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Biogenic Amines in Wine: Understanding the Headache


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The presence of biogenic amines in wine is becoming increasingly important to consumers and producers alike, due to the potential threats of toxicity to humans and consequent trade implications. In the scientific field, biogenic amines have the potential to be applied as indicators of food spoilage and/or authenticity. Biogenic amines can be formed from their respective amino acid precursors by various microorganisms present in the wine, at any stage of production, ageing or storage. To understand the large number of factors that could influence the formation of biogenic amines, the chemical, biochemical, enzymatic and genetic properties relating to these compounds have to be considered. Analytical and molecular methods to detect biogenic amines in wine, as well as possibilities that could enable better control over their production levels in wine will also be explored in this review.
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S. Afr. J. Enol. Vitic., Vol. 29, No. 2, 2008
Biogenic amines are a group of organic nitrogenous compounds
formed and degraded by the metabolisms of living organisms
(microorganisms, plants and animals). The main biogenic amines
associated with wine are putrescine, histamine, tyramine and
cadaverine, followed by phenylethylamine, spermidine, spermine,
agmatine and tryptamine. Biogenic amines in wine can be formed
by various microorganisms associated with the different stages of
wine production and storage.
The vinication of grapes consists of two main fermentation
steps. Alcoholic fermentation of grape must, usually performed by
the wine yeast Saccharomyces cerevisiae, involves the conversion
of grape sugar to ethanol and CO2. Malolactic fermentation
is conducted in most red and some white wines by lactic acid
bacteria of the genera Lactobacillus, Leuconostoc, Pediococcus
and Oenococcus; principally to deacidify the medium by
the conversion of l-malic acid to l-lactic acid. Malolactic
fermentation also ensures a certain degree of microbial stability
to the wine and modies the sensory characteristics of the wine
by the production of secondary bacterial metabolites (Lonvaud-
Funel, 1999). Spoilage organisms such as acetic acid bacteria
and the yeast Brettanomyces bruxellensis can grow during
fermentations or storage and produce compounds that impart
negative characteristics to the wine.
Apart from the primary metabolic products and many avour
compounds (both desirable and undesirable) released during
the fermentations, some microorganisms produce secondary
metabolic products that may affect the wholesomeness of the
wine. Biogenic amines are one such group of compounds. Other
secondary metabolites associated with winemaking that could
pose a health risk to humans include sulphur dioxide (SO2), ethyl
carbamate and the mycotoxin, Ochratoxin A.
The focus of this review will be biogenic amines. Their formation
by various wine microorganisms will be discussed with specic
reference to the enzymes and genes involved. The chemical,
biochemical and toxicological properties of biogenic amines are
considered. Factors inuencing the formation of biogenic amines,
covering the entire production chain, are critically reviewed.
Analytical and molecular methods used in biogenic amine
identication and quantication are also discussed.
Lactic acid bacteria
Lactic acid bacteria are present on healthy grapes in low numbers
and the population generally declines during alcoholic fermentation.
Lactic acid bacteria are also transferred to winery equipment, where
they can be present in signicant numbers (Wibowo et al., 1985).
Oenococcus oeni is normally the dominant species that survives to
the end of alcoholic fermentation and is predominantly responsible
for malolactic fermentation. However, if the pH of the wine is
above pH 3.5 species of Pediococcus and Lactobacillus, generally
associated with spoilage, may survive and grow to levels of 106
to 108 cells/mL and have antagonistic interactions with O. oeni
(Wibowo et al., 1985; Lonvaud-Funel, 1999).
Extensive research has been done to correlate biogenic amine
production in wine with species of lactic acid bacteria involved
in the winemaking process. In the past, spoilage bacteria, mainly
Pediococcus spp. such as Pediococcus cerevisiae, were held
responsible for histamine production in wine (Delni 1989). Recent
results by Landete et al. (2007b) show an agreement that, although
the percentage of Pediococcus spp. capable of producing histamine
seems to be low, some strains (for example Pediococcus parvulus
in this study) can be responsible for the highest concentrations of
histamine. It is widely known that Lactobacillus, Leuconostoc and
Oenococcus spp. are also implicated in biogenic amine production
in wine. Different strains of Lactobacillus hilgardii, Lactobacillus
buchneri, Lactobacillus brevis and Lactobacillus mali produce a
variety of biogenic amines in wine (Moreno-Arribas & Lonvaud-
Funel, 1999; Moreno-Arribas et al., 2000; Downing, 2003; Moreno-
Arribas et al., 2003; Costantini et al., 2006; Landete et al., 2007b).
Biogenic Amines in Wine: Understanding the Headache
A.Y. Smit1, W.J. du Toit1 and M. du Toit1,2*
(1) Department of Viticulture and Oenology Stellenbosch University, Private Bag X1, Matieland (Stellenbosch), South Africa
(2) Institute for Wine Biotechnology, Stellenbosch University, Private Bag X1, Matieland (Stellenbosch), South Africa
Submitted for publication: August 2008
Accepted for publication: September 2008
Key words: Biogenic amines; wine; bacteria; yeast; wine factors; decarboxylase enzymes; detection methods; toxicology
The presence of biogenic amines in wine is becoming increasingly important to consumers and producers alike,
due to the potential threats of toxicity to humans and consequent trade implications. In the scientic eld, biogenic
amines have the potential to be applied as indicators of food spoilage and/or authenticity. Biogenic amines can be
formed from their respective amino acid precursors by various microorganisms present in the wine, at any stage
of production, ageing or storage. To understand the large number of factors that could inuence the formation of
biogenic amines, the chemical, biochemical, enzymatic and genetic properties relating to these compounds have to
be considered. Analytical and molecular methods to detect biogenic amines in wine, as well as possibilities that could
enable better control over their production levels in wine will also be explored in this review.
*Corresponding author: E-mail address:
S. Afr. J. Enol. Vitic., Vol. 29, No. 2, 2008
Biogenic Amines in Wine
Until 2003 there had been no reports on the role of Leuconostoc
strains in the formation of biogenic amines in wine. Moreno-Arribas
et al. (2003) have shown that strains of Leuconostoc mesenteroides
have a high potential to produce tyramine. Another strain of Leuc.
mesenteroides isolated from wine, capable of producing relatively
high levels of histamine, has been identied by Landete et al.
Commercial O. oeni strains are selected for their oenological
parameters, including the absence of amino acid decarboxylases.
According to in vitro studies conducted by Moreno-Arribas et
al. (2003), none of four commercial malolactic starter cultures
examined could produce histamine, tyramine or putrescine.
Martín-Álvarez et al. (2006) also compared inoculated with
spontaneous malolactic fermentation in 224 samples of Spanish
red wine. The authors found that inoculation with a commercial
starter culture of lactic acid bacteria could reduce the incidence
of biogenic amines compared to spontaneous malolactic
fermentation in wines. Starter cultures could eliminate indigenous
bacteria, or might be able to degrade biogenic amines produced
by undesirable strains.
No biogenic amine production was observed by Moreno-
Arribas et al. (2003), who tested 39 strains in a decarboxylase
assay broth medium, using HPLC to quantify biogenic amine
concentrations. Similar results were also obtained by Straub et
al. (1995), who tested for histamine producers among 88 strains
of O. oeni. Costantini et al. (2006) found no O. oeni among 92
strains able to produce biogenic amines in a broth medium. PCR
screening was used by these authors to conrm the absence of the
respective decarboxylase genes.
In contrast, Guerrini et al. (2002) found that O. oeni is able
to signicantly contribute to the overall biogenic amine content
of wines and that the ability of O. oeni to produce biogenic
amines varies among strains. Twenty six of 44 strains tested were
able to produce up to 33 mg/L histamine under optimal growth
conditions in a synthetic medium. Putrescine and cadaverine were
also produced by some strains. The high frequency of histamine-
producing strains found in this study is in accordance with a study
done by Coton et al. (1998a), who reported that most O. oeni
strains isolated from wine have histamine producing capability.
The latter research group found that almost half of the 118 wines
tested from the Southwest of France contained histamine in
varying amounts and contained histidine decarboxylase positive
bacteria belonging to O. oeni.
Thus, the ability of lactic acid bacteria to produce amines seems
to be strain-dependent, and not a species specic characteristic.
It may be that decarboxylase activities are randomly distributed
within the different species of Lactobacillus, Leuconostoc,
Pediococcus and Oenococcus. Some lactic acid bacteria strains
have the ability to simultaneously produce different amines
(Coton et al., 1998a; Moreno-Arribas et al., 2000; Guerrini et al.,
2002) supporting the suggestion by Gale (1946) that some strains
might possess more than one amino acid decarboxylase activity
under specic culture conditions.
According to literature (Lonvaud-Funel, 2001) only lactic
acid bacterial strains with histidine-, tyrosine- and ornithine
decarboxylase activities have been isolated from wine. The
properties of each of the decarboxylase enzymes will be discussed
Histidine decarboxylase
Two different kinds of decarboxylase enzymes exist. The majority
of amino acid decarboxylase enzymes - those belonging to animals
and Gram-negative bacteria - require pyridoxal 5’-phosphate
as a cofactor. However, some bacterial histidine decarboxylase
enzymes use a covalently-bound pyruvoyl group as a cofactor in
the reaction and are pyridoxal 5’-phosphate independent (Cotton
et al., 1998b; Lonvaud-Funel, 2001). The latter group include
well-studied Gram-positive bacteria: Lactobacillus 30a (Chang &
Snell, 1968); L. buchneri, Clostridium perfringens (Recsei et al.,
1983); Micrococcus sp. (Prozorouski & Jörnvall, 1975) and O.
oeni (Rollan et al., 1995). Yet, Landete et al. (2006) demonstrated
experimentally that pyridoxal 5’-phosphate does enhance amino
acid decarboxylase activity in Gram-positive lactic acid bacteria
and could therefore also be considered a decarboxylase cofactor
for this group of bacteria.
The rst histidine decarboxylase (HDC) enzyme of a wine
lactic acid bacterium was isolated from a histamine producing
strain (O. oeni 9204) by Lonvaud-Funel & Joyeux (1994) and
has since been studied by various authors. Coton et al. (1998b)
puried this HDC enzyme to homogeneity and provided valuable
molecular data. This HDC enzyme is a single polypeptide of 315
amino acids, comprised of α and β subunits. The gene sequence
aided researchers to develop rapid and specic detection systems
based on polymerase chain reaction (PCR) to detect potential
histamine-producing bacteria from wine (Le Jeune et al., 1995;
Coton et al., 1998a).
The HDC enzyme has a high degree of cooperativity; at low
histidine concentrations HDC has a low substrate afnity, but
as histidine concentration increases, binding to the active site is
favoured. The product, histamine, acts as a competitive inhibitor
of the antiport histidine/histamine at the cell membrane and
decreases the HDC activity (Rollan et al., 1995). The pH optimum
of HDC is 4.8. These ndings are the same as described previously
for HDC of Lactobacillus 30a (Chang & Snell, 1968).
Lucas et al. (2005) related HDC enzyme activity of L. hilgardii
0006 (isolated from wine), to the presence of an 80 kb plasmid
on which the decarboxylase gene was located as part of a four-
gene cluster. They proposed that this discovery is possibly the rst
described histamine-producing pathway. The four genes, hdcP,
hdcA, hisRS and hdcB code for a histidine/histamine exchanger,
a HDC, a histidyl-tRNA synthetase and an unknown product
respectively. There is evidence to suggest that this same gene
cluster may also be present in other histamine producers, such as
Lactobacillus 30a and O. oeni 9204. Also, lactobacilli are able to
transfer a conjugative plasmid to bacteria of the same or different
genera (Gevers et al., 2003). Lucas et al. (2005) suggest that a
plasmid-encoded HDC system could be transferred horizontally;
and that the location of the gene on an unstable plasmid may
explain the random distribution of HDC positive bacteria.
Some authors reported the presence of histamine decarboxylase
activity amongst a high proportion of wine lactic acid bacteria,
even if the levels of histamine produced in wine are generally low
(Lonvaud-Funel & Joyeux, 1994; Le Jeune et al., 1995; Coton et
al., 1998a; Guerrini et al., 2002; Landete et al., 2005a, 2007b).
Other authors found the frequency of histamine producers in wine
to be very low (Moreno-Arribas et al., 2003).
