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Isolation and characterization of tyramine-producing Enterococcus faecium strains from red wine
Food Microbiology 28 (2011) 434e439
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Food Microbiology journal homepage: www.elsevier.com/locate/fm
Isolation and characterization of tyramine-producing Enterococcus faecium strains from red wine Vittorio Capozzi a, Victor Ladero b, Luciano Beneduce a, María Fernández b, Miguel A. Alvarez b, Bach Benoit d, Barnavon Laurent d, Francesco Grieco c, Giuseppe Spano a, * a
Dipartimento di Scienze degli Alimenti, Università di Foggia, via Napoli 25, 71100 Foggia, Italy IPLA-CSIC, Carretera de Infiesto s/n, 33300 Villaviciosa, Asturias, Spain Istituto di Scienze delle Produzioni Alimentari (ISPA), via Provinciale Lecce-Monteroni, 73100 Lecce, Italy d Inter Rhône, Scientific and Technical Services, 2260 route du grès, 84100 Orange, France b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2010 Received in revised form 12 October 2010 Accepted 19 October 2010 Available online 26 October 2010
Enterococcus faecium strains were isolated from red wines undergoing malolactic fermentation and identified by comparison of their 16S rDNA gene sequences with those included in the GenEMBL Databases. The tyrosine decarboxylase gene was identified in all the strains analysed by PCR using genespecific primers and the ability to produce tyramine in a synthetic media was analysed by RP-HPLC. Survival of an E. faecium strain was also evaluated in microvinification assays using two different musts with different ethanol concentrations (10% and 12% (v/v)). Tyramine production was monitored during the vinification trials. Our results suggest that E. faecium strains isolated from wine are able to produce tyramine and tolerate wine conditions following a pre-acidic stress. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Biogenic amines Wine tdc Enterococcus faecium Tyramine
1. Introduction Biogenic amines (BA) are low molecular weight organic bases, can be formed in fermented food and beverages by bacterial metabolism via the activity of specific amino acid decarboxylases or as a spontaneous chemical reaction. The presence of BA in foods has traditionally been used as an indicator of undesired microbial activity. In the case of non fermented food products, relatively high levels of certain BA have been reported to correlate with deterioration of food products and/or their defective manufacturing. However, in fermented foods and beverages, BA-producing microorganisms can be present in the raw material or be part of the starter or secondary microbiota (Linares et al., 2010). BA are considered as a threat for human health and their toxicity has led to the general agreement that they should not be allowed to accumulate in food (Ladero et al., 2010). BA production in foods requires the availability of precursors (i.e. amino acids), the presence of microorganisms with amino acid decarboxylases, and favourable conditions for their growth and decarboxylating activity (Arena
* Corresponding author. Tel.: þ39 (0) 881 589234; fax: þ39 (0) 881 740211. E-mail address: [email protected] (G. Spano). 0740-0020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2010.10.005
et al., 2008). The most important in foods and beverages are histamine, tyramine, putrescine, cadaverine and b-phenylethylamine, which are respectively produced by the decarboxylation of histidine, tyrosine, ornithine, lysine and b-phenylalanine (LonvaudFunel, 2001; Fernandez and Zúñiga, 2006; Landete et al., 2007). However, putrescine, together with agmatine, can also derive from the arginine metabolism. Several food fermenting lactic acid bacteria (LAB) are able to produce biogenic amines and genes of diverse BA-producing pathways have been identified in LAB. Interestingly, the presence of genes seems to be rather straindependant than species-specific, suggesting that horizontal gene transfer may account for their dissemination in LAB (Lucas et al., 2005; Marcobal et al., 2006; Coton and Coton, 2009). In addition, enzymes involved in BA production can be encoded by unstable plasmids (Lucas et al., 2005; Satomi et al., 2008). The amount and type of BA formed is strongly influenced by the food composition, microbiota and by other parameters which allow bacterial growth during food processing and storage. Several BA have been identified in wine and their total concentration has been reported to range from 0.5 mg l1 to about 50 mg l1 depending on the quality and type of the wine analysed (Lonvaud-Funel, 2001; Landete et al., 2005a). Extensive research has been done to correlate BA production in wine with the presence
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of LAB involved in the winemaking process, since it is known that Lactobacillus, Leuconostoc, Pediococcus and Oenococcus spp. are implicated in biogenic amine production in wine. Leuconostoc mesenteroides has a high potential to produce tyramine or histamine in wine (Moreno-Arribas et al., 2003; Landete et al., 2005b, 2007). Oenococcus oeni is able to significantly contribute to the overall BA content of wines, mainly producing histamine, and the ability of O. oeni to produce BA varies among strains (Coton et al., 1998; Guerrini et al., 2002). Some Pediococcus parvulus strains contribute to histamine accumulation in wine (Landete et al., 2005b). Moreover, some LAB strains have the ability to simultaneously produce different BA (Coton et al., 1998; Moreno-Arribas et al., 2000; Lonvaud-Funel, 2001; Guerrini et al., 2002) suggesting that such strains might possess more than one amino acid decarboxylase activity under specific culture conditions. Different strains of Lactobacillus hilgardii, Lactobacillus buchneri, Lactobacillus brevis and Lactobacillus mali produce a variety of BA in wine (Moreno-Arribas and Lonvaud-Funel, 1999; Moreno-Arribas et al., 2000, 2003; Martín-Álvarez et al., 2006; Costantini et al., 2006; Landete et al., 2007). In addition to Oenococcus and Lactobacillus species, LAB rarely found in wine may be a potential source of BA. For example, histamine producing L. parabuchneri or L. