Multiciencias

ISSN: 1317-2255
Revistamulticiencias@gmail.com
University of the Western Cape
Venezuela

Camacho, Maria; Torres, Gabriel; left, Peter; Benítez, Betty; Allara, Maria; Céspedes, Euclimar
Determination of biogenic amines in beers that are expended in Maracaibo city-
Venezuela
Multiciencias, vol. 7, No. 3, September to December, 2007, pp. 276-287
University of the Western Cape
Fixed point, Venezuela

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Biological Sciences

MULTICIENCIAS , Vol. 7, No. 3, 2007 (276 - 281)

ISSN 1317-2255 / Dep. legal pp. 200002FA828

Determination of biogenic amines in beer

That are expended in the city of Maracaibo

Maria Camacho 1 , Gabriel Torres 3 2 , Pedro Izquierdo , Betty Benitez 5

4 , Maria Allara
and Euclimar Grasses 6
1 Associate Professor of the Faculty of Medicine. School of Bioanalysis.
Department of Morfofisiopatologia. University of Zulia. E-mail: mcarolca@gmail.com. 2
Associate Professor of the Faculty of Veterinary Science. Department of Biochemistry.
University of Zulia. 3 Professor of the Faculty of Veterinary Science. Department of
Biochemistry. University of Zulia. 4 Associate Professor of the Faculty of Medicine. School of
Bioanalysis. Departamentro of Morfofisiopatologia.
University of Zulia. 5 Associate Professor of the Faculty of Veterinary Science.
Department of Biochemistry. University of Zulia. 6 MgSc. in Food Technology.

Summar
y

The amines are produced in the beers by micro-organisms capable of development-rrollarse

in conditions of low pH and dissolved oxygen, which characterize these drank-alcoholic
beverages. The objective of this research was to determine the concentration of amines bio-in
different types and brands of beers that are produced and sold in Venezuela. It is-leccionaron
bottled beers type Light and Pilsen, three processing plants (A, B and C). We analyzed 36
samples (6 per plant and type), and it was determined the concentration of amines by High
Performance Liquid Chromatography. Only two amines were detected: Histamine, present in
83.3% of the analyzed samples, while Tryptamine is found in a 94.4% of these. In the plants B
and C was detected a frequency of 100% of histamine and Triptami-na for the types Light and
Pilsen, while the plant to 50% presented 83.3% of histamine and of tryptamine. The Plant "A"
revealed the lowest concentration of histamine corresponding to 7.766 parts per million (ppm),
the Plant "B" showed the highest concentration of histamine (25.678 ppm), and tryptamine
(22.508 ppm), while the 18.198 tryptamine concentration (ppm) was lower in the plant "C". No
significant differences were observed with regard to the concentration of histamine and
tryptamine for Light and Pilsen. It was concluded that the content of tryptamine-na and Histami
exceeded the limits reported in the literature for alcoholic beverages (2 ppm), which could cause
health problems in the consumer.

Keyword: Beer, amines, high-performance liquid chromatography.

Received: 09-02-2006 / Accepted: 08-11-2007

MULTICIENCIAS VOL. 7, No. 3, 2007 (276 - 281) / CORE FIXED POINT - TheThere
Cape
Introduction
The beers have been commonly reported as a health risk
due to the content of biogenic amines. These substances are
derived from the des-carboxylation of amino acids or by the
transamination of aldehydes by aminoacyl trasnsaminasas,
being normal troop constitution of many foods and drinks
and have been found in wines, beer, fish and fish products
car-trials as a result of the processes of degradation and fer-
mentation enzyme preparation. Its presence in high amounts
in foods is associated with deterioration. However, low
concentrations of amines are essential to many
physiological functions, however, at high concentrations
can cause some harmful effects such as hypo- and
hypertension, nausea, heart-throbs, poisoning kidney and in
more severe cases of intracranial hemorrhage and death [8,
14].
University
are ofseveral
the Western
factors that influence the content of
amines in beer, as are the variety of barley and germination
conditions, addition of potassium bromate, drying
procedures-roasting of Malta, percentage of complementary
flours, concentration of hops and wine, as well as procedure
of filling and bacterial growth [1].
Under normal conditions on human bio-exogenous
amines absorbed from food are quickly detoxification-
decades by the action of the enzyme monoamine oxidase
(MAO) inhibitors or diamine oxidase (DAO), but in the case
of people who are allergic or treated with a MAO inhibitor,
such as ethanol, the detoxification process is affected and
the amines are accumulated in the body [6, 7].
On the other hand, it has been reported that ethanol can
po-tenciar The effect of histamine because it makes the di-
fusion of the amines through the wall of the stomach and in-
terfiere in the degradation of histamine [5]. This could
explain ex-that the limit of histamine in food is of 100
278 CAMACHO et al. / DETERMINATION OF BIOGENIC AMINES IN BEERS THAT ARE SOLD IN THE CITY OF MARACAIBO, Venezuela

Parts per million (ppm) [15], while drinks al-coholicas has A standard of 40 ppm of amines, dissolved in perchloric
suggested a limit of 2 ppm [9]. acid 0.4 M. The derivatization of the standards was made in
The consumption of beer in Venezuela is similar to the the same way as samples.
high consumption of wine in Europe, some authors indicate The chromatography equipment specifications
that it is important to control and monitor the with-had of We used a team of High Performance Liquid
these amines in these drinks, because they can au-increase ®
Chromatography (HPLC) Shimadzu , constituted by a
the problems of public health [4]. The aim of this research val-uvula automatic injection, a controller system SCL-6B,
was to determine the concentration of amines in different two pumps LC-6A, a reverse phase column RP-18
types and brands of beers that are produced and sold in ®
LICHROSPHER Merck , of 12.5 cm in length by 4.5 mm
Venezuela. internal diameter placed at room temperature environment,
a UV-VIS detector SPD-10A, 254 nm and 0.08 AUFS. For
Materials and Methods the analysis of the data was used the soft-ware
chromatographic Class VP.
Sampling The mobile phase to was composed of master-Acetate nio
Samples were selected from two types of beers em- 0.1 M, and acetonitrile (50:50) and the mobile phase B only by
botelladas: Light and Pilsen, obtained from three plants acetonitrile. Using a linear elution program with gra-tooth,
process-sadoras of the country, called plant, plant B and where the mobile phase was in a percentage of 80% in the first
plant C, acquired in supermarkets, liquor and abastos ubi- 19 min, 19 to 22 min in a 20 per cent and 22 to 32 min at 80%,
tered in the city of Maracaibo, Venezuela. The beers were with a flow rate of 0.7 mL/min. [6].
collected from different batches of production between the Quantification of Amines
months of October 2002 to March 2003. For the study gave To determine the concentration of amines
a total of 36 samples at a rate of 6 per plant and type of In the samples, comparing the retention time of each
beer, and triplicate were analyzed to determine the Sample and reference standard for the area,
concentration of amines. By performing the calculation according to the following
formula:
Determination of the amines CM= AM /Astd x Cstd x Fd
Removal of the amines Where:
It was carried out following the methodology of Moret et CM = concentration of the sample, expressed in ppm Am
al = Area of the sample
[17]. A volume of 10 mL of beer was degassed by Standard = concentration of the CSTD, expressed in ppm
ultrasonication, adding 10 mL of a mixture of n-butanol and Astd = Area of the standard
chloroform 1:1 (v/v). The same was placed 4
Fd = dilution factor (5 x10 )
®
In a mixer Lab-Line for 30 minutes, and subsequently
transferred to a separating funnel. To 1mL of the organic Statistical Analysis
phase is added 2 drops of HCl 1M, aforan-dose to 10 ml
with acetonitrile. The data obtained were analyzed using the statistical
Formation of Fluorescent Derivatives package SAS-pa. Analysis of variance (ANOVA) on the
Of the sample previously removed is measured 1mL, the basis of a completely randomized experimental design, with
Which were added 200 L of a solution of NaOH 2N and 300 the aim of determining differences between treatments
L of a buffer of NaHCO 3 saturated. Followed-mind were applied, with a significance of 0.05 [19].
added 2 mL of a solution of chloride dansilo in acetone Statistical Model:
(1mg/mL) and the mixture was carried out at 40°C for 45 And ijk= μ + Pi + Tj + (PT)ij + ijk
minutes. where
Subsequently removed the chloride dansilo resi-dual Yijk = response variable (amines)
adding 100 L of pure ammonia. The 30 minutes-cough this Tj = treatment effect (type of beer)
mixture was adjusted to a volume of 5 mL with acetonitrile Ijk = experimental error
and centrifuged for 5 minutes at 2.500 rpm P= Breweries
®
A centrifuge Dynac Model brand Clay Adams . This
derivative is injected 20L in the HPLC.
MULTICIENCIAS VOL. 7, No. 3, 2007 (276 - 281) / CORE FIXED POINT - The University of the Western
Cape
Results and Discussion
Of the eight amines investigated histamine, tryptamine,
tyramine, cadaverine, feniletila, putrescine, spermidine and
spermine-only histamine, and tryptamine were de-tectadas
by HPLC in samples of beer analyzes, for both types Light
as Pilsen. Various research-Researchers have reported on
beers mainly the presence of histamine and tyramine [2],
however short-zero et al.; [3] and Cortacero et al. [4],
detected histamine, tryptamine, putrescine, cadaverine and
tyramine in different types of beers.
The Table 1, indicates the percentage of histamine and
triptami-na in the 36 samples analyzed. The histamine was
pre-sat on 30 of the samples studied, corresponding to
83.3% of the total, with an average concentration of 19.20
ppm. This concentration can be considered high if
compared with the reports of left- Puli-do et al. [10], in
Spanish beers, where ron is detected concentrations of 0,30-
4.90 ppm. On the other hand these amines exceed the limits
reported by Halasz et al., [9] and Loret et al., [15], as toxic
to alcoholic beverages at concentrations greater than 2 ppm.
The tryptamine was present in 34 of the total number of
samples analyzed, which corresponds to a 94.4%, with an
average concentration of 20.87 ppm, these concentration-
tions of amines obtained in these types of drinks were
higher than those found by Izquierdo-Pulido et al., [11],
Buiatti et al .,[1], and Gloria et al ., [7] who analyzed
samples of beers to the ligh and Pilsen, whose con-
centrations average were under the age of 2 ppm.
The amines in beer could be related to the bacterial
contamination ( Lactobacillus, Pe-diococus ) of the product
during its preparation and storage operation; it has been
pointed out that there is a relationship between the content
of amines and hygienic defects in food processing, and its
possible role as a-dor indicates poor quality of the raw
material [16].
The Table 2, shows the means and significance of the
concentration of histamine and tryptamine variables for the
three plants of beers studied. The administration of
histamine concentration ranged from 7.77 to 25.68 to the
plant and in the plant B. The statistical analysis pointed out
that in the beers of the plant to-concentrations were detected
significantly lower (p< 0.05) with regard to the plants B and
C. These results could be related-related mainly with the
working conditions of each processing plant, either in the
process of
279
Table 1. Concentration of amines in beer
Amines ¯ppm* %
Histamine 19.20 83.30
Tryptamine 20.87 94.4
N = 36.
* Average values expressed in mg/L
Table 2. Concentration* media of histamine and triptami-na
by effect of breweries.
Plant Histamine Tryptamine
To 7.77 to 21.91 to
B 25.68 b 22.51 to
C 24.18 b 18.20 to
Means followed by different letters in the same column, are
significantly different (P<0.05).
* Average values expressed in ppm.
Fermentation or pollution produced by levadu-ras, or to
other factors such as the quality of the raw pri-ma (malt or
barley) [1].
The concentrations of tryptamine ranged between 18.20
ppm for the Plant 22,51C and ppm for the plant A. No
significant differences were found for the concentra-tion of
this amine. The results obtained for tripta-mine differ with
those reported by Kalac et al., [13], whose average range of
tryptamine for beer was of 0,56-1.65 ppm. Equally Buiatti
et al ., [1], reported a tryptamine concentration of 0.1-2.6
ppm in beer-zas alcoholic beverages. The presence and
concentration of this ami-na is associated also to human-
health conditions, the activity of the yeast, microbial
contamination during the preparation of the beer [12], and
the fermentation process [13, 14, 15].
The Table 3, shows the means and significance for the va-
riable histamine, and tryptamine by effect of type (Light and
Pil-sen). No significant differences were observed (p<0.05) in
the concentration of histamine to the type Light (19.01 ppm)
and to the Pilsen (19.40 ppm), which suggests that the
concentration of this amine is the same for both types, indicate-
do perhaps a similar process in which influ-ye the fermentation
process and the amino acids that may be present in the wort
Brewer [12].
Histamine concentrations obtained for the effect type
were compared with Kalac et al., [13], who point to
concentrations up to 1.19
280 CAMACHO et al. / DETERMINATION OF BIOGENIC AMINES IN BEERS THAT ARE SOLD IN THE CITY OF MARACAIBO, Venezuela

Mg/L in the beer type Light and 0.59 mg/L in the Pilsner Table 3. Concentration* media of histamine and triptami-na
beers, in contrast to the results reported by Zee et al [20] by effect of types of beer.
who found in European and Canadian beers a range of 4,50-
7.27 mg/L in the same types of beers. Type Histamine Tryptamine
In both types of beers (Light and Pilsen) The concentration Light 19.01 18.82
of histamine found exceeds 6 mg/L. This concentration can be Pilsen 19.40 22.92
a predisposing factor for the appearance of headaches, Means followed by different letters in the same column, they are mean-
hypotension, and other toxic effects if ingested in quantities of Significantly different (P< 0.05).
more than 10mg/L [15]. * Average values expressed in ppm.
The Table 4, shows the average and significance for the
va-variables histamine, and tryptamine by effect of plant
and type. The site for the types Light and Pilsen submitted Table 4. Concentration* media of histamine and tryptamine
the lowest concentration of histamine (4.62 and 10.91 ppm), by effect of plants and different types of beer.
respectively not detected significant differences among
them, but, if with regard to the plants B and C, which Plant Type Histamine Tryptamine
presented the highest values of histamine. The plants A and To Light 4.62
to
23.13
to

B showed no significant differences between the types of Pilsen 10.91

to
20.68
to

beers, while the C-di-presented significant differences B Light 23.88

bc
16.41
to

(P<0.05) between the Ligth and Pil-sen (28.55 and 19.81 Pilsen 27.48
c
28.61
to

ppm). The variability in the concentration of histamine by C Light 28.54

c
16.93
to
b to
plant-type effect, it may be due to the fact that the samples Pilsen 19.81 19.47
included different brands as well as different batches of the Means followed by different letters in the same column, they are mean-
same brand [14]. This is based on the fact that the Significantly different (P<0.05).
coefficient of variation was high CV= 32.11%. Similar * Average values expressed in ppm.

results to those reported by Gloria and col. [7], where the

concentration of Histami-na presented considerable Conclusions
variations in the different ti-pos of brazilian beers analyzed
(CV=73%, CV=58% and CV= 41%) for the types Ligh and Of the eight amines investigated only two ami-nas,
Pilsen. histamine, and tryptamine were detected in the samples of
beers analyzed.
In connection with the media and significance for the
The histamine was present in 83.3% of the total beer
variable Tryptamine effect types of beer (Table 3), no
samples analyzed, while Tryptamine was in the 94.4 %.
significant differences were observed for the concentra-tion
Of the three breweries studied, the plant, was
of tryptamine for Light and Pilsen (18.82 and 22.92 ppm),
significantly different from the other two, since the beer
respectively, although it has been reported that the
produced presented the lowest concentration of histamine.
tryptamine concentration depends on the type of beer, in
The concentration of histamine and tryptamine present in
addition to the process of brewing, malting, crushing and
fermentation [9]. the finished beer exceeded the maximum limits reported in
the literature which could affect the health of the consumer
During the malting process detected tripta-mine at
to exist a high consumption of beer.
concentrations above 3.5 ppm. In the same vein-tea during
the process of fermentation with concentrations of -4,40
3.00 ppm [15]. Recommendations
The existence of these amines into the beers analyzed is
Determine the capacity of the microorganisms we used
due possibly to the presence of the lac-acid bacteria in the
as starter cultures in the production of biogenic amines.
fermentation process. During the drafting process,
anaerobic conditions can be opti-more for the synthesis of
enzymes descarboxilasas teria-lactic acid bacteria
Lactobacillus and Micrococcus spp . [11].
MULTICIENCIAS VOL. 7, No. 3, 2007 (276 - 281) / CORE FIXED POINT - The University of the Western
Cape
Apply good manufacturing practices and include
election-quality control from the collection of the raw
material and during the brewing process.
Establish concentration limits for Bio-amines in
Venezuelan Beers.
Determine the microorganisms involved in the
production of biogenic amines in beers.
Thanks
The authors wish to express their appreciation to the
Council of Scientific and Humanistic Development of the
Univer-sity of Zulia (counts-light), by the contribution of
the equipment used, and financing of research.
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