S. Afr. J. Enol. Vitic., Vol. 29, No. 2, 2008
Biogenic Amines in Wine
Landete et al. (2005a) screened 136 strains of wine lactic acid
bacteria for the presence of the HDC gene and the ability to
produce histamine in a synthetic medium. These included strains
of Lactobacillus (54), Oenococcus (32), Pediococcus (34) and
Leuconostoc (16). Their results showed that Lactobacillus (13%),
Oenococcus (78%), Pediococcus (12%) and Leuconostoc (6%)
were able to produce histamine. O. oeni had the highest frequency
of histamine production, but produced the lowest concentrations.
L. hilgardii and P. parvulus produced histamine at the highest
concentrations (up to 200 mg/L) and can therefore be regarded as
potential spoilage bacteria in wine. According to this study, other
strains able to contribute to histamine in wine are O. oeni, L. mali
and Leuc. mesenteroides.
Tyrosine decarboxylase
Tyrosine decarboxylase (TDC) in bacteria had only been
investigated in Enterococcus faecalis (Allenmark & Servenius,
1978) until 1999, whereafter a tyramine-producing strain was
isolated from wine and identied as L. brevis IOEB 9809 (Moreno-
Arribas & Lonvaud-Funel, 1999).
In 2002 and 2003 the TDC gene of L. brevis IOEB 9809 was
puried and sequenced (Lucas & Lonvaud-Funel, 2002; Lucas et
al., 2003). The sequence (7979 bp) contained four complete genes
encoding a tyrosyl-tRNA synthetase, the tyrosine decarboxylase,
a probable tyrosine permease and a Na+/H+ antiporter. The
authors suggest that the TDC gene of L. brevis 9809 is the rst
well-characterised bacterial TDC gene. The TDC gene encodes
for the 264 amino acids of the enzyme.
TDC was found to be active in a pH range of 3.0 to 7.0 with
an optimum at pH 5.0. TDC enzyme activity was reported to be
pyridoxal 5’-phosphate dependent (Moreno-Arribas & Lonvaud-
Funel, 1999; Coton & Coton, 2005). Its activity is enhanced by the
substrate (L-tyrosine) and the coenzyme (pyridoxal 5’-phosphate).
These ndings are similar to the properties described for TDC of
Ent. faecalis (Allenmark & Servenius, 1978). As with histamine
and HDC activity, tyramine acts as a competitive inhibitor of TDC
and therefore the presence of the formed product will decrease the
enzyme activity.
A rapid PCR assay was developed to detect L. brevis strains
carrying the TDC gene (Lucas & Lonvaud-Funel, 2002). Downing
(2003) determined that the described primers could be used to
amplify a fragment of the TDC gene in tyrosine decarboxylating
L. brevis as well as L. hilgardii strains from South African wines.
A number of wine lactic acid bacteria strains have since been
identied as tyramine producers. Moreno-Arribas et al. (2000)
isolated and identied a number of tyramine producing lactic
acid bacteria in wine that had undergone malolactic fermentation.
None of the strains in their study were identied as O. oeni, but
all as L. brevis and L. hilgardii. Downing (2003) found tyramine
to be the main amine formed in synthetic medium by L. brevis and
L. hilgardii strains isolated from South African wines. Arena et
al. (2007) found that Lactobacillus plantarum strains harbouring
the TDC gene were able to produce tyramine in wine. As noted
elsewhere in this review, Leuc. mesenteroides was also found to be
a tyramine producer (Moreno-Arribas et al., 2003). O. oeni seems
to have a low distribution of the metabolic ability to produce
tyramine (Moreno-Arribas et al., 2000; Geurrini et al., 2002). As
far as literature suggested in 2003, no tyramine producing O. oeni
strain had yet been reported (Moreno-Arribas et al., 2003), with
the exception of a strain (O. oeni DSM 2025) that was shown to
be able to produce tyramine in a laboratory medium (Choudhury
et al., 1990).
Ornithine decarboxylase
Marcobal et al. (2004) reported that a putrescine producing
strain of O. oeni (IOEB 8419) was isolated from ropy red wine
by Coton et al. (1999) and was suspected of having ornithine
decarboxylase (ODC) activity. Later, seven strains of O. oeni (out
of 44) were reported to be able to produce putrescine in culture
media (Guerrini et al., 2002). Then, a putrescine producing strain
(O. oeni BIFI-83) was isolated from the fermentation lees of a
Spanish wine which showed a high concentration of putrescine.
This led to the rst report of the ODC gene to be present in the
genome O. oeni and being detectable by PCR (Marcobal et al.,
2004, 2005). The ODC gene encodes a 745 amino acid residue
protein. ODC is also a pyridoxal 5’-phosphate dependent enzyme.
The amino acid sequence of this ODC gene shares a 67% identity
with that of Lactobacillus 30a (Marcobal et al., 2004).
Putrescine is reported to be the most abundant biogenic amine
in wine, both qualitatively and quantitatively. Putrescine could be
observed in high concentrations (up to 200 mg/L), especially after
malolactic fermentation (Glória et al., 1998). Although putrescine
has been found to be present in almost 100% of samples analysed by
many authors (Glória et al., 1998; Soueros et al., 1998; Vasquez-
Lasa et al., 1998; Soleas et al., 1999; Moreno & Azpilicueta, 2004;
Landete et al., 2005b; Bover-Cid et al., 2006; González Marco &
Ancín Azpilicueta, 2006; Alcaide-Hidalgo et al., 2007), only one
strain of wine lactic acid bacteria has been reported in literature to
possess the ODC gene: O. oeni BIFI-83 (Marcobal et al., 2004).
Marcobal et al. (2004) found that the ODC gene is rarely present
in the genome of O. oeni. The authors found none of 42 strains
tested to possess the gene. Also, ornithine is usually only present
at low levels in wine and it would be expected that the levels of
putrescine would be correspondingly low.
The arginine deiminase pathway
A possible explanation for the production of high levels of putrescine
in wine has been proposed by Mangani et al. (2005). The authors
proved that putrescine, the most abundant biogenic amine in wine,
could be produced by O. oeni from ornithine and also from arginine
at wine pH (3.2). Some strains that can produce putrescine possess
the complete enzyme system to convert arginine, a major amino
acid in wine, to putrescine. In this case arginine is catabolised
via the arginine deiminase (ADI) pathway. For this to occur, all
three enzymes of this pathway must be present in the bacterial
strain and be active under wine conditions. These enzymes are
arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and
carbamate kinase (CK). Some O. oeni strains can be decient in one
or two of these enzymes or arginine catabolism could be inhibited
by low pH (Mangani et al., 2005). However, metabiosis can occur
among O. oeni species. This process is an association and exchange
between strains able to metabolise arginine to ornithine, but unable
to produce putrescine and strains that cannot degrade arginine
but can produce putrescine from ornithine. Putrescine production
by metabiostic association can occur slowly after the completion
of malolactic fermentation, whereas conversion of ornithine to
putrescine by the ODC enzyme of a single O. oeni strain was found
to occur simultaneous with malic acid degradation and at a faster
S. Afr. J. Enol. Vitic., Vol. 29, No. 2, 2008
Biogenic Amines in Wine
rate (Mangani et al., 2005). Thus, the authors concluded that despite
commercial starter cultures being unable to use arginine or possess
no ODC, metabiosis can occur with indigenous strains and may
play an important role in putrescine accumulation in wine.
Similarly, Arena & Manca de Nadra (2001) showed previously
that a Lactobacillus species (L. hilgardii X1B), isolated from wine
was able to degrade arginine via the arginine deiminase pathway
and the arginine decarboxylase pathway. This enables the bacteria to
produce putrescine from the intermediates ornithine and agmatine.
Other decarboxylases
De Las Rivas et al. (2006) described the rst oligonucleotides
for the PCR detection of lysine decarboxylase encoding genes
in foodborne bacteria. Primers were designed to amplify the
genes coding for lysine decarboxylase by alignment of lysine
decarboxylase proteins in two groups: rst for Enterobacteriaceae
and second for bacteria from the genera Bacillus, Clostridium,
Listeria and Staphylococcus. The possibility exists to extend this
PCR method to detect cadaverine-producing bacteria in wine.
It is known that an assosiation exists between phenylethylamine
and tyramine production in lactic acid bacteria. This could be
explained by the fact that phenylalanine is also a substrate for
TDC; producing phenylethylamine in a secondary reaction
(Landete et al., 2007c). The ability of wine lactic acid bacteria
to form phenylethylamine seems to be rare, with L. brevis and L.
hilgardii being the only strains to date associated with high levels
of phenylethylamine in wines (Moreno-Arribas et al., 2000;
Landete et al., 2007c). According to Landete et al. (2007c) there
have been no reports on studies regarding other phenylethylamine
decarboxylase enzymes.
In general, lactic acid bacteria are the main microorganisms held
responsible for biogenic amine production in wine. Soueros et al.
(1998) determined that the levels of biogenic amines (histamine,
tyramine and putrescine) are low after alcoholic fermentations and
increase in most wines during and after spontaneous malolactic
fermentations with a corresponding decrease in the respective
precursor amino acid concentration. Izquierdo Cañas et al.
(2007) also determined that histamine, tyramine and putrescine
concentrations increased by 106% to 174% in Spanish wines due
to spontaneous MLF.
Granchi et al. (2005) examined biogenic amine production
and the dynamics of microorganisms throughout the whole
winemaking process of commercial vinications. They found a
decrease of biogenic amines (especially putrescine) during both
spontaneous and induced alcoholic fermentations while yeast
dominated. Biogenic amine concentrations (putrescine and
histamine and/or cadaverine and tyramine) increased signicantly
during both spontaneous and inoculated malolactic fermentations
conducted by O. oeni strains. Other authors who observed the
most important increase of biogenic amines during malolactic
fermentation, when compared to the contributions by alcoholic
fermentation and/or ageing, include Cilliers & Van Wyk (1985),
Landete et al. (2005b), Marcobal et al. (2006b) and Alcaide-
Hidalgo et al. (2007). According to the results of Landete et al.
(2007b), lactic acid bacteria can be considered the microorganisms
responsible for the production of biogenic amines in wine, since
in this study (involving 231 microorganisms), no yeasts or acetic
acid bacteria were found capable of producing biogenic amines.
A large variety of indigenous yeast species can grow and perform
the alcoholic fermentation in wine, along with commercial S.
cerevisiae strains. Few studies have been conducted on the
formation of biogenic amines by yeasts, of which most only
compared different yeasts species and quantied only histamine
(Torrea & Ancín, 2002).
A number of authors reported that no remarkable increase in
the concentration of biogenic amines could be observed during
alcoholic fermentation, with the conclusion that yeasts do not
appear to be responsible for the production of most amines found
in industrial commercial red wines (Herbert et al., 2005; Marcobal
et al., 2006b). Granchi et al. (2005) even reported a decrease
of biogenic amines (especially putrescine) during alcoholic
fermentation while yeast dominates, for both spontaneous and
induced commercial vinications. In another study, no potential
to produce biogenic amines in synthetic medium, grape must or
wine was found among 36 strains of yeast isolated from grape
must and wine. The yeasts tested included strains belonging to
the genera Aureobasidium, Candida, Hanseniaspora, Hansenula,
Kloeckera, Metschnikowia, Pichia, Rhodotorula and strains of the
species S. cerevisiae, S. cerevisiae var. bayanus, S. cerevisiae var.
chevalieri and S. cerevisiae var. steiner (Landete et al., 2007b).
Any contribution of yeast to biogenic amine production could
therefore be indirect: yeasts can alter the composition of grape
musts by using some amino acids and secreting others during
alcoholic fermentation and autolysis, thereby changing the
concentration of precursor amino acids in the wine that can be
used by other microorganisms in subsequent fermentation steps
(Soueros et al., 1998).
In contrast, a number of studies show that an increase in biogenic
amine concentration in wine by the direct action of yeast during
both spontaneous and inoculated fermentations on experimental
and commercial scale is possible. The results of a study performed
by Buteau et al. (1984) disagree with the notion that biogenic
amines (and particularly histamine) are formed by lactic acid
bacteria during malolactic fermentation. These authors found that
agmatine, cadaverine, ethanolamine, histamine, putrescine and
tyramine were produced in the highest quantities during alcoholic
fermentation. Moreover, some biogenic amines decreased during
malolactic fermentation.
Goñi & Ancín Azpilicueta (2001) examined the concentration
of biogenic amines produced by different S. cerevisiae strains in
rosé wines. They found a slight increase in biogenic amines (pu-
trescine, spermine, spermidine, phenylethylamine and tyramine)
depending on the strain. Wines fermented by killer strains showed
the highest concentration of biogenic amines, when compared to
those with a killer neutral phenotype. The variation in the con-
centrations of the biogenic amines in this study was ascribed to
differences in the extent of production or use of the amines by the
yeasts as a source of nitrogen.
Torrea & Ancín (2002) proceeded with similar studies and found
that inoculated musts produced wines with higher concentration
of biogenic amines compared to spontaneous fermentations
performed by indigenous yeasts. They attribute this to the greater
consumption of precursor amino acids during fermentation by the
commercial yeasts, and the lower yeast population in spontaneous
S. Afr. J. Enol. Vitic., Vol. 29, No. 2, 2008
Biogenic Amines in Wine
In a study conducted by Caruso et al. (2002), 50 yeast strains of
ve different genera isolated from grapes and wine were screened
for biogenic amine production. B. bruxellensis formed the highest
concentration of total biogenic amines, followed by S. cerevisiae.
The ability of S. cerevisiae to produce biogenic amines seems to
be strain dependent and not a constant characteristic of the species.
Some strains of S. cerevisiae, particularly those present during
spontaneous alcoholic fermentations, can therefore be considered
as wine spoilage microorganisms due to their ability to produce
biogenic amines. S. cerevisiae, Kloeckera apiculata, Candida
stellata, Metschnikowia pulcherrima and B. bruxellensis were also
tested by Granchi et al. (2005) in another study. Table 1 gives an
indication of the total biogenic amines produced by these yeasts,
of which B. bruxellensis produced the highest concentration in
both studies, followed by S. cerevisiae. Agmatine, followed by
phenylethylamine and ethanolamine were produced in the highest
amounts by most species.
It is clear that yeasts form different biogenic amines than
lactic acid bacteria. Little is available in literature regarding the
biochemistry, genetics and regulation of biogenic amine production
of wine yeasts. The polyamines, spermine and spermidine, are
formed from putrescine. Unlike putrescine itself, these polyamines
have been found to be an absolute requirement for optimal growth
in yeasts, in particular for meiotic sporulation and for maintenance
of the double-stranded RNA killer plasmid (Cohn et al., 1978).
Yeast mutants with decreased ODC activity do not decarboxylate
sufcient ornithine to putrescine and consequently grow slower
because they synthesise fewer polyamines (Cohn et al., 1980).
Cohn et al. (1980) also suggest that putrescine in yeast serves
primarily as precursor for spermidine and spermine biosynthesis.
ODC in yeast is the key regulatory enzyme of the polyamine
biosynthetic pathway (Gupta et al., 2001). Yeast ODC activity
requires a thiol and pyridoxal 5’-phosphate for activity and is
rapidly decreased by the presence of spermine and spermidine
(Tyagi et al., 1981).
No mention could be found in literature of other decarboxylase
enzymes studied in wine yeasts or yeasts from related elds.
Biotic stresses to the grapevine, such as those caused by the fungus
Botrytis cinerea, can lead to an increase of amine content of the
grape berries (Hajós et al., 2000). B. cinerea is responsible for the
formation of aszu grapes, which are characteristic of the famous
Tokaj wines from Hungary. The fungus penetrates the grape skin
and signicantly alters the composition and concentration of amino
acids, carbohydrates and amines in the grape berries by increasing
its concentrations while the water content of the grape decreases
and the berry becomes raisin-like. Aszu wines are made by adding
different quantities (butts) of these noble rot grapes to the ordinary
wine grapes. Amine concentrations in aszu wines were found to
be higher in three different cultivars studied when compared to
corresponding normal wines from the same varieties. A positive
correlation could be made between the biogenic amine content
and the number of butts used for the aszu wine (Sass-Kiss et al.,
Marques et al. (2007) tested the potential of the fungicides
carbendazyme, iprodione and procymidone (applied every three
weeks to vines) to reduce the incidence of biogenic amines in grape
musts and wine. It was found that control wine (that received no
Production of biogenic amines by different wine yeast species in
sterile grape must under laboratory conditions in two different
Yeast species
Average total biogenic amines (mg/L)
Caruso et al.
Granchi et al.
Saccharomyces cerevisiae 12.14 13.7
Kloeckera apiculata 6.21 9.7
Candida stellata 7.73 7.8
Metschnikowia pulcherrima 9.6 13.3
Brettanomyces bruxellensis 15.01 20
fungicide treatments) presented the highest mean concentrations
of biogenic amines at the end of malolactic fermentation. They
concluded that fungal metabolic activity could directly contribute
to biogenic amine formation (especially for isoamylamine) or
lead to the activity of bacteria other than those normally present
in healthy grapes.
Acetic acid bacteria and other bacteria
Forty strains of acetic acid bacteria isolated from grape must and
wine were screened in synthetic medium and wine by Landete
et al. (2007b) for their ability to form histamine, tyramine,
phenylethylamine, putrescine, cadaverine and tryptamine, but
no positive results were obtained. Histamine-producing Bacillus
species (two strains) and acetic acid bacteria (one strain of
Acetobacter pasteuriamus) are reported to have been isolated
from Taiwanese fruit wines (Chang et al., 2008).
No further mention regarding the formation of biogenic amines
by acetic acid bacteria in wine could be found in literature.
Biogenic amines are compounds of low molecular weight, derived
from aromatic or cationic amino acids and all have one or more
positive charge and a hydrophobic skeleton (ten Brink et al.,
1990; Shalaby, 1996). The chemical structure of biogenic amines
can be aliphatic (putrescine, cadaverine, spermine, spermidine),
aromatic (tyramine, phenylethylamine) or heterocyclic (histamine,
tryptamine) (ten Brink et al., 1990). Fig. 1 shows the chemical
structures of some biogenic amines.
Putrescine, spermine and spermidine are present in plants,
where they are important for physiological processes such as
owering and fruit development, cell division, stress responses
and senescence (Halász et al., 1994). A large number of volatile
amines (for example ethylamine, methylamine, dimethylamine,
pyrrolidine) are also present in grapes (Ouch et al., 1981).
Biogenic amines are likely to be found in food and beverages
that contain proteins or free amino acids; if conditions persist that
favour microbial or biochemical activity. Such foodstuffs include
sh, sh products, meat products (sausages), eggs, cheeses, nuts,
fermented and fresh fruits and vegetables such as sauerkraut and
soy bean products, beers and wines (Halász et al., 1994; Silla
Santos, 1996).
S. Afr. J. Enol. Vitic., Vol. 29, No. 2, 2008
Biogenic Amines in Wine
In fermented foods, the non-volatile or biogenic amines (histamine,
putrescine, cadaverine, spermine, spermidine, agmatine, tyramine,
tryptamine) and phenylethylamine (a volatile amine) are formed
mainly by the microbial decarboxylation of the corresponding
amino acids (Zee et al., 1983; ten Brink et al., 1990; Halász et al.,
1994). Volatile amines are believed to be formed by the reductive
amination or transamination of the corresponding aldehyde or
ketone (Smith, 1980; Ouch et al., 1981).
Gram-positive bacteria are usually associated with spoilage of
fermented foods (Silla Santos, 1996). In non-fermented foods, the
presence of biogenic amines above a certain level can indicate
microbial spoilage (ten Brink et al., 1990). In raw sh and meat,
Gram-negative bacteria are known to produce histamine when
exposed to inappropriate temperatures.
Biogenic amines in wine can originate from the plant (grape
berries) itself or be produced during the fermentation processes,
ageing or storage when wine is exposed to the activity of
decarboxylase positive microorganisms. Contamination may
occur due to poor sanitary conditions of both grapes and processing
equipment (Zee et al., 1983; ten Brink et al., 1990; Shalaby, 1996;
Leitão et al., 2005). Most biogenic amine contamination of wine
is believed to take place during malolactic fermentation.
Many genera of bacteria are able to decarboxylate free amino
acids by the action of decarboxylase enzymes. Because the
amino acids are obtained by the bacterium from the extracellular
medium (wine), it is believed that decarboxylase enzymes operate
together with a transporter protein (Lucas et al., 2005). During
amino acid uptake, a membrane potential is generated by the
Chemical structures of some of the most oenologically important biogenic amines.
UAliphatic aminesU
UAromatic aminesU
UHeterocyclic aminesU
S. Afr. J. Enol. Vitic., Vol. 29, No. 2, 2008
Biogenic Amines in Wine
transporter because there is a net charge difference between the
precursor (for example monovalent histidine) and the product
(divalent histamine). A pH gradient is generated when a proton
is consumed in the decarboxylation reaction. These two steps
together generate a proton motive force. This reaction is thought
to favour growth and survival in acidic media such as wine, since
it produces metabolic energy by the described precursor/product
antiport (Molenaar et al., 1993), and regulates (increases) the pH -
thereby extending the growth period by rendering the extracellular
medium less toxic to the cell (Gale, 1946).
Histamine is present at low levels in the human body and
is involved in key functions such as the allergic response,
neurotransmission and vascular permeability (Ohtsu & Watanabe,
2003). Other normal physiological functions of biogenic amines
in humans include the regulation of body temperature, stomach
volume, stomach pH and brain activity (ten Brink et al., 1990).
Normally, if a low concentration of biogenic amines is ingested,
they are quickly detoxied in the human body by amine oxidases
or through conjugation. Amine oxidases catalyse the oxidative
deamination of biogenic amines to produce an aldehyde,
hydrogen peroxide and ammonia (Gardini et al., 2005). However,
if an excessive amount of biogenic amines is ingested or if the
normal catabolic routes are inhibited or genetically decient,
several physiological disorders can occur (ten Brink et al., 1990).
Histamine poisoning is sometimes referred to as “scombroid sh
poisoning” due to illness resulting from consumption of sh such
as tuna, mackerel and sardines; while high levels of tyramine in
cheese causes a phenomenon known as the “cheese reaction”
(Taylor, 1986; ten Brink et al., 1990). These false food allergies
are of particular importance in wine, because the presence of
ethanol, acetaldehyde and other biogenic amines may promote
the harmful effects of histamine and tyramine by inhibiting their
normal metabolism in humans (Landete et al., 2006). Histamine
is often described as the most important biogenic amine since it is
one of the most biologically active amines (Halász et al., 1994).
Histamine causes dilation of peripheral blood vessels, capillaries
and arteries, thus resulting in hypotension, ushing and headache
(Silla Santos, 1996). It also causes contraction of intestinal smooth
muscle, resulting in abdominal cramps, diarrhoea and vomiting
(Taylor, 1986).
Apart from allergic response, other serious human pathologies
caused by biogenic amines include carcinogenesis and tumor
invasion (ornithine-derived polyamines and histamine), immune
response and neurological disorders (histamine), the formation
of carcinogenic nitrosamines by reaction between nitrite and
secondary amines (putrescine, cadaverine, agmatine), migraines
and hypertension (tyramine and phenylethylamine) and
Parkinson’s disease, Schizophrenia and mood disorders (tyramine)
(Smith, 1980; ten Brink et al., 1990; Silla Santos, 1996; Medina
et al., 1999).
The toxic level of biogenic amines depends on the tolerance of
the individual for the compound, the concentration of total biogenic
amines and the consumption of ethanol and/or drugs. The toxicity of
histamine and tyramine depends on the effectivity of the catabolic
path which employs monoamine oxidase (MAO) and diamine
oxidase (DAO) enzymes, which again varies in individuals (ten
Brink et al., 1990). Biogenic amines such as tyramine, putrescine
and cadaverine that may also be present in the wine can inhibit
the metabolism of histamine. These amines favour the passage
of histamine from the intestines into the systemic circulation
by competing for binding sites in the gastrointestinal tract or by
interfering with the catabolism of histamine by saturating the activity
of mono- or diamine oxidases (Kanny et al., 2001). The amine
oxidase enzymes are not very specic and alcohol, acetaldehyde
and anti-depressive drugs can also cause interference (ten Brink et
al., 1990; Straub et al., 1995).
