Journal of Food Engineering 73 (2006) 93–100

www.elsevier.com/locate/jfoodeng

Amylase production by Aspergillus niger in submerged cultivation

on two wastes from food industries
Mabel Salas Hernández a, Marilú Rodrı́guez Rodrı́guez a,
Nelson Pérez Guerra b,*, Renato Pérez Rosés c
a
Departamento de Fundamentos Quı́micos y Biológicos, Facultad de Ingenierı́a Quı́mica, Universidad de Oriente Sede ‘‘Julio A. Mella’’,
90900 Santiago de Cuba, Cuba
b
Departamento de Bioquı́mica, Genètica e Inmunologı́a, Facultad de Ciencias de Ourense, Universidad de Vigo, As Lagoas,
s/n. CP 32004 Ourense, Spain
c
Facultad de Farmacia, Universidad de Oriente, Sede Central, 90500 Santiago de Cuba, Cuba

Received 6 December 2004; accepted 14 January 2005

Available online 5 March 2005

Abstract

Synthesis of amylase and protease by Aspergillus niger strain UO-1 was followed in media prepared with brewery (BW) and meat
(MPW) wastewaters supplemented with different starch concentrations. The highest amylase (70.29 and 60.12 EU/mL) and protease
(6.11 and 6.03 EU/mL) production were, respectively, obtained in the BW and MPW media supplemented with 40 g of starch/L of
medium after 88 h of fermentation. In addition, the initial chemical oxygen demand (COD) in both wastes was reduced by more
than 92%.
High amylase and protease activities were found in the BW medium supplemented with casaminoacids, peptone or yeast extract,
but ammonium nitrate and sodium nitrate were also good nitrogen sources for amylase production. The stabilities of amylase and
protease were higher at 50
C and pH 4.95 and at 53.4
C and pH 3.87, respectively, but they were highly sensitive at temperatures of
70
C or higher.
 2005 Elsevier Ltd. All rights reserved.

Keywords: Aspergillus; Amylase; Protease; Meat processing wastes; Brewery wastes; Chemical oxygen demand (COD)

1. Introduction represent a serious environmental problem in Santiago

The meat and brewery industries produce large quan-
tities of wastewaters with a high chemical oxygen de-
mand (COD) and biochemical oxygen demand (BOD).
The untreated effluents of the ‘‘Hatuey’’ brewery with
a BOD (500–2600 mg/L), COD (780–3500 mg/L) and
untreated wastes from the meat processing plant with
a BOD (600–3000 mg/L) and COD (800–4000 mg/L)
*
Corresponding author. Tel.: +34 88 387 062/62 697 0159; fax: +34
88 387 001.
E-mail address: nelsonpg@uvigo.es (N.P. Guerra).
de Cuba. Therefore, it is necessary to develop clean tech-
nologies for treating these effluents.
The possibility of depuration of other wastes from
food industry, by using them as substrates for various
bioproductions of potential economic interest has been
reported before (Guerra & Pastrana, 2003; Murado
et al., 1993; Roukas, 1999). Mussel processing wastes
were satisfactorily used for producing single cell protein
and a highly stable amylolytic preparation from different
Aspergillus strains (Murado et al., 1993). The biological
treatment of these wastes was found to be an efficient
way for reducing their initial COD by more than 90%
(Murado et al., 1993).

