HPLC quantification of biogenic amines in cheeses: correlation with PCR-detection of tyramine-producing microorganisms.

Abstract

The consumption of food and beverages containing high amounts of biogenic amines (BA) can have toxicological effects. BA found in foods and beverages are synthesized by the microbial decarboxylation of certain amino acids. This paper reports the concentrations of BAs in a number of commercial cheeses, as determined by HPLC. The cheeses studied were made from raw and pasteurized milk of different origin, and were subjected to different ripening periods. BA concentrations were lower in short ripening period than in long ripening period cheeses, and higher in cheeses made from raw milk than in those made from pasteurized milk. The highest BA concentrations were recorded in blue cheeses made from raw milk. Tyramine was the most commonly recorded and abundant BA. The presence of tyramine-producing bacteria was determined by PCR, and a good correlation obtained between the results of this method and tyramine detection by HPLC. These methods could be used to complement one another in the detection and quantification of tyramine in cheese prevention of tyramine accumulation in cheese.

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HPLC quantification of biogenic amines in cheeses: correlation
with PCR-detection of tyramine-producing microorganisms
Marı ´a Ferna ´ndez, Daniel M Linares, Beatriz del Rı ´o, Victor Ladero and Miguel A Alvarez*
Instituto de Productos La ´cteos de Asturias (IPLA-CSIC), Carretera de Infiesto s/n, 33300 Villaviciosa, Spain
Received 24 July 2006 and accepted for publication 20 November 2006
The consumption of food and beverages containing high amounts of biogenic amines (BA) can
have toxicological effects. BA found in foods and beverages are synthesized by the microbial
decarboxylation of certain amino acids. This paper reports the concentrations of BAs in a number
of commercial cheeses, as determined by HPLC. The cheeses studied were made from raw and
pasteurized milk of different origin, and were subjected to different ripening periods. BA
concentrations were lower in short ripening period than in long ripening period cheeses, and
higher in cheeses made from raw milk than in those made from pasteurized milk. The highest
BA concentrations were recorded in blue cheeses made from raw milk. Tyramine was the most
commonly recorded and abundant BA. The presence of tyramine-producing bacteria was
determined by PCR, and a good correlation obtained between the results of this method and
tyramine detection by HPLC. These methods could be used to complement one another in the
detection and quantification of tyramine in cheese prevention of tyramine accumulation in cheese.
Keywords: Biogenic amines, tyramine, cheese, HPLC, PCR.
Biogenic amines (BAs) are low molecular weight organic
bases sometimes present in foods. They are mainly pro-
duced by the microbial decarboxylation of certain amino
acids. BA formation in foods should be controlled since
these compounds are associated with respiratory distress,
headache, hyper- and hypotension, and allergies. These
problems are particularly severe in people with low levels
of monoamine and diamino-oxidase (enzymes belonging
to the BA detoxification system); such deficiencies are as-
sociated with genetic background and certain medical
treatments (Joosten & Northolt, 1987, Hala ´sz et al. 1994).
Although there are no regulations governing the BA con-
tent in most foodstuffs, the European legislation (Directive
91/493/EEC) set a limit for the histamine levels in fishery
products of 100–200 mg Kg–1for fresh fish and up to
400 mg Kg–1for cured products. The US Food and Drug
Administration set a limit for histamine in canned tuna
of 500 ppm (http.//www.fda.gov). Some authors have
suggested a general limit for histamine levels in food of
100 mg Kg–1(Ten Brick et al. 1990).
Histamine, tyramine, putrescine and cadaverine are the
most common BAs in fermented foods. They are mainly
produced by lactic acid bacteria (LAB; Lonvaud-Funel,
2001); their appearance and accumulation are therefore
influenced by the environmental factors (temperature, pH,
availability of substrate, etc.) that affect the growth and/or
decarboxylase activity of these organisms (Valsamaki et al.
2000). Methods that can rapidly detect BA-producing
strains in foodstuffs are required if food quality and safety
is to be assured. Such a capability would also help the
dairy industry inspect raw materials destined for use in
food production.