Generally the toxic dose in alcoholic beverages is considered
to be between 8 and 20 mg/L for histamine, 25 and 40 mg/L for
tyramine, while as little as 3 mg/L phenylethylamine can cause
negative physiological effects (Soueros et al., 1998). Kanny et
al. (2001) reports that a normal individual can tolerate 120 mg/L
of histamine taken orally before symptoms occur, but only 7 μg
administered intravenously.
However, there are studies that conclude that no relationship
exists between the oral ingestion of biogenic amines and wine
intolerance (Kanny et al., 1999, 2001; Jansen et al., 2003). Rather,
these authors propose that wine may contain compounds (ethanol
and acetaldehyde) that stimulate the release of histamine within
the body.
Other than toxic effects, some biogenic amines also have
other negative consequences, particularly regarding the sensory
characteristics of the wine and economic implications.
A study by Rohn et al. (2005) suggests that histamine can be
identied at high concentrations in commercial wines by well
trained wine assessors. The study employed mouthfeel descriptors
such as “irritation at the deep throat” and “crawling of the tongue”.
No specic taste could be attributed to histamine. Putrescine,
the most abundant biogenic amine in wine can reduce sensorial
quality at 15 to 20 mg/L and 20 to 30 mg/L in white and red wines
respectively (Arena & Manca de Nadra, 2001). In contrast, Wantke
et al. (2008) determined that sensory wine quality is unrelated to
histamine levels. During their study, 100 Austrian red wines and
26 sparkling wines judged by a professional wine taster were also
analysed for histamine content.
In wine at a low pH, volatile amines occur as odourless salts, but
in the mouth they are partially liberated and their avours become
apparent (Lehtonen, 1996). Volatile amines could therefore also
inuence wine aroma.
Biogenic amines in wine could also cause commercial import
and export difculties. Certain countries will reject wines that
contain more than a certain concentration of histamine. The upper
limits for histamine in wine in some European countries are (mg/L
histamine): Germany (2), Holland (3), Finland (5), Belgium (5 to
6), France (8), Switzerland and Austria (10) (Lehtonen, 1996).
The levels of biogenic amines produced in wine largely depend on
the abundance of amino acid precursors in the medium, since on the
whole, biogenic amines increase with an increase in amino acids.
Amino acid content may be inuenced by vinication methods,
grape variety, geographical region and vintage (Lonvaud-Funel &
Joyeux, 1994; Soueros et al., 1998; Moreno Arribas et al., 2000).
While some factors increase the precursor amino acid
concentration, other factors inuence the growth and enzyme
activity of microorganisms that can form the biogenic amines.
S. Afr. J. Enol. Vitic., Vol. 29, No. 2, 2008
Biogenic Amines in Wine
Viticultural inuences
Some amines, such as putrescine and other polyamines, may
already be present in grape berries (Halász et al., 1994; Bover-Cid
et al., 2006). Cabernet Sauvignon, for example, was found to have
high concentrations of putrescine, cadaverine and spermidine
in the pericarp of the berries (Glória et al., 1998). Putrescine,
cadaverine and spermidine have also been found present in high
concentrations in the seeds of grape berries (Kiss et al., 2006).
Therefore, putrescine concentration in wine may be inuenced
more by geographical region and grape variety than by winemaking
practices (Landete et al., 2005). Potassium deciencies in the
soil have been linked to an increase in putrescine concentration
in plants (Adams, 1991); while water deciencies do not seem
to inuence the content of biogenic amines in grape berries and
wines (Bover-Cid et al., 2006). The degree of maturation of the
grape and the soil type can also inuence amine levels in the nal
product (Glória et al., 1998).
Biogenic amines are also dependent on grape variety and vine
nutrition, which will determine the concentration and composition
of precursor amino acids in grape must and nally, together with
yeast metabolisms, in the wine (Soueros et al., 1998). Nitrogen
fertilization treatments can cause an increase in grape amino acids
and amine concentrations (Spayd et al., 1994; Soueros et al.,
2007). Grape varieties with higher levels of assimilable amino
acids have been found to yield the highest nal concentrations of
biogenic amines (Herbert et al., 2005).
An experiment was conducted by Cecchini et al. (2005) to
determine whether red grape cultivars have an effect on the
content of biogenic amines in wines. Merlot, Syrah, Sangiovese,
Cesanese d’Afile and Cabernet Franc were studied. Analysis of
variance showed a signicant difference between individual and
total amine contents in wines obtained from musts of the different
cultivars. Cabernet Franc was found to have signicantly higher
total amine content than any of the other cultivars studied.
More than one study has reported that the grape variety Pinot
noir contains higher levels of biogenic amines when compared
to Cabernet Sauvignon. Ough (1971) observed that signicantly
higher histamine concentrations were present in Pinot noir
in California compared to Cabernet Sauvignon, while Glória
et al. (1998) found that Pinot noir (Oregon, USA) contained
signicantly more total biogenic amines compared to Cabernet
Sauvignon. Soleas et al. (1999) also observed higher amine
concentrations in Pinot noir from Ontario (Canada) compared
to other red wines. Other authors have observed cultivar related
differences in biogenic amine content in Hungarian (Hajós et al.,
2000), Greek (Soueros et al., 2007) and Italian grapes and wines
(Del Prete et al., 2009).
The vintage and the region of production can also affect the
free amino acid and amine content in must and wine (Herbert et
al., 2005). The concentrations of precursor amino acids can vary
signicantly over years. The inuence of vintage on biogenic
amine levels could also be attributed to the diversity of wine
microorganisms that are naturally selected - their growth could
again be correlated to the pH of the grapes and wines that can
differ accordingly with the vintage (Martín-Álvarez et al., 2006).
These authors found Spanish Tempranillo wines from a specic
region had signicantly more biogenic amines in 2001 than in
2002. Amine levels of aszu wines were also found to be inuenced
by vintage and were signicantly different for the 1993, 1997 and
1998 vintages (Sass-Kiss et al., 2000). On the contrary, Glória et
al. (1998), found no difference in amine concentrations from two
vintages (1991 and 1992).
Grape skin maceration practices
Grape skin maceration promotes extraction of grape components
such as phenolic compounds, proteins, amino acids and
polysaccharides. During cold maceration, grape must is left in
contact with the grape skins at a cold temperature prior to alcoholic
fermentation. Most red wines undergo alcoholic fermentation in
contact with grape skins. Extended maceration after alcoholic
fermentation can also be applied at cool temperature to extent the
extraction period. Pectolytic enzymes are added to grape musts to
increase the yield of juice, to clarify the must or wine, to extract
more grape derived compounds such as phenols and to facilitate
pressing and ltration.
Some authors could nd no connection between grape skin
maceration practices and the levels of biogenic amines in wines.
Soleas et al., (1999) found no correlation between length of skin
contact and concentration of biogenic amines, while Martín-
Álvarez et al., (2006) concluded that the addition of pectolytic
enzymes to the grapes at 2 g/100 kg did not promote biogenic
amine accumulation in wine in their study.
However, others found that the duration of skin maceration to
be a very important variable that affects biogenic amine content
in wines, and that longer maceration time could favour increased
production of biogenic amines (Bauza et al., 1995; Martín-
Álvarez et al., 2006). Results from the latter research group are
represented in Table 2.
An investigation on musts and wines of elderberry fruit
showed that histamine content was dependent on the method of
pulp treatment prior to processing, namely hot maceration, hot
maceration and depectinisation and fermentation on the pulp.
Their results show that histamine is formed by the action of native
HDC of the berries, as well as by enzymes present in the Polish
pectolytic preparation (Pectopol PT), and during fermentation
by the action of decarboxylases produced by yeasts. The highest
decarboxylase enzyme activity was found in Bordeaux yeast,
followed by Burgundy, Malga, Tokay and Syrena yeasts. (These
yeasts seem to be unspecied S. cerevisiae wine yeast starter
cultures.) None of the wines had undergone any malolactic
fermentation, thus excluding the possibility of biogenic amine
formation by the action of bacterial decarboxylases (Pogorzelski,
1992). The presence of and possible contribution by indigenous
lactic acid bacteria on the elderberry fruit was not reported.
Phenolic compounds
Grape phenolics are also extracted from the grape skins (and
seeds) during maceration and fermentation. These compounds
are responsible for many chemical reactions in grape must and
wine. They are involved in oxidation reactions (they are strong
antioxidants), and inuence the avour and colour of wine.
Even though it has been known for some time that phenolic
compounds may have an effect (inhibiting or stimulatory) on
the growth, metabolism and malolactic activity of lactic acid
bacteria (Vivas et al., 1997; Alberto et al., 2001, 2007), the
rst study to ascertain whether phenolic compounds can affect
biogenic amine production by wine lactic acid bacteria was done
S. Afr. J. Enol. Vitic., Vol. 29, No. 2, 2008
Biogenic Amines in Wine
in 2007 by Alberto et al. with specic focus on the inuence of
phenolic compounds on agmatine metabolism of L. hilgardii
X1B. The formation of putrescine from agmatine does not involve
amino acid decarboxylase. Alberto et al. (2007) observed the
conversion of agmatine into putrescine was lower in the presence
of all phenolic compounds, with the exception of gallic acid
and quercetin. Gallic acid and quercetin also had no inuence
on bacterial growth or survival in the presence of agmatine and
therefore had no interactions with agmatine metabolism.
Putrescine, the end product of this pathway, is able to protect DNA
damaged by reactive oxygen species and increase cell survival of
Escherichia coli under conditions of oxidative stress (Tkachenko,
2004). Phenolics are powerful antioxidants and the reduction of
putrescine formation in the presence of phenolics is attributed to
the ability of the phenolics to protect the cells against oxidative
stress themselves (Alberto et al., 2007). Phenolic decarboxylation
could also compete with the enzyme involved in the conversion of
agmatine into putrescine, N-carbamoylputrescine decarboxylase,
and thus inhibits the formation of putrescine. According to this
study, the presence of phenolic compounds could pose a natural
solution to reduce putrescine formation in red wine.
Wine physiochemical composition
Wine physiochemical factors such as pH, temperature, SO2 and a
variety of substrates and products of fermentation can inuence
the concentration and diversity of microorganisms in the wine,
but can also affect decarboxylase enzyme activity and gene
Inuence of wine composition on enzyme activity and decar
boxy lase gene expression
In wine, the presence of the HDC gene does not mean that the
enzyme is functional and that histamine will necessarily be
formed (Coton et al., 1998a). As discussed earlier in this review,
histidine decarboxylation can be used by bacterial cells to generate
additional energy under poor growth conditions (Konings et al.,
1997). It seems as if though the production of histamine by lactic
acid bacteria is always enhanced under poor growth conditions,
for example when the medium has a shortage of fermentable
substrates such as l-malic acid and glucose (Lonvaud-Funel &
Joyeux, 1994). However, the presence of glucose and l-malic
acid can have a stimulating effect on decarboxylation. Moreno-
Arribas et al. (2000) reported that more tyramine was produced
by a TDC positive Lactobacillus strain in the presence of glucose,
possibly due to the energy provided to aid the enzyme activity. In
contrast, Arena et al. (2007) showed that tyramine formation was
decreased by increasing concentrations of glucose, fructose and
l-malic acid. Glucose was found to have no effect on the HDC
enzyme of O. oeni (Rollan et al., 1995). Malic acid was found
to play a key role in activating arginine catabolism by O. oeni,
increasing the production of putrescine (Mangani et al., 2005).
The product of malolactic fermentation, lactic acid, was found to
inhibit HDC activity (Rollan et al., 1995; Lonvaud-Funel, 2001);
while on the contrary, lactic acid does not appear to inhibit ODC
activity (Mangani et al., 2005). Citric acid may also inhibit HDC
and TDC activity to a small extent at levels normally present in
Average biogenic amine concentrations (mg/L) for different levels of vintage, pectolytic enzymes, ageing on lees, maceration time and
bacteria inoculation (Martín-Álvarez et al., 2006).