0260-8774/$ - see front matter  2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2005.01.009
94 M.S. Hernández et al. / Journal of Food Engineering 73 (2006) 93–100
The Aspergillus species produce a large variety of
extracellular enzymes of which amylases and proteases
are of significant industrial importance (Pandey et al.,
2000). Amylases have not only been used in fermenta-
tion processes, but also in processed food industry
and in the textile and paper industries (Ellaiah,
Adinarayana, Bhavani, Padmaja, & Srinivasulu, 2002;
Gigras, Sahai, & Gupta, 2002). Proteases have found a
wide application in several industrial processes such as
in cheese production, meat tenderization, in the baking
industry, and in many other fields, including textiles
and leather industries and as an additive to detergents
(Bai, Ge, & Zhang, 1999).
Considering the substantial availability of highly rich
wastewasters at very low prices by local brewery and
meat processing plants of Santiago de Cuba, the use of
these wastes as culture media for different bioproduc-
tions could provide a profitable substrate for a low cost
production, with advantage of an effective reduction of
their initial COD. However, there is not information
available on the use of brewery and meat processing
wastes for the production of industrial enzymes by
Aspergillus niger.
The aim of this study was to examine the possible uti-
lization of both brewery and meat processing wastes of
Santiago de Cuba for amylase production by A. niger
strain UO-1 under submerged culture conditions.
Because Aspergillus strains are also capable of produc-
ing proteases (Punt et al., 2003), dynamic changes of
these enzymes were followed along with amylase
production in both media. The effects of the initial
starch levels and the type of nitrogen source on the
production of both enzymes were studied. The effects
of pH, temperature and metal ions on the stability of
both enzymes were also investigated. In addition, the
reduction in COD in both wastes after biological treat-
ment was determined.
2. Materials and methods
2.1. Microorganisms
A. niger strain UO-1, was acquired from the Biotech-
nology Center of the University of Oriente (Santiago de
Cuba, Cuba) and it was maintained on potato dextrose
agar slants at 4
C (Ellaiah et al., 2002).
2.2. Inoculum preparation, culture media and
fermentation conditions
Inocula were prepared by transferring 2-mL of 60-h
old slant culture in 50 mL of medium (250 mL Erlen-
meyer) composed by (g/L): glucose, 20; (NH4)2SO4,
6.6; KH2PO4, 3.5; FeSO4 Æ 7H2 O, 0.15; MgSO4 Æ 7H2O,
0.10; MnCl2 Æ 2H2O, 0.45 and mycological peptone, 3.0;
at pH 6.8. The culture was incubated at 30
C/48 h
(220 rpm).
Brewery (BW) and meat processing (MPW) wastewa-
ters, which were used as a base of the culture media,
were obtained from a local brewery and a local meat
processing plant. Both wastes were centrifuged at
12,000·g/15 min to remove the solids in suspension.
The supernatant obtained from BW contained (g/L):
COD, 3.40; total sugars, 1.98; reducing sugars, 1.46;
total nitrogen, 0.095; total phosphorous, 0.034. The
composition of the supernatant obtained from MPW
(g/L) was: COD, 3.00; total sugars, 1.82; reducing sugars,
0.99; total nitrogen, 0.172; total phosphorous, 0.028.
The production media were obtained by supplement-
ing these supernatants with the following nutrients (g/
L): mycological peptone, 3.0; (NH4)2SO4, 6.6; CaCO3,
8.0; NaCl, 5; KH2PO4, 3.5; FeSO4 Æ 7H2O, 0.15;
MgSO4 Æ 7H2O, 0.10. To study the influence of the initial
concentration of starch on amylase and protease pro-
duction, the media were supplemented with soluble po-
tato starch to obtain initial starch concentrations of 10,
20, 30 and 40 g/L. Media without starch were used as
controls. The media were adjusted at pH 6.0 and steril-
ized (121
C/15 min), inoculated with a 2% inoculum
level (3 · 107 spores/mL) and incubated at 30
C/88 h.
All submerged cultures were carried out in 250-mL
Erlenmeyer with 50 mL of production media in an
orbital shaker (200 rpm), using eight flasks in each fer-
mentation series.
The culture samples, which comprises an experimen-
tal unit (one flask) were collected each 12 h. Mycelial
mass was harvested by paper filtration using a pre-dried
and pre-weighted Whatman filter paper No. 1, washed
with distilled water and dried to constant weight at
105
C. The growth of the organism was determined as
dry weight. Paper-filtered media were used to perform
analytical determinations (pH, enzyme production, total
sugars, nitrogen and phosphorous and COD).
Methods for determination of total sugars, nitrogen
and phosphorous were described or referred to in a pre-
vious work (Guerra & Pastrana, 2003). COD analyses
were carried out in the paper-filtered media at the end
of each fermentation using the closed reflux colorimetric
method as described previously (Greenberg, Connors, &
Jenkins, 1980). All determinations were carried out in
triplicate.
2.3. Assays of amylase and protease activity
Total amylase activity (TAA) was determined accord-
ing to Murado et al. (1993) mixing 80 lL of paper-
filtered medium (suitably diluted) with 400 lL of
0.15 M citrate-phosphate buffer; pH 5.0 (1 volume)
and 4% soluble starch (1.5 volumes) previously main-
tained at 40
C/15 min. The reaction mixture was incu-
bated at 40
C for 10 min. The reaction was stopped