Cheese is one of the foods most commonly associated
with BA poisoning; indeed, the term ‘cheese reaction’
has been coined to refer to it (Ten Brick et al. 1990). The
microbiota present in cheese has different origins (milk,
starters, and contaminating microorganisms) and its de-
velopment is affected by factors such as the treatment the
milk receives and the ripening conditions. Several authors
have shown the BA content of cheese made from raw milk
to be higher than that of cheese made from pasteurized
milk (Joosten & Northolt, 1987; Ordon ˜ez et al. 1997;
Novella-Rodrı ´guez et al. 2004). Since BAs accumulate in
food products, the duration of ripening is a critical factor
affecting the final BA content (Ordon ˜ez et al. 1997;
Novella-Rodrı ´guez et al. 2003a). In addition, the high level
of proteolysis that occurs in cheeses (which provides the
amino acid substrates for the production of BAs), com-
bined with the acidic environment of this food, favors the
synthesis and activity of decarboxylation enzymes (Joosten
& Northolt, 1987).
*For correspondence; e-mail: maag@ipla.csic.es
Journal of Dairy Research (2007) 74 276–282.
doi:10.1017/S0022029907002488
f Proprietors of Journal of Dairy Research 2007
First published online 30 April 2007
276
Printed in the United Kingdom

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In recent years, a number of techniques based on HPLC,
capillary electrophoresis, etc. have been developed for the
detection of BAs (Cinquina et al. 2004). These methods can
identify the different types in food samples and accurately
quantify them. However, they have the drawback of long
sample preparation times, and are tedious if large numbers
of samples must be examined. Further, raw materials sel-
dom contain BAs, and although these may develop over
time, these methods cannot predict this. Methods for the
detection of BA-producing strains have been proposed
based on differential media that demonstrate their de-
carboxylase activity. However, these methods require the
isolation of the strains and days may pass before a reliable
result is obtained. PCR has also been proposed as a tech-
nique for detecting BA-producing LAB strains, and several
sets of primers for the detection of histamine, tyramine and
pustrescine producers have been proposed (Le Jeune et al.
1995; Coton et al. 2004; Ferna ´ndez et al. 2004; de las
Rivas et al. 2005). Recently, PCR was proposed for the
early detection of tyramine-producing bacteria during
cheese production (Ferna ´ndez et al. 2006).
In this work, the BA content of different commercial
cheeses was analysed by HPLC. PCR detection of tyramine-
producing bacteria was also performed and the results
were compared with those obtained by HPLC.
Materials and Methods
Samples
Sixty one randomly purchased commercial cheeses 48
of them of different Spanish regions and 13 imported
from Europe (France, Switzerland and Italy) were analysed
for their BA content and for the presence of tyramine-
producing bacterial strains. These commercial samples
included cheeses made from different types of milk
(goat, cow and sheep), from raw or pasteurized milk, and
which were subject to different ripening periods (short
<3 months, long o3 months).
Analysis of biogenic amines by HPLC
One gram of cheese was homogenized with 10 ml 0.1 M-
hydrochloric acid solution containing 0.2% 3,3k-thio-
dipropionic acid (TDPA) (Fluka, Madrid Spain) using Ultra-
Turrax homogenizer (OMNI International, Watersburry
USA) for 2 min at 20000 rpm. This mixture was kept in
an ultrasonic bath for maximal 30 min, centrifuged at
5000 g for 20 min, and any top fat layer was removed. The
supernatant was filtered through a 0.45 mm membrane
and 3 ml of the filtrate was deproteinized by passing
through ultrafiltration inserts (Amicon Biomax 5; Milli-
pore) via centrifugation at maximal 3500 g for about 1 h.
Membrane-filtrated liquid food samples were diluted with
0.1 M-HCI 0.2%TDPA 250 mM internal standard and ultra-
filtrated as described above. Twenty microlitres of the
sample were derivatized following the protocol described
by Krause et al. (1995), and 10 ml of the derivatized sample
was injected. The quantitative analysis of the BA content
extracted was carried out by reverse-phase (RP)-HPLC
using a Waters liquid chromatograph controlled by
Millenium 32 Software (Waters Milford, MA, USA) fol-
lowing the protocol of Krause et al. (1995). All the sep-
arations were carried out on Waters Nova-pack C18
column (150r3.9 mm) and detection was performed at
436 nm. Gradient and detection conditions were similar to
those described by Krause et al. (1995).