Histamine Methylamine Ethylamine Tyramine Phenylethyl-
amine Putrescine Cadaverine
Vintage ** ** ** ** * **
2001 117 4.87±0.67 1.36±0.12 3.07±0.20 2.12±0.55 3.36±0.50 9.69±1.24 1.16±0.44
2002 107 1.44±0.69 0.39±0.12 0.44±0.20 0.62±0.57 2.29±0.52 4.70±1.29 1.60±0.46
Pectolytic enzymes ** *
No 162 2.76±0.59 1.01±0.11 1.66±0.17 0.96±0.49 3.86±0.44 8.08±1.10 1.93±0.39
Yes 62 3.55±0.81 0.74±0.14 1.85±0.24 1.77±0.67 1.79±0.61 10.46±1.45 1.55±0.51
Ageing with lees * ** **
No 159 3.82±0.63 0.71±0.11 1.92±0.19 2.30±0.52 2.68±0.47 3.93±1.1 1.21±0.42
Yes 65 2.49±0.78 1.05±0.14 1.58±0.23 0.43±0.64 2.97±0.58 10.46±1.45 1.55±0.51
Skin maceration (time) ** * ** **
<10 days 123 1.83±0.58 0.71±0.10 1.83±0.17 0.51±0.47 2.70±0.43 3.32±1.07 1.02±0.38
>10 days 101 4.89±0.40 1.04±0.14 1.68±0.23 2.22±0.65 2.95±0.59 11.07±1.46 1.74±0.52
MLF inoculation ** * *
No 193 4.89±0.40 1.01±0.07 2.00±0.12 2.37±0.33 2.67±0.30 7.34±0.74 2.09±0.26
Yes 31 1.42±1.04 0.75±0.19 1.50±0.3 0.37±0.86 2.99±0.78 7.05±1.94 0.67±0.69
*The factor has a statistically signicant effect on the variable, P<0.05.
**The factor has a statistically signicant effect on the variable, P<0.01.
S. Afr. J. Enol. Vitic., Vol. 29, No. 2, 2008
Biogenic Amines in Wine
wines after malolactic fermentation (Rollan et al., 1995; Moreno-
Arribas & Lonvaud-Funel, 1999). Other compounds found
to inhibit TDC activity to different extents include glycerol,
β-mercaptoethanol, lactic acid and ethanol. However, Moreno-
Arribas & Lonvaud-Funel (1999) conclude that even the highest
concentrations of these compounds likely to be present in wine
will not be sufcient to prevent the formation of tyramine.
pH and ethanol at levels found in wine could inhibit decarboxylase
enzyme activity (Leitão et al., 2000). HDC activity and consequent
histamine production is enhanced at pH 3.5 and by ethanol
concentrations up to 10%, where the conditions for histidine
transport inside the cells are more favourable due to the uidication
of the cell membrane by ethanol (Lonvaud-Funel & Joyeux, 1994).
A high ethanol concentration (12% or more), as most often found
in wine, reduces the HDC activity by altering the physicochemical
properties of the membrane and slowing down histidine transport
(Rollan et al., 1995).
Histamine production was further found to be regulated by the
presence of histidine, histamine, pyridoxal 5’-phosphate and the
bacterial growth phase (Landete et al., 2006). It was observed
that HDC gene expression is induced by histidine (at 1 or 2
g/L) and decreased by histamine (at 1 or 2 g/L), while pyridoxal
5’-phosphate enhanced HDC activity (at 0.5 g/L).
During a study performed by Lucas et al. (2005) it was
discovered that the HDC positive phenotype disappeared under
certain culture conditions in L. hilgardii 0006, isolated from
wine. In a poor, acidic medium the number of HDC positive cells
increased, presumable due to the energetic and growth advantage
of these cells. In a rich medium with a higher pH, the number
of mutant cells lacking HDC activity increased. Loss of enzyme
activity was found to correspond to the loss of a large plasmid (80
kb) on which the HDC gene was located.
In addition to decarboxylase activity, a small number of O. oeni
strains (six of 220 strains tested) were found to also have proteolytic
activity – the ability to release amino acids from peptides and pro-
teins. The proteolytic activity is also dependent on nutritional and
energetic composition of the medium, and generally increases when
high-energy nutrients are exhausted in the late exponential growth
phase (Leitão et al., 2000). Decarboxylase activity is also expressed
when the cells need the additional energy produced during amino
acid transport. Northern blot analysis conrmed that HDC expres-
sion appeared during the early growth phase, reached a peak during
the exponential growth phase (because the decarboxylation gener-
ates metabolic energy for this time of growth and cell division) and
decreased signicantly during the stationary phase when growth
and cell division decrease (Molenaar et al., 1993; Landete et al.,
2006). Other researchers also found that decarboxylase activity in-
creases or could be biosynthesised towards the end of the active
growth phase and during the exponential phase of bacterial growth
under experimental and industrial winemaking conditions (Gale,
1946; Moreno-Arribas et al., 2000; Marcobal et al., 2006b). O. oeni
expresses proteolytic and decarboxylase activities only when there
is no easier strategy for cell survival (Molenaar et al., 1993).
Inuence of wine composition on bacterial growth and survival
A study was performed to analyse the effects of ve physiochemical
factors (incubation temperature, incubation time, environmental
pH, added tyrosine concentration and pyridoxal 5’-phosphate
supplementation) on cell growth and tyramine production of L.
brevis CECT 4669 and Ent. faecium BIFI-58 (isolated from grape
must) under anaerobic and aerobic conditions. A multiple linear
regression model was used to predict that the optimum conditions
for growth and tyramine production was anaerobic incubation at
acidic pH (4.4) in the presence of a high tyrosine concentration
(Marcobal et al., 2006a).
Studies of the complete vinication of industrial wines indi-
cated that SO2 does prevent the formation of biogenic amines by
reducing lactic acid bacterial numbers in the wine (Marcobal et
al., 2006b). Vidal-Carou et al. (1990) found that the highest levels
of biogenic amines are found in red wines with low SO2 levels
and that increased levels of SO2 was correlated with a decrease in
the concentration of histamine and tyramine. The effect of SO2 on
tyramine accumulation was also found to be dependent on pH. At
a higher pH, an increase of SO2 was found to cause a decrease in
tyramine concentration, while at a lower pH tyramine increased
with an increase of SO2 (Gardini et al., 2005). This response to pH
is contrary to what is usually encountered during biogenic amine
production. Normally, at a higher wine pH, the bacterial microo-
ra is more diverse and the growth and survival of decarboxylase
positive bacteria becomes more likely. Hence, at higher pH, high-
er levels of biogenic amines are produced in most cases (Wibowo
et al., 1985; Lonvaud-Funel & Joyeux, 1994; Gardini et al., 2005;
Landete et al., 2005b; Martín-Álvarez et al., 2006). Landete et al.
(2005b) observed that wines from La Rioja with a high histamine
content had a pH of 3.6 or higher. Cilliers & Van Wyk (1985) also
noted that the pH of all the red wines containing large amounts of
histamine (>10 mg/L) in their study exceeded 3.7.
Lower levels of biogenic amines were produced in conditions
of high ethanol concentrations and low pyridoxal 5’-phosphate
concentrations (Gardini et al., 2005). The decrease in tyramine
production by O. oeni T56 (a tyramine producer) under conditions
of high ethanol and low pH was attributed to reduced metabolic
activity and cellular viability and not to the specic decarboxylase
activity in this study.
Acetic acid (volatile acidity) has been correlated with high
levels of histamine in white and rosé wines in one study. However,
the reason for this correlation was not determined (Vidal-Carou et
al., 1990).
Conditions during ageing and storage of wine
After malolactic fermentation Landete et al. (2005b) noticed
a further increase in histamine during the rst six months of
storage in bottles. Gerbaux & Monamy (2000) also found that the
concentration of histamine increases between four and eight months
after malolactic fermentation in Pinot Noir and Chardonnay. A
third study (Herbert et al., 2005) showed a consistent increase of
histamine in red and white wines 18 months after the completion
of malolactic fermentation, whilst tyramine and putrescine seemed
to increase immediately following malolactic fermentation in red
wines in this study.
The reason for the initial increase following the completion of
malolactic fermentation could be that SO2 added to the wine after
malolactic fermentation does not completely stop all biochemical
reactions and enzyme activity. Also, due to the high pH of many
wines, SO2 is less effective and hence biogenic amines can increase
in sulphated wines during ageing (Gerbaux & Monamy, 2000).
S. Afr. J. Enol. Vitic., Vol. 29, No. 2, 2008
Biogenic Amines in Wine
Another reason for increase in biogenic amines following
fermentations is derived from the winemaking practice of ageing
wine in contact with yeast lees. Yeast autolysis favours the growth
and activity of lactic acid bacteria due to the release of vitamins
and nitrogenous compounds. Lactic acid bacteria are able to
hydrolyse and metabolise proteins and peptides and use the
released amino acids as nutrients or energy sources. These amino
acids may include the precursors of biogenic amines (Lonvaud-
Funel & Joyeux, 1994). Yeast and bacterial lees can also be the
source of decarboxylase positive lactic acid bacteria. Bauza et
al. (1995) observed an increased production of tyramine and
putrescine when wines are inoculated with bacteria through the
addition of lees. Incidentally, the rst ODC gene from a putrescine
producing wine lactic acid bacterium was isolated from wine lees
(Marcobal et al., 2004).
Martín-Álvarez et al. (2006) left their wines in contact with lees
for two months after alcoholic fermentation, before ageing in barrels.
The mean concentrations of methylamine and putrescine were
higher in wines aged on lees. In contrast, tyramine concentrations
were signicantly lower (Table 2). The authors postulate that
tyramine could be consumed by residual microorganisms from
lees for the production of carbon skeletons or amino groups. Coton
et al. (1998a) also noted that even when no more culturable cells
were detectable, HDC could still be active – thus biogenic amines
can be produced during ageing. Moreover, most amines as well
as decarboxylase enzymes are heat stable and will not be reduced
during processing (such as pasteurisation), and can for this reason
even increase during storage (ten Brink et al., 1990). The inuence
of lees on the presence of biogenic amines in wine was recently
reviewed by Pérez-Serradilla & Luque de Castro (2008).
After the initial increase of biogenic amines during storage,
a general decrease or stabilisation in concentration could be
observed by various research groups (Gerbaux & Monamy, 2000;
Landete et al., 2005b; Marcobal et al., 2006b). Biogenic amines
can be degraded by oxidase enzymes present in some bacteria
towards the end of the ageing period, even at wine pH (Vidal-
Carou et al., 1991; Moreno & Azpilicueta, 2004). The general
decrease of biogenic amines during ageing could explain why
the highest histamine content (average 8.72 mg/L) was found by
Vasquez-Lasa et al. (1998) in young red wines compared to red
wines subjected to different traditional ageing prescriptions in
Rioja, Spain. No statistically signicant differences were found
between histamine levels in crianza red wines (6.67 mg/L), reserva
red wines (6.92 mg/L) or gran reserva red wines (5.12 mg/L).
Other factors present during ageing may or may not play a
role in the accumulation of biogenic amines. Hernández-Orte et
al. (2008) examined a number of factors that inuence biogenic
amine evolution, particularly during storage of wine in oak barrels.
Moreno & Azpilicueta (2004) compared the biogenic amine
concentrations of ltered and unltered wines aged in barrels for
243 days. Diatomaceous earth can adsorb cationic amino acids
and proteins on its surface; which can inuence the evolution of
biogenic amines during ageing. Unltered wine can contain skin
residues which can be rich in amino acid precursors. However,
it was found that the degree of turbidity did not inuence the
accumulation of biogenic amines during ageing. Also, the type
of barrel (American, French Allier and French Nevers oak) did
not inuence the content of biogenic amines. In another study,
the highest mean values for histamine were acquired in wines
where malolactic fermentation was performed in tanks (not
barrels), followed by ageing in the presence of lees stirred weekly
or monthly. In this study, putrescine increased during ageing in
wines aged in presence of yeast lees, but remained stable in wines
without lees (Alcaide-Hidalgo et al., 2007).
Normally, the storage of wine at elevated or uctuating tempera-
tures can cause unwanted chemical, microbial or enzymatic reac-
tions of wine components and seriously decrease product quality.
However, it was found that wine storage temperature only has a
small effect on amine concentration. Histamine concentration was
found to increase slightly when wines were stored for 105 days,
more so at 20°C than at the more extreme temperatures of 4°C
or 35°C. The formation or degradation of amines in wine mainly
took place during the rst 45 days of storage for all temperatures
studied, due to the presence of residual decarboxylase activity after
alcoholic and malolactic fermentations (González Marco & Ancín
Azpilicueta, 2006). Vidal-Carou et al. (1991) found no formation or
increase in biogenic amines (histamine or tyramine) at various tem-
peratures ranging from 4°C to 35°C in wines stored for 93 to 125
days under spoilage conditions. The only changes observed in bio-
genic amine content were the decrease in histamine and tyramine,
independent of temperature. The authors could not explain this phe-
Ancín-Azpilicueta et al. (2008) reviewed some of the factors
that inuence biogenic amine concentration in wine, including
their evolution at different winemaking stages and during storage
of the product.
Wine style and type
Red wines generally show a higher concentration of biogenic
amines than white and rosé wines. The higher values are attributed
to the presence of lactic acid bacteria and malolactic fermentation
(Landete et al., 2005b); since in the case where white and rosé
wines did undergo malolactic fermentation in this study, the
amine values were close to those observed in red wines after
malolactic fermentation. White wines, in general, also contain
fewer amino acid precursors and have lower pH due to the absence
of skin contact during fermentation and the absence of malolactic
fermentation, and may consequently have lower biogenic amine
concentrations than red wines (Zee et al., 1983; Cilliers & Van
Wyk, 1985; Vazquez-Lasa et al., 1998; Leitão et al., 2005). Buteau
et al. (1984) attributed the higher levels found in red wines due to
the lack of bentonite treatments (which adsorbs amines) and the
release of cellular amines by autolysing yeast cells in lees during
malolactic fermentation.
Rupasinghe & Clegg (2007) analysed and compared the
biogenic amine content of ten different types of fruit wines (apple,
black currant, blueberry, cherry, cranberry, elderberry, peach,
pear, plum, raspberry) to wines made from grapes (Cabernet
Sauvignon, Chardonnay, Riesling and icewine made from Vidal
Blanc). The results indicate that Cabernet Sauvignon grapes
contain signicantly higher levels of biogenic amines (11 143 μg/L
histamine) than all other fruit wines. Again, this was attributed to
the fact that this red wine was the only wine that had undergone
malolactic fermentation.
In Chinese rice wines, the average amount of total biogenic
amines was found to be 107 mg/L, which is higher than average
S. Afr. J. Enol. Vitic., Vol. 29, No. 2, 2008
Biogenic Amines in Wine
levels reported in wine made from grapes. Interestingly, no
putrescine was detected in any of the 42 samples of rice wines
tested, while histamine was detected in all samples in the range
of 5.02 to 78.50 mg/L, followed by spermine (present in 93%
of samples), cadaverine (87%), tyramine (79%) and spermidine
(79%). The highest levels of total biogenic amines (100 to 241
mg/L) were observed in Shaoxing rice wine. The vinication
procedure followed in Shaoxing includes a soaking step where the
rice is soaked in a fermentation substrate (soaking water) containing
a large amount of free amino acids and active decarboxylase
positive lactic acid bacteria. No signicant correlation was found
between the pH of rice wines (4.04 to 4.33) and biogenic amines
in this work (Yongmei et al., 2007).
More studies on the levels of biogenic amines in traditional
alcoholic beverages (Lasekan & Lasekan, 2000), natural ciders
(Garai et al., 2006), beer and biologically aged sherry-type wines
(Moreno-Arribas & Polo, 2008) have also been published. For the
purpose of this review it is only important to note that the growth
and activity of lactic acid bacteria are implicated in biogenic
amine production in all these products.
Analysis of biogenic amines, individually or simultaneously, is
important because of their toxicity potential and their potential to
be applied as indicators of food spoilage or authenticity. Biogenic
amines can be quantied by a variety of analytical methods that
require sophisticated equipment. Qualitative measurements can
also indicate the presence of amines in wine. For review papers on
analytical methods used in the determination of biogenic amines,
refer to Lehtonen (1996) and Önal (2007). The potential of
biogenic amines to appear in wine can also be determined by using
molecular tools which can detect the presence of decarboxylase
positive microorganisms.
Qualitative and semi-quantitative methods
Screening methods using selective media
Some of the rst methods developed for qualitative biogenic amine
detection (initially for histamine) are microbiological screening
methods that involve the use of a differential agar medium with a
pH indicator (bromocresol purple), where an increase in pH as a
result of amine formation can be easily observed by a change in
colour. Amino acid precursors are contained in the decarboxylase
assay medium. Modications of the screening plate medium have
been made by various researchers to make it more suitable for
the growth of lactic acid bacteria and activity of decarboxylase
enzymes. Improvements have also led to greater sensitivity and
reliability of the screening plate method, and it has shown good
correlation with other chemical analytical methods (Choudhury,
1990; Maijala, 1993; Bover-Cid & Holzapfel, 1999). Yet, it is still
advisable to conrm the results of simple screening methods by
another analytical method (such as HPLC), because false positive
results can occur due to the formation of other alkaline compounds
(such as ammonia), and failure to detect amine production has
also been reported (Moreno-Arribas et al., 2003).
Enzymatic methods
Enzymatic methods to quantify histamine were rst reported for
use in sh. When these enzymatic methods were applied to musts
and wines, many false positive results were recorded. A direct
enzymatic test for the use in wine was developed by Landete et al.
(2004). This test is performed by the sequential activity of the two
enzymes, diamine oxidase (to break down histamine) and peroxidase
(to produce a colour change). A linear correlation between optical
density and histamine concentration is used for quantication.
A very good correlation (r2=0.9984) could also be established
between biogenic amine quantication by this enzymatic method
versus HPLC analysis. The advantage of this method is its limited
sample preparation and time-consumption, and it does not require
expensive or sophisticated equipment or training.
Enzyme-linked immunosorbent assays (ELISA) are commonly
used for the quantitative analysis of histamine in scombroid sh.
Such a test was applied to wine samples for the rst time by
Marcobal et al., (2005b). No false negative results were obtained
by the ELISA test, although there was a slight overestimation of
histamine in a few samples when correlated to the results obtained
by reverse phase-HPLC (r=0.91). This rapid, easy method could
be used for screening in laboratories that are not equipped with
an HPLC, in order to distinguish between wines with a histamine
content of more or less than 10 mg/L.
Thinlayer chromatography
Thin-layer chromatography (TLC) was one of the rst techniques
used for the determination of biogenic amines in foods (Halász
et al., 1994). Despite the advantage of not requiring special or
expensive equipment, TLC is known to be time consuming and
only semi-quantitative.
A simple and rapid qualitative TLC method for the determination
of the ability of bacteria to produce biogenic amines in liquid
culture media containing the amino acid precursor is described by
Gárcia-Moruno et al. (2005). This method improves on previous
TLC methods due to the omition of an extraction step from the
bacterial supernatants. The uorescent dansyl derivatives of
histamine, tyramine, putrescine and phenylethylamine can be
separated and identied using TLC. This method is not known to
give false positive results. The only equipment involved is a photo
camera and a UV transilluminator.
Quantitative methods
Wine is a complex matrix and biogenic amines are present at low
concentrations, hindering the ease of determination. Biogenic
amines are non-volatile, polar compounds, which makes their
isolation from wine (an aqueous matrix) complicated. In addition,
the aliphatic biogenic amines are difcult to detect as they exhibit
no characteristic ultraviolet (UV) absorption, uorescent properties
or electrochemical activity. These properties prohibit the direct
detection of biogenic amines, for example by spectrophotometric
or uorimetric methods. To solve this problem, biogenic amines
have to be derivatised to enable their detection by higher
absorbance wavelengths. Samples usually also require a clean-up
and pre-concentration step. Solid phase extraction (SPE) is widely
used and has even been automated. Liquid-liquid extraction of
the analyte with organic solvent can also be exploited. Both these
method have their disadvantages such as losses of amines and low
recoveries (García-Villar et al., 2006).
Liquid chromatography
Presently, high-performance liquid chromatography (HPLC)
methods seem to still be the most widely used analytical approach
to test for the presence of biogenic amines. These methods usually
S. Afr. J. Enol. Vitic., Vol. 29, No. 2, 2008
Biogenic Amines in Wine
include pre- or post-column derivatisation and uorimetric
detection of the corresponding derivatives. Various HPLC
methods have been described. Modications and improvements
are consistently being made to pre- and post column treatments
and reagents in order to reduce preparation and analysis time and to
improve resolution of biogenic amine peaks in the chromatogram
(Soleas et al., 1999; Marcobal et al., 2005b).
O-phthalaldehyde (OPA) is one of the preferred derivatising
agents, giving rise to highly uorescent derivatives. Vidal-Carou
et al. (2003) optimised a method using OPA for the determination
of 12 biogenic amines (both primary amines and polyamines) in
wine and other alcoholic beverages. Dansyl-chloride, the other
popular derivatising reagent, can react with primary, secondary
and tertiary amino groups under selected conditions. These
derivatives are stable and also uorescent and therefore detectable
in the UV region.
Fluorenylmethylchloroformate (FMOC) can be employed as
derivatisation reagent to determine spermine and spermidine
concentrations in addition to other biogenic amines, along with
the simultaneous detection of their precursor amino acids in wine
(Bauza et al., 1995). Another derivatisation reagent that can react
with primary, secondary and aromatic primary amino groups is
1,2-naphthoquinone-4-sulfonate (NQS). García-Villar et al. (2006)
used liquid-liquid extraction with chloroform to preconcentrate
biogenic amines and to remove polar compounds such as amino
acid derivatives which interfere with subsequent separation steps.
8-phenyl-(4-oxy-acetic acid N-hydroxysuccinimide ester)-
(TMPAB-OSu) is another recently synthesised uorescent
reagent that can be used for rapid pre-column derivatisation for
HPLC analysis of primary as well as secondary biogenic amine
concentrations in wine (Li et al., 2006).
Another HPLC method proposes the direct injection of samples
derivatised with 6-aminoquinolyl-N-hydroxysuccinimidyl carba-
mate (AQC). No other pre-treatment (extraction) is required. His-
ta mine, putrescine, cadaverine and tyramine in musts and wines
can be analysed with this method (Hernández-Orte et al., 2006).
Gómez-Alonso et al. (2007) use diethyl ethoxymethylene-
malonate (DEEMM) as derivatising agent to form aminoenones
which can be detected in the UV-visible region. By this method,
nine biogenic amines, 24 amino acids and the ammonium ion can
be analysed simultaneously in a single injection by reverse phase-
HPLC. The authors claim that this method is an improvement to
previous HPLC methods that can simultaneously quantify amines
and amino acids.
Loukou & Zotou (2003) presented the rst HPLC-uorescence
method (as opposed to HPLC-UV) to simultaneously assay 11
oenologically important biogenic amines. An HLPC-diode
array detection-atmospheric pressure chemical ionisation mass
spectrometry system (HPLC-DAD-APCI-MS) was used to
characterise dansylamides for the rst time in wine and other
alcoholic beverages after pretreatment with polyvinylpyrrolidone
(PVP). Pretreatment is used to remove substances from the matrix
that can interfere in the derivatisation and quantication. A similar
method (reverse phase HPLC-DAD) was described by mo Dugo
et al. (2006) that does not require any sample pretreatment before
derivatisation with dansyl chloride.
Alberto et al. (2002) described two reverse phase HPLC
methods, one of which uses pre-column derivatisation with OPA
to separate and quantify both biogenic amines and amino acids in
a single run.
Micellar liquid chromatography (MLC) uses a surfactant
solution instead of aqueous-organic solvents as is used by HPLC.
The method described by Gil-Agustí et al. (2007) does not require
pre-treatment of samples (other than ltration) or an extraction
procedure prior to analysis, and can be performed with a single
direct injection. With this method, tryptamine and tyramine
and their precursors concentrations, tryptophane and tyrosine,
can be determined simultaneously in wine samples. Micellar
electrokinetic chromatography (MECC) separation with laser-
induced uorescence (LIF) detection is another method that can
be applied for the quantication of a large number of biogenic
amines and amino acids in wine (Nouadje et al., 1997).