 M.S. Hernández et al. / Journal of Food Engineering 73 (2006) 93–100
by addition of 480 lL of dinitrosalicylic acid, and the re-
leased glucose was determined by 3,5-dinitrosalicylic
acid reaction (Bernfeld, 1951). One unit of amylase
activity (enzymatic units (EU)/mL) was defined as the
amount of enzyme that releases 1 mg/mL of reducing
sugars (glucose equivalents) under the assay conditions.
Protease activity of cell-free medium was determined
by a modified Ansons method (Yang & Wang, 1999).
The reaction mixture with 1 mL of 1% (w/v) casein in
0.02 M NaOH and 2 mL of 0.4 M phosphate buffer
pH 6.0 and 1 mL of cell-free medium (suitably diluted)
were incubated at 30
C/10 min. The reaction was
stopped with 3 mL of 10% trichloroacetic acid, mixed
and after 5 min, centrifuged at 12,000· for 5 min, then
0.5 mL of the supernatants were incubated with
2.5 mL of 0.1 M NaOH in 2% (w/v) Na2CO3 for
10 min. Thereafter 0.25 mL of Folin phenolic reagent
(commercial solution diluted 1:1 in distilled water) were
added, mixed and stayed for 30 min at room tempera-
ture. The absorbance measured at 750 nm was converted
to mg of tyrosine/L using a calibration curve (mg tyro-
sine/L vs absorbance). The tyrosine solutions dissolved
in 0.01 M HCl, were treated in the same conditions as
the cell-free medium. One unit of protease (enzymatic
units (EU)/mL) was defined as the amount of enzyme
that produced an absorbance at 750 nm equivalent to
1 lmol of tyrosine in 1 min under the assay conditions.
2.4. Effect of temperature and pH on stability of
amylase and protease
A second-order factorial plan, using a rotatable de-
sign (Box, Hunter, & Hunter, 1989) based on five levels
and two variables was used to study the combined influ-
ence of pH and temperature on the stability of both
amylase and protease. The design consisted of 13 exper-
iments with four (22) factorial points, four axial points
to form a central composite design with a = 1.267 and
five center points for replication.
Samples of cell-free medium containing the enzymes
were buffered at different pH with the appropriate buffer
(0.1 M potassium hydrogen phthalate-HCl buffer for pH
2.3, 3.0 and 5.5; 0.1 M Tris–HCl buffer for pH 8.0 and
8.7). The buffering agents were prepared as concentrated
stock solutions. Then, appropriate volumes of them
were mixed with the supernatant samples to obtain a
final buffer concentration of 0.1 M. The samples were
incubated for 10 min (for protease) and 5 min (for amy-
lase) at the corresponding temperature according to the
experimental matrix defined by the design used (Table
2). The enzyme activities obtained were corrected with
the corresponding dilution factor.
Results were analyzed by Experimental Design
Module of the Statistica sofware package (Statistica
for Windows computer program manual; StatSoft Inc.
Tulsa, OK, USA). The response surfaces were plotted
95
using the DeltaGraph sofware, version 4.0 (SPSS, Inc.,
Chicago, IL, USA).
2.5. Effect of metal ions on amylase and protease activity
Samples of cell-free medium were incubated with
each compound (at 10 mM final concentration) for
30 min in the optimum conditions of temperature and
pH determined in the previous assay. After the incuba-
tion the remaining activity was determined by the corre-
sponding enzyme assay. The residual enzyme activity
was expressed as a percentage of the activity in control
samples without reagent (Aquino, Jorge, Terenzi, &
Polizeli, 2003). Individual experiments were performed
in triplicate. Data sets were analyzed by analysis of
variance (ANOVA) on SPSS 8.0 for Windows.
3. Results and discussion
3.1. Amylases and proteases production in media prepared
with brewery and meat processing wastewaters
supplemented with starch
Amylase production by A. niger strain UO-1 was fol-
lowed in media prepared with brewery and meat pro-
cessing wastewaters supplemented with starch. The
time course of growth, pH, total sugars and enzymes
(amylases and proteases) production is shown in Fig. 1.
The levels of amylase and protease enzymes obtained
were markedly dependent on the concentration of initial
starch concentration in the culture media (Fig. 1).
Increased amylase and protease levels were obtained in
the media supplemented with starch when compared
to the control cultures (BW and MPW media without
starch).
The high initial concentration of substrate (40 g of
starch/L) in the media favored cell growth, as the pro-
duction of both enzymes. Although the maximum level
of amylase was reached in BW medium (70.29 EU/
mL), which was higher than in MPW medium
(60.12 EU/mL), the maximum protease levels (ffi6 EU/
mL) were similar in both media.
During fermentation, the pH initially dropped from
6.0 to 4.0 after 24 h followed by its increase to 7.4
approximately (48 h), and remained constant thereafter.
The concentration of total sugars, nitrogen and phos-
phorous decreased during fermentation, coinciding with
an increase of biomass and enzyme production leading
to a final COD of 0.24 g/L (in the media supplemented
with 40 g of starch/L) or lower (in the media with lower
initial starch concentrations). This indicates that at least
92% and 93% of the initial COD were removed from the
brewery and meat processing wastes, respectively.
Although the culture media prepared with both
wastes were capable of supporting the growth and
96 M.S. Hernández et al. / Journal of Food Engineering 73 (2006) 93–100

