100 ppm NH3).
Dissolve 314.1 mg of solid NH4Cl in 100 mL of water and then dilute
10-fold. Prepare a working standard solution (0.5 ppm NH3) daily.
The APR assay sequence. Place 1 mL of sample (or standard) in a test
tube. Add EDTA solution (100 mL) with gentle shaking. Next, add
APR (1 mL) and hypochlorite (0.5 mL) in quick succession, mixing
after each addition. Finally, add 2.5 mL of water and incubate at
258C for 60 minutes. Take A625 readings for samples. Prepare a
reagent blank as described next.
Reagent blank (reverse addition method). First, mix hypochlorite
(0.5 mL) and APR (1 mL) solutions and allow to react for 510
minutes. Next add EDTA (100 mL) followed by 3.4 mL of water (or
the designated blank solution).
When reverse addition is used, hypochlorite reacts with phenol rst.
Traces of NH3 present in the blank are not detected (48,49). Reverse addition
is useful where ammonia-free water is not available for sample preparation.
After optimization, the linear dynamic range for ammonia analysis was 50
500 ppb. Assay sensitivity was 200% greater than the results shown in Fig. 2.
Color formation with 50800 ppb NH3 was virtually complete after 15
minutes at 14308C. Temperature variations had little effect on the reaction.
Thermostating at 258C for 60 minutes improved the precision.
Addition of acetone to APR increased the response to ammonia by 10fold. The color yield with 500 ppb ammonia declined by 2.65%, 4.8%, and
6.8% for 4.5-hour-, 1-day- or 5-day-old APR. Addition of EDTA prevented
interference from 100 ppb copper. The intervals between addition of various
reagents must not exceed 1 minute to ensure optimum precision. Comparing
results for normal and reverse addition provides a means for detecting very
small amounts of NH3 in samplessuch as water. Otherwise, ammonia-free
water is needed for preparing reagents and blanks. The calibration curve for
the APR assay is described by the equation A625 0.7120X, where X is the
concentration of nitrogen (ppm). The analytical sensitivity was 0.7120
(absorbance units) for 1 ppm ammonia. The SD for the reagent blank was
+0:0005 ppm. These values lead to an expected LLD for ammonia of 1.6
Kjeldahl Method
21
TABLE 9 The Comparative Costs of Manual APR Assay and Other Techniquesa
Technique
Manual APR
method
Micro-Kjeldahl
AutoAnalyzer
APR test
Capital
costb
Running cost
per yearc
Analysis
per year
Cost per
analysisd
6000 (1)
5200 (1)
8000
0.72 (1)
12000 (2)
81,000 (13.5)
7000 (1.3)
17,000 (3.3)
2000
32,000
4.1 (5.4)
0.78 (1)
Costs are given in deutsche marks. At current exchange rate 2.8 DM $1.4 1. All methods
employ a digestion unit costing DM6000.
b
Capital costs for the micro-Kjeldahl method include the cost of a distillation unit and an
autotitrator.
c
The running cost includes DM5000 for miscellaneous chemicals.
d
Calculated for a 10-year period as capital cost/10 running cost)/no. of samples. Ratios of
costs are given in parentheses for each column.
ppb. Assuming a default FK of 6.25, the LLD for protein is 10 ppb. The
APR assay is widely used in conjunction with the AutoAnalyzer.The
composition of the APR used in CFA is pretty much the same as described
earlier (45,52).
Kaul and Sharma (25) describe a rare attempt to deploy a manual
Kjeldahl-APR assay for protein analysis. They used a Tectator heating
block for micro-Kjeldahl digestion of grain. Sample nitrogen was then
analyzed by the APR assay. The analytical performance was similar to
results obtained with the AutoAnalyzer-APR assay or the conventional
micro-Kjeldahl analysis. From Table 9, the capital cost for the manual
Kjeldahl-APR assay was 2 times lower than for the micro-Kjeldahl and 13.5
times lower than for the AutoAnalyzer method. Running costs were also
lowest for the manual APR assay.
For laboratories handling 40 or more analyses per day, it may be
worth investing in an automated technique. The manual Kjeldahl-APR
analysis was advantageous for small laboratories lacking the wherewithal to
purchase an AutoAnalyzer. Mohyuddin and Mazza (53) used the manual
Kjeldahl-APR assay to analyze potatoes (see Sec. II.B.2). The mean protein
content for 14 potato cultivars was 10.65 (+1.23)% by the manual APR
assay and 10.53 (+1.13)% using the AutoAnalyzer.
3.2.
Nessler's Reagent
22
Chapter 1
18
3.3.
Acetylacetone-Formaldehyde Reagent
19
Kjeldahl Method
23
Method 2
Determination of ammonia using acetylacetone-formaldehyde reagent (55).
Reagents
Acetylacetone-formaldehyde reagent. Place 15 mL of formaldehyde
(37% w/v) and acetylacetone (7.8 mL) into a 100-mL ask. Make up
to 100 mL with distilled water.
Sodium acetate (2M). Dissolve sodium acetate (82 g) in 1 L of distilled
water.
Procedure
Add prediluted Kjeldahl digest (< 2 mL; 25100 mg N) to a 25-mL
conical ask followed by sodium acetate solution (3 mL) and
acetylacetone-formaldehyde reagent (3 mL). Incubate the mixture at
97.88C for 15 minutes and cool to room temperature.
Bring the total volume to 25 mL and record A412 values using a 1-cm
cuvette.
The linear dynamic range for the preceding assay was 0.56.0 mg N
(per nal reaction mixture). The calibration graph was described by
A412 9:8610 2 X 4:2610 3 R2 0:9999, where X is the amount of
nitrogen present in the nal (25-mL) reaction mixture. The new method
shows levels of accuracy and precision equal to those of the micro-Kjeldahl
method.
3.4.
Ninhydrin* reacts with amino acids (Fig. 4) in two stages: (a) the amino acid
is oxidized to aldehyde and ammonia while ninhydrin is converted to
hydrindantin and (b) hydrindantin and 1 mole of ninhydrin react with
ammonia to form Ruhemann's purple (56,57). For ammonia determination
an added reducing agent is necessary to convert ninhydrin to hydrindantin.
Ninhydrin solution is available commercially. Results from the ninhydrinKjeldahl assay agree closely with those from Kjeldahl analysis (5658). The
linear dynamic range for colorimetric Kjeldahl assay depends on the extent
of sample dilution just before ninhydrin analysis. A 2-mL standard
ammonium sulfate solution containing 5.6 mg ( or 2.8 ppm) reacted with
2 mL of ninhydrin solution yields an A570 reading of 0.805. Interference was
noted for concentrations of selenium above 86 mg mL 1 (prediluted digest).
No interferences were observed with Fe, Zn, Pb, Cu, Ca, Ba, Al, Mg, Co, or
* Ninhydrin is often encountered in detective novels as a reagent for ngerprint analysis.
24
Chapter 1
(a)
(b)
FIGURE 4
Kjeldahl Method
FIGURE 5
25
26
4.1.
Chapter 1
Crude protein (cP) is expressed by Eq. (20), where Ci (mole) is the amount of
each amino acid in the sample and bi (g mole 1), is the formula weight for
each amino acid.
cP
20
X
Ci bi
20
i1
However, amino acid proles are reported in terms of mole fraction of each
amino acid (Xi):
Xi Ci =Cnet
21
where Cnet is the net concentration of amino acids found in the sample.
Substituting Ci Cnet Xi in Eq. (20),
cP Cnet
20
X
bi Xi
22
i1
P
The term (biXi) is called the mean residue weight, W ( g mole 1).
This is the average formula weight for all amino acids in the sample adjusted
for their frequency.*
X
W g mole 1
bi Xi
23
and
cP WCnet
or
cP FCnet
24
Cnet
i1
18
P
bi Ci
CPro
CTrp
or
i1
bi Xi
XPro
XTrp
25
where XPro and XTrp are the mole fractions of proline and tryptophan. For a
* Horstmann called this parameter the weight equivalent (WE).
Kjeldahl Method
27
range of meat products, F (g mole 1) was 1020% larger than W [Eq. (23)].
Calculations of F (g mole 1) appear in last three columns of Table 5. Typical
values for F (g mole 1) are given in Table 10. Values for F range from 100 to
125 g mole 1 for most proteins.Most protein sources are now routinely
analyzed for amino acid scores during nutritional evaluations (Chapter 14).
This yields all the information necessary for total protein estimation.
Zarkadas and co-workers in Canada are strong exponents of quantitative
amino acid analysis (Table 11).
4.2.
1000Wj
nj M i
26
F (g mole 1)
129.84
119.82
118.85
112.70
107.06109.01
94.02
104.21
114.43
114.48
115.69
113.13116.00
108.4108.52
118.43
108.52
112.98
28
Chapter 1
TABLE 11
Sample/comments
NASASkylab meals
Mushroom total protein
Porcine muscle and connective tissue
proteins (myosin, actin, elastin, and
collagen
Actin, myosin, and collagen in
composite meat products; mixed meat
sausages, bologna, frankfurters,
sausages, hamburgers
Additives and ingredients for meat
products including soybean, wheat
products, potato protein
Porcine skin (rind)
Chicken meat and connective tissue
Apple ower buds
Bone isolates
New soybean cultivars
Reference
Heidelbaugh et al. (59)
Weaver et al. (62), Braaksma and
Schaap (63)
Zarkadas et al. (64), Zarkadas et al. (65)
Karatzas and Zarkadas (66)
27
Similarly, the concentration of Nt-methylhistidine and 4-hydroxyproline was the basis for assessing the amount of myobrillar protein and
connective tissue (collagen and elastin) in meat. These methods are
satisfactory for meat and meat products. They may have questionable
validity for composite foods. Plant foods may contain 4-hydroxyproline
rich glycoproteins (extensins, lectins, salt-extractable glycoproteins). For
example, alfalfa protein and potato protein contain signicant levels of
4OH-Pro.
Kjeldahl Method
4.3.
29
30
Chapter 1
Samplea
Animal feedstuffs, fertilizers
Beer
Brewing grainsbarley, malt, rice
Cereal grainswheat, barley, corn,
sorghum
Dairy productsskimmed, powdered
milk etc. chocolate milkshake,
cheeses, etc.
Fruitguava, peaches, plum
Infant food
Meat and meat products, sh (raw, sh
in oil, tuna)
Oilseeds (soybean, canola, sunower,
corn)
Potatoes
Vegetablescabbages, broccoli,
ketchup, tomato
a
Reference
Sweeney and Rexroad (78), Schmitter
and Rhihs (79), Sweeney (80), Sachen
and Thiex (81), Tate (82)
ASBC (83), Johnson and Johansson
(84,85)
ASBC (86), Buckee (28,87), Krotz et al.
(88), Johansson (89), Angelino et al.
(90)
Bicsak (91), Bicsak (92), Williams et al.
(93)
Wiles and Gray (94), Wiles et al. (95),
Simonne et al. (96,97)
Simonne et al. (96,97), Huang et al. (98)
Bellemonte et al. (99)
King-Brink and Sebranek (100),
Simonne et al. (96,97), Buschmann
and Westphal (101)
Bicsak (91), Duan and DeClercq (102),
Berner and Brown (103)
Young et al. (104)
Simonne et al. (96,97)
Kjeldahl Method
TABLE 13
1.
2.
3.
4.
5.
6.
7.
8.
31
1994 and 1996, respectively (9193). Trials for the combustion method
usually follow guidelines described by Youden and Sleiner (105):
1. The number of laboratories ranges from 7 to 12. Studies involving
as few as three laboratories have been reported.
2. All studies compare CNAs with Kjeldahl analysis.
3. Interlaboratory studies focus on a single food group. Therefore,
CNAs tend to receive approval for one food group at a time
(Table 12).
4. Trials usually test a ``generic combustion method'' and are
independent of the choice of instruments.
Minimum performance guidelines for CNA instruments include (a) a
furnace temperature of 9508C, (b) a separation system for trapping CO2 and
water, (c) a thermal conductivity detector for nitrogen, (d) sufcient
accuracy to produce results within 0.15% of the mean (% nitrogen) results
for 10 successive measurements using a standard compound, and (e)
sufcient precision to produce a relative standard deviation of 0.01%.The
LECO FP-428 analyzer was used by about 80% of the laboratories involved
in collaborative trials. The Foss-Heraue Macro-N analyzer, Carlo Erba
NA-5000, and Perkin Elmer PE2410 also feature. The LECO FP-2000
combustion analyzer appears in the latest trials.
5.2.
The modern CNA has advantages over the Kjeldahl method (Table 13).
There is greater speed of analysis and greater operator safety stemming from
the nonuse of aggressive chemicals. The estimated cost for analysis is $0.37
$0.50 per sample with the LECO FP-2000 protein analyzer (LECO
Corporation, Saint Joseph, MI) compared with $1.0 per test for the
Kjeldahl method (106109).
32
Chapter 1
Kjeldahl
Dumas
Chemical
requirements
Other suppliesa
Ancillary equipment
Disposal of chemical
Time per analysis
Degree of hazardc
Accuracy
Precision (CV %)
a
Does not include main equipment (Kjeldahl digester and distillation apparatus, or CNA
instrument
b
Optional, but advisable for large-scale testing.
c
Arbitrary scale of 110, with 10 being extremely hazardous and 1 completely safe. There may
be a risk of burns when maintaining the combustion instrument.
Kjeldahl Method
5.3.
33
Combustion analysis rst received AOAC approval for feeds in 1968. The
classical instruments (Coleman model 29A nitrogen analyzer) used a
manometer for the volumetric assay of nitrogen (110). Comprehensive
testing using modern TCD-based CNAs appeared in 1987 (78). A ninelaboratory collaborative trial to determine nitrogen in feeds was successfully
completed in 1989 (80). The AOAC approved CNAs for animal feed testing
in 1990.
The small sample sizes (150500 mg) used with modern CNAs raised
concerns about sampling. Extensive grinding and mixing are essential to
ensure sample homogeneity and representative sampling. Sweeney and
Rexroad (78) analyzed 14 different animal feeds using the LECO FP-228
instrument with a prescribed sample size of <150 mg. Estimates of feed
nitrogen agreed closely with results from Kjeldahl analysis. The precision of
analysis was signicantly lower (0.0130.052%) for the combustion method
as compared with Kjeldahl analysis (0.0060.035%). Schmitter and Rihs (79)
increased the sample size for the LECO-F228 analyzer from 150 mg to 1 g by
palletizing before loading into the instrument port. Adding a few drops of
polyethylene (2% w/w in ethyl acetate solvent) prevented aking of the
pellets. Table 15 shows nitrogen and protein data for feeds determined using
the CNA (78,79). Results have been averaged for samples of sizes 0.151.0 g.
Protein values are calculated as %N 6 6.25. The results agree favorably with
Kjeldahl analysis (Fig. 6). There appeared to be signicant positive bias for
feedstuffs having < 2% nitrogen (Fig. 7). The bias was ascribed to plantderived materials containing high levels of nitrate. The Kjeldahl method
achieves low recoveries of nitrogen from refractory compounds (75) with
N22O or N22N bonds (nitrite; nitrate; oximes; azo-, nitro-, nitrosocompounds).
Sachen and Thiex (81) found that CNAs showed a 1.38% (protein)
bias for hay samples. They attributed such results to ``atmospheric error''
arising from air being trapped in the interstices of the (uffy) hay samples.
Compressing samples to remove trapped air led to agreement between the
Kjeldahl and CNA results (81). The LECO FP-2000 nitrogen analyzer was
not subject to an atmospheric blank because of improvements in instrument
design and efcient purging of atmospheric gases before sample combustion.
Sachen and Thiex examined a range of pelleting equipment and procedures
for eliminating the atmospheric blank for the LECO FP-428 instrument.
They proposed that powdered cellulose could be analyzed to check for an
atmospheric error (81). Not all investigators agree about the nature of the
atmospheric error.
34
TABLE 15
Chapter 1
Nitrogen and Protein in Feedstuffs Determined by Combustion Method
Sample
Straw
Corn silage
Porc soup
Hay
Corn grain
Barley
Grass silage
Oats
Triticale
Wheat
Dried grass
Cow premix
Alfafa pellets
Hog feed
Broiler nisher
Milk powder
Bone meal
Rapeseed
Protein conc.
Soybean meal
Cattle conc.
Yeast
Peanut meal
Meat meal
Fish meal
Gluten
Feather meal
Soy protein conc.
N (g kg 1)
Protein (%)
0.620
1.175
1.185
1.280
1.375
1.515
1.835
1.760
1.835
1.985
2.240
2.755
2.770
3.390
3.420
4.365
4.325
5.710
6.045
6.485
6.690
6.895
8.450
9.300
9.960
11.730
13.610
14.020
3.88
7.34
7.41
8.00
8.59
9.47
11.47
11.00
11.47
12.41
14.00
17.22
17.31
21.19
21.38
27.28
27.03
35.69
37.78
40.53
20.91
43.09
52.81
58.13
62.25
73.31
85.06
87.63
Kjeldahl Method
35
FIGURE 6 Comparison of protein results for feeds determined using the combustion
method and the Kjeldahl method. Micro-CNA and macro-CNA refer to
the use of 150-mg and 1-g sample sizes with the combustion nitrogen
analyzer. List of feedstuffs is given in Table 15. (Data derived from Ref.
78.)
36
Chapter 1
5.4.
Kjeldahl Method
5.5.
37
Despite the recent widespread use of CNAs for food protein analysis, few
applications in the dairy eld have been published. Wiles et al. (95)
described an 11-member interlaboratory study from New Zealand. They
compared milk protein analysis by CNAs and the Kjeldahl method. Samples
included ultra-heat-treated (UHT) whole milk, infant formulas, whole milk
powder, skimmed milk powder, whey protein concentrate, casein, and
sodium caseinate. Nine of the eleven laboratories employed the LECO FP428 instrument. Results for CNAs agreed closely with Kjeldahl ndings.
There was no systematic bias associated with the former results. Indeed, no
evidence was found for a systematic difference between CNA and Kjeldahl
results reported between 1968 and 1997 (95).
5.6.
Bellemonte et al. (99) analyzed ve categories of baby foods using the Carlo
Erba model 1500 nitrogen analyzer. This instrument uses a high furnace
temperature (18008C) combined with an oxygen-rich atmosphere to achieve
complete sample combustion. Nitrogen is quantied using a TCD. The
analysis time for this instrument is reportedly 3 minutes. All sample types
were analyzed successfully. Results obtained by the Kjeldahl method were
14% lower than those obtained with CNAs (Table 16). Results from both
techniques compared favorably with protein values declared by food
manufacturers. Compared with the Kjeldahl method, CNAs are convenient
for baby food analysis. The sample throughput and safety considerations
favor the Dumas method as described in Sec. 5.2.
5.7.
Meat
38
Chapter 1
TABLE 16
Samplea
Declared
protein
Kjeldahlb
Dumasb
15.55
10.28
14.94
10.12
15.36
10.35
9.50
54.20
9.40
52.15
14.70
53.20
9.52
9.62
9.72
a
Numbers in parentheses represent number of different foods in the category analyzed. Protein
values are averaged for each food category
b
Protein values were calculated with a conversion factor (Fk) 6.38 for milk formula or 6.25 for
all other categories of baby food.
Germany. This trial, too, concluded that the performance of the Dumas
method was comparable to that of the Kjeldahl assay but that the former
method was quicker and more environment friendly (101).
REFERENCES
1.
2.
3.
4.
5.
6.
Kjeldahl Method
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
39
40
Chapter 1
Kjeldahl Method
41
42
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
Chapter 1
tissue proteins in the bovine diaphragm. J Agric Food Chem 36:11051108,
1988.
CG Zarkadas, N Karatzas, AD Khalili, S Khanizadeh, G Morin. Quantitative
determination of the myobrillar proteins and connective tissue content in
selected porcine skeletal muscles. J Agric Food Chem 36:11311146, 1988.
CN Karatzas, CG Zakardas. Determination of the myobrillar and
connective tissue protein contents and amino acid composition of selected
composite meat products. J Agric Food Chem 36:11091121, 1988.
CG Zarkadas, NJ Drouliscos, CN Karatzas. Comparison of the total protein,
nitrogen and amino acid composition of selected additives and ingredients
used in composite meat products. J Agric Food Chem 36:11211131, 1988.
Q Nguyen, MA Fanous, LH Kamma, AD Khalili, PH Schuepp, CG
Zarkadas. Comparison of the amino acid composition of two commercial
porcine skins (rind). J Agric Food Chem 34:565572, 1989.
CN Karatzas, CG Zarkadas. Comparison of the amino acid composition of
the intracellular and extracellular matrix protein fractions isolated from avian
skeletal muscles. Poult Sci 68:811824, 1989.
S Khanizadeh, D Buszar, CG Zarkadas. Comparison of three methods for
calculating protein content in developing apple ower buds. J Assoc Off Anal
Chem 75:734737, 1992.
CG Zarkadas, Y Ziran, GC Zarkadas, A Minero-Amador. Assessment of the
protein quality of beefstock bone isolates for use as an ingredient in meat and
poultry products. J Agric Food Chem 43:7783, 1995.
CG Zarkadas, HD Voldeng, YI Ziran, Keijin-Shang, PL Pattsion, Comparison of the protein quality of ve new northern adapted natto soybean
cultivars by amino acid analysis. J Agric Food Chem 45:20132019, 1997.
S Khanizadeh, D Buszard, CG Zarkadas. Misuse of the Kjeldahl method for
estimating protein content in plant tissue. HortScience 30:13411342, 1995.
R Fiedler, G Proksch, A Koepf. The determination of total nitrogen in plant
materials with an automatic nitrogen analyzer. Anal Chim Acta 63:435443,
1973.
DW Nelson, LL Sommers. Total nitrogen analysis of soil and plant tissue. J
Assoc Off Anal Chem 63:770778, 1980.
YS Lee, CE Damon, JP Crisler. A micro method for the determination of
protein and screening of added water in meat and meat products. Mikrochim
Acta 4:477483, 1972.
TL Lunder. Determination of total nitrogen in foodstuffs according to
Dumas, by means of the Micro-Rapid N automatic analyzer. Lab Pract
23(4):170172, 1974.
RA Sweeney, PR Rexroad. Comparison of LECO FP-228 ``Nitrogen
Determinator'' with AOAC copper catalyst Kjeldahl method for crude
protein. J Assoc Off Anal Chem 70:10281030, 1987.
BM Schmitter, R Rihs. Evaluation of a macrocombustion method for total
nitrogen determination in feedstuffs. J Agric Food Chem 37:992994, 1989.
Kjeldahl Method
43
44
Chapter 1
Kjeldahl Method
45
110. ME Ebeling. The Dumas method for nitrogen in feeds. J Assoc Off Anal
Chem 51:766770, 1968.
111. R Winker, S Botterbrodt, E Rabe, MG Linhauer. Nitrogen/protein
determination in wheat and wheat products (our and meal) with the Dumas
method. Getreide Mehl Brot 54(2):8691, 2000.
112. MG D'Egidio, C Cecchini, G Novembre. Comparison of Dumas and Kjeldahl
methods for determination of crude protein in cereal. Tec Molitoria 50:1189
1195.
2
The Alkaline Copper Reagent:
Biuret Assay
1. INTRODUCTION
Copper (Cu2) ions react with proteins to form a blue complex at high pH.
The intensity of the color is proportional to the amount of protein present.
The oldest copper binding technique for protein analysis is called the biuret
method. The name, although quite deeply entrenched in the literature, is
unfortunate. The assay has little to do with biuret. A more accurate name is
the alkaline copper reagent (ACR) test. Older literature features almost
exclusively the ``biuret reagent.'' Depending on the context, both these
names will be used. First we will discuss the history and developments of the
ACR assay. Modern and (where relevant) old protocols involving ACR are
described. Applications in food protein analysis are then considered.
Berthelot reported that biuret (NH2CONHCONH2) forms a blue
complex with copper ions under alkaline conditions. Proteins were reported
to undergo a similar reaction in 1873. Early developments of the biuret
method are described by Hiller (1) and also by Robinson and Hodgen (2).
The two publications are some of the earliest accounts of the ACR assay.
Blood plasma was diluted with 25 volumes of 0.9% saline and adjusted to
10% (w/v) TCA. The protein precipitate formed was recovered by ltration
and redissolved in 3% sodium hydroxide solution (5 mL). Then copper
47
48
Chapter 2
sulfate solution (20% w/w; 2.5 mL) was added. After 12 hours the brown
precipitate (Cu2 hydroxide) produced was removed by centrifugation. A
linear relationship was found between DA560 readings and protein
concentration (00.2 mg mL 1). The assay sensitivity was 6 DA560 units
per (mg mL 1) protein. The formation of copper (hydro)oxide precipitate
was a nuisance. Samples had to be centrifuged before DA560 readings could
be taken.
2.
Mehl (3) added ethylene glycol to ACR to arrest the formation of Cu2
hydroxide. Ethylene glycol (100 mL), 60% (w/v) sodium hydroxide (40 mL),
and 4% (w/v) copper sulfate were mixed and the solution was diluted to
400 mL with water. The mixture was heated to precipitate copper hydroxide
and then ltered. Extra sodium hydroxide was added to bring the nal
concentration to 1011% (w/w). The resulting ACR was stable for several
months. Dissolved egg albumin ( 3 mg mL 1) was analyzed by mixing with
an equal volume of ACR and taking DA550 readings after 90 minutes. The
blue color, once formed, was stable for about 20 hours. Sols (4) used
glycerin as a stabilizer for ACR. The amount of glycerin required was 100
times lower than ethylene glycol. ACR solution was prepared by mixing
glycerin (2 mL), copper sulfate (5% w/v; 80 mL), and sodium hydroxide
(20% w/v; 400 mL) and making up to 1 L with water. To perform analysis,
1 mL of test solution (< 12% w/v protein) was mixed with an equal volume
of ACR solution. Colorimetric readings were recorded at two wavelengths,
accounting for the unpopularity of the procedure.
Weichselbaum (5) produced a modied ACR by adding potassium
sodium tartrate as the stabilizer for Cu2. Gornall et al. (6) evaluated several
modied ACR solutions, nding Mehl's copper reagent difcult to
reproduce. Weichelsbaum also considered the effect of changing copper
sulfate and sodium hydroxide concentrations and the reaction temperature
on color formation. This work revealed some tolerance for variations in
regent composition and experimental conditions.
The ACR developed by Weichselbaum was employed by Gornall et al.
for protein analysis in clinical samples. This variant ACR became more
popular after Layne's account of it appeared in Methods in Enzymology in
1957 (7). ACR is now widely used for food protein analysis. A summary of
this popular method is given next.
49
Method 1
Protein analysis using alkaline copper reagent (5,6)
Reagents
1. Copper sulfate CuSO4 5H2 O
2. Rochelle salt (potassium sodium tartrate)
3. Sodium hydroxide (10% w/w solution)
Preparation of ACR. Dissolve copper sulfate (1.5 g) and potassium
sodium tartrate (6 g) in 500 mL of water. Add 300 mL of sodium
hydroxide (10% w/w) solution with constant mixing. Adjust the
total volume to 1 L with distilled water. This solution should keep
indenitely. Discard if it shows signs of forming a black or reddish
precipitate. The nal concentrations of copper sulfate and sodium
hydroxide are 0.015% and 3% w/v, respectively.
Procedure
Add 8 mL of ACR solution to 2 mL of protein solution. Mix and allow
to react for 30 minutes. Record DA540 using a 1-cm cuvette.
Dilute plasma (protein) in 0.9% saline before analysis. To separate the
albumin and globulin, dilute 0.5 mL of plasma with 9.5 mL of
sodium sulfate solution (22.6% w/v). Remove 2 mL of the solution
for biuret analysis of total plasma protein. Add 3 mL of ether to the
remaining solution, mix for 30 seconds, and centrifuge. Remove
2 mL of the supernatant and analyze to nd the plasma albumin
concentration. The difference (total protein plasma albumin) is
the plasma globulin concentration.
The linear range for protein analysis with Method 1 was 110% (w/v)
protein. The results were strongly correlated with Kjeldahl nitrogen.
TABLE 1 Effect of Different Proteins on
the Color Yield from the ACR Assay
Protein
Serum albumin
Serum globulin
Egg albumin
Casein
Gelatin
Zein
Source: Ref. 6.
Relative color
intensity (%)
100
98.8
98.6
88.1
74.2
92.1
50
Chapter 2
Different proteins gave slightly different color yields (Table 1). Casein, zein,
and gelatin produce low amounts of color compared with serum albumin.
The biuret method cannot be used in the presence of ammonium salts. The
ACR formulation is one of the most popular modications of the biuret
reagent and is the standard method for clinical samples (8). The ACR
formulation is also widely used for the analysis of animal proteins.
3.
Strickland et al. (9) titrated proteins with increasing amounts of ACR. After
each addition, the absorption spectrum was recorded between 425 and
850 nm. Sparingly soluble proteins were dissolved in 8 M urea, which had no
deleterious effects on color formation. By plotting the maximum absorbance
change versus copper concentration, the maximum amount of protein
required to bind all Cu2 was found.
Table 2 shows that lmax for ACR-protein mixtures is 535590 nm. The
extinction coefcient for the Cu2-protein complex (De, M 1 cm 1 with
respect to protein concentration) increased with the molecular weight and
the number of peptide groups per protein. The amount of Cu2 bound to a
xed weight of protein is shown in Table 2. Note that gliadin and gelatin
exhibit low color yields per unit weight of protein. This characteristic is
partly related to the high proline content in these proteins. Other amino acid
side chains also alter the specic color yield from protein-Cu2 binding.
Cysteine binds copper strongly, forming Cu-mercaptide. Therefore cysteinerich proteins show a reduced color yield during ACR analysis. High
concentrations of glutamate and aspartate compete with the peptide
backbone for Cu2 binding.
Cu2 binding with biuret, glycinamide, and glycine oligopeptides was
reviewed by Sigel and Martin (10). Cu2 binding reects the acid-base
properties of the peptide bond. Delocalization of the peptide nitrogen lonepair electron leads to a planar geometry with partial double-bond character.
The resulting canonical structures have a high electron density at the peptide
oxygen. The amide hydrogen acts as a weak acid, becoming ionized only
under very alkaline conditions (Fig. 1). Binding of Cu2 to the peptide
group occurs via oxygen at neutral pH. In a highly alkaline medium
(pH 13), complexation involves the nitrogen group. Under alkaline
conditions, 2 moles of biuret bind with each Cu2 atom, forming a sixmember ring (Fig. 2). Cu2 forms a tetradentate complex. The amide
nitrogen becomes deprotonated in the process. Peptide-metal ion binding at
high pH values occurs in competition with metal ion hydrolysis. Cu2
515
590
555
544
535
554
545
550
45,000
75,000
180,000
310,000
max
103
189
27,000
36,800
Molecular
weight
163.6
150.0
144.2
154.0
27.8
90.3
143.4
151.4
"a
per M
Biuret
Triglycine
Gliadin
b-Lactoglobulin
Zein
Gelatin
Serum
globulin
Edestin
Test
sample
TABLE 2
635
638
670
586
206
189
700
620
[P] (g/Cu)
6.0
6.0
6.3
5.0
4.0
3.0
6.0
6.0
Peptides
per Cu
79,867.7
10,579.9
16,141.8
47,303.8
13.9
90.3
5,531.1
8,986.3
"c
per M
488.2
70.5
111.9
307.2
0.5
1.0
38.6
59.4
Cu atoms
(mole
ligand) 1
257.64
235.11
215.22
262.80
134.95
477.78
204.86
244.19
"d
per kg
52
FIGURE 1
Chapter 2
Resonance hybrid forms of the peptide bond. Structures (1) and (2)
predominate under physiological conditions at a ratio of 60:40. At neutral
pH the electron density is highest at the oxygen atom. At the pH of the
biuret reaction (pH 14), deprotonation of the amide nitrogen leads to
signicant amounts of (3) with high electron density at the nitrogen atom.
binding with the OH ion leads to an insoluble precipitate. A model for the
2:1 biuret- Cu2 complex at high pH is shown in Fig. 2.
Three kinds of Cu2 complexes are formed with polypeptides. At low
pH Cu2 interacts exclusively with side-chain residues forming ``type S''
complexes. At intermediate pH values there is binding to both side chains
53
4. INTERFERENCE COMPOUNDS
ACR dissolves extraneous plant colors and dyes with a high absorbance
at 530550 nm (11).* To overcome this problem during ACR analysis,
Jennings (11) diluted samples with high backgroud readings 100-fold using
standard Lowry reagent A (see Chapter 4). The samples were then
analyzed by treating with Lowry reagent B (11). Pinckney (12) reduced the
interference from phopholipid by adding carbon tetrachloride to ground
meals before analysis. Johnson and Craney (13) noted that isopropanol
reduced the interfering effects from plant dyes. Some of the substances
capable of interfering with the ACR method are listed in Table 3.
Possible interferences during wheat protein analysis using ACR were
investigated by Mitsuda and Mitsunaga (14). They extracted whole wheat
our or bran with 0.5% (w/v) sodium hydroxide. The alkali-soluble extracts
were identied as starch, glucose, pigment, and lipids (Table 4). The effects
of each component were evaluated by adding known amounts to model
solutions consisting of 0.5 or 5 mg mL 1 ovalbumin and then assaying the
mixture by the ACR method. Glucose (<5 mg mL 1) reduced the color yield
for the ACR assay, probably by competing with the peptide group for Cu2
binding. The interference was less pronounced when colorimetric readings
were taken after 30 minutes rather than 2.5 hours. The Cu2 reaction with
glucose takes place more slowly than the Cu2 binding to protein. The
different rates of reaction offer an opportunity for improving analytical
performance (see later). Lipid and starch suspensions cause light scattering
and an apparent increase in absorbance readings. Starch is also hydrolyzed
* Examples of strongly colored grain include barely and oats, which have blue or black chaff
(pericarp, aleurone layer, and outer endosperm).
54
Chapter 2
TABLE 3 Some Interfering and Noninterfering Compounds for ACR Protein Assay
Reactive compounds
Colors
Anthocyanins and other avonol
Lycopenes (carotenoids and related)
Pigmentschlorophyll and related
Biopolymers
Lignin and low molecular-weight
derivatives
Nonpeptide compounds
DNA
Glucose
Glucoseamine
Starch
Amino acids (histidine and cysteine)
Buffer components
Ethanolamine, ethylenediamine, Tris
Unreactive compounds
Amino acids
Gly, Tyr, Arg, Met, Phe, Asp, Glu,
Iso, Lys
Organic acids
Formic, acetic, lactic
Others
Creatine, betaine, EDTA
Amides
Acetamide, sulfanilic acid,
dimethylformamide, urea
N-methylacetamide
Amines
Ammonia, ethylamine
TABLE 4
Component
Glucose
Starch
Protein
Pigment
Lipid
a
Whole wheat
Bran
9.0
670.0
130.0
0.0
121.0
8.2
120.0
140.0
7.4
32.5
A 1-g sample was extracted with 0.5% NaOH (100 mL) (14).
55
Regression
equationa
SE (%)
Y 13:3x 7:73
Y 40:8x 3:60
0.54
0.79
1.24
1.18
Y 49:3x
Y 58:5x
0.99
0.98
0.21
0.28
0:48
0:14
56
Chapter 2
Protein
Insulin
Bovine serum globulin
Bovine serum albumin
Histone
Ribonuclease
Ovalbumin
A310a
Protein
Gelatin
Histone
Casein (Hammersten)
Ovalbumin
Trypsin
Bovine serum albumin
Lysozyme
a
Value for 1 mg mL
5.2
6.7
5.3
6.3
5.9
5.0
1.56
1.71
1.74
1.98
2.17
2.21
2.20
protein solution.
6.
The UV ACR assay is performed at 255320 nm. The technique is 10- to 15fold more sensitive than the conventional ACR protein analysis. The use of
UV absorption measurements in conjunction with the ACR method dates
back to 1957. The method became popular owing to its rediscovery by
Ellman (17). Itzhaki and Gill (18) used a similar approach with minor
modications. More recently, Kanaya and Hiromi (19) used a stopped-ow
version of the UV biuret assay.
The sensitivity of the UV biuret assay to gelatin was lower than
observed with other proteins (17) (Table 6). Similar sensitivity differences
are observed at visible wavelengths (see Table 1). The dipeptide L-ProGly
had a high molar extinction coefcient e 176 M 1 cm 1 when analyzed
by the UV biuret method. In contrast, L-GlyPro had zero extinction. The
biuret-positive dipeptide has a ``normal'' peptide nitrogen atom, whereas the
peptide bond in L-GlyPro is formed from a secondary amine nitrogen
57
Cereal Proteins
Wheat Proteins
Pinckney (12) described a method for extracting wheat protein from our
using a 1:10 volume mixture of carbon tetrachloride and dilute (0.05 N)
potassium hydroxide. The mixture was centrifuged and the aqueous phase
analyzed using ACR stabilized with 0.32% glycerol. About 100 samples of
58
Chapter 2
hard red winter wheat, 36 samples of hard spring wheat, and 28 samples of
hard white wheat were analyzed by Pinckney (12). The A550 readings were
highly correlated with Kjeldahl protein (R 0.9250.976). The standard
error for the ACR assay was 0.300.32 with a reproducibility of 0.1%. Sources
of error include suboptimal peptization by dilute potassium hydroxide. The
rate of color formation was also undesirably slow, requiring 2040 hours.
Jennings (11) developed a one-step assay by adding ACR directly to
our. Protein extraction and color development occurred simultaneously as
the our was agitated with ACR. This innovation, by eliminating a separate
peptization stage, led to a signicant increase in the speed of ACR analysis.
Jennings also found that K-Na tartrate was a better stabilizer for Cu2 than
glycerol. Samples of the former seemed to contain fewer impurities and the
likelihood of copper oxide formation was reduced. The Cu2-tartrate
complex (lmax 675 nm) also interfered less with A550 readings compared
with the Cu2 glycerol chelate (lmax 630 nm). During the one-step assay
the protein extraction efciency was 8497%. With barley varieties having a
black or blue aleurone layer, there was interference from extraneous plant
colors. Unidentied chromogenic species with lmax 500800 nm were
dissolved at high pH. The interference was especially acute for the analysis
of whole-meal wheat our or unhulled oats. The extraneous color was
associated with oat bran (29).
A one-step ACR method was employed to assess protein levels in 45
varieties of brown and milled rice (24). There was a high correlation between
A550 readings and crude protein levels (N 6 5.95).* The calibration equation
for milled rice was cP 15:48A550 0:063, where cP is the % Kjeldahl
protein. The linear range extended to A550 1.0 with a regression coefcient
of 0.964 and a standard error of 0.46. A further ACR analysis of 42 brown
rice samples led to the regression equation cP 16:04A550 0:233
(R 0.981) with a standard error of analysis of 0.30. Other results for
ground rice samples are summarized in Table 7.
Further improvements of the ACR assay were introduced by Johnson
and Craney (13). First, they modied the ACR assay for use with strongly
colored cereals such as barley, oats, and grain sorghum. Next, solid copper
carbonate was added directly to our suspended in alkaline-isopropanol
solution, thereby doing away with a need for a prepared ACR solution.
These changes led to a method that was signicantly faster than any
previous ACR assay (Method 2).
* Ground rice (1 g) and 2 mL of carbon tetrachloride were added to several (25 150 mm) test
tubes along with ACR (40 mL). Samples were shaken with a mechanical shaker for 90 minutes.
Aliquots (15 mL) from each sample were centrifuged and A550 readings were recoreded.
59
TABLE 7 Protein Content of Ground Rice Determined Using the Biuret Assay
Milled rice
Method
MicroKjeldahl
ACR or
biuret
Brown rice
Mean (%)
Range (%)
Mean (%)
Range (%)
7.50
5.7411.69
8.25
6.1511.74
8.18
5.9512.50
8.85
6.3012.45
Method 2
Rapid analysis of wheat proteins (13).
Reagents
1. Copper carbonate (solid).
2. Potassium hydroxide pellets
3. Isopropanol
Alkaline isopropanol solvent. Add potassium hydroxide (5.61 g) to
isopropanol (600 mL). Make up to 1 L with water.
Procedure
Place 1.00 (+ 0.001) g of our in a 250-mL Erlenmeyer (conical) ask
and add solid copper carbonate (1.0 + 0.1 g). Suspend the mixture
in alkaline isopropanol solution (50 mL) and shake vigorously using
a mechanical shaker for 15 minutes. Allow the sample to stand for a
further 15 minutes and lter through a glass ber lter with vacuum
suction. Take A550 readings against a reagent blank.
About 391 cereal samples were analyzed, including grain sorghum (47
samples), corn (48 samples), oats (40 samples), barley (44 samples), wheat
(165 samples), and hard and soft wheat our (47 samples). The ACR assay
results are highly correlated with crude protein levels determined by
Kjeldahl analysis (Table 8). The former technique could be applied to a wide
range of cereal grains. Using multiple sample shakers, a throughput of
0.2 min 1 could be achieved. Compared with the preprepared ACR
solution, solid copper carbonate is more stable for prolonged storage. The
isopropanol reduced interference by extraneous plant dyes and also reduced
the solubility of starch in the alkaline reaction medium. By eliminating a
peptization stage, the time for the ACR analysis was reduced from 3540
60
Chapter 2
TABLE 8 Analysis of Cereal Proteins Using the Method of Johnson and Craney
Cereal
Number of
samples
Grain
sorghum
Corn
48
Oats
40
Barley
44
Wheat
(whole
meal)
Wheat four
(rened)
47
165
47
Regression
equationa
(Yi )
13:36Xi
2:64
11:486Xi
4:0
15:93Xi
1:84
20:36Xi
1:33
16:07Xi
1:47
16:68Xi
0:26
Sensitivityb
A550 per
(%)
Analytical
error (%)
0.98
0.0748
0.19
0.95
0.0871
0.14
0.99
0.0627
0.17
0.97
0.0491
0.23
0.99
0.0622
0.18
0.99
0.0599
0.14
61
protein using the biuret and Kjeldahl methods is also described by Belavady
et al. (31).
7.2.
Meat Proteins
62
Chapter 2
TABLE 9
Sample (n)a
ACRb / biuret
Kjeldahl
% Moisture
% Fatc
Beef (10)
Pork (11)
Chicken (9)
Cod (8)
24.2 0.3
23.1 0.3
25.2 0.3
18.8 0.2
24.3 0.3
22.4 0.3
24.6 0.2
19.2 0.2
60.3 0.11
55.2 0.18
58.8 0.13
64.5 0.07
20.3
26.7
21.2
20.3
Meat sample and number replicate analysis.bACR alkaline copper reagent or biuret method.
Approximate fat content estimated as difference between protein and moisture.
Source: Ref. 32.
c
(35) showed that the ACR method is probably the most accurate of
colorimetric method for assaying whole-body protein concentration in testanimal carcasses during feeding trials. Silgjnic and Samardzija (36)
considered the best ways to dissolve meat samples for biuret analysis.
Beef, pork, chicken meat, frankfurters, sausage, and sh did not dissolve
fully at high pH, leading to losses during the subsequent analysis. Dissolving
samples at lower pH using concentrated urea solutions provided a better
alternative.
Protease action affects the results of the ACR assay for meat protein.
Errors may arise for meat samples with high amounts of endogenous
proteases. Horse muscle is thought to contain unusually high levels of
catheptic activity. Autolysis leading to loss of some peptide bonds may
affect results obtained using ACR analysis. Turgut (37) has shown that
differently treated sh muscle gives different results with ACR analysis,
probably as a result of sh muscle autolysis. Prusa and Bowers (38) used the
biuret assay to determine the solubility of turkey muscle protein under the
inuence of nonmeat ingredients (sodium nitrite, sodium chloride, and
phosphate salts).
7.3.
63
7.4.
Dairy Proteins
7.5.
64
Chapter 2
Method 4
Analysis of yeast protein by the ACR method.
Reagents
1. ACR (Method 1)
2. Aqueous toluene solution (water containing 10 ppm toluene)
3. Sodium hydroxide solid
4. Torula yeast
Procedure
Yeast protein solubilization. Lyse dried yeast (3 g) by suspending in
100 mL of aqueous toluene solution. Shake or stir the suspension of
cells in a water bath at 508C for 6 hours. Add sodium hydroxide to
bring to 0.5 M and heat at 75808C for 30 minutes. Freeze the
sample at 208C overnight, thaw, and reheat for a further 30
minutes.
Protein analysis. To 1 mL of yeast protein extract add 4 mL of ACR
solution and allow 30 minutes for color formation. Record A540
readings.
Yeast protein reacted with the ACR solution forming a purple-violet color
with lmax 540 nm. The ACR assay results were strongly correlated with
Kjeldahl results (%N 6 6.25). About 12% of nitrogen in dried yeast was
NPN. Protein monitoring during fermenter operation was addressed by
Nielsen et al. (46). They set up a laboratory scale fermenter and examined
various ow analyses for monitoring sugars, lactic acid, biomass, and
protein in the feed stream. Growth media components (yeast extract and
peptone) contain high levels of NPN and glucose. Despite such difculties,
the ACR assay was successfully used to monitor the utilization of feed
protein during the fermentation by lactic acid bacteria.
REFERENCES
1.
2.
3.
4.
A Hiller. Determination of albumin and globulin in urine. Proc Soc Exp Biol
Med 24:385386, 1927.
HW Robinson, CH Hodgen. The biuret reaction in the determination of
serum proteins. 1. A study of the conditions necessary for the production of a
stable color which bears a quantitative relationship to the protein concentration. J Biol Chem 135:707724, 1940.
JWC Mehl. The biuret reaction of proteins in the presence of ethylene glycol. J
Biol Chem 157:173180, 1945.
A Sols. An improved biuret reaction of proteins and the two-standard
colorimetry. Nature 160:89, 1947.
65
66
Chapter 2
24. LC Parial, LW Rooney, BD Webb. Use of dye-binding and biuret techniques for estimating protein in brown and milled rice. Cereal Chem 47:3843,
1970.
25. M Gullord. Studies on the biuret method for determination of protein in
cereals. Meld Nor Landbrukshoegsk 51(13):6 pp 1972.
26. Y Pomeranz, RB Moore, FS Lai. Reliability of ve methods for protein
determination in barley and malt. J Am Soc Brew Chem 35(2):8693, 1977.
27. PS Misra, R Barba-Ho, ET Mertz, DV Glover. Studies on corn proteins. V.
Reduced color response of opakue-2 corn protein to the biuret reagent, and its
use for the rapid identication of opaque-2 corn. Cereal Chem 50:184190,
1973.
28. YG Doesthale, K Visveswara-Rao. Application of rapid biuret technique for
protein estimation in sorghum and pearl millet. Indian J Nutr Diet 14(3):65
69, 1977.
29. PC Williams. The determination of proteins in whole wheatmeal and our by
the biuret method. J Sci Food Agric 12:5861, 1961.
30. FC Strong, AMA Duate. A room temperature, rapid method for the
determination of protein in wheat and other grains by the biuret reaction.
Cereal Chem 69:659664,1992.
31. B Belavady, MM Subramanya, KNRK Murthy, S Ramachandra, AN Bagali,
SA Hosamani. Appropriate sample for determination of protein in sorghum
(Sorghum bicolor (L) Moench) raised in agricultural trials. J Sci Food Agric
37:207210, 1986.
32. J Torten, JR Whitaker. Evaluation of the biuret and dye-binding methods for
protein determination in meats. J Food Sci 29:1681174, 1964.
33. R Lasztity, D Torley, F Orsi. Contribution to the protein determination in
meat products. Proceedings of the European Meeting of Meat Research
Workers 24:L1:1L1:6, 1978.
34. W Reichardt, J Mueller, S Mueller, B Eckhert. Beef and pork. Direct
determination of connective-tissue-protein-free pure protein content [Rind
und Schweineeisch. Zur direkten Bestimmung des bindegewebseiweissfreien
Reineiweissgehaltes (Rein-BEFFE)]. Fleischwirtschaft 74:13271329, 1994.
35. SPJ Brooks, BJ Lampi, G Sarwar, HG Botting. A comparison of methods for
determining total body protein. Anal Biochem 226:2630, 1995.
36. D Smiljanic, S Samardzija. Biuret method for determination of proteins in
food. I. Conditions of dissolving the sample. Technol Mesa 38:153157, 1997.
37. H Turgut. Drawbacks in the use of the biuret method for determination of
the same protein in differently treated sh samples. Food Chem 4:161165,
1979.
38. KJ Prusa, JA Bowers. Protein extraction from frozen, thawed turkey muscle
with sodium nitrite, sodium chloride, and selected phosphate salts. J Food Sci
49:709713, 720, 1984.
39. WE Townend, JE Thomson, JR Hutchins. ``Coagulation test'' for cooked
meat temperature: effect of sample preparation methods. J Food Sci 50:1179
1180, 1186, 1985.
67
3
The Lowry Method
1. INTRODUCTION
The protein assay of Lowry et al. (1) is simply called the Lowry assay. The
prototype assay has a number of drawbacks. The standard curve is
nonlinear. The technique uses unstable reagents, which are generally
prepared daily. The classical method is also subject to a variety of
interference compounds. Nevertheless, the Lowry assay remains highly
important. Peterson's modication of the Lowry assay (2) is robust,
sensitive, and impervious to most interferences. This chapter contains
descriptions of the Lowry assay, the underlying principles (Sec. 3),
calibration features (Sec. 4), interference compounds, and common sample
pretreatment strategies for ensuring accurate results (Sec. 5 and 6).
Applications of the Lowry method to food protein analysis are reviewed
in Sec. 7.
The design of the Lowry protein assay can be traced to the
investigations of Wu (3,4). These were concerned with the use of tungstatemolybdate reagent for protein analysis. Folin and Ciocalteu (5) also
determined tyrosine and tryptophan in protein hydrolysates using tungstate-molybdate. Herriot (6) achieved a 3- to 15-fold increase in sensitivity of
the tungstate-molybdate assay in the presence of copper (Cu2) ions.
69
70
2.
Chapter 3
71
72
Chapter 3
protein-DOC precipitate was resuspended in distilled water and reprecipitated for a second time. Peterson (2) used DOC-TCA precipitation to
develop an assay, now available in kit form (Sigma-Aldrich Ltd.), that
rendered the classical Lowry method obsolete.*
Method 3
Peterson's modication of the Lowry protein assay (2,8,9)
Reagents
1. Copper sulfate
2. Sodium potassium tartrate
3. Sodium carbonate
4. Sodium dodecyl sulfate (SDS) stock solution (5% w/v)
5. Sodium deoxycholate solution (0.15% w/v)
6. Sodium hydroxide stock solution (0.8 M)
7. Trichloroacetic acid (72% w/v)
8. Folin-Ciocalteu reagent (2 N)
9. Bovine serum albumin (BSA) (0.5 mg mL 1) as protein standard.
Add 1 mg mL 1 sodium azide as preservative and store frozen as
small aliquots.