PCR analysis
Five grams of each cheese sample were homogenized
mechanically in 40 ml 2% sodium citrate in a Stomacher
Lab-Blender 400 (Seward Medical, London, UK) for 1 min.
DNA was extracted from the homogenate following the
method of Ogier et al. (2002); 20 ng of total DNA were
used in each PCR reaction. Two hundred nanomoles of the
oligonucleotides tdc1 and tdc2, designed on the basis of
LAB tyrosine decarboxylase gene sequences (Ferna ´ndez
et al. 2004), were used as primers for the amplification of
an internal fragment of the tyrosine decarboxylase gene.
The PCR conditions involved an initial denaturation
step (95 8C for 5 min), 35 amplification cycles (95 8C
for 45 s, 50 8C for 1 min, and 72 8C for 1 min), and a final
extension step at 72 8C for 7 min. All amplifications
were performed with puRe Taq Ready-To-Go PCR beads
following the manufacturer’s instructions (Amersham-
Biosciences, Buckinghamshire, UK) The amplicons were
separated and visualized on 0.7% agarose gel as described
by Sambrook et al. (1989).
Results
Biogenic amine content of the cheeses
BAs were detected in over 70% of the samples, but
the concentrations were very variable (Table 1). The
average tyramine content of the positive samples was
close to 187.47 mg Kg–1; for histamine the value was
130.92 mg Kg–1. Putrescine, histamine and particularly
tyramine (detected in 42, 52 and 54% of the samples re-
spectively) were the most common and abundant BAs,
reaching maximum values of 876, 1042, and 1052 mg Kg–1
respectively in some cheeses. Other BAs were much less
common, e.g., spermine was detected in only five sam-
ples, and b-phenylethylamine in just three. More than one
type of BA was usually detected in positive samples; only
three cheeses had one type of BA alone (always tyramine).
Table 1 shows the maximum and minimun values, and
averages for each BA and cheese type.
Influence of milk treatment on BA formation
Some 87.5% cheeses made from raw milk were BA-
positive, compared with 68.9% of those made from
Biogenic amines in cheeses
277

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Table 1. Biogenic amines: Maximum and average content (mg Kg–1) in the different classes of cheese analysed. Cheeses were organized based on milk type and length of
ripening period. n: number of cheeses analysed in the category. The averages (shown in grey) were calculated including the samples that had not detected (nd) values. The
data presented in columns ‘tyramine HPLC’ and ‘tdc PCR detection’ correspond to number of positive samples.=not calculated.
n
Tyramine
Max average
Histamine
Max average
Putrescine
Max average
Cadaverine
Max average
b-Phenylalanine
Max average
Spermine
Max average
Tyramine
HPLC
tdc PCR
detection
Short
ripening
period
Raw milkCow
Goat
3nd
63.94
=
233.33
=
nd
nd–233.33
49.54
0–22.02
4.4
nd
nd
110.8
=
102.56
=
nd
nd–110.8
35.36
nd
38.75
=
10.4
=
nd
nd–38.75
6.14
nd
38.9
=
96.34
=
nd
nd–96.34
25.87
ndnd02
1 nd nd11
Sheep
1
48.4
=
nd
11
Mixture1nd01
6
Pasteurized
milk
Cow
5ndnd nd ndnd12
Goat
Sheep
Mixture
1
0
ndnd ndnd nd12
3nd
0–60.2
20.06
0–60.2
6.68
nd–110.8
18.23
nd–96.54
22.84
0–510.2
171.3
0–118.6
49.84
65.18
=
0–510.2
59.47
0–65.42
21.8
0–27.68
13.84
ndndnd nd01
9
0–22.02
2.44
nd–233.33
21.28
nd–279.49
67.66
0–453.77
152.6
0–296.89
132.66
216.85
=
0–453.77
121.98
0–80.9
26.96
0–30.48
15.24
0–301.06
150.53
15
nd–38.75
3.27
nd–176.32
160.99
0–387.4
138.36
0–197.85
127.03
97.68
=
0–176.32
90.27
0–175.39
58.46
0–18.12
9.06
nd–96.34
10.34
nd–135.87
44.81
Long
ripening
period
Raw milkCow
7
nd–40.7
5.81
nd–18.3
12.4
34
Goat
3 nd ndnd22
Sheep
10
0–328.45
85.8
194.2
=
0–328.45
65.04
ndnd 108
Mixture
1 nd
5.7
=
11
21
0–40.7
1.93
Pasteurized
milk
Cow
3ndnd
nd–6.8
2.2
12
Goat
2 nd ndnd22
Sheep
2 nd nd nd nd nd11
278
M Ferna ´ndez and others

Page 4

pasteurized milk. In addition, the BA concentration of
the raw milk cheeses was higher. For example, the
average tyramine content of the long ripening period
raw milk cheeses was 121.98 mg kg–1compared with
33.19 mg kg–1in long ripening period, pasteurized milk
cheeses. For histamine the values were 59.47 mg kg–1and
13.18 mg kg–1respectively. Cadaverine and b-phenyl-
ethylamine were only detected in cheeses made from raw
milk. Moreover, the highest concentrations of all BAs were
recorded in raw milk cheeses (Table 1), with tyramine the
most common. The highest concentrations of this BA were
found in raw milk blue cheeses, both on average
(444.8 mg kg–1) and in terms of maximum concentration
(1051.98 mg kg–1).
Influence of milk origin
To determine the importance of milk origin on the BA
content, the cheese samples were grouped into four cat-
egories: those made from cows’ milk (n=19), sheep’s milk
(n=13), goats’ milk (n=7), or a mixture of milks (n=22).
BAs were detected in almost 70% of the goats’ milk and
milk mixture cheeses, a value very similar to the mean for
all cheeses. In the cheeses made from sheep’s milk, how-
ever, the percentage of positive samples was much higher
at 92%. However, it should be borne in mind that 85% of
the sheep’s cheeses were made from raw milk. BAs were
detected in only 32% of the cheeses made from cows’
milk (only 45% of these were raw milk cheeses). Figure 1
summarizes the results of the BA content of these different
types of cheese. The number of BA-positive cheeses made
from pasteurized milk of all types was similar. However,
the number of positive raw milk cheeses varied signifi-
cantly depending on the origin of milk (sheep>goat>cow).
Pasteurization of the milk is therefore an important first
step in the production of safe cheese.
Mixture
5
0–21.79
0–48.4
0–16.58
nd
nd
1
1
4.34
10.62
3.31
13
0–301.06
0–65.42
0–175.39
33.19
13.18
15.00
33
0–453.77
0–510.2
0–176.32
0–328.45
0–40.7
88.15
41.93
73.33
41.39
1.23
Blue
cheeses
Raw milk
Cow
1
0–188.82
0–210.8
15.4
137.63
nd
1
1
=
=
=
=
Mixture
4
0–1051.98
0–1041.81
0–875.8
0–756.78
0–27.42
4
4
508.89
462.38
236.07
320.85
=
5
0–1051.98
0–1041.81
0–875.8
0–756.78
0–27.42
5
5
444.8
412.06
191.93
284.01
3.04
Pasteurized
milk
Mixture
8
0–526.63
0–127.2
0–237.56
0–489.4
nd
5
5
117.16
56.51
46.74
61.15
13
0–1051.98
0–1041.81
0–875.8
0–756.78
0–27.42
229.57
253.87
95.25
173.57
2.10
0
10
20
30
40
50
60
70
80
90
100
CGSCGS
Type of milk
% of samples with BA
rawpasteurized
Fig. 1. Percentage of samples containing biogenic amines
according to the treatment and the type of milk used. CGS:
cheeses made from a mixture of cows’, sheep’s and goats’ milk;
C cheeses made from cows’ milk; G cheeses elaborated with
goats’ milk, S cheeses made from sheep’s milk.
Biogenic amines in cheeses
279

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Influence of ripening period
To check whether the ripening period influenced the final
BA content, the cheeses were grouped as either short or
long ripening period cheeses. Some 66.7% of long ripen-
ing period cheeses were BA-positive, while only 33.3% of
short ripening period cheeses had detectable BA con-
centrations. The highest BA concentrations were observed
in long ripening period cheeses (more than double that
seen in the short ripening period cheeses). These samples
hadthehighest average
21.28 mg kg–1for short ripening period cheeses) and ab-
solute values (453.77 compared with 233.33 mg kg–1),
both for individual BAs and total BA content (Table 1).
This suggests that proteolysis, and the availability of conse-
quent amino acid precursors, is required for BA pro-
duction, as reported by other authors (Pinho et al. 2004).
The long ripening period raw milk cheeses had the
highest BA concentrations. Remarkably, although in the
long ripening period cheeses the putrescine concentration
was higher than that of cadaverine, in the short ripening
period cheeses the cadaverine concentration was higher
than that of putrescine.
The highest BA concentrations of all were reached in
raw milk blue cheeses (1051.98 and 1041.81 mg kg–1of
tyramine and histamine respectively). In these cheeses,
fungi with strong proteolytic activity might make more
amino acid substrates available to the decarboxylating
enzymes.
(88.15comparedwith
Detection of tyramine producing strains by PCR
Clearly, the presence of BA-producing strains is necessary
for the accumulation of these compounds in cheese. Since
tyramine was the most abundant BA detected, BA-produc-
ing strains were sought by PCR. PCR was used to detect the
presence of tyramine-producing strains in all 61 cheese
samples and the results were compared with the HPLC tyr-
amine detection results. A band of the expected size was
obtained with 41 samples (Table 1). In nine of these, tyr-
amine had not been detected by HPLC; these samples cor-
responded to short ripening period cheeses (all showed a
low concentration of tyrosine, the precursor of tyramine, as
determined by HPLC [30 mg kg–1; data not shown]). In
three cases, tyramine-producing strains were not detected
by PCR although tyramine had been detected by HPLC.
Remarkably, these samples corresponded to cheeses made
from raw milk. Since the primers used in the PCR reaction
were only designed to detect LAB, other microorganisms
might be responsible for this tyramine production.
Discussion
The BA content of cheeses has been analysed by several
authors, although most have only focused on one type of
cheese (Valsamaki et al. 2000; Novella-Rodrı ´guez et al.
2003b; Pinho et al. 2004). In this work, random samples
of commercial cheese samples, including different types
made from different varieties of milk, differently treated
milks, and which were subject to different ripening
periods, were analysed. As described by other authors
(Gennaro et al. 2003; Novella-Rodrı ´guez et al. 2003a),
tyramine, histamine, putrescine and cadaverine were the
most common and abundant BAs detected. It is note-
worthy that 46% of the samples contained tyramine or
histamine in concentrations that exceed 100 mg kg–1; in-
deed, two samples had concentrations of over 1 g kg–1. In
blue cheeses, the average content of tyramine was
448 mg kg–1. These results indicate that more tools are
needed, not only for detection and quantification of BA in
food products, but also for its early detection and the pre-
vention of its accumulation.
Several authors have related the presence and concen-
tration of BA in cheeses with the treatment the milk under-
goes (Joosten & Northolt, 1987; Ordon ˜ez et al. 1997;
Novella-Rodrı ´guez et al. 2004). The present results
confirm that pasteurization of the milk reduces the BA
content of cheese, perhaps due to a reduction in the
number ofdecarboxylating
Enterobacteriaceae and enterococci (Ordon ˜ez et al. 1997;
Gennaro et al. 2003; Novella-Rodrı ´guez et al. 2004). This
effect was particularly noticeable with respect to cadaver-
ine. Certainly, it has been shown that pasteurization
reduces the number of Enterobacteriaceae strains (Marino
et al. 2000). Other authors attribute the differences in the
BA content of pasteurized and non-pasteurized cheeses to
the heat sensitivity of certain cofactors, such as pyridoxal
5-phosphate, needed for the amino acid decarboxylation
reaction (Joosten & Northolt, 1987).