Fig. 1. Time course of growth and enzyme synthesis by A. niger strain UO-1 grown in media prepared with brewery (BW) and meat processing
(MPW) wastewaters supplemented with different initial concentrations of total sugars ((d) 0, (s) 10, (h) 20; (n) 30; () 40 g/L). TS: total sugars;
TAA: total amylolytic activity; PA: protease activity; Nt: total nitrogen; P: total phosphorous. The cultures were carried out at 30
C/88 h in an
orbital shaker at 200 rpm.

enzyme production by A. niger strain UO-1 (Fig. 1),
probably the starch was not the best carbon source for
protease production (Prasad, Malik, & Mathur, 1984;
Walker & Campbell, 1983). These authors reported that
the use of glucose and maltose as carbon and energy
source resulted in good growth but low protease produc-
tion, suggesting the use of slow metabolisable carbon
sources to improve protease production.
To determine whether increasing concentrations of
starch caused substrate inhibition on enzyme produc-
tion, the experimental data of total amylolytic and pro-
tease activities obtained at the end of each fermentation
were adjusted to an Andrews-type model:
EAm  S
EA ¼ ð1Þ
K s þ S þ K I  S2
 M.S. Hernández et al. / Journal of Food Engineering 73 (2006) 93–100 97

Fig. 2. Total amylolytic (TAA) and protease (PA) activity levels produced by A. niger strain UO-1 after 88 h of incubation as a function of initial
concentrations of total sugars on BW and MPW media.

where EA (enzyme activity) and S (initial sugars concen- Table 1

tration) are the variables, and EAm (maximum enzyme Effect of the nitrogen source on production of amylase and protease by
activity), KI (inhibition constant) and KS (saturation A. niger strain UO-1
constant) are the parameters in Eq. (1). The values of Nitrogen sources Amylase activity Protease activity
the parameters obtained from this model are showed (0.21 g N/L) (EU/mL) (EU/mL)
in Fig. 2. As can be seen, the Eq. (1) yielded good (NH4)2SO4 (control) 60.0 6.0
fits for the experimental enzyme production data with Yeast extract 81.2 14.1
Casein 19.7 12.1
higher values for the correlation coefficients (R2). Casaminoacids 89.8 11.6
The relationship between the initial starch concentra- Peptone 89.9 13.1
tion and both amylase and protease production was sim- Alanine 40.9 5.9
ply hyperbolic (decreasing yields) and does not show, NaNO3 61.2 5.6
for the initial starch concentrations selected (0–40 g/L), NH4NO3 80.0 5.3
effects of substrate inhibition (KI = 0 in all cases). Simi-
lar results were obtained by Murado, González, Tor-
rado, and Pastrana (1997) for amylase production by in A. oryzae in a medium prepared with mussel process-
A. oryzae in culture media prepared with mussel pro- ing wastes.
cessing wastes. For protease production, the best nitrogen sources
were the yeast extract or peptone followed by the casein
3.2. Effect of nitrogen source on amylase and protease and the casaminoacids in decreasing order (Table 1).
production The protease levels obtained with these nitrogen sources
were two fold higher than those obtained with inorganic
The effect of nitrogen sources on amylase and prote- nitrogen sources ((NH4)2SO4, NH4NO3 and NaNO3)
ase production by A. niger strain UO-1 was studied in and alanine.
BW medium, where previously showed the highest levels
of amylase. In this assay, the nitrogen source 3.3. Effect of pH and temperature on the stability of
((NH4)2SO4) was substituted by yeast extract, casein, enzymes
casaminoacids, peptone, alanine, NH4NO3 and NaNO3
at a nitrogen concentration of 0.21 g/L. Samples of amylase and protease (produced in BW
The highest levels of amylase were obtained when the medium supplemented with 40 g of starch/L), were used
medium was supplemented with peptone, casaminoacids to test their heat and pH stability using a second-order
or yeast extract though, both ammonium nitrate and so- rotatable design. The pH and temperature (T) values,
dium nitrate (commercially cheaper) also increased amy- as well as the time of treatment were ranged at levels
lase production (Table 1). Mahmoud (1993) obtained commonly used in food treatments (Table 2).
the higher amylase production by A. flavus and A. Table 3 summarizes the responses for each individual
fumigatus using glycine and yeast extract as N sources experiment along with the predicted responses. The
whereas Kundu, Das, and Grupta (1973) reported model equations fitted by regression analysis are given
NH4NO3 and NaNO3 as best nitrogen sources for by
maximum amylase production by A. oryzae. Torrado,
TAA ¼ 63:78 þ 3:56T þ 7:13 pH  19:39T 2  15:21 pH2
González, and Murado (1998) reported NaNO3 and
(NH4)2 HPO4 as best N sources for amylase production ð2Þ
98 M.S. Hernández et al. / Journal of Food Engineering 73 (2006) 93–100