Procedurey
Copper-tartrate-carbonate (CTC) stock solution. Dissolve copper
sulfate (0.1 g) as well as sodium potassium tartrate (0.2 g) each in
about 10 mL of distilled water. Add both solutions to sodium
carbonate (10 g in 50 mL of distilled water) and make up to a nal
volume of 100 mL.
Preparation of reagent A. Mix CTC, SDS, and sodium hydroxide stock
solutions in a volume ration of 1:2:1. Reagent A is stable at room
temperature for 23 weeks. Refrigeration at 48C will double the
useful life. SDS precipitates in the refrigerator. Redissolve by
holding the reagent bottle under a running hot-water tap.
Preparation of reagent B. Dilute commercial 2 N Folin-Ciocalteu
reagent 1:1 with distilled water. This is stable for many months at
room temperature.
Protein analysis. Add sufcient water to bring the protein sample (5
100 mg) volume to 1 mL. Now, add 0.1 mL of DOC solution,
followed 10 minutes later with 0.1 mL of TCA solution. Centrifuge
the mixture at 10,000 g (mark 12 on a microcentrifuge) for 10
minutes. Decant the supernatant and place the upturned Eppendorf
* Peterson's modication of the Lowry assay had received 6130 citations as of June 2001.
{ It is convenient to employ (1.5-mL) microcentrifuge (Eppendorf) tubes for this procedure.
73
The following account is based on Refs. 1114. The Lowry assay involves
two reactions. First, a protein-Cu2 complex forms. Six peptide bonds
surround a central Cu2 atom. The high-pH solvent (OH &0:1M; pH 13)
induces protein denaturation. Loss of native structure precedes binding with
Cu2 to form a type B protein-Cu2 complex (Chapter 2). Protein
denaturation at high pH also exposes tyrosine and tryptophan residues,
which then ionize.
The second stage of the Lowry assay is a redox reaction with FolinCiocalteu reagent via two pathways. First, Mo6/W6 reacts directly with
amino acid side chains (histidine, cysteine, asparagine, tyrosine, tryptophan). A high pH is not required for these reactions. Second, Cu2 mediates
the dehydrogenation of the polypeptide via metal ioncatalyzed oxidation.
The electrons are transferred to Mo6/W6, leading to a color change.
Features of the Mo6/W6 reaction with Cu2 and protein are summarized
in Table 1.
3.2.
74
Chapter 3
E
RH22Cu3 II
k2
FIGURE 1
75
With molecular oxygen as oxidant, reaction (2) would lead to the superoxide
radical [Eq. (5)], which probably remains protein bound as a ternary
complex (III).
1
RH22Cu2 I O2 k?
RH2
2Cu3 II O2
/ ?O222R22Cu1 III H
76
Chapter 3
1
R22Cu2 II Qxn k?
Qxn
R22Cu3 ?R22Cu2
7
8
Evidence for Eq. (8) comes from the deployment of 2.20 -biquinoline
(2,2 DQ), which should form a purple complex with Cu1. Addition of
2,20 DQ failed to detect the presence of Cu1. Therefore, the copper ion
probably cycles between oxidation states 2 , 3 , and 4 under strongly
oxidizing conditions. Failure to detect free Cu1 shows that the sequence of
reactions during the Lowry assay is probably Eq. (2)?, Eq. (3)?, Eq. (7)?
0
77
Cu2 Qxn H2 O
?Cu2 Qxn
hexapeptide fragments
The duration of the Lowry assay is 40 minutes. Proteins react with Cu2
(reagent C) in 10 minutes. A further 30 minutes is needed for the reaction
with reagent E before A750 readings are recorded. The speed of the Lowry
assay depends on one or more of the reactions described in Sec. 3.2.
1. The time course for the alkali denaturation of proteins can be
prolonged for stable proteins (e.g., lysozyme).
2. The oxidation of RH22Cu2 by atmospheric oxygen [Eq. (1)]
occurs with the rate constant (k1) of 5.5 6 10 4 (s 1). The time for
99.9% completion (5/k1) is 151 minutes.
3. Oxidation of RH22Cu2 by IrCl6 (and presumably by Mo6/
W6) occurs with a rate close to the diffusion-controlled limit.
Reduction of Mo6/W6 by simple reductants is nearly instantaneous, being delayed only by the reagent mixing time.
4. Reduced tungstate-molybdate undergoes slow deprotonation and
structural changes at high pH. At 258C this process takes about 30
minutes.
5. Rearrangement of RH22Cu3 to form a radical species [Eq. (2)] is
a slow process. With IrCl26 as oxidant, k2 is 1.66 6 10 3 (s 1) with
a 99.9% completion time of 36 minutes. Clearly, it is not possible
to identify a single rate-limiting reaction for the Lowry assay of
proteins.
A high reaction rate is essential for automated protein analysis. Anderson
and Marshall (15) and also Huang et al. (16) adapted the Lowry method for
continuous ow analysis. The sample throughput was 30 per hour.
4. CALIBRATION FEATURES
Calibration graphs for the Lowry assay are usually curved. A linear
response is obtained for simple compounds (Sn2, tyrosine, etc.) (14). One
explanation for the nonlinear calibration graphs for proteins is that Mo6/
78
Chapter 3
10
where F is the slope of the calibration graph. The line described by Eq. (10)
passes through the origin and hence
P DA750 =F
11
Stauffer (17) tted Eq. (12) or its logarithmic form [Eq. (13)] to data from
the Lowry assay.
A700 oPF
12
13
w=F
14
Notice that Y log A750 is the value for the unknown sample. Coakley
and Jones (18) used reagent volumes three times higher than in the standard
method.* Their calibration results were described by a hyperbolic curve.
* A 0.6-mL portion of protein standard solutions was reacted with 3.0 mL of Lowry reagent C.
After standing for 10 minutes, 0.3 mL of Folin-Ciocalteu regent was added. The A750 readings
were taken 30 minutes later.
A750
79
P
wP F
15
FA750
1 wA750
16
A750 P
17
y1
x1
y2
x2
18
where y1 1/A750 (1) and y2 1/A750 (2). Likewise, x1 1/ [P]1 and x2 [P]2.
The concentrations for the protein standards should be [P]1 0.1 mg mL 1
and [P]2 510 mg mL 1 BSA. The intercept of Eq. (17) (w) is found from
averages for y1, y2 and x1, x2.
w y
F
x
where
y
y1 y2
2
and
x
x1 x2
2
19
The previous treatment applies for a protein concentration between 0.1 and
10 mg mL 1. With protein concentrations below 0.2 mg mL 1, a straightforward calibration graph can be used with little loss of accuracy.
* Allowing for the 6.5 times dilution during the Lowry assay, a protein stock solution of 0.065
5 mg mL 1 leads to a ``within-cuvette'' concentration of 0.010.77 mg mL 1. Investigators are
liable to cite either of these protein concentrations in their work.
80
Chapter 3
TABLE 2
Biochemical classication
1.
2.
3.
4.
5.
Amine derivatives
Amino acids
Buffers
Chelating agents
Detergents (e.g., Triton X-100,
Tween)
6. Drugs
7. Hexosamines
8. Lipids and fatty acids
9. Miscellaneous compounds
10. Cryoprotectants
11. Polyvinylpyrrolidone
12. Nucleic acids
13. Organic solvents
14. Phenols and polyphenols
15. Polysaccharides
16. Reducing agents
17. Salts
18. Sugars
5.
Food additives
Acids and acidulants
Amino acids
Colors
Dyes
Sweeteners
Articial antioxidants (BHA, BHT, etc.)
Starch
Polysaccharides
Sulfur dioxide, sultes
Uric acid
Fe2
INTERFERENCE COMPOUNDS
Interference compounds for the Lowry assay are either chelators or reducing
agents. Chelators diminish color formation by sequestering Cu2. Reducing
agents react with the Folin reagent to create extra color. Some interfering
compounds act as both chelators and reducing agents. Dyes having an
absorbance maximum near 600750 nm are also interfering compounds.
Peterson classied interfering compounds into more than 13 classes
comprising over 180 chemicals (Table 2).
Many of these compounds are encountered in raw and processed
foods.* Ascorbic acid and other reducing compounds are found in foods of
plant origin (fruits, juices, pastes, concentrates). Wines and certain
beverages have high concentrations of phenols and related compounds.
Carbohydrates (simple sugarssucrose, glucose, and fructose) and polysaccharides (pectin or starch) occur in foods including jams, preserves, and
* However, the analyst may not be aware of the presence of interfering compounds in a given
sample.
81
Carbohydrates
Reducing sugars (mainly hexoses) react with Cu2 and the Folin reagent.
Nonreducing sugars (sucrose, oligosaccharides, polysaccharides) form
complexes with Cu2. Tagatose, sucrose, and inulin interfered with the
Lowry assay after exposure to hot alkali or acid (21). These solvents are
routinely used to dissolve proteins. As well as reducing Cu2 to Cu1, sugars
reduce tungstate-molybdate. The effects of simple sugars are signicant at
concentrations above 1 mM. The order of effectiveness is fructose >
sorbose > xylose > rhamnose > mannose > glucose (22).
Toldra (23) considered protein analysis in connection with the
production of high-fructose syrup. Effective control of this process requires
the determination of a-amylase and glucoamylase specic activity (hence
protein concentration) in samples containing up to 3040% sugar. A
solution of 1% (w/v) glucose or maltose produced an A750 response
equivalent to that of 18.8 or 16.6 mg mL 1 BSA. Protein analysis is also
necessary to determine the activity of fungal pectinases produced in
submerged culture using high concentrations of pectin as inducer (24).
Concentration of 0.11.0% (w/v) pectin interfered with both the classical
and modied Lowry assays. Calibration graphs showed a decrease in the
82
Chapter 3
slope and increased intercept. Assay sensitivity decreased while the LLD
increased with increasing pectin concentration (Chapter 1). The color with
protein-free samples increased linearly with the amount of added pectin.
These results imply complex formation between pectin and Cu2. The
Bradford method (Chapters 6 and 7) coped better with samples containing
< 0.5% (w/v) pectin. In our experience, pectin forms a gelatinous mass
during the Lowry assay. To avoid this problem, samples are exposed 0.1 M
CaCl2 and then centrifuged before protein analysis. Berg (25) warned that
``anyone attempting to determine protein contamination in polysaccharide
preparations by the Lowry procedure could be misled by positive results
which may be attributed to the carbohydrate.''
5.3.
Chlorophyll
5.4.
83
FIGURE 2
84
5.5.
Chapter 3
Lipids
Lipids increase sample turbidity and A750 values. The products of lipid
autoxidation also react directly with the Folin reagent. Heating lipids with
alkali (a common method of protein solubilization) leads to Lowry-positive
products. The problem has been examined with arachidonic acid,
phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol
(33). Samples may be delipidated by shaking with chloroform or petroleum
ether and then redissolved with 0.5 M NaOH by storing at 378C overnight
or by heating at 1008C for 30 minutes. Solvent extraction will not be wholly
successful if the protein and proteolipid complexes dissolve within an
organic solvent phase. Emulsion formation can also lead to apparent loss of
protein from the sample.
Lees and Paxman (34) modied the Lowry assay for proteolipids and
lipoproteins. Their method is fast and does not require heating.* Markwell
et al. (35) added 1% SDS directly to Lowry reagent C to dissolve membranes
and other lipid. The concentration of copper sulfate was also increased from
0.5 to 4% (w/w) to reduce interferences from sucrose and EDTA. The
modied method gave results identical to those obtained with lipoprotein
samples delipidated using petroleum ether. However, the new method
allowed higher rates of analysis and greater convenience. There was no
interference from 40200 mM sucrose. SDS also features in Method 3,
which is therefore able to deal with membrane lipids routinely. According to
Kirazov et al. (36), treating membrane-containing fractions with 1 M NaOH
or 0.5% SDS reduced the stability of A750 readings and did not reduce the
interference from lipids.
5.6.
RT
ln C
nF
20
85
constant, and R the gas constant. For dilute solutions, C becomes equal to
concentration. Therefore, the effect of reducing compounds on the Lowry
assay is related to the ``intrinsic'' redox properties (E8 and n) and the
concentration of reducing agent.
Dithiothreitol (DTT), 2-mercaptoethanol (2ME), reduced glutathione
(GSH), and oxidized gluthathione (GSSG) produce color with the Lowry
assay (37). Reducing compounds react directly with the Folin-Ciocalteu
reagent. Table 3 shows the degree of color formation with some SH
compounds. As the underlying reaction is a redox process, calibration
graphs for SH compounds will be nonlinear. For each SH compound De
(M 1 cm 1) was determined from the steepest slope in Fig. 3. Values for De
determined in this manner (Table 3, top half) agree with results from Ref.
14. DTT is one of the most effective interfering SH compounds. Samples
with > 0.2 mM DTT have high A750 high background absorbances.
TABLE 3 The Color Yield from the Lowry Assay of Some Reducing Compounds
Reducing agent
Dithiothreitol
2-Mecaptoethanol
Glutathione
(reduced)
Cysteine
Glutathione
(oxidized)
Cystine
Cysteine
Fe2
Sn2
Ascorbic acid
Phenol
Tyrosine
Indole
Tryptophan
" (M
cm 1)a
nb
7625 (176)
3900
3333
2
1
1
3400
1870 (760)
1
0.5
1702 (822)
0.5
3150
3150
6100
6700
12400
12800
13000
13200
1
1
2
2
4
2 (4)
2 (4)
2 (2)
E1/2(mV)c
0.398
0.398
0.253
0.205
a
The De (M 1 cm 1) values in the top half of Table 3 are calculated from the maximum slope in
Fig. 3. Data in the bottom half of Table 3 from Ref. 14.
b
n electrons transferred to Mo6/W6 from one molecule of reducing compound. Values for n
were determined by colorimetry,c Half-wave electrode potentials were determined by cyclic
voltammetry, using standard calomel electrode (i.e., 0.242 V vs. NHE), from Ref. 78. Note
that for dilute solutions E1/2 E8 Eref.
86
Chapter 3
FIGURE 3
General Strategies
87
Destruction of SH Compounds
88
Chapter 3
7.1.
Reference
Hii and Herwig (42), Williams et al. (43)
Padhye and Salunke (44), Sathe and Salunkhe
(45), Sebecic et al. (46), Paredes-Lopez et al.
(47), Seguchi (48,49)
Eze and Dumbroff (25)
Huang et al. (50), Kroening et al. (51)
Ogunbunmi and Bassir (52)
Hoff (53), Vigue and Li (54)
Latlief and Knorr (55)
Vananuvat and Kinsella (56), Gierhart and Potter
(57), Abramov et al. (58), Kovar et al. (59),
Oliveira et al. (60)
Galluzzo and Regenstein (61), Tran and Einerson
(62)
Juffs (63), Ory and Sekul (64), Kwan et al. (65),
Siddiqui et al. (66), Ramana-Murthy et al. (67)
Sathe and Salunkhe (45) extracted proteins from the great northern bean
(Phaseolus vulgaris L) before assaying by the Lowry method.* Results were
standardized against the Kjeldahl method (%N 6 6.25). Whole bean our
had 26.1% (w/w) protein (per dry weight) or 22.17% (w/w) crude protein.
About 21.8% of the total protein was water soluble (albumin). Another
73.4% could be extracted with dilute salt solutions (globulin). Table 5 shows
virtually quantitative recovery of bean protein using solutions of sodium
carbonate (0.5%), potassium sulfate (5%), SDS (5%), or sodium hydroxide
(0.02 N or 1 N). Sodium phosphate buffer was not an efcient extractant for
bean our protein. Urea and SDS, also commonly used as proteinsolubilizing agents, did not perform so well.
The extent of protein recovery by extraction is usually uncertain.
Depending on the solvent, different protein fractions (albumin, globulins,
prolamins, etc.) are extracted to different extents. The direct addition of
Lowry reagent C to our samples should be investigated. This approach
* Suspend whole bean our (1 g) in 25 mL of solvent with thorough mixing. Let stand for 12
hours and then centrifuge (5000g) for 45 minutes. Analyze for protein content using Method 1.
89
TABLE 5 Analysis of Bean Flour Protein Extracts Using the Lowry Assay
Solventa
Sodium chloride
Sodium sulfate
Sodium acetate
sodium carbonate
Sodium dihydrogen phosphate
Sodium phosphate
Potassium chloride
Potassium sulfate
Urea
SDS
HCl
Dimethylformamide
pH
6.2
6.3
6.5
10.0
6.75
5.0
6.7
5.9
7.2
7.1
1.5
6.0
61.9
65.3
52.9
93.4
55.2
38.3
57.4
75.9 (100)
69.8 (100)
60.2 (100)
75.6
41.6
a
All salts were used at concentrations of 0.5% (w/v) aqueous solution. Values in parentheses are
% extraction using 5% (w/v) solutions.
Source: Adapted from Ref. 45.
worked well for biuret analysis of our (Chapter 2). After centrifugation,
Lowry analysis could then be completed by adding Folin-Ciocalteu reagent.
Sebecic (46) considered the Lowry procedure as a ``new'' method for
wheat protein determination. Forty-ve varieties of Yugoslav wheat were
analyzed.* The Lowry and Kjeldahl methods were highly correlated. The
sensitivity of these methods was 100-fold higher than that of the biuret
assay. Sebecic concluded that the Lowry procedure was a good substitute
for the Kjeldahl or Kjel-Foss method. It was certainly faster, simpler, less
expensive, and easier to perform than the Kjeldahl method.
Seguchi (49) analyzed starch granule surface proteins after extraction
with 1% SDS overnight. Although present in very small amounts
(0.06% w/w), the specically bound protein may have important effects on
the functional properties of wheat our. Treating wheat our at high
temperatures (e.g., 1008C for 40 hours or 608C for 24 days) or storing at
room temperature for long periods (130 days) caused greater than a 300%
increase in wheat starch granule surface proteins.
* Wheat our (0.4 g) was suspended in 50 mL of distilled water and heated at 958C for 2 minutes
to gelatinize starch. The mixture was cooled and brought to a total volume of 100 mL. To the
prediluted samples (with 417 mg mL 1 protein) were added 10 mL of Lowry reagent C. FolinCiocalteu reagent (1 mL) was added after 30 minutes and A540 readings recorded 30 minutes
later.
90
7.2.
Chapter 3
91
reagent.* Hull (70) used this approach to monitor proteolysis in milk. The
results were expressed as tyrosine equivalents by referring to a calibration
graph for tyrosine. Juffs (63) found that tyrosine equivalents were
signicantly correlated with milk bacterial count, age of the cow, period
of lactation, and milk yield. There were variations in tyrosine equivalence
for normal milk, and it was not possible to set a value for proteolyzed milk
samples.
The concentration of phosphotyrosine residues can also affect milk
tyrosine values. Phosphotyrosine does not react with the Folin-Ciocalteu
reagent (71). The total tyrosine value was determined after treating samples
with added alkaline phosphates. It is therefore suggested that the FolicCiocalteu phenol reagent be used as basis for assaying phosphotyrosine
phosphatase. Finally, milk proteolysis was monitored using the Lowry,
uorescamine, or trinitrobenzene sulfonate method or by A280 readings (65).
The Lowry and Anson methods were highly correlated (R 0.99). Although
10-times more sensitive, the Lowry assay showed less agreement with
techniques that monitor free amino groups. Ory and Sekul (64) recommended that milk proteolysis should be routinely measured with quantitative SDSpolyacrylamide gel electropho (SDS-PAGE) as well as
spectrophotometry.
Gut uid is a challenging milieu for protein analysis. Crossman et al.
(72) compared the modied Lowry assay and other techniques (quantitative
amino acid analysis,{ BCA, and Bradford method) for monitoring protein
levels in the gut uid from the marine herbivorous sh Kyphosus sydneyanus.
The Lowry and BCA assays gave similar results. All spectrophotometric
methods showed a low correlation with quantitative amino acid analysis.
The results were also dependent on the mode of sample collection and
pretreatment. In was important to freeze sh digesta immediately after
collection. The sample could be thawed and gut uid removed closer to the
time for protein analysis. The Lowry assay was also applied in the study of
dietary protein effects on sulde production by bacteria in the human large
intestines (73).
7.5.
92
Chapter 3
media contain a range of interferences for protein assay. Two examples will
illustrate the protein analysis issues. Vananuvat and Kinsella (56) grew
Saccharomyces fragilis on spray-dried crude lactose (2% w/w) supplemented
with ammonium sulfate (0.5%), peptone (0.5%), yeast extract (0.5%), and
urea (0.03%) as nitrogen source. Microbial cell numbers and protein and
nucleic acid concentrations were monitored during the fermentation.
Protein concentrations from the Lowry method were always lower than
Kjeldahl protein (56). Sample pretreatment to remove low-molecular-weight
substances is recommended. Given the complexity of growth media,
quantitative SDS-PAGE is probably a good idea for protein quantitation.
Orban et al. (76) examined the production of yeast autolysates from
Kluyveromyces fragilis grown on pasteurized whey (2%) supplemented with
ammonium sulfate (0.4% w/v), potassium dihydrogen phosphate (0.2% w/v),
and yeast extract. The cells were grown for about 8 hours in a batch
fermenter and harvested by centrifugation. Autolysis was initiated by
suspending freeze-dried cells (10% w/v) in dilute saline (5% w/v NaCl)
solution. Sample results are given in Table 6. Determinations of SCP are
usually performed after washing the wet cells free from growth medium. By
washing the cells thoroughly, interferences from culture medium components (sugars, peptides, and proteins) can be avoided. However, Lowry and
Kjeldahl results were consistently different because of interferences by the
high levels of ribonucleic acid found in yeast cells.
Dried yeast
(% dw)
Autolysatea
(% dw)
49.7
40.4
2.0b
2.0
7.7
41.0
2.2
37.3
19.7
5.8b
3.9
9.0
17.0
2.92
Method
Modied
Enhanced
a
93
LLDa (g)
4
3
Linear
range (g)
Color
stabilityb
Sample
volume (L)
4100
380
0.85
0.85
200
200
7.6.
Collagen
REFERENCES
1.
94
Chapter 3
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
95
96
Chapter 3
97
98
Chapter 3
4
The Bicinchoninic Acid Protein Assay
1. INTRODUCTION
The bicinchoninic acid (BCA) protein assay was developed by Smith et al.
(1). The plan was to substitute BCA for the Folin-Ciocalteu reagent in the
Lowry assay. The advantages of the BCA method include decreased
sensitivity to interferences, a need for one working reagent, and color
stability. The BCA protein assay has the same sensitivity as the Lowry
method. Reagents for the BCA assay are available commercially. As yet,
the BCA assay does not feature greatly in the food science literature. As
of June 2001, there were 16 references to bicinchoninic acid or
bicinchoninate in the Food Science and Technology abstracts. General
citations in the Science Citation Index number over 8500. The BCA assay
is without a doubt more popular than indicated by the low number of
formal citations.
The characteristics of the BCA protein assay are reviewed in this
chapter. Section 1 is an account of the history of BCA reagent and its use for
chemical analysis. In Sec. 2, the basic BCA procedure is presented, followed,
in Sec. 3, by a discussion of the chemistry underlying color formation. In
Secs. 46 are descriptions of calibration features, interfering compounds,
and sample pretreatment strategies for avoiding error. After a discussion of
99
100
Chapter 4
automated formats (Sec. 7), we turn to application of the BCA assay to food
protein analysis in Sec. 8.
1.1.
BCA is the trivial name for 2,20 -diquinolyl-4,40 -dicarboxylic acid. Early
applications include the analysis of Cu2 in metal alloys and blood sugars.
Hoste (2) evaluated 2,20 -diquinolyl (2,20 DQ) and nine related heterocyclic
compounds for Cu2 analysis after reducing Cu2 to Cu1 with
hydroxylamine hydrochloride. He found that 2,20 DQ forms a 2:1 complex
with Cu1 (Fig. 1) having maximum absorbance (lmax) at 540 nm and a
molar extinction coefcient (De540) of 5490 M 1 cm 1. To determine Cu1,
the working solution of 2,20 DQ (0.02% w/v in ethanol, 10 mL) was added to
3 mL of sample. The mixture was diluted with 25 mL of ethanol and A540
measurements were recorded. The Cu1 was quantitatively determined in
the presence of Cu2, with which there is no reaction.
Kertesz (3) employed 2,20 DQ (0.05% w/v in glacial acetic acid) for the
analysis of copper at the active site of the enzyme tyrosinase. To calibrate
the assay, 2 mL of 2,20 DQ was added to enzyme-free samples containing
101
max (nm)
560565
565
566
560
561
556
561
576
576
" (M
cm 1)
79208000
3100
7100
6130
6200
28100
5150
6600
6020
102
Chapter 4
"354.5 /104 (M
3.95
4.58
4.43
4.73
3.88
(0.1)
(0.1)
(0.2)
(0.2)
(0.2)
cm 1)
103
104
Chapter 4
105
Two reactions occur between proteins and Cu2 during the BCA assay. The
rst is a temperature-insensitive reaction between Cu2 and oxidizable
protein side chains (tyrosine, tryptophan, cysteine). The second reaction is a
temperature-sensitive process involving the binding of Cu2 to the peptide
backbone. Once bound, Cu2 undergoes reduction to Cu1. Assays
performed at high temperatures assist the peptide pathway. Because the
peptide backbone is the same for different proteins, a high-temperature
BCA assay lessens protein-protein variations in results. Wiechelman et al.
106
Chapter 4
(16) assessed the reactivity of various model compounds with BCA and
Cu2. They also determined the redox potential for some of these
compounds. Their ndings are summarized in Table 3. Clearly, the twotier reaction scheme proposed by Smith et al. (1) is correct. However,
oxidizable side chains do not react easily at either 37 or 608C. The following
half-reactions probably lead to the reduction of 2 moles of Cu2.
2Cys?Cys
Cys 2e 2H
1
dopa 2e H
Tyr?semiquinone radical?L
TABLE 4
Compound
Fructose
Glucose
Indole
Tryptophan
Tyrosine
Cysteine
Acetol
Ascorbic acid
2,4-Dinitrophenyl
hydrazine
Hydroxylamine
a
" (M
cm 1)a
43,000
6,120
3,621
1,589
1,172
1,357
10,000
3,030
16,400
0.2
0.8
2.2
5.0
6.8
5.8
0.8
0.26
0.5
3,230
2.5
Values for the apparent extinction coefcient are from Ref. 16 and 22.
Moles of each compound necessary to reduce 1 mole of Cu2 to Cu1. Value is calculated
as De (M 1 cm 1) divided by 8000 (M 1 cm 1).
b
107
H2 O2 Cu1 ? OH OH Cu2
Attack by .O2 or .OH can initiate a free radical reaction leading to covalent
modications of amino acid side chains. Depending on the reaction
conditions, MCO can result in subtle changes in protein tertiary and
quaternary structure, aggregation, or fragmentation into small polypeptides. Madurawe et al. (19) and also Bush and Lumpkin (20) showed that
lactate dehydrogenase (LDH) is inactivated by MCO in the presence of
added ascorbic acid, Cu2, and hydrogen peroxide. Inactivation of LDH
by MCO also occurred without added ascorbic acid. Under such
* DHA=dehydro ascorbic acid.
108
Chapter 4
3.3.
E 0:153
In the presence of BCA, the apparent redox potential (E8*) for the
Cu2/Cu1 redox couple can be estimated from Eq. (7) (21).
E * E 0:059 log1=Kd
109
From the Kd value given for Cu1 (BCA)2 the E8* calculated from Eq. (7) is
approximately 0.802. The BCA increases the oxidizing power of the Cu2/
Cu1 system. By comparison, Cu2 is a relatively mild oxidizing agent
(E8 0.153). This suggests that BCA is not a passive ligand for Cu2 but
also plays an integral role in the protein detection mechanism.
4. CALIBRATION FEATURES
The linear dynamic range for the BCA protein assay extends to 40 mg of
protein. The standard graph for 20120 mg of BSA is curvilinear. The linear
range is approximately the same for gelatin, avidin, immunoglobulin G,
chymotrypsinogen, insulin, ribonuclease, and soybean trypsin inhibitor (1).
The limit of linearity is probably an intrinsic feature of the BCA assay.
Depending on the protein, the maximum DA560 12 units 120 mg 1 of
protein.
The calibration graph for the BCA protein assay is nonlinear just like
the standard curve for the Lowry assay (Chapter 3). The two assays have
roughly equal sensitivities. Protein-protein variations in color yield are
also similar. The Lowry assay for inorganic reductants leads to straightline graphs. Nonlinearity was ascribed to a slow reaction with proteins
that allows time for the degradation of Mo6/W6 at high pH. With the
BCA assay, the instability explanation for nonlinearty is less tenable. In
contrast to Mo6/W6, BCA is stable in alkaline media. What is clear is
that the reduction of Cu2 to Cu1 becomes less quantitative at high
protein concentrations. Some possible causes for nonlinearity are listed in
Table 5.
A hyperbolic standard curve can be readily explained in terms of
saturation phenomenon. Protein binding to a xed concentration of Cu2 is
according to Eq. (8)
P Cu2 ?PCu2
110
Chapter 4
TABLE 5 Plausible Explanations for Nonlinear BCA Protein Assay and Their
Rebuttal
Explanations
1. Incomplete reduction of Cu2 by proteins.
2. Cu1 is bound to the protein and unavailable to BCA,
3. Cu1 is lost as the insoluble hydroxide,
4. Cu2 concentration is limiting in the BCA assay.
Rebuttals for points 13
1. High temperature increases sensitivity or extent of reaction. There is no effect on
linear range for analysis.
2. The binding afnity of BCA for Cu1 is 10,000 times greater than the afnity of
proteins for Cu1.
3. Formation of insoluble copper precipitates is curtailed by tartrate or BCA.
function.
b
Cu2 0 P
P Kd
10
11
As with the Lowry assay (19), the calibration graph for the BSA-protein
assay can be linearized using a range of transformations (Chapter 3).
5.
INTERFERENCE COMPOUNDS
Smith et al. (1) evaluated 41 potential interferences for the BCA assay.
These are either chelators or reducing agents. A further group of
miscellaneous compounds disrupt the assay by virtue of their strong color.
Glucose (100 mM) caused a large increase in the reagent blank. Sucrose
(10%) had little effect on the BCA assay. EDTA, guanidine hydrochloride,
sorbitol, and ammonium sulfate produced signicant color losses compared
with control samples. A list of additional interfering compounds includes
biogenic amines, pharmaceutical agents, and Benedict-positive compounds
(Table 6). The Benedict test is a method for determining easily oxidizable
compounds in biological samples. Upon heating with the test sample, Cu2
111
Reference
Slocum and Duepree (23)
( ) With the exception of Tricine, buffers showed no response with BCA reagent.
Effects have been carefully documented in food systems (see Sec. 5.2).
112
Chapter 4
The TCA-DOC precipitation method (31) was adapted for the BCA assay
by Brown et al. (32). Protein pellets were prepared as described in Method 3
of Chapter 3 and resuspended in 50 mL of alkaline SDS solution (5% w/v
SDS dissolved in 0.1 N NaOH). Then 1 mL of a standard BCA working
solution was added, followed by sample incubation at 378C for 30 minutes.
Interferences by glucose, DTT, 2ME, and ammonium sulfate were
eliminated after the TCA-DOC procedure. Protein was recovered quantitatively from media containing various proprietary ampholytes or polybuffer.
Shahabi and Dyers (33) proposed a two-point kinetic assay for dealing
with interferences. Their approach exploits differences in the kinetics of the
BCA reaction with proteins and interfering compounds. Many interferences
(ascorbic acid, cysteine, uric acid ) react with BCA according to rst-order
kinetics. By contrast, the reaction with proteins is usually zero order. This
means that interfering compounds produce a xed amount of background
color rapidly. In contrast, the absorbance change in the presence of proteins
increases linearly with time. Using the rate of color formation as the index of
protein concentration eliminated the effect of interferences.
Solid-phase assays are another means for avoiding interferences. Gates
(34) adsorbed protein samples (50 mL) on 1 6 2-cm strips of cationized
nylon membrane (Zeta-Probe, Biorad Ltd.). The bound protein was rinsed
free of interferences and then air dried. Each membrane was placed in a
1.2 6 10-cm test tube and 2 mL of BCA working solution was added. The
reaction was incubated at 608C for 30 minutes and the concentration of
nylon-immobilized protein determined from DA562 measurements. The
sensitivity of the solid-phase protein assay was 78 (+5)93 (+3.4)% of the
sensitivity obtained with the conventional solution assay. The limits of
detection (*10 mg protein) and the precision of analysis were the same as for
the conventional BCA assay. Accurate results were obtained in the presence
of glucose (20 mM), Tris-HCl buffer (200 mM), or DTT (0.1% w/v). Drying
the protein solution facilitated binding to the nylon support.
113
A microwell plate BCA assay was applied for the rapid analysis of protein
fractions from sucrose gradient centrifugation by Redinbaugh and Turley
(35). The assay involved a commercial BCA reagent kit from Pierce
Chemical Company with BSA as the standard protein. Protein samples
(10 mL) were placed in the 96-well microwell plate, to which BCA working
reagent (200 mL) was added. The samples were incubated at 228C for about
14 hours (i.e., overnight) or at 608C for 2 hours. Absorbance readings were
recorded at 570 nm. Lane et al. (36) also described a BCA microwell plate
protein assay, as have Sorensen and Brodbeck (37). Typically, color
formation took place at 378C for 30 minutes. Absorbance readings for 96
samples could be completed in 5 minutes.
The performance of microwell plate-based BCA protein assays
depends on a range of factors including sample size, volume of protein to
BCA working reagent, incubation temperature, and time. With the standard
assay (1:20 ratio of protein to BCA reagent), the linear dynamic range for
analysis was between 1 and 12 mg of protein (compare with a linear range of
10120 mg for the conventional assay). The calibration graph remains
curvilinear (1). The proposal that microwell plate-based BCA assays are 10fold more sensitive than the conventional assay is not supported by the
available evidence when results are normalized for differences in the assay
temperature or for proteinprotein variations in the response.
The maximum absorbance expected for a given amount of protein can
be estimated for the BCA assay. Consider 10 mg of BSA in a 210-mL assay
volume. First, six peptide bonds bind each Cu2 ion. Taking the molecular
weights of BSA and amino acids as 66,000 and 110, then 100 moles of Cu2
ions will bind to 1 mole of BSA (see Chapter 2, Table 2). Consequently,
10 mg of protein (1.51 6 10 10 mole BSA) will bind with 15.1 6 10 9 mole of
114
Chapter 4
7.2.
0.113 (0.003)
0.093 (0.002)
0.0547
0.0245
10:50B
10:200C
0.52.5
212
Sensitivity A/g
(BSA)
10:200A
10:200A
BSA/BCA volume
(L)
0.22
110
Linear range
(g)
0.9940
0.9967
3.3
3.5
%
Error
Results with bovine serum albumin as standard. Samples were incubated at (A) 228C overnight, (B) 608C for 30 minutes, or (C) 378C for 30
minutes.
Redingbaugh and
Turley (35)
Microassay
Standard assay
Lane et al. (36)
Microassay
Standard assay
Assay
format
116
Chapter 4
There are only a small number of reports describing the use of the BCA
assay for food protein analysis. No doubt, the numbers of applications will
117
FIGURE 2 Correlation between Kjeldahl protein and BCA assay of corn meal
protein and samples of zein. (Drawn from Ref. 44.)
118
Chapter 4
Some 23 forage plant (leaf) samples were analyzed using the BCA assay by
Messman and Weiss (45). These included alfalfa (fresh, wilted, hay, silage,
leaves), crown vetch (fresh, wilted, silage), spinach (fresh), perennial
ryegrass (fresh), orchard grass (fresh, wilted), corn plant (silage), and peal
millet (fresh). For sample pretreatment, leaves were lyophilized and ground
using a 1-mm Wiley mill. The resulting powders were extracted with boratephosphate buffer (0.1 M ionic strength) containing SDS (1% w/w). Sonicating the suspension of leaf powder for up to 2 minutes facilitated protein
extraction. In most cases, protein recovery was 85%. The protein extracts
were analyzed with the BCA and Kjeldahl methods (N 6 6.25).
The BCA method gave unreliable estimates of leaf proteins. There was
poor agreement between BCA and Kjeldhal results. Leaf samples contain
numerous interfering compounds (plant pigments, peptides, sulfhydryl
compounds, and phenol derivatives) that can interfere with both the BCA
and Kjeldahl methods. Attempts to circumvent interferences using the
DOC-TCA procedure were not successful. The yield of leaf protein
recovered by precipitation with DOC-TCA ranged from 40 to 80%. Protein
recovery by cold-acetone precipitation was not signicantly higher.
8.3.
Salt-soluble proteins from soybean, tamarind, ragi, jack fruit, mango kernel,
and sorghum were analyzed by Kamath and Pattabiraman (29). Whole
meals were extracted with 0.3 M NaCl (buffered with 20 mM phosphate
buffer, pH 6.9). Protein extracts were then analyzed using the BCA,
Bradford, and Lowry assays. A range of pure proteins (BSA, casein,
chymotrypsinogen, lysozyme, myoglobin, trypsin, zein) were also analyzed.
The BCA, Bradford, and Lowry assays showed differences in proteinprotein variations in color yield. Apparently, endogenous seed compounds
affected the results. High responses were obtained with sorghum, mango
kernel, and other samples known to have high concentrations of total
phenol. The BCA reagent was more sensitive to phenolic substances than to
protein. On a scale of 1.0 for BSA, the color yields from a range of phenolic
compounds were pyrogallic acid (86), gallic acid (2.1), pyrocatechol (106.0),
tannic acid (9.3), and phenol (0.8). The BCA response to protein and
phenolic substances was additive. There was a linear response between A567
119
120
Chapter 4
Extraction solvent
Water
SDS-NaOH
GnHCl
Water
SDS-NaOH
GnHCl
Water
SDS-NaOH
GnHCl
2.05
12.03
10.43
3.41
14.70
12.11
7.30
102.00
6.10
other animal carcasses with roughly similar fat content. This work might be
usefully compared with that of Toten and Whitaker (50) described in
Chapter 2. Carcasses were passed through a Horbart meat grinder (model
4812) once. The skin (including hair) was cut into pieces with scissors and
added to the meat. Homogenizing in a Waring blender and a Brinkman
polytron homogenizer further reduced the meat particle size. Finally, 1 g of
protein was solubilized by homogenizing with (a) water, (b) 5% SDS
dissolved in 0.5 M sodium hydroxide, or (c) 6 M guanidine hydrochloride
solution.
Samples were analyzed by the BCA method, biuret assay, or Bradford
assay. Protein extracts were also analyzed by three so-called absolute
methods, i.e., quantitative amino acid analysis, protein hydrolysis followed
by ninhydrin analysis, and Kjeldahl analysis. Quantitative amino acid
analysis yields a value of 16.3 (+0.5) g protein/100 g sample (n 15
replicates). However, Kjeldahl results (N 6 6.25) were 34% higher than
expected. Accurate results were obtained by using Fk 5.51. The ninhydrin
assay results depended on the choice of amino acid standard. The BCA and
biuret methods were not adversely affected by the presence of SDS or
guanidine hydrochloride. The Bradford procedure was unusable in the
presence of SDS (Chapters 6 and 7). These results are summarized in the
following.
Apparently, SDS-NaOH was the most efcient protein extraction
solvent tested. Thereafter, the accuracy of results varied in the order biuret
> BCA >> Bradford. The performance of the BCA assay may be better
than indicated from results in Table 8. First, the BCA assay was not
adversely affected by SDS-NaOH or GnHCl when serum albumin was
121
employed as the standard protein. Inadequate explanation was given for the
ability of the BCA assay to detect only 75% of the carcass protein.
Calibration graphs for the BCA are nonlinear. However, a hyperbolic
function was not applied by the authors although such an equation was
tted to results from the Bradford assay. There are good prospects that the
BCA method can be adapted for animal protein analysis, perhaps with SDSNaOH as the extraction solvent.
8.7.
There were several sources of error during attempts to analyze proteins from
freshwater algae. Meijer and Wijffels (51) noted that the efciency of protein
extraction from cells was variable. Attempts to facilitate extraction using
chemical means led to interferences with the Bradford and BCA protein
assays. Proteins could also undergo severe damage under harsh extraction
conditions such as boiling with alkali. Such harsh treatments could lead to
standard proteins being less representative of the sample.
Boiling Chlorella cells with 1 M NaOH for 30 minutes led to a recovery
of between 3% (Bradford assay) and 14% (BCA assay) of the crude protein.
By comparison, extracting yeast cells under similar conditions produced
protein recoveries between 76% (Bradford method) and 85% (BCA method).
When BSA standard protein was exposed to boiling 1 M NaOH for 30
minutes, there was 32% (Bradford method) or 85% (BCA assay) of the
response recorded for the untreated proteins. Apparently, chemical damage
due to heating at high pH is only partly responsible for the poor assay
results. Chlorella protein was efciently extracted by sonicating 50-mL
samples of fresh algae suspended in sodium phosphate buffer (25 mM)
containing 1% SDS. After sonication for 0.5, 1, and 3 minutes, there was 36,
80, and 104% recovery of crude protein as determined by the BCA assay. To
avoid foaming and a rise in sample temperature, the period of sonication
was divided into six 30-second intervals. In the presence of SDS, the
Bradford assay could not be used.
REFERENCES
1.
2.
3.
122
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Chapter 4
G Felsenfeld. The determination of cuprous ion in copper proteins. Arch
Biochem Biophys 87:247251, 1960.
AL Gershuns, AA Verezubova, ZhA Tolstykh. Photocolorimetric determination of copper with 2,20 -bicinchoninic acid. Izv Vyssh Uchebn Zaved Khim
Khim Technol 4 (1):2527, 1961.
S Nakano. 2,20 -Biquinoline derivatives VI. Copper (I) chelates of the 4,40 substituted 2,20 -biquinoline derivatives and the determination of copper by 2,20 bicinchoninic acid. Yakugaku Zasshi 82:486491, 1962.
IS Mustan, NS Frumina, VS Kovashova. Determination of copper in various
samples with 2,20 -bicinchoninic acid. Zavod Lab 29:782785, 1963.
VN Tikhonov. Highly selective titration method for determining copper with
2,20 -bicinchoninic acid. Zh Anal Khim 27:673677, 1972.
NN Noskova. Photometric determination of copper in blood using 2,20 bicinchoninic acid. Mikroelem Sib 9:159161, 1974.
F Buhl, Z Gregorowicz, B Piwowarska. Complex compound of 2,20 bicinchoninic acid with copper (I) ions. Pr Nauk Uniw Slask Katowicach
91:2733, 1975.
F Capitan, JMR Navarro, LF Capitanvallvey, Solid-phase spectrophotometric
microdetermination of copper. Anal Lett 24:2011217, 1991.
AJ Brenner, ED Harris. A quantitative test for copper using bicinchoninic acid.
Anal Biochem 226:8084, 1995 [see Anal Biochem 230:360, 1995 for erratum].
RF McFeeters. A manual method for reducing sugar determination with 2,20 bicinchoninic acid reagent. Anal Biochem 103:302306, 1980.
EM Grindler. Automated determination of glucose via reductive formation of
lavender Cu(I)-2,20 -bicinchoninate chelate. Clin Chem 16:519, 536, 1970.
K Mopper, EM Grindler. A new noncorrosive dye reagent for automatic
chromatography. Anal Biochem 56:440442, 1973.
KJ Wiechelman, RD Braun, JD Fitzpatrick. Investigation of the bicinchoninic
acid protein assay: identication of the groups responsible for color formation.
Anal Biochem 175:231237, 1988.
RL Levine, D Garland, CN Oliver, A Amici, I Climent, A-G Lenz, B-W Ahn, S
Shaltiel, ER Stadtman. Determination of carbonyl content in oxidatively
modied proteins. Methods Enzymol 186:464478, 1990.
RE Pacici, KJA Davies. Protein degradation as an index of oxidative stress.
Methods Enzymol 186:485502, 1990.
RD Madurawe, Z Lin, PK Dryden, JA Lumpkin. Stability of lactate
dehydrogenase in metal-catalyzed oxidation solutions containing chelated
metals. Biotechnol Prog 13:179184, 1997.
KD Bush, JA Lumpkin. Structural damage to lactate dehydrogenase during
copper iminodiacetic acid metal afnity chromatography. Biotechnol Prog
14:943950, 1998.
DA Skoog, DM West. Fundamentals of Analytical Chemistry. 3rd ed. New
York: Holt, Rinehart and Winston, 1976, p 305.
Q Chen, N Klemm, I Jeng. Quantitative Benedict test using bicinchoninic acid.
Anal Biochem 182:5457, 1989.
123
124
Chapter 4
5
The Udy Method
1. INTRODUCTION
Proteins react with certain organic dyes to produce insoluble complexes. The
quantity of dye bound is proportional to (a) the dye-binding capacity
(DBC)* and (b) the protein concentration. Farm-gate prices for milk (in
Australia, Denmark, France, Netherlands, New Zealand, United States) is
partly determined by its protein content. Dye-binding assays are widely used
for milk protein determination. Amido Black 10B (C.I. 20470), Acid Orange
12 (C.I. 15970), and Orange-G (C.I. 16230) are the three main dyes used.
Dye-binding assays are presented in Sections 13 of this chapter. Section 4
covers the chemistry of protein binding with azo dyes. Section 5 is a review
of interference compounds and other sources of error. Section 6 covers
applications of dye-binding assays in food protein analysis.
Protein-dye interactions are of interest in relation to (a) dyeing of
natural bers and textiles, (b) dyeing of tissue sections for microscopic
observation, (c) the use of dyes to evaluate renal function, (d) application of
dyes as pH indicators in protein-rich media, and (e) uptake of dyes by
photographic materials. In 1925 Grollman (1) described phenol red
125
126
Chapter 5
Db =cP KDf
127
McGann (16). The literature through to 1967 was discussed by Cole (17).
Sherbon (18) reviewed dye-binding assays for milk proteins.
2. THE UDY METHOD
The Udy assay using Acid Orange 12 is described here (Method 1).
Information in the public domain deals with milk and dairy products. Other
applications developed by Udy Corporation during the 1960s were probably
commercially sensitive and not published (Table 1). The Udy method is the
basis of Udy Protein Systems2.
2.1.
FIGURE 1
128
Chapter 5
TABLE 1
Alfafa
Barley
Beans
Bermuda grass
Caseinate
Cheese (hard)
Chickpeas
Corn silage
Cottonseed meal
Cowpeas
Fish meal
Gaines burger
Gram
Grass peas
Lentils
Linseed meal
Malted barley
Meat
Milkuid
Milkpowders
Mung beans
Mustard meal
Oats
Oat groats
Peanut meal
Peas
Pigeon peas
Rapeseed meal
Rice
Rye
Safower meal
Sesame meal
Sorghum (milo)
Soybean
Soybean hulls
Soybean meals
Sunower meal
Triticale
Urd beans
Wheat
Wheat germ
Wheatgluten
Wheydelactose
Wheyfresh
Wheypowdered
129
Phosphate buffer (0.05 M, pH 1.81.9). Dissolve potassium dihydrogen phosphate (3.4 g) and oxalic acid (2 g) in 100 mL of warm water.
Add to 800 mL of water containing phosphoric acid (3.4 mL), acetic
acid (60 mL), and propionic acid (1 mL) and dilute the mixture to
1 L.
Working Acid Orange 12 dye reagent (0.13% w/v). Dissolve 1.3 g of
Acid Orange 12 in 100 mL of warm phosphate buffer. Allow to cool
and dilute to 1 L with phosphate buffer.
Reference dye solution (0.06% w/v). Dilute the working dye reagent
with phosphate buffer. Prepare further dilutions and produce a
calibration curve of free dye concentration versus A480 readings.
Performing a dye-binding assay. Place 1.52.4 mL of liquid sample (or
0.250.5 g of solid) in a 2-oz plastic polyethylene bottle. Add 40.44 g
(40 mL) of working dye solution and shake vigorously for 30
seconds. Solid samples may be shaken for 5 minutes.
Centrifuge at 3500 rpm for 30 minutes or lter to remove insoluble
dye-protein complex. Dilute* the supernatant 100-fold with
phosphate buffer and record A480 readings versus an appropriate
buffer blank. Determine the amount of free dye from the calibration
graph.
Calibration for protein determination. Determine dye binding for
samples with known amounts of crude protein (%N 6 6.25).
Establish the regression equation relating Db versus crude protein.
Analyses of 73 whole milk or 34 spray-dried milk samples by Orange
G binding led to a highly signicant correlation between crude protein
(%N 6 6.38) and the amount of free dye (23);
cP 100
V1 D V2 Df
kEm
* Colorimetric measurements are possible without dilution when using a very short (0.3 mm)
path-length ow-through cuvette.
{ The equivalent weight (k) for an ionic species (g mole 1) is the molecular weight divided by the
number of charges per molecule; k 226 (g mole 1) assuming that this dye has two positive
charges. Note that the number of equivalents of dye bound is DBC/k.
130
Chapter 5
V1 D Df
kEm
Rearranging Equation (2) leads to Eq. (4) or Eq. (5) for liquid or solid
samples, respectively.
Df
V1
D
V2
Df D
cPEkm
V2
cPEkm
V1
2.2.
2.3.
Milk protein analysis using Amido Black 10B is important in both North
America and Europe (2730). Commercial instruments such as the ProMilk
Mark II or ProMilk PMA (manufactured by Foss Food Technology Corp.)
use Amido Black 10B. Several investigators reported difculties with Amido
Black 10B staining of plant proteins; some Amido Black 10B samples may
contain impurities with different afnities for plant proteins (31). [Method 2
is adapted from Sherbon (32,33).]
* This view is incorrect (Section 3.2). Proteins bind equal amounts of Orange-G and Acid
Orange 12. The molar extinction coefcients for Orange-G and Acid Orange 12 are also similar.
131
Method 2
Protein analysis using Amido Black 10B dye binding* (32,33).
Reagents
1. Amido Black 10B
2. Citric acid
3. Disodium hydrogen phosphate
4. Thymol blue (optional preservative)
Procedure
0.05M Citrate0.01 M phosphate buffer. Dissolve citric acid (52.6 g),
sodium dihydrogen phosphate (3.3 g), and thymol blue (1 g) in
660 mL of water.
Working dye solution (0.075% w/v). Dissolve Amido Black 10B (3 g) in
1 L of water by heating to 708C. Mix with citrate-phosphate buffer
and add 3.33 kg of water.
Reference dye solution. Dilute the working dye solution (1 volume)
with 2.5 volumes of distilled water.
Add 1 mL of sample to 20 mL of dye solution. Mix for 0.53 minutes.
Filter to remove insoluble dye-protein complex. Dilute the supernatant 100-fold with phosphate buffer. Record A620 readings.
Determine the amount of free dye from a calibration graph of A620
plotted against reference dye solutions.{
For calibration analyze at least 10 samples of known protein
concentration in duplicate.
Methods 1 and 2 were readily scaled down by mixing 50100 mL of
sample with 1 mL of dye reagent in a polyethylene microcentrifuge tube. The
protein-dye precipitate was then removed by microcentrifugation (13,000
rpm; 5 minutes). Absorbance readings were recorded after diluting the dyesupernatant solution 100-fold. For strongly colored samples, the dry weight
for the protein-dye complex was recorded. These micro-dye-binding assays
led to signicant savings of reagent and improved convenience (34).
3. SOLID-PHASE DYE-BINDING ASSAYS
Protein is adsorbed on a lter support such as nitrocellulose, lter paper, or
a glass ber lter. Sometimes, the adsorbed protein is ``xed'' by treating
* Amido Black 10B dye binding is also called the ProMilk method.
{ Absorbance measurements can be taken without dilution if using the purpose-made 0.3 mm
path length cuvette. Specic instructions are given for use with commercial instruments.
132
Chapter 5
133
lipid, respectively. There was compatibility with a great many buffer salts
(200 mM): NaCl, KCl, Na phosphate, K phosphate, HEPES, Tris-HCl,
MES, MOPS. However, protein-protein variations in the dye response were
evident. The proceding technique was apparently more accurate than the
modied Lowry assay or biuret method.
The speed for solid-phase analysis was increased by Nakamura and
co-workers (39). They used microltration apparatus* to apply multiple
samples to nitrocellulose membranes. Stained protein spots were measured
directly via a densitometer. The linear dynamic range for analysis was 110
mg. Sensitivity using Amido Black 10B staining was twofold higher than
with Ponceau Red. For both dyes the order of increasing sensitivity was
trypsin < lysozyme < cytochrome c < bovine serum albumin < human serum
albumin < concanavalin A < histone II < human g-globulin. Assay results
were unaffected at pH 3.69. There was no interference from a range of
salts, sugars, amino acids, nucleotides, polyols or EDTA. Detergents (SDS,
Triton X-100, Tweens) or high concentrations of denaturants reduced
protein binding to the nitrocellulose membrane.
3.2.
Azo dyes represent 60% of all known dye structures (44). The rst azo
compounds were synthesized by Peter Gries in 1858 using building units
designated A, D, E, M, and Z (Fig. 2). Mono-azo dyes were formed via
electrophilic attack of a diazotized species (A) on a sulfonated amino* BIO-DOT apparatus; Bio-Rad Laboratories, Richmond, CA. Membrane-bound protein was
stained using Amido Black 10B or Ponceau Red (0.1% w/v) dissolved in 7% acetic acid and
destained with 7% (w/v) acetic acid.
134
FIGURE 2
Chapter 5
135
4.2.
Azo dyes bind with the guanidino, imidazole, and the e-NH2 side chain of
arginine, histidine, and lysine, respectively (4,5,7,4548). Interactions with
wool (composed of the protein keratin) occur via ionic bonding. Further
136
Chapter 5
137
TABLE 2 Characteristics of Some Acid Azo Dyes Used for Protein Assay
Dye
C.I. No.
Molecular weight
Net charge
lmax
e (Abs mL/mg)a
AB 10B
AO 12
OG
20470
616.50
1
620
81.5 (43,684)
15790
350
1
482
59.0 (22,066)
16230
452.38
2
480
46.9 (20,683)
AB10, Amido Black 10B; AO12, Acid Orange 12; OG, Orange G.
a
Extinction coefcient, absorbance per unit concentration of dye bound (mg mL 1). Value in
parentheses is molar extinction coefcient.
Source: Refs. 21 and 26.
138
Chapter 5
TABLE 3 Dye-Binding Capacity for a Range of Samples for Orange G (OG), Acid
Orange 12 (AO 12), and Amido Black 10B (AB 10B)
DBC (mmoles g
Sample
BSA
HSA
HGG
k-Casein
Meat meal
Fish meal
Milk protein
Soybean
Average
Ratiob
cP)a
OG
AO12
AB 10B
1365.0
1466.8
1028.8
692.5
630.5
708.0
800.9
984.5
959.6
0.8
1775.7
1780.0
1354.3
857.1
905.7
920.0
1094.3
1345.7
1254.1
1.0
743.5
834.4
582.8
274.4
339.3
336.0
490.3
555.2
519.5
0.54
Dye-binding capacity (mmoles of dye bound per gram protein) from Ref. 50.
Average dye: BAA ratio. BSA, bovine serum albumin; HSA, human serum albimin; HGG,
human gamma globulin.
b
4.3.
139
involving the p orbital and lowers the energy of the p* state. Lysine,
arginine, or histidyl (auxochromic) groups donate electrons to the dye
molecule, thereby increasing its conjugation extent. A further explanation
centers on the transfer of free dye molecules from a polar low-viscosity
solvent phase to a relatively nonpolar or restricted protein phase. Dye
transfer to more nonpolar solvents and micelles leads to spectral changes
resembling those observed during protein binding (6264).
Usually, a xed concentration of dye is exposed to increasing amounts
of protein. Absorbance readings are recorded with a reference cuvette
containing a dye solution of the same concentration as the sample cuvette
(Table 4). Measuring the ``difference absorption'' (DA) is useful where a dye
solution has a high background. The absorbance change for dye reagent
depends on the total dye concentration (D), extinction coefcient (ef), and
the cuvette path length (1 cm) as described in Equation (6).
A1 ef D
Df, protein, and the protein-dye complex are in equilibrium. Db has its own
extinction coefcient (eb). The net absorption change is described by
Denition
Absorbance for dye and dye protein
Difference absorbance
Fraction of dye bound
Total concentration of dye
Concentration of bound dye
Concentration of free dye
Extinction coefcient for free dye
Extinction coefcient for bound dye
Extinction coefcient difference for the free and
bound dye
Apparent molar extinction change when a fraction of
dye is bound
Added, free concentration of protein
Conditional dissociation constant
Wavelength for maximum absorbance
Isobestic wavelength where De 0
Number of dye molecules bound per molecule
protein; ns strong sites
140
Chapter 5
Equation (7).
A2 ef D
Db eb Db
From Eqs (6) and (7) it can be seen that A2A1 DA Db(eb
A2
A1
D
Db eb ef
D
or
eapp aeb
ef ef
and therefore
a
eapp ef
eb ef
where a is the fraction of dye bound and eapp ( A2/D) is the apparent
extinction coefcient change when dye is bound.
The isobestic point (liso) is the wavelength at which bound and free
dye molecules have equal absorptivity (ef eb). By running absorbance
spectra with increasing dye or protein concentration, liso can be identied as
the wavelength at which there is no absorbance change (DA 0). The
existence of an isobestic point is indication that the dye exists as two
interconvertible forms (e.g., bound and free). No isobestic point will appear
if ef = eb over the wavelength range examined. The corollary is that proteindye binding will not generate an absorbance change if De 0.
4.4.
10
DA=DenP
DA=De
DA=De
11
141
Kd
DA=DenP
DA=De
and
DA
nDePD
Kd nP
12
DAmax
D
13
Equation (13) is the chief means by which De and also eb may be determined
(65). First, invert all terms in Eq. (12). The resulting double-reciprocal
relation [Eq. (14)]* allows the determination of DAmax by graphical means
(see the following).
1
Kd
1
DA DeDnP DeD
14
Multiplying the former relation by DADeD gives Eq. (15). Other linearized
forms result from multiplying Eq. (15) by 1/(DeKd) or 1/(DeKdD).
DA DeD
DAKd
nP
15
DA
D
nPDe Kd
DA
DeKd
16
DA
n
PDDe Kd
nDA
DeDKd
17
142
Chapter 5
nD
Kd
nGP
Kd
18
where G Db/P.
To evaluate DAmax, De, and Kd/n, proceed as follows:
1. Add varying concentrations of protein to a xed concentration of
dye (D).
2. For each sample measure DA.
3. Using Equation (14), plot a graph of 1/DA (Y-axis) versus 1/P
(X-axis). From the X 0 intercept nd 1/DAmax ( 1/DeD).
4. Use the estimate for DAmax and nd De from Eq. (13).
5. From the slope and known values for De and D calculate Kd/n. It
is not possible to determine Kd independently using Eqs (14)(17).
An alternative stratagem is to translate DA values to Db (e.g.,
Db DA/De) and Df ( D DA/De). Thereafter, use Eq. (18) to
evaluate all binding parameters.
Note that Eqs (12)(18) are valid only at high protein/dye ratios.
Under these circumstances, only high-afnity protein sites (strong sites) are
occupied. Binding parameters therefore relate to strong sites. The number of
strong binding sites (ns) is distinct from the total number of sites (n).
Case 2. High dye/protein ratio.
Eq. (11); therefore,
DA
(DA/De) & D in
DenPD
Kd D
19
Eq. (19) describes protein ligand binding when a small xed concentration
of protein is exposed to varying concentrations of dye. As the concentration
of dye increases (e.g., D > 10Kd), DA increases to a maximum value (DAmax)
and Eq. (19) becomes*
DAmax nDeP
20
Using the De value determined before (see Case 1), nd the total number of
binding sites (n) as follows:
1. Add varying concentrations of dye to a xed concentration of
protein.
* A high dye concentration is dened in relation to Kd and not protein concentration.
143
21
4.5.
144
FIGURE 6
Chapter 5
145
FIGURE 7 Analysis of T-azo-R binding with bovine serum albumin. Same data as
shown in Fig. 6. (Top graph) Determination of binding parameters in
accordance with Eq. (15). DA is plotted versus DA/P. The slope is Kd and
intercept is DAmax. (Bottom graph) Determination of binding parameters
according to Eq. (18). As P ? 0, then Db ? nP and Kd * D. Under such
conditions it follows that G n (see Refs. 58 and 59).
146
Chapter 5
Dye, equation
T-azo-R
Eq. (14)
Eq. (15)
Eq. (18)
Bromophenol blue
Eq. (14)
Eq. (15)
Eq. (18)
Thymol blue
Eq. (18)
a
mM 1
De (M
cm 1)
0.162
0.177
0.181
4709
5024
3.11
2.03
2.23
87787
69250
69.0
1430
62.a
6.0
26
[PQ]S.
P D PDAQ ;
PDAQ PDS ;
Kd P D =PDAQ
K2 PDAQ =PDS
22
23
24
The overall process is comparable to isoelectric precipitation (4). Precipitation occurs when sufcient numbers of dye molecules bind to neutralize all
protein charges. In contrast, excess protein produces a ``colloidal protective
effect'' that maintains the solubility of protein-dye complexes. From the
denition of solubility product (KS) we have
KS Kd K2 P D
25
Ka Pf H =P
26
The total protein concentration (P) [P ) [Pf] and after substituting for
Pf,
Ka P
P H =P
27
147
and
P
P
Ka =H 1
28
PD
Ka =H 1
29
To attain very low concentrations of soluble protein (in equilibrium with the
protein-dye precipitate) requires a high dye concentrations at a low pH.
Strong protein-dye binding (small Kd) will also facilitate quantitative
precipitation of protein from solution. At pH 4.84 the gelatin complex with
Amido Black 10B yields KS & 4 6 10 12. Under higher acidity conditions
the value KS was too low to determine (4). Refer to Skoog and West (66) for
more information on KS-solubility relations.
5. INTERFERENCE COMPOUNDS AND THEIR AVOIDANCE
There was no interference from low-molecular-weight compounds, including amino acids and peptides. Lipids do not affect protein-dye binding.
Ionic surfactants and benzoic acid derivatives (e.g., p-aminobenzoic acid)
might interfere if present in high concentrations. Chaotropic agents such as
urea are also likely to affect dye-binding results. Dye binding with
nonprotein food components is possible. Calibration graphs for wheat
our protein had a nonzero intercept owing to dye binding with wheat bglucan (67). Mass transfer or diffusion limitations may be important for
solid or powdered food materials. Physical effects can be overcome by
achieving high sample agitation, increasing the mixing time, and reducing
sample particle size.
6. APPLICATIONS OF DYE-BINDING ASSAYS FOR FOOD
PROTEIN ANALYSIS
6.1.
Udy (9) analyzed whole milk and spray-dried milk samples by Orange-G
binding. The milk samples and Kjeldahl protein values were supplied by
Ashworth and co-workers at the Department of Dairy Science, Washington
State University (Pullman, WA). Dye-binding studies at Ashworth's
148
Chapter 5
laboratory led to one of the rst Ph.D. dissertations in this area (68).
Literature covering milk or dairy protein analysis using Orange-G and Acid
Orange 12 is summarized in Table 6.
Ashworth et al. (24) analyzed 354 milk samples from six breeds of
cows. Milk powders were also analyzed. The average protein content for
milks was 3.49 (+ 0.273)%. Some 95% of protein determinations were
within + 0.67% of the crude protein content. NPN, proteose peptone, milk
fat, and lactose caused little or no interference. Sample preservatives
(hydrogen peroxide, formaldehyde, or mercuric hydrochloride) also did not
affect the results. Adding mercuric chloride (1.35 mg %) to milk samples
allowed room temperature storage before analysis. Antibiotics were not
effective preservatives.
Reference
Udy (9), Ashworth et al. (24), Dolby
(25), Ashworth and Chaudry (26), and
Conetta et al. (69), Park and King (70)
Ashworth (20)
Kroger et al. (71)
Ashworth (20)
Sherbon (21)
Sherbon and Luke (72)
Sherbon and Luke (73)
Sherbon (74)
Conetta et al. (69), Lakin (75,76),
Wilkinson and Richardson (77),
Kristoffersen (78)
Sherbon and Fleming (79),
Bruhn et al. (80)
Rawson and Mahoney (81,82)
149
The DBC for milk protein fractions was assessed by Ashworth and
Chaudry (26). Milk protein fractions should have similar DBC values;
otherwise, assays may be affected by variations in milk composition.
Compared with whey proteins, caseins had a lower DBC (Table 7).
Presumably the proportions of casein and whey protein remain fairly
constant in different milk samples. The quantity of protein in several brands
of milk drink (chocolate milk, two-ten, half-and-half, vitamin D milk, etc.)
were determined by Ashworth (20).
The Orange-G binding capacity for milk proteins was remarkably
constant, notwithstanding processing into products such as ice cream and
evaporated milk (Table 8). Fresh milk had a protein content of 3.5%,
whereas evaporated milk contained 7% protein. Cheese manufacture had a
signicant lowering effect on DBC, probably because of proteolysis. Notice
that the values for the DBC are 50% lower than those reported in Table 3
for reasons discussed earlier.
6.2.
AB 10B (mmoles g
566.6
561.7
595.8
589.3
556.8
577.9
592.5
504.9
730.5
763.0
584.4
602.1
76.1
cP)
Orange G (mmoles g
cP)
389.4
396.0
407.1
446.9
415.9
398.2
420.4
376.1
539.8
557.5
278.8
420.6
76.2
a
DBC was calculated from Ref. 26 using dye molecular weights from Table 2. Values are
between 50 and 100% lower than expected from the basic amino acid content in milk proteins
(see Table 3).
150
Chapter 5
TABLE 8 Dye-Binding Capacity and Protein Content for Different Milk Productsa
Product
Milk
Fresh
Evaporated
Powdered, nonfat
Powdered, whole
Buttermilk
Cottage cheeses
Creamed
Dry curd
Cheddar cheese
Mild
Sharp
Cream
2050% fat
Ice cream
Vanilla
Chocolate
OG (mmoles g 1)
AO12 (mmoles g 1)
Protein (%)
393.5
386.8
394.6
366.9
381.1
1000.0
932.8
1000.0
963.6
902.0
3.5
7.0
36.0
26.0
4.0
394.8
394.8
966.4
966.4
15.0
18.0
306.8
245.4
801.1
700.3
24.0
24.0
394.8
1000.06
2.5
394.8
362.3
1000.0
963.6
4.0
4.0
a
A comparison with data from Table 3 shows that the values for OG are lower than expected.
Source: DBC values calucalated from Ref. 21.
* One lot of sterile, canned milk was analyzed by ve laboratories. Three laboratories carried
out crude protein determination by the Kjeldahl method. A further 25 fresh milk samples were
analyzed via 140 replicate determinations.
151
Ice cream mix had 3.852% protein or 3.968% protein by the Kjeldhal and
dye-binding analysis, respectively. Consequently, dye binding received
AOAC approval as a method for protein determination in ice cream and
nonfat dry milk Analysis of ice cream mix and ice cream proteins using dye
binding is further described by Kleyn (84) and by Bruhn (85). Further
examples of these studies are listed in Table 9.
Sherbon (32) compared the Pro-Milk and Udy methods for the
analysis of milk proteins. The two techniques gave comparable results.
Greater care was needed in calibrating the Pro-Milk instrument. A
recommendation to grant ofcial status to the Pro-Milk method was
deferred pending further work. A year later, a six-laboratory study of the
Pro-Milk method led to AOAC approval (33). In addition to reports cited in
Table 9 the Pro-Milk Analyzer is discussed in Refs. 8691.
6.3.
Udy (8) found that wheat albumin, gluten, albumin gluten, and residue
proteins bound constant amounts of Orange-G regardless of the variety of
seed. These observations paved the way for a quantitative analysis of wheat
TABLE 9 Application of Amido Black 10B for Milk and Dairy Protein Analysisa
Amido Black 10B
Cheese
Condensed milk
Ice cream, frozen dessert
Milk
Reference
Kroger and Weaver (92)
Lueck (93)
Kroger et al. (71)
Dolby (25), Ashworth and Chaudry
(26), Radcliffe (94), O'Connell (86),
Conetta et al. (69), Sherbon (95),
Kroger (96), Uzonyi (97), Patel et al.
(98), Ng-Kwai-Hang and Hayes (99),
van Reusel and Klijn (100)
Roper and Dolby (101), McGann et al.
(102), Renner and Ando (103),
Reimerdes and Flegel (104)
Grappin et al. (105), Mabon and
Brechany (106)
Waite and Smith (107)
Sanderson (108), O'Connell and
McGann (109)
152
Chapter 5
protein using dye binding (9). In all, 128 samples of whole wheat our and
218 samples of rened wheat our (from 58 known and 34 unknown wheat
varieties) were examined for Orange-G binding capacity at pH 2.2. The
samples were also analyzed for crude protein content by the Kjeldahl
method. The correlation between amounts of dye bound and crude protein
(%N 6 6.25) is described by Equation (30) (rened wheat our) or Equation
(31) (whole wheat our).
cP 1:092Db
4:62
30
cP 1:000Db
5:53
31
R 0:988
32
153
Parameter
Milled rice
Regression linea
Protein (%)
SY.X (%)
R
Y 14.67 13.60A485
5.5511.65
+ 0.48
0.961
Brown rice
Y 14.78 14.12A485
6.0011.95
+ 0.26
0.984
a
Y crude protein content (%). Protein (%) is range for 45 samples. SY.X (%) standard
deviation from the regression line. Data are from experiments using a laboratory shaker (112).
(22). The correlation coefcient for dye-binding and Kjeldhal results was
0.974 (barley) or 0.984 (malt). The average mean squared error for analysis
(with the Kjeldahl method as reference) was 0.897%. Using commercial
apparatus, 200 protein determinations were completed daily.
Baker and Hunt (114) evaluated the Pro-meter instrument (Foss
America Inc) for dye-binding analysis of wheat protein. About 107 wheat
samples were ground to pass 20-mesh screen and then analyzed according
the instrument manufacturer's instructions. The graph of instrument
response versus protein content was curvilinear for 50 samples of red wheat
(hard red spring, hard red winter, durum wheat). By contrast, a linear
calibration graph was obtained for 57 white wheat samples. The Pro-meter
instrument was judged satisfactory despite some mechanical difculties. The
lter system was periodically clogged, necessitating dismantling and
cleaning of the measuring unit.
6.4.
4:559
R 0:989
33
The standard error of analysis was 1.8213%. Flour particle size had
negligible effects on protein results. Mild heat did not affect dye-binding
154
Chapter 5
results although other studies show that severe heating reduces the DBC of
soy proteins (116120).
Romo et al. (121) assessed seed protein extractability using the Udy
method. The following DA482 changes were noted for the different seed
protein solutions (10 mg mL 1): 1.363 (eld bean), 1.197 (cowpea), 2.976
(rapeseed), 2.2454 (sesame seed), and 1.203 (cotton seed). Clearly, the assay
sensitivity is different for different seed proteins. Medina et al. (122)
proposed that a single calibration graph might be used for cereal, legume,
and oilseed protein analysis. A composite graph would save time. Sesame
our, rapeseed meal, and rapeseed our were analyzed by the standard Udy
(shaker mixing) method. Fig. 8 shows a composite calibration graph for
Acid Orange 12 binding to cereal, legume, and oilseed proteins. The leastsquares equation for the composite graph is*
cP 0:2152Db 4:7333
R 0:981
34
FIGURE 8 A composite calibration graph relating dye binding (X-axis) and crude
protein content for *28 samples of legumes, cereals, and oilseeds.
(Drawn from Table IV in Ref. 123.)
* Actually, the regression equation reported in the literature was Y 0.245X 2.532
(R 0.995). In contrast, Fig. 1 was drawn using only 50% of the experimental data.
155
R 0:93
35
R 0:98
36
From such data it may be shown that the apparent DBC for Orange-G is
204 mg (dye) g 1 (cP) or 490 mmoles (dye) g 1 (cP). With Acid Orange 12 the
DBC is 357 mg g 1 (cP) or 580 mmoles (dye) g 1 (cP). The calibration data
were not affected by our particle size (40 or 60 mesh). However, DBC was
higher for a protein/dye ratio of 2:1 as compared with a ratio of 4:1. Values
for the DBC were proportional to the net concentration of arginine,
histidine, and lysine. The standard deviation for analysis was 1.3% (Orange
G) or 0.80% (Acid Orange). From the higher DBC (per weight), precision,
TABLE 11 Protein Content in Sesame and Rapeseed Products
Determined from Acid Orange 12 Dye Binding and Kjeldahl
Analysis
Protein (% w/w)a
Sample
Sesame our
Rapeseed our
Rapeseed meal
Dye binding
Kjeldahl
59.4 (+ 0.524)
59.4 (+ 1.743)
36.1 (+ 0.595)
58.9 (+ 1.093)
60 (+ 2.91)
36 (+ 0.338)
a
Values are mean (+ SD).
Source: Summarized from Ref. 122.
156
Chapter 5
and sensitivity of analysis, Goh and Clandinin (123) concluded that Acid
Orange 12 was a more suitable dye reagent for rapeseed protein
determination.
6.5.
A.
TABLE 12
Binding
Sample (na)
Meat meal
(21)
Whale meat
meal (12)
Fish meal (8)
Soy bean (8)
or Groundnut
meals (6)
Miscellaneous
foodsb
a
Regression line
DBC
(mmole g 1 cP)
% Error (CV)
cP 0.278Db 30
796925
6.4
cP 0.216Db 30
842770
2.0
cP 0.325Db 24
cP 0.217Db 28
675
1020
2.3
2.0
cP 0.414Db 12
157
cP 0.301Db 8.18
cP 0.602Db 2.50
cP 0.367Db 5.45
cP 0.632Db 3.00
0.90
0.94
0.80
0.95
Sample
Beef
Chicken breast
Pork loin
Cod llet
DBC (mg g
cP)
a
Symbols cP and Db are as dened previously.
Source: Summarized from Ref. 125.
B.
Meat Proteins
Raw beef, chicken, pork (loin), and cod llets were analyzed using OrangeG or Amido Black 10B* dye binding by Torten and Whitaker (125). Their
procedure was described in Chapter 4. A signicant correlation was
observed between crude protein values (Kjeldhal-N 6 6.25) and Db (Table
13).
The DBC for raw meat proteins decreased linearly with increasing
sample protein (see last column of Table 13). The amount of dye bound
depended on the dye/protein ratio. In general, inadequate amounts of
Orange-G were used in many early studies. Dye limitations and inadvertent
changes in protein/dye ratio for different assays reduced the reliability of
dye-binding assays. The regression equation (cP 0.301Db 8.18) for beef
applies over a restricted range of protein content. The effect of a changing
DBC is shown in the simulations reported in Fig. 9. One set of results are
computed on the basis that the DBC is xed. Where DBC varies the dye/
protein ratio the simulated calibration graphs were nonlinear (Fig. 9). The
curves are remarkably like actual calibration curves reported for ground
pork and chicken (125). These samples showed a high dependence of DBC
on protein content and large deviations from linearity. A linear equation did
t the data but only over a highly restricted range of protein content.
Ground chicken, pork loin, or cod llet having greater than 50% crude
protein content should probably not be analyzed by Orange-G dye binding.
It was on account of the dependence of DBC on protein content that Amido
Black 10B was judged unsuitable for meat protein analysis.
* As Amido Black 10B was found to be unsuitable for raw meat analysis, the following
discussion focuses on results obtained with Orange-G.
158
FIGURE 9
Chapter 5
159
Meat product
Egg (whole)
Egg albumin (egg white)
Chicken meat
Chicken liver
Beef or pork (ground)
Beef liver
Proteose peptone
Gelatin
DBC (mg g
cP)a
410440
390410
460480
360390
430440
420440
90145
310350
a
Ranges of values are given for analysis performed in the presence of
excess of dye concentration of 0.40.6 mg mL 1.
Source: Ref. 126.
C.
Egg, chicken, and meat products were analyzed by Ashworth (126) (Table 14).
As he was one of the rst investigators to apply dye-binding assays to foods,
his approach merits attention. Reliable results were obtained provided that
the free Acid Orange 12 concentration (after shaking with the protein sample)
was kept within the range of 0.40.6 mg mL 1. To achieve this, the initial the
dye/protein ratio was kept within a range of 0.640.92. Pork had the same
DBC as beef, which was lower than the value of chicken. The DBC for
mixtures of meat could be deduced from values for individual components.
Dye binding was not affected by the presence of fat or by normal cooking
(1608C, 40 minutes). It was concluded that dye binding is useful method for
composition control in ground meats, eggs, and prepared mixes.
D.
Sausage Protein
Seperich and Price (127) determined protein in model sausage emulsions and
muscle components (myobrillar protein, sarcoplasmic protein, and stroma)
from which they were produced. The approach was modied from Ref.
128.* These studies conrmed that protein dye binding was not affected by
sausage emulsion fat content from 20 to 40%. The DBC was a function of
* Sausage emulsion samples (3.5 g) were homogenized with 51 mL of citrate (0.2 M)phosphate
(0.1 M) buffer (pH 5.5). Ten milliliters of the resulting homogenate was retained for Kjeldahl
analysis. The remainder was shaken with 80 mL of Acid Orange 12 (0.563.64 mM; 0.2
1.27 mg mL 1) in a 250-mL centrifuge tube for 30 minutes and then centrifuged (5.680g; 5
minutes).
160
Chapter 5
dye/protein ratio. At the highest dye concentration examined the DBC was
of the order of 400 mg g 1 (cP), in line with values reported by other
investigators. However, DBC decreased to about 3334 mg g 1 (cP) at a dye
concentration of 0.2 mg mL 1.
6.6.
Mushrooms
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
* Mushrooms were diced, freeze dried, and then oven dried to a constant weight. A 100-mg
portion of mushroom powder was mixed with 20 mL of dye solution. BSA was used as the
standard protein. Amido Black 10B was used in conjunction with the Pro-Milk Mk II
instrument.
20.
21.
22.
23.
24.
25.
26.
27.
28.
161
162
Chapter 5
163
48. I Molnar-Perl, M Pinter-Szakacs, D Medzihradszky. Dye-binding ``stoichiometry'' and selectivity of cresol red with various proteins. Food Chem 35:69
80, 1990.
49. D Racusen. Stoichiometry of the Amido Black reaction with proteins. Anal
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50. I Molnar-Perl, M Pinter-Szakacs, A Kovago, I Petroczy, UP Kralovanszky, I
Matyas. Dye-binding stoichiometry of AO12, AB 10B and OG with etalon
proteins, feed and feedingstuffs and its application for reactive lysine
determination. Food Chem 20:2138, 1986.
51. WAB McBryde. Spectrophotometric determinations of equilibrium constants
in solution. Talanta 21:9791004, 1974.
52. IM Klotz, FM Walker, RB Pivan. The binding of organic ions by proteins. J
Am Chem Soc 68:14871490, 1946.
53. IM Klotz. Spectrophotometric investigations of the interactions of proteins
with organic anions. J Am Chem Soc 68:22992304, 1946.
54. SE Sheppard, RC Houck, C Dittmar. The sorption of soluble dyes by gelatin.
J Phys Chem 46:158176, 1942.
55. M Pesavento, A Profumo. Interaction of serum albumin with a sulphonated
azo dye in acidic solution. Talanta 38:10991106, 1991.
56. KE Lind, U Kragh-Hansen, JV Moller. Protein binding to small molecules. V.
Binding of bromophenol blue by chemical modications of human serum
albumin. Biochim Biophys Acta 371:451461, 1974.
57. Y-J Wei, K Li, S-Y Tong. The interaction of bromophenol blue with proteins
in acidic solution. Talanta 43:110, 1996.
58. Y-J Wei, K Li, S-Y Tong. Spectral study of interaction of thymol blue with
protein in acidic solution. Anal Chim Acta 341:97104, 1997.
59. AN Glazer. On the prevalence of ``nonspecic'' binding at the specic binding
sites of globular proteins. Proc Natl Acad Sci USA 65:10571063, 1970.
60. AG Mayes, R Eisenthal, J Hubble. Binding isotherms for soluble immobilized
afnity ligands from spectral titration. Biotechnol Bioeng 40:12631270, 1992.
61. J Hubble, AG Mayes, R Eisenthal. Spectral analysis of interactions between
proteins and dye ligands. Anal Chim Acta 279:167177, 1993.
62. GS Hartley. The effect of long-chain salts on indicators: the valency-type of
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63. L Michaelis, S Granick. Metachromasy of basic dyestuffs. J Am Chem Soc
67:12121219, 1945.
64. SE Sheppard, AL Geddes. Amphipathic character of proteins and certain
lyophile colloids as indicated by absorption spectra of dyes. J Chem Phys
13:6365, 1945.
65. RW Congdon, GW Muth, AG Splittgerber. The binding interaction of
Coomassie Blue with proteins. Anal Biochem 213:407413, 1993.
66. DA Skoog, DM West. Fundamentals of Analytical Chemistry. 3rd ed. New
York: Holt, Rinehart & Winston, 1976.
67. PJ Wood, RG Fulcher. Interaction of some dyes with cereal beta-glucans.
Cereal Chem 55:952966, 1978.
164
Chapter 5
68. RG Seals. Some aspects of dye binding of milk and milk powder proteins.
PhD thesis, Washington State University, 1960.
69. A Conetta, L Stooker, H Zehnder. An automated system for the determination of milkfat, protein and lactose in milk. Advances in Automated Analysis.
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70. DL Park, RL King. Evaluation of automated dye-binding determination of
protein in milk. J Assoc Of Anal Chem 57:4246, 1974.
71. M Kroger, EE Katz, JC Weaver. Determining protein content of ice cream
and frozen desserts. J Dairy Sci 61:274277, 1978.
72. JW Sherbon, HA Luke. Collaborative study of the dye binding method
applied to chocolate milk drinks, cultured buttermilk, and half-and-half. J
Assoc Of Anal Chem 51:811816, 1968.
73. JW Sherbon, HA Luke. Comparison of the dye binding and Kjeldahl methods
for protein analysis of non-fat dry milk and ice cream. J Assoc Of Anal Chem
52:138142, 1969.
74. JW Sherbon. Dye binding method for protein content of dairy products. J
Assoc Of Anal Chem 53:862864, 1970.
75. AL Lakin. The estimation of protein and the evaluation of protein quality by
dye-binding procedures. ISFT Proc 6:8083, 1973.
76. AL Lakin. Comparison of the amounts of dyes bound by milk proteins under
the conditions employed in dye-binding procedures. XIX International Dairy
Congress 1E:277278, 1974.
77. RF Wilkinson, GH Richardson. Continuous ow analysis of milk proteins
using ultra-violet spectroscopy. J Dairy Sci 58:798, 1975.
78. T Kristoffersen, KH Koo, WL Slatter. Determination of casein by the dye
method for estimation of cottage cheese curd yield. Cult Dairy Prod J 9:1214,
1974.
79. JW Sherbon, R Fleming. Comparison of two formulations of Acid Orange 12
for the determination of protein in milk. J Assoc Of Anal Chem 58:773776,
1975.
80. JC Bruhn, S Pecore, AA Franke. Measuring protein in frozen dairy desserts
by dye binding. J Food Prot 43:753755, 1980.
81. N Rawson, RR Mahoney. Effect of processing and storage on the protein
quality of spray-dried lactose-hydrolyzed milk powder. Lebensm Wiss
Technol 16:313316, 1983.
82. N Rawson, RR Mahoney. A modied method for determination of reactive
lysine in milk powder using Remazol Brilliant Blue R. Lebensm Wiss Technol
16:14, 1983.
83. AH Luke. Collaborative testing of the dye binding method for milk protein. J
Assoc Anal Chem 50:560564, 1967.
84. RH Kleyn. Frozen desserts under protein analysis. Dairy Ice Cream Field
159(7):44, 46, 1976.
85. JC Bruhn. Protein determinations in ice cream. Am Dairy Rev 40(2):34B
34D, 1978.
165
166
Chapter 5
103. E Renner, S Ando. Determination of the casein and whey protein contents of
milk by Amido Black methods. XIX International Dairy Congress E:459460,
1974.
104. EH Reimerdes, B Flegel. Casein micelles: heat-induced changes of the dyebinding capacity. XX International Dairy Congress E:22432244, 1978.
105. R Grappin, R Juenet, D Ale. Determination of the protein content of cows'
and goats' milk by dye-binding and infrared methods. J Dairy Sci 62(Suppl
1):3839, 1979.
106. RM Mabon, EY Brechany. The measurement of protein in fresh and stored
goats' milk by a dye-binding technique. Lab Pract 31:2627, 1982.
107. R Waite, GM Smith. Measurement of the protein content of milk from
mastitic quarters by the Amido Black method. J Dairy Res 39:195201, 1972.
108. WB Sanderson. Determination of undenatured whey protein nitrogen in skim
milk powder by dye binding. N Z J Dairy Sci Technol 5:4648, 1970.
109. JA O'Connell, TCA McGann. Rapid estimation of protein in skim milk
powders. Ir Agric Cream Rev 25(110):1719, 1972.
110. WT Greenaway. Comparisons of the Kjeldahl, dye binding, and biuret
methods for wheat protein content. Cereal Chem 49:609615, 1972.
111. Y Pomeranz, RB More. Reliability of several methods for protein determination in wheat. Bakers Dig 49:4448, 58, 1975.
112. LC Parial, LW Rooney, BD Webb. Use of dye-binding and biuret techniques
for estimating protein in brown and milled rice. Cereal Chem 47:3843, 1970.
113. Y Pomeranz, RB Moore, FS Lai. Reliability of ve methods for protein
determination in barley and malt. J Am Soc Brew Chem 35:8693, 1977.
114. D Baker, WH Hunt. Pro-Meter evaluation. Cereal Foods World 20:246247,
1975.
115. Y Pomeranz. Evaluation of factors affecting the determination of nitrogen in
soya products by the biuret and Orange-G dye-binding methods. J Food Sci
30:307311, 1965.
116. T Hymowitz, FI Collins, SJ Gibbons. A modied dye-binding method for
estimating soybean protein. Agron J 61:601603, 1969.
117. L Sandler, FL Warren. Effect of ethyl chloroformate on the DBC of protein.
Anal Chem 46:18701872, 1974.
118. IM Perl, MP Szakacs, A Koevago, J Petroczy. Stoichiometric dye-binding
procedure for the determination of the reactive lysine content of soya bean
protein. Food Chem 16:163174, 1985.
119. I Molnar-Peal, M Pinter-Szakacs, D Medzihradszky. Dye-binding ``stoichiometry'' and selectivity of cresol red with various proteins. Food Chem 35:69
80, 1990.
120. S Lin, AL Lakin. Thermal denaturation of soy proteins as related to their dyebinding characteristics and functionality. J Am Oil Chem Assoc 67:872878,
1990.
121. CR Romo, AL Lakin, EF Rolfe. Properties of protein isolates prepared from
ground seeds. I. Development and evaluation of a dye binding procedure for
the measurement of protein solubility. J Food Technol 10:541546, 1975.
167
6
The Bradford MethodPrinciples
1. INTRODUCTION
Proteins bind with Coomassie Brilliant Blue G250 (CBBG,* C.I. 42655) to
produce a sparingly soluble complex (1). Protein-dye binding alters the
absorption spectrum for CBBG. This is the basis of the assay developed by
Bradford in 1976 (2). The Bradford assay has several advantages compared
with the Lowry test: (a) four- to tenfold greater sensitivity, (b) tenfold
greater speed, (c) decreased susceptibility to interferences, (d) requirement
for a single reagent, and (e) lower cost. The Bradford assay is quicker than
dye-protein precipitation (Udy assay) as no ltration step is required.
Ready-to-use CBBG dye reagent is available from Bio-Rad Laboratory
Ltd., Pierce Warriner Ltd., and the Sigma-Aldrich Chemical Company.
Principles of the Bradford assay are described in this chapter. Applications
for food protein analysis are discussed in Chapter 7.
Coomassie Blue is the trade name for a group of dyes rst produced by
Imperial Chemical Industries (ICI) Ltd. (UK). Weiler discovered CBBG in
1913. The 1971 edition of the Colour Index (3) lists 40 Coomassie dyes
including Coomassie Blue FF (C.I. 42645), Coomassie Blue R (C.I. 42660),
Coomassie Blue BL (C.I. 50315), Coomassie Brilliant Blue G (C.I. 42655),
Coomassie Blue GL (C.I. 50320), and Coomassie Blue RL (C.I. 13390). The
* Abbreviations: CBBG, Coomassie Brilliant Blue G250; CBBR, Coomassie Brilliant Blue
R250.
169
170
Chapter 6
G and R labels refer to dyes having a greenish blue or reddish blue hue.
Samples of dye with 2.5 times greater purity than the standard grade (19
22% purity) are labeled ``250.'' CBBG is also known as C.I. Acid Blue 90,
Xylene Brilliant Cyanine G, or Brilliant Blue G (4).
CBBR* was rst used as a protein stain in 1963 by Fazekas de St.
Groth et al. (5). Cellulose acetate electrophoresis support was soaked in
sulfosalicylic acid to x protein bands and then transferred to the CBBR
solution (0.25% w/v in water). Blue protein bands form against a clear
background. Nonspecic staining increased if CBBR dye was prepared with
methanol rather than water. From a densitometric analysis of polyacrylaminde gels, the blue color was proportional to protein amount (020 mg). In
later developments, polyacrylamide gels were stained with CBBR dissolved
with a 5:1:5 mixture of methanol, acetic acid, and water or 12.5% (w/v) TCA
(6).
Diezel et al. (1) introduced CBBG as a protein stain for electrophoresis. CBBG has two methyl groups more than CBBR (Fig. 1) and is
therefore less soluble in 12% TCA. This lowers dye penetration into
polyacrylamide gels and reduces background staining. The sensitivity of
* CBBR (CI 42660) is also known as Acid Blue 83, Coomassie Blue R, Xylene Brilliant Cyanine
6b, or Supranolcyanin 6B.
Bradford MethodPrinciples
171
CBBG for protein is also signicantly greater than CBBR.* Reisner et al. (7)
used perchloric acid (3.5%) as solvent for CBBG. The free dye exists as a
colorless (leuco) molecule in perchloric acid solvent. Binding to protein
leads to a blue protein-dye complex. There was virtually no background
staining for polyacrylamide gels. Assay sensitivity was comparable to that
obtained with Naphthylamine Black 10 (0.5% w/v) stain.{
2. THEORY OF THE BRADFORD ASSAY
Protein-protein variations in the sensitivity of the Bradford assay (8,9) led to
interest in CBB-protein interactions. Coomassie Brilliant Blue binds with
proteins by electrostatic interactions. The complex is also held together by
van der Waals forces (5). The rst quantitative investigation of CBBR
binding with proteins was reported by Tal et al. (10). Dye binding occurs
only with polypeptides larger than about 3000 daltons. The number of dye
molecules bound per molecule of protein (n) was strongly correlated with
the total number of arginine, histidine, and lysine (Arg His Lys)
residues. However, the average DBC was 100% greater than combined
numbers of basic amino acid residues. Hydrophobic interactions may
account for dye binding when charged protein sites were saturated.
Rosenthal and Koussale (11) determined the critical micelle concentration
(cmc) of nonionic surfactants with the Bradford reagent. Hydrophobic
solubilization of CBBG within micelles led to marked increases in
absorption at 620 nm.
2.1.
Compton and Jones (12) assessed the effect of pH, protein, and surfactants
on the absorption spectrum of CBBG. They concluded that CBBG exists in
three ionized forms rather than two. The absorptivity of protein-CBBG
complexes increased with protein molecular weight (13). Signcantly higher
color yields were obtained for CBBG binding with polyarginine, polylysine,
or polyhistidine. There was no dye binding with polyaspartic acid,
polyglutamic acid, and polyproline. Lea et al. (14) recorded anomalous
results with the Bradford assay for highly basic proteins. Chemical
* A polyacrylamide slab gel (10 6 15 cm) is soaked in 40 mL of 12.5% TCA for 5 minutes to x
protein. Then 2.5 mL of CBBG solution (0.25% w/v dissolved in water) is added and the gel is
incubated for 1530 minutes. Transferring the gel to a 5% (w/v) acetic acid solution for 12 hours
increases protein band intensity and reduces background staining.
{ This dye is the same as Amido Black 10B (Chapter 5).
172
Chapter 6
CBBG dye shows two lmax values at 470 and 650 nm at pH 0.8* (12).
Adjusting the dye reagent to pH 1.2 produced the following changes: (a)
decreased absorbance at 650 and 470 nm and (b) a new absorbance peak at
595 nm. There was no isobestic point, meaning that more than two
interconverting dye species occurred over the pH range examined. In a
different experiment, addition of 140 mg of BSA to CBBG dye reagent
produced a difference spectra (dye protein versus dye) with lmax at 595
nm. Exposure of CBBG to excess SDS diminished the 470 peak and
produced a new peak at 650 nm.
Shareef and Shetty (20) identied a fourth CBBG charged species. The
purple bi-ionic form (CBBG2 , lmax 515 nm) appears at pH 11.5. With
acidic conditions (pH 1.25) Coomassie Blue dye is a positively charged/
cationic/red (CBBGH2)1 species (lmax 475 nm). This is in equilibrium
with other CBBG forms. (CBBGH2)1 is converted to the zero-charge/
neutral/green (CBBGH)0 species (lmax 650 nm) at about pH 1.6. Thereafter (CBBG)1 , which is the anionic blue form (lmax 595), is produced
at pH 1.8pH 7. The Compton-Jones scheme for CBBG ionization is
summarized in Table 1. Crystal violet shows a similar three-state ionization
as it changes color from violet to green to yellow at pH 8 to pH 2.4 and
pH 0.6 (21);
R1 violet?RH2 green?RH2 3 yellow
Bradford MethodPrinciples
173
Anion
Structure
Net charge
Color
lmax(nm)
ve charges
ve charges
pH*
(CBBG)1
1
Blue
595
1
2
1.87
2.3.
Neutral
(CBBGH)0
0
Green
650
2
2
1.6
Cation
(CBBGH2) 1
1
Red/leuco
470475
3
2
1.25
Just prior to binding there is the anionic form (CBBG)1 in solution. Dye
binding is with the positively charged protein site (RNH3)1. Protein-bound
CBBG has a net charge of zero.
CBBG1 RNH3 1 ?CBBG:RNH3 0
Table 1 shows that lmax is 650 nm for the neutral dye species (CBBGH)0 in
174
Chapter 6
Just prior to binding, the free dye form is the blue (CBBG)1 species. Dye
binding involves hydrophobic interactions with one of two types of neutral
protein sites, Ro or (CBBGRNH3)0. Protein-bound CBBG has a net charge
of 1.
CBBG1 R0 ?CBBGR1
or
CBBG1 CBBGRNH3 0 ?CBBG2 RNH3 1
Dye binding shifts the dye ionization equilibrium toward (CBBG)1 and
produces an absorbance increase at 595 nm. Equation (4) accounts for the
use of A595 readings for the Bradford assay. Dye binding with R0 does not
account for the correlation between DBC and the number of positively
charged basic amino acid residues. In contrast, the number of
(CBBGRNH3)0 sites is determined by the number of basic amino acids.
The Bradford assay can be monitored at either 620 or 595 nm. The
alternative dye-binding scenarios are not mutually exclusive. As described in
Chapter 5, dye binding involves both nonpolar and ionic sites. CBBR binds
to proteins with a dye basic amino acid ratio ranging from 1:1.5 to 1:2 (10).
Compton and Jones (12) suggest that 60% of the Gibbs free energy change
for protein-CBBG binding (*40 kJ mol 1) is due to the nonpolar structure
(benzene and methylene groups) of the dye. The remaining binding free
energy (*30 kJ mol 1) arises from protein interactions with the sulfonate
groups of CBBG.
The metachromatic properties of crystal violet provide relevant
insights (23). In contemporary terms, metachromasia is a change in the
dye absorption spectra due to changes in the dye environment. The lmax
Bradford MethodPrinciples
175
DA=DenP
DA=De
DA=De
5
Db and
176
Chapter 6
therefore
Kd
Df nP Db
Db
Db Kd Df nPDf
nPDf
Kd Df
and
Db
Db Df nP nP
PDf Kd
Db
PKd
10
Df Kd
G
Kd
11
The more familiar version of Eq. (11) (Db/Df nP/Kd Db/Kd) is usable
where the protein concentration is kept constant. Most dye-binding studies
employ a constant concentration of dye while the protein concentration is
varied.
Two studies of CBBG-protein binding have been published. Congdon
et al. (17) segregated protein binding sites for CBBG into ``strong'' and
``weak'' sites. To characterize strong binding sites, different amounts of BSA
(041 mM) were added to a xed concentration (20 mM)* of CBBG. From a
graph of 1/DA versus 1/P the x 0 yields a reciprocal for the maximum
* The concentration of CBBG was 0.0166% (w/v) or 200 mM. To a xed volume (100 mL) of dye
reagent solution was added 02.66 mg of BSA. The nal volume of mixture was brought to
1 mL where needed with distilled water. Then DA620 was recorded for each protein
concentration.
Bradford MethodPrinciples
177
12
The left-hand side of Eq. (12) is supposedly the maximum gradient from the
Bradford assay standard curve.* The results for CBBG binding to seven
proteins are reported (Table 2).
For BSA binding with T-azo-R, Eq. (11) gives a poor t to the results.
The presence of two classes of binding sites (e.g., strong and weak sites)
should lead to curvature in the Scatchard plot (28,29) although this has not
been demonstrated. Because few graphs for CBBG-protein binding have
been published, I have reexamined the data for BSA binding with T-azo-R
TABLE 2 Parameters for Coomassie Brilliant Blue G250 Binding to Selected
Proteinsa
Protein
BSA
Alcohol dehydrogenase
a-Lactalbumin
Glutamate dehydrogenase
Chymotrypsin
Ovalbumin
Carbonic anhydrase
Kd (mM)
n(ns)
18.6
40.0
110.0
8.9
80.0
23.0
35.0
105 (2.7)
30 (7)
14 (2.4)
? (2.2)
13 (2.3)
33 (1.9)
28 (2.8)
eb(M
cm 1)
48,000
57,700
55,400
57,900
55,700
41,900
51,900
a
nS number of strong binding sites (in parentheses) and Kd are average values from the
modied Scatchard plot [Eq. (11)] and Hill plot [Eq. (15)].
Source: Based on results from Ref. 18.
* Eq. (12) is not an appropriate expression for the assay sensitivity. The right-hand expression
should nDeD/Kd (Section 3 of this chapter). The consequence of using Equation (12) to
estimate n is described in Section 5.
178
Chapter 6
(see Chapter 5, Figs 5 and 6 and associated text). The data from Ref. 30
were replotted using the Scatchard equation [Eq. (11)] or modied
Scatchard relations [Eqs (13)(15)].
1
n
Df GKd
1
Kd
G nDf
G
1
G
1
Kd
13
1
n
n
14
1
Kd
nDf
15
Equations for the straight lines and binding parameters are reported in
Table 3.
The results show a poor t to the Scatchard plot; the regression
coefcient (R) for the graph shown in Fig. 2 was 0.7310. From the equation
of the straight line Kd 11.6 mM and nS 106. Other equations led to more
gratifying transformations of the data. Using Eqs (13)(15), R 0.9941. In
Table 3, two data entries are shown for each graph. In the rst case, values
for Kd and nS were assessed assuming that all data conform to a straight line
for a single class of binding sites.
Figs 25 show deviations from linearity at the extremes. The second
data entry in Table 3 is derived from results tted to the main linear phase in
each graph. Eqs (13) and (15) emphasize data collected at high protein
TABLE 3 Parameters for T-Azo-R Binding to Bovine Serum Albumin Analyzed
Using Scatchard and Modied Scatchard Plots
Linearization
equation
Eq. (11)
Eq. (13)
(strong sites)a
Eq. (14)
(weak strong sites)a
Eq. (15)
(strong sites)a
a
Equation
6
Y 9.21 6 10 8.62 6 10 X
Y 1.06 6 107 X 164 6 105
Y 9.26 6 10
X 164 6 10
Y 0.984 9.26 6 10
Kd (mM)
11.6
6.1
3.8
5.6
19.61
5.8
4.2
106
64
43
61
76
63
48
Different equations emphasize data collected at a high protein/dye ratio (strong sites) or a low
protein/dye ratio (weak strong binding sites). Second data entries are calculated using the
major linear phase of each graph.
Bradford MethodPrinciples
179
FIGURE 2 Scatchard plot for T-Azo-R binding with bovine serum albumin. Dye (10
mM) was titrated with 06 mM BSA. Study was performed at pH 2.3.
Data from Ref. 31 plotted in accordance with Eq. (8).
180
Chapter 6
FIGURE 3 Modied Scatchard plot for T-Azo-R binding with bovine serum
albumin. Dye (10 mM) was titrated with 06 mM BSA. Study was
performed at pH 2.3. Data from Ref. 31 plotted in accordance with
Eq. (10).
FIGURE 4
Bradford MethodPrinciples
181
FIGURE 5 Modied Scatchard plot for T-Azo-R binding with bovine serum
albumin. Dye (10 mM) was titrated with 06 mM BSA. Data from Ref.
31 plotted according to Eq. (15).
The relative color yield for CBBG binding to different poly-L-amino acids
was poly-L-lysine (1), poly-L-histidine (4.2), poly-L-tryptophan (4.4), poly-Ltyrosine (4.7), and poly-L-arginine (36) (12). With increasing dye concentration there was a more similar CBBG response to different basic amino acids
* It is not certain whether these calculations were corrected for changes in the dye ionization
with pH. The low ef,20nm value at pH 1 could be due to a sixfold lower concentration of blue
(neutral) dye species at pH 1 compared with pH 7.
