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Biogenic amine production by lactic acid bacteria, acetic bacteria and yeast isolated from wine
Food Control 18 (2007) 1569–1574 www.elsevier.com/locate/foodcont
Biogenic amine production by lactic acid bacteria, acetic bacteria and yeast isolated from wine J.M. Landete ¤, S. Ferrer, I. Pardo ENOLAB – Laboratori de Microbiologia Enològica, Departament de Microbiologia i Ecologia, Facultat de Biologia, Universitat de València, C/Dr. Moliner 50, 46100 Burjassot, València, Spain Received 14 June 2006; received in revised form 13 December 2006; accepted 19 December 2006
Abstract Biogenic amines are undesirable in all foods and beverages because if consumed at too high concentration, they may induce foodborne intoxications. The biogenic amine production by 155 strains of lactic acid bacteria, 40 strains of acetic bacteria and 36 strains of yeast isolated from wine were analysed in wine, grape must and synthetic media by HPLC. We did not observe biogenic amine production by acetic bacteria and yeast; however, we found production of histamine, tyramine, phenylethylamine and putrescine by LAB. A correlation of 100% was observed between biogenic amine production in synthetic medium and wine and between activity and presence of gene. With the results expose in this paper, we can consider than the lactic acid bacteria are the microorganisms responsible of histamine, tyramine and phenylethylamine production in wine. However, we cannot consider the microorganisms analysed in this work to be those responsible for tryptamine, cadaverine and putrescine levels in wine. These results could lead to future applications for preventing excessive amounts of histamine, tyramine and phenylethylamine forming during viniWcation and storage. © 2007 Elsevier Ltd. All rights reserved. Keywords: Lactic acid bacteria; Acetic bacteria; Yeast; Biogenic amine; Wine; HPLC
1. Introduction Biogenic amines are organic bases endowed with biological activity, frequently found in fermented foods and beverages. They are produced mainly as a consequence of decarboxylation of amino acids. High concentration of biogenic amines can cause undesirable physiological eVects in sensitive humans, especially when alcohol and acetaldehyde are present (Bauza et al., 1995; Maynard & Schenker, 1996). More speciWcally, histamine is known to cause headaches, low blood pressure, heart palpitations, edema, vomiting, diarrhea, and other symptoms (Bauza et al., 1995). Tyramine and phenylethylamine can produce hypertension through the release of noradrenaline and norephedrine, respectively, which are vasoconstrictors (Forsythe & Redmond, 1974). Putrescine
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and cadaverine, although not toxic themselves, aggravate the adverse eVects of histamine, tyramine and phenylethylamine, as they interfere with the enzymes that metabolize them (ten Brink, Damink, Joosten, & Huis in‘t Veld, 1990). The presence of biogenic amines in must and wines is well documented in the literature (Landete, Ferrer, Polo, & Pardo, 2005; Lehtonen, 1996; SouXeros, Marie-Lyse, & Bertrand, 1998). The production of histamine, tyramine and putrescine by lactic acid bacteria (LAB) isolated from wine has also been studied by diVerent authors (Landete, Ferrer, & Pardo, 2005; Le Jeune, Lonvaud-Funel, ten Brink, Hofstra, & van der Vossen, 1995; Moreno-Arribas, Polo, Jorganes, & Muñoz, 2003). However, other microorganisms (yeast and acetic bacteria (AB)) and other biogenic amines (phenylethylamine, cadaverine and tryptamine) present in must and wines have received little or no attention. Only a few studies on the production of non-volatile amines by yeast during alcoholic fermentation are known (Buteau, Duitschaever, & Ashton, 1984). Torrea and Ancin (2002) observed
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the production of isoamylamine and ethylamine, and Caruso et al. (2002) observed the production of ethanolamine and agmatine by yeast. No studies on biogenic amine production by acetic bacteria were found in the literature. Some authors have reported higher biogenic amines levels after malolactic fermentation (Aerny, 1985; Cilliers & van Wyk, 1985); however, other authors have not (Lafon-Lafourcade, 1975; Ough, Crowell, Kunke, Vilas, & Lagier, 1987). Information about of microbial Xora responsible of biogenic amine production would contribute to the knowledge of when the biogenic amine concentration increases and when it is thus necessary to take precautions during winemaking. The aim of this work was to study microorganisms isolated from wine for their ability to produce biogenic amines, and as such we analysed the production of histamine, tyramine, phenylethylamine, putrescine, cadaverine and tryptamine by LAB, yeast and AB in synthetic medium, wine and grape must.
0.1 g, tyrosine 2 g, histidine 2 g, phenylalanine 2 g, ornithine 2 g, lysine 2 g, tryptophane 2 g, pyridoxal phosphate 0.25 g and pH 5.5. All the products used were purchased from Sigma Chemical Co. (St. Louis, MO, USA). To analyse the biogenic amine production by AB, 0.1 ml (OD600 nm D 0.50) of a pre-culture grown in MA were inoculated into 10 ml of MA supplemented with tyrosine 2 g, histidine 2 g, phenylalanine 2 g, ornithine 2 g, lysine 2 g, tryptophane 2 g, pyridoxal phosphate 0.25 g and pH 5.5. In the case of yeast, 0.1 ml of pre-culture grown in YPD was inoculated into 10 ml of YPD supplemented with tyrosine 2 g, histidine 2 g, phenylalanine 2 g, ornithine 2 g, lysine 2 g, tryptophane 2 g, pyridoxal phosphate 0.25 g and pH 5.5. All the microorganisms were incubated at 28 °C during 5 days in aerobic conditions; moreover, the AB were incubated with agitation. 2.3. Analyses of production of biogenic amines by microorganisms in wine
2. Materials and methods 2.1. Strains and growth conditions A total of 155 strains of LAB, 40 strains of AB and 36 strains of yeast were examined in this study (Table 1). The diVerent microorganisms were isolated from wine or must in our laboratory (Blasco, Pardo, & Ferrer, submitted for publication; Landete, Ferrer, Polo et al., 2005; Pardo, 1987; Rodas, Ferrer, & Pardo, 2003) or provided by diVerent culture collection. The other microorganisms were provided by the Spanish Type Culture Collection (CECT), by the German Collection of Microorganisms and Cell Culture (DSMZ) and by the Belgian Co-ordenated Collections of Microorganisms (BCCM/LMG). In this work, we used positive controls isolated from wine Lactobacillus hilgardii 5 W and L. hilgardii X1B (Strasser de Saad & Manca de Nadra, 1987) and Oenococcus oeni BIF83 (Marcobal, de las Rivas, Moreno-Arribas, & Muñoz, 2004). All LAB were grown in MRS broth (Scharlau Chemie S.A. Barcelona, Spain) supplemented with 0.5% L-cysteine, except O. oeni, which was cultivated in MLO (Zúñiga, Pardo, & Ferrer, 1993). All AB were grown in Mannitol Agar (MA) and all yeast in Yeast Peptone Dextran (YPD). MA and YPD were recommended by the CECT. All organisms were incubated at 28 °C until mid-log phase in aerobic conditions; moreover, the AB were incubated with agitation.