Table 2
Experimental domain and codification of the variables used in the
factorial design analysis for the combined influence of temperature and
pH on the stability of amylases and proteases produced in BW medium
Codified values Natural values
T (
C)
1.267 22
1 30
0 60
+1 90
+1.267 98
Increments 1 30
PA ¼ 6:12  0:25T  1:11 pH  1:13T 2  0:98 pH2
where TAA and PA are the residual total amylolytic
activity and proteolytic activity (EU/mL), respectively
and T is the temperature (
C). In both cases, the model
terms T, pH, T2 and pH2 were found to be significant
according to the Student t-test (a = 0.05) and P-values,
meanwhile the interaction term between T and pH vari-
ables (T pH) was found to be non-significant in both
equations (Table 3). Since the P-values in the ANOVA
table (Table 4) are less than 0.05 for both models, there
is a statistically significant relationship between the
variables at the 95% confidence level. The value of the
adjusted determination coefficient (adj. R2) was calcu-
lated to be 0.977 and 0.969 for models (2) and (3),
respectively (Fig. 3), which indicated a high significance
of both models. All of the above considerations indicate
an excellent adequacy of the quadratic models to the
experimental data.
pH The response surfaces obtained from the empirical
2.3 models (2) and (3) are depicted in Fig. 3. Optimal amy-
3.0 lase activity was at pH 4.95 (coded value of 0.22) and
5.5
8.0
50
C (coded value of 0), with visible decrease toward
8.7 low or high values of both variables (Fig. 3). However,
2.5 the maximal protease activity was obtained at pH 3.87
(coded value of 0.65) and 53.4
C (coded value of
0.22).
At optimal conditions both enzymes retained approx-
imately 85% of initial activities after 1 h of incubation.
ð3Þ
Nevertheless, when the amylase and protease samples
were adjusted for optimum pH value but incubated at
70, 90 and 100
C, their initial activities were completely
lost at 30, 22 and 18 min, respectively (Fig. 4).
3.4. Effects of metal ions on stability of amylase and
protease
Table 5 shows the effect of various metal ions on the
enzymic activity of amylase and protease. Amylase
activity was slightly activated by metal ions such as
Ca2+ and Na+, but was strongly inhibited by Cu2+,
Hg2+ and Zn2+ (P < 0.05). Similar results have been

Table 3
Comparison of the observed and the predicted responses (amylase and protease activity after pH and heat treatment) and coefficients of regression
analysis
T pH Amylase activity (EU/mL) Protease activity (EU/mL)
Observed response Predicted value Observed response Predicted value
1 1 39.81 39.87 2.54 2.64
1 1 26.43 25.61 4.94 4.86
1 1 29.15 32.75 3.59 3.15
1 1 18.93 18.49 5.22 5.37
1.267 0 36.37 37.16 4.02 3.97
1.267 0 30.43 28.14 4.40 4.61
0 1.267 51.09 48.40 2.90 3.14
0 1.267 29.12 30.32 6.03 5.95
0 0 60.53 63.78 5.94 6.12
0 0 62.36 63.78 6.12 6.12
0 0 67.53 63.78 6.33 6.12
0 0 63.48 63.78 6.23 6.12
0 0 64.40 63.78 6.02 6.12
Significance of factors
Factor Model (2) Model (3)
Coefficient t-value P-value Coefficient t-value P-value
*
Constant 63.78 55.34 0.000000 6.11 66.98 0.000000*
T 3.56 3.67 0.010328* 0.25 3.27 0.013601*
pH 7.13 7.36 0.000219* 1.11 14.45 0.000002*
T Æ pH 0.79 0.61 0.583885 1.13 12.49 0.103900
T2 19.39 16.92 0.000001* 0.98 10.75 0.000005*
pH2 15.21 13.28 0.000005* 0.19 1.87 0.000013*
*
Significant at P < 0.05 or t (a = 0.05; t = 4) > 2.78.
 M.S. Hernández et al. / Journal of Food Engineering 73 (2006) 93–100 99

Table 4
Summarized data of analysis of variance (ANOVA) of the empirical models obtained for amylase and protease stability after pH and temperature
treatment according to the experimental design defined in Table 2
Source Sum of squares Degrees of Mean of squares F-value P-value
freedom
TAA PA TAA PA TAA PA TAA PA
*
Regression 3617.65 21.22 8 452.21 2.65 66.82 106.66 0.0005 0.0002*
Residual 27.07 0.10 4 6.77 0.03
Total 3644.72 21.32 12
TAA: total amylase activity, PA: protease activity.
*
Significant at P < 0.05.

Table 5
Effect of metal ions on amylase and protease activity
Chemical Relative amylase Relative protease
activity (%) activity(%)
None (control) 100 100
CaCl2 105.3 116.5
CuCl2 8.6 15.3
HgCl2 6.2 42.5
MgCl2 93.8 111.3
MnCl2 87.1 98.9
NaCl 107.9 102.4
ZnCl2 12.6 34.9

Fig. 3. Response surface corresponding to the stability of amylase

(TAA) and protease (PA) produced on BW medium after temperature
(T) and pH treatment according to the experimental plan defined in
Table 2. T and pH are in coded values.
lus awamori (Anindyawati, Melliawati, Ito, Iizuka, &
Minamiura, 1998), Talaromyces emersonii (Bunni,
McHale, & McHale, 1989), Scytalidium sp. (Odibo,
Okafor, & Okafor, 1992) and Scytalidium thermophilum
(Aquino et al., 2003).
Ca2+, Mg2+ and Na+ ions slightly enhanced A. niger
protease activity as has been observed for Scedosporium
apiospermum proteases (Larcher et al., 1996). Mn2+ ion
had only a slight inhibitory effect, whereas Cu2+, Hg2+
and Zn2+ ions exhibited the highest inhibitory effects
(P <0.05) on protease activity.
The activator metal ions (Ca2+ and Na+) protect the
enzymes against thermal denaturation and play a vital
role in maintaining the active configuration of the
enzyme at high temperatures (Gupta, Beg, Khan, &
Chauchan, 2002).