The BA content of cheese depends more on the micro-
biological quality of the milk than on the type of milk
used. For example, raw sheep’s milk cheese had the
highest percentage of BA-positive samples, whereas the
pasteurized sheep’s milk cheeses had a number of BA-
positive samples similar to that of other pasteurized milk
cheeses (Fig. 1).
Higher BA contents were detected in long ripening
period than in short ripening period cheeses. The influence
of proteolysis on BA formation has been reported by others
authors (Ordon ˜ez et al. 1997; Ferna ´ndez-Garcı ´a et al.
2000; Novella-Rodrı ´guez et al. 2004). Proteolysis links the
ripening period with BA formation since the amino acids
that form the substrate of the decarboxylating enzymes
have to be released from casein. Innocente & D’Agostin,
(2002) showed that the total amine content tends to in-
crease with advancing proteolytic maturation. Similarly,
Ferna ´ndez-Garcı ´a et al. (2000) recorded the positive in-
fluence on BA formation of adding proteinase to the
cheese matrix. Proteolytic activity also explains the high
BA concentration detected in the blue cheeses, in this case
enhanced by the presence of fungi with strong proteolytic
activity.
An essential factor in BA production in cheese is the
presence of microorganisms with decarboxylation activity;
microorganismssuch as
280
M Ferna ´ndez and others

Page 6

the correlation between the presence of bacteria with the
tyrosine decarboxylase gene (tdcA) and the capability of
strains to synthesize tyramine has been demonstrated, and
PCR has been proposed by a number of authors as a
method for identifying tyramine-producing strains (Coton
et al. 2004; Ferna ´ndez et al. 2004). PCR provides a rapid
means of detecting tyramine-producing microorganism in
fermented foodstuffs, and for predicting tyramine ac-
cumulation. The present results show an acceptable cor-
relation between the PCR and HPLC results. The three
cheeses in which tyramine was detected by HPLC, yet no
amplification product was obtained, were all made from
raw milk. The tyramine-producing strains responsible may
have been Gram negative bacteria, which would not have
been detected by the primers used (it should be re-
membered, of course, that Gram negative bacteria are
highly undesirable in any foodstuffs; many are pathogens
or spoilage agents). The low number of samples with these
characteristics appears to indicate that the main tyramine-
producing strains in cheeses are LAB, which are easily
detected by PCR.
In nine samples, tyramine was not detected by HPLC,
although a tdcA PCR product was obtained. Seven of these
samples corresponded to short ripening period cheeses.
The absence of tyramine is probably related to low level
proteolytic activity and therefore to a low tyrosine con-
centration in the cheese matrix (confirmed by HPLC).
However, these seven cheeses are potentially able to ac-
cumulate tyramine (Ferna ´ndez et al. 2006).
In the remaining two samples of cheese, although tyro-
sine was detected by HPLC and tyramine-producing bac-
teria were identified by PCR, no tyramine was detected.
The alkaline pH of this type of cheese might be related to
a weaker expression and/or activity of the decarboxylase
enzyme. In any event, cheeses containing both the pre-
cursor amino acid and decarboxylating microorganisms
are potentially dangerous to consumers since tyramine
could be synthesized at a later time during the ripening
period or during storage before consumption.
Although there is no legislation regarding the tyramine
content of cheese, the reduction of its concentration
in food is recommended. HPLC and PCR are comp-
lementary technologies that can be used to help attain
this goal: HPLC analysis is essential for determining
the exact concentration of tyramine in the samples, while
PCR is an easy and rapid method for analyzing large
numbers of samples for the presence of tyramine-produc-
ing microorganisms when tyramine itself would be un-
detectable.
This research was performed with financial support from the
Commission of the European Communities (QLK1-CT2002-
02389) and MEC, Spain (AGL2006-01024). We thank Adrian
Burton for proofreading the English. DM Linares was the recipient
of a fellowship from the Spanish Ministry of Education and
Science. M Ferna ´ndez and B del Rı ´o are beneficiaries of I3P CSIC
contracts financed by the European Social Fund.
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