To analyse the biogenic amine production in wine, 0.1 ml (OD600 nm D 0.50) of a pre-culture grown with all the microorganisms was inoculated into 10 ml of wine supplemented with 0.5 g l¡1 of histidine, tyrosine, phenylalanine, ornithine, tryptophane and lysine. All of them were incubated at 28 °C for 10 days, and the AB were incubated with agitation. The wines used in this work came from red Bobal (average pH 3.6) and red Tempranillo (average pH 3.65) varieties from Utiel-Requena (Spain). Moreover, we analysed the biogenic amine production by yeast in grape must, 0.1 ml (OD600 nm D 0.50) of a pre-culture grown of yeast was inoculated into 10 ml of grape must supplemented with 0.5 g l¡1 of histidine, tyrosine, phenylalanine, ornithine, tryptophane and lysine. All of them were incubated at 28 °C for 10 days. The grape must used in this work came from red Tempranillo (average pH 3.45) varieties from Utiel-Requena (Spain). A sample of wine and grape must without inoculation was included in the experiments as blank. The wine and grape must used in this work were sterilized before inoculum; they were Wltered through a membrane of cellulose and 0.22 m pore size. The microorganisms were streaked on adequate plate medium for having evidence on the survival of the bacterial cells after their inoculation into wine or grape must. 2.4. Biogenic amine analyses by HPLC
2.2. Analyses of biogenic amines produced by microorganisms in synthetic medium The ability to produce diVerent biogenic amines was analysed in LAB. An aliquot (0.1 ml; OD600 nm D 0.50) of a pre-culture grown in MRS or MLO medium was inoculated in 10 ml of biogenic amine production medium (BAPM), consisting (per litre) of: meat extract 8 g, tryptone 5 g, yeast extract 4 g, glucose 1.5 g, fructose 1 g, Tween 80 0.5 g, MgSO4 0.2 g, FeSO4 0.04 g, MnSO4 0.05 g, CaCO3
The biogenic amines content was determined by the HPLC method proposed by Landete, Ferrer, Polo et al. (2005). 2.5. Detection of histidine decarboxylase, tyrosine decarboxylase and ornithine decarboxylase gene Bacterial DNA for partial hdc, tdc and odc gene ampliWcation was obtained by using the Microbial DNA isola-
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Table 1 Microorganisms used in this study Microorganisms
Strains no.
References
Lactic acid bacteria Lactobacillus brevis Lactobacillus casei Lactobacillus collinoides
21 2 1
Rodas et al. (2003), Landete et al. (2005) and Landete, Ferrer, Polo et al. (2005) Rodas et al. (2003) Rodas et al. (2003) Rodas et al. (2003), Landete et al. (2005) and Landete, Ferrer, Polo et al. (2005) Strasser de Saad and Manca de Nadra (1987) Rodas et al. (2003) Rodas et al. (2003) Rodas et al. (2003) Rodas et al. (2003) Rodas et al. (2003) Rodas et al. (2003), Landete et al. (2005), Landete, Ferrer, Polo et al. (2005), Marcobal et al. (2004) Rodas et al. (2003), Landete et al. (2005) and Landete, Ferrer, Polo et al. (2005) Rodas et al. (2003)
Lactobacillus hilgardii Lactobacillus mali Lactobacillus paracasei Lactobacillus plantarum Lactobacillus vini Leuconostoc mesenteroides Oenococcus oeni
17 3 11 5 1 15 39
Pediococcus parvulus Pediococcus pentosaceus
37 3
Acetic bacteria Acetobacter aceti Acetobacter malorum Acetobacter pasteurianus
7 1 6
Acetobacter pomorum Acetobacter sp. Acetobacter tropicales Gluconacetobacter xylinus Gluconacetobacter diazotrophicus Gluconacetobacter europaeus Gluconacetobacter hansenii Gluconacetobacter liquefaciens Gluconacetobacter oboediens Gluconacetobacter oxydans Gluconacetobacter sacchari Gluconobacter cerinus Gluconobacter asaii Gluconobacter frateurii Yeast Aureobasidium pullulans Candida boidinii Hanseniaspora guilliermondii Hanseniaspora uvarum Hansenula mrakii Kloeckera apiculata Metschnikowia pulcherrima Pichia kluyveri Pichia membranaefaciens Pichia pinus Rhodotorula rubra Saccharomyces cerevisiae Saccharomyces cerevisiae var. bayanus Saccharomyces cerevisiae var.chevalieri Saccharomyces cerevisiae var. Steiner
1 1 1 4 1 1 4 2 1 5 1 1 1 2 1 1 2 1 1 2 1 2 2 1 2 14 2 3 1
Blasco et al. (submitted for publication), 298t CECT, 1693, 1372 and 1531 LMG Blasco et al. (submitted for publication) Blasco et al. (submitted for publication), 474 and 824 CECT, 1553 and 1601 LMG, 3509 DSMZ 18884 LMG 944 CECT Blasco et al. (submitted for publication) 315t and 473 CECT, 1515t LMG, 2004 DSMZ 7603 LMG 6160 DSMZ Blasco et al. (submitted for publication), 1524 and 1529 LMG 1348 LMG, 5603 DSMZ 11826 DSMZ Blasco et al. (submitted for publication), 360t and 4009 CECT, 1408 and 1414 LMG 2117t DSMZ 1368 LMG 1390 LMG Blasco et al. (submitted for publication), 1365 LMG Pardo (1987) Pardo (1987) Pardo (1987) Pardo (1987), 10389 CECT Pardo (1987) Pardo (1987) Pardo (1987) Pardo (1987) Pardo (1987) Pardo (1987) Pardo (1987) Pardo (1987), T73 CECT Pardo (1987) Pardo (1987) Pardo (1987)
tion Kit (MoBio Laboratories, Inc). We used diVerent primer pairs for amplifying the internal part of histidine decarboxylase A gene of LAB. These primer pairs JV16HC/JV17HC and CL1mod/JV17HC were used by Landete et al. (2005). The detection of tdc gene in LAB was made by partial ampliWcation of this gene using the primers p0303 (Lucas, Landete, Coton, Coton, & Lonvaud-Funel, 2003) and P1-rev (Lucas & Lonvaud-Funel, 2002). The oligonucleotides primers and conditions used
to detection of tdc gene were the proposed by Marcobal et al. (2004). 3. Results Cultures of 231 microorganisms representing LAB, AB and yeast were investigated for their potential to form histamine, tyramine, phenylethylamine, putrescine, cadaverine and tryptamine. Table 2 shows the number of
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Table 2 Microorganisms (from Table 1) capable to produce biogenic amines in synthetic medium, wine and grape must (in the case of yeast) Microorganisms
Strains Total strains forming tested Hist Tyr Phen Putr
Lactic acid bacteria 155 Acetic bacteria 40 Yeast 36
Cad
Trypt
53/53 32/32 32/32 2/2 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0