A more recent liquid chromatography method to simultaneously
analyse ten oenologically important biogenic amines has
been proposed (Hernández-Borges et al., 2007). Nano-liquid
chromatography with UV detection was used to quantify the biogenic
amines with dansyl-chloride as derivatising agent and employing
SPE for post-derivatisation clean-up and to extract the analytes
from the wine. Other liquid chromatography techniques that are
applicable to biogenic amine analysis in wine have been described
by Ibe et al. (1991); Hlabangana et al. (2006) (ion-pair liquid
chromatography) and Millán et al. (2007) (liquid chromatography-
electronspray ionisation ion trap mass spectrometry).
Capillary electrophoresis
Capillary electrophoresis (CE) methods are attractive due to their
short analysis time and high resolution. One problem is their
lack of sensitivity, which can be overcome by coupling the CE
to mass spectrometry (MS) detection, instead of UV detection.
Other detection systems are also available. For example, a CE
method using conductometric detection and which requires no
derivatisation or sample cleaning steps was developed recently
by Kvasnicka & Voldrich (2006). This direct method is sensitive
and can detect biogenic amines in foods and wine in less than 15
minutes. A high-performance capillary electrophoresis (HPCE)
method exists to determine biogenic amines in wine, amongst
other foodstuffs (Kovács et al., 1999).
An automated method for the determination of biogenic amines
in wine has been described in order to exclude manual pretreatment
of the sample (Acre et al., 1998). This method employs the use of
a minicolumn for solid-phase extraction (SPE) to simultaneously
clean-up and concentrate the sample prior to analysis by capillary
electrophoresis (CE) with indirect UV detection, in a ow system.
This method allows for the separation and determination of a
wide range of biogenic amines present in wine in less than 15
minutes, compared to 25 minutes by HPLC. Another advantage
over HPLC is the direct detection. As noted, HPLC determination
requires derivatisation of the amine due to aliphatic biogenic
amines containing no chromophores that absorb signicantly in
the UV-visual region.
Another automated method is described by Santos et al. (2004)
for biogenic amine determination in white and red wines. They
proposed the use of capillary electrophoresis-electronspray mass
spectrometry (CE-ESI-MS) for the separation and quantication of
S. Afr. J. Enol. Vitic., Vol. 29, No. 2, 2008
Biogenic Amines in Wine
nine biogenic amines. Simó et al. (2008) compared the suitability of
capillary electrophoresis–ion-trap mass spectrometry (CE–IT-MS)
and capillary electrophoresis–time-of-ight mass spectrometry
(CE–TOF-MS) to analyse biogenic amines in wine. Biogenic
amines could be quantied in wines using both methods, without
any previous treatment or derivatisation of wine samples. CE–
TOF-MS showed the better capability to analyse ten oenological
biogenic amines in a single analysis. Concentrations as low as 10
ng/mL could be detected and the analysis time of this method is fast
(8 minutes by CE–TOF-MS compared to 40 minutes by HPLC).
Gas chromatography
Volatile amines have been measured in the past by gas chromato-
graphy. A very sensitive and accurate gas-chromatography/mass
spectrometry method was developed by Fernandes & Ferreira
(2000), with a run time of only 18 minutes. This method is the rst
GC-MS method that allows for the simultaneous determination
of diamines, polyamines and aromatic amines in grape juice and
various wine styles tested. The conversion of biogenic amines to
their corresponding volatile (o-heptauorobutyryl) derivatives
allows for determination by gas chromatography.
Polymerase chain reaction
The use of methods that can rapidly detect the presence of biogenic
amines and biogenic amine producing lactic acid bacteria at an
early stage is important for preventing the accumulation of biogenic
amines in wine and other food products. PCR techniques have
been developed to detect bacterial amino acid decarboxylases in a
rapid, sensitive and accurate manner. Thus, PCR cannot determine
quantitative (or qualitative) amounts of biogenic amines; but it can
be used to estimate the potential risk of amine formation (Coton et
al., 1998a). Since molecular methods are independent of the culture
conditions, it can be used to detect biogenic amine producers
even under circumstances where the lactic acid bacteria had lost
the ability to produce biogenic amines. Such instances have been
reported after prolonged storage or cultivation in synthetic media
(Lonvaud-Funel & Joyeux, 1994; Leitão et al., 2000).
In principle, some DNA sequences of some genes are highly
conserved, so PCR can be applied to detect specic genes in
various organisms. Specic oligonucleotides for the amplication
of histidine-, tyrosine-, ornithine- and lysine decarboxylase genes
have been designed to detect the presence of bacteria which can
possibly produce the corresponding biogenic amine in wine.
Le Jeune et al. (1995) developed degenerate primers to detect
HDC positive lactic acid bacteria. These primers were based on
nucleotide and amino acid sequences of Lactobacillus 30a and
C. perfringens and the amino acid sequences of L. buchneri and
Micrococcus sp. The primers allowed for amplication of a HDC
gene fragment using DNA of these bacterial strains. The HDC
gene of O. oeni has also been described (Coton et al., 1998b).
Another set of universal primers for the detection of HDC genes in
Gram-positive bacteria was developed by Coton & Coton (2005).
These primers are based on HDC gene nucleotide sequences
from a variety of lactic acid bacteria such as O. oeni IOEB 9204,
Lactobacillus 30a and C. perfringens.
By a similar approach, universal primers were designed by Coton
et al. (2004) for the early detection of potential tyramine-producing
bacteria in wine. Marcobal et al. (2005) designed the rst complete
set of primers to amplify the gene coding for ODC by aligning
amino acid sequences of ODCs from Gram-positive and Gram-
negative bacteria. De las Rivas et al. (2006) described the rst PCR
assay to determine the presence of lysine decarboxylase positive,
cadaverine producing microorganisms in food.
Multiplex amplication methods were developed to reduce the
quantity of reagent, labour costs and time, because more than
one gene species (target amines) can be detected simultaneously
using multiplex PCR, as opposed to uniplex PCR (Coton &
Coton, 2005; De las Rivas et al., 2005; Marcobal et al., 2005a).
Molecular methods for the detection of biogenic amine producing
bacteria have been reviewed recently by Landete et al. (2007a).
Using biogenic amines as an analytic tool
Different chemometric procedures have been applied to establish
criteria for differentiation in wines (Rius & Massart, 1991).
Principal Component Analysis (PCA) has proved to be a useful
tool for pattern recognition, classication, modelling and data
evaluation. In a number of recent studies, wines have been
successfully classied on the basis of biogenic amines.
Soueros et al. (1998) used PCA to classify French wines of four
regions according to wine style and origin by analyses of amino
acids, biogenic amines, volatile compounds, and organic acids.
Wines made from botrytised grapes formed a separate group.
Csomós et al. (2002) distinguished Hungarian red and white
wines from the same geographic origin and vintage using PCA,
according to their biogenic amine and polyphenol content. A further
study concerning Hungarian wines concluded that, on the basis of
the results of chemometric analyses (PCA and linear discriminant
analysis), free amino acid and biogenic amine contents seem to be
useful to differentiate wines according to winemaking technology,
geographic origin, grape variety, and year of vintage (Héberger
et al., 2003). García-Villar et al. (2007) also propose the use of
simple chemometric techniques such as PCA and partial least-
squares regression to characterise wines. Their results indicate
that biogenic amines could be used as descriptors of enological
practices and ageing patterns in Spanish wines.
Kiss et al. (2006) performed a study with the objective to
compare the amine composition of Aszu grapes (noble rotten
grapes) with that of gray rotten berries (infected mainly with
B. cinerea) and of berries infected with other local green molds
(mainly Penicillium). Using multivariate statistical analyses
(principal component analysis and linear discriminant analysis)
intact grapes, Aszu grapes, and grape berries infected mainly with
Penicillium species could be separated from each other in grape
samples with the same origin. The composition and concentration
of biogenic amines might also provide useful information on the
authentication of the Tokaj aszu wines (Hajós et al., 2000) and
could be used in grapes, wines and aszu wines for quality control
purposes (Sass-Kiss et al., 2000; Kiss & Sass-Kiss, 2005).
Due to indigenous lactic acid bacteria, including O. oeni, being
able to produce biogenic amines during malolactic fermentation,
the application of a genetically engineered malolactic wine yeast
strain, capable of the complete degradation of l-malic acid in
wine, has been proposed (Husnik et al., 2006). According to
the authors, this yeast could prevent the formation of biogenic
amines in wine by omitting the need for lactic acid bacteria to
perform malolactic fermentation. Currently, consumer rejection
S. Afr. J. Enol. Vitic., Vol. 29, No. 2, 2008
Biogenic Amines in Wine
of genetically modied organisms in wine producing countries
still renders this option unavailable in most countries.
It is known that some microorganisms, among them lactic acid
bacteria, are capable of degrading biogenic amines by amine
oxidase enzyme activity. Consequently, Leuschner et al. (1998)
studied a large number of food fermenting organisms to screen for
the potential to degrade histamine and tyramine. Unfortunately,
at this stage, amine degradation seems to be restricted to aerobic
microorganisms which are of limited use in fermented foods such
as wine which harbours an anaerobic environment.
Presently, the only realistic option to control the potential
problem of biogenic amines is by inhibiting the growth of
decarboxylase positive indigenous bacteria and other spoilage
microorganisms. Sulphur dioxide is known to have antimicrobial
properties. Molecular SO2 can act to inhibit lactic acid bacteria
in wine. The antibacterial activity of SO2 is pH-dependent and
will decrease with an increase in pH. Lysozyme is an enzyme
that can cause lysis of the cell walls of Gram-positive bacteria,
including wine lactic acid bacteria. Lysozyme retains its activity
in higher pH wines. It can be successfully used to delay or inhibit
the growth of most lactic acid bacteria, especially when used in
combination with SO2 (Delni et al., 2004; Ribérau-Gayon et al.,
2006). Bacteriocins are antimicrobial peptides produced by some
strains of lactic acid bacteria. Nisin is a commercially available
bacteriocin that acts on the cytoplasmic membrane of Gram-
positive bacteria. The possibility exists to exploit the synergistic
effect of nisin and metabisulphite (SO2) on growth inhibition of
wine spoilage lactic acid bacteria for wine preservation (Rojo-
Bezares et al., 2007) but it has not been authorised for application
in wine. The potential use of natural phenolic compounds as
antimicrobial agents to control the growth of lactic acid bacteria
in wine has also been proposed (García-Ruiz et al., 2007).
Inoculation with O. oeni starter cultures that are unable to produce
biogenic amines is a viable option for the control of these compounds
in wine (Martín-Álvarez et al., 2006). It seems that co-inoculation
of O. oeni starter cultures together with alcoholic fermentation has
the potential to curb biogenic amine formation even more than
conventional inoculation for malolactic fermentation after the
completion of alcoholic fermentation (Van der Merwe, 2007).
While regulating the soil and vine nutritional status are the
only viticultural manipulations that could be made to control
the accumulation of biogenic amines in grapes, their occurrence
in musts or wine could possibly also be controlled by reducing
practices that increase amino acid extraction such as grape skin
maceration and lees contact.
The distribution of biogenic amine producers amongst wine
microorganisms seems to be random and not a species specic
quality. Lactic acid bacteria are the wine microorganisms that are,
for the most part, associated with amino acid decarboxylation and
biogenic amine formation. However, O. oeni seems to have a low
distribution of tyrosine-, histidine-, and ODC genes in its genome.
The most important increase of biogenic amines takes place during
malolactic fermentation, when compared to the contributions by
alcoholic fermentation and ageing respectively. The contribution
of yeast to biogenic amine production could be indirect (by amino
acid secretion and autolysis) or direct, where killer positive strains
produce the highest concentration of biogenic amines. Strains of
B. bruxellensis, followed by strains of S. cerevisiae were found to
produce signicant concentrations of biogenic amines.
Biogenic amine concentrations in wine may be inuenced by
their presence in grape berries; dictated by factors such as soil
potassium deciencies, grape variety, geographical region and
vintage. The concentrations of precursor amino acids are inuenced
by winemaking practices such as grape skin maceration. Biogenic
amine formation is also determined by wine parameters and
components of which pH, ethanol, SO2 and pyridoxal 5’-phosphate
have the most important effect on the diversity of microorganisms,
decarboxylase enzyme activity and decarboxylase gene expression.
The concentration of biogenic amines is dependent on wine type
and style, but the presence of biogenic amines seems to be attributed
to the presence of lactic acid bacteria in all cases.
PCR reactions can detect the presence of biogenic amine producing
lactic acid bacteria and thereby estimate the potential risk of
formation of histamine, tyrosine, cadaverine and putrescine in wine.
Biogenic amines can be measured qualitatively (with screening and
enzymatic methods), semi-quantitatively (by thin-layer chroma-
tography) or quantitatively (using liquid chromatography, capillary
electrophoresis or gas chromatography).
Biogenic amine formation in wine is most likely to be prevented
by inhibition of indigenous lactic acid bacteria and other spoilage
microorganisms that could possess decarboxylase activity.
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... The main metabolism of amino acids determines the production of many aromatic compounds that are important for the final sensory quality of wine [21], which are themselves produced by yeasts during the fermentation process [22]. Another fate of amino acids is found in decarboxylation via exogenous enzymes released by various microorganisms, including yeasts, giving rise to low molecular weight organic compounds (which in high concentrations are detrimental to health [23]). The concentration in wine of these latter compounds is influenced by the abundance of amino acid precursors, the presence of decarboxylase positive microorganisms, and by many oenological parameters-such as alcohol and sulfur dioxide content, pH, and temperature [23,24]-that can increase the concentration of precursor amino acids or favor the growth of positive decarboxylase microorganisms [25]. ...
... Another fate of amino acids is found in decarboxylation via exogenous enzymes released by various microorganisms, including yeasts, giving rise to low molecular weight organic compounds (which in high concentrations are detrimental to health [23]). The concentration in wine of these latter compounds is influenced by the abundance of amino acid precursors, the presence of decarboxylase positive microorganisms, and by many oenological parameters-such as alcohol and sulfur dioxide content, pH, and temperature [23,24]-that can increase the concentration of precursor amino acids or favor the growth of positive decarboxylase microorganisms [25]. ...
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The type and quantity of precursor amino acids present in grape must that are used by wine yeasts affect the organoleptic and health properties of wine. The aim of this work was to conduct a preliminary screening among Saccharomyces and non-Saccharomyces indigenous strains, which were previously isolated from different Italian regional grape varieties. This was performed in order to evaluate their decarboxylase activity on certain important amino acids—such as arginine, proline, serine, and tyrosine—that are present in grape must. In particular, a qualitative test on 122 wine yeasts was performed on a decarboxylase medium using arginine, proline, serine, and tyrosine as precursor amino acids. Our results showed a considerable variability among the microbial species tested for this parameter. Indeed, Saccharomyces cerevisiae strains exhibited a high decarboxylase capability of the four amino acids tested; moreover, only 10% of the total (i.e., a total of 81) did not show this trait. A high recovery of decarboxylation ability for at least one amino acid was also found for Zygosaccharomyces bailii and Hanseniaspora spp. These findings can, therefore, promote the inclusion of decarboxylase activity as an additional characteristic in a wine yeast selection program in order to choose starter cultures that possess desirable technological traits; moreover, this also can contribute to the safeguarding of consumer health.
... As highlighted by different authors [31,32], the ability of S. cerevisiae to produce biogenic amines seems to be strain-dependent and not a species-specific quality. ...
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Background: Non-conventional yeasts (NCY) (i.e., non-Saccharomyces) may be used as alternative starters to promote biodiversity and quality of fermented foods and beverages (e.g., wine, beer, bakery products). Methods: A total of 32 wine-associated yeasts (Campania region, Italy) were genetically identified and screened for decarboxylase activity and leavening ability. The best selected strains were used to study the leavening kinetics in model doughs (MDs). A commercial strain of Saccharomyces cerevisiae was used as the control. The volatile organic profiles of the inoculated MDs were analyzed by solid phase microextraction/gas chromatography-mass spectrometry (SPME/GC-MS). Results: Most of strains belonged to the NCY species Hanseniaspora uvarum, Metschnikowia pulcherrima, Pichia kudriavzevii, Torulaspora delbruekii, and Zygotorulaspora florentina, while a few strains were S. cerevisiae. Most strains of H. uvarum lacked decarboxylase activity and showed a high leaving activity after 24 h of incubation that was comparable to the S. cerevisiae strains. The selected H. uvarum strains generated a different flavor profile of the doughs compared to the S. cerevisiae strains. In particular, NCY reduced the fraction of aldehydes that were potentially involved in oxidative phenomena. Conclusions: The use of NCY could be advantageous in the bakery industry, as they can provide greater diversity than S. cerevisiae-based products, and may be useful in reducing and avoiding yeast intolerance.
... BAs are formed by microorganisms associated with different stages of winemaking (Guo et al., 2015;Smit et al., 2008). Two species isolated during spontaneous MLF, Lactobacillus rhamnosus and Oenococcus oeni, were producers of biogenic amines (Henríquez-Aedo et al., 2016). ...
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The purpose of this study was to evaluate the content of biogenic amines (BAs) in wines using dispersive liquid-liquid microextraction-gas chromatography-mass spectrometry (DLLME-GC-MS). An additional objective was to assess the correlations between selected parameters characterizing the samples such as the content of BAs, sugars, and organic acids, pH, and total acidity. Wines produced from the same grape variety in which alcoholic fermentation (AF) was carried out by different yeast strains and in which malolactic fermentation (MLF) was spontaneous, differed in the content of biogenic amines. The concentrations of putrescine, cadaverine and tryptamine were higher in the Rondo wines (237-405, 34.04-61.11, <LOD-12.456 µg/L, respectively) and Zweigelt wines (416-489, 72.67-88.43, <LOD-13.083 µg/L, respectively) subjected to spontaneous MLF than in the wines subjected to induced MLF. Chemometric analysis allowed us to determine correlations between selected wine parameters. The wine samples are well separated into two patterns depending on the grape variety. Despite the fact that information on BAs is not included in databases of wine composition, information on their concentration as well as knowledge of existing correlations between BAs and other wine parameters is crucial and may be useful for the food industry, health professionals and consumers.
... In parallel, methods for detection of BAproducing bacteria have been developed to estimate the risk of BA-food content and to prevent their accumulation in food products. The control of parameters such as temperature, presence of organic acids is extensively reported to influence bacterial growth and their metabolic activities Alberto et al., (2007) andSmit et al., (2008) prolonged storage periods make the food more susceptible to amine formation(Hernandez et al., 2008). So that, low temperature should be applied during storage to inhibit proteolytic and decarboxylase activity of bacteria.Komprda et al., (2007) andChong et al., (2011) mentioned that temperature is the major factor for controlling the biogenic amines formation in food. ...
Herein, we demonstrated that bentonites can be incisively used to reduce wine BAs content, especially putrescine molecules. Pioneering kinetic and thermodynamic studies of putrescine adsorption onto two commercially available bentonites (optimal concentration of 0.40 g dm-3) were performed resulting in ca. 60% removal by physisorption mechanism. Both bentonites showed also promising results in more complex systems, resulting in a lower putrescine adsorption due to the competition with other molecules (as proteins, polyphenols), typically present in wines. Nonetheless, we managed to reduce the putrescine content below 10 ppm both in red and white wines.
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Viticulture is one of the traditional industries in Slovakia, where there are six wine-growing regions: Malokarpatska, Southern Slovakia, Central Slovakia, Nitra, Eastern Slovakia, and Tokaj. This study focuses on the detection of microbiota in soil samples, grape leaves and berries, and samples taken from fermenting must and young wine (the variety Tramín červený) in relation to the detected concentrations of biogenic amines during the fermentation process. In the examined samples, the number of yeasts and molds (from 3.8 to 6.8 log cfu/g or mL) and TVC (from 3.7 to 6.5 log cfu/g or mL) were determined via culture examination. At the same time, the number of LAB (from ˂3.0 to 4.4 log cfu/g or mL) was determined, which was the highest on day 4 of the must fermentation process and was related to the detected of the highest concentration of biogenic amines (histamine and tyramine) on day 6 in the investigated must samples using the UHPLC system. Mycobiota species were identified by MALDI-TOF MS, PCR, ITS-PCR-RFLP, and PCR sequencing of the amplified products. The study confirmed the presence of the yeasts Saccharomyces cerevisiae, Metschnikowia pulcherrima, Hanseniospora uvarum, Pichia kudriavzevii, Pichia kluyveri, Pichia fermentas, Torulaspora delbrueckii, and Candida tenuis. At the same time, the presence of molds (Cladosporium herbarum, Cladosporium cladosporioides, Penicillium granulatum, Penicillium mononematosum, Botritis cinerea, and Penicillium glabrum) was also confirmed in soil samples, leaves, grape berries, and fresh grape must. The study confirmed the reduction in the species diversity of the microbiota during the must fermentation process, which resulted in decreases in the concentrations of the monitored biogenic amines in the early stages of the must fermentation process and young wine of the variety Tramín červený.
Biogenic amines produced during fermentation are harmful and deteriorate the sensory experience of food. However, methods for overcoming the impact of biogenic amines in Huangjiu, a traditional Chinese food, have not been considered for research. This review analyzes various studies that have evaluated the content of biogenic amine content in Huangjiu, the formation of biogenic amines, and associated factors. Furthermore, the study discusses the health hazards of biogenic amines, and summarizes the prevalent approaches to determine their content in Huangjiu. Finally, the strategies to reduce the content of biogenic amines in Huangjiu have been listed. Although the analysis of biogenic amines in food products is crucial, government legislation and stringent recommended limits for biogenic amines in Huangjiu are nonexistent. This necessitates advanced research on regulating biogenic amines in Huangjiu production, along with the development of real-time monitoring techniques. Further efforts are required to accelerate the establishment of relevant regulations and ensure the safety of fermented foods.
A relevant trend in winemaking is to reduce the use of chemical compounds in both the vineyard and winery. In organic productions, synthetic chemical fertilizers and pesticides must be avoided, aiming to achieve the production of a “safer wine”. Also, in line with consumer preferences and due to the effects of global climate change, new trends have emerged in wine fermentation and wine technology, namely new genetic engineering methods, such as CRISPR/Cas9, described as efficient, versatile, cheap, easy-to-use, and able to target multiple sites. Consumers are also looking for wines with less ethanol and fruitier aromas, but with a good balance in terms of acidity and mouthfeel. An appropriate acidity, pleasant to the palate is more difficult to achieve in wines that have high acidity. Non-conventional yeasts, in the past considered spoilage yeasts, now can be used to enhance the aroma profile of wine or to modulate wine composition, namely wine acidity, reinforcing the role of non-Saccharomyces as selected strains for controlling fermentations mostly in co-fermentation with Saccharomyces cerevisiae.
In Rioja Alavesa, a worldwide recognized wine region, it has been identified a lack of data about the situation of biogenic amines in its wines. Due to the relevance of the topic and the interest of regional wine stakeholders it was determined the situation of biogenic amines levels on 70 commercial red wines from Rioja Alavesa region. The most abundant was putrescine (15.12 ± 8.31 mg/L) followed by histamine (4.60 ± 2.60 mg/L), tyramine (3.19 ± 2.51 mg/L) and cadaverine (2.19 ± 2.25 mg/L), respectively. The absence of significant differences between different types of wines (young, “crianza” and “reserva” wines) indicated that biogenic amines production occurred mainly during fermentation processes and not ageing period. Although the situation was not considered alarming, it is still necessary to keep working in order to reduce biogenic amines levels to minimum. Any strategy aimed at reducing biogenic amines levels will increase the competitiveness of Rioja Alavesa red wines in global market by giving them added value.
In spite of the low quantities of biogenic amines present in wines compared to other fermented foods, these compounds represent one of the most important risks for wine quality and safety. In addition, biogenic amines may constitute an economic problem in commercial transactions of wines. The aim of this chapter is to present an overview of the current knowledge about the incidence of biogenic amines in wines, the factors that affect their formation, their toxicological impact on humans, the main analytical tools for their identification and quantification in wines or the molecular approaches for the decarboxylase positive detection of microorganisms. Finally, the most suitable preventive measurements during the wine chain production are emphasized.
Biogenic amines are important nitrogen compounds of biological importance in vegetable, microbial and animal cells. They can be detected in both raw and processed foods. In food microbiology they have sometimes been related to spoilage and fermentation processes. Some toxicological characteristics and outbreaks of food poisoning are associated with histamine and tyramine. Secondary amines may undergo nitrosation and form nitrosamines. A better knowledge of the factors controlling their formation is necessary in order to improve the quality and safety of food.