182
TABLE 4
Protein
(pH 1.0)
Alcohol dehydrogenase
Bovine serumalbumin
Carbonic anhydrase
Chymotrypsin
a-Lactalbumin
b-Lactoglobulin
Ovalbumin
(pH 7.0)
Alcohol dehydrogenase
Bovine serumalbumin
Carbonic anhydrase
Chymotrypsin
a-Lactalbumin
b-Lactoglobulin
Ovalbumin
1
cm
(pH 7) or 8800 M
De620(M
cm 1)
11,300
18,300
7,300
1,300
9,300
12,300
8,300
2.6
4.8
0.9
2.1
15.0
3.0
0.9
49,200
39,200
43,200
47,200
44,200
25,200
34,200
127.0
193.0
125.0
101.0
143.0
84.0
87.0
cm
n
28
100
25
16
14
18
33
Lys Arg
32
86
27
17
13
18
36
2
5
1
2
6
12
2
(pH 1).b Sensitivity as determined from the maximum slope of the calibration graph.
Chapter 6
a
ef (620) 43,700 M
Source: Ref. 17.
Bradford MethodPrinciples
183
(10); DA595 readings were 0.92, 1.2, and 1.5 for poly-L-lysine, poly-Lhistidine, and poly-L-arginine, respectively. These studies agree on the
importance of poly-L-arginine as a CBBG binding site. Moreno et al. (13)
reported a relative color yield from different polyamino acids as poly-Llysine (1), poly-L-tyrosine (1.9), poly-L-arginine (3), poly-L-histidine (>5.5).
CBBG binding increased with the polypeptide molecular weight, but
differences in color yield are less marked when absorbance changes are
expressed per unit mass (microgram) of material analyzed.
In summary, studies involving CBBG/R binding to poly-L-amino
acids indicate that these dyes bind to basic groups. Nonpolar amino acid
residues, notably tyrosine and tryptophan, are also important binding sites.
Additional nonpolar sites for CBBG binding are created as the anionic dye
molecule binds to positively charged protein sites. However, poly-L-amino
acid results should be treated with caution. Proteins rarely feature the high
density of sites associated with homopolymers. The correlation between
CBBG binding and the number of basic amino acids occurs when ionic
bonding predominates and the dye species is limiting (Chapter 5). The
relations between protein-binding parameters (n, Kd, De) and characteristics
of the Bradford assay are discussed in the next section.
3. EFFECT OF PROTEIN-DYE BINDING PARAMETERS ON
THE BRADFORD ASSAY
The plot of DA versus protein concentration leads to a hyperbolic
calibration graph.*
DA
nDePD
Kd nP
16
Eq. (16) is a result of the equilibrium between free dye, protein, and the
bound dye. At low protein concentrations nP << Kd and
DA nDeDP=Kd
17
18
A linear calibration graph will be obtained when nP << Kd. For a linear
relationship between DA and [P] it is necceary that Kd exceeds protein
* This relation is the same as Equation (12) of Chapter 5.
184
Chapter 6
19
4.
A double reciprocal plot (1/DA versus 1/[P]) or log(DA) versus log[P] will
extend the linear range of a hyperbolic Bradford calibration graph
(Chapters 3, Sections 3 and 4). Other linearization schemes have been
proposed for the Bradford assay. Sedmak and Grossberg (31) increased the
upper limit of linearity from 10 mg BSA to 50 mg by plotting A620/A456 versus
protein amount. Bearden (32) obtained an upper limit of linearity of 40 mg
BSA by plotting the difference between A595 and A465 readings against the
amount of BSA. Zor and Selinger (33) plotted A595/A450 versus protein
concentration. These linearization schemes allow for (a) the hyperbolic
protein-dye binding prole and (b) the overlap of the absorbance spectra for
the free dye and bound dye.
5.
Eq. (18), which properly describes the sensitivity of the Bradford assay,
reduces to Eq. (12) only when [D] & Kd. Otherwise, the total number of
binding sites calculated from Eq. (12) will be in error by the factor D/Kd.
Bradford MethodPrinciples
185
This error appears in estimates for the total number of sites reported in
Table 2 and Table 4. The CBBG concentration used in the studies was *113
mM and hence values for n need revising downward by 113/Kd. A further
error appears with respect to pH 1 data in Tables 2 and 4. Allowing for a
shift from (CBBGH)0 to the (CBBGH2) species at low pH, ef,620 and hence
De probably remain unchanged with pH (De620 is approximately 18,300). Eq.
(12) then gives n 214 for BSA at pH 1. The corrected number of binding
sites is 35 (i.e., 214 6 Kd/D). A further independent estimate comes from
Fig. 1 of Chapter 7. A plot of DA/P versus D gives the regression line
Y 5.94 6 1010D. The gradient of this graph is n De/Kd [see Eq. (18)].
Assuming that Kd 18.6 6 10 6 M and De 18,300 M 1 cm 1 for BSA (see
Tables 4 and 6), we obtain n 56.
The preceding corrections lead to new estimates for the maximum
number of CBBG binding sites for BSA (n 3556). As BSA has
approximately 117 Arg His Lys sites, only 3048% of available cationic
sites bind CBBG. In Table 4 the reported agreement between numbers of
CBBG binding sites and (Arg Lys) residues is fortuitous.
In any case, the n estimate from Equation (12) (Table 4) exceeds the
total of Arg Lys residues. Hydrophobic interactions were invoked to
explain excess dye binding. CBBR-protein complexes precipitate within the
interstices of polyacrylamide gels. The number of dye binding sites exceeds
the number of protein cationic sites (Lys Arg His) up to 150% (10).
However, Kd values for protein-CBBR binding are similar to those in Table
6.4: lysozyme (35.7 mM), cytochrome c (83.3 mM), RNAse (125 mM), trypsin
(83.3 mM), pepsinogen (37 mM), pepsin (77 mM), and gramicidin S (111 mM).
Between 1.3 and 3.0 CBBR molecules bind for each basic amino acid
residue. Initially CBBR molecules bind to protein via 1:1 ionic interactions.
There then follows the uptake of a second CBBR molecule via nonpolar
interactions. This binding scheme accounts for (a) the correlation between
DBC and numbers of basic residues and (b) the involvement of nonpolar
interactions in protein-dye binding. Wilson (4) suggested the same idea.
6. SOLID-PHASE DYE-BINDING ASSAYS
To perform a solid-phase assay, the protein is rst bound to lter discs and
stained with CBBG dye reagent. Destaining is carried out to remove
nonspecically bound dye and the stained protein spot is excised using a
cork borer and immersed in a solubilization solvent. The amount of dye
solubilized is measured from DA readings at 600630 nm. A calibration
graph can be produced as usual by analyzing standard concentrations of
protein. Solid-phase dye-binding assays are described in Chapter 5.
186
7.
7.1.
Chapter 6
Compounds that interfere with the Bradford protein assay are listed in
Table 5. These include food constituents such as chlorophyll, pectin, and
ethanol and low-molecular-weight surface-active agents. Reducing compounds such as ascorbic acid have a fading effect on triphenylmethane
colors (34). The action is slow and requires about 7 days. Compounds
thought to be compatible with the Bradford method are listed in Table 6.
Many buffer salts, EDTA, hydroxycinnamic acid derivatives, and low
concentrations of avanols apparently do not interfere with protein-CBBG
interactions (Section 7.3).
The preceding information is intended only as a rough guide. Food
components not listed should be compared with their nearest listed relative.
For example, pectin and gum arabic are interfering compounds. Expect
interference from other structural polysaccharides (alginate, carrageenans)
and plant gums (acacia, Tara gum, etc.). Tables 5 and Table 6 are not
denitive guides: (a) potential interferences are usually tested in the absence
of protein, (b) many interferences were tested using the prototype Bradford
method (involving low dye concentrations), and (c) some interferences have
Bradford MethodPrinciples
187
Nonionic detergent concentrations above the cmc produce a shift in the lmax
value for CBBG from 650 nm to 600620 nm. Thereafter A620 increases at
the expense of A470 and A650 values. CBBG binding to nonionic detergents
leads to an increase in A595 compared with A650. The reverse is observed for
CBBG binding to anionic detergents (SDS). At concentrations below
the cmc, SDS reduces the assay sensitivity by competing with dye molecules
Potassium chloride (1 M)
Potassium phosphate (pH 7; 1 M)
rRNA (0.25 mg/mL)
Rutin
Sinapic acid
Sodium acetate (0.6 M)
Sodium citrate (0.05 M)
Streptomycin sulfate (20%)
Thymidine (1 mM)
tRNA (0.4 mg/mL)
Tyrosine (1 mM)
Valine
Glucuronic acid
Sitosterol
Stigmasterol
Maltol
Chlorogenic acid
Apigenin
Phloretin
Chrystin
Fisetin
188
Chapter 6
for charged and noncharged protein sites. The spectral changes are due to
the solubilization of neutral (CBBG)0 species inside detergent micelles (11).
The positively charged dye species (CBBG)1 binds to SDS, forming a
neutral complex, which is then solubilized within detergent micelles (35).
To avoid interference, Zaman and Verwilghen (36) reduced the
concentration of SDS in protein samples by precipitating with potassium
salts. The SDS-depleted sample is then assayed using the Sedmak-Grossberg
assay (Chapter 7, Section 2.5). Kapp and Vinogradov (37) removed SDS
from protein samples using a column lled with the ion-exchange resin
AG11A8 (Bio-Rad Laboratories). The SDS binding capacity was 1.7 mg/
mL (wet resin). Binding of the detergent was extremely rm and no simple
techniques were found to regenerate the support. The recovery of protein
from this support ranged from 52 to 90%. SDS may be removed from
samples using charcoal cartridges made in house (38). Preextracting SDS
from PAGE gels (with charcoal placed within a dialysis bag) allows gel
staining using the conventional Bradford reagent. This can therefore serve
as a dual-purpose reagent for protein analysis in solution as well as for
PAGE.
Alkyl-b-D-glucopyranoside detergents are compatible with the Bradford assay. Fanger (39) screened a number of commercially available
detergents for solubilizing membrane proteins.* Low interferences were
obtained with octyl-b-D-glucopyranose and related alkyl-b-D-glucopyranosides. There was tolerance for up to 10% (w/w) detergent in protein samples.
Detergents with low cmc have more adverse effects on the Bradford
assay (Fig. 6). Nonionic detergents showed a bell-shaped concentration
response. Friedenauer and Berlet (40) found the optimum color yield for a
Triton X-100 concentration of 0.008% in the nal assay mixture; the cmc for
Triton X-100 is about 0.01% (w/w). Assay sensitivity increased by 11 to
128% with an average increase of 33% for 15 different proteins examined.
Apparently, Triton X-100 facilitates protein-dye interactions via noncovalent bonding. The detergent had no effect on protein conformation. To
enhance sensitivity the detergent should be added to the protein sample
before dye.
7.3.
Bradford MethodPrinciples
189
ferulic acid, and sinapic acid are also compatible solutes. Flavanoids and
related substances possessing a chalcone skeleton (anthocyanins, anthoxanthins, proanthocyanadins, and tannin) can interfere (41). Other potential
interferences from plants include products of the mevalonate pathway
(carotenoids, lycopenes, terpenes, etc.) and the molanic acid pathway (lipids,
phospholipids, and lipid hydroperoxides). Some phenol-CBBG complexes
have an absorption spectrum that overlaps that for the protein-CBBG
complex. There is no evidence that protein precipitation with tannins is a
source of error. Techniques for preparing plant proteins, to minimize
interactions with phenolic compounds, are reviewed by Loomis and Battaile
(42) and Loomis (43).
Phenolic metabolites bind to proteins via (a) hydrogen bonding to the
peptide oxygen, (b) oxidation to quinones followed by covalent reactions
with the protein e-NH2 or SH group, (c) ionic interactions with phenolate or
carboxylate groups, and (d) hydrophobic interactions between the phenolic
benzene ring and aromatic amino acid residues. Some techniques for
controlling protein-phenol interactions include (a) maintaining sample pH
at just below neutral pH; (b) addition of phenolic adsorbents, e.g.,
polyvinylpryrrolidone (PVP), Polyclar AT, Amberlite XAD-2, XAD-4, or
XAD-7; (c) use of antioxidants and phenol oxidase inhibitors including
190
Chapter 6
chelators, sulte, and ascorbic acid; (d) addition of protective proteins such
as BSA; and (e) use of inert atmospheres such as argon, carbon dioxide, or
nitrogen (42,43).
The Bradford assay is more resistant to interference by phenolics than
the biuret, Lowry, and BCA assays. Indeed, Folin Ciocalteu and BCA
reagents are used for the quantitative analysis of phenolics and other food
antioxidants. These assays are therefore not suited to plant-derived samples
including leafy vegetables, fruits, stem, and tubers. Processed foods such as
beverages (tea, coffee, beer, wine, chocolate) are also not readily analyzed by
the Lowry or BCA assays. The Bradford assay is widely applied to beer and
wine protein analysis (Chapter 7, Sec. 4). Sample pretreatment involves size
exclusion chromatography to remove low-molecular-weight interferences
from beer or wine. Dialysis or treatment with phenolic adsorbents is also
feasible.
7.4.
Bradford MethodPrinciples
191
and soybean trypsin inhibitor) (44). In some cases, the relative sensitivity of
the Bradford assay was related to differences in protein hydrophobicity. The
rate of color formation was also marginally reduced for some glycosylated
versus nonglycosylated proteins.
Rocher et al. (46) identied six distinct g- and o-secalins by
fractionation of rye storage proteins some of which were glycosylated. A
40-kDa rye protein was exceptional in having signicant absorbance in the
visible region, low CBBG binding, and immunoreactivity. Other naturally
glycosylated proteins [peanut conarachin, soybean conglycinin (47), lupin
conglutin (48)] might show abnormally low reactivity with CBBG. The total
protein content of rapeseed our determined by the Bradford method did
not agree with results of Kjeldahl method (49).
The mechanism by which sugars interfere with the Bradford assay is
uncertain. Sugars have no direct interactions with CBBG. Glycosylated
proteins contain oligosaccharide units linked to asparagine (Asn-X-Ser or
Asn-X-Thr), serine, or threonine residues that are not obviously involved in
protein-CBBG binding (50). Perhaps steric hindrance and the generally
lower hydrophobicity of glycosylated proteins may account for the
reduction in CBBG binding.
7.5.
REFERENCES
1.
2.
192
Chapter 6
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Bradford MethodPrinciples
193
194
Chapter 6
7
Bradford AssayApplications
1. INTRODUCTION
The principles of the Bradford assay (1) are discussed in Chapter 6.
Applications of the Bradford assay for food protein analysis are discussed in
this chapter. Section 2.2 introduces several modications of the Bradford
assay designed to increase its compatibility with buffer salts, reduce proteinprotein variations in results, and enable analysis of detergent-containing
samples. Protein precipitation using the TCA-DOC method is not suitable
for the Bradford assay. Section 2.3 describes several protein precipitation
methods not using detergents. The performance characteristics of the
different assays are summarized in Section 3. Section 4 of this chapter is a
review of applications of the Bradford assay for food protein analysis.
The design of the Bradford assay owes much to classical dye-binding assays.
There are similarities in the choice of buffer and the relative volumes for
protein and dye reagent:
Method 1
CBBG dye binding assay for proteins (1).
195
196
Chapter 7
Reagents
1. Coomassie Brilliant Blue G250 (Sigma)
2. Ethanol (95% w/v)
3. Phosphoric acid (85% w/v)
4. Bovine serum albumin
Procedure
Preparation of CBBG dye reagent. Dissolve CBBG (100 mg) in 50 mL
of ethanol. Add 100 mL of phosphoric acid (85% w/v) and dilute to
1 L with distilled water. Filter through Whatman No. 1 paper and
store in a stoppered bottle.*
Standard protein assay. Pipette 100 mL of protein (1001000 mg mL 1)
into several 1.2 6 10 cm test tubes. Add 5 mL of dye reagent to each.
Mix and record A595 readings after about 5 minutes. Prepare a
reagent blank with 100 mL of solvent and 5 mL of dye reagent.
Microassay. Pipette 100 mL of protein standard (10100 mg mL 1) into
small test tubes.{ Add 1 mL of dye solution to each, mix, and record
A595 readings against an appropriate reagent blank.
Calibration graph. Plot DA595 (i.e., absorbance corrected for the blank
reading) versus amount of protein (10100 mg).
Determine the concentration of protein in 0.1mL of the unknown
sample by referring to the calibration graph.
A blue color develops within 2 minutes of mixing protein and dye
solutions. The color remains virtually constant for up to 20 minutes and
decreases slowly between 20 and 50 minutes. Assay reproducibility can be
improved by measuring A595 readings within 5 and 60 minutes after mixing
dye and protein. The calibration features of the Bradford assay are
described in Chapter 6.
2.2.
The Sedmak-Grossberg (2) assay employs perchloric acid (3.5% w/w) as the
solvent for CBBG. The sensitivity is higher than with Method 1 but the new
method is subject to interferences by strongly buffered samples. The use of
perchloric acid solvent for CBBG was rst suggested by Reisner et al. (3).
CBBG is red (lmax 465) when dissolved in 3.5% (w/v) perchloric acid
solution. Addition to protein generates a blue protein-dye complex with
* The nal reagent composition is nominally 0.01% CBBG in 8.5% phosphoric acid and 4.5%
ethanol.
{ Plastic microcentrifuge tubes are convenient vessels for microassay.
Bradford AssayApplications
197
lmax 595 nm. Using a low concentration of mineral acid (nitric or sulfuric
acid) as solvent leads to reagent instability with regard to small changes in
sample ionic strength. If CBBG is dissolved with high concentrations of
mineral acid, there is poor color change when the dye is added to protein.
Weak organic acids (e.g., acetic, formic, and isobutyric acid) are not suitable
solvents for CBBG because very high concentrations are needed to maintain
the dye pH (2).
The Sedmak-Grossberg reagent gives an immediate color reaction
with proteins. The absorbance change is stable for 6090 minutes. With
0.6 M HCl as solvent, the color was stable for 34 hours. Thereafter the
protein-dye complex formed a precipitate. The main disadvantage of this
assay is its susceptibility to pH variations. Samples containing 0.1 M
phosphate buffer (pH 7.0) or Tris-HCl buffer (pH 8.0) are not usable. Gel
ltration fractions with low buffer capacity (*10 mM) can be analyzed
accurately.
Bearden (4) modied the Sedmak-Grossberg and Bradford assays to
(a) increase the color stability, (b) increase assay sensitivity, and (c) reduce
interference by buffer salts. The new dye reagent was prepared as described
in Method 1 but without ethanol. Upon mixing equal volumes of protein
and dye solutions (thereby avoiding the 10- to 50-fold dilution of samples by
the dye reagent), a blue complex is formed in 12 minutes. The absorbance
increases slowly over 6090 minutes, then decreases from 90 to 180 minutes.
After 3 hours, A595 readings were within +8% of the maximum. The
increase in color stability was attributed to the omission of ethanol from the
reagent formulation. The protein-dye complex may be less soluble in
perchloric acid plus organic solvent (ethanol or methanol). Plastic cuvettes
are to be avoided as they may catalyze protein-dye precipitation.
The Read and Northcote (5) assay reduced protein-protein variations
in assay results by increasing the CBBG dye reagent concentration.
The order of color intensity for Method 1 with different proteins is
RNAse < ovalbumin < lysozyme < BSA cytochrome c (6); trypsin < chymotrypsin < pepsinogen < lysozyme < BSA < cytochrome c (7). More uniform
responses were achieved by increasing the CBBG concentration. Table 1
lists a range of CBBG dyes from various suppliers (5,8). Commercial
samples vary greatly in dye content because different users require different
levels of purity. Only the highest purity CBBG (98% purity) samples
should be used for protein analysis. The technical grades have 2050% (w/w)
dye matter. Variations in dye purity makes it impossible to compare the
performance of Bradford reagents (prepared with CBBG) from different
suppliers. Ideally, dye reagent formulations should be standardized as
suggested by Sedmak and Grossberg (2). Reagents prepared from different
batches of dye should be diluted to provide a CBBG content of 0.01% (w/v)
198
Chapter 7
TABLE 1 Composition of Coomassie Brilliant Blue G250 Samples Used for Protein
Analysis
Dye (designation and supplier)
Coomassie Brilliant Blue G250, Sigma
(Poole, UK or St. Louis, MO)
Serva Coomassie Blue G, Serva
(Heidelberg, Germany)
Serva Blue G, Serva (Heidelberg,
Germany)
Coomassie Blue G, Eastman (Rochester,
NY)
Dye purity is given as percent dry weight (% DW). This information has not been checked
against current dye grades from suppliers. The formula weight of CBBB 854 Da. Saturating
concentrations correspond to &117 mM.
Source: Compiled from Refs. 5 and 8.
with A550 1.18 (Table 1). This is actually twofold greater than the CBBG
concentration in Method 1.
Stoscheck (9,10) reduced protein-protein variations in assay results by
15-fold. She achieved this by adding NaOH to the assay mixture. Alkali also
increased assay sensitivity by 2.7-fold. Either 520 mL of 10 M NaOH or 50
200 mL of 1 M NaOH should be added to 1 mL of Bradford reagent. The
modest (0.020.12) pH change leads to no changes in dye ionization. NaOH
is thought to produce a salting-out effect leading to enhanced protein-dye
binding by hydrophobic interactions. The quantity of NaOH needed
decreases with increasing CBBG dye concentration. At low dye concentrations about 200 mL of 1.0 M NaOH was needed for optimum effect. Dye
reagent prepared from high-purity Serva Blue G dye required 50 mL of
1.0 M NaOH per mL of dye solution. The NaOH can be added to the
protein sample or to the Bradford dye reagent. The effect of adding other
salts (e.g., ammonium sulfate) on the Bradford assay should be examined. In
summary, the Read-Northcote-Stoschek modication can be implemented
by simply using high-purity CBBG grade (such as Serva grade; Table 1) for
routine analysis. The nal dye reagent should be alkalinized with 50 mL (of
1.0 M NaOH) per mL (dye reagent) before use.
SDS, which is commonly added to electrophoresis buffers, lowers the
sensitivity of the Bradford assay (Chapter 6). With the Zaman and
Verwilghen (11) version of the Bradford assay, SDS is removed from
Bradford AssayApplications
199
samples by precipitation with potassium phosphate. Boccaccio and QesadaAllue (12) used potassium chloride as the precipitant for SDS. After
centrifugation, the SDS-depleted samples are analyzed using Method 1. The
linear range for analysis was 050 mg BSA with a sensitivity 10-fold lower
than observed with SDS-free samples. The sensitivity for SDS-depleted
samples reached 80% of the value for control samples when Boccaccio and
Quesada-Allue (12) analyzed insect cuticular protein extracted with the aid
of SDS. This approach should work for crustaceacrabs, lobster, and
related commodities.
Proteins dissolved in electrophoresis buffers (normally containing 2%
w/w SDS and 5% w/w 2-mercaptoethanol) were also successfully analyzed
after simple dilution (13). Typically, 10 mL of sample was diluted to 100 mL
with distilled water. Then 5 mL of Bradford dye reagent was added.
Calibrations with BSA dissolved in electrophoresis sample buffer showed a
linear dynamic range up to 100 mg BSA. Once more the assay sensitivity was
10-fold lower in the presence of SDS but comparable to the sensitivity of the
Lowry assay.
2.3.
* To 100 mL of protein solution add 25 mL of TCA (72% w/w). Incubate the sample at 08C on ice
for 30 minutes and then centrifuge at 10,000g using a microcentrifuge. Discard the supernatant
and remove excess TCA using absorbent paper. Redissolve the protein pellet with 50 mL of 1 M
NaOH. For refractory samples incubate at 378C for 23 hours. Add 1 mL of Bradford reagent
and record absorbance values at 595620 nm.
200
Chapter 7
2.4.
* Adjust the sample (100 mg protein) volume to 200 mL with distilled water, if necessary. Add
10 mL of potassium phosphate buffer (0.5 M pH 7.4), 10 mL of calcium chloride (0.25 M), and
1 mL of ethanol (80% v/v); mix after each addition. Centrifuge at 7000 g and remove the
supernatant by aspiration. Wash the protein pellet by adding 100 mL of water and 1 mL of
ethanol. Centrifuge and remove the supernatant as before. Repeat the washing step if necessary.
Add CBCG dye concentrate (100 mL) and allow at least 5 minutes for the pellet to dissolve. Next
add 400 mL of distilled water and record absorbance readings at 595 nm.
{ Dissolve CBBG or CBBR in a minimum volume of water-methanol (2:1) solvent. Add an
equal volume of sodium chloride (5 M). Filter and dry CBBG precipitate at 808C. Alternatively,
dissolve the CBBG solid with methanol. Dry the methanol solution to recover solid CBBG.
Bradford AssayApplications
201
Sensitivityb
DA595 mg 1
Sensitivityc
DA595 (mL mg 1)
Linear range
(mg)
0.011
0.028
0.057
0.099
0.058
0.059
0.063
0.059
550
220
110
0.55
202
Chapter 7
Sedmak-Grossberg Modication
A plot of A620 versus the amount of BSA (060 mg) produced a curvilinear
graph. A linear response was obtained for 010 mg BSA. An extended linear
graph was produced by plotting A620/A465 versus protein. The reproducibility of the standard and microassay formats was 45% for 10 replicate
measurements with 10 mg of BSA. The sensitivity was 0.044 (DA595 mg 1).
There was no color change with polypeptides smaller than 3000 Da. This socalled molecular weight selectivity is an important feature of CBBG dyebinding assays (Section 4). Many low-molecular-weight nonprotein constituents do not interfere with the Bradford assay (2).
C.
Bearden's Modication
The linear dynamic range was 040 mg protein. For samples containing
0.021 mg of protein the assay sensitivity was 0.045 (DA mg 1), which is four
times greater than that for the conventional Bradford method (4).
3.2.
Increasing the CBBG dye reagent concentration led to the following effects:
(a) a two- to four fold increase in assay sensitivity, (b) increased rate of color
formation, and (c) reduced protein-protein variations in assay sensitivity.
Optimum assay sensitivity was attained using Serva Blue G dye of 98%
purity (Table 1). Addition of 50 mL of 1.0 M NaOH per mL dye reagent
reduced protein-protein variations by 15-fold. Assay sensitivity increased by
a further 2.7-fold (5).
Bradford AssayApplications
203
204
Chapter 7
acid and 2.5% (w/w) ethanol.* Making the dye reagent 0.008% (w/v) with
respect to Triton X-100 increased assay sensitivity 25-fold compared with
the standard Bradford assay. The effect of Triton X-100 and other
detergents on the Bradford assay is described further in the next section.
3.3.
4.
4.1.
Adequate levels of protein ensure beer foam stability. However, too much
protein leads to haze formation in cold beer. Rapid methods for beer protein
analysis are necessary for improved quality assurance and for monitoring
brewing processes. Beer is an aqueous extract from malted barley to which
hops are added for avor. The nished product contains protein,
polypeptides, NPN, polyphenolic dyes, carbohydrates, and nucleic acids.
The Kjeldahl method, although approved for beer protein analysis, is
subject to interferences by NPN. Plant-derived substances interfere with the
biuret, Lowry and BCA methods. Lewis et al. (25) were rst to use the
Bradford assay for beer protein analysis (Table 3).{
* A dye concentration of 0.01% (w/w) appears to be saturating for a solvent comprising 8.5%
phosphoric acid and 4.5% ethanol. The saturating concentration of CBBG appears to have
doubled for a solvent with higher (12.5%) phosphoric acid and lower (2.5%) ethanol.
{ Beer and wort samples were subjected to ultraltration using a 10 kDa molecular size cutoff
membrane to remove interferents. Pretreatment is also possible by size exclusion chromatography (SEC) with a Sephadex G50 (2.2 6 40 cm) column. Then 0.10.5 mL of pretreated beer
or wort was added to 5 mL of Bradford reagent with mixing. A595 or A620 readings were
recorded after 1040 minutes.
Bradford AssayApplications
205
Reference
Lewis et al. (25)
Hii and Herwig (26)
American Society of Brewing Chemists
(27)
Dale and Young (28)
American Society of Brewing Chemists
(29)
Kano and Kamimura (30)
Onishi and Proudlove (31)
Williams et al. (32)
* Samples were eluted with 0.05 M NaCl (ow rate 84 mL hr 1). Fractions eluting from the
column void volume (>2 kDa molecular size) were collected for analysis; 0.4 mL of sample was
added to 5 mL of Bradford reagent and the assay performed as described in Method 1.
206
Chapter 7
0:02857
The CV for analysis was 5.8%. Sensitivity toward beer proteins was
lower than for lysozyme. Beer total nitrogen had 6380% NPN. The protein
standard should be chosen carefully. Compared with other protein assays,
the Bradford technique had advantages of greater speed, higher resistance to
interferences, and high molecular weight specicity. Dale and Young (28)
examined the correlation between Bradford assay results and beer foam
stability (head retention index). Beer was pretreated by SEC using FPLC*
with Sephadex G25, G50, and G57 columns. Fractions eluted in the void
volume were collected for analysis. There was a signicant correlation
(R 0.91) between foam stability and Bradford assay results. Foam stability
was related to the quantity of polypeptides with sizes >5, >30, or >80 kDa.
Beer contained polypeptides ranging from 2 to 100 kDa.
A collaborative test for the Bradford assay organized by the American
Society of Brewing Chemists was reported in 1987 (27,29).{ The major
ndings of the ASBC study were:
1. The Bradford method is rapid, convenient, and highly sensitive for
beer proteins.
2. The precision of analysis for within-laboratory errors is 2.64.5%.
The between-laboratory errors range from 15.5 to 35.5%.
3. Degassing (14 hours vs. 1 hour standing) and the degree of mixing
beer and dye reagent (3 vs. 5 seconds) have a signicant effect on
the test results.
4. Temperature (208C versus 258C), color development time (1055
minutes), or light exposure has no signicant effect on the
Bradford assay.
5. Results with different dye reagents are unacceptably different
(Fig. 2).
The recommendation to grant the Bradford method ``approved'' status
was deferred in 1987. Between-laboratory differences were considered too
great. Variation arose from the use of different commercial samples of
CBBG in different laboratories (Fig. 2). Ready-made dye reagents were
* FPLC=Fast protein liquid chromatography.
{ Two samples each of premium beer, light beer, and malt liquor were analyzed by 13
laboratories.
Bradford AssayApplications
FIGURE 2
207
from Piece Ltd. or Bio-Rad Ltd. Other CBBG samples were purchased as
solids and the dye reagent solutions were prepared in house. Within the
remit of the test, it was not possible to determine which dye reagent gave
accurate results. However, the ``true'' protein content of the beers was not
determined. I discussed problems likely to arise from batch-to-batch
differences in CBBG dye purity (Table 1). To improve assay repeatability
(between-laboratory reproducibility), strictly dened dye formulations
should be used.
Williams et al. (32) analyzed protein levels for stout beer, bitter, and
lager from British retail outlets. The CBBG reagent from Bio-Rad Ltd. was
used. Beer was analyzed as is or after exhaustive dialysis to remove lowmolecular-weight components. Results from this investigation are illustrated
in Fig. 1 of Chapter 1. Reliable estimates for beer protein were obtained with
the Bradford and the PMR* assays (Table 4). Both techniques had a CV of
1.7%. Kjeldahl, Dumas, biuret, Lowry, and BCA methods were subject to
error due to the presence of dialyzable inteference compounds in beer.
* PMR=Pyrogallol-red molybdate.
208
Chapter 7
Bradford
PRM
460
430
200
160
270
250
1050
800
630
500
570
500
a
Lower values show protein values after
dialyzing samples.
Source: Summarized from Ref. 32.
Foam
fractions
BCA
Bradford
1
2
3
4
5
Total
356
84
181
56
356
1036
66
32
114
22
144
378
a
Fractions 15 are listed in order of increasing
hydrophobicity.
Source: Values estimated from Ref. 35.
Bradford AssayApplications
TABLE 6
209
Reference
Hsu and Heatherbell (33)
Murphy et al. (34)
Murphy et al. (35)
Brenna and de Vecchi (36)
Waters et al. (37)
Marchal et al. (38)
Boyes et al. (39)
5 were equal when normalized for protein concentration. The BCA assay
results were affected by reducing substances such as melanoidins.
4.2.
Wine protein is frequently analyzed using the Bradford assay (Table 6). Like
beer, wine contains endogenous polyphenols and reducing compounds. The
wide range of protein values reported for wine (1 mg L 11 g L 1) probably
reects inherent differences between types of wines, grape cultivars and
maturity, and processing methods. Some variations are due to simple
measurement error.
The Bradford assay is more resistant to interfering compounds from
wine (see Lowry, biuret, BCA, and Kjeldahl methods). However, claims that
high concentrations of polyphenols (<400 mg L 1) do not interfere with dye
binding were challenged (36). The choice of standard proteins for wine
analysis is crucial.
Research on wine proteins is mainly concerned with haze formation
and identifying the protein fraction(s) responsible for wine instability. Hsu
and Heatherbell (33)* in 1987 were probably the rst to analyze grape and
* Grapes were homogenized with liquid nitrogen using a Waring blender and then freeze dried.
The resultant powder (3 g) was extracted for 2 hours with 30 mL of 0.1 M citrate, 0.2 M
phosphate buffer (pH 5) containing Amberlite XAD-4 (6 g), PVP (3 g), and two protease
inhibitors (diethyldithiocarbamate, 5 mM, and phenylmethylsulfonyl uoride, 5 mM). The
resultant mixture was ltered through muslin cloth and then centrifuged. The clear supernatant
was analyzed by the Bradford assay. Then A595 readings were recorded after 15 minutes.
210
Chapter 7
wine proteins using the Bradford method. Treating grape juice or wine with
ion-exchange resin (XAD-4) and PVP reduced total phenols by 44 and 57%
(Table 7). However, protein-quinone interactions were already complete
during the wine manufacturing process. Electrophoresis analysis achieved
poor resolution of wine proteins despite treatment with Amberlite and PVP.
There was also signicant adsorption of wine proteins to phenol adsorbents.
In particular, it is thought that hydrophilic proteins adsorbed strongly on
tannins, polyphenols, and phenol adsorbents.
The rate of A595 increase during wine protein analysis was examined
by Murphy et al. (34). The A595 reached a maximum after 90 minutes for
wine proteins and after 210 minutes with BSA. The incubation time for
wine protein analysis using the Bradford assay has to be extended and
strictly controlled. Dissolving BSA with protein-depleted wine reproduced
the slow reaction with CBBG; addition of polyphenols to BSA in idealized
buffer solutions did not.
Wine protein values depend on the maturity of the grapes used by the
manufacturer. Gewurztraminer and white Riesling grapes were found to
contain 50120 mg (protein) L 1; such results are usually cited as mg L 1
equivalent to BSA (35). Interestingly, grape juice protein levels were highly
correlated with acidity (R > 0.86). A highly signicant relationship also
existed between wine soluble solids content (1025% w/w) and wine proteins
(25105 mg L 1 eq. BSA). For wines having a solids content of 1820% the
protein value was 40 mg L 1 (BSA eq.). Adsorption of wine proteins with
Protein
(mg L 1) eq. BSA
Phenola
(mg L 1)
102 + 1.8
34%
77 + 1.5
25%
29.4 + 0.5
29%
98 + 4.2
44%
298 + 14
49%
124 + 6.3
57%
Bradford AssayApplications
211
0:00941
212
Chapter 7
wine and sauvignon blanc wine. Kiwi fruit juice and corn protein extract
were also analyzed using a new ``alkali'' Bradford assay.* Fruit juice and
gewurztraminer wine protein estimates were *10 times higher with the new
method (Table 8). The alkali Bradford assay is similar to the procedure of
Stoschek (9,10). In both instances 50 mL of NaOH (1.0 M) is added to the
sample before the Bradford reagent. The effect of pH adjustment may be to
reduce protein H-bonding interactions with phenolic compounds.
4.3.
Cereal Products
Protein from wheat our, dough, bread crumb, or gluten was estimated with
the Bradford assay by Eynard et al. (40). Samples were preextracted with a
range of solvents before analysis. The calibration graph (with 50350 mg
gluten as standard protein) was curved and described by a second-order
equation, A595 ax2 bx c, where x is the amount of protein. As an
alternative, plotting A595/A460 versus protein gave a linear graph. Proteinprotein variations in sensitivity followed the series serum albumin > watersoluble protein * acid-soluble protein > gluten * gliadin. Assay sensitivity
matched the total number of CBBG-reactive amino acids
(Arg Lys His Tyr Trp Phe). Water-extractable materials from
our, dough, and bread crumb had protein contents of 19.4%, 22.3%, and
6.9 g%, respectively. By comparison, the acetic acidextractable material
from our, bread dough, and bread crumb has 57.7% protein, 58.2%
protein, and 8.3% protein. Baking led to a large decrease in the solubility of
bread crumb protein.
TABLE 8 Effect of NaOH Addition on Bradford Assay of Fruit Juice and
Wine Protein
Protein (mg L 1)
Sample
Pressed kiwi fruit juice
Heat-treated kiwi fruit juice
As above bentonite treated
Gewurztraminer wine
( bentonite treatment)
Sauvignon blanc wine
Source: Summarized from Ref. 39.
Bradford assay
( NaOH)
Bradford assay
2000
560830
120
75250
150410
0
9.733
<50
<7
Bradford AssayApplications
213
Legumes
Dhillon and Nainawatee (43) found that whole or defatted mungbeen our
had 24% or 24.9% protein (per dry weight, DW) using the Bradford assay
and Kjeldahl (N 6 6.25) analysis.* As a form of sample pretreatment,
legume protein was rst extracted using Tris-SDS-2ME buffer (0.05 M TrisHCl buffer, pH 7.0, with 10 mM 2-mecaptoethanol and 2% SDS). The
calibration graph had a linear range of 0100 mg BSA.{ In agreement with
Rubin and Warren (13), assay sensitivity was reduced 10-fold due to the
presence of SDS (<0.05%).
The maturation process for beans (Phaseolus vulgaris L. cv. or de
mayo) was monitored by the Bradford, Kjeldahl, and Lowry protein assays
(44). Results from micro-Kjeldahl analysis of bean our and the dialyzed
our homogenate are shown in Fig. 3. Fig. 4 shows the changes in PBSsoluble bean protein fractions. Broad beans had an apparent crude protein
content of 40% DW. Dialysis removed approximate 45% of the nitrogen,
which is therefore low-molecular-weight NPN.
Bean protein levels (15% DW) decreased slightly with maturation.
Results in Fig. 4 are for samples freed from NPN by dialysis. There were
inherent differences between results from different assay methods. Using
casein as a standard, the Bradford, micro-Kjeldahl, Lowry, and biuret
* Shake 100 mg of our with 1 mL of Tris-SDS-2ME for 60 minutes. Centrifuge (5000g) and
collect the supernatant. Wash the pellet with 2 6 2 mL of Tris-SDS-2ME buffer and combine
with the supernatant. Dilute (5 mL) pooled extract with water to a nal volume of 40 mL.
Determined protein content by adding 0.1 mL of extract to 5 mL of standard Bradford reagent.
{ Dissolve BSA standards in 0.05 M Tris-HCl buffer containing 0.05% (w/v) SDS.
214
Chapter 7
Potatoes
Potatoes contain high amounts of NPN. The amount and quality of protein
also differ for different potato varieties and states of maturation. Rapid
methods for protein analysis are necessary to assist in attempts to breed
potato varieties having higher protein content. Criteria for selecting
Bradford AssayApplications
215
convenient assays for potato breeding include (45) (a) efcient extraction
and solubilization of protein from potato tissue, (b) compatibility with
different potato varieties and tubers at different stages of maturation, (c)
lack of interferences from NPN, and (d) low expense and high throughput
compared with the Kjeldahl method.
Snyder and Desborough (45) determined potato tuber protein using
the Bradford assay.* Bradford test results for potato tubers were strongly
correlated with Kjeldahl results (R 0.93). The six hybrid potato varieties
studied had 315.6% protein per DW in agreement with quantitative amino
acid analysis or micro-Kjeldahl results. The cost per analysis was 100 times
lower and the sample throughput 6 times higher for the Bradford assay
* Dice, freeze, and lyophilize fresh potato. Suspend the ground potato powder (15 g) in 2.5 mL
of water and then add 2.5 mL of 1 M sodium hydroxide. Incubate at room temperature for 2.5
hours. Remove 0.4 mL of extract to another test tube and add 5 mL of Bradford reagent.
Record A595 readings as usual.
216
Chapter 7
4.6.
Mushrooms
Bradford AssayApplications
4.7.
217
Honey
4.8.
Richard and Paquin (50) compared the Bradford and Kjeldahl assays for
whey protein concentrate (WPC). It was thought desirable to control both
the ionic strength and pH of the solvent. Thus, 1 g of WPC (33.8% protein)
was dissolved in 100 mL of McIlvine buffer adjusted to various pH values.
The solutions were centrifuged (40,000g) and then ltered using Whatman
No. 1 paper. Solubility results from the Bradford and Kjeldahl assays were
3% different (*83% solubility) at pH 69. At pH 3 protein solubility
determined via the Bradford assay was 7% lower than the value from
Kjeldahl analysis (78%). Compared with Kjeldahl analysis, the Bradford
assay was a simpler, faster, and a more affordable technique for monitoring
the solubility of WPC.
4.9.
Insoluble Proteins
218
Chapter 7
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Bradford AssayApplications
219
220
Chapter 7
8
Immunological Assay: General
Principles and the Agar Diffusion
Assay
1. INTRODUCTION
1.1.
222
Chapter 8
Implications of Adulteration
223
The potential ill gain from adulteration is partly related to (a) the frequency
of species substitution, (b) the percent weight of adulterant added, and (c)
the price dividend from substitution. Multiplying PAPI by the net value of
224
Chapter 8
trade gives the nancial cost to the consumer arising from undeclared
substitutions.
PAPI can be estimated only crudely for several reasons. First,
information about the pattern of protein substitution worldwide is scarce.
Wide variations will occur in the frequency and extent of undeclared
substitutions in different countries and states and regions within any given
country. Next, the incentive for adulteration is probably related to the net
value of trade in that particular commodity. Most protein commodities
could be subject to adulteration but higher value animal proteins are more
likely targets. The estimated rate of undeclared substitutions for meat is
about 16% in the state of Florida (7). For either raw or cooked meat, the
weight of adulterant added was 15% (average 2.5%). The price dividend for
the added meat can be estimated from the formula 1 (Y/X), where Y is the
real price of the adulterant and X is the advertised price. When the added
protein has no intrinsic value, the price dividend is 1.0. Assuming a price
dividend of 0.5, the PAPI estimate for the U.S. meat and poultry market is
then 0.2%. The upper PAPI can be as high as 5%. Thus, the frequency of
substitution is easily 50% in some sectors of the meat industry. A percent
weight substitution of 10% also seems credible. For the worst-case scenario,
we assume a maximum price dividend of 1, leading to PAPI value of 5%.
The value of the U.S. meat and poultry trade is about US$120 billion
according to FSIS gures from 1995. Consumer spending on meat and
poultry products accounts for one third of the annual food budget. Meat
purchases included raw beef, pork, lamb, chicken, turkey, and approximately 250,000 processed meat products, which are processed foods
containing >2% poultry or >3% meat. Examples include ham, sausages,
sources, soups, stews, pizzas, and frozen dinners. From the preceding PAPI
and net trade values, we may suppose that the U.S. meat consumer is
overcharged by between US$250 million and US$6 billion annually.
The most efcient techniques for detecting protein adulteration are
immunological. Other well-established approaches include quantitative
sodium dodecyl sulfate polyacrylamide gel electrophoresis (QSDS-PAGE),
isoelectric focusing (IEF), and capillary electrophoresis (8). High-pressure
liquid chromatography (9) and fast protein liquid chromatography (10)
have also been employed for the differentiation of milk from different
species. Finally, mention must be made of nonprotein-based methods.
Detection of amino acids, sugars, or fats associated with particular species
can be a clue to adulteration. Species-specic DNA testing using
hybridization probes or polymerase chain reaction (PCR) is also increasing.
The interested reader is referred to papers by Hunt et al. (11), Fairbrother
et al. (12), Lenstra and Buntjer (13), and Wolf et al. (14); the subject is
reviewed by Meyer and Candrian (15).
FIGURE 2
225
Methods for food protein immunoassay are divided into marker-free and
marker-linked methods.
2. IMMUNOLOGICAL METHODS
Immunoassays are based on the use of antibodies (Fig. 2). The range of
techniques includes marker-free techniques in solution or agar, e.g.,
solution-phase precipitation, agar gel immunodiffusion, immunoelectrophoresis, and counterelectrophoresis. The marker-linked techniques involve
enzyme immunoassay (EIA) or radioimmunoassay (RIA). Application of
immunoassays in food analysis and authenticity testing are reviewed by
Samarajeewa et al. (16), Gazzaz et al. (17), Allen and Smith (18), Barai et al.
(19), Lee and Morgan (20), Hernandez et al. (21), Taylor et al. (22), Smith
(23), and Mandokhot and Kotwal (24).
2.1.
226
Chapter 8
2.2.
Principles of Immunoassay
B
B
IgP IgT BP
B
PKa
A graph of [B] plotted versus [B]/[P] yields a straight line with a slope of 1/Ka
and the intercept IgT. Dividing the value of the intercept by the known
molar concentration of antibody gives the number of antigen binding sites
per Ig molecule; ordinarily, the answer should be 2. The Scatchard plot
is usually nonlinear, showing the presence of different populations of
antibodies. Adsorbing Ig to a solid support will also obscure some antigen
binding sites, leading to a range of Ig-antigen binding afnity.
We turn to the effect of antibody-antigen binding parameters on the
immunoassay characteristics. The various terms in Eq. (3) can be expressed
in terms of the fraction of antigen bound, X:
Ka
IgT
X
PX1
227
FIGURE 3
228
Chapter 8
2.3.
Background Immunology
229
230
Chapter 8
plates and the cell supernatant examined for the mAb. It was possible to
isolate a single hybridoma cell line (or clone) that produces the required Ig.
Hybridomas can be grown indenitely and large amounts of mAbs
produced for analytical use. Only a few of the available genes for the H
and L regions are expressed owing to a process called allelic exclusion (25).
Fusion is needed for the production of mAbs. The mere mixing of myeloma
cells and spleen cells (polyethylene glycol as a fusion agent) will not lead to
new Ig.
3.
AGID assays are an extension of the test tube precipitation test rst
demonstrated by Uhlenauth in 1901 (cited in Refs. 5 and 31). Further work
between 1902 and 1928 led to the production of less complex antigen
(inoculation) mixtures designed to improve specicity and reduce toxicity to
231
232
Chapter 8
233
234
Chapter 8
2. PBS buffer
3. 6.5-mm-diameter lter discsoptional
Procedure
Prepare 1% (w/v) molten agar in PBS by heating on a water bath. Pour
about 4 mL of molten agar into a petri dish and allow to set at room
temperature. Refrigerating for a few hours will harden the gel. Cut
out several 6-mm-diameter holes or wells using a cork borer. Place
one well in the center of the petri dish and then surround this with a
quartet of wells each placed 6 mm from the central well. Remove the
circular piece of gel using a Pasteur pipette attached to a vacuum
line. Place a drop of molten agar into each well to act as a seal,
thereby avoiding the migration of samples below the well.
To perform a standard AGID assay, place pAb in the central well. In
the surrounding wells, place one each of the test samples (*20 mL).
Cover the petri dishes to avoid dehydration and allow to stand at
room temperature for 24 hours.
3.2.
The rst attempts to identify raw meat using the AGID assays were
described by Warnecke and Safe (38). They found that actomyosin was a
poor antigen, with injections of 20150 mg leading to only moderate pAb
production in rabbits. Rabbit pAB for beef whole serum showed crossreactivity for lamb extract. No reactivity was seen for horse meat or pork.
The crude pAb was rendered monospecic for beef by immunoadsorption.
An AGID assay with immunoadsorbed pAb allowed the detection of beef.
Many of the methods described in this report are still in use at the present
time.
Further developments in AGID assays for meat speciation occurred in
the laboratories of the U.S. Department of Agriculture, Beltsville, MD.
Fugate and Penn (39) used AGID assays to identify meat from beef, horse,
pig, and sheep. Of 12 meat samples examined, 11 were correctly identied.
They recommended that the AGID test should be subjected to collaborative
testing. AGID assays using pAb for residual serum albumin from meat were
produced by Hayden (40). Hers was probably the rst dissertation on this
subject. At the Department of Food Science, University of Georgia (Athens,
GA), Helm et al. (41) compared the AGID assay and a simple test tube
precipitin test using (rabbit) pAb for beef, horse, lamb, and pork. The
AGID test detected adulteration at the 2% level.The solution precipitin test
was *three times faster and four times more sensitive. In Australia, Swart
235
and Wilks (42) differentiated beef, horse, kangaroo, and mutton by AGID
assay. Species identication eld tests (SIFTs) were developed in 1984 by
Mageau et al. (43) at the USDA. Finally, Darwish et al. (44) also described
an AGID assay for detecting beef adulteration with camel and pork. Some
examples of the application of immunodiffusion assays include the detection
of pork in beef mince meat (45), Alaska pollack surimi analysis in meat
products (46), and the detection of various meat types (beef, pork, horse,
poultry) in hamburger (47).
3.3.
SIFTs are designed for eld testing in abattoirs and meat inspection
stations. The rst of these test kits is called ORBIT (43). The acronym
stands for the Overnight Rapid Bovine Identication Test. ORBIT was
followed by PROFIT (Poultry Rapid Overnight Field Identication Test)
(48), SOFT (Serological Ovine Field Test) (49), PRIME (Porcine Rapid
Identication Method) (50), REST (Rapid Equine Serological Test) (51),
DRIFT (Deer Rapid Identication Field Test) (52), and MULTI-SIFT
(Multispecies Identication Field Test) (53). The last kit can simultaneously
test for beef, poultry, pork, sheep, horse, and deer meat. After successful
collaborative trials (26), ORBIT and PROFIT received approval from the
AOAC. These methods can detect meat adulteration levels of about 10% or
greater (27).
SIFTs are not radically different from classical AGID assays.
However, considerable design effort has gone into making SIFTs attractive
and easy to use. Antibody and meat sample extract are adsorbed onto lter
paper discs. Both are then freeze dried, providing stabilized reagent discs
that can be stored for up to 12 months at refrigeration temperature. The
antibody lter discs and all materials needed to perform SIFTs are available
commercially. The tests are used by the USDA and meat inspection services
in the United States.
According to the team responsible for SIFTs, conventional methods
for meat speciation (e.g., electrophoresis, chromatography) share many of
the following disadvantages: (a) tests are usually performed within a formal
laboratory, (b) relatively sophisticated equipment is needed, (c) high levels
of staff expertise and training are necessary, (d) time delays arise due to offsite testing with an attendant need for transmitting samples and assay results
to and from the analyst, and (e) relatively labile reagents are used. SIFTs
were developed with the aim of avoiding such disadvantages. The assays are
highly reliable, fast, easy to use, accurate, and sensitive. Little expertise or
previous experience is needed for successful testing. Finally, SIFTs use
236
Chapter 8
Ideal antigens for cooked meat analysis survive thermal treatment at 708C
or autoclaving temperatures of 1208C for 15 minutes. Hayden (54)
considered troponin as a heat-stable meat antigen (Table 2). Extensive
research since 1977 has shown that thermostable meat antigens are usually
troponin C, troponin I, or troponin T. Schweiger et al. (55) used puried
turkey troponin T for AGID assay. Thermostable antigens were initially
developed from adrenal gland extracts (56,57) by Milgrom and Witebsky
(58).* Rabbit pAb for boiling-resistant, ethanol-insoluble (BE) antigen from
beef showed cross-reactivity with BE antigen from sheep. There was no
reaction with heated adrenal extracts from pig, rat, guinea pigs, or humans.
The method for BE antigen preparation has remained unchanged for nearly
40 years. Hayden (56,57) used BE antigen for detecting cooked beef sausage
adulteration with horse, sheep, and pig meat.
Radhakrishna et al. (62) showed that (buffalo, goat, oxen) muscle BE
antigen had SDS-PAGE bands corresponding to troponin (C, I, and T) and
tropomyosin. Bhilegaonkar et al. (67) found that the concentration of
(buffalo, sheep, goat, pig) BE antigen was 21130% higher in adrenal tissue
* Boiling-resistant ethanol-insoluble (BE) antigen is produced by boiling an aqueous extract
from bovine adrenal glands or muscle tissue followed by centrifugation. The supernatant is
autoclaved at 1208C for 30 minutes and centrifuged. Then three volumes of 95% ethanol are
added to the soluble fraction. A whitish precipitate forms after *1214 hours at 378C and is
then dissolved in normal saline for immunization.
237
Immunized
host
ORBIT,
beef (39)
Rabbit,
goat, sheep
PROFIT,
poultry (40)
Goat
PRIME,
pork (42)
Goat
SOFT,
sheep (41)
REST,
horse (43)
Calf
DRIFT,
deer (44)
Goat
MULTI-SIFT,
six different
species (45)
Various
Sheep
Performance
70 samples. No false
positives/negatives, Specicity: bison
( ), bovine ( ), water buffalo ( ),
deer ( ), elk ( ), goat ( ), horse ( ).
66 samples. No false
/ results. Specicity: chicken ( ),
turkey ( ), goose ( ), quail ( ),
partridge ( ), bovine ( ), deer ( ),
horse ( ), pig ( ), sheep ( ).LLD 3%
or 5% in pork or beef
83 samples. No false /
results. LLD 5% pork (in beef), 3% pork
in lamb.
90 samples. No false /
results. LLD 3% mutton in beef.
101 samples. No false /
results. Specicity: deer, ( ), donkey
( ), mule ( ), beef ( ), pork ( ),
sheep ( ), chicken ( ), turkey ( ),
kangaroo ( ). LLD 3% horse
100 samples. No false /
results. Specicity: mule deer ( ), elk
( ), moose ( ), reindeer ( ), beef ( ),
horse ( ), sheep ( ), chicken/turkey
( ).
No false / results.
Specicity (as above)
LLD, Lower limit of detection or minimum % weight of adulterant detectable. All antiserum lter
discs are stable for 45 months at room temperature and 12 months at 48C. ( ) positive test,
( ) negative test.
238
Chapter 8
Reference
Hayden (54)
Hayden (56,57)
Schweiger et al. (55)
Kang'ethe et al. (59)
Sherikar et al. (60,61)
Radhakrishna et al. (62);
Bhilegaonkar et al. (67);
Sherikar et al. (63)
Reddy and Giridhar-Reddy (64);
Saisekhar and Reddy (65)
Levieux and Levieux (66)
Saisekhar and Reddy (65) isolated troponin T from raw beef and
buffalo meat. An AGID assay based on native troponin T antigen from
buffalo cross-reacted with cattle, goat, and sheep meat. There was no
reaction with chicken or pork. The (rabbit) pAb for buffalo was rendered
monospecic by immunoadsorption with cattle, goat, and sheep antigen.
The AGID assay using monospecic buffalo pAb could detect beef or
mutton adulteration with 1% of buffalo meat. Interestingly, (rabbit) pAb
for bovine troponin T was monospecic for beef without prior immunopurication. No cross-reactivity occurred with buffalo, goat, sheep, or chevon
meat. The LLD was 10% beef addition to samples of buffalo, chevon, or
mutton. These tests based on pAb for native troponin T did not detect cooked
meat. In contrast, pAb for native troponin T from chicken or turkey was
sensitive to (poultry meat ) antigen in fried sausages (54,55). Clearly,
troponin T is not heat resistant. Heating may generate a denatured but
soluble troponin T that functions as a BE antigen.
Much evidence points to troponin T being the major BE antigen.
However, other muscle proteins might also play this role. SDS-PAGE
analysis of muscle proteins extracted by 0.6 M KCl shows bands for myosin
(200 kDa), actinin, actin (42 kDa), and troponin T (37 kDa). In the 3228
kDa region appeared bands for troponin I, tropomyosin, and myosin light
239
3.5.
240
Chapter 8
wildlife game species.* The tests developed by Kang'ethe et al. (59) from the
University of Nairobi used (goat) pAb. The crude pAb was surprisingly
monospecic in most cases. Cross-reactivity was observed for some closely
related species: buffalo and cattle, bushbuck and cattle, Grant's gazelle and
sheep, and Grant's gazelle and Thomson's gazelle. After immunoadsorbtion, each pAb was rendered monospecic for thermostable antigen, cooked
meat extracts, or fresh meat. The domesticated species were clearly
distinguished from the game species. Differentiating between Grant's and
Thomson's gazelle, kongoni and topi, and kongoni and wildebeest remained
problematic. Using goat as the host for pAb production probably accounts
for low cross-reactivity. By comparison, (rabbit) pAb shows lower
specicity and a greater likelihood of cross-reactivity between closely
related species.
Chicken meat is in high demand in parts of Asia owing to religious
restrictions related to the consumption of beef. Sherikar et al. (61,63)
produced an AGID assay for chicken meat adulteration with beef, buffalo,
goat, mutton, or pork. BE antigen was prepared from heart, kidney, liver,
spleen, or lung tissue. Crude (rabbit) pAb for pork was monospecic. The
other (rabbit) pAbs were puried by immunoadsorption. Partially puried
pAbs reacted only with homologous antigen from raw tissue or tissue mildly
heated at 708C. Fully cooked meat could not be detected unless further
processed to BE antigen (by autoclaving, centrifugation, and ethanol
precipitation). Apparently, components in the relatively complex cooked
meat extract interfered with antibody-antigen binding and/or precipitin
formation. AGID assays were performed on samples with BE antigen.
Adulteration of chicken with 10% (w/w) beef, buffalo, goat, or sheep meat
was detectable. The LLD for pork was 5% (w/w).
Reddy and Giridhar-Reddy (64) also produced an AGID assay for
cooked pork. Porcine muscle BE antigen and the corresponding (rabbit)
pAb were prepared as usual. The crude (rabbit) pAb was monospecic for
pork. Samples for analysis were extracted from raw pork or after heating at
1208C for 30 minutes. The AGID assay detected 10% (w/w) pork in
cooked meat from buffalo, cattle, chicken, goat, and sheep. The LLD for
pork in raw meat mixtures was 20% (w/w).
The frequency of undeclared meat substitutions was referred to earlier.
Hsieh et al. (7) examined 806 raw meat and 96 cooked meat samples (mostly
* The species include buffalo (Syncerus caffer), bushbuck (Tragelaphus scriptus), cattle (Bos
indicus), eland (Taurotragus oryx), goat (Capra aegagrus hircus), Grant's gazelle (Gazella
granti), impala (Aepyceros malampus), kongoni (Alcelaphus buselaphus cokii), oryx (Oryx spp.),
sheep (Ovis ammonaires), Thomson's gazelle (Gazella thomsoni), topi (Damaliscis lunatus),
waterbuck (Kobus spp.), and wildebeest (Connochaetes taurinus).
241
beef and ground veal). The AGID tests involved commercially available
pAb specic for raw sheep, pork, beef, and horse meat. Enzyme-linked
immunosorbent assay (ELISA) kits utilizing pAb for thermostable antigen
were used for the analysis of cooked meats. About 16% of raw meat samples
were adulterated with meat from another species. The frequency of
adulteration increased to 23% for cooked meat. The most frequent
adulterants for ground veal or beef were sheep (47%), pork (42%), or
poultry (31%). There were no substitutions involving horse meat. Crosscontamination via (improperly cleaned) processing equipment was not
signicant. Ground lamb and pork had adulteration frequencies of 66.7%
and 53%, respectively. So far, immunological methods cannot distinguish
beef from veal or mutton from lamb.
3.6.
AGID assays have not proved popular for the analysis of nonmeat proteins.
The analysis of soy protein in meat is reviewed by Llewellyn (5). Hammond
et al. (72) found that cross-reactivity with other legume proteins was
widespread. The reliability of AGID tests for vegetable proteins is poor
because of the effect of heat, extrusion, texturization, and other forms of
processing. More promising are methods based on ELISA (Chapter 10).
REFERENCES
1.
2.
3.
4.
5.
6.
7.
242
Chapter 8
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
243
244
Chapter 8
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
245
9
Speciation of Meat Proteins by
Enzyme-Linked Immunosorbent
Assay
1. INTRODUCTION
Enzyme-linked immunosorbent assay (ELISA) was invented by Eva Engvall
during her Ph.D. studies in Stockholm in 1971 (1). A similar assay was
produced by Van Weemen and Shuurs (2). Developments between 1971 to
1981 are reviewed in reference 3. ELISA is an example of an enzyme
immunoassay (EIA). These are immunological tests ending with enzymatic
analysis. Enzyme multiplied immunoassay tests (EMITs) are performed in
solution. ELISA employs immunoglobins attached to a solid surface. EIA is
further categorized as competitive or noncompetitive (Table 1). Secs 25 of
this chapter describe authenticity testing for raw and cooked meat by
ELISA. The meat antigens detected with such tests are mostly residual
serum proteins from the blood or else muscle proteins. Sec. 6 covers ELISA
for meat protein using monoclonal antibodies (mAbs). Seafood speciation is
discussed in Sec. 7, followed by a comparison of the different ELISA
formats in Sec. 8. Attempts to detect meat and bone meal in animal feeds are
described in Sec. 9, as are some new ELISA tests for the bovine spongiform
encephalopathy (BSE) agent.
During EMIT an enzyme-antigen conjugate (Enz-antigen) reacts with
antibody (Ig). Steric hindrance from Ig binding reduces enzyme activity. The
247
248
Chapter 9
Solid-phase EIA
ELISA
Competitive
Enz-antigen, Enz-Ig
Noncompetitive
Direct, indirect, sandwich
added sample (with known concentration of antigen) competes with the Enzantigen for Ig. This process relieves steric inhibition of the Enz-antigen.
EMITs are suitable for analyzing small molecular weight antigens. The
measured enzyme activity is proportional to the added analyte concentration.
To perform EMIT requires access to pure antigen and Enz-antigen conjugate.
Competitive ELISA uses enzyme-labeled antibody (Enz-Ig or EnzIg0 )* or Enz-Antigen conjugate as reagent:
1. Adsorb pure antigen on microwell plates (Fig. 1)
2. Block excess adsorption sites with milk protein.
3. In a separate step, preincubate Enz-Ig with the sample (containing
an unknown concentration of antigen).
4. Transfer the mixture to the antigen-coated microwell plate.
5. Wash to remove excess reagent.
6. Assay the microwell plate for bound Enz-Ig using enzyme
substrate.
The enzymatic activity is inversely related to the concentration of antigen in
step 3. An alternative one-step competitive ELISA is performed as follows:
1. Coat microwell plates with antibody.
2. Block nonspecic sites (as described above).
3. Add Enz-antigen and sample and allow competition for the
microwell-bound Ig.
4. Wash microwell plates and assay with enzyme substrate.
The enzyme activity is inversely related to sample antigen concentration.
* Enz-Ig enzyme linked to a primary antibody specic for a food protein. Eng-Ig0 enzyme
conjugate with antibody (Ig0 ) specic for the primary antibody, e.g., goat antibody for (rabbit)
Ig.
249
250
FIGURE 2
Chapter 9
* They were afliated with Unilever Research Laboratories, Colworth House, Bedford (UK).
251
Examples
Ochratoxin A1, aatoxins
Clostridium, Staphylococcus, E. coli
Natural, synthetic reproductive or
growth-affecting hormones
Solanine, trypsin inhibitor
Indoleacetic acid, abscisic acid
Colors, avors
Gums, stabilizers, emulsiers
Beef, buffalo, camel, poultry
Cow, ewe, goat's milk (speciation),
specic milk proteins
Serum protein speciation
Single-cell proteins
Soya, wheat gluten, pea, potato
252
Chapter 9
Reference
Engvall and Perlman (1)
Kang'ethe et al. (19)
Whittaker et al. (18,20)
Patterson et al. (21)
Jones and Patterson (22)
Patterson and Spencer (23)
Jones and Patterson (24)
Pelly and Tindle (25)
Martin et al. (26,27)
Martin et al. (27)
Ayob et al. (29)
Stevenson et al. (30)
Taylor et al. (31)
Studies based on muscle protein antigen. Indirect ELISAs are noncompetitive methods unless
stated.
plate reader enables colorimetric readings from microwell plates in situ. The
96 wells can be read within a space of 1.5 minutes. Several precision
micropipettes are necessary for dispensing reagents; most essential are 50-mL
and 100-mL pipettes. A continually adjustable (50200 mL) multiwell pipette
is convenient for rapid dispensing. Polystyrene microwell plates appear to be
the solid phase of choice.
2.
253
2. Antigen standard
3. Coating buffer (0.1 M sodium carbonate buffer, pH 9)
4. PBST (phosphate-buffered saline with 0.05% Tween 20)wash
and diluent buffer
5. Enzyme assay buffer (citrate-phosphate buffer, pH 4.2)
6. Enzyme substrate (varioussee the following)
7. Enzyme stopping solution (varioussee the following)
Procedure
1. Coat microwell plates. Add 100 mL of meat extract (diluted in
PBST) to microwell plates and incubate for 60 minutes at room
temperature. Wash with PBST (100 mL) three times.
2. First antibody. Add 100 mL of rabbit antibody (diluted in PBST).
Incubate for 3060 minutes and wash wells with PBST (100 mL)
three times.
3. Detection antibody. Enzyme conjugate. Add 100 mL of Enzantibody conjugate. Incubate for 60 minutes. Wash with PBST
(100 mL) three times.
4. Enzyme assay. Add 100 mL of enzyme substrate. Incubate for 30
minutes. Add stopping solution and record absorbency reading
with the plate reader.
Not counting the PBST washing steps, indirect ELISA involves four steps.
Perform preliminary experiments to establish the optimum sample (antigen)
dilution as well as the required concentrations of antibody and Enz-Ig
conjugate. Incubation times ranging from 30 minutes to 3 hours have been
employed. Horseradish peroxidase (HRP) is the most common enzyme label
for ELISA. Suitable substrates for HRP are ABTS [2,20 -azino-di(3ethylbenzthiazoline sulfonate)] and hydrogen peroxide. Other HRP
substrates are listed in Table 4. Alkaline phosphatase, urease, and glucose
oxidase have also been used as labels.
Kang'ethe et al. (19) developed indirect ELISA for horse meat.
Polyclonal antibody (pAb) for horse serum albumin (HrSA) was raised by
immunizing rabbits. Preliminary tests using AGID assay showed that
(rabbit) pAb cross-reacted with BSA and sheep serum albumin (SSA).
Therefore crude (rabbit) pAb for HrSA was puried by column
immunoadsorption. Antibody samples were diluted by about 100 1 and
meat extracts diluted by 200 1 and 3200 1 before assay. Indirect ELISA was
performed essentially as described in Method 1. Substitution of beef with 5
80% horse meat produced the calibration response
1
DA492 K 1
%Horse ln
1
C
K2
254
TABLE 4
Chapter 9
Enzymes and Substrates for ELISA
Enzyme
Horseradish peroxidase
Horseradish peroxidase
Horseradish peroxidase
Horseradish peroxidase
Horseradish peroxidase
Alkaline phosphatase
Stopping solution
30 mL of NaCN (37 mM)
or 50 mL citric acid
(0.1 M)
50 mL of H2SO4 (4 M)
50 mL of H2SO4 (12.7 M)
50 mL of H2SO4 (12.7 M)
100 mL of H2SO4 (3 M)
25 mL of NaOH (0.4 N
NaOH)
where C, K1, and K2 are constants and A492 is the absorbance reading. A
simple straight-line equation applied for 060% substitution of beef by horse
meat. The assay precision was 2.38%.
Whittaker et al. (18,20) employed indirect ELISA for the identication
of uncooked meat from cattle, camel, horse, kangaroo, and sheep. To
improve specicity, (rabbit) pAbs for serum proteins were puried via
afnity chromatography. Antigen adsorption to microwell plates was
optimum at pH 56. The working range for analysis was 1080% (w/w)
adulteration.
255
FIGURE 3
* The American plains bison is also buffalo. The buffalo referred to in this chapter is the water
buffalo of Asia and Africa called simply buffalo in the literature.
256
Chapter 9
Tests with 8 pAbs versus 8 meat extracts (64 tests) showed three false
positives. The (cow) pAb for sheep cross-reacted with goat meat. However
(sheep) pAb for goat did not cross-react with sheep. The (sheep) pAb for
beef cross-reacted with buffalo meat, but (sheep) pAb for buffalo did not
react with beef. By choosing the host animal for antibody production
carefully, specic pAb could be produced for sandwich-ELISA. The
relatively high pAb specicity was ascribed to the following factors: (a)
choice of host species (sheep or goats produced more specic antibodies
than rabbit or mice) and (b) choice of antigen. Using whole serum protein
for immunization, rather than a single pure protein, introduces many
antigenic determinants. The pAbs are produced that are more discriminating between species. Meat samples (1 g) were extracted by 10 mL of solvent
and diluted by a factor of 10 15000 1 before analysis. The LLD was 1%
(w/w) kangaroo meat substitution for beef or 1% (w/w) substitution of goat
meat for sheep. Cross-reactivity between closely related species (beefbuffalo, goat-sheep, and donkey-horse) was evident.
Patterson and Spencer (23) also produced monospecic pAbs for
buffalo, goat, or donkey using cattle, sheep, or horse as host, respectively.
Each pAb was then puried by immunoafnity chromatography. Thus,
(sheep) pAb for goat was puried with a column of Sepharosegoat serum
protein. Bound pAb was eluted with ammonium thiocyanate (2.5 M, pH
7.0), desalted by gel ltration with Sephadex G25, and concentrated by
ultraltration. The pAb sample containing 8 mg mL 1 protein was divided
into two portions; half was covalently conjugated to HRP for detection
and the other half was used for capture. Enzymatic detection was via
o-toluidinehydrogen peroxide (Table 4). From visual inspection the LLD
was 0.1% (w/w) donkey meat added to horse meat or 0.1% (w/w) goat meat
added to mutton. Beef adulteration with > 1.0% (w/w) buffalo meat was
detectable.
Jones and Patterson (22) showed that it was possible to detect 0.51%
(w/w) adulteration of beef by pork using a sandwich ELISA. The linear
range of analysis was 13%. The capture antibody was (rabbit) pAb for
porcine serum albumin (PSA). The detector pAb was produced with a sheep
host. Both (rabbit) pAb and (sheep) pAb for PSA were puried by a twostage afnity procedure. The order of reagent addition was (rabbit) pAb for
PSA, meat extract, (sheep) pAb for PSA, and HRP-pAb conjugate for sheep
Ig. The (sheep) pAb for PSA was unstable when directly adsorbed on
microwell plates.* Presumably, instability prevents the preparation of HRP
* Reagent stability is an important feature of sandwich ELISA. Once coated with capture
antibody, microwell plates may be dried and stored at refrigerator temperatures for 6 months.
257
conjugate with (sheep) pAb for PSA. Also important for assay design is the
low specicity of (rabbit) pAb for PSA. Using (sheep) pAb for PSA to
complete the ``sandwich'' improved assay sensitivity. For details of the
sample pretreatment see footnote.* The LLD was 1% (w/w) pork in minced
beef, beef sausage mix, or beef burger mix. For 110% (w/w) substitution,
the assay response was described by the relation
A492 0:301 0:153 log%Pork
258
Chapter 9
The corresponding assay for pork adulteration with 110% (w/w) chicken
meat led to the following performance.
A492 0:590 0:154 ln%Chicken
FIGURE 4
259
Blood versus muscle protein antigen for meat speciation using sandwich
ELISA. Adulteration of beef with pork was analyzed using (rabbit) pAb
for porcine muscle protein (open circles) or pAb for porcine serum
albumin (closed circles).
260
Chapter 9
5.
5.1.
Kang'ethe and Lindqvist (35) found that BE antigen was not wholly suitable
for indirect ELISA. The antigen showed irregular adsorption to microwell
plates because of the presence of extraneous proteins (probably gelatin).
Samples of BE antigen gelled at 48C. Notwithstanding partial purication
by size exclusion chromatography, indirect ELISA using BE antigen yielded
poor sensitivity (36). Tests involved (goat) pAb for partially puried BE
antigen from 4 domesticated species (cattle, camel, pig, and sheep) and 14
games species.* With a total of 324 tests (18 antibody 6 18 meat samples),
no cross-reactivity was observed using pAbs for water buffalo, camel, horse,
topi, and pig. The (goat) pAb for cattle BE antigen cross-reacted with
virtually every species tested. Using the appropriate species-specic (goat)
pAb between 1 and 10% (w/w) adulteration of beef (with buffalo), pork
(with warthog), or goat (with impala) was detectable.
The sensitivity of meat tests using BE antigen was improved 100-fold
by adopting the sandwich ELISA format (37). The higher performance was
attributed to BE antigen binding to capture antibody rather than to a
``bare'' microwell plate. Direct binding to surfaces alters antigen conformation and can reduce antibody binding (38,39). In all, six sandwich ELISA
tests were developed using (goat) pAb for BE antigen from buffalo, bushpig,
camel, cattle, horse, and pig. Each assay was tested with meat samples from
14 species.* The ELISA test for beef showed cross-reactivity for buffalo,
horse, and bushbuck meat. All other assays were species specic.
Adulteration of beef or pork with 120% (w/w) buffalo or camel meat
was readily detected using the appropriate sandwich ELISA for these
adulterants.
* Buffalo (Syncerus caffer), bushbuck (Tragelaphus scriptus), cattle (Bos indicus), eland
(Taurotragus oryx), goat (Capra aegagrus hircus), Grant's gazelle (Gazella granti), impala
(Aepyceros malampus), kongoni (Alcelaphus buselaphus cokei), oryx (Oryx spp.), sheep (Ovis
ammonaires), Thomson's gazelle (Gazella thomsoni), topi (Damaliscus linatus), waterbuck
(Kobus spp.), and wildebeest (Connochaetes taurinus).
5.2.
261
262
Chapter 9
FIGURE 6
263
The identity of the 50-kDa protein associated with nTA has not been
established. Antibody for nTA did not react with a-acid glycoprotein (also
called a-HS-glycoprotein) (45,46). The list of blood serum proteins includes
immunoglobins (160 kDa), transferrin (85 kDa), albumin (66 kDa), Ig
fragment (45 kDa), a1-antitrypsin (45 kDa), orosomucoid (44 kDa), GC
globulin (51 kDa), a-HS-glycoprotein (49 kDa), g-globulin (25 kDa) and
b2-microglobulin (11.8 kDa). The a-HS-glycoprotein is probably the most
heat-stable serum protein. However, several serum proteins have molecular
sizes around the region 4550 kDa (51).
The thermal stability characteristics of nTAs are unlike those of
other muscle proteins (Table 5, Fig. 7). Levieux et al. (52) heated readily
extractable muscle proteins and analyzed the residual soluble proteins
by QSDS-PAGE. The order of thermal resistance was albumin
> myoglobin lactate dehydrogenase (M4) > IgG > transferrin. All proteins were completely denatured by heating at > 808C for 30 minutes. I
estimate that the half-life (t1/2) of chicken nTA is 213.6 or 52 minutes at 100
or 1258C, respectively. Turkey or beef nTAs were relatively less heat
resistant by comparison with t1/2 of 51.3 or 103 minutes at 1008C. These t1/2
values were reduced to 35 minutes (turkey nTA) or 68 minutes (beef nTA) at
1208C. The heat deactivation mechanism for nTA was also different from
264
TABLE 5
Chapter 9
Thermal Inactivation Parameters for Some Soluble Muscle Proteins
DH#
(kJ mol 1)
DS#
(J mol 1 K 1)
IgG
184.3
280
LDH (M4)
340.7
740
Myoglobin
455.7
1040
Albumin
475.3
1130
Muscle
protein
Chkn-nTA
86
49
Trk-nTA or
pork-nTA
22
201
Arrhenius equation
ln k 58.6
(1/T)
ln k 113.0
(1/T)
ln k 136.4
(1/T)
ln k 150.6
(1/T)
ln k 17.9
(1/T)
ln k 1.25
(1/T)
2.23 6 104
4.05 6 104
4.90 6 104
5.3 6 104
1.03 6 104
2.67 6 103
FIGURE 7 Simulated thermal inactivation proles for bovine muscle proteins and
native-thermostable antigen from chicken (Chkn-nTA) or turkey
(Trk-nTa). Samples are held at 20958C for 30 minutes. (Calculated
from data in Table 5.)
265
that for the other muscle proteins. The activation enthalpy change (DH#)
and entropy change (DS#) for heat deactivation were large and positive for
most of the muscle proteins. This is indicative of large conformational
changes being the rate-limiting step during heat denaturation.
For nTA we nd that DH# < 100 and DS# was negative. Low
transition state parameters are characteristic of conformationally plastic
proteins (53). Such proteins survive heat treatment owing to the ability to
refold once the thermal stress is removed. A low DH# can also arise where
bioactivity (antigenicity) resides in lower order (primary or secondary)
protein structure. Simulated thermal inactivation proles for nTA and some
other muscle proteins are compared in Fig. 7. I have assumed that (a) the
Arrhenius equation (Table 5) applies over a temperature range of 201208C
and (b) thermal deactivation kinetics are rst order (54). Caution is also
warranted because the initial data (45,60) for modeling came from a limited
temperature range of 54668C for muscle proteins and 1001208C for nTA.
5.4.
266
Chapter 9
Between 1989 and 1999 two research groups explored the use of mAbs
for meat speciation. One group (from Spain) utilized afnity-puried
(unheated) meat antigen for immunization. Hsieh's group at the Department of Nutrition and Food Science, Auburn University initially used
(crude) thermostable meat antigens. The comparatively small number of
publications describing ELISA with mAbs are summarized in Table 6.
6.1.
Afnity-puried chicken muscle antigen (28) (Section 4) was used for mAb
production. Three hybridoma cell lines (designated AH4, BC9, and CF2)
were identied that produced mAb for chicken muscle antigen. SDS-PAGE
and immunoblotting revealed that mAb-CF2 was specic for chicken muscle
pyruvate kinase (58 kDa). Both mAb-AH4 and mAb-BC9 bound to a
47-kDa protein tentatively identied as 3-phosphoglycerate kinase. Martin
et al. (62) puried mAb-BC9 by ion-exchange chromatography with a
Mono-Q column for use as capture antibody. The detection antibody for
their sandwich-ELISA was (rabbit) pAb for chicken muscle antigen.
Visualization was by commercial HRP-labeled (goat) antibody for rabbit
IgG.
A sandwich ELISA using mAb-BC9 was selective for raw poultry
(chicken and turkey) meat. No cross-reactivity was observed with beef,
horse, pork, rabbit, or mutton. There was also no reaction with puried
proteins such as casein, gelatin, or soy protein. The sandwich ELISA test
TABLE 6 Monoclonal Antibody for the Speciation of Raw or Heated Meat Proteins
by ELISA
Analysis, commentsa
Chicken
Chicken
Porku,c
Horse
Chicken LDHc
Turkey LDHc
Poultryc
Meat (beef, pork, etc.)c
Central nervous system tissue
a
Reference
Martin et al. (61)
Martin et al. (62)
Morales et al. (63), Chen et al. (41),
Chen and Hsieh (42)
Garcia et al. (64)
Abouzied et al. (55)
Wang and Smith (57)
Sheu and Hsieh (40)
Hsieh et al. (43), Chen et al. (41)
O'Callaghan (65), Schmidt et al. (66)
267
A pork-specic mAb-DD9 did not react with beef, chicken, horse, casein,
soy, gelatin, or BSA (63). The mAb-DD9 was prepared from unheated
muscle protein antigen puried by immunoafnity chromatography (26,27).
Indirect ELISA utilizing mAb-DD9 detected beef or chicken adulteration by
1100% (w/w) pork. The calibration response was described by
A405 0:0692 0:7989 ln%Pork
for beef and chicken samples, respectively. The LLD was 0.1% (w/w), which
is below levels probably economically advantageous to the retailer. The
assay sensitivity compares favourably with results expressed in Eq. (3).
Heating meat samples to 658C for 30 minutes had no adverse on the assay
reponse, but autoclaving samples at 1208C for 20 minutes led to loss of assay
sensitivity.
Chen and Hsieh (42) have recently described an mAb-based ELISA
for detecting the presence of pork within cooked or processed meat
products. The assay employs porcine thermostable antigen (Sec. 5.3). The
LLD for pork was 0.5% (w/w) with intrassay and interassay precision of
5.8% and 7.9%. The highly accurate method was able to identify pork in 45
commercial processed meat samples. Sawaya and co-workers (67) also
produced an ELISA test sensitive to cooked pork although they used pAb.
Horse meatspecic mAb-DD3 (64) showed no cross-reactivity for
beef, chicken, pork, soy proteins, casein, gelatin, or BSA. Addition of
050% (w/w) horse meat to beef led to the following ELISA response.
A405 0:4626 0:0314 %Horse
10
The LLD for horse meat was 2% (w/w). The Spanish group suggest that
268
Chapter 9
The mAb-2F8 had selectivity for beef, sheep, or lamb and reduced
sensitivity for deer meat (43). There was no cross-reactivity with raw
pork, beef, lamb, mutton, or deer. Chicken or turkey was not detected in
either the raw or cooked state. Indirect ELISA using mAb-2F8 was specic
for red meat. Adulteration of poultry (chicken and/or turkey) with 0.515%
(w/w) beef, horse, sheep, or pork was easily detectable. The LLD for deer
meat was * 5% (w/w) addition to poultry. Multiple adulterants produced a
cumulative signal. Red meat is often more expensive than chicken. However,
the preceding test will be useful for detecting the substitution of chicken
meat with less valuable beef or pork trimmings (49). Chicken is also in
higher demand in some parts of Asia where the consumption of beef is
restricted by religious stricture.
7.
Taylor and Jones (72) and also Taylor et al. (73) described an indirect
ELISA using (rabbit) pAb for soluble antigens from canned sardine, bonito,
yellown tuna, or skipjack tuna. Crude (rabbit) pAb for canned sh antigen
was nonspecic. High responses were obtained for most canned sh samples
(Fig. 8). To improve the specicity, pAb was puried via (a) batch
immunoadsorption, (b) immunoadsorption using antigens immobilized on
269
Rock Shrimp
270
Chapter 9
identied rock shrimp from three geographic locations in the United States.
These samples were also correctly differentiated from 23 other seafood
samples including white shrimp (from Colombia, Ecuador, Honduras, and
Peru) and blue shrimp (from Ecuador). The specicity for rock shrimp was
attributed to the use of puried antigen for mAb production. The test was
more sensitive for heated samples probably because protein M was heated
during isolation by SDS-PAGE. The antigen had molecular size of
17.718.5 kDa and made up *20% of the total water-soluble protein (75).
7.3.
Red Snapper
Factors affecting assay performance include (a) the ELISA format, (b) the
type of enzyme label, (c) antibody characteristics, and (d) the nature of the
antigen used. Calibration graphs for sandwich ELISA [Eqs (6) and (7)] or
indirect ELISA [Eqs (8) and (9)] show that the indirect format is more
sensitive. Using pAb for capture can lead to loss of assay response for
sandwich ELISA. Multiple interactions involved in pAb-antigen binding
may leave few epitopes for the detector antibody. Poor assay sensitivity also
arises when a sandwich ELISA utilizes the same mAb for both capture and
detection. Under such circumstances, highly specic epitopes become
occupied by the capture mAb with few left for binding with the (same)
mAb for detection. Comparing the sandwich ELISA tests for muscle antigen
271
shows that using mAb for capture enhances assay sensitivity by about an
order of magnitude compared with the use of pAb. Finally, whether puried
antigens are necessary for mAb production is contentious. With mAb for
unheated samples it seems necessary to use puried antigen. However,
thermostable antigens appear to induce specic mAb formation (see earlier).
9. MEAT TESTING FOR TRANSMISSIBLE SPONGIFORM
ENCEPHALOPATHY AGENTS
9.1.
272
Chapter 9
Concentrations of the BSE agent are higher in the central nervous system
(CNS) tissue compared with peripheral nerves. A sandwich ELISA for CNS
tissue was developed using mAb for GFAP (glial brillary acidic protein)
(66). This protein is found only in astrocyte cells in the CNS.* The ELISA
for GFAP had an approximate LLD of 1 ng (GFAP) with a linear range
extending to 40 ng. The within-assay precision was 3.254%. While assay
sensitivity remained constant for different matrices, the LLD increased in
the order ground beef brain tissue > ground beef spinal cord tissue
> puried GFAP standards > brain > spinal cord. The ELISA gave the
concentrations of GFAP in spinal cord tissue (55,000220,000 ng mg 1
tissue), brain tissue (900055,000 ng mg 1 tissue), and cerebral cortex
(17,000 ng mg 1 tissue). Neck muscle and ground beef were free of GFAP.
The antigen is not very stable; therefore CNS tissue could be detected for
only up to 8 days when samples were stored at 48C. CNS tissue was also
analyzed using immunoblot analysis (84). The tests were directed at two
antigens: GFAP and neuron-specic enolase. Immunoblot analysis did
detect CNS tissue if samples were subjected to extreme temperature
processing.
9.3.
273
ID Lalyated, Netherlands
Imperial College of Science and Technology, UK
Institute of Neurodegenerative Diseases, University of California, San
Francisco
Perkin Elmer Life Sciences, UK
Prionics AG, Switzerland
The BSE test produced by Prionics AG, Switzerland (Prionics AG,
University of Zurich, 8057 Zurich, Switzerland) appears to be the favorite.
Prionics-checkTM employs immunoblot analysis. Brain tissue extract is rst
exposed to a protease solution followed by SDS-PAGE and then transferred
to a nitrocellulose membrane by Western transfer. The membrane-bound
proteins are detected immunologically using mAb specic for prion
particles. The test is able to differentiate between the benign prion protein
(PrPC) and the disease-causing PrPSc because the former is susceptible to
protease attack but the latter is not.
According to the advertising literature, Prionics-check is intended for
(a) identication of suspected BSE cases, (b) diagnostic testing in abattoirs
and slaughterhouses, and (c) general monitoring for scrapie and BSE.
Validation of the Prionics-Check tests has been documented (86). Prionicscheck will be used for mandatory BSE testing in the European Union from
2001. Some important characteristics of the Prionic-checks include (a) high
selectivity and specicity, (b) ability to distinguish cattle with BSE from
those with other neurological disease states, (c) detection of subclinical cases
of BSE, (d) ease of use and availability in a kit form, (e) suitability for eld
use, (f) high sample throughput (the time of analysis is reportedly 68 hours
from tissue extraction to nal test results), and (g) current use for Swiss BSE
surveillance for all sick and falling cattle. At this time, Prionics AG
manufacture at least two prion-specic mAbs (6H4 and 34C9) as well as
pAb (RO29). The antibodies are suitable for developing ELISA. The
company also has available the full kit for BSE detection. The life science
diagnostics company Bio-Rad Ltd. currently manufactures an ELISA test
for BSE (PLATELIA2 BSE test). This test is commercially available in the
United Kindom, France, Germany, Belgium, Luxembourg, Norway,
Sweden, Switzerland, Italy, and Spain. At this time the test is not being
sold in the United States (87). The list of companies now entering the
BSE diagnostics market is growing rapidly as shown by the following list
(88).
274
Chapter 9
Abbeymoy Ltd.
Bayer
Boehringer-Ingelheim AG
Commissariat a l'Energie
Atomique
Genesis Biventures
Mary Jo Schmerr
New York State Basic
Research Institute for
Neurological Disorders
Prionics AG
Altegen Inc.
Caprion Pharmaceuticals Inc.
Centre Suisse
d'Electronique et de
Microtechnique SA
Enfer Scientic Ltd.
Anonyx Inc.
Bio-Rad Inc.
IGEN International
Paradigm Genetics Inc.
Celcus Inc.
Disease Sciences Ltd.
For further discussions of immunological tests for the BSE agent see
Refs. 89 and 90.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
275
276
Chapter 9
277
278
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
Chapter 9
inuenced by endpoint cooking temperature. J Agric Food Chem 42:1829
1833, 1994.
CH Wang, DM Smith. Lactate dehydrogenase monoclonal antibody
immunoassay for detection of turkey meat in beef and pork. J Food Sci
60:253256, 1995.
DM Smith, LD Desrocher, AM Booren, CH Wang, MM Abouzied, JJ Pestka,
J Veeramuthu. Cooking temperature of turkey ham affects lactate dehydrogenase, serum albumin and immunoglobulin G as determined by ELISA. J
Food Sci 61:209212, 1996.
DS Smith, LD Desrocher. Immunoassays for determination of endpoint
processing temperatures in poultry and beef products. J Muscle Foods 7:335
344, 1996.
D Levieux, A Levieux. Immunochemical quantication of myoglobin heat
denaturation: comparative studies with monoclonal and pAb. Food Agric
Immunol 8:111120, 1996.
R Martin, RJ Wardale, SJ Jones, PE Hernandez, RLS Patterson. Production
and characterization of monoclonal antibodies specic to chicken muscle
soluble proteins. Meat Sci 25:199207, 1989.
R Martin, RJ Wardale, SJ Jones, PE Hernandez, RLS Patterson. Monoclonal
antibody sandwich ELISA for the potential detection of chicken meat in
mixtures of raw beef and pork. Meat Sci 30:2331, 1991.
P Morales, T Garcia, I Gonzalez, R Martin, B Sanz, PE Hernandez.
Monoclonal antibody detection of porcine meat. J Food Prot 57:146149,
1994.
T Garcia, R Martin, P Morales, AI Haza, G Anguita, I Gonzalez, B Sanz, PE
Hernandez. Production of a horse-specic monoclonal antibody and detection
of horse meat in raw meat mixtures by an indirect ELISA. J Sci Food Agric
66:411415, 1994.
JP O'Callaghan. Quantitation of glial brillary acidic protein: comparison of
slot-immunoassay with a novel sandwich ELISA. Neurotoxicol Teratol
13:275281, 1991.
GR Schmidt, KL Hossner, RS Yemm, DH Gould, JP O'Callaghan. An
enzyme-linked immunosorbent assay for glial brillary acidic protein as an
indicator of the presence of brain and spinal cord in meat. J Food Prot
62:394397, 1999.
WN Sawaya, MS Mameesh, E El-Rayes, A Husain, B Dashti. Detection of
pork in processed meat by an enzyme-linked immunosorbent assay using
antiswine antisera. J Food Sci 55:293297, 1990.
V Venugopal. Mince from low cost sh species. Trends Food Sci Technol 3:2
5, 1992.
SR Tannenbaum, BB Stillings, NS Scrimshaw, eds. The Economics, Marketing, and Technology of Fish Protein Concentrate. Cambridge, MA: MIT
Press, 1974.
W Sidwell. Fish speciation by immunochemical techniques. In: MRA Morgan,
CJ Smith, PA Williams, eds. Food Safety and Quality Assurance. Applica-
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
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Chapter 9
89. LKJ van Keulen, JPM Langeveld, GJ Garssen, JG Jacobs, BEC Schreuder,
MA Smits. Diagnosis of bovine spongiform encephalopathy: a review. Vet Q
22:197200, 2000.
90. D Momocilovic, A Rasooly. Detection and analysis of animal materials in
food and feed. J Food Prot 63:16021609, 2000.
10
Speciation of Soya Protein by
Enzyme-Linked Immunoassay
1. INTRODUCTION
There are usually guidelines for adding plant and other nonmeat protein to
meat products. Food technologists use such ingredients legitimately to
enhance functional properties such as water holding, fat binding, and
gelation. Nevertheless, levels of nonanimal proteins in meat should be
monitored. Much research has appeared in connection with soybean
protein, this being the most important nonmeat protein ingredient. This
chapter describes immunological methods for detecting bulk quantities
(>0.5% w/w) of soybean protein in meat and meat products. The topic is
dominated by methods of sample pretreatment designed to ensure accurate
results no matter the sample processing history.
282
Chapter 10
Reference
Hitchcock et al. (1), Grifths et al. (2)
Grifths et al. (3)
Crimes et al. (4)
Olsman et al. (5)
Ravestein and Driedonks (6)
Hall et al. (7)
McNeal (8)
Hewedy and Smith (9)
* The following steps are involved: (a) Pretreat soy sample and standards by denaturationrenaturation protocol. (b) React soy samples or standards with (rabbit) pAb for soy protein. (c)
Add mixture to a microwell plate precoated with bound (renatured) soy protein. (d) Incubate to
allow antibody-antigen reaction. (e) Wash thoroughly with PBST. (f) Add enzyme-labeled pAb
for rabbit IgG. (g) Wash thoroughly with PBST. (h) Add enzyme substrate. (i) Stop reaction
after a xed incubation time and record absorbance readings.
FIGURE 1
283
284
Chapter 10
285
Soya beans contain between 40 and 50% protein by dry weight. The major
protein groups are (a) storage proteins (7080% total), (b) enzymes (notably
lipoxygenase, lactate dehydrogenase), (c) protease inhibitors (notably the
Bowman-Birk and the Kunitz inhibitors), and (d) other storage proteins
(e.g., lectin). The foremost storage protein (*40% total protein) is 11S
glycinin. It is a member of the leguminin family of proteins. The second
storage protein (*30% total protein) is the 7S conglycinin.
Glycinin (molecular mass of 350 kDa) is a hexamer of A-B subunits.
The six acidic (A) and basic (B) subunit pairs are each joined by a disulde
286
Chapter 10
3.2.
Thermal Denaturation
287
;
B Subunits solid-aggregates
Processes in Equation (1) will be modied by soy protein interactions with
muscle proteins, lipid, rusk, and other ingredients. Indeed, TD for soy
protein depends on the moisture level, ionic strength, pH, and the presence
of sulfhydryl compounds including other meat proteins. Within a defatted
our matrix containing about 60% (w/w) moisture, TD for glycinin was
908C. The TD value increased to 1608C for absolutely dry our (28,29).
3.3.
288
Chapter 10
The antigenicity of soy protein is dominated by the 7S protein (Fig. 1). This
is the result of a more efcient renaturation of conglycinin compared with
glycinin. After exposure to urea, extreme pH or high temperature, glycinin
dissociates into its constituent subunits (3739). German et al. (19) showed
that heating then disrupts S22S bonds between A-B subunits. Disulde bond
cleavange is catalyzed by indigenous free sulfhydryl compounds associated
with soy protein. Isolated glycinin B subunits then aggregate via SH/S22S
exchange while the A chains remain soluble (20). On the other hand
conglycinin does not aggregate extensively upon heating. This protein lacks
S22S bonds and also has low levels of SH groups. Extensive heating
eventually produces soluble conglycinin aggregates although SDS-PAGE
analysis shows little irreversible damage.
Conglycinin and glycinin refold with *80% and *70% efciency after
exposure to 8 M urea and dialysis, respectively. However, treatment with
urea2-ME (which disrupts A-B disulde bonds) leads to a glycinin
renaturing efciency of *20%. Breaking the A-B disulde linkage produces
a marked reduction in glycinin renaturation efciency and enhanced
289
290
Chapter 10
Protease Inhibitors
Peptide Antigens
Peptide antigens with continuous epitopes are potentially useful thermostable antigens. Yasumoto, et al. (46) developed a noncompetitive indirect
ELISA for soybean protein predigested with trypsin. The assay was highly
specic with no cross-reactivity for pork, beef, egg, or azuki bean proteins.
Soya protein (10.420.8% w/w) was quantitatively determined for pork
sausages cooked at 808C for 20 minutes. The LLD was 0.4%. The pAbs used
for this assay were raised by immunizing rabbits with intact glycinin.
As a form of pretreatment, glycinin standards were autoclaved at
1208C for 180 minutes and then digested with trypsin for 24 hours.
Microwell plates were coated with the peptide digest and blocked with BSA
to avoid nonspecic binding. Food samples, e.g., cooked sausages, were
pretreated by homogenization with acidic ethanol, followed by acetone
precipitation and drying. The powders were suspended in buffer, autoclaved
for 1208C for 180 minutes, and digested with trypsin for 24 hours at 378C.
Proteolysis was terminated by boiling briey. The supernatant was removed
for indirect ELISA.
Carter et al. (47) used glycinin peptides as antigens for pAb and mAb
production. The absence of discrete protein bands during SDS-PAGE
analysis conrmed a complete hydrolysis of glycinin (1 mg mL 1) by
treatment with 20 mg of subtilisin (in ammonium bicarbonate buffer,
50 mM, pH 8) overnight. The (rabbit) pAbs for glycinin peptides were
nonspecic (Fig. 3). Avidity for intact glycinin was attributed to surfacelocated antigenic determinants. There was cross-reactivity with other seed
globulins (b-conglycinin, pea 11S globulin), probably due to partial amino
acid sequence homology. Three mAbs (designated mAbs IFRN024,
IFRN025, and IFRN026) were also produced with interesting specicity
characteristics (Fig. 4). The mAb-IFRN024 was nonspecic and recognized
an epitope that was susceptible to denaturation by SDS treatment. Both
mAb-IFRN025 and mAb-IFRN026 were specic for both intact glycinin
and glycinin peptides. The former mAb showed a 100-fold greater avidity
291
FIGURE 3
Indirect ELISA for soya bean and other seed globulins using polyclonal
antibodies for glycinin peptides. (Drawn from Ref. 47.)
FIGURE 4
Indirect ELISA for soybean and other seed globulins using mAbs for
glycinin peptides. Results for mAb-IFRN0024 and mAb-IFRN0026
were multiplied by 10 before plotting. (Drawn from Ref. 47.)
292
Chapter 10
for these samples. The epitopes recognized by mAb-IFRN025 and mAbIFRN026 were resistant to combined SDS and thermal treatment. One
should expect that peptide antigens will be resistant to processing effects
owing to their small size.
5.3.
Conglycinin
Nonmeat protein additives from cereal sources, milk, and egg have been
analyzed by ELISA. However, there is far greater interest in the detection of
trace amounts of these proteins in relation to their ability to cause allergic
reactions. The analysis of protein allergens is discussed in Chapter 11.
REFERENCES
1.
2.
* Glycinin (4 mg mL 1 in 0.35 mM K-phosphate buffer 0.1 M NaCl) was heated and then
centrifuged to remove insoluble aggregates. The soluble fraction was analyzed by ELISA.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
293
294
Chapter 10
295
11
Determination of Trace Protein
Allergens in Foods
1. INTRODUCTION
Food allergy is one of a number of adverse reactions to foods (Table 1). The
nontoxic adverse reactions are mediated by the immune system (allergy).
Nonimmune system mediated adverse reaction is termed a food intolerance. Some allergic reactions to food, for example, anaphylactic shock, may
be severe and even fatal. General symptoms of food allergy are reviewed by
Anderson (1), Blades (2), Jones (3), and also Burks and Sampson (4).
Allergenic foods are discussed by Hee et al. (5) and also Taylor et al. (6).
Food allergens are reviewed by Matsuda and Nakamura (7), Bush and Hee
(8), and Hee (9). This chapter describes the analysis of trace amounts of
protein allergens in food. Allergens are associated with eight or nine major
food groups (Sec. 1.1). Soybean, peanut, and gluten allergies are described
in Secs 2, 3, and 4. In each instance we also consider the structure of the
protein allegens and the effect of processing on assay results.
1.1.
Articles by Campbell (10) and Amor (11) provide summaries of current food
labeling regulations related to allergens. It is believed that food may be
rendered safe by providing information related to allergen content.
297
298
Chapter 11
Non-IgE mediated
Non-immune mediated
Food intolerance
Food aversion
Compounds
Adverse effects
Natural food
components, food
additives
Protein allergens
Protein allergens
Lactase deciency, inborn
errors of
Metabolism, pharmacological compounds
Psychological avoidance
299
(wheat, rye, barley, oats) and derived products; and (h) sulte in
concentrations of 10 mg kg 1 or greater. Future FDA and Codex Commission policy may require the declaration of foods containing allergenic
proteins produced through genetic engineering. The concern is that genetic
modication may result in the inadvertent transfer of allergenic proteins
from one food source to another via genetic modication (12). Food
regulations and mandatory labeling requirements related to allergens are
also discussed in articles by Nestle (13) and others (14,15).
1.2.
Some of the major protein allergens in foods have been isolated and
partially characterized. Many are heat resistant and survive common food
processing operations. They also tend to be resistant to digestion. The
presence of antibodies in the circulation suggests that allergens pass through
the digestive tract (16). Allergenic peptides and perhaps whole proteins may
be absorbed intact, especially in infants. Some well-characterized food
allergens are listed in Table 2. The detection of food allergens plays a vital
role in the management of diets for susceptible subjects.
Two groups of analyses are employed for food allergens: (a) in vivo
tests using live patients, in which mild symptoms are induced in the subject
during a skin prick test or the double-blind, placebo-controlled food
challenge (DBPCFC) test, and (b) in vitro detection, which covers three
types of tests involving isolated pAbs. A well-known procedure is the
radioallergoadsorbent test (RAST) for circulating antibody. The food
sample containing antigen is mixed with serum (collected from the allergic
subject). The mixture is then added to a xed amount of antigen
immobilized on CNBr-activated Sepharose. After washing the support,
any bound pAb is determined by reacting with iodine-125-labeled (rabbit)
antibody for human IgE. The amount of radioactivity bound to the solid
phase, measured with a scintillation counter, is inversely related to the
concentration of circulating pAb as well as to the food allergen content.
RAST is essentially a competitive immunosorbent assay using iodine-125labeled (rabbit) pAb for ``visualization'' (17,18). The second in vitro assay
for food allergens involves immunoblotting (19,20); examples of these
studies are listed in Table 3. The food sample is analyzed by SDS-PAGE,
transferred to a nitrocellulose membrane, and exposed to serum from the
allergic subject. The bound IgE is visualized using iodine-125-labeled
(rabbit) pAb for human IgE followed by radiography (Table 3). The nal
group of in vitro tests for food allergens involves classical ELISA. Included
in this group are tests described in Chapter 10 for bulk food proteins.
300
TABLE 2
Chapter 11
Some Food Allergens Involved in IgE-Mediated Adverse Reactions
Food group
Milk
Egg white
Soybean
Peanut
Castor bean
Codsh
Shrimp and other
crustacea
References
Shibasaki et al. (21), Bucks et al. (22), Bush et al. (23), Herian
et al. (24), Ogawa et al. (25), E-F Ebabiker et al. (26)
Sachs et al. (27), Barnett et al. (28), Bucks et al. (29), Bucks et
al. (30), Uhlemann et al. (31), Hee et al. (32), de Jong et al.
(33)
Hoffmann et al. (34), Nagpal et al. (35), Lehrer et al. (36),
Reese et al. (37)
Hide (38), Morgan et al. (39), Nordlee et al. (12), Borja et al.
(40)
Bargman et al. (41), Hlywka et al. (42)
Hoffman (43), Langeland (44), Leduc et al. (45)
Ball et al. (46), Restani et al. (47), Rosendal and Barkholt (48)
301
302
Chapter 11
Identity
7SF, Gly m Bd 30K
b-Conglycinin asubunit
7SF, Gly m Bd 28K
7SF
7SF
7SF
7S globulin, b-unit
7SF
11S globulin, a-unit
2S, KSTI
Patients with
antibody (%)
65.0
23.3
23.3
18.8
14.5
10.1
10.1
15.9
1.4
2.9
N-terminal 15-amino-acid sequences for Gly m Bd 30K and 34-kDa oil body
protein were the same. The mAb for either protein cross-reacted with the
other. Following established convention, Gly m Bd 30K, being the rst
soybean allergen identied, was designated Gly m 1.
Kalinski et al. (53) independently characterized the 34-kDa oil body
protein. Within intact cells, the protein was a vacuolar protein designated
P34. Like other storage proteins, protein P34 undergoes post-translational
glycosylation and proteolysis. During processing, P34 appears along the
endoplasmic recticulum, Golgi bodies, and eventually within vacuoles or
protein bodies. Protein P34 had partial sequence homology with cysteine
proteases from the papain superfamily. It is not certain that P34 has
proteolytic activity (54). Its association with soybean oil bodies was an
experimental artifact produced by cell disruption. A survey of a large
number of soybean strains shows that P34 is widespread. The possibility of
eliminating P34 from soybean lines by breeding seems doubtful (55).
The next important soybean allergen (Gly m Bd 70K) is the 70-kDa
b-conglycinin a-subunit. Antibody specic for the a-subunit did not crossreact with the other two (a0 or b) subunits of b-conclycinin despite some
sequence homology between these polypeptides (56). The third soybean
allergen (Gly m Bd 28K) is a 26-kDa glycosylated protein with a pI of 6.1
(57). It is apparently unstable and present in very small quantities in
defatted soy our (*15 ppm). Perhaps for these reasons, Gly m Bd 28K was
difcult to detect in processed foods containing soy protein. Finally, pAbs
from some soybean-sensitive individuals recognized the A-chain residue
303
2.2.
Quantitative ELISA for Gly m Bd 30K was developed by Tsuji et al. (60)
using mAb produced by conventional means. Eventually, two cell lines
producing mAb (mAb-F5 and mAb-H6) were isolated. Immunoblot
analysis showed that mAb-F5 and mAb-H6 were both specic for Gly m
Bd 30K. Sandwich ELISA for Gly m Bd 30K employed mAb-H6 as the
capture antibody and an HRP conjugate with mAb-F5 as detector. The
linear range for analysis was 2200, 5500, or 10500 ng protein for reduced/
carboxymethylated-allergen, SDS/2-MEtreated sample, or native soy
protein, respectively. Ten soybean products and ve meat products
containing soy protein isolate (SPI) were analyzed by a sandwich ELISA
for Gly m Bd 30K (61). Results from this study are summarized in Table 5.*
From such data it may be surmised that (a) high concentrations of Gly
m Bd 30K are detectable within a range of processed soybean products, (b)
fermented soy products (miso, soyu, and natto) are free of allergen, (c) Gly
m Bd 30K can be accurately determined in cooked meat products known to
contain soy protein isolate, (d) sandwich ELISA results were corroborated
by immunoblot analysis using pAbs from soybean-sensitive patients, and (e)
Gly m Bd 30K is heat resistant, accounting for its detection in cooked
products. The thermal stability of soybean antigens is discussed in Chapter
10. The allergen was also resistant to digestion by chymotrypsin and trypsin.
Levels of Gly m Bd 30K declined to negligible values in fermented soy
products, probably as a result of digestion by microbial (acid) proteases.
Treating soybean samples with added microbial proteases reduced the level
of allergen, leading to hypoallergenic soybean products (62). Clearly, the
preceding assay has potential uses for monitoring soybean allergen in
processed foods. The ELISA test could also nd use as a quality control tool
for hypoallergenic soybean products.
* As pretreatment each (5-g) sample was homogenized with Na- phosphate buffer (50 mM, pH
8.0 with 1% w/v SDS and 20 mM 2-ME). The extract was centrifuged and SDS was precipitated
with KCl (1 M nal concentration). After centrifugation, the SDS-depleted sample was
analyzed by sandwich ELISA.
304
Chapter 11
Product
Soybean
Soymilk
Tofu (kinugoshi)
Tofu (momen)
Kori-dofu
Kinako
Abura-age
Yuba
Miso
Shoyu
Natto
Meatballs
Beef croquettes
Fried chicken
Fish sausages
Hamburger
Sandwich ELISA
results (mg allergen
g 1 N)
126
106
89
65
64
29
59
66
npb
np
np
17
21
9
np
np
Immunoblot
analysisa
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
2.3.
ELISA for bulk soy protein analysis (Chapter 10) may be useful for
monitoring soybean allergens indirectly. Yeung and Collins (63) described a
competitive ELISA using (rabbit) pAb for whole soybean extract. The
assay, which was virtually identical to those used by Hitchcock and coworkers (6467), was intended to detect bulk soy protein as opposed to trace
allergen. The antigen for immunizing rabbits was prepared from soybean
our defatted with cold acetone.* The competitive ELISA for soy protein
(63) had the following characteristics:
* Soy our was extracted with cold Tris-HCl buffer (10 mM, pH 8, with 1% w/v SDS and
10 mM 2-ME) at 48C for 16 hours.
305
Peanut Allergy
306
Chapter 11
anaphylactic response can arise from topical contact with peanut residue on
another individual, by inhalation (74), or by exposure to peanuts in
vegetarian ``beef '' burgers (75). Small amounts of peanut protein associated
with peanut oil can also induce an allergic episode. Peanut protein was
found in some infant formula prepared from peanut oil (76,77). Highly
rened protein-free oil apparently poses little threat (51).
Reported fatalities due to peanut allergy are of the order of 125
persons per year in the United States (84). Figures from Pumphrey et al. (78)
suggest that there were 21 deaths from peanut and tree nut allergy in the
United Kingdom during the 5- to 6-year period following 1992. Of 541
patients showing sensitivity to nuts, 90% had serum IgE specic for peanut.
About 67% of patients showed serum IgE for another nut besides peanut,
while 34% of patients had immunoglobins for all nuts tested. The threshold
quantity necessary to induce allergy varies for different subjects. Exposure
to peanut allergens in early infancy (<1 year old) or before birth may
contribute to sensitization (70). It is not yet certain whether pregnant and
lactating women should avoid peanuts and tree nuts in their diet (7880).
Avoidance of peanuts is the main strategy for managing the diet of
sensitive individuals. Hidden allergens from peanut-derived ingredients are a
major issue. Eating outside the home setting can also be problematic.
Restaurateurs cannot provide strict assurances that their ingredients or
recipes are free from peanut allergens. People thought to be highly sensitive
to peanut protein are also advised to have rapid access to epinephrine
(adrenaline) by injection. In summary, peanut and nut allergies present
serious health threats. Reviews by Sampson (81), Hourihane (82), Burks et
al. (83,84), and also Warner (85) deal with the clinical and public health
aspects of peanut allergy. Some distinctive features of peanut allergy are
(a) high incidence in the population, (b) speed and severity of the symptoms,
(c) lack of an effective treatment, (d) widespread use of peanuts and peanut
ingredients in the food chain, and (e) persistence of peanut sensitivity with
age. The availability of tests for trace amounts of peanut will reduce the risk
of exposure to peanut allergens.
3.2.
307
28.0, 16.0
6064, 17.0
40.0, 33.0
20.0, 1718
308
Chapter 11
3.3.
3.4.
309
310
Chapter 11
3.6.
311
nuts: soybean, green pea, chick pea, lupine, hazelnuts, Brazil nuts, pine nut,
pecan, almonds, walnut, and cashew nut. Also, no interferences occurred
with general foodstuffs such as corn, cocoa, chocolate, milk, sugar, soy
lecithin, peanut lectin, and coconut. Peanut proteins were quantitatively
determined in over 40 processed food items belonging to the following food
groups: potato chips (crisps), cooking oil, pasta sources, plain cookies, ice
cream, and milk chocolate bars. All products labeled as containing peanut
protein were correctly identied with no false-positive results.
A sandwich ELISA using (rabbit) pAb for puried Ara h 1 was
described by Koppleman and co-workers (101). The assay characteristics
can be summarized as follows:
1. Range. The linear dynamic range for peanut protein was
0.0051 mg mL 1. Fried peanut samples gave 4580% of the
response obtained from raw samples.
2. Specicity. No cross-reactivity occurred with 40 food products
including, 9 legumes, 9 nuts, 7 cereals, 4 oil seeds, and 9 animal
proteins. In tests involving 31 processed goods (10 cookie brands,
6 chocolates, 3 cereals, 3 baby foods, and 8 sources and soups) all
products labeled as containing peanuts were accurately identied.
3. Recovery. For samples spiked with 0.00011.0% peanut protein,
recoveries were typically 85105%. Reproducible recoveries were
obtained from high-fat foods.
The competitive indirect ELISA described by Holzhauser and Vieths
(104) employed commercial pAbs for unheated peanut protein. Before use,
pAbs were puried by immunoadsorption with soy protein, white bean, and
marzipan. Precooking peanuts had little effect on the levels of Ara h 1 and
its binding by both human and rabbit pAbs. The nal assay was successfully
applied to over 30 commercial food products.
Three commercial ELISA kits for peanut allergen were evaluated (105)
at the Nestle Research Center (Lausanne, Switzerland). The kits were those
from Cortecs Ltd. (UK), Prolab Ltd. (Canada), and the TNO (Netherlands). The last two kits appear to be derivatives of the assays developed by
Yeung and Collins (102) and Koppleman and co-workers (101). Detailed
results concerning the preliminary evaluation were not reported. The
Cortecs kit was further evaluated for a semiquantitative determination of
trace amounts of peanuts in a range of confectionery products. The
particular kit employs a sandwich ELISA format with antibody for
concanavalin A for capture. Biotinylated antibody for concanavalin is
used for detection. Bound antigen is visualized with streptavidin-labeled
HRP assayed using tetramethylbenzidine. Recovery of peanut allergen from
dark chocolate was only 23% when using the manufacturer's extraction
312
Chapter 11
4.
4.1.
313
consists of couscous and bread. Catassi and co-workers (122) suggested that
celiac disease has adaptive value in preventing intestinal adhesion and
infection by pathogenic microorganisms such as Vibrio cholerae.
The major lesion accompanying celiac disease is atrophy of microvilli
from the small intestine, duodenum, and jejunum. Microscopic observation
shows a attening of the mucosal lining and reduced surface area. There is
malabsorption of most nutrients. The intestinal absorptive layer, being
compromised, may allow the uptake of a greater range of psychoactive
peptides. Theories explaining the etiology of gluten-sensitive enteropathy
are summarized by Cornell (115), who also describes in vitro methods for
assaying celiac-reactive polypeptides or allergens. Bruzzone and Asp (116)
provide an informative introduction to gluten-sensitive enteropathy starting
from its discovery in 19501953 by Dickie and covering possible links
between diet and the immune system. Progress in the study of glutensensitive enteropathy has suffered owing to the lack of reliable assays for the
condition. Current methods for diagnosis for this condition involve
1. Use of organ culture. Intestinal biopsy spceimens when grown in
cell-tissue culture show normal microvilli within 24 hours.
Addition of gluten (peptides) to culture media reintroduces
abnormalities in microvilli.
2. Detection of circulating antigliadin antibodies. Blood serum from
sufferers from gluten-sensitive enteropathy is used as the basis of
indirect ELISA.
The symptoms of celiac disease in infants and young children include
growth impairment, abnormal stool, abdominal distention, and psychological effects, which show up as generally poor temperament. Adults
sometimes exhibit diarrhea, atulence, bulky stools, anemia, fatigue, weight
loss, bone weakness, and neurological disorders including depression and
schizophrenia. Treatment for celiac disease involves the exclusion of gluten
from the diet. United Kingdom regulations state that gluten-free foods
should contain no more than 300 mg (gliadin) per 100 g foodstuff (i.e.,
300 mg % or 0.3% w/w). European Union and WHO limits for gluten-free
foods are between 10 and 1 mg %.
Wheat gluten is an inexpensive protein and therefore widely used as a
``vegetable protein'' ingredient for meat products (123). Wheat our is used
for thickening soups and other processed foods. Gluten appears in
confectionery products and is also used as a binder for pharmaceutical
tablets. Beer contains barley protein. Rye bread is widely consumed in parts
of Eastern Europe. Raw meat products such as sausages may contain wheat
proteins derived from rusk. Hidden allergens need to be considered in
attempts to reduce celiac-active proteins and peptides in the diet. There is
314
Chapter 11
4.2.
A.
Thomas Burr Osborne divided plant seed proteins into ve classes according
to their solubility in water, 0.1 M sodium chloride, alcohol, or alkali.
Sequentially extracting our using this range of solvents recovers albumins
(in water), globulins (with 0.1 M salt), prolamins (in 4070% ethanol, and
glutelins (with 0.1 M sodium hydroxide). A fth class of protein (called
residue protein) can be extracted only with severe solvents. The glutelins are
extractable in alcohol and 2-ME or DTT. About 7090% of the storage
proteins in cereals consist of nearly equal proportions of prolamin and
glutelins.* An important exception is cereal oat, which contains 80%
globulins. Rice has 15% prolamin and 8090% glutelin. The allergens
responsible for celiac disease are thought to be amino acid repeat sequences
found in cereal storage proteins.
Tatham et al. (124) devised a new classication scheme for cereal
proteins (Table 7). The new scheme acknowledges fundamental structural
TABLE 7 Cereal Prolamins Thought to Be Involved in Celiac Disease
Prolamin
Sulfur rich
Monomeric
Polymeric
Sulfur poor
HMW
Wheat
g-Gliadin
b-Gliadin
a-Gliadin
LMW glutenin
o-Gliadin
HMW glutenin
Rye
Barley
g-Secalin
g-Hordein
o-Secalin
HMW secalin
B hordein
C hordein
D hordein
* By contrast, legume seeds (soya bean, pea, kidney bean, and peanut) contain globulins as the
major storage proteins. There is relatively little (010%) prolamin or glutelin present.
315
relationships between the different protein fractions. First, all cereal protein
fractions (besides albumins) are soluble in 70% alcohol and are therefore
classed as prolamins. Next, the storage proteins are grouped according to
similarities in their amino acid and cDNA sequences into three classes:
(a) sulfur-rich prolamins (a-, b-, g-gliadin), (b) sulfur-poor prolamins
(o-gliadin), and (c) high-molecular-weight (HMW) prolamins. The sulfurpoor prolamins are monomers possessing only intramolecular disulde
bonds. This group includes o-gliadin (wheat), o-secalin (rye), and C hordein
(barley). Some sulfur-rich prolamins form HMW polymers. By contrast, a-,
b-, and g-gliadins represent monomeric sulfur-rich prolamins. The three
classes of prolamins have related architectures.
B.
Gluten
Gluten is prepared by washing wheat our with water to remove the starch.
This leaves a viscoelastic protein network (gluten) that is responsible for the
gas-retentive properties of wheat dough. Gluten is a network formed by
interacting wheat prolamins (Table 7). Each of the four gliadins can be
prepared on a large scale by ion-exchange chromatography (125).* A single
chromatographic run involving about 54 g crude gliadin yields 1 g of ogliadin and approximately 10 g each of g-, b-, and a-gliadin. Electrophoresis
at low pH separates of gliadin into four subtypes. SDS-PAGE analysis
shows the following order of increasing electrophoretic mobility: a-gliadin
> b-gliadin >, g-gliadin > o-gliadin. Glutelin is highly polymerized by S22S
bonds.
The sulfur-rich prolamins possess N-terminal repetitive amino acid
sequences containing high numbers of three amino acids: glutamine (Q),
proline (P), and phenylalanine (F) (Figure 1). The C-terminal areas have
variable sequences bearing a number of half cysteine residues normally
involved in intramolecular S22S bonding. Also noteworthy is the rare
occurrence of SH groups in o-gliadin. Regularities are also evident in the 28
structure of cereal proteins (126,127). Thus, a-gliadin, b-gliadin, and
g-gliadin have 3637% a-helix, 1112% b-sheet, and 5253% aperiodic
structure plus b-turns. The precise quantity of b-turn or hairpin loop
structure is uncertain because this does not produce a distinct circular
* Stir 1 kg of our with water-saturated n-butanol to remove lipids. Next, extract with four
volumes of 70% (v/v) aqueous ethanol. Dry the extract by rotary evaporation and redissolve the
protein in 0.1 M acetic acid solvent. Dialyze against the same solvent and fractionate with a
CMC-cellulose column (10 cm 6 22 cm, containing 1.7 kg support) equilibrated with sodium
acetate buffer (5 mM 1 M dimethylformamide and adjusted to pH 3.5 with acetic acid). Elute
the bound protein using stepwise changes in NaCl concentration gradients.
316
Chapter 11
FIGURE 1 The primary structure of cereal prolamins. (Adapted from Refs. 124 and
126.)
317
4.3.
The thermal denaturation of gliadin was examined using 70% (v/v) ethanol
as solvent. Heating g-gliadin led to a conformational change at 20608C as
monitored by far-UV circular dichroism.The spectral changes showed an
isobestic point implying that denaturation was a two-state (helix-coil type)
transition. At elevated temperatures the proportion of a-helix declined by
7% and the structure of gliadin loosened to expose phenylalanyl residues to
the solvent. Puried a-, b-, and o-gliadin behaved similarly. Conformational
changes produced by the relatively short (5-minute) thermal treatments were
reversible (126,127). Heating wheat our matrix revealed that o-gliadin is
more heat resistant than the sulfur-rich gliadins.* The levels of a/b-gliadin
decreased substantially after 20 minutes of heating. The g-gliadin was
resistant for up to 50 minutes of heating at 1008C. There was no change in
the quantity of o-gliadin recovered after 0100 minutes of heating. This
result is due to the virtual absence of half cystine residues in o-gliadin. This
protein can not undergo sulfhydryl-disulde exchange during heating (133).
4.4.
Immunological assays for cereal proteins are generally concerned with the
detection of celiac-active peptides. Some attempts to assess grain varietal
differences by EIA were reported (134). Also of interest is the identication
of (bread making) quality-related polypeptides by immunoassay (135,136);
literature in this area was reviewed by Skerritt et al. (137). Sections 4.4
through to 4.10 consider ELISA tests for trace (nanogram to microgram)
quantities of cereal proteins. ELISAs for wheat allergens and bulk protein
adulterants are not fundamentally different in their design. However, the
former techniques possess greater acuity. SDS-PAGEimmunoblot analysis
is another ELISA format.
* A suspension of wheat our in water was heated at 1008C for 0, 10, 20, 30, 50, and 100
minutes. Flour proteins were then extracted with 1 M urea solution and analyzed by gradient gel
electrophoresis.
318
4.5.
Chapter 11
319
Antigen
Sandwich, pAb
a-Gliadin, gliadin
Sandwich, Competitive,
pAb
dot blotting, mAb
Gliadin
Sandwich, Competitive,
pAb
Sandwich, mAb
Immunoblotting
Sandwich, pAb
Competitive, pAb
Gliadin
o-Gliadin, gliadin
Gliadin
Gliadin
Gliadin
Gliadin
Sandwich, mAb
Sandwich, mAb AOAC
approved laboratory
test
a-Gliadin, g-gliadin
o-Gliadin, glutenin
subunits
o-Gliadin, glutenin
subunits
Gliadin
Sandwich-ELISA
Sandwich, mAb
Competitive
Gliadin peptides
References
Windemann et al. (138),
Meier et al. (139),
Fritschy et al. (140)
McKillop et al. (141)
Skerritt and Smith (133),
Skerritt (142), Skerritt
et al. (143), Freedman
et al. (144)
Troncone et al. (145)
Freedman et al. (146)
Janssen et al. (147)
Ayob et al. (148)
Friis (149), Chirdo et al.
(150)
Mills et al. (151)
Hill and Skerritt (152),
Skerritt et al. (153),
Skerritt and Hill
(154,155)
Skerritt and Hill (156)
Hekkens and Twist de
Graaf (157)
Ellis et al. (158), DeneryPapini (159), Ellis et al.
(160), Nicolas et al.
(161)
320
Chapter 11
The AOAC-approved test for gluten employs mAbs for capture and
detection (154). Skerritt and Smith (133) and Skerritt (142) found two mAbs
(mAb-401/21 and mAb-304/13) with specicity for o-gliadin, HMW and
LMW glutenin subunits. Because o-gliadin is heat resistant (Section 4.3),
the preceding tests may be suitable for the analysis of heat-processed foods
(see later). Either mAb-401/21 or mAb-304/13 could be used for capture or
detection, leading to similar results. Preliminary tests (153) showed
specicity for wheat (bread or durum) > rye*barley 4 maize > oats. No
responses were observed for oat, maize, or rice protein at levels comparable
321
to those for wheat gluten. Sorghum was not tested. Assay performance
depended on these factors (152,153,154):
1. Extraction solvent. The optimal solvent for gluten extraction from
a range of food samples was 40 % (v/v) ethanol. Using 70% (v/v)
ethanol, 1 M urea, or 1 mM HCl as solvent led to underestimation
or overestimation of the gluten content.
2. Form of extraction. Homogenization of our samples using an
omnimixer or Ultraturrax mixer for about 30 seconds led to
accurate analysis. Vortexing for 30 or 60 seconds duration (four
times per hour) led to inaccuracy, probably due to shear-induced
precipitation of gluten.
3. Choice of gluten standard. Normal gluten is a suitable standard.
This is relatively soluble, stable in the freeze-dried form, and
usable over a wide concentration range (dilutions of up to
10,000 1 were used for analysis).
4. Choice of solid phase. Polystyrene microwell plates were preferable
to PVC plates, which produced high nonspecic binding. Background adsorption of gluten was not ameliorated by a range of
blocking solutions including PBST with 0.055% BSA, 0.65 M
NaCl, or 2% Tween.
5. mAb characteristics. The nature and concentration of the capture
and detector antibody and coating conditions (time and temperature of coating) affected the assay.
Following optimization, the linear dynamic range of the AOACapproved ELISA test was 0.00755 mg mL 1 (15 mg % to 10% w/w gluten in
actual foods). The LLD was 0.10.2 mg mL 1, which is equivalent to
0.20.38-mg gliadin per 100 g of our (0.2 0.38 mg %). A partial list of food
types successfully analyzed includes starches (wheat, maize), cake ours
(plain, self-raisin, bread crumb, cookie), gluten-free bread mixes, processed
meat (cooked beef, ham sausages, salami), baby foods (beef or chicken
based with and without our thickener), breakfast cereals, baked goods
(bread crumb mix, sweet cookies, crisp bread), soups, confectionery
(caramel, chocolate), and others (lentils, eggs, milk powder).
322
4.8.
Chapter 11
323
functioned as the detector antibody. The mAb-IFRN 033 was specic for agliadin and g-gliadin, which together constitute *85% of gliadin. The linear
range for gluten was 0.252.5 mg mL 1 with the LLD being 0.1 mg mL 1.
The preceding tests may be compared with the 1987 sandwich ELISA
test developed by Freedman et al. (146) from Guys and St Thomas's
Hospital, London. They used (rabbit) pAb for capture and mAb for
unfractionated gliadin for detection. The bound mAb was visualized with
alkaline phosphataselabeled (goat) pAb for murine IgG/IgM. The linear
dynamic range for this assay was 10 ng mL 1 to 1 mg mL 1. Strong wheat
our contained 3.7% (w/w) gliadin-equivalent proteins. Several brands of
``gluten-free'' our produced from wheat starch had 1.93.3 mg % of gliadin.
The day-to-day precision of the assay was better than 5%.
4.9.
The Skerritt and Hill (156) home test kit for gluten performed well with
ordinary citizens, who successfully identied gluten-free foods with 82
100% accuracy. The home test agreed closely with results from the AOACapproved laboratory test kit. The home test kit can be usefully compared
324
Chapter 11
with the home pregnancy test kits, which are now commonplace. Similar
self-use kits are possible for other food allergens. The home-gluten test
consists of the following components:
1. Polystyrene test tubes (Nunc, Denmark) precoated with mAb-401/
21 and blocked with 1% BSA. The antibody-coated test tubes are
stable at 48C for 12 months or at 208C for 6 months.
2. Graduated tubes for sample extraction.
3. Enzyme-labeled mAb-402/21 for detection.
4. Substrate solution (TMB/hydrogen peroxide).
The kit was eld tested in eastern Australia by 5 dieticians or food
technologists and 47 ordinary citizens registered with the Celiac Society.
Participants were 10- to 77-year-old urban and rural dwellers of varied
educational background. The testers were given six food samples to grade as
having ``low,'' ``borderline,'' or ``high'' gluten. Samples classed in the
borderline (40 mg %) or high (>150 mg %) gluten categories were not
acceptable as ``gluten-free'' foods, whereas those with <10 mg % were
placed in the gluten-free category. For sample pretreatment, 0.5 g of ground
or nely chopped foodstuff was shaken with 5 mL of dilute hydrochloric
acid (2 mM) for 1 minute. There was 100% protein recovery from wheat
starch, 62% recovery from wheat starch having 100 mg % gluten, and 18%
recovery from wheat our with 11% gluten.
Home testing for gluten involved ve simple steps: (a) add 0.8 mL of
reaction buffer (1% BSA solution in PBST) to the antibody-coated test tubes
provided, (b) add one drop (30 mL) of food extract and mix gently for 30
seconds, (c) add three drops of HRP-mAb conjugate, (d) wait about 11=2
minutes and rinse the tubes with running tap water, and (e) add enzyme
substrate solution. A positive test gives a blue coloration within 2 minutes.
Classify tests results as high, low, or borderline by comparing results with
color produced by standard samples.
4.11.
325
3. Poor limit of detection. The LLD achieved by the AOACapproved gluten test may be inadequate to deal with future,
more stringent legislation regarding gluten content.
4. Unproven range of applicability. Although maize, rice, and oats
were tested by the approved AOAC method, it may be desirable to
test a wider range of materials.
5. Low reliability for certain foods. A commercial ELISA kit based
on the approved method gave discordant results for 11 out of 24
foods measured in ve laboratories (Section 4.8).
Peptides are useful antigens for developing gluten ELISA tests. A
sandwich ELISA for gluten was developed using mAb-WB8 with specicity
for peptide B3144 as the detector (158). B3144 consists of the celiac-toxic Nterminal residues 356 from a-gliadin (129). The capture antibody was
(rabbit) pAb for unfractionated gliadin. The mAb-WB8 bound a-gliadin, bgliadin, g-gliadin, o-gliadin, glutenin subunits, and unfractionated gliadin.
There was also high reactivity toward rye and barley prolamins. The
response for celiac-active proteins was 1254000 times greater than that with
celiac-negative cereals such as oats, maize, millet, or sorghum. Very low
binding occurred with ovalbumin or BSA (Fig. 2).
FIGURE 2
326
Chapter 11
FIGURE 3
Sandwich ELISA tests for gluten developed with mAbs for peptide
antigens. The capture antibody is (rabbit) pAb. The dectector antibody
is either mAb-WB8 or mAb-PN3 with specicity for a-gliadin Nterminal residues 356 or 3149, respectively. (Drawn using results from
Refs. 158 and 160.)
327
extracting with a reducing buffer was unsuccessful. Residual mercaptoethanol affected the stability of antibody during ELISA. The inability to assay
gliadin in heat-processed samples is a serious shortcoming. The test using
mAb-PN3 is also likely to underestimate celiac-toxic potential when a
product contains ingredients from barley or rye. This is because the limit of
detection for wheat protein was 250-fold lower than values for hordein or
secalin. The latter proteins will remain ``hidden'' in foods exhibiting
relatively low amounts of celiacactive proteins.
Denery-Papini and co-workers (159) produced pAbs for a number of
synthetic peptides. Each peptide matched a unique amino acid sequence
found in one of the four classes of gliadins. Each 8- to 12-residue peptide
was covalently coupled to a protein carrier for immunization. The (rabbit)
pAbs for these antigens were evaluated by ELISA as well as SDS-PAGE
and immunoblot analysis. A pAb specic for the N-terminal sequence from
g-gliadin or o-gliadin bound only to the corresponding protein. A particular
pAb specic for the N-terminal sequence of a/b-gliadin was used as the basis
for a competitive ELISA test for gliadin (161). Gliadin levels were found to
be 23335040 mg % using our from 20 wheat cultivars. The results were
highly correlated with gliadin determinations by reverse-phase HPLC
(R 0.82) and Kjeldahl analysis (0.86). The linear range for analysis was
45250 mg mL 1 (extract) with an LLD of *9 mg mL 1. Stirring wheat ours
with 70% (w/w) ethanol for 14 hours led to the solubilization of 48% to the
total protein. The solubilized protein was gliadin with little contamination
from glutenin.
Results from ELISA tests for gluten are summarized in Table 9. This
information was compiled from the limited number of studies involving real
foods (140,141,144,146,149,150,154,160). The agreement between gluten
values is reasonable. Results are routinely reported in milligrams of gliadin
(equivalent protein) per 100 g dry food (mg %). Measuring gluten in this
fashion is convenient because ELISA tests are usually calibrated using
whole gliadin extract. The ideal assay for gluten should show equal
responsivity toward prolamins from all celiac-active cereals. It is necessary
to test samples of alcohol-soluble proteins from each cereal. The results in
Table 9 do not include contributions from glutelin subunits, which
(although celiac active) dissolve poorly in 4070% (v/v) aqueous alcohol.
Glutenin could be more effectively solubilized for immunoassay using
solvents containing SDS or guanidine hydrochloride. Conroy and Esen
(169) found that zein was efciently adsorbed by polystyrene microwell
plates from acetic acid (60% v/v) or 6 M urea (dissolved in carbonate coating
buffer). Sample extraction with these solvents could improve the recovery of
glutenin.
328
TABLE 9
Gliadin
Chapter 11
Apparent Gluten Levels in Range of Foods Determined by ELISA for
Foodstuff or commodity
Gluten-negative foods
Tropical cereals
Millet, sorghum, rice, jawar
Legumes
Soya our, chickpea our, buckwheat, quinoa
Miscellaneous
Potato our, molasses, skimmed milk powder, sugar
beet
Gluten-free (wheat starches)
Kinderm, Glutan, Wheatex, Nutregen
Wheat starch-based products
Cooked foodsa
Glutan GF ber loaf, Glutan GF white bread with
soya, Lopron white bread, low-protein chocolate chip
cookies
Uncooked products
Glutan GF white mix, ber mix, Rite Diet lowprotein our mix
Wheat-free products
Cooked products
Chocolate digestive biscuits, custard creams, tea biscuits,
orange avor cream wafers, mince pies
Uncooked products
Glutan pasta spirals, vermicelli, spaghetti, macaroni
Flours
Buckwheatb
Sorghumb
Maizeb
Riceb
Oatsb
Barley
Rye
Wheat (self-raising, plain)
a
b
Gliadin (mg %)
Negative
0.57.75
0.120.24
0.91.4
0.1441.18
0.076.7
4
13
46
4757
100240
400
580
40008100
329
relevant. The most sensitive tests are ELISA using pAb. The LLD for such
assays is about 0.51 ng mL 1. To achieve high acuity it is necessary to avoid
nonspecic binding of gliadin to microwell plates. Exposing microwell plates
to 3% skimmed milk powder or BSA for 60120 minutes reduces nonspecic
protein binding. Precoating with extraneous protein is better than adding
the coating protein together with the sample. Coating of microwell plates at
48C overnight leads to better assay performance in contrast to coating at
378C for 90 minutes. Using mAb in place of pAb increases the cost of
ELISA tests by an estimated 10-fold. On the other hand, mAbs exhibit
increased specicity and decreased likelihood of detecting celiac-negative
cereals. Immunoafntity-puried pAbs show high selectivity for celiacactive cereals and are more frequently employed than mAbs.
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124. AS Tatham, PR Shewry, PS Belton. Structural studies of cereal prolamins,
including wheat gluten. Adv Cereal Sci Technol 10:178, 1990.
125. AL Patey, DJ Evans. Large-scale preparation of gliadin proteins. J Sci Food
Agric 24:12291233, 1973.
126. AS Thatham, BJ Miin, PR Shewry. The beta-turn conformation in wheat
gluten proteins: relationship to gluten proteins. Cereal Chem 62:405442,
1985.
127. AS Tatham, PR Shewry. The conformation of wheat gluten proteins. The
secondary structures and thermal stabilities of a-, b-, g-, and o-gliadins. J
Cereal Sci 3:103113, 1985.
128. JM Devery, V Bender, I Penttila, JH Skerritt. Identication of reactive
synthetic gliadin peptides specic for celiac disease. Int Arch Allergy Appl
Immunol 95:356362, 1991.
129. HJ Ellis, AP Doyle, H Sieser, RP Sturgess, PJ Ciclitira. Specicity of
monoclonal antibodies to domain I of a-gliadins. Scand J Gastroenterol
28:212216, 1993.
130. S Tanabe, S Arai, Y Yanagihara, H Mita, K Takahashi, M Watanabe. A
major wheat allergen has a Gln-Gln-Gln-Pro-Pro motif identied as an IgEbinding epitope. Biochem Biophys Res Commun 219:290293, 1996.
131. A Tanabe, S Arai, M Watanabe. Modication of wheat our with bromelain
and baking hypoallergenic bread with added ingredients. Biosci Biotechnol
Biochem 60:12691272, 1996.
132. KH Choi, RA Laursen. Amino-acid sequence and glycan structures of
cysteine protease with proline specicity from ginger rhizome Zingiber
ofcinale. Eur J Biochem 267:15161526, 2000.
133. JH Skerritt, RA Smith. A sensitive monoclonal-antibody-based test for gluten
detection: studies with cooked or processed foods. J Sci Food Agric 36:980
986, 1985.
337
338
Chapter 11
339
12
Biological and Chemical Tests for
Protein Nutrient Value
1. INTRODUCTION
The quality of dietary protein should meet the physiological needs of the
consumer. Protein with a high nutrient value is digestible and provides a full
complement of the essential amino acids. Protein nutrient value (PNV)
raises issues beyond the normal terms of reference for chemical analysis. A
great deal of progress was made in this eld recently and a consensus was
reached about procedures for assessing protein quality.This chapter
considers PNV and its evaluation. Sec. 1 describes some of the most
important indicators of protein quality, factors affecting these indicators,
and an outline of the literature. Human bioassays for protein quality are
discussed in Sec. 2. Investigations using human subjects are slow and
expensive. Alternative tests involve rats, chicks, and other small animals
(Sec. 3). A number of chemical tests for protein quality are also available
(Secs. 4 and 5) that are fast and inexpensive but have yet to undergo
rigorous interlaboratory evaluation. The range of in vitro tests for protein
quality includes measurements of reactive lysine, dye binding, and the rate
of hydrolysis by proteolytic enzymes.
341
342
1.1.
Chapter 12
Indices of protein quality are listed in Table 1. The protein efciency ratio
(PER) and the net protein ratio (NPR) are determined noninvasively by
measuring the body weight and the amount of protein consumed. Another
group of noninvasive indices includes nitrogen balance (B), nitrogen balance
index (NBI), and biological value (BV). They are derived from the amount
of protein ingested (nitrogen intake, I) and excreted in the feces (fecal
nitrogen, F) or urine (urinary nitrogen, U). Finally, the net protein
utilization (NPU) and gross protein value (GPV) require the determination
of total body or carcass nitrogen.
The purpose of dietary protein is to provide amino acids for protein
synthesis. This process occurs at a higher rate in young, actively growing,
animals. Gender, physiological status, and natural differences between
individuals are also important (Table 2). There is some recycling of amino
acids as a consequence of the constant breakdown of old protein and its
transformation into new protein; indeed, the amount of protein resynthesized may exceed the daily intake. Amino acid recycling is not 100%
TABLE 1
Quality index
Protein efciency ratio (PER)
weight gained
PER protein
consumed
groupnonprotein group
NPR weight change protein
protein consumed
nonprotein group
NPU body N proteinNgroup
consumed
test protein
GPV NPU
NPU casein
TDC I
B I (F U)
NBI (B B0) / I
BV 100(B B0) / A
F F0
I
Letters with subscript 0 refer to parameters for a protein-free diet. For example, F0 fecal
nitrogen concentration for rats fed a nonprotein diet, also called metabolic nitrogen.
343
Sociological
Protein related
Processing history
Dietary
efcient. Over time there is a gradual net loss of body protein, which must be
recovered from the diet. A maintenance diet provides protein at a level that
is just sufcient to avoid a net loss of body mass. Excess dietary amino acids
are deaminated and then oxidized to supply energy. Such processes lead to a
complicated relationship between protein (nitrogen) intake and utilization.
The large number of variables affecting PNV makes it difcult to measure
accurately.
1.2.
344
Chapter 12
345
FIGURE 1 The effect of 5 days of storage on residual amino acid levels for a caseinglucose mixture or an egg albuminglucose mixture. Samples were stored
dry at 378C, pH 6.3 at an RH of 6870%. (Drawn from data from Refs. 1
and 2.)
346
2.
Chapter 12
44
347
FIGURE 2 An N balance curve for egg or whole wheat protein. Young men were
rst fed a protein-free diet (I 0) followed by a single diet (e.g., with
40 mg N/kg/day) or at multiple levels of nitrogen. The equation of the
straight line is B 0.5(I) 44 for egg protein and B 0.27(I) 35 for
whole wheat protein. Each data point is averaged from seven test
subjects.*
whether the nitrogen balance curves are linear or not. For completely linear
responses the ``I 0'' intercept is more likely to be independent of the type
of protein sample.
Protein quality can also be determined at a single level of N intake.
PNV is then calculated using two experimental points [e.g., I 0 and
I 40 g kg 1 (body weight) day 1]. This approach is prone to error if the N
balance plot is not a straight line. It is preferable to determine PNV by
feeding several diets with different levels of N intake. According to the slope
method, Eq. (1) parameters and associated PNV indices are determined
from several experimental points. From Table 3, the slope method is more
sensitive and shows clearly that the order of increasing quality is egg
protein > soy isolate > whole wheat protein. The level of protein required
for maintenance is also a sensitive index of PNV. A feeding trial using one
level of nitrogen failed to identify egg protein as possessing superior quality.
* Where B Nitrogen balance and I Nitrogen intake as protein (see Table 1).
348
Chapter 12
TABLE 3 Estimation of Protein Nutrient Value from N Balance Studies with Young
Adult Mena
Protein source,
levels of N
Single test
Egg
Soy isolate
Whole wheat
Slope method
Egg
Soy isolate
Whole wheat
N utilization efciency
(slope)*
N required for
maintenance (intercept)**
0.63
0.54
0.65
81
94
78
0.50
0.43
0.27
88
107
131
Protein quality was determined as the (*) the dimensionless slope or (**) intercept [mg N kg
(body weight) day 1] of the N balance curve.
3.
Before 1994 there was a statutory requirement in the United States and
Canada for food labeling information based on the PER. Protein quality is
now measured by a new technique described in Chapter 14. The classic PER
assay remains important. This is usually determined using 4-week-old rats.
The PER technique was thoroughly discussed in the proceedings from the
38th Annual Meeting of the Institute of Food Technologist Dallas, Texas
349
2:5PERSAMPLE
PERCASEIN
Values for cPER allow more reliable comparisons of assay results from
different laboratories. Bender and Doell (60) incorporated weight loss for
rats fed on a protein-free diet into the PER equation. The resulting index,
called the net protein ratio (NPR), is a measure of the protein requirement
for growth and maintenance. In contrast, PER measures only the protein
requirement for growth. Dividing the NPR by the corresponding value for a
standard protein (casein or lactalbumin) yields the relative NPR value
(RNPR). PER, cPER, NPR, and RNPR represent increasingly robust
indices for protein quality determined using the rat bioassay.
In Europe, the preferred rat bioassay involves the measurement of
carcass nitrogen. To determine net protein utilization (NPU), rats are fed
for 10 days on a protein-free diet. Another group of rats is fed the same
basal diet supplemented with 10% protein. Both groups of rats are sacriced
and their carcasses are dried at 1058C or dissolved in concentrated sulfuric
acid. Whole-body nitrogen is determined by Kjeldahl analysis or else
estimated from the known body moisture content. Rats have a xed
water/nitrogen ratio (61).
The PER, NPR, and NPU assays are single-point assays. These tests
involve a single level of N intake. A slope method or multiple-level tests
350
Chapter 12
involving diets with different levels of protein yield more reliable estimates
of quality. PER measurements based on multiple levels of N intake were
introduced by Hegsted and Chang (62) and Hegsted et al. (63). Feeding
multiple levels of nitrogen led to two slope measurements for protein quality
called the relative nutritive value (RNV) and the relative protein value
(RPV).
3.2.
Sources of Error
According to Bodwell (56), the chief merits of animal testing are their
convenience and low cost. According to this investigator,
351
3.4.
352
Chapter 12
indicators (Table 1). In vivo digestibility of the major food protein groups
has been reviewed by Hopkins (66) and Sarwar (67).
Method 1
An outline of AOAC guidelines for conducting a rat bioassay.
True protein digestibility was determined for six samples (casein,
canned macaroni and cheese, tuna sh in water, rolled oats, pinto beans,
and heat-treated milk powder).
Sample Pretreatment
1. Freeze dry food samples and (where necessary) grind to pass
through a 20-mesh screen.
2. Determine the proximate composition (e.g., % nitrogen, fat,
water, ber) for each sample.
3. Assess the nitrogen content for each sample by Kjeldhal analysis.
4. Formulate standard rat diets to provide equal levels of protein-N
(*1.6% or 10% true protein).
5. From the known proximate composition, normalize the level of
fat and ber in each diet.
Table 4 shows the composition of some of the diets used in AOAC
study (65). Notice that diet 1 is a nonprotein diet. Butylated hydroxyanisole
(BHA) was added as antioxidant. Dietary constituents were mixed
thoroughly using a mechanical mixer to avoid selection by rats. Male
Sprague-Dawley rats (5070 g) were acclimated in wire net cages (at 18
268C and 4070% relative humidity) for 2 days on normal rat food. Each
test diet was fed (15 g per day, water available ad libitum) to two groups of
four rats (i.e., eight rats per diet) for a total of 9 days. The rst 4 days was an
adaptation period. The next 5 days constituted the balance period, during
which the weight of food eaten was recorded. Rat fecal output was also
collected, dried, and stored for nitrogen determination. Nitrogen intake was
determined from the weight of feed consumed and the percent nitrogen
content of each diet. TPD was calculated from Eq. (3).
TPD
IP
FP FO;P
IP
where IP, FP, and FO,P are protein intake, fecal protein, and metabolic
protein. Eq. (3) is a specic form of the digestibility equation (see Table 1)
applied to dietary protein. Digestibility can also be dened in relation to
amino acids (Chapter 14).
353
Component
(g/100 g)
Casein
Macaroni and
cheese
Tuna sh
Rolled oats
Vitamins
Minerals
Corn oil
Cornstarch
Cellulose/ber
BHA
10.8
54.8
2.0
3.5
10.0
68.7
5.0
0.005
2.0
3.5
1.5
34.5
3.7
0.005
11.0
2.0
3.5
9.7
68.8
5.0
0.005
57.8
2.0
3.5
6.6
29.4
0.7
0.005
2.0
3.5
10.0
79.5
5.0
0.005
a
The plan conforms to the AOAC test diets for rat bioassays.
Source: Adapted from Refs. 65 and 121.
3.5.
Choppe and Kratzer (68) used White Plymouth Rock chicks to evaluate
PNV for meat and bone meal. Chicks were fed with normal starter mash
rations until 5 days old. They were then divided into groups of 10 and fed a
basal diet (Table 5) with or without a supplement of test sample. Sufcient
meat and bone meal were added to supply 24% of protein (Kjeldahl,
%N 6 6.25). The chicks were weighed at 5, 10, and 15 days old. Moran et al.
(69) used New Hampshire 6 Delaware chicks to evaluate the effect of
TABLE 5 An Example Basal Diet for Chick Feeding Trials
Basal diet
Cornstarch
Mineral mixture
Soybean oil (crude,
degummed)
Cellulose
Choline chloride
Vitamin supplements
Source: Adapted from Ref. 68.
Composition (%)
*75
2
5
4
12.5
2.16
354
Chapter 12
4.
4.1.
The use of amino acid data to determine PNV was suggested in 1946 by
Mitchell and Block (70). They are also credited with introducing the concept
of protein chemical score. To determine the chemical score, divide the
quantity of each amino acid from the test sample by the amount found in
egg protein or another reference protein [Eq. (4)]. Repeat the calculation for
each of the 910 essential amino acids (EAAs).
Chemical score
The chemical score is the smallest ratio found; this also identies the limiting
amino acid. Oser (71) introduced the integrated chemical score, which is the
geometric mean of the ``egg ratio'' for all essential amino acids. By the 1970s
the egg protein standard had fallen into disuse. Protein quality is now
determined in relation to the essential amino acid requirements for humans
(Table 6).
Amino acid scores (AASs) are estimated using the FAO/WHO/UNU
(1985) amino acid requirement pattern for 2- to 5-year-old preschool
children (Table 6). The calculation of AAS follows the same principles as
shown in Eq. (4). First, determine the essential amino acid prole for the test
sample. Next, divide the amount of each essential amino acid by the
corresponding value from the FAO/WHO/UNU (1985) list. The smallest
ratio (a) identies the limiting amino acid and (b) yields a numerical value
for the AAS. It is a relatively simple matter to calculate the AAS value for a
hypothetical mixture of foods. Some investigators have taken the sum of the
chemical scores for the nine essential amino acids (EAA9) as the index for
quality.
355
Infanta
mean
Infanta
range
25
years
1012
years
Adult
26
46
93
66
42
72
1836
4753
83107
5376
2960
68118
19
28
66
58
25
63
19
28
44
44
22
22
16
13
19
16
17
19
43
17
55
460
540
4045
1617
4477
398589
411602
34
11
35
339
661
28
9
25
241
759
9
5
13
127
873
1000
1000
1000
1000
1000
a
Mean and range of values found in human milk.
Source: From FAO/WHO/UNU (1985) [see Refs. 75, 76 of Chapter 14].
4.2.
Sources of Error
Errors in amino acid analysis (Chapter 1) will affect values of the AAS.
Ideally, the hydrolysis of proteins using 6 M HCl should produce efcient
release of all amino acids followed by accurate analysis by column
chromatography. In practice, the sulfur amino acids and tryptophan are
partially destroyed during acid hydrolysis. The release of valine and
isoleucine is dependent on the hydrolysis time. In order to obtain data for all
amino acids, multiple protein hydrolysis is necessary. The majority of amino
acids are analyzed after protein hydrolysis in 6 N HCl at 1108C for 24 hours.
The sulfur amino acids are rst oxidized by performic acid treatment to
cysteic acid or methionine sulfone. Tryptophan determination is performed
after alkaline hydrolysis.
Many criticisms of amino acid analysis appear to have been addressed
with the advent of more sophisticated equipment. The precision of amino
acid analysis was thought to be questionable in the past. However, recent
collaborative studies have shown a within-laboratory precision of about 3%.
The interlaboratory precision for assaying labile amino acids was 1617%.
356
Chapter 12
4.3.
Lysine is the rst limiting essential amino acid in cereals. The most reactive
essential amino acid also appears to be lysine (Fig. 1). Protein-bound lysine
residues with low chemical reactivity tend to be nutritionally unavailable.
Therefore available lysine is widely monitored as an index of protein quality
(Table 7).*
To measure lysine reactivity, the food sample is exposed to FDNB,
TNBS, MIU, or EVS at high pH for periods ranging from 2 hours (FDNB)
to 4 days (MIU). The chemically modied protein is hydrolyzed by reuxing
with 6 M HCl. The hydrolysate is analyzed by colorimetry to determine the
concentration of reactive lysine. The FDNB (difference) or EVS (difference)
methods involve conventional amino acid analysis. Chemically modied
TABLE 7
Reagent
1-Fluoro-2,4-dinitrobenzene (FDNB)
1-Fluoro-2,4-dinitrobenzene (FDNB)
(difference method)
Trinitrobenzenesulfonic acid (TNBS)
o-Methylisourea (MIU)
o-Phthalaldehyde (OPA)
Ethyl vinyl sulfone (EVS) (difference
method)
Differential dye-binding capacity
References
Sanger (72), Carpenter (73), Hurrell and
Carpenter (74), Booth (75)
Booth (75), Roach et al. (76), Ostrowski
et al. (77), Couch (78)
Kakade and Liener (79)
Hurrell and Carpenter (74)
Gondo et al. (80)
Friedman and Finley (81)
Sandler and Warren (82)
* Carpenter suggests that ``reactive lysine'' is a more accurate description. Lysine chemical
reactivity is not synonymous with nutritional availabilitysee Section 5.1. However, reactive
lysine is not an effective search keyword in the food science literature. We use ``available lysine''
to imply chemically reactive protein lysine groups.
357
lysine is eluted with a different retention time, allowing unmodied (socalled bound) lysine to be found.
4.4.
FDNB introduced by Sanger (72) is the most widely used reagent for
assaying available lysine. Lea and Hannan (1,83) used FDNB to monitor
changes in reactive lysine concentration for a casein-glucose mixture stored
in the dry state. They also followed changes in the reactive lysine content of
dry milk powder. Later, Carpenter and co-workers (73,74) from the School
of Agriculture, University of Cambridge (UK) extended the FDNB
procedure for protein-bound lysine in foods. In Method 2, the sample is
rst modied with FDNB and then hydrolyzed by heating with 6 M HCl.
The hydrolysate is extracted with diethyl ether and the absorbance reading is
recorded for the aqueous phase (Tube A). A regent blank (Tube B) is
prepared by neutralizing the hydrolysate, treating with an acylating agent,
and then extracting with diethyl ether. The reactive lysine concentration is
proportional to the absorbance of Tube A minus the absorbance for Tube
B.
Method 2
Determination of FDNB-reactive lysine (73,75).