4. Conclusions

The data showed the potential use of wastes from

Fig. 4. Effect of temperature on the stability of both the amylase and
protease produced by A. niger strain UO-1 in BW medium. The
amylase samples were buffered at pH 4.95 (with 0.1 M potassium
hydrogen phthalate-HCl buffer) and incubated at 50
C (s), 70
C (h),
90
C (,) and 100
C (). The protease samples were buffered at pH
3.87 (with 0.1 M potassium hydrogen phthalate-HCl buffer) and
incubated at 50
C (s), 70
C (h), 90
C (,) and 100
C ().
reported with amylases from strains of Aspergillus ta-
marii (Moreira, Lenartovicz, & Peralta, 2004), Aspergil-
meat processing and brewery plants as culture media
for a large-scale production of both amylase and prote-
ase by A. niger UO-1 besides substantially reducing the
organic load of wastes. However, studies based on the
use of carbon sources from agro-industrial residues
(wheat bran, rice bran, rice husk, maize bran, etc.) for
amylase and protease production are necessary to opti-
mize the production of both enzymes in media prepared
with brewery and meat processing wastes.
100 M.S. Hernández et al. / Journal of Food Engineering 73 (2006) 93–100
References
Anindyawati, T., Melliawati, R., Ito, K., Iizuka, M., & Minamiura, N.
(1998). Three different types of a-amylases from Aspergillus
awamori KT-11: Their purifications, properties, and specificities.
Bioscience, Biotechnology and Biochemistry, 62, 1351–1357.
Aquino, A. C. M. M., Jorge, J. A., Terenzi, H. F., & Polizeli, M. L. T.
M. (2003). Studies on a thermostable a-amylase from the thermo-
philic fungus Scytalidium thermophilum. Applied Microbiology and
Biotechnology, 61, 323–328.
Bai, H., Ge, S. J., & Zhang, L. X. (1999). Total hydrolysis of food
proteins by the combined use of soluble and immobilized protease.
International Journal of Food Science and Technology, 34, 95–99.
Bernfeld, P. (1951). Enzymes of starch degradation and synthesis.
Advances in Enzymology, 12, 397–427.
Box, G. E. P., Hunter, W. G., & Hunter, J. S. (1989). In S. A. Reverte
(Ed.), Estadı́stica para investigadores (pp. 317–361). Barcelona,
España.
Bunni, L., McHale, L., & McHale, A. P. (1989). Production, isolation
and partial characterization of an amylase system produced by
Talaromyces emersonii CBS 814.70. Enzyme and Microbial Tech-
nology, 11, 370–375.
Ellaiah, P., Adinarayana, K., Bhavani, Y., Padmaja, P., & Srinivasulu,
B. (2002). Optimization of process parameters for glucoamylase
production under solid state fermentation by a new isolated
Aspergillus species. Process Biochemistry, 38, 615–620.
Gigras, P., Sahai, V., & Gupta, R. (2002). Statistical media optimi-
zation and production of ITS a-amylase from Aspergillus oryzae in
a bioreactor. Current Microbiology, 45, 203–208.
Greenberg, A. E., Connors, J. J., & Jenkins, D. (1980). Standard
methods for the examination of water and wastewater (15th ed.).
Washington, DC: American Public Health Association.
Guerra, N. P., & Pastrana, L. (2003). Enhancement of nisin produc-
tion by Lactococcus lactis in periodically re-alkalized cultures.
Biotechnology and Applied Biochemistry, 38, 157–167.
Gupta, R., Beg, Q. K., Khan, S., & Chauchan, B. (2002). An overview
on fermentation, downstream processing and properties of micro-
bial alkaline proteases. Applied Microbiology and Biotechnology,
60, 381–395.
Kundu, A. K., Das, S., & Grupta, T. K. (1973). Influence of culture
and nutritional conditions on the production of amylase by the
submerged culture of Aspergillus oryzae. Journal of Fermentation
Technology, 51, 142–150.
Larcher, G., Cimon, B., Symoens, F., Tronchin, G., Chabasse, D., &
Bouchara, J. P. (1996). A 33 kDa serine protease from
Scedosporium apiospermum. Biochemical Journal, 315, 119–126.
Mahmoud, A. L. E. (1993). Different factors affecting growth and
amylase production by fungi inhabiting poultry feeds. Journal of
Basic Microbiology, 33, 187–192.
Moreira, F. B., Lenartovicz, V., & Peralta, R. M. (2004). A
thermostable maltose-tolerant a-amylase from Aspergillus tamarii.
Journal of Basic Microbiology, 44, 29–35.
Murado, M. A., González, M. P., Torrado, A., & Pastrana, L. M.
(1997). Amylase production by solid state culture of Aspergillus
oryzae on polyurethane foams. Some mechanistic approaches from
an empirical model. Process Biochemistry, 32, 35–42.
Murado, M. A., Siso, M. I. G., González, M. P., Montemayor, M. I.,
Pastrana, L., & Mirón, J. (1993). Characterization of microbial
biomasses and amylolytic preparations obtained from mussel-
processing waste treatment. Bioresource Technology, 43,
117–125.
Odibo, F. J. C., Okafor, N., & Okafor, B. U. (1992). Purification and
immobilization of Scytalidium sp. a-amylase and its general
properties. Journal of General and Applied Microbiology, 38,
1–11.
Pandey, A., Nigam, P., Soccol, C. R., Soccol, V. T., Singh, D., &
Mohan, R. (2000). Advances in microbial amylases. Biotechnology
and Applied Biochemistry, 31, 135–152.
Prasad, R., Malik, P. K., & Mathur, D. K. (1984). Optimization of
nutritional and environmental factors for the production of
caseinolytic enzyme of Micrococcus sp. isolated from cheddar
cheese. Asian Journal of Dairy Research, 3, 25–36.
Punt, P. J., Drint-Kuijvenhoven, A., Lokman, B. C., Spencer, J. A.,
Jeenes, D., Archer, D. A., & van den Hondel, C. A. M. J. J. (2003).
The role of Aspergillus niger furin-type protease gene in processing
of fungal proproteins and fusion proteins. Evidence for alternative
processing of recombinant (fusion-) proteins. Journal of Biotech-
nology, 106, 23–32.
Roukas, T. (1999). Pullulan production from brewery wastes by
Aureobasidium pullulans. World Journal and Biotechnology, 15,
447–450.
Torrado, A., González, M. P., & Murado, M. A. (1998). pH regulation
in solid state culture through the initial ratio between oxidized and
reduced sources of nitrogen. A model applicable to the amylase
production by Aspergillus oryzae. Biotechnology Techniques, 12,
411–415.
Walker, W. E., & Campbell, L. L. (1983). Induction of a-amylase of
Bacillus sterothermophilus by maltodextrin. Journal of Bacteriology,
86, 687–691.
Yang, S. S., & Wang, J. Y. (1999). Protease and amylase production of
Streptomyces rimosus in submerged and solid state cultivations.
Botanical Bulletin of Academia Sinica, 40, 259–265.