positive strains out of the total number of strains investigated. No strains of AB or yeast were able to produce the biogenic amines studied in this work. Strains of LAB were able to produce histamine, tyramine, phenylethylamine and putrescine (Table 2). However, no LAB were able to produce cadaverine or tryptamine (Table 2). In the LAB isolated from wine, Pediococcus parvulus, Lact. mali and Leuconostoc mesenteroides only could produce histamine, Lact. brevis can produce tyramine and phenylethylamine, O. oeni can produce histamine and putrescine, while Lact. hilgardii was able to produce histamine, tyramine, phenylethylamine and putrescine (Table 3). A correlation of 100% was observed between biogenic amine production in synthetic medium and wine; moreover, the biogenic amines produced in synthetic medium were always lower than in wine. All strains showing ability to produce histamine, tyramine and putrescine harboured the hdc, tdc and odc gene respectively. However, this gene was not present in strains that were unable to produce these amines. We observed that all the Pediococcus produced levels higher than 200 mg l¡1 in synthetic medium; however 83 mg l¡1 was the highest histamine production in wine. Pediococcus was the genus with the highest histamine production (Table 3). We also observed that although Oenococcus is the genus that showed the highest percentage of histidine decarboxylase positive strains, they also showed lower levels of histamine production, very low levels of histamine were produced by O. oeni in wine (Table 3). In the
case of Lactobacillus genus, Lact. hilgardii produced higher levels of histamine; in contrast, Lact. mali showed a low histamine production in synthetic medium and wine. This ability to produce tyramine appears to be a general character of Lact. brevis: all assayed strains produced this amine in synthetic medium and wine, although the tyramine production in wine was very low, being 14 mg l¡1 the highest tyramine production in wine by Lact. brevis (Table 3). However, the tyramine production is a strain-dependent character in Lact. hilgardii, as only 3 of 17 strains produced it. Lact. hilgardii strains were able to produce higher levels of tyramine in wine that Lact. brevis. We can observe that tyramine producer strains were also able to produce phenylethylamine, although the quantity of this last one was always lower in both synthetic medium and wine. In our laboratory, we did not Wnd any microorganisms able to produce putrescine; however, Lact. hilgardii X1B and the O. oeni BIF83, coming on from others laboratories, were able to produce putrescine (Table 3). 4. Discussion Throughout the years, investigators have questioned which microorganisms are responsible of biogenic amine production, because it is important to know the ability to produce amines of microorganisms involved in fermented foods in order to establish the potential risk of toxicological disorders in consumers. This work is the most extensive study about biogenic amine production by microorganisms and the Wrst exhaustive study of biogenic amine production by yeast and AB. Biogenic amine production has been analysed in the synthetic media appropriate for each microorganisms, in wine and grape must. The biogenic amine production by LABs had been analysed in others works (Landete et al., 2005; Moreno-Arribas et al., 2003) but this work is the Wrst exhaustive study of the biogenic amine production by LABs in wine, natural medium of these microorganisms. Therefore, the data obtained from wine supply higher information.
Table 3 QuantiWcation of the ability of lactic acid bacteria to generate histamine, tyramine, phenylethylamine and putrescine in synthetic medium and wine: number of strains producing biogenic amines (mean § SD values) Lactic acid bacteria
Lactobacillus brevis Lactobacillus casei Lactobacillus collinoides Lactobacillus hilgardii Lactobacillus mali Lactobacillus paracasei Lactobacillus plantarum Lactobacillus vini Leuconostoc mesenteroides Oenococcus ovni Pediococcus parvulus Pediococcus pentosaceus
Strains Histamine (mg l¡1) no. BAPM Wine
Tyramine (mg l¡1) BAPM
21 2 1 17 3 11 5 1 15 39 37 3
21 (755.8 § 314.2) 0 0 3 (482 § 50.3) 0 0 0 0 0 0 0 0
0 0 0 5 (135.8 § 80.4) 2 (43.5 § 3.5) 0 0 0 1 (43) 33 (22.3 § 17.7) 8 (260 § 58.6) 0
0 0 0 5 (24.2 § 14.5) 2 (10.1 § 1.1) 0 0 0 1 (17) 33 (4.2 § 3.2) 8 (37.3 § 8.6) 0
Phenylethylamine (mg l¡1)
Putrescine (mg l¡1)
Wine
BAPM
Wine
BAPM
Wine
21 (7.1 § 4.2) 0 0 3 (23.2 § 7.8) 0 0 0 0 0 0 0 0
21 (169.2 § 67.4) 0 0 3 (92.5 § 21.8) 0 0 0 0 0 0 0 0
21 (4.2 § 3.1) 0 0 3 (7.8 § 4.1) 0 0 0 0 0 0 0 0
0 0 0 1 (221) 0 0 0 0 0 1 (129) 0 0
0 0 0 1 (67) 0 0 0 0 0 1 (42) 0 0
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Histamine and tyramine are, besides putrescine, the most abundant amines in wine. Phenylethylamine is also frequently found. Although tryptamine and cadaverine are also found in wine, they are in much lower concentrations than the others mentioned (Landete, Ferrer, Polo et al., 2005). Some strains of LAB are unique microorganisms responsible for the histamine, tyramine and phenylethylamine concentrations in wine. We propose that the LAB is not responsible for putrescine levels in wine, because this is not a frequent character of the LAB analysed. The concentration of putrescine, tryptamine and cadaverine cannot be attributed to the microorganisms involved in the viniWcation, it is possible that some biogenic amine concentration is from the grapes and Vitis vinifera, with some amines being normal constituents of grapes in variable amounts in diVerent varieties. Among the biogenic amines detected in grapes, putrescine and spermidine are usually abundant (20 and 45 mg/kg of fresh fruit, respectively) (Baucom, Tabacchi, Cottrell, & Richmond, 1996; Buteau et al., 1984; Ough et al., 1987; Radler & Fáth, 1991). Low potassium concentrations in soil have been reported to be responsible for high putrescine concentrations in plants (Landete, Ferrer, Polo et al., 2005; Vaz de Arruda Silveira, Malavolta, & Broetto, 2001). As the histamine, tyramine and phenylethylamine concentrations found in must are very low or non-existent (Landete, Ferrer, Polo et al., 2005). So, it is normal that the concentrations of histamine, tyramine and phenylethylamine must be attributed to strains of LAB. We have observed that O. oeni, Lact. hilgardii, Lact. mali, Leuc. mesenteroides and Ped. parvulus can contribute to the histamine synthesis in wine, but the main species responsible of high histamine production in wines seem to be Lact. hilgardii and Ped. parvulus. We demonstrate in this work that histamine-producing strains of O. oeni are very frequent in wine, in contrast with the paper of Moreno-Arribas et al. (2003), where no Oenococcus histamine producer strains were detected. However, our work agrees with Guerrini, Mangani, Granchi, and Vincenzini (2002) who found a high number of Oenococcus histamine producers in wine, but low levels of histamine production in general. Histamine-producing strains of Lactobacillus, Pediococcus and Leuconostoc are also detected, but with lower frequencies. Our results do not disagree with the most common idea that Pediococcus spp. (DelWni, 1989) is the main organism responsible for histamine production, because although the percentage of Pediococcus histamine producers is low, some strains can produce the highest concentration of histamine. In addition, Lact. hilgardii was also capable of producing high levels of histamine. We have demonstrated that phenylethylamine production is associated with tyramine production in LAB. Others authors, such as Gonzales del Llano, Cuesta, and Rodríguez (1998), have already reported this correlation in some high tyramine producing Leuconostoc strains isolated from dairy products. Moreno-Arribas, Torlois, Joyeux, Bertrand, and Lonvaud-Funel (2000) found similar results in Lact. brevis and Lact. hilgardii isolated from wine. This correla-
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tion could be explained by the fact that phenylalanine is also a substrate for tyrosine decarboxylase, producing phenylethylamine in a secondary reaction, as demonstrated by Boeker and Snell (1972). It has been demonstrated that the ability to form tyramine and phenylethylamine is not widespread among wine LAB. They are held mainly by Lact. brevis and some Lact. hilgardii strains, but not by other common LAB found in wine. Other authors have reported that all strains of Lact. brevis isolated, from wine and from meat, were able to produce tyramine (Moreno-Arribas et al., 2000). The variability of this character in Lact. hilgardii identiWed in this work has been already been reported by Moreno-Arribas et al. (2000). The inability of O. oeni, Ped. pentosaceus, Lact. plantarum, Lact. casei to produce tyramine, as shown by our strains, agrees with the results obtained by Moreno-Arribas et al. (2003). Marcobal et al. (2004) identiWed the only putrescine-producer O. oeni strains. They also showed that the presence of the ornithine decarboxylase gene is a rare event in Spanish wine O. oeni strains. Therefore, it is in accord with the data presented in this study. Landete, Ferrer, Polo et al. (2005) showed an increase in histamine, tyramine and phenylethylamine, during the malolactic fermentation; however, they did not observe an increase in the case of putrescine, cadaverine and tryptamine. This is also in accord with LAB being responsible of histamine, tyramine and phenylethylamine levels in wine. From this work, we emphasize the importance of the 100% correlation between biogenic amine production in synthetic medium and in wine, which allow us to use the synthetic medium in further experiments. In fact, this has several advantages in comparison with wine, due to its constant chemical composition and its cheaper price. As 100% correlation between the presence of hdc, tdc and odc genes and the histamine, tyramine and putrescine production has been demonstrated we aYrm that PCR speciWc is a easy genetic useful for the screening of dangerous LAB in foods. One way to prevent the problem of high biogenic amine concentration would be in reducing to a minimum the length of the processes that incorporate amino acids to must or wine as grape skin maceration and the contact with lees, but this is impossible when aged wines are intended. Others factors that are not susceptible to change are grape variety and the kind of soil. For these reasons, the control of this problem could be easily solved by inhibiting the growth of indigenous LAB and inoculating commercial selected O. oeni strains unable to produce biogenic amine. In support of this idea, Landete, Ferrer, Polo et al. (2005) found lower concentrations of histamine, tyramine and phenylethylamine in wines in which malolactic fermentation was performed with a commercial starter culture. Acknowledgements The authors gratefully acknowledge to the Comisión Interministerial de Ciencia y Tecnología (CICYT), Ministerio
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de Educación y Ciencia, Spain (Project AGL2000-0827C02-01) by Wnancial support of this work. References Aerny, J. (1985). Origine de l’histamine des vins. Connaissances actuelles. Bulletin de l’O.I V., 656–657, 1016–1019. Baucom, T. L., Tabacchi, M. H., Cottrell, T. H. E., & Richmond, B. S. (1996). Biogenic amine content of New York state wines. Journal of Food Science, 51, 1376–1377. Bauza, T., Blaisse, A., Teissedre, P. L., Cabanis, J. C., Kanny, G., & Moneret-Vautrin, D. A. (1995). Les amines biogenes du vin, métabolisme et toxicité. Bulletin de l’O.I V., 767–768, 42–67. Blasco, L., Pardo, I., & Ferrer, S. (submitted for publication). Development of speciWc Xuorescent oligonucleotide probes for in situ identiWcation of wine acetic acid bacteria. International Journal of Food Microbiology. Boeker, E. A., & Snell, E. E. (1972). Amino acid decarboxylases. In P. D. Boyer (Ed.), The enzymes (Vol .VI, third ed., p. 217). NY: Academic Press. Buteau, C., Duitschaever, C. L., & Ashton, G. C. (1984). High-performance liquid chromatographic detection and quantitation of amines in must and wine. Journal of Chromatography, 284, 201–210. Caruso, M., Fiore, C., Contursi, M., Salzano, G., Paparella, A., & Romano, P. (2002). Formation of biogenic amines as criteria for the selection of wine yeasts. Journal of Microbiology and Biotechnology, 18, 159–163. Cilliers, J. D., & van Wyk, C. J. (1985). Histamine and tyramine content of South African wine. South African Journal of Enology and Viticulture, 6, 35–40. DelWni, C. (1989). Ability of wine malolactic bacteria to produce histamine. Sciences des Aliments, 9, 413–416. Forsythe, W. L., & Redmond, A. (1974). Two controlled trials of tyramine in children with migraine. Developmental Medicine and Child Neurology, 16, 794–799. Gonzales del Llano, D., Cuesta, P., & Rodríguez, A. (1998). Biogenic amine production by wild lactococal and leuconostoc strains. Letters in Applied Microbiology, 26, 270–274. Guerrini, S., Mangani, S., Granchi, L., & Vincenzini, M. (2002). Biogenic amine production by Oenococcus oeni. Current Microbiology, 44, 374–378. Lafon-Lafourcade, S. (1975). L‘histamine des vins. Connaissance de la Vigne et du Vin, 22, 11–24. Landete, J. M., Ferrer, S., & Pardo, I. (2005). Which are the lactic acid bacteria responsible of histamine production in wine? Journal of Applied Microbiology, 99, 580–586. Landete, J. M., Ferrer, S., Polo, L., & Pardo, I. (2005). Biogenic amines in wines from three Spanish regions. Journal of Agriculture and Food Chemistry, 53, 119–1124. Le Jeune, C., Lonvaud-Funel, A., ten Brink, Hofstra, H., & van der Vossen, J. M. B. M. (1995). Development of a detection system for histidine decarboxylating lactic acid bacteria on DNA probes, PCR and activity test. Journal of Applied Bacteriology, 78, 316–326. Lehtonen, P. (1996). Determination of amines and amino acids in wine – A review. American Journal of Enology and Viticulture, 47, 127–133.
Lucas, P., Landete, J., Coton, M., Coton, E., & Lonvaud-Funel, A. (2003). The tyrosine decarboxylase operon of Lactobacillus brevis IOEB 9809: Characterization and conservation in tyramine-producing bacteria. FEMS Microbiology Letters, 229, 65–71. Lucas, P., & Lonvaud-Funel, A. (2002). PuriWcation and partial gene sequence of the tyrosine decarboxylase of Lactobacillus brevis IOEB 9809. FEMS Microbiolgy Letters, 211, 85–89. Marcobal, A., de las Rivas, B., Moreno-Arribas, M. V., & Muñoz, R. (2004). IdentiWcation of the ornithine decarboxylase gene in the putrescine-producer Oenococcus oeni BIFI-83. FEMS Microbiology Letters, 239, 213–220. Maynard, L. S., & Schenker, V. J. (1996). Monoamine-oxidase inhibition by ethanol in vitro. Nature, 196, 575–576. Moreno-Arribas, M. V., Polo, M. C., Jorganes, F., & Muñoz, R. (2003). Screening of biogenic amine production by lactic acid bacteria isolated from grape must and wine. International Journal of Food Microbiology, 84, 117–123. Moreno-Arribas, V., Torlois, S., Joyeux, A., Bertrand, A., & LonvaudFunel, A. (2000). Isolation, properties and behaviour of tyramine-producing lactic acid bacteria from wine. Journal of Applied Microbiology, 88, 584–593. Ough, C. S., Crowell, E. A., Kunke, R. E., Vilas, M. R. S., & Lagier, S. (1987). A study of histamine production by various wine bacteria in model solution and in wine. Journal of Food Processing and Preservation, 12, 63–70. Pardo (1987). Estudio de la microXora presente en mostos y vinos de la D.O. Utiel-Requena. Doctoral Thesis. Universidad de Valencia. Radler, F., & Fáth, K. P. (1991). Histamine and other biogenic amines in wines. In J. Rantz (Ed.), Proceedings of the International Symposium on Nitrogen in Grapes and Wine (pp. 5–195). Davis, CA: American Society for Enology and Viticulture. Rodas, A. M., Ferrer, S., & Pardo, I. (2003). 16S-ARDRA, a tool for identiWcation of lactic acid bacteria isolated from grape must and wine. Systematic Applied Microbiology, 26, 412–422. SouXeros, E., Marie-Lyse, B., & Bertrand, A. (1998). Correlation between the content of biogenic amines and other wine compounds. American Journal of Enology and Viticulture, 49, 266–277. Strasser de Saad, A. M., & Manca de Nadra, M. C. (1987). Isolation and identiWcation of the lactic acid bacteria from Cafayate (Argentina) wines. Microbiologie-Alimants-Nutrition, 5, 45–49. ten Brink, B., Damink, C., Joosten, H. M. L. J., & Huis in‘t Veld, J. H. J. (1990). Ocurrence and formation of biologically active amines in foods. International Journal of Food Microbiology, 11, 73–84. Torrea, D., & Ancin, C. (2002). Content of biogenic amines in a Chardonnay wine obtained through spontaneous and inoculated fermentations. Journal of Agriculture and Food Chemistry, 50, 4895–4899. Vaz de Arruda Silveira, R. L., Malavolta, E., & Broetto, F. (2001). EVect of potassium on dry matter production and concentration of putrescine, spermidine and spermine in Eucaliptus grandis progenies. Scientia Florestalis, 59, 13–25. Zúñiga, M., Pardo, I., & Ferrer, S. (1993). A improved medium for distinguishing between homofermentative and heterofermentative lactic acid bacteria. International Journal of Food Microbiology, 18, 37–42.
Biogenic amine production by lactic acid bacteria, acetic bacteria and yeast isolated from wine J.M. Landete ¤, S. Ferrer, I. Pardo ENOLAB – Laboratori de Microbiologia Enològica, Departament de Microbiologia i Ecologia, Facultat de Biologia, Universitat de València, C/Dr. Moliner 50, 46100 Burjassot, València, Spain Received 14 June 2006; received in revised form 13 December 2006; accepted 19 December 2006
Abstract Biogenic amines are undesirable in all foods and beverages because if consumed at too high concentration, they may induce foodborne intoxications. The biogenic amine production by 155 strains of lactic acid bacteria, 40 strains of acetic bacteria and 36 strains of yeast isolated from wine were analysed in wine, grape must and synthetic media by HPLC. We did not observe biogenic amine production by acetic bacteria and yeast; however, we found production of histamine, tyramine, phenylethylamine and putrescine by LAB. A correlation of 100% was observed between biogenic amine production in synthetic medium and wine and between activity and presence of gene. With the results expose in this paper, we can consider than the lactic acid bacteria are the microorganisms responsible of histamine, tyramine and phenylethylamine production in wine. However, we cannot consider the microorganisms analysed in this work to be those responsible for tryptamine, cadaverine and putrescine levels in wine. These results could lead to future applications for preventing excessive amounts of histamine, tyramine and phenylethylamine forming during viniWcation and storage. © 2007 Elsevier Ltd. All rights reserved. Keywords: Lactic acid bacteria; Acetic bacteria; Yeast; Biogenic amine; Wine; HPLC
1. Introduction Biogenic amines are organic bases endowed with biological activity, frequently found in fermented foods and beverages. They are produced mainly as a consequence of decarboxylation of amino acids. High concentration of biogenic amines can cause undesirable physiological eVects in sensitive humans, especially when alcohol and acetaldehyde are present (Bauza et al., 1995; Maynard & Schenker, 1996). More speciWcally, histamine is known to cause headaches, low blood pressure, heart palpitations, edema, vomiting, diarrhea, and other symptoms (Bauza et al., 1995). Tyramine and phenylethylamine can produce hypertension through the release of noradrenaline and norephedrine, respectively, which are vasoconstrictors (Forsythe & Redmond, 1974). Putrescine
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and cadaverine, although not toxic themselves, aggravate the adverse eVects of histamine, tyramine and phenylethylamine, as they interfere with the enzymes that metabolize them (ten Brink, Damink, Joosten, & Huis in‘t Veld, 1990). The presence of biogenic amines in must and wines is well documented in the literature (Landete, Ferrer, Polo, & Pardo, 2005; Lehtonen, 1996; SouXeros, Marie-Lyse, & Bertrand, 1998). The production of histamine, tyramine and putrescine by lactic acid bacteria (LAB) isolated from wine has also been studied by diVerent authors (Landete, Ferrer, & Pardo, 2005; Le Jeune, Lonvaud-Funel, ten Brink, Hofstra, & van der Vossen, 1995; Moreno-Arribas, Polo, Jorganes, & Muñoz, 2003). However, other microorganisms (yeast and acetic bacteria (AB)) and other biogenic amines (phenylethylamine, cadaverine and tryptamine) present in must and wines have received little or no attention. Only a few studies on the production of non-volatile amines by yeast during alcoholic fermentation are known (Buteau, Duitschaever, & Ashton, 1984). Torrea and Ancin (2002) observed
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the production of isoamylamine and ethylamine, and Caruso et al. (2002) observed the production of ethanolamine and agmatine by yeast. No studies on biogenic amine production by acetic bacteria were found in the literature. Some authors have reported higher biogenic amines levels after malolactic fermentation (Aerny, 1985; Cilliers & van Wyk, 1985); however, other authors have not (Lafon-Lafourcade, 1975; Ough, Crowell, Kunke, Vilas, & Lagier, 1987). Information about of microbial Xora responsible of biogenic amine production would contribute to the knowledge of when the biogenic amine concentration increases and when it is thus necessary to take precautions during winemaking. The aim of this work was to study microorganisms isolated from wine for their ability to produce biogenic amines, and as such we analysed the production of histamine, tyramine, phenylethylamine, putrescine, cadaverine and tryptamine by LAB, yeast and AB in synthetic medium, wine and grape must.
0.1 g, tyrosine 2 g, histidine 2 g, phenylalanine 2 g, ornithine 2 g, lysine 2 g, tryptophane 2 g, pyridoxal phosphate 0.25 g and pH 5.5. All the products used were purchased from Sigma Chemical Co. (St. Louis, MO, USA). To analyse the biogenic amine production by AB, 0.1 ml (OD600 nm D 0.50) of a pre-culture grown in MA were inoculated into 10 ml of MA supplemented with tyrosine 2 g, histidine 2 g, phenylalanine 2 g, ornithine 2 g, lysine 2 g, tryptophane 2 g, pyridoxal phosphate 0.25 g and pH 5.5. In the case of yeast, 0.1 ml of pre-culture grown in YPD was inoculated into 10 ml of YPD supplemented with tyrosine 2 g, histidine 2 g, phenylalanine 2 g, ornithine 2 g, lysine 2 g, tryptophane 2 g, pyridoxal phosphate 0.25 g and pH 5.5. All the microorganisms were incubated at 28 °C during 5 days in aerobic conditions; moreover, the AB were incubated with agitation. 2.3. Analyses of production of biogenic amines by microorganisms in wine
2. Materials and methods 2.1. Strains and growth conditions A total of 155 strains of LAB, 40 strains of AB and 36 strains of yeast were examined in this study (Table 1). The diVerent microorganisms were isolated from wine or must in our laboratory (Blasco, Pardo, & Ferrer, submitted for publication; Landete, Ferrer, Polo et al., 2005; Pardo, 1987; Rodas, Ferrer, & Pardo, 2003) or provided by diVerent culture collection. The other microorganisms were provided by the Spanish Type Culture Collection (CECT), by the German Collection of Microorganisms and Cell Culture (DSMZ) and by the Belgian Co-ordenated Collections of Microorganisms (BCCM/LMG). In this work, we used positive controls isolated from wine Lactobacillus hilgardii 5 W and L. hilgardii X1B (Strasser de Saad & Manca de Nadra, 1987) and Oenococcus oeni BIF83 (Marcobal, de las Rivas, Moreno-Arribas, & Muñoz, 2004). All LAB were grown in MRS broth (Scharlau Chemie S.A. Barcelona, Spain) supplemented with 0.5% L-cysteine, except O. oeni, which was cultivated in MLO (Zúñiga, Pardo, & Ferrer, 1993). All AB were grown in Mannitol Agar (MA) and all yeast in Yeast Peptone Dextran (YPD). MA and YPD were recommended by the CECT. All organisms were incubated at 28 °C until mid-log phase in aerobic conditions; moreover, the AB were incubated with agitation.
To analyse the biogenic amine production in wine, 0.1 ml (OD600 nm D 0.50) of a pre-culture grown with all the microorganisms was inoculated into 10 ml of wine supplemented with 0.5 g l¡1 of histidine, tyrosine, phenylalanine, ornithine, tryptophane and lysine. All of them were incubated at 28 °C for 10 days, and the AB were incubated with agitation. The wines used in this work came from red Bobal (average pH 3.6) and red Tempranillo (average pH 3.65) varieties from Utiel-Requena (Spain). Moreover, we analysed the biogenic amine production by yeast in grape must, 0.1 ml (OD600 nm D 0.50) of a pre-culture grown of yeast was inoculated into 10 ml of grape must supplemented with 0.5 g l¡1 of histidine, tyrosine, phenylalanine, ornithine, tryptophane and lysine. All of them were incubated at 28 °C for 10 days. The grape must used in this work came from red Tempranillo (average pH 3.45) varieties from Utiel-Requena (Spain). A sample of wine and grape must without inoculation was included in the experiments as blank. The wine and grape must used in this work were sterilized before inoculum; they were Wltered through a membrane of cellulose and 0.22 m pore size. The microorganisms were streaked on adequate plate medium for having evidence on the survival of the bacterial cells after their inoculation into wine or grape must. 2.4. Biogenic amine analyses by HPLC
2.2. Analyses of biogenic amines produced by microorganisms in synthetic medium The ability to produce diVerent biogenic amines was analysed in LAB. An aliquot (0.1 ml; OD600 nm D 0.50) of a pre-culture grown in MRS or MLO medium was inoculated in 10 ml of biogenic amine production medium (BAPM), consisting (per litre) of: meat extract 8 g, tryptone 5 g, yeast extract 4 g, glucose 1.5 g, fructose 1 g, Tween 80 0.5 g, MgSO4 0.2 g, FeSO4 0.04 g, MnSO4 0.05 g, CaCO3
The biogenic amines content was determined by the HPLC method proposed by Landete, Ferrer, Polo et al. (2005). 2.5. Detection of histidine decarboxylase, tyrosine decarboxylase and ornithine decarboxylase gene Bacterial DNA for partial hdc, tdc and odc gene ampliWcation was obtained by using the Microbial DNA isola-
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Table 1 Microorganisms used in this study Microorganisms
Strains no.
References
Lactic acid bacteria Lactobacillus brevis Lactobacillus casei Lactobacillus collinoides
21 2 1
Rodas et al. (2003), Landete et al. (2005) and Landete, Ferrer, Polo et al. (2005) Rodas et al. (2003) Rodas et al. (2003) Rodas et al. (2003), Landete et al. (2005) and Landete, Ferrer, Polo et al. (2005) Strasser de Saad and Manca de Nadra (1987) Rodas et al. (2003) Rodas et al. (2003) Rodas et al. (2003) Rodas et al. (2003) Rodas et al. (2003) Rodas et al. (2003), Landete et al. (2005), Landete, Ferrer, Polo et al. (2005), Marcobal et al. (2004) Rodas et al. (2003), Landete et al. (2005) and Landete, Ferrer, Polo et al. (2005) Rodas et al. (2003)
Lactobacillus hilgardii Lactobacillus mali Lactobacillus paracasei Lactobacillus plantarum Lactobacillus vini Leuconostoc mesenteroides Oenococcus oeni
17 3 11 5 1 15 39
Pediococcus parvulus Pediococcus pentosaceus
37 3
Acetic bacteria Acetobacter aceti Acetobacter malorum Acetobacter pasteurianus
7 1 6
Acetobacter pomorum Acetobacter sp. Acetobacter tropicales Gluconacetobacter xylinus Gluconacetobacter diazotrophicus Gluconacetobacter europaeus Gluconacetobacter hansenii Gluconacetobacter liquefaciens Gluconacetobacter oboediens Gluconacetobacter oxydans Gluconacetobacter sacchari Gluconobacter cerinus Gluconobacter asaii Gluconobacter frateurii Yeast Aureobasidium pullulans Candida boidinii Hanseniaspora guilliermondii Hanseniaspora uvarum Hansenula mrakii Kloeckera apiculata Metschnikowia pulcherrima Pichia kluyveri Pichia membranaefaciens Pichia pinus Rhodotorula rubra Saccharomyces cerevisiae Saccharomyces cerevisiae var. bayanus Saccharomyces cerevisiae var.chevalieri Saccharomyces cerevisiae var. Steiner
1 1 1 4 1 1 4 2 1 5 1 1 1 2 1 1 2 1 1 2 1 2 2 1 2 14 2 3 1
Blasco et al. (submitted for publication), 298t CECT, 1693, 1372 and 1531 LMG Blasco et al. (submitted for publication) Blasco et al. (submitted for publication), 474 and 824 CECT, 1553 and 1601 LMG, 3509 DSMZ 18884 LMG 944 CECT Blasco et al. (submitted for publication) 315t and 473 CECT, 1515t LMG, 2004 DSMZ 7603 LMG 6160 DSMZ Blasco et al. (submitted for publication), 1524 and 1529 LMG 1348 LMG, 5603 DSMZ 11826 DSMZ Blasco et al. (submitted for publication), 360t and 4009 CECT, 1408 and 1414 LMG 2117t DSMZ 1368 LMG 1390 LMG Blasco et al. (submitted for publication), 1365 LMG Pardo (1987) Pardo (1987) Pardo (1987) Pardo (1987), 10389 CECT Pardo (1987) Pardo (1987) Pardo (1987) Pardo (1987) Pardo (1987) Pardo (1987) Pardo (1987) Pardo (1987), T73 CECT Pardo (1987) Pardo (1987) Pardo (1987)
tion Kit (MoBio Laboratories, Inc). We used diVerent primer pairs for amplifying the internal part of histidine decarboxylase A gene of LAB. These primer pairs JV16HC/JV17HC and CL1mod/JV17HC were used by Landete et al. (2005). The detection of tdc gene in LAB was made by partial ampliWcation of this gene using the primers p0303 (Lucas, Landete, Coton, Coton, & Lonvaud-Funel, 2003) and P1-rev (Lucas & Lonvaud-Funel, 2002). The oligonucleotides primers and conditions used
to detection of tdc gene were the proposed by Marcobal et al. (2004). 3. Results Cultures of 231 microorganisms representing LAB, AB and yeast were investigated for their potential to form histamine, tyramine, phenylethylamine, putrescine, cadaverine and tryptamine. Table 2 shows the number of
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Table 2 Microorganisms (from Table 1) capable to produce biogenic amines in synthetic medium, wine and grape must (in the case of yeast) Microorganisms
Strains Total strains forming tested Hist Tyr Phen Putr
Lactic acid bacteria 155 Acetic bacteria 40 Yeast 36
Cad
Trypt
53/53 32/32 32/32 2/2 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0
positive strains out of the total number of strains investigated. No strains of AB or yeast were able to produce the biogenic amines studied in this work. Strains of LAB were able to produce histamine, tyramine, phenylethylamine and putrescine (Table 2). However, no LAB were able to produce cadaverine or tryptamine (Table 2). In the LAB isolated from wine, Pediococcus parvulus, Lact. mali and Leuconostoc mesenteroides only could produce histamine, Lact. brevis can produce tyramine and phenylethylamine, O. oeni can produce histamine and putrescine, while Lact. hilgardii was able to produce histamine, tyramine, phenylethylamine and putrescine (Table 3). A correlation of 100% was observed between biogenic amine production in synthetic medium and wine; moreover, the biogenic amines produced in synthetic medium were always lower than in wine. All strains showing ability to produce histamine, tyramine and putrescine harboured the hdc, tdc and odc gene respectively. However, this gene was not present in strains that were unable to produce these amines. We observed that all the Pediococcus produced levels higher than 200 mg l¡1 in synthetic medium; however 83 mg l¡1 was the highest histamine production in wine. Pediococcus was the genus with the highest histamine production (Table 3). We also observed that although Oenococcus is the genus that showed the highest percentage of histidine decarboxylase positive strains, they also showed lower levels of histamine production, very low levels of histamine were produced by O. oeni in wine (Table 3). In the
case of Lactobacillus genus, Lact. hilgardii produced higher levels of histamine; in contrast, Lact. mali showed a low histamine production in synthetic medium and wine. This ability to produce tyramine appears to be a general character of Lact. brevis: all assayed strains produced this amine in synthetic medium and wine, although the tyramine production in wine was very low, being 14 mg l¡1 the highest tyramine production in wine by Lact. brevis (Table 3). However, the tyramine production is a strain-dependent character in Lact. hilgardii, as only 3 of 17 strains produced it. Lact. hilgardii strains were able to produce higher levels of tyramine in wine that Lact. brevis. We can observe that tyramine producer strains were also able to produce phenylethylamine, although the quantity of this last one was always lower in both synthetic medium and wine. In our laboratory, we did not Wnd any microorganisms able to produce putrescine; however, Lact. hilgardii X1B and the O. oeni BIF83, coming on from others laboratories, were able to produce putrescine (Table 3). 4. Discussion Throughout the years, investigators have questioned which microorganisms are responsible of biogenic amine production, because it is important to know the ability to produce amines of microorganisms involved in fermented foods in order to establish the potential risk of toxicological disorders in consumers. This work is the most extensive study about biogenic amine production by microorganisms and the Wrst exhaustive study of biogenic amine production by yeast and AB. Biogenic amine production has been analysed in the synthetic media appropriate for each microorganisms, in wine and grape must. The biogenic amine production by LABs had been analysed in others works (Landete et al., 2005; Moreno-Arribas et al., 2003) but this work is the Wrst exhaustive study of the biogenic amine production by LABs in wine, natural medium of these microorganisms. Therefore, the data obtained from wine supply higher information.
Table 3 QuantiWcation of the ability of lactic acid bacteria to generate histamine, tyramine, phenylethylamine and putrescine in synthetic medium and wine: number of strains producing biogenic amines (mean § SD values) Lactic acid bacteria
Lactobacillus brevis Lactobacillus casei Lactobacillus collinoides Lactobacillus hilgardii Lactobacillus mali Lactobacillus paracasei Lactobacillus plantarum Lactobacillus vini Leuconostoc mesenteroides Oenococcus ovni Pediococcus parvulus Pediococcus pentosaceus
Strains Histamine (mg l¡1) no. BAPM Wine
Tyramine (mg l¡1) BAPM
21 2 1 17 3 11 5 1 15 39 37 3
21 (755.8 § 314.2) 0 0 3 (482 § 50.3) 0 0 0 0 0 0 0 0
0 0 0 5 (135.8 § 80.4) 2 (43.5 § 3.5) 0 0 0 1 (43) 33 (22.3 § 17.7) 8 (260 § 58.6) 0
0 0 0 5 (24.2 § 14.5) 2 (10.1 § 1.1) 0 0 0 1 (17) 33 (4.2 § 3.2) 8 (37.3 § 8.6) 0
Phenylethylamine (mg l¡1)
Putrescine (mg l¡1)
Wine
BAPM
Wine
BAPM
Wine
21 (7.1 § 4.2) 0 0 3 (23.2 § 7.8) 0 0 0 0 0 0 0 0
21 (169.2 § 67.4) 0 0 3 (92.5 § 21.8) 0 0 0 0 0 0 0 0
21 (4.2 § 3.1) 0 0 3 (7.8 § 4.1) 0 0 0 0 0 0 0 0
0 0 0 1 (221) 0 0 0 0 0 1 (129) 0 0
0 0 0 1 (67) 0 0 0 0 0 1 (42) 0 0
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Histamine and tyramine are, besides putrescine, the most abundant amines in wine. Phenylethylamine is also frequently found. Although tryptamine and cadaverine are also found in wine, they are in much lower concentrations than the others mentioned (Landete, Ferrer, Polo et al., 2005). Some strains of LAB are unique microorganisms responsible for the histamine, tyramine and phenylethylamine concentrations in wine. We propose that the LAB is not responsible for putrescine levels in wine, because this is not a frequent character of the LAB analysed. The concentration of putrescine, tryptamine and cadaverine cannot be attributed to the microorganisms involved in the viniWcation, it is possible that some biogenic amine concentration is from the grapes and Vitis vinifera, with some amines being normal constituents of grapes in variable amounts in diVerent varieties. Among the biogenic amines detected in grapes, putrescine and spermidine are usually abundant (20 and 45 mg/kg of fresh fruit, respectively) (Baucom, Tabacchi, Cottrell, & Richmond, 1996; Buteau et al., 1984; Ough et al., 1987; Radler & Fáth, 1991). Low potassium concentrations in soil have been reported to be responsible for high putrescine concentrations in plants (Landete, Ferrer, Polo et al., 2005; Vaz de Arruda Silveira, Malavolta, & Broetto, 2001). As the histamine, tyramine and phenylethylamine concentrations found in must are very low or non-existent (Landete, Ferrer, Polo et al., 2005). So, it is normal that the concentrations of histamine, tyramine and phenylethylamine must be attributed to strains of LAB. We have observed that O. oeni, Lact. hilgardii, Lact. mali, Leuc. mesenteroides and Ped. parvulus can contribute to the histamine synthesis in wine, but the main species responsible of high histamine production in wines seem to be Lact. hilgardii and Ped. parvulus. We demonstrate in this work that histamine-producing strains of O. oeni are very frequent in wine, in contrast with the paper of Moreno-Arribas et al. (2003), where no Oenococcus histamine producer strains were detected. However, our work agrees with Guerrini, Mangani, Granchi, and Vincenzini (2002) who found a high number of Oenococcus histamine producers in wine, but low levels of histamine production in general. Histamine-producing strains of Lactobacillus, Pediococcus and Leuconostoc are also detected, but with lower frequencies. Our results do not disagree with the most common idea that Pediococcus spp. (DelWni, 1989) is the main organism responsible for histamine production, because although the percentage of Pediococcus histamine producers is low, some strains can produce the highest concentration of histamine. In addition, Lact. hilgardii was also capable of producing high levels of histamine. We have demonstrated that phenylethylamine production is associated with tyramine production in LAB. Others authors, such as Gonzales del Llano, Cuesta, and Rodríguez (1998), have already reported this correlation in some high tyramine producing Leuconostoc strains isolated from dairy products. Moreno-Arribas, Torlois, Joyeux, Bertrand, and Lonvaud-Funel (2000) found similar results in Lact. brevis and Lact. hilgardii isolated from wine. This correla-
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tion could be explained by the fact that phenylalanine is also a substrate for tyrosine decarboxylase, producing phenylethylamine in a secondary reaction, as demonstrated by Boeker and Snell (1972). It has been demonstrated that the ability to form tyramine and phenylethylamine is not widespread among wine LAB. They are held mainly by Lact. brevis and some Lact. hilgardii strains, but not by other common LAB found in wine. Other authors have reported that all strains of Lact. brevis isolated, from wine and from meat, were able to produce tyramine (Moreno-Arribas et al., 2000). The variability of this character in Lact. hilgardii identiWed in this work has been already been reported by Moreno-Arribas et al. (2000). The inability of O. oeni, Ped. pentosaceus, Lact. plantarum, Lact. casei to produce tyramine, as shown by our strains, agrees with the results obtained by Moreno-Arribas et al. (2003). Marcobal et al. (2004) identiWed the only putrescine-producer O. oeni strains. They also showed that the presence of the ornithine decarboxylase gene is a rare event in Spanish wine O. oeni strains. Therefore, it is in accord with the data presented in this study. Landete, Ferrer, Polo et al. (2005) showed an increase in histamine, tyramine and phenylethylamine, during the malolactic fermentation; however, they did not observe an increase in the case of putrescine, cadaverine and tryptamine. This is also in accord with LAB being responsible of histamine, tyramine and phenylethylamine levels in wine. From this work, we emphasize the importance of the 100% correlation between biogenic amine production in synthetic medium and in wine, which allow us to use the synthetic medium in further experiments. In fact, this has several advantages in comparison with wine, due to its constant chemical composition and its cheaper price. As 100% correlation between the presence of hdc, tdc and odc genes and the histamine, tyramine and putrescine production has been demonstrated we aYrm that PCR speciWc is a easy genetic useful for the screening of dangerous LAB in foods. One way to prevent the problem of high biogenic amine concentration would be in reducing to a minimum the length of the processes that incorporate amino acids to must or wine as grape skin maceration and the contact with lees, but this is impossible when aged wines are intended. Others factors that are not susceptible to change are grape variety and the kind of soil. For these reasons, the control of this problem could be easily solved by inhibiting the growth of indigenous LAB and inoculating commercial selected O. oeni strains unable to produce biogenic amine. In support of this idea, Landete, Ferrer, Polo et al. (2005) found lower concentrations of histamine, tyramine and phenylethylamine in wines in which malolactic fermentation was performed with a commercial starter culture. Acknowledgements The authors gratefully acknowledge to the Comisión Interministerial de Ciencia y Tecnología (CICYT), Ministerio
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