Reagent
1. 1-Fluoro-2,4-dinitrobenzene (2.5% w/w in ethanol). Melt solid
FDNB by warming to about 408C and dispense using an
automatic pipette. About 12 mL of FDNB solution is required
per sample.
358
Chapter 12
359
Tube A (aq)a
Tube B (aq)
Tube B (eth)
0.203
0.090
0.002
0.002
0.089
0.007
0.201
0.008
0.138
a
Tube A (ether phase) discarded.
Source: Based on results from Ref. 73.
B (aq)
0.201
0.001
0.005
360
Chapter 12
4.6.
Protein source
Various including oilseeds
(cottonseed, sesame,
peanut, soybean), sh
meal, meat meal,
feather
Various including maize,
gluten, milk replacer,
soybean
Groundnut meal, arachin,
conarachin
Various including sh
meal, meat meal, milk
powder, wheat, maize,
soybean, sunower
meals
Casein, soybean, gluten,
zein, sh meal,
albumin, lysozyme
Column, comments
References
Amberlite IR-120
(*1 6 6 cm) wash
3 N HCl, eluent 3 N
HCl Me-Et-ketone
Matheson (89,90)
Roach et al. (76), Booth
(75), Ostrowski et al.
(77), Bailey (91)
361
products were detected at 436 nm. Two peaks for 2,4-DNP and e-DNP-Lys
were well separated with retention times of 10 and 12 minutes. Comparing
Carpenter's technique (Method 1) with the HPLC method showed that the
former gave signicantly higher lysine readings for high-carbohydrate
samples.
Rabasseda and co-workers (93) attempted to optimize the RP- HPLC
method using product detection at 274 nm. The LLD was 4.5 6 10 9 g of eDNP-Lys or 2 6 10 9 g for unmodied lysine. The precision of analysis was
0.95% for sh meal and 1.66% for soybean meal. Castillo and co-workers
(94) criticized the previous HPLC analysis for the arbitrary choice of assay
conditions (e.g., conditions for sample hydrolysis, choice of mobile phase
pH, choice of detector wavelength). The optimal RP-HPLC assay involved a
C18 column operated at 408C to avoid precipitation of DNP during analysis.
Peak elution was with 22:77 (v/v) acetonitrile0.01 M acetic acid (pH 5)
solvent at a ow rate of 2 mL min 1. DNP-protein was more efciently
hydrolyzed by treating solid samples with an excess of relatively dilute HCl
acid (1 mL 6 M HCl per 1 mg sample) as compared with highly concentrated
acid (1 mL of 10 M HCl per 1 mg sample). Apparently, e-DNP-Lys was
degraded by 10 M HCl. Analytical precision varied with sample type; the
CV ranged from 1.41 to 3.19% for enteral formula, milk, and lentil.
4.7.
The analysis of free amino acids and peptides using TNBS (2,4,6trinitrobenzene sulfonic acid) was introduced by Okuyama and co-workers
(96,97). TNBS reacts with primary amines in a manner similar to FDNB.
Habeeb (98) used TNBS to monitor protein-bound lysine under relatively
mild conditions. The BSA reaction with formaldehyde could be followed
using TNBS. Binding of SDS to lysine residues interfered with the TNBS
reaction. Mokrasch (99) used TNBS for the coestimation of amines, amino
acids, and proteins. Kakade and Liener (79) were rst to determine available
lysine using TNBS. The trinitrophenyl (TNP) protein derivatives can be
hydrolyzed by autoclaving with concentrated HCl for 60 minutes as
compared with a minimum hydrolysis time of 4 hours for DNP-proteins.
Analysis of trinitrophenylated products is by spectrophotometry (see
Method 2) or RP-HPLC (100).
Method 3
Determination of reactive lysine using TNBS (79).
Reagents
1. 2,4,6-Trinitrobenzene sulfonic acid (0.1% w/w solution in water).
362
Chapter 12
4.8.
Sandler and Warren (82) measured DBC before and after acylating protein
samples using ethyl chloroformate. The differential dye binding capacity
(dDBC) is the change in dye-binding capacity produced when lysine residues
are blocked by chemical modication. Hurrell et al. (106) used propionic
anhydride as an acylating agent and called their measurement the dyebinding lysine procedure. As described in Chapters 57, protein DBC is
determined by the net concentration of basic amino acids. Values for the
dDBC are directly proportional to lysine levels alone and therefore provide
a sensitive indicator of protein quality. The dDBC procedure is summarized
next.
363
Method 4
Determination of differential dye-binding capacity using propionic anhydride (106,107).
Reagents
1. Propionic anhydride
2. As listed for the Udy method (Method 1, Chapter 5)
3. Sodium acetate (0.2 M)
Procedure
Dye-binding capacity. To 20500 mg of ground sample (*2090%
w/w protein) in a 100-mL ask, add three 6-mm glass beads and
2 mL of sodium acetate (0.2 M). Shake for 10 minutes to wet. Add
40 mL of dye solution and shake for a further 60 minutes. Filter the
sample, dilute the supernatant as needed, and read its absorbency.
Determine the DBC (mg g 1 protein) from a dye calibration graph.
DBC for a chemically modied sample. Weigh 20500 mg of sample
and wet with sodium acetate as above. Add 0.4 mL of propionic
anhydride, mix, and allow to react for 1015 minutes. Add 40 mL of
dye solution and treat as before.
Calculate the dDBC as the difference between DBC values for the
modied and unmodied protein.
Protein acylation with propionic anhydride is straightforward and
quantitative. The procedure for determining dDBC is no more protracted
than a conventional dye-binding assay. The lysine content of 24 different
varieties of grain legumes was rapidly determined by the dDBC method.
Hurrell et al. (106) found that the dDBC method had potential for
monitoring plant breeding. The technique was successfully applied for
monitoring processing damage. Values for dDBC (mmoles of dye bound per
16 g N) are numerically equal to the concentration of total lysine (mmoles
per 16 g N) determined by amino acid analysis or using the FDNP technique
(Fig. 3). The dDBC measurements are free from interference by starch, lipid,
or free lysine (up to 30% w/v).
Sandler and Warren (82) found that carbethoxylation of sh meal
required 3060 minutes, with 40 minutes being the least time needed to
ensure complete reaction. The rate of dye binding was slow at 208C
although equilibrium is reached in about 20 minutes at 408C. There was
good agreement between values for DBC and the total basic amino acid
concentration (TBAA) and also between dDBC and lysine values measured
by amino acid analysis (Table 10). Pearl and co-workers (107) determined
the dDBC values for soybean meal using Method 3 with minor
modications. The amount of Orange G dye bound to soybean protein
364
Chapter 12
FIGURE 3 Relationship between the total lysine and the differential dye-binding
capacity (dDBC) for a range of commodities: bovine serum albumin,
skim milk powder, sh meal, pea our, soya our, meat and bone meal,
barley, peanut our, corn gluten, as well as a variety of legumes (winged
bean, pea, green gram, chickpea, lentil, cowpea, dry bean, soybean,
runner bean, and pigeon pea). The equation of the line Y mX C,
where m 1.0328 (+0.013), C 0.071 (+0.3810). R2 0.9967. For
statistical testing, F 6287.38 with 21 degrees of freedom.
TBAA
(mmoles)
DBC
(mmoles)
Lys*
(mmoles)
dDBC
(mmoles)
Fish-a
Fish-b
Soya
Cashew
79 (+8.6)
74 (+8.1)
57.7
47.0
87
73.6
59.4
52.1
43 (+4.7)
40 (+4.4)
24.1
13.7
44 (+4.8)
38 (+3.6)
36.4
17.6
a
TBAA, total basic amino acids, i.e., Lys His Arg concentration (mmole/120 mg of sample);
DBC, dye-binding capacity (mmole dye bound/120 mg sample); Lys, results from amino acid
analysis (mmole/120 mg sample); dDBC, differential dye-binding capacity. Samples were sh
meal (sh-a), defatted sh meal (sh-b), soya meal (soya), or cashew nut meal (cashew).
365
was determined before and after acylation with propionic anhydride. Slight
changes in protein-bound lysine due to thermal treatment were detected.
These results (82,106,107) conrm that the dDBC method is a highly
affordable alternative to amino acid analysis when one wants to measure
lysine levels.
4.9.
One advantage of the TNBS method (compared with the FDNB technique)
is the shorter time for protein hydrolysis. The solvent extraction steps for
both techniques reduce interference. Hurrell and Carpenter (74) compared
FDNB, TNBS, and other reagents for reactive lysine determination in
model systems comprising ovalbumin, lactalbumin, and glucose (3:2:5
weight ratio).* Samples were stored at 378C for up to 30 days or autoclaved
at 1218C for 15 minutes. The reacted protein-sugar mixtures underwent
large changes in the ability to sustain growth in chicks or rats.
The MIU and TNBS methods underestimate total lysine levels for the
unheated (control) sample (Table 11). The ability to detect mild levels of
damage (378C, 30 days storage) also varies for different techniques. Amino
acid analysis and FDNP and TNBS methods did not accurately measure
TABLE 11 Total Lysine and FDNB-Reactive Lysine in Ovalbumin, Lactalbumin,
and Glucose Mixture by Different Methods
Lysine (mg/g protein)
Methoda
Total Lys
Total Lys (NaBH4)
FDNB (diff)
FDNB (dir)
TNBS
MIU
Control
378C, 30 d
1218C, 15 min
86.3
81.1
84.7
81.6
53.6b
71.9b
50.9
12.2
45.7
19.6
35.9
15.8
29.3
18.7
19.5
12.2
11.2
6.5
Total lysine, amino acid analysis. Total Lys (NaBH4), amino acid analysis after sample
reduction by sodium borohydride. Other abbreviations are dened in the text.
b
Results are signicantly different from the control.
Source: Summarized from Ref. 74.
* Lactalbumin is spray dried whey protein. It has low solubility owing to heat damage by spray
drying.
366
Chapter 12
mild heat damage. The errors were ascribed to two factors: (a) products of
early Maillard reactions are not acid stable and some glycosylated lysine
may be degraded to free lysine during protein hydrolysis in 6 M HCl, and (b)
early Maillard reaction products may react with TNBS or FDNB. This may
be because the nucleophilic character of the lysine derivatives formed during
the early Maillard reaction is similar to that for unmodied lysine. Both
errors can be ameliorated by the addition of sodium borohydride
(74,101,108). The Schiff base formed between lysine e-NH2 and sugars is
rendered acid stable by treatment with sodium borohydride. Without this
precaution, available lysine assays involving acid hydrolysis at high
temperatures will be subject to error.
By comparison with Method 1, the chromatographic detection of eDNP-Lys leads to some of the following improvements:
1. Reduction of interference from other yellow substances formed
during protein chemical modication (e.g., dinitrophenol and oDNP tyrosine). Plants also contain brown humin and other dyes
that can act as interferences.
2. Ability to measure free lysine and N-terminal lysine residues in
one experiment.
3. Decreased error in the case of high-carbohydrate samples. The
color yield for Method 1 can be impaired when e-DNP-Lys is
reduced to the diaminophenol derivative. Alternatively, carbohydrate may react with FDNB to generate water-soluble derivatives
that interfere with the analysis of e-DNP-Lys.
4. Absence of sample pretreatment. Protein hydrolysate can be
subjected to chromatographic analysis directly without an ether
extraction step.
5. High sample throughput.
5.
PROTEIN DIGESTIBILITY
367
5.1.
Denition of Terms
Term
Equationa
Apparent protein
digestibility (APD)
True protein digestibility
(TPD)
Availability/bioavailability
(I
True adsorption
Retention (R)
Nitrogen balance (B)
(I
(I
(F
F)/I
F0))/I
(I (F F0))/I or
(F F0) (U U0)/I
I (F F0)
I (F U)
I (F U)
Expressed in terms of
retention
(R U)/I
(R U F0)/I
(R F0 U0)/I
a
I, intake; F, total fecal excretion; R, retention; U, total urinary excretion. Subscript 0 refers to
metabolic values determined with a protein-free diet.
* These relations also apply to dietary nitrogen, which is assumed to be proportional to crude
protein (N 6 6.25). See Chapter 1 for a more detailed description of Kjeldahl analysis.
368
Chapter 12
subjects or rats. Dietary protein levels probably do not affect the amount of
protein-N secreted via healthy kidneys and hence U U0 is close to zero.
For amino acids and other low-molecular-weight nutrients, ignoring urinary
losses may lead to signicant error (109). The importance of ``digestibility''
can be seen by comparing relations in Tables 1 and 12; notice that
parameters such as NBI and BV (Table 3) also feature digestibility.
Digestibility can be measured for specic amino acids and is called
amino acid availability (AAAv), amino acid digestibility, or simply
availability. The three terms are used interchangeably. The AAAv (unlike
TPD) allows for different adsorption rates for specic amino acids.
AAAv
IAA
FAA FO;AA
IAA
5.2.
369
The food sample is exposed to one or more proteases and the degree of
proteolysis is monitored. Single enzymes or combinations of enzymes can be
used including pepsin, chymotrypsin, trypsin, papain, and peptidase. The
degree of protein hydrolysis is generally measured as TCA-soluble products
at 275280 nm, by following the fall in system pH, or by monitoring the
release of free amino groups using TNBS. Techniques for monitoring
protein digestibility were reviewed by Swaisgood and Catignani (115,116)
and by Boisen and Eggum (117). The AOAC-approved test for in vitro
protein digestibility is based on the three-enzyme method of Hsu et al. (118)
or the four-enzyme approach (119,120). A pH meter or pH-Stat instrument
is used to monitor these multienzyme reactions (see Method 5).
Method 5
Determination of in vitro protein digestibility using three or four enzymes
(118120).
Reagents
1. Porcine pancreatic trypsin (14,190 units per mg)
2. Bovine pancreatic a-chymotrypsin (60 BAEE units per mg)
3. Porcine intestinal peptidase (40 units per g powder)
4. Pronase
5. Dilute (0.1 M) hydrochloric acid and sodium hydroxide
Enzyme solution A. Dissolve trypsin (16 mg), chymotrypsin (31 mg),
and peptidase (13 mg) in distilled water at 378C. Adjust to pH 8
using dilute HCl or NaOH and bring to a nal volume of 10 mL.
Enzyme solution B. Dissolve 65 units of pronase in distilled water at
378C. Adjust to pH 8 using dilute HCl or NaOH and bring to a nal
volume of 10 mL. Store the enzyme solution in ice.
Procedure*
Determination of in vitro protein digestibility via a three-enzyme test.
Add the food sample (62.5 mg or equivalent to 10 mg N) to about 6
8 mL of distilled water and soak for 60 minutes at 378C. Adjust to
pH 8 using dilute HCl or NaOH and bring to a nal volume of
* Pronase (enzyme solution B) is not needed in the three-enzyme method.
370
Chapter 12
APD% 234:84
Fig. 4 shows the relation between in vitro protein digestibility and in vivo
results for over 20 processed and unprocessed food protein sources. The
right-hand axis in Fig. 4 shows the residuals for pH (10 min) results, i.e.,
difference between in vitro and in vivo test results. These is no systematic
trend in the residuals. In vitro protein digestibility estimates using the
multienzyme method were not affected by the buffer capacity of normal
food samples.
In a collaborative test for the three-enzyme method, pH was
monitored with a pH-Stat instrument (121).* For sample pretreatment
fresh or canned foods were freeze-dried and ground into a powder. Highlipid foods were defatted by extracting with anhydrous cold ether. In vitro
protein digestibility was examined essentially as described in Method 4 with
minor modications. Reaction progress was monitored from the volume of
0.1 N NaOH added to the reaction vessel by the pH-Stat set at pH 8. In
vitro protein digestibility{ was calculated from
IVPD% 79:28 40:74V mL
* The test using a pH-Stat is AOAC approved. Participants in the interlaboratory tests were
afliated with the USDA (Beltsville) Health and Welfare Canada (Ottawa), Protein
Technologies International (St. Louis, MO), CIVO-TNO Food Analysis InstituteAJ (Zeist,
The Netherlands), Kraft Inc. (Glenview, IL), National Institute of Animal Science (Tjele,
Denmark).
{ The term in vitro protein digestibility has been substituted for ``percent true digestibility'' in
the original reference to avoid confusion with true protein digestibility (TPD).
371
where IVPD stands for in vitro protein digestibility and V (mL) is the
volume of alkali added to the enzyme reaction over a 5-minute period. In
vitro protein digestibility values were adjusted according to a scale with
casein assigned an arbitrary value of 100. From 204 independent analyses
(17 samples* 6 2 replicates 6 6 laboratories) the between-laboratory reproducibility was 0.835.0%. The within-laboratory repeatability ranged
from 0.35 to 1.4%. The multienzyme in vitro protein digestibility method
allows a precise and rapid assessment of digestibility. The correlation
between TPD and in vitro protein digestibility implies that protein
* The 17 protein sources examined were casein, nonfat dried milk, nonfat dried milkheated,
tuna sh, salami, canned sausage, chicken frankfurters, beef stew, macaroni and cheese, peanut
butter, soy isolate, pea concentrate, chick peas, rolled oats, pinto beans, whole wheat cereal, and
rice-wheat gluten cereal.
372
Chapter 12
5.4.
5.5.
100h
hTOT
The htot is also equal to the net concentration of amino acids (mmoles) per
gram of protein. The DH was determined using a pH-Stat instrument to
measure the volume of alkali required to maintain a static pH value during
proteolysis (V):
DH%
VN b
a MhTOT
10
10pH pK
1 10pH pK
11
373
and
pK 7:8
298 T
2400
298:T
12
Under standard conditions used for the approved test (pH 8, T 378C
and pK 6.91) we may suppose that a & 0.91. For a wide range of proteins
the typical value for htot is about 89 meq g 1 protein (122), that is, htot
1/F where F (&112 g mole 1) is the average formula weight for all 20
naturally occurring amino acids (see Chapter 1).
5.6.
N dialysate
6100
N dialysate N dialysis bag
13
374
Chapter 12
attempts to correct amino acid scores for digestibility are discussed further
in Chapter 14.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
375
376
Chapter 12
377
378
Chapter 12
379
380
Chapter 12
113. NR Eldred, G Rodney. The effect of proteolytic enzymes on raw and heated
casein. J Biol Chem 162:261265, 1946.
114. D Melnick, BL Oser. The inuence of heat-processing on the function and
nutritive properties of protein. Food Res 3(2):5771, 1949.
115. HE Swaisgood, GL Catignani. In vitro measurement of effects of processing
on protein nutritional quality. J Food Prot 45:12481256, 1982.
116. HE Swaisgood, GL Catignani. Protein digestibility: in vitro methods of
assessment. Adv Food Nutr Res 35:185236, 1982.
117. S Boisen, BO Eggum. Critical evaluation of in vitro methods for estimating
digestibility in simple-stomach animals. Nutr Res Rev 4:141162, 1991.
118. HW Hsu, DL Vavak, LD Satterlee, GA Miller. A multienzyme technique for
estimating protein digestibility. J Food Sci 42:12691273, 1977.
119. LD Satterlee, HF Marshall, JM Temmyson. Measuring protein quality. J Am
Oil Chem Soc 56:103106, 1976.
120. LD Satterlee, JG Kendrick, HF Marshall, DK Jewell, RA Ali, MM Heckman,
F Steinke, P Larson, RD Philips, G Sarwar, P Slump. In vitro assay for
predicting protein efciency ratio as measured by rat bioassay: collaborative
study. J Assoc Off Anal Chem 65:798806, 1982.
121. FE McDonough, G Sarwar, FH Steinke, P Slump, S Garcia, S Boisen. In vitro
assay for protein digestibility: inter-laboratory study. J Assoc Off Anal Chem
73:622625, 1990.
122. J Adler-Nissen. Enzymic Hydrolysis of Food Proteins. London: Elsevier
Applied Science, 1986, chapters 2 and 6.
123. J Mauron, F Mottu, E Bujard, RH Egli. The availability of lysine, methionine
and tryptophan in condensed milk and milk powder. In vitro digestion studies.
Arch Biochem Biophys 59:433451, 1955.
124. SF Gauthier, C Vachon, JD Jones, L Savoie. Assessment of protein
digestibility by in vitro enzymatic hydrolysis with simultaneous dialysis. J
Nutr 112:17181725, 1982.
125. L Savoie, SF Gauthier. Dialysis cell for the in vitro measurement of protein
digestibility. J Food Sci 51:496498, 1982.
126. JF Kennedy, RJ Noy, JA Stead, CA White. A new rapid enzyme digestion
method for predicting in vitro protein quality (PDD index). Food Chem
32:277295, 1989.
13
Effect of Processing on Protein
Nutrient Value
1. INTRODUCTION
Chemical and biological techniques for determining protein nutrient value
(PNV) are described in Chapter 12. The effect of food processing on PNV
for specic food commodities is discussed here. Up until 19931994 the
protein effeciency ratio (PER) assay was the reference method for assessing
protein quality (1). It has been superseded by a new measure of protein
quality called protein digestibilitycorrected amino acid score, PDCAAS
(Chapter 14). The PER remains important for comparative purposes and is
still widely used. The present discussion is broadly arranged according to
different food commodity groups.
382
Chapter 13
boiling (30 seconds), or UHT treatment (1351508C for a few seconds) did
not reduce the PER or digestibility determined using the rat bioassay (7).
From the standpoint of protein quality, there is no nutritional reason for
discouraging heat processing of milk for human consumption. The total
lysine content of milk ranges from 6.2 to 11.1 g 100 g 1 (protein). Roller
drying milk that had been preprocessed by UHT treatment produced 2.5
6.4% reduction in available lysine (8). Scorching during roller drying
produced up to 35% loss of lysine (6), whereas storing dried milk for 6
months produced 15.4% decline in the available lysine (9).
2.1.
The effect of prolonged storage on the PNV of freeze-dried versus spraydried lactose-hydrolyzed milk powder was examined by Rawson and
* Measures of protein quality are dened in Chapter 12.
383
FIGURE 1 Effect of processing on milk protein quality. Y-axis values are compared
with freeze-dried normal milk powder as control. X-axis abbreviations:
N-SD, spray-dried normal milk; LH SD, lactose-hydrolyzed and spray
dried; LH EV SD, lactose hydrolyzedevaporatedspray dried;
LH SD ( Lys), lactose hydrolyzed and spray dried with 1% (w/w)
lysine added. Insert shows TDC, total digestibility; BV, biological value;
NPU, net protein utilization determined with a rat bioassay. (Data from
Ref. 10.)
384
Chapter 13
FDNB-Lys
(mg g 1)
DBCb
(mg g 1)
Total Lys
(mg g 1)
Rat assay
NPR
28
25
25
12
23
23
17
218
178
200
120
195
235
150
29.1
28.9
29.1
15.7
23.4
23.6
17.8
4.8
3.0
4.0
3.2
a
Processing and storage conditions. bDye-binding capacity determined using Remazo Brilliant
Blue R as described in Ref. 11.
were then stored for 112 months. Protein quality was assessed using the rat
bioassays. Available lysine was assayed using the o-methylisourea method
(13). The loss of available lysine was greater for lactose-hydrolyzed milk
powder compared with normal milk powder. Chemical and biological assays
of available lysine were highly correlated. There was a bell-shaped
relationship between AW and quality loss. Maximum losses of PNV
occurred at AW 0.55. These observations agree with results for the dry
caseinate-glucose system (3). Lea and Hannan (3) investigated the reaction
between dry sodium caseinate and glucose at temperatures of 0708C and
AW < 0.95. The system was adjusted to pH 310 before drying. The decline
in free amino groups after 65 days storage at 378C, followed a bell-shaped
curve with a maximum at AW 0.60.8. At low AW the reaction rate may be
limited by the restricted diffusion of reagents. At high AW reactant dilution
or inhibition by water accounts for the decreased rate of deterioration. The
loss of lysine increased steadily from pH 3 to pH 8 and leveled off at pH 8
10. The effect of temperature (AW 0.75) on the kinetics of lysine loss
conformed with the simple Arrhenius equation ln(k) 14446.7(1/T) 38.0
(r 0.9999), where k is the percent loss of lysine per second and
T temperature in kelvin (Fig. 2). Alternative models involving nonlinear
temperature dependence are described in Chapter 14.
3.
INFANT FORMULAS
Mitchell and Grundel (14) compared several chemical methods (total lysine
determination by amino acid analysis, differential dye-binding capacity, in
FIGURE 2
385
Arrhenius plot showing the effect of temperature on the rate of Lys loss
for casein-glucose mixture at 70% RH. The temperature range studied
was 0708C. Reaction pH (not given) is probably pH 6.3 or near the
natural pH for dried milk. (Drawn from data in Refs. 35.)
vitro protein digestibility) and bioassays (TPD, PER) for evaluating protein
quality for ve infant formulas.* These protein quality indices were
evaluated for their sensitivity to ``humidity-related'' damage. For all ve
infant formulas, in vitro protein digestibility was said to give a poor estimate
of TPD. For example, the in vitro digestibility was was 8889% for soybased infant formulas compared with the TPD of 9194%. For milk-based
formulas, the in vitro protein digestibility was 8588% compared with a
preadjusted TPD of 8588%; the latter TPD value was adjusted to 9697%
to allow for lactose intolerance in rats. Apparently there is a digestibility
decit of 11% when rats are fed diets with lactose levels comparable to the
concentration of lactose in infant formulas. The dDBC values agreed with
total lysine results. The rat bioassay showed no protein quality changes for
30-day-old infant formulas; however, there were large drops for in vitro
* The designation and composition of the ve commercial infant formulas were: milk-based I
(skimmed milk, reduced minerals, whey, lactose, oleo, coconut, soy, oleic oils, and lecithin),
milk-based II (skimmed milk, lactose, coconut and corn oils), milk-based III (skimmed milk,
lactose, soy and coconut oils), soy-based I (soy protein isolate, corn syrup solids, coconut and
corn oils), and soy-based II (soy protein isolate, soy oil, lecithin, sucrose, tapioca dextrin).
386
Chapter 13
protein digestibility and dDBC. Mitchell and Grundel (14) concluded that
(a) agreement among the various assays is poor although different in vitro
methods show qualitative similarities in some cases, (b) changes in dDBC
and browning of the stored milk-based and soy-based formulas do not
reect changes in relative PER, (c) in vitro methods are not accurate
predictors of protein quality for the rat, and (d) only certain humidityrelated protein nutritional damage in infant formulas could be predicted by
monitoring dDBC or in vitro protein digestibility (14). An interesting
feature of this study is the assumption that lysine is the limiting amino acid
for soy-based infant formulas (see later).
Sarwar et al. (15) evaluated the impact of amino acid supplementation
on protein quality for four commercial soy-based infant formulas. The
samples were fed to 2-week-old weanling rats as the sole source of protein.
Each diet contained net 8% protein, 20% fat, and adequate amounts of
minerals and vitamins. The RPER and RNPR for casein plus methionine
were assigned a value of 100. By comparison, the RPER and RNPR values
for the infant formulas were 7181 and 7885, respectively. There was no
improvement in RPER or RNPR values when diets were supplemented with
either lysine (0.2%), methionine (0.2%), threonine (0.1%), or tryptophan
(0.05%). Increased levels of amino acids appeared in the serum, indicating
efcient adsorption. Protein quality improved following the supplementation with four essential amino acids (RPER or RNPR values 100).
Apparently several amino acids are colimiting for soy-based formulas.
Measuring available lysine is a useful way to monitor protein quality where
it has been demonstated that lysine is the (only) limiting amino acid.
Otherwise, focusing on a single amino acid will lead to errors of the type
described in the preceding paragraph. Processing losses and shelf-life studies
for infant formulas have also been reported by other investigators (16,17).
Under optimal conditions, infant formulas can be stored for prolonged
periods (1620 weeks) without losses of available lysine. Poor storage
conditions can lead to a 2030% loss of available lysine.
4.
Protein concentrates and dietary supplements for livestock are derived from
both animal and plant sources (Table 2). The commercial value for feed
concentrates depends on their total nitrogen content as determined by the
Kjeldahl analysis. Differences in feed protein quality arise due to (a)
intrinsic differences in the quality of raw materials and (b) effects of
processing. Heat processing renders feed material more portable and
palatable. It also improves the chemical and microbiological stability of
387
388
Chapter 13
trates.* A range of chemical methods were compared with the chick and rat
bioassays for protein quality. Techniques of interest had to be simple
enough to be performed in the average analytical laboratory. They also had
to be rapid, allowing (a) on-line monitoring of quality during feedstuff
production and (b) quality assurance at storage or shipment depots.
4.1.
Table 2 lists some of the chemical methods for assessing feed protein quality
examined in the ARC study. The tests were compared with a chick bioassay
for gross protein value (GPV) and the rat bioassay for NPU. The test
samples were protein feedstuffs commercially important for feeding
nonruminant livestock. To foster cooperation from participants, the names
of feed manufacturers and sources of feed materials were not recorded.
About 130 samples covering seven classes of feedstuffs were investigated.
Key ndings of this study were that (a) rankings of feed protein quality
based on the GPV and NPU tests did not agree with each other except in the
case of whale meat meal; (b) when all samples taken together there was no
correlation between GPV and the crude protein, nitrogen solubility, or
Orange G binding; and (c) no chemical method was positively correlated
with feeding trials for all protein concentrates.
However, available lysine showed a positive correlation with the GPV
test for meat and bone meals (R 0.82), sh meal (R 0.96), and whale
meat meal (0.94). Orange G binding was a useful index for quality, being
correlated with available lysine. With oilseed protein concentrates, GPV was
correlated with the nitrogen solubility index. No chemical test showed any
correlation with NPU or GP. The presence of antinutritional factors
(trypsin inhibitors in groundnuts and soybean, gossypol in cottonseed) may
be partly to blame, as was bacterial contamination of feedstuffs. In applying
PNV testing to practical situations, the ARC group referred to many issues
summarized in Table 2 of Chapter 12. For example, protein meals are
usually fed as compound rations containing different protein types.
Livestock fed with these materials differed in terms of their variety, strain,
* The participants include J. Bibby & Sons Ltd., Liverpool (UK); Bovril Ltd., London (UK);
Croseld & Calthrop Ltd., Liverpool (UK); Department of the Government Chemist, London
(UK); Glaxo Laboratories Ltd., Greenford (UK); National Institute for Medical Research
(UK); National Institute for Research in Dairying, Shineld (UK); Rowett Research Institute,
Aberdeen (UK); Tory Research Station, Aberdeen (UK); Unilever Ltd., Shanbrook (UK);
University of Cambridge School of Agriculture (UK); U.S. Department of Agriculture
Southern Utilization Research Division, New Orleans; Walton Oaks Experimental Station,
Vitamins Ltd., Tadworth (UK).
389
and age; older pigs or chickens were less sensitive to low-quality protein.
Chemical methods for assessing the quality of protein concentrates/feeds
were also assessed by Bunyan and Price (19). The meat meal (26 batches),
whale meat meal (16 batches), and sh meal (13 batches) samples were
similar to those described for the ARC collaborative study. However, this
study considered only animal-derived protein concentrates. Results for each
class of protein concentrate were considered separately (Table 3).
Orange G binding capacity was a useful quality indicator for most
protein concentrates. FDNB-reactive lysine was not correlated with
bioassay results for samples other than whale meat meal. Three meat
meal samples with unusually high crude protein content (N 6 6.25) had
somewhat anomalous properties. For a crude protein content > 79% the
true protein value was only 2128%. At least one of the samples appeared to
be adulterated with feather meal judging from the low methionine content
and high amounts of gelatin. The NPR value was 0.42.2 for most meat
meals with a value of 1.6 signifying weight loss during the rat bioassay.
4.2.
Orange G Binding
Meat meal
(n 26)
Fish meal
(n 13)
4090
938
7090
5.47.8
3.04.6
0.92.6
2595
1762
7588
3469
4.07.3
1.62.6
6073
2266
7490
2780
3.46.9
1.32.5
Y1 0.278Db 30
Y1 0.261Db 30
Y2 0.239Db 27
Y3 3.86Db0.43
None
Y2 0.287D 17
Y3 6.37Db19.8
BV 7.52X1 4.0
0.325
Db 24
None
a
Y1 crude protein, Y2 true protein, Y3 NPU, Db amount of dye bound (acid equivalents
of dye bound/104 grams of protein), X1 reactive Lys.
390
Chapter 13
4.3.
391
These relations were generated from data in Table 5 of Ref. 26. The ranges
of values used were 2.545.9 g (lysine) 100 g 1 protein, 2.432.74 g (histidine)
100 g 1 protein, and 2.985.65 g (arginine) 100 g 1 protein. The total basic
amino acid range is therefore 7.9514.3 g per 100 g 1 protein. The regression
coefcients for Eq. (1), Eq. (2), and Eq. (3) were 0.9201, 0.9533, and 0.9532,
respectively. The nonzero intercept from Eq. (1) suggests that Acid Orange
12 binds to sites other than lysine. Eq. (2) conrms that the DBC is strongly
392
Chapter 13
correlated with TBAA and that Acid Orange 12 binds no sites other than the
three basic amino acids. Eq. (3) conrms the previous results. The coefcient
for arginine, histidine, and lysine shows their relative contributions to DBC.
Apparently, the order of dye binding to basic amino acids is histidine
> lysine 4 arginine. Nevertheless, heating produced the greatest decline in
lysine with histidine being the least heat susceptible.
The extent of heat damage to rapeseed meal is also a function of the
sample moisture content (Fig. 4). The DBC was reduced to a greater extent
by heating rapeseed meal at 1020% moisture as compared with 2% or 40%
moisture. Reactions producing quality loss are limited by lack of reagent
mobility in low-moisture systems and by dilution effects in high-moisture
systems. Changes in available lysine show a more complex dependence on
moisture levels. With prolonged heating, losses in available lysine were
higher for samples having 2% moisture as compared with 40%.
The assessment protein heat damage from DBC is described further by
Hook (28,29), Peal et al. (30), Randall et al. (31), and also by Kratzer et al.
(32). Protein samples investigated include wheat, soybean, defatted milk,
whole egg, and whole blood proteins. In general, changes in DBC tended to
FIGURE 4
393
lag behind PER. Quality loss was strongly correlated with decreases in
available lysine. Lin and Lakin (33) reported disparities between the DBC
and FDNB-reactive lysine for heated soy meal samples. Steaming soy meal
at atmospheric pressure led to the progressive loss of the nitrogen solubility
index (NSI) due to protein denaturation and insolubilization by covalent
(sulfur-disulde exchange) and noncovalent aggregation. Urease activity
decreased because of enzyme inactivation after 6080 minutes of heating. In
vitro protein digestibility increased, probably due to the inactivation of
soybean trypsin inhibitors. The level of unreactive lysine increased gradually
from 0.14 g (lysine) g 1 protein and leveled off at 0.26 g (lysine) g 1 protein
after heating of soybean meal for 120 minutes. Assuming an initial lysine
content of 6.3 g per 100 g, then 9598% of lysine residues remained available
after heating. Steam treatment led to an increase in DBC, probably due to
heat denaturation of soy protein.
4.4.
Meat and bone meal samples had high levels of gelatin measured as the
amount of hot watersoluble protein. There was a considerable difference in
the PNV for individual samples owing to their varied thermal history. For
20 different samples, Choppe and Kratzer (21) found a strong (negative)
correlation between the amount of hot watersoluble bone meal gelatin and
PNV. El (34) suggested a regression equation for predicting PER values for
meat or sh based on the collagen content. Calculated PER values agreed
closely with experimental values for sardine, lamb, bovine liver, chicken
meat, or beef sausages. Collagen content may provide a rapid and
inexpensive assay for estimating PNV for meat.
5. LEGUMES AND OILSEEDS
The structure and characteristics of legume proteins and effect of processing
on their quality were reviewed by Vanderstoep (35), Chang and Satterlee
(36), Sathe et al. (37), Friedman (38), and de Lumen and Uchimiya (39).
Common processing operations for legumes include baking/roasting,
dehulling, cooking, canning, extrusion cooking, fermentation, germination,
and hydrothermal treatment. A list of some legumes for which the protein
quality has been investigated over the last decade is given in Table 4. Most
of these processes improve the quality of legume proteins through the
reduction of protein antinutritional factors (trypsin inhibitors, hemagglutinins/lectins) and chemical antinutrients (oxalates, phytic acid, and
tannins).
394
Chapter 13
5.1.
References
Ortega-Nieblas et al. (40)
Available Lysine
395
396
Chapter 13
5.4.
5.5.
397
Weaning foods are produced from mixtures of cereal and legumes (and
occasionally milk). The multiple protein sources provide complementary
supplies of essential amino acids cysteine/methionine and lysine. A recent
emphasis is on the production of low-cost weaning foods using materials
locally available in developing countries. Gupta and Sehgal (61), Dahiya
and Kapoor (62), Gahlawat and Sehgal (63) from Haryana University
(Hisar State, India) describe a number of weaning food formulations based
on local cereals and legumes including wheat, pearl millet (bajra), Bengal
gram, green gram (mung beans), groundnuts, peal millet, rice, kangini
(Setaria italica), and sanwak (Echinochloa frumentacea).
Formulations containing two or three components were generally
subjected to a range of processing techniques including sprouting, roasting,
and malting. For commodities such as bajra (peal millet), barley, green
gram, amaranth grain (Amaranthus sp.), and jaggery, malting and/or
roasting led to protein quality indices comparable to those for a commercial
weaning food; PER 2.042.13, BV 79.5680.68, NPU 66.7567.86,
NPR 2.132.76, and PRE (protein retention efciency) 34.1844.18.
Dahiya and Kapoor (62) produced food supplements for preschool children
using malted and/or roasted bajra, Bengal gram, green gram (mung beans),
groundnuts, jaggery, or amaranth leaves. Bajra-based food supplements had
quality indices (PER, food efciency ratio, BV, NPU, NPR, and PER)
signicantly higher than those of wheat-based supplements (P < 0.05). The
authors suggest that the quality of their formulations was equal to that of
Cerelac2, a commercial supplement. However, PNV was found to be lower
(P < 0.05) than the value for casein (standard protein). Rats fed on bajrabased supplements showed an excellent growth pattern throughout the
feeding trial.
Santos et al. (69) prepared extruded weaning foods using a mixture of
rice, mung bean, and milk (70:25:5). Protein quality was determined by a rat
bioassay. The quality of the weaning food was signicantly improved if rice
and mung bean were extruded rst before milk was added. The optimal PER
was 2.25, which is comparable to the growth-promoting effect of casein.
Extrusion cooking the complete mixture led to a PER value of 1.93.
Supplementation with lysine increased the PER value to 2.10. Obviously,
extrusion cooking destroyed some lysine. Fermentation and supplementation of a traditional Ghanaian cornmeal weaning food with soybean meal
improved its protein quality (70). Studies also suggest that roasted cowpea,
widely grown in the Sahel, may be suitable as a weaning food supplement
(64,71).
398
Chapter 13
6.
Cereal products (bread, biscuits, cooked rice, noodles, pasta, etc.) are part
of the staple diet in much of North and South America, North Africa, and
Asia. Cereals also provide proteins indirectly when used as animal
feedstuffs. We now consider the nutritional quality of cereal proteins.
Bread, biscuit, and pasta making quality etc. are not discussed. The reader
should refer to the following reviews for information on protein functional
quality (7476).
The effect of extrusion cooking on protein quality was reviewed by
Bjoerck and Asp (77), Cheftel (78), and Mercier (79). Salunkhe et al. (80)
reviewed the nutritional quality of cereal proteins in general. Lorenz (81)
considered the effect of sprouting on cereal protein quality. Dixon-Philip
(82) discussed the consequences of milling, baking, extrusion, hydrothermal
processing, and fermentation on the quality of cereal products. Examples of
processing effects on PNV in cereal-based products are described here.
6.1.
Amaranth
399
Maize
Bressani et al. (84) examined the effect of processing maize into tortillas on
their nutritional characteristics. Eleven ordinary maize cultivars and one
variety of quality-protein maize (QPM) called `Nutricta' were processed into
cooked maize or tortillas according to methods used in rural Guatemala.
Protein quality was signicantly higher (P < 0.03) in tortillas than in raw
maize. The QPM cultivar had superior PNV both as raw grain and as
tortilla. Gupta (85) reported increased protein quality for maize after
sprouting provided that radicals and plumules were removed from corn
kernels. The true digestibility was unaffected by sprouting although BV,
NPU, and utilizable protein increased. Gupta and Eggum (86) developed a
process for transforming the by-product from corn oil production into a
food-grade protein meal. Commercial oil cake was extracted with hexane
and 80% ethanol and then sieved to remove undesirable materials. The
defatted maize germ oil cake had 24.7% protein. Albumin, globulin, and
zein decreased while glutelin and residue protein fraction increased. The
meal protein had higher levels of lysine and tryptophan than whole maize
grain. Protein digestibility and BV were improved as compared with the
starting material.
6.3.
Rice, Millet-Sorghum
Eggum and Juliano (87) found that rice protein quality was not adversely
affected by simple cooking or parboiling. Extrusion cooking led to adverse
effects on protein quality. Lysine is the limiting essential amino acid in millet
(88). The digestibility of all cultivars was high (Table 5). Autoclaving led to
a 1925% decrease in the digestibility and an overall increase in BV.
Fortication with lysine led to greater increases in BV as compared with the
effect of heating. Geervani (89) reviewed effects of processing on protein
quality in millet sorghum and other cereals important for developing
countries. Changes in amino acid composition and protein quality
characteristics for millet (as well as barley, oats, wheat, rye, and maize)
before and after boiling are discussed. Dry heat processing (e.g., as used for
baking biscuits and bread), frying, fermentation (especially sorghum and
millet products), and germination are also discussed. Pawar et al. (90)
obtained a PER increase from 2.14 to 2.32 for pearl millet by soaking in
0.2 N HCl for 15 hours. Cooking for 20 minutes led to further improvements
400
Chapter 13
Value
Protein (%)
Lysine (g 16 g 1 N)
True digestibility (%)
Biological value (%)
10.7817.3
0.9951.39
9599.3
48.356.5
in protein quality. The TPD, BV, NPU, and utilizable protein were also
increased for soaked and/or scaried pearl millet.
6.4.
401
References
Country
Denmark
Canada
Finland
India
Norway
USA
USA
India
Philippines
Vietnam
Thailand
India
USA
Sweden
Germany
Pakistan
Czechoslovakia
402
Chapter 13
MA Burnette III, II Rosof. GMA test protocol for protein quality assays.
Food Technol 32(12):6668, 1978.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
403
404
Chapter 13
405
406
Chapter 13
407
408
Chapter 13
409
410
Chapter 13
14
Protein DigestibilityCorrected Amino
Acid Scores
1. INTRODUCTION
The protein digestibilitycorrected amino acid score (PDCAAS) was
adopted as the AOAC-approved index for protein quality in 19931994.
The PDCAAS is the amino acid score (AAS) multiplied by protein
digestibility. Alterations in either parameter changes protein nutrient value
(PNV). PDCAAS is discussed in this chapter. In Sec. 2 considration is given
to digestibility and its relation to protein structure. In Secs 3 and 4 is a
review of protein denaturation and chemical deterioration during food
processing and their effect on the PDCAAS. There is also increasing
realization that moisture-temperature-time relations affect the food matrix
and protein quality. Some possible links between Tg (glass transition
temperature) and PNV are explored in Sec. 5. The determination of
PDCAAS for a range of foods is discussed in Sec. 6.
2. PROTEIN DIGESTIBILITY
True protein digestibility (TPD) measured using a rat bioassay agrees with
in vitro protein digestibility (Chapter 13). This results has two implications:
411
412
Chapter 14
2.1.
ki PKeq
1 Keq
2.2.
413
* Under nondenaturing conditions, the small proportion of the U state in equilibrium with the
native state is attacked by proteases. It is a moot point whether the N state itself undergoes
proteolytic attack.
414
2.3.
Chapter 14
Enzyme Specicity
PROTEIN DENATURATION
415
NU
N!I
D. Processing variables
High temperatures (sterilization, cooking, drying)
Low temperature (cold storage, freezing)
Moisture control (dehydrated storage, drying)
High pressure (sterilization, low-temperature gelation)
High or low pH (solubilization, texturization)
Procesing chemicals (alcohols, alkali, nitrate, reducing compounds, etc.)
Exposure to interfaces (emulcation, foaming, etc.)
High shear treatment
a
Notes: A, Two forms of protein denaturation; B, conditions that facilitate reversible and
irreversible denaturation; C, types of reversible and irreversible denaturation; D, forms of food
processes leading to denaturation.
416
4.
Chapter 14
Carbonyl-Amine Reactions
Within a polypeptide all except three essential amino acids are protected
from the Maillard reaction. Luise Maillard (21) discovered the reactions
between amino acids and sugars. Nonenzymatic browning leads to the
formation of melanoidins and avor precursors. The reactive protein
residues are lysine (e-NH2), methionine (22S22CH3), and tryptophan (indole
group). With high-carbohydrate (plant) foods, lysine is the most reactive
essential amino acid and the most limiting. Methionine or tryptophan is
usually limiting for animal proteins.
417
The effect of pH, water activity (Aw), temperature, and different sugars on
the rate of the carbonyl-amine reaction was studied by Labuza and
Saltmarch (23,26). Maillard browning shows a bell-shaped pH dependence
with a maximum at about pH 8. Extremes of pH lead to a decline in the rate
of browning by (de)protonating the carbonyl and amino functions. The
dependence on AW is also bell shaped. Browning decreases with decreasing
moisture content due to the high medium viscosity and diffusion
restrictions. With increasing AW the reaction rate increases until sufcient
water is available for monolayer coverage. With still higher moisture
content the rate of reaction decreases due to the dilution of reactants. The
temperature dependence of the Maillard reaction was a described with linear
Arrhenius equation. The activation energy for lysine loss was about 80 kJ
mol 1 (Section 5.3). The relative rates of reaction with different sugars
follow the order lactose > D-ribose > D-fructose > D-glucose; pentose
> hexose > disaccharide.
4.3.
418
Chapter 14
Cysteine, serine, and phosphothreonine residues are converted to dehydroalanine at high pH. The process involves the elimination of hydrogen
sulde, water, or the phosphate group, respectively. Dehydroalanine then
reacts with a range of protein groups forming cross-links (Fig. 1).
Racemization also takes place under alkaline conditions (33). The two
reactions proceed via the following common steps: (a) abstraction of a
proton from an a-C atom by a base (B) produces a carbanion ion, (b)
readdition of H to the a-C then generates the corresponding D-amino acid,
(c) b-elimination from the carbanion ion produces dehydroalanine, and (d)
addition of protein side chains (histidine, cysteine, lysine) to dehydroalaninie forms cross-links. The subject is reviewed by Hurrell (28), Maga (34),
Otterburn (35), and Swaisgood and Catignani (36).
4.5.
Friedman and Liardon (37) found that the rate of racemization is sensitive
to ( ) or ( ) inductive effects. For simple amino acids the rate of
racemization was predicted using linear free energy relationships. Free
amino acids are *10-fold less reactive than protein-bound amino acids. The
presence of an adjacent carboxyl group destabilizes the intermediate
carbanion ion. A high pH (>9) increases the rate of racemization by
facilitating the initial proton abstraction. Proline residues (especially when
next to aspartate or aparagine) are more reactive than other amino acid
residues. Polypeptide sequences containing serine and cysteine (e.g., Ser/
Cys22X22Lys or Ser/Cys22X22X) are also highly reactive.
Racemization and cross-linking can be observed by heating model
proteins in alkaline solution (38,39). Similar reactions take place in foods
when proteins are exposed to high pH and temperature. A traditional
method for producing tortilla involves steeping corn in 1% lime solution at
808C for 2045 minutes. The preparation of sh protein concentrates,
texturization of soy protein, recovery of residual protein from bone, alkaline
extraction of plant or yeast proteins, and lye peeling also exposes food
proteins to high pH values. Cross-linking and racemization reduce in vitro
protein digestibility (40,41) as well as PNV (42).
419
420
5.
Chapter 14
5.2.
421
The Tg for gluten was reported by several workers (4752). The Tg has also
been reported for beef proteins (53), caseinate (54), and gliadin (5557).
Noel et al. (58) determined the Tg for fractionated wheat gluten proteins (a-,
g-, and o-gliadins and HMW glutenin) using the Perkin-Elmer DSC2
422
Chapter 14
w1 Tg 1 w2 Tg 2 k
w1 w2 k
423
Yg
1 expT Tc=A
TC TC;0 exp kw
A A0 expk00 w
424
Chapter 14
moisture content (w). We have replicated these simulations (Fig. 3) with the
following two results: (a) the inection temperature decreases with
increasing moisture, and (b) the gradient of each graph increases
with increasing moisture. Therefore, sensitivity to temperature
increases with increasing moisture. To preserve high-moisture foods requires
a low storage temperature and improved temperature control. Drying
allows higher storage temperatures and increased tolerance with respect to
temperature variations.
So far, only a few investigators have applied the polymer science
approach to food protein deterioration. The physical-mechanical basis for
glass-liquid transitions for proteins has not been extensively discussed.
According to Ferry (64), Tg is the temperature below which wriggling
motions and conformational rearrangements within a polymer cease.
Liquids and polymers have a constitutive volume (determined by van der
Waals contacts) and free volume arising from packing irregularities or
defects. During cooling the free volume changes in accordance with the
FIGURE 3
425
426
Chapter 14
DE #
RT
427
10
2c
b
T 1
11
DCp#
2Rc
T 2
12
and
Fig. 4 and Table 2 show results for the deterioration of pasta (74). The loss
of FNDB-lysine and browning reactions were adequately described by
Equation (10) (R2 0:98 0:99).
Close to the Tg (1208C) for a dry protein the magnitude of DH # is
large, implying that quality loss may be limited by conformational
transitions. The low DH # value near room temperature is a consequence
of the temperature dependence of this parameter.
* The heat capacity (Cp; J g 1 K 1) is the amount of heat required to raise the temperature of 1 g
of material by 1 K. The reaction A B ! C D will lead to a heat capacity change (DCp ) if the
reactants and products interact differently with the solvent.
428
Chapter 14
212.3
1.328 6 105
1.968 6 107
0.991
42.2a (272.2)
202.8 (419.1)
104.79 (107.6)
4395
192.7
1.22 6 105
1.825 6 107
0.980
40.1 (252.6)
229.1 (264.5)
110.7 (146.6)
2359
The rst and second values are for temperatures of 378C and 1208C.
429
and suitability for routine use, and (f) low cost not exceeding $200 per test at
1991 prices. The panel of eminent nutritionists selected a new test involving
protein digestibility-corrected amino acid score (PDCAAS). They further
agreed that PDCAAS should be based on the FAO/WHO/UNU 1985 list
showing the essential amino acid requirements for 25 yr. preschool children
(76). The values are given in Chapter 12 (p. 355). In 1993 the FDA (USA)
adopted the PDCAAS procedure for the routine evaluation of PNV and for
food labeling purposes. Developments leading to the adoption of the
PDCAAS test are reviewed by Sarwar and McDonough (77), Boutrif (78),
Madi (79), Henley and Kuster (80), and also Kuntz (81). The basic
principles for evaluating PDCAAS and examples of its use are discussed in
Section 6.5. First, however, we review older methods for PNV evaluation
and how the PDCAAS index evolved (Sections 6.16.4).
6.1.
Mendel (82) stated that a protein's quality is related to its minimum quantity
of essential amino acids. In 1946 Mitchell and Block (83) dened the
chemical score for protein quality by calculating the decit of essential
amino acids as compared with essential amino acids from whole egg.
Samples with equal amounts of crude protein (%N 6 6.25) were analyzed
for their essential amino acid content. The results were each divided by the
essential amino acid content for whole egg. The result having the largest
decit compared with egg protein shows the limiting essential amino acid.
Chemical score
EAASAMPLE
EAAWHOLE EGG
13
430
Chapter 14
TABLE 3 Chemical Scores for a Range of Food Proteins Determined Using Whole
Egg Standard
Protein source
Beef muscle
Beef liver
Egg albumin
Cow's milk
Lactalbumin
Beef kidney
Beef heart
Casein
Sunower seed
Soybean (heated)
Rolled oats
Yeast (average)
White rice
Corn germ
Sesame seed
Wheat germ
Whole wheat
Cottonseed
Whole corn
White our
Peanut
Pea
Gelatin
Human milk
Blood serum
Hemoglobin
Flax seed
Alfalfa
Limiting
EAA
Chemical
score
Cys Met
Ile
Lys
Cys Met
Met
Cys Met
Ile
Cys Met
Lys
Met
Lys
Cys Met
Lys
Met
Lys
Ile
Lys
Lys
Lys
Met
Met
Met
Trp
Met
Ile
Ile
Lys
Ile
0.71
0.70
0.69
0.68
0.66
0.65
0.65
0.58
0.53
0.49
0.46
0.45
0.44
0.39
0.39
0.38
0.37
0.37
0.28
0.28
0.24
0.24
0.0
0.86
0.44
0.10
0.35
0.45
BV (%)
Digestibility
(%)
Chemical
score 6
Dig/100
76
77
82
90
84
77
74
73
65
75
66
69
66
78
71
75
70
61
60
52
58
48
25
100
97
100
95
98
99
100
99
94
96
93
93
78
85
92
95
91
90
94
100
97
91
95
71.0
67.9
69.0
64.6
64.7
64.4
65.0
57.4
49.8
47.0
42.8
41.9
34.3
33.2
35.9
36.1
33.7
33.3
26.3
28.0
23.3
21.8
0.0
FIGURE 5
431
The chemical score as an index for protein quality. (Top) Chemical score
is strongly correlated with the biological value. (Bottom) Showing the
correlation between biological value and ``chemical score corrected for
digestibility'' (open circles). A low correlation is observed between BV
and digestibility (see closed symbols).
432
6.2.
Chapter 14
Sheffner et al. (88) introduced the pepsin digest residue (PDR) index in 1956.
The PDR is determined by combining in vitro digestibility with the essential
amino acid pattern. The food sample (containing 1 g of protein) was
incubated with pepsin (25 mg) in 30 mL of sulfuric acid (0.1 N) solution for
24 hours.* Precipitating with sodium tungstate and 0.66 M sulfuric acid
separated undigested protein from the products. The essential amino acid
proles for the substrate and soluble digest (products) are determined by
microbiological assay. Subtraction of these values gave the essential amino
acid pattern for the nonhydrolysed protein residue. For both the digest and
residue, each essential amino acid is expressed as a percentage of the total
content of essential amino acids. The two columns of results were then
divided by the corresponding values for egg protein. Next, the geometric
mean was determined for the ``egg ratios'' and the resulting values were
multiplied by a factor that takes into account the relative amounts of digest
and residue formed by the action of pepsin on the sample and the egg
protein. The PDR index was not easy to calculate. Sheffner et al. noted that
any process that decreases pepsin digestibility will also lower protein
nutritional value.
6.3.
433
for only one enzyme, (c) use of modern amino acid analysis instrumentation,
(d) higher reproducibility, and (e) use of computerized calculations.
6.4.
Computed-PER Index
6.5.
434
Chapter 14
5. Determine digestibility
6. Calculate PDCAAS Lowest AAS 6 digestibility
14
All proteins having a PDCAAS in excess of 100% (or > 1) are assigned a
value of 100%. A protein with a PDCAAS value above 100% does not
provide further benet as excess amino acids are utilized for energy.
According to Henley and Kuster (80), the PDCAAS method provides a
measure of protein quality that is directly correlated with human
requirements. The PDCAAS method also has considerable exibility.
Manufacturers and diet planners can provide larger quantities of lower
quality dietary protein in order to meet the recommended daily requirements.
6.6.
Applications
Sarwar et al. (94) showed that a number of rat bioassays (PER, PER,
RPER, NPR, or RNPR) ranked 20 food products in the same order of
protein quality. The correlations between different rat assays were highly
signicant (r 0.980.99). Chemical scores were also correlated with the
results of rat bioassays. Correcting the chemical score for the digestibility
improved the observed correlation. Carnovale et al. (95) compared PNV for
wild and cultivated species of Vigna. The wild type had a signicantly higher
protein content, trypsin inhibitory activity, and tannin content. Protein
digestibility was lower although PNV assessed in terms of the PDCAAS was
not signicantly different.
A.
435
Quinoa
Quinoa (Chenopodium quinoa Wild) seed protein quality was evaluated using
amino acid analysis and animal feeding trials. The rst limiting amino acids
were tyrosine and phenylalanine with a chemical score of 0.86. Quinoa
protein (14% w/w of the seed) had levels of lysine, methionine, and cysteine
superior to those found in most other plant proteins. From animal feeding
experiments NPU was 75.7, BV was 82.6, and digestibility was 91.7%. These
results yield an estimated PDCAAS of 79%. This value is higher than the
value for meat (87).
B.
Rice
The quality of protein associated with cooked milled rice and a typical ricebased menu for Filipino preschool children and adults was assessed by
Eggum et al. (96). Digestibility, BV, and NPU were assessed with growing
rats. Digestibility was 88.8% for the preschool child diet, compared with a
BV of 90.0 and NPU of 79.9. For the adult diet digestibility was 87.3%, BV
was 86.6, and NPU was 75.5. On its own, cooked rice had a digestibility of
90.0%, BV equal to 82.5, and an NPU value of 74.3. The availability of
lysine (the limiting essential amino acid) was 95.4% for the preschool child
diet, 95.7 for the adult diet, and 100.0 for rice. For whole diets the chemical
scores were 1.00 for the preschool child diet, 0.92 for the adult diet, and 0.62
for rice. From such gures the PDCAAS may be estimated as 88.8% for the
preschool diet, 80.4% for the adult diet, and 56.0% for cooked rice.
C.
Maize
436
Chapter 14
PDCAAS for IAPO-13 of 3738%. It was concluded from such studies that
Canadian QPM maize variety had higher quality than common maize and
that breeding maize for high protein quality showed a great deal of promise.
D.
Oats
Hull-less or naked oats (Avena sativa var. nuda) from temperate zones in
Asia were studied by a Canadian group for their potential use for both
human and animal nutrition. Zarkadas et al. (99) determined the protein
content for three high-yielding and rust-resistant naked oat cultivars. The
protein levels were 13.67 (+ 0.60)%, 13.93 (+ 0.53)%, and 14.40 (+ 0.55)%
for varieties AC Percy, AC Hill, and AC Lotta. All cultivars had a good
balance of nine essential amino acids. Lysine was limiting, followed by
threonine. Assuming a human protein digestibility of 86%, estimates for
PDCAAS are 54.9% (AC Hill), 56.3% (AC Lotta), and 59.3% (AC Percy).
The PDCAAS values for other protein sources are mechanically dehulled
oats (62%), maize (29%), soybean (86%), and egg (95%).
Zarkadas et al. (100) also evaluated the PNV of two newly released
Canadian oat cultivars (Newman and AC Stewart). Oat wholemeal had a
total protein content of 10.75 (+ 0.23)% and 11.92 (+ 0.06)% for strains
Newman and AC Stewart, respectively. The corresponding total protein
content for the oat groats (dehulled grains) was 13.27 (+ 0.24)% and 12.61
(+ 0.94)%. The limiting amino acid was lysine for oat wholemeal and lysine
plus threonine for groats. Values for PDCAAS based on the FAO/WHO/
UNU pattern (2-year-old preschool children) were reportedly 5862.3%
(strain Newman) or 66.762.3% (strain AC Stewart). Dehulling had no
easily predictable effect on the PNV.
E.
Collagen
437
FAO/WHO/UNUa
(mg/1 g protein)
11
16
35
335
15
23
1222
11
27
184195
805816
19
28
66
58
25
63
34
11
35
339
661
438
Chapter 14
sensitivity to heating when corrected for digestibility. For example, raw and
home-cooked beans had available lysine contents of 6.21 and 6.19 g per
100 g protein, respectively. This yields corresponding in vitro protein
digestibilitycorrected available lysine scores 2.68 and 5.1 g per 100 g
protein. After normalization with the FAO/WHO/UNU (1985) pattern, the
protein digestibilitycorrected available lysine score (PDCALS) was 40% for
raw beans and 74% for cooked beans. Apparently, lysine as well as cysteine
plus methionine may be limiting for red kidney beans.
6.7.
439
notional PDCAAS were highly correlated with the RNPR for 17 diets
(R 0.92). However, values for PDCAAS were consistently higher than
AvCAAS by between 2% and 8% (Fig. 6).
Wu and co-workers (106,107) found much more signicant differences
in PNV after correcting amino acid scores for TPD and AAAv. With raw
kidney beans, cysteine plus methionine was limiting with a chemical score of
0.944. The TPD value was 15.7% for raw beans, increasing to 7287% for
heat-processed beans. By comparison, AAAv was negative ( 18.6%) for raw
bean protein and positive (39.868.0%) for heat-processed beans. Rats fed
with raw beans had higher concentrations of cysteine plus methionine in their
feces as compared with amounts initially present in their diets. The AAAv
values for several other amino acids (alanine, proline, valine, luecine, and
threonine) were also negative. The AvCAAS for raw kidney bean protein was
therefore 17.6% compared with a PDCAAS of 13.9%. The relative merits
of the PDCAAS and AvCAAS are discussed by Darragh et al. (108).
Estimating TPD using a rat assay is a slow process, requiring about 8
days for completion. In view of the high correlation between TPD and in
vitro protein digestibility, correcting AAS using in vitro protein digestibility
is more cost effective. Rozan and co-workers (109) considered AAS for
soybean, lypine, and rapeseed meal proteins using the FAO/WHO/UNU
(1995) pattern for 2- to 5-year-old preschool children. The AAS values were
then corrected using in vitro protein digestibility measured as the degree of
FIGURE 6
Protein nutrient value estimates based on amino acid scores corrected for
true protein digestibility (PDCAAS) or amino acid availability (AAS).
[Drawn from the data of Sarwar (93).] Y-axis shows PDCAAS minus
AAS.
440
Chapter 14
441
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120, 1954.
17. C Tanford. Protein denaturation. Adv Protein Chem 23:121282, 1968.
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19. TJ Ahern, AM Klibanov. Analysis of processes causing thermal inactivation
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20. JE Kinsella. Texturized proteins: fabrication, avoring, and nutrition. CRC
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21. LC Maillard. Action des acides amines sur les sucres: formation des
melanoidines par voie methodique. C R Hebd Seances Acad Sci 154:6668,
1912.
22. E Dworschak. Nonenzyme browning and its effect on protein nutrition. CRC
Crit Rev Food Sci Nutr 13:140, 1980.
23. TP Labuza, M Saltmarch. The nonenzymatic browning reaction as affected by
water in foods. In: LB Rockland, GF Stewart, eds. Water Activity: Inuence
on Food Quality. New York: Academic Press, 1981, pp. 605650.
24. M Friedman. Lysinoalanine formation in soybean proteins: kinetics and
mechanisms. In: J Cherry ed. Food Protein Deterioration. Mechanisms and
Functionality. Washington, DC: American Chemical Society, 1982, pp. 231
273.
25. M Friedman. Chemically reactive and unreactive lysine as an index of
browning. Diabetes 31(Suppl 3):514, 1982.
26. M Saltmarch, TP Labuza. Nonenzymatic browning via the Maillard reaction
in foods. Diabetes 31(Suppl 3):2936, 1982.
27. RE Feeney, JR Whitaker. The Maillard reaction and its prevention. In: J
Cherry ed. Food Protein Deterioration. Mechanisms and Functionality.
Washington, DC: American Chemical Society, 1982, pp 201229.
28. RF Hurrell. Reactions of food proteins during processing and storage and
their nutritional consequences. In: BFJ Hudson, ed. Developments of Food
Proteins3. London: Elsevier Applied Science, 1984, pp 213244.
29. MS Feather. D-Glucose-L-alanine interactions: their role in Maillard polymer
formation and Strecker degradation reactions. In: RD Philips, JW Finley, eds.
Protein Quality and the Effects of Processing. New York: Marcel Dekker,
1989, pp 263272.
30. PA Finot. Nonenzymic browning products: physiological effects and
metabolic transit in relation to chemical structure: a review. Diabetes
31(Suppl 3):2228, 1982.
31. K Lorenz. Microwave heating of foodschanges in nutrient and chemical
composition. CRC Crit Rev Food Sci Nutr 7:339370, 1976.
32. GA Cross, DYC Fung. A review of the effects of microwave cooking on
foods. J Environ Health 44:88193, 1982.
442
Chapter 14
443
444
Chapter 14
445
446
Chapter 14
Index
[Adulteration]
PCR analysis and, 224
US meat trade and, 224
Advanced glycation products, Bradford
assay and, 190
Adulteration frequency, 240
Adverse reaction, food and, 298
Agar gel double immunodiffusion assay,
230246
Agaricus, Udy assay, 160
Aggregated protein, Bradford assay,
217
AGIDagar gel double
immunodiffusion assay, 230246
Albumin, biuret assay, 4849
Alkali treatment, digestibility and, 373
Alkaline copper reagent, 47
Alkali-phenol reagent, Kjeldahl assay,
18
Allergens, 299300
castor bean, 300
egg-white, 300
447
448
[Allergens]
milk, 300
peanuts, 305312
shrimp, 300
soybean, 301305
wheat, 312329
Allergy
infants and, 306
pregnancy and, 306
Amaranth, 398399
Amido Black 10B, 130131
Amino acid analysis (see Quantitative
amino acid analysis, QAAA)
Amino acid availability (AAAv), 368,
438
Amino acid availability, protein quality
and, 438440
Amino acid score, 354355, 433
errors in, 355
Ammonia determination, 1823
Anaphylactic shock, 297, 305
Anilino-naphthalene-8-sulfonic acid,
413
ANS uorescence, 413
Antigen
actomysin, 234
binding curves, 227, 261
boiling and ethanol resistant, 236, 239,
240, 260
cooked meat and, 236, 238
peptides as, 324327
thermostable proteins as, 236, 261
troponin T as, 236, 238, 239
AOAC, Association of Ofcial
Analytical Chemists
digestibility, 369
Dumas assay, 30, 34,36
dye binding assay, 149, 151
gluten ELISA, 320321, 324325
guidelines, rat bioassay and,
351353
immunodiffusion assay, 230
Kjeldahl assay, 15
factors and, 10
Udy assay, 149
Index
Applications
biuret assay, 5767
Bradford assay, 195
Udy assay, 147
Ara h 2, peanut allergens and, 308
Arachin, 306
Arginine, dye binding and, 135, 139,
212
Arrhenius equation, 384, 426427
PNV and, 384
Assay performance, 5
Association of ofcial Analytical
chemists (see AOAC)
Atmospheric error, Dumas assay, 33
Atopic dermatitis, 305
Authenticity, proteins and, 222
Autoanalyzer, 21, 16
Autoclaving, effects on digestibility, 368
Automated, Kjeldahl assay, 1516
Availability corrected amino acid score,
438
Available amino acid score (see AAS)
Available lysine, 356366
chromatographic analysis, 360361
comparison of assays, 365
cottonseed, 360
interferences, 359, 362
lactalbumin, 365
legumes, 394395
moisture and, 426
ovalbumin, 365
reagents for, 356
sodium borohydride and, 365366
temperature and, 426427
AvCAAS (see Availability corrected
amino acid score)
Azo dyes, 133134,137
B lymphocytes, 228229
Baby foods
Dumas assay, 38
meat protein detection in, 262
peanut allergens in, 311
Baby formula, 3738
Bacteria, feedstuffs quality and, 388
Index
Baker's asthma, 312
Barley
biuret assay, 55
Dumas assay, 36
dye binding assay, 153
Kjeldahl analysis, 8
protein analysis, 7, 8
rapid assays for analysis, 7
Udy assay, 152
BCA assay, 99122
algae protein, 121
animal carcass protein, 119121
automated, 113
calibration features, 109
copper analysis, 100
ow injection analysis, 114116
interferences, 110111
lysine and, 119
mechanisms, 105
metal ion catalyzed oxidation, 107
method, 104
microwell plate format, 113115
phenolic compounds effects, 118
reducing compounds and, 106
sample pretreatment for, 112
serum copper, 102
solid phase assay, 117
sugars and, 103
TCA-DOC precipitation and, 112
BCA, derivatives, 101
BE antigen (boiling and ethanol resistant
antigen), 236, 239, 240, 260
Bean protein, Bradford assay, 213
Beans, PDCAAS value, 437
Beef
analysis, ORBIT, 237
croquettes, soy protein detection in,
304
protein, dye binding assay, 157
Beer
Bradford assay, 190, 204206
celiac disease and, 312
Dumas analysis, 30, 36
Kjeldahl analysis, 8, 14
Lowry assay, 88
449
b-Lactoglobulin, 149, 182,
digestibility, 309
dye binding, 149
Binding constant, protein-CBBG, 177
Binding sites, dyes, 140
Binding, T-azo-R, 177181
Bioassay, 387, 388
chick, 354
human, 341
protein quality and, 346354
rat 348353
Biological value (BV), 342
Bio-Rad LTD, 207
Biotin-streptavidin detection, 261262
2,20 -Biqinoline, Lowry assay and, 76
Birds nest soup, allergy, 305
Bitter, Bradford assay, 207
Biuret assay, 47
barley analysis, 55
casein determination, 49
cereal proteins, 57
protein solubility determinations by,
63
Biuret, structure, 47
Boiling and ethanol resistant antigen,
236, 239, 240, 260
Bone meal, 271
Bovine spongiform encephalopathy (see
BSE agent)
Bradford assay
advantages, 169
beer protein, 204205
beverages and, 190
bread crumb, 212
calibration features, 201
carotenoids and, 189
Chardonnay wine, 211
compatible solutes, 186, 187
effect of SDS, 187
interferences, 186
legumes, 213
lipids and, 191
mechanisms, 172
microassay format, 196
modied, 196
450
[Bradford assay]
monosaccharides and, 190
mungbean our, 213
mushrooms, 216
pinot noir, 211
polypeptide selectivity, 171
polyphenols and, 209210
polysaccharides and, 186, 191, 199
reagent pH, 172
sensitivity, 202203
solid phase, 185
standard method, 196
wavelength of, 173175
Bran protein, 55
Bread crumb, Bradford assay and, 212
Bread making quality, 212213
Bread mix, gluten free, 321
Bread wheat, 57
Brest milk, 318
Brewing grains, Dumas assay, 30, 33, 36
British beers, Bradford assay, 208
BSA binding, CBBG, 185
BSE agent, 271273
bone meal and, 271
immunological detection, 272
test kits for, 271273
Buckwheat, celiacs and, 320
Buffalo, antigen, 238
Buffer effects, Lowry assay, 81
Butter milk, dye binding assay, 148
BV (see Biological value)
Cake our, gluten free, 321
Calibration, 37
ammonia analysis and, 19, 20, 23
BCA assay and, 109110, 113
biuret assay for rice, 5859
Bradford assay and, 201
denitions, 3
Dumas assay and, 35
dye binding assay, 129, 131, 154, 155
ELISA format and, 270
gliadin ELISA, 323
gluten ELISA, 323
horse meat ELISA, 267
Index
[Calibration]
Lowry assay, 72, 7780
meat ELISA, 253
pork ELISA, 257, 267
soy ELISA, 284
Udy assay, 154
Camel, ELISA, 252, 254, 255, 260
Canned baby foods, ELISA and, 262
Canned sh, ELISA, 268269
Canned foods
digestibility, 370
Kjeldahl analysis, 16
Canned milk, 150
Canned tuna, 268
Carbethoxylation, sh meal, 363
Carbohydrates
available lysine determination and,
359, 362, 366
Lowry assay and, 81
Carbonyl-amine reaction, (see also
Maillard Reaction), 416418
Caroteinoids, Bradford assay and, 189
Casein
biuret assay, 49
digestibility, 370
dye binding assay, 126
PER value, 349
protein quality, 344
Udy assay, 138, 149
Catalysts, Kjeldahl assay and, 9
CBBC sources, Bradford assay and,
197198
CBBG, bi-ionic form, 172
Celiac disease, 312313
Celiac Society of Great Britain, 322
Celiac-negative cereals, 323
Cereal products
Bradford assay, 212
PNV, 398401
Cereal proteins, classication, 314
Cereals
allergens, 312329
biuret assay, 57
Lowry assay, 88
Udy assay, 151
Index
Cerelac, 397
Chardonnay wine, Bradford assay, 211
Cheddar cheese, dye binding, 150
Cheese, 151
Chemical deterioration, proteins,
416420
Chemical score, 429431
egg protein and, 354
Chemistry
biuret assay, 50
Lowry assay, 77
Udy assay, 133147
Chick bioassay, protein quality, 353354
Chick pea, Udy assay, 153
Chicken
antigen for, 238
determination amino acid analysis, 27
dye binding assay, 157, 159
immunodiffusion assay, 240
Chlorophyll, Lowry assay and, 8182
Chocolate
bars, ELISA, 310
gluten ELISA and, 320, 321
milk, dye binding assay, 148
peanut allergens in, 310311
Chromatographic determination,
available lysine, 360361
Circular dichroism, 317
Citations off, Lowry assay, 71
CNS tissue in feed, 272
Cod llet, dye binding assay, 157
Codex Alimentarius Commission,
allergens, 298
Collaborative testing
Bradford assay, 205206
Dumas assay, 30, 34, 36
gluten ELISA, 322
ice cream analysis dye binding, 150
PNV, 390
soya protein ELISA, 282
three-enzyme assay for digestibility,
370
Collagen
Lowry assay, 93
PDCAAS value, 436437
451
[Collagen]
quantitative amino acid analysis,
2728
Colorimetric assay, ammonia, 1823
Colorimetric Kjeldahl analysis, 1825
Combustion analysis, milk products, 37
Combustion nitrogen analyzer (see
Dumas assay)
Commodities, Udy assay, 128
Compatible solutes, Bradford assay,
186, 187
Compton-Jones scheme, CBBG,
172173
ComputedPER index (see c-PER)
Conarachin, 306
Condensed milk, 151
Confectionary, gluten ELISA, 322
Confectionary products, ELISA, 311
Conformational stability, 425
Conglycinin, 285289, 292
renaturing efciency, 288
Conversion factor, Kjeldahl assay, 10,
12
Cooked beef, ELISA, 260
Cooked meat
antigens for, 236, 238
immunoassay, 239, 247, 260, 268
soy allergens and, 303
Cooked poultry, ELISA, 261, 262
Cooking oil, peanut allergens and,
310
Cooking, gluten analysis and, 326
Coomassie Brilliant Blue G250, 169
Copper analysis, BCA assay and, 101
Copper complex, Biuret assay, 5052
Copper concentration, Lowry assay and,
82
Copper hydroxide, biuret assay, 48
Corn our, gluten detection in, 322
Corn meal, BCA assay, 117
Corn protein, Bradford assay, 212, 213
Correlation coefcient, denition, 5
Cortecs, 262, 271
gluten ELISA kit, 319
peanut ELISA kit, 311
452
[Cortecs]
pork ELISA kit, 271
Costs
Dumas assay, 32
PNV tests, 429
Cottage cheese, dye binding, 150
Cottonseed, available lysine, 360
Cowpea, Udy assay, 154155
c-PER, 433
Creutzfeldt-Jacob disease, 271
Cross reactivity (see Specicity)
Cross-linking, 418419
Crystal violet, protein binding and,
172
Crystalline-amorphous transition, 425
Cyoprotectants, Lowry assay and, 82
Cysteine, racemization, 418
Dairy products
Dumas assay, 30
dye binding, 147
Dairy proteins, Biuret assay, 63
DBC (see Dye binding capacity)
d DBC, differential dye-binding capacity
available lysine determination and,
362365
legumes and, 364
soybean meal and, 363
total lysine and, 364
Deer analysis, DRIFT, 237
Defatting, meat, 61
Degree of hydrolysis, 373
Dehulling, PNV and 395
Dehydrogenation, Lowry assay, 75
Denaturation, 414419
meat antigens, 263265
peanut allergens, 308309
processing and, 415
Denaturation temperature (TD), soy
proteins, 286287
Denaturation-renaturation, soy
antigens, 284
Denaturing agents, 414
Dent CO251 (see also Maize), 435
Design, Lowry assay, 69
Index
Detergent effect, Bradford assay,
187188
Dialysis assay, digestibility, 373
Differential dye-binding capacity
(d DBC), 362365
Differential scanning calorimetry (see
DSC)
Difculties, Udy assay, 130
Digestibility
denitions of, 367368
peanut allergens, 309310
three enzyme method for, 369372
Dimethyl sulfoxide and Lowry assay, 82
Diphtheria toxin, 231
Disulde bonding
gluten and, 315
in soybean protein, 285, 288
Donkey, ELISA, 256, 262
Double immunodiffusion, 230
Dried milk, 344
Kjeldahl assay, 13
PNV for, 382384
DRIFT, 235
DSC, soy protein, 286
DSC2, Tg measurements, 422
Dumas assay, 29
Advantages of, 31, 36
atmospheric error and, 33
beer protein, 30, 36
feedstuffs, 34
instrumentation, 3133
ketchup, 30
malt, 30
materials, 32
milk products, 37
oilseed protein, 33
semolina, 38
Dye binding assay
beef protein, 157
calibration with Kjeldahl, 150,
152155
chicken meat, 157, 159
cod llet, 157
Dye binding lysine, 362
Dye equivalent weight, 129
Index
Dye purity, Bradford assay and,
197198, 200
Effect of dye volume, Bradford, 201
Egg albumin, 344
biuret assay, 48, 63
Bradford assay, 217
Egg allergens, 305, 309
Egg products, dye binding assay, 159
Egg protein, 290, 292, 346, 347, 392
Udy assay, 159
Egg ratio, 354
Egg white allergens, 300
Egg yolk, Bradford assay and, 201
Egyptian legumes, PNV, 394
Electrophoresis (see also SDS-PAGE)
CBBG, 170
samples, Bradford assay, 198, 199
ELISA
aatoxins, 251
bacterial toxins, 25
boiling resistant antigen, 260
BSE agent, 271273
buffalo, 256
meat, 260
cattle, 256
commercial, 282
competitive, 248
confectionary products, 311
cooked beef, 262
meat, 260265
pork, 262
poultry, 262
enzyme substrates, 254
format, 248250, 255
game meat, 260
gliadin, 317329
gluten, 317329
horse meat, 252256, 258, 260263,
266268
muscle antigen for, 257260
Ochratoxin, 251
performance characteristics, 270
peroxidase assay and, 254
pork, 260
453
[ELISA]
analysis, 256, 257
pyruvate kinase as antigen, 258
raw meat, 252, 255
red snapper, 270
reviews, 250
rock shrimp, 269270
sardine, 268
sea food, 268270
sheep, 256
soy protein, 281296
ELISA test kit
BSE, 273
deer meat, 262
gluten, 323
meat, 262
allergens, 310
peanut, 311
pork, 262
soybean, 283284
tuna, 268
working range, 254, 320
Emulsion proteins, Lowry assay, 90
End-point temperature, meat ELISA,
265
End-point temperatures, Biuret assay,
62
Enzyme linked immunosorbent assay
(see ELISA)
Enzyme, protein digestibility and, 366
Equations, Udy assay, 141142
Equilibrium, Udy assay, 138, 156
Errors
chemical score, 355
PER determination, 350
Essential amino acids, 355
Ethyl vinyl sulfone (EVS), 356
Evaporated milk, protein analysis,
149
FAO/WHO/UNU, 433
Fatalities, peanut allergy, 306
FDA, 298
FDNB, 1-Fluoro-2,4-dinitrobenzene
assay, 356
454
[FDNB]
available lysine determination,
356359
correction factor, 359
Fecal nitrogen, 367, 368
Feed supplements, 386
Feedstuffs and concentrates, 386393
Feedstuffs, 387
Dumas assay, 30, 33, 34, 35
dye binding assay, 156, 390
Kjeldahl analysis, 16
Orange G binding, 388390
PNV evaluation, 346, 387393
Udy assay, 156
Fermented soybean products,
allergenicity, 304
Fertilizer, Dumas assay, 30
FIA, BCA assay using, 114
Ficol, Lowry assay, 82, 83
Fish, allergens, 298
Fish and seafood identication, 268271
Fish gelatin, 311
Fish meal, 387, 388, 390
Acid Orange 12 binding, 390
DBC, 389
Kjeldahl analysis and, 15
Udy assay, 138, 156
Fish protein, Tg, 423
Fish protein concentrate, 268
Fish sausages, soy protein detection in,
304
Flint CO255 (see Maize), 435
Flow injection analysis (see FIA)
Flower bud protein, quantitative amino
acid analysis, 27
1-Fluoro-2,4-dinitrobenzene (see
FDNB)
Folin-Ciocalteu reagent, 71
Food allergy, 297
intolerance, 297
matrix, protein quality and, 420425
Foods, Dumas assay, 30
Freeze dried proteins, 425
Fringe-micelle model, 421
Frozen dessert, 151
Index
Fruit juice, Bradford assay, 211
Game meat, ELISA, 260
Gel immunodiffusion assay, cooked
meat, 239241
Gelatin
BCA assay, 105
Biuret assay, 49, 56
complexes with Amido Black 10B, 147
feedstuffs and, 156
Lowry assay, 93
Gelatin granules, 126
Gel-ltration, beer, 206
Glass transition temperature (see also
Tg), 420425
Gliadin, 314317
in human milk, 318
Tg value for, 424
Gluten, 315
available lysine, 360
confectionary products and, 322
ELISA, 317329
home test, 323324
preparation, 315
standards for ELISA, 321
Tg value for, 421
Gluten-free foods, 313, 318, 322, 323
Gly m Bd 30k, as soybean allergen,
301304
Glycation, Bradford assay, 190
Glycinin, 285289
Gordon-Taylor equation, 422425
GPV (Gross protein value), 342
Grain and cereal, Kjeldahl assay, 13
Grains, Dumas assay, 33
Grape juice, Bradford assay, 210
Graphical analysis, Udy assay, 144145
Gross protein value (see GPV)
Ground rice, biuret assay, 59
Half and half milk, protein assay, 149
Hamburger, meat identication and, 235
Heat effects, soy antigens, 287
Heat resistance, conglycinin, 288
Histidine, dye binding and, 135, 139
Index
Home test, gluten, 323324
Honey
Bradford assay, 217
Kjeldahl analysis, 217
Horse
antigen production using, 231
ELISA, 267
immunoassay, 234
immunodiffusion assay, 239
meat, 252256, 258, 260263, 266268
meat protein, immunoassay, 252256,
258, 260263, 266268
muscle antigen, identity, 258, 261
muscle protease, biuret assay and, 62
protein assay, 235
Horseradish peroxidase, 253254
Human milk, gliadin detection in, 318
Hybridoma, 229
Hydrogen peroxide
Biuret assay, 55
Kjeldahl assay and, 9
Hypoallergenic bread, 316
Ice cream dye binding, 148
protein, 150157
Udy assay, 148, 150151
Immunization schedule, 231, 233
Immunoabsorption, 232
Immunoassay, principles, 226
Immunoblotting, 299300, 307
Immunodiffusion assay
chicken meat, 240
meat, 230
Immunological assays, disadvantages,
250
Immunology, 228
Imperial Chemical Industries (ICI), 169
In vitro digestibility, 368374
protein stability and, 413
protein structure and, 413
Incidental additives, 298
Indanetrione assay, Kjeldahl assay, 23
Indophenol reagent, Kjeldahl assay, 18
Infant formula, 384386
amino acid supplementation, 386
455
[Infant formula]
Dumas assay, 3738
peanut protein detection in, 306
PER, 350
PNV bioassay, 385
rat bioassay, 385
Infrared analysis, cereal proteins, 7
Insect protein, Bradford assay, 199
Insoluble protein, Bradford assay, 217
Instant breakfasts, Kjeldahl assay
analysis, 7
Interferences
available lysine, 362, 363, 366
BCA assay list for, 111
biuret assay, 5355
Bradford assay, 186191
d DBC, 363
Lowry assay, 80
ninhydrin assay, 23
plant dyes as, 57, 58, 59
Udy assay, 147
Isobestic point, 140, 172
IVPD (see In vitro digestibility)
Kangaroo meat, immunoassay, 234, 235
Ketchup, Dumas assay, 30
Kinetics, 395
Lowry assay, 77
proteolysis, 412414
Kiwi fruit juice, Bradford assay, 211,
212
Kjeldahl analysis, 1, 725, 344
barley, 8
beer, 8, 14
catalysts, 9
colorimetric, acetylacetone and, 22
comparison with dye binding assay,
150, 152, 153
honey, 217
gliadin, 327
Kjeldahl factor and, 10, 12
nitrogen-protein conversion factor,
10, 12
PER values and, 349
PNV determination, 349
456
[Kjeldahl analysis]
reactions of, 9
reliability, 7
sausages, 17
Kjel-foss, Instrument, 15
Labeling, food allergens, 297
Lactalbumin, PER value, 349
Lactose, protein quality and, 382,
385
Larger, Bradford assay, 208
Leaf protein, Lowry assay, 88, 90
LECO FP-2000, Dumas assay, 31
LECO FP-228, Dumas assay, 33
LECO FP-428, Dumas assay, 3132
Legumes
available lysine, 394395
baking and PNV of, 395
Bradford assay, 213
dehulling and PNV, 395
dye biding, 153156
ELISA and, 312
Lowry assay, 88
PNV, 393398
roasting and PNV, 396
sprouting and PNV, 395
steaming and hydrothermal
treatment, 395
Lesions, celiac disease, 312313
Linear dynamic range, 5
Linear free energy relations, amino
acids, 418
Linearity, Bradford assay, 201
Lipid
Bradford assay and, 191, 200
interferences by, 84
Lower limit of detection (LLD)
denition, 3, 6
ELISA, 265
gluten ELISA, 320, 324
soy bean ELISA, 284, 290
Udy assay, 152
Lowry assay
buffers and, 81
carbohydrates and, 81
Index
[Lowry assay]
cereals, 88
chlorophyll and, 8182
citations of, 71
cryoprotectants and, 82
design, 69
emulsion proteins, 90
leaf protein, 90
legumes, 88
mechanisms of, 77
nondairy creamers, 90
sample pretreatments for, 8687
single cell protein, 91
Lysine
BCA assay and, 119
bioassay and, 344
CBBG binding and, 171
determination, 356366
dye binding and, 135, 138
glycation and Bradford assay, 190
loss, Arrhenius plot 385
Maillard reaction, 382, 416418
available lysine, 366
pH and, 417
Maize
available lysine, 360
cultivars, 435
Dumas analysis, 29
PNV, 399
Malt
Dumas assay, 30, 36
dye biding, 152
Materials, Dumas assay, 32
Matrix effects, protein deterioration
and, 420
Mean residue weight, tables of values, 27
Meat, 247281, 284, 303
analysis for soybean, 285
antigenic components, 232, 251
BCA assay and, 119121
biuret assay, 6163
Dumas analysis, 29
dye binding assay, 157
gel immunodiffusion assay, 230
Index
[Meat]
quantitative amino acid analysis, 27
sample pretreatment for ELISA, 284
soy protein detection in, 303
Udy assay, 156157
Meat analogue, Kjeldahl analysis, 16
Meat and bone meal, PNV, 393
Meat and meat products, Kjeldahl
analysis, 15
Meat antigen, 231
Meat rendering plants, 272
Meat speciation, 247280
Meatballs, soy protein detection in, 304
Mechanism
biuret assay, 50
Lowry assay, 73, 77
Udy assay, 133147
Melanoma, 229
Metachromasia, Crystal violet, 174
Metal ion catalyzed oxidation, Lowry
assay, 73
Michaelis-Menten kinetics, 414
Microassay, Bradford, 196
Microbiuret assay, 56
Micro-Kjeldahl analysis, 21, 13
Microwave heating, 396
Microwell plate, BCA assay using, 113
Milk, 282
allergens, 298, 300
ELISA, 282
powder, 381
powder, PER, 350
prices and protein content, 125
products, Dumas assay, 37
protein, dye binding assay, 147, 148
protein, ELISA plate coating, 248,
272
protein, Udy assay, 147151
soymilk detection in, 281
Millet
PNV, 399400
protein, biuret assay, 60
Mince, soy protein detection in, 284
Model wine solution, Bradford assay,
211
457
Moist heating, allergen stability and,
308
Moisture
food deterioration and, 416
peanut allergens and, 308
PER values and, 350
PNV and, 383384
rapeseed heat damage and, 392
Moisture-temperature-time relations,
411
Molten globule and digestibility,
412415
Monoclonal antibodies
development for ELISA, 265268
gluten ELISA and, 325327
meat analysis and, 265267
pork ELISA and, 267
soy bean ELISA and, 290292
turkey analysis, 266
Monosaccharides, Bradford assay and,
190
Multiple processing, legumes, 397
Mungbean, Bradford assay, 213
PNV, 394
Muscle lactate dehydrogenase, ELISA,
264, 265
Mushrooms
Bradford assay, 216
Kjeldahl analysis, 160
quantitative amino acid analysis, 27
Udy assay, 160
Myoglobin, thermostable antigen and,
238, 239
Naphthylamine brown, 126
NB, nitrogen balance, 342, 346347, 367
NDI, nitrogen digestibility index, 373
Nessler's reagent, Kjeldahl method and,
21
Net protein utilization (NPU), 342
Ninhydrin reagent, Kjeldahl method
and, 2324
Nitrogen balance (NB), 342, 346347,
367
Nitrogen digestibility index (NDI), 373
458
Nondairy creamers, Lowry assay, 90
Nonanimal protein ingredients, 281
Nonfat milk, 149
NPU, net protein utilization, 342
Nucleic acid, Bradford assay and, 191
Nuts
allergens and, 300301
ELISA, 310312
Oats
Ac Hill, 436
Ac Lotta, 436
Ac Percy 436
biuret assay, 60
PDCAAS value, 436
Oilseeds, Dumas assay, 30, 33
OPA (see o-Phthaldehyde)
o-Phthaldehyde, 356
Orange G
binding, PNV, feedstuffs, 389390
structure of, 136
Udy assay using, 130
ORBIT, 235
Ovalbumin, 182
pAb, for detection of donkey muscle
protein, 256, 262
PAGE, 81, 402
Pavalbumin, allegen and, 300
PDCAAS, 411, 427, 429, 433438
applications, 434439
beans, 437
benets, 434
calculation, 434
collagen, 436437
maize, 434
oats, 436
rice, 434
Vigna, 434
PDR index (see Pepsin, digest residue
index)
Pea globulin, ELISA, 290, 291
Peanut allergens, 300, 306308
cooking oil and, 310
preparation, 307
Index
Peanut allergy, 305312
average fatalities, 306
in infants, 306
in pregnancy, 306
Peanut protein, in confectionary, 311
Peanuts, chemical score, 429
Pepsin digest dialysate index (PDD
index), 432
digest residue index (PDR index), 432
pancreatin digestion index (PPD
index), 432
Peptide antigens
gluten ELISA and, 324327
soybean ELISA and, 290
Peptidyl sites, digestibility and, 414
PER (Protein efciency ratio), 342,
348351
AOAC guidelines, 351
complex foods and, 350
rat acclimation period, 350
sweetened foods and, 350
Perilla, 396
Peterson's Lowry assay, 72
Phenol red, 125
Phenol
Bradford assay and, 188
BCA assay and, 119
Phospholipids, Lowry assay, 84
Phosphothreonine, racemization,
418
Pierce Warriner LTD, 207
Pinot Noir, Bradford assay, 211
Pioneer 3953 (see Maize), 435
Plant colors, Biuret assay and, 55
Plant metabolites, Bradford assay,
188189
Plant proteins, 221
Amido Black 10B binding, 130
Plasticizer, water as, 420
PNV (protein nutrient value), 341380,
411
amino acid losses and, 344345
animal tests, 348354
assessment by PDCAAS, 434
bioassays, 346354
Index
[PNV (protein nutrient value)]
consumer factors and, 343
factors affecting, 343
food labeling and, 344
human assays and, 346348
hygiene and, 343
indicators for, 342
infant formulas and, 384
insect infestation and, 400
Kjeldahl analysis and, 349
legumes, 393398
literature, 345, 348349
meat and bone meal, 393
pregnant and lactating women and,
344
processing and, 381402
quality control and, 344
rat bioassay, 348
soaking and, 395
Polyacrylamide gels, CBBG stain, 170
Polyamino acids, dye binding and, 171,
181, 183
Polyclonal antibody production, 232
Polyethylene glycol, 132
Polymer chemistry, 420
Polymer science approach, 424
Polymerase chain reaction, 224
Polyols, 133
Polypeptide selectivity, Bradford assay
and, 171
Polyphenols, Bradford assay and,
209210
Polysaccharide interference, Bradford
assay, 186, 191, 199
Polyvalent antigen, 229
Porcine rind protein, 27
Pork
analysis, PRIME, 237
dye binding assay, 157
ELISA, 267268
thermostable antigen, 238
Potato protein
Bradford assay, 214216
chemical score, 429
Kjeldahl analysis, 13
459
[Potato protein]
Lowry assay, 88
Poultry analysis, PROFIT, 237
Poultry, ELISA for, 266267, 268
PPPD index, 432
Precision, protein assays and, 5
Preservatives, milk, 148
Prionics AG, 273
Procedure
biuret assay, 49
Bradford assay, 195
Lowry assay, 70
PDCAAS and, 433434
soy protein antigen preparation, 284
Udy assay, 128
Processed foods, 2
digestibility, 370371
gluten detection in, 310, 320
interferences in, 80
Kjeldahl analysis, 16
PROFIT, 235
Pro-meter instrument, Udy assay, 153
Propionic anhydride, acylation, 363
Protein
analysis, signicance, 2
assay, characteristics, 2
binding, CBBG, 173, 175, 177
binding, dyes, 135
concentrates, 386393
digestibility, 366, 411
digestibility corrected amino acid
score, (see PDCAAS)
efciency ratio (see PER)
haze, 209
immunoassay, 225
M, shrimp antigen, 269
molecular weights, 57
nutrient value (see PNV)
precipitation, Bradford assay, 199
carbonyl-amine reaction and,
417418
factors inuencing, 343
milk, 381
segmental mobility, digestibility and,
413
460
[Protein]
stain, CBBR, 170
Protein-protein-variation,
BCA assay, 105
Bradford assay, 197, 201, 212
Protein-quinone interactions, 210
Proteolysis, Lowry assay, 9091
Purication, Coomassie Brilliant Blue,
200
QPM (see Maize), 435
QSDS-PAGE, 7
Quantitative amino acid analysis, pork
rind, 27
Quantitative sodium dodecylsulfate
polyacrylamide gel
electrophoresis (see QSDSPAGE)
Racemization
amino acids and, 418419
cysteine, 418
factors controlling, 418419
rate prediction, 418
Radcliff Inrmary, 322
Radioallergo absorbent test (see RAST)
Rapeseed
heat damage, 392
meal, PNV, 390392
protein, Udy assay, 154155
RAST, 299
Rat bioassay, 411
Raw meat, ELISA, 252, 255
R-Biopharm, ELISA test kit, gluten,
320
Reactive lysine (see also Available
lysine), 356
Red snapper, 270
Red wheat, Udy assay, 153
Relative nutritive value (see RNV)
Relative protein value (see RPV)
Reliability
Kjeldahl assay, 7
Udy assay, 152
Renaturing buffer, 284
Index
Reverse phase high pressure
chromatography (see RP-HPLC)
Reviews
celiac disease, 313
dye binding, 126127
peanut allergy, 306
protein quality, 345
Rice, 402, 435
beans, protein, 395
protein
biuret assay, 59
Udy assay, 153
PDCAAS value, 435
PNV and, 399
RNV, 342
Rock shrimp, 260270
RP-HPLC
available lysine, 361
gliadin analysis, 327
RPV, 342
Salt effects, Bradford assay, 201
Sample calibration, 4, 7
Sample pelleting, Dumas assay, 33
Sample pretreatment
beer, Bradford assay, 205
cereals, 55
corn meal, BCA assay 117
Lowry assay and, 8687
meat, 261
Biuret analysis, 61
ELISA, 285
Plasma, 49
soy bean product, 284
Sardine, 268
Sausages
dye binding, 159
gluten and, 313, 321
Immunodiffusion assay, 239
Kjeldahl analysis, 17
soy protein, 284, 289, 290
Udy assay, 159
Scenario, dye binding, 141143
Schecter and Berger scheme, 414
Screening, high-lysine cereals, 401402
Index
SDS, Bradford assay, 187
SDS-PAGE, 300, 307, 309
antigen analysis, 237
peanut allergens, 309
wheat allergen, 315317
Seal meat, immunodiffusion, 239
Secondary structure, Gliadin, 317
Seed globulins, ELISA and, 291
Seharawi, celiac disease and, 312
Semolina, Dumas assay, 38
Sensitivity, 1, 5
BCA assay of copper, 102
biuret assay, 55, 56
Bradford assay, 177, 179, 183185,
197, 201204
colorimetric Kjeldahl assay, 18
dye binding, 131,132, 133
gluten ELISA, 329
immunoassay, 227
Lowry assay for wheat protein, 89
meat ELISA, 260
protein analysis, 1, 2, 56
Udy assay, 137
Serine, racemization, 418
Sesame our, Udy assay, 155
Sheep antigen, 231
Sheep, ELISA, 249, 252, 255256, 257,
260, 261, 262, 268
Sheep serum albumin (SSA), 253
Shrimps, 269, 300, 301
Single cell protein, Lowry assay, 91
Skimmed milk
dye binding assay, 151
Kjeldahl factor for 12
Slope assay, protein quality, 347
Small animal bioassay, 348
Soaking, PNV and, 395
Sodium borohydride, available lysine,
365366
Sodium dodecyl sulfate (see SDS)
Solid phase assay
Bradford assay, 185
Udy assay, 131133, 143
Solubility relations, Udy assay, 143
Soluble complexes, Udy assay, 138140
461
Sorghum, 399400
biuret assay, 60
Soup, gluten detection in, 322
Soy
bean allergens, analysis, 303305
ELISA
cooking and, 283
specicity, 283, 291
our, Udy assay, 153
products, ELISA, 289
protein, 285286
in sausages, 289
thermal denaturation, 286287
protein antigen, 284, 285
Soybean
ELISA, 281296
7S protein, 285289
Udy assay, 138
Soybean meal, Orange G binding, 389
Soybean protein, available lysine, 360
Soymilk, 282
Species identication eld test (see
SIFTS), 235
Specicity
denition, 3
gluten, ELISA, 318, 325, 326
meat ELISA, 256, 263
peanut ELISA, 311
soya bean ELISA, 283
Spray dried milk, 382383
dye binding, 129, 130,147
PNV, 382
Udy assay, 129
Sprouted cereals, protein quality, 345
St. Bartholomew's Hospital, 322
St. James University Hospital, 322
Standard assay, Bradford, 196
Starch interferences, 55
Statistical principles, 4
Steaming, PNV and, 395
Stiffness parameter, 423
Stout, Bradford assay 207
Structure
Acid Orange 12 dye, 136
Acid Red 1, 135
462
[Structure]
Amido Black 10B, 137
bicinchoninic acid, 100
biuret, 47
CBBG, 170
CBBR, 170
copper-biuret complex, 47
dehydropeptide, 75
iminopeptide, 75
Orange G, 136
T-azo-R, 135
Sudanese legumes, PNV, 394
Sulfhydryl compounds
BCA assay and, 118
Lowry assay and, 8485
Sulfhydryl group, dye binding, 126
Sulfhydryl-disulde exchange, 317, 415
Symptoms
celiac disease, 312313
food allergy, 298
peanut allergy, 305
T-azo-R, 134
TCA-DOC precipitation
Bradford assay and, 118, 195, 199200
Lowry assay and, 72
TDC (total digestibility coefcient), 342
Tectator heating block, 21
Test kits, species identication and,
235236
Tg (glass transition temperature),
420425
sh protein hydrolysate, 423
gluten, 421
moisture and, 422
protein quality and, 420425
proteins and peptides, 421
Theory (see Mechanism)
Thermal
conductivity detectors, 30
denaturation, peanut allergen,
308309
stability, meat antigens, 263265
Thermostable antigen, 238
meat and, 261
Index
[Thermostable antigen]
soybean and, 289292
Three enzyme assay, in vitro
digestibility, 369372
TNBS assay, 119, 356, 361362, 365,
369
Tomato protein, Dumas assay, 30
Tomato seed, Lowry assay, 88
Tortillas, 399
Total digestibility co-efcient (see
TDC)
TPD (see True protein digestibility)
Trace allergens, 297
Trinitrobenzene Sulfonic acid, (see
TNBS)
Troponin, meat antigens and, 236,
238239
Troponin T, ELISA antigens and, 261
True protein digestibility, 367, 433
Trypsin inhibitors, protein digestibility,
372
Tuna, 268
PER value for, 352
soy protein in, 305
Turkey sausages, 239
Two-state denaturation, 412, 414415,
425
Tyrosinase, copper detection in, 99
Udy assay, 125126, 138
barley, 152
cereal proteins, 151
difculties, 130
dye-protein solubility and, 143
equations for, 141142
sh meal, 138, 156
mechanisms of, 133137
milk protein, 147151
reliability, 152
sausages, 159
various commodities, 128
UHT milk, protein quality and, 382
Ultraltration, 211
Uncooked meat, ELISA, 254257
immunoassay, 234
Index
Water
ammonia determination in, 1920
PER and, 350, 352
Water activity, 383384
Maillard reaction and, 383384, 417
Water holding, 281
Water supply, Kjel-Foss instrument and,
15
Weaning foods, PNV, 397398
Western transfer, 273
Whale meal, 387
Wheat, 345
allergens of, 314317
allergy, 312313
463
[Wheat]
biuret assay, 53, 5760
dye binding assay, 151
reliability of analysis, 7
Udy assay, 151
Whey protein, Bradford assay, 217
Whole milk, dye binding, 149
William-Landel-Ferry kinetics, 420
Wine, Bradford assay, 190, 209212
Yeast protein, Lowry assay,
6364, 91
Zein, biuret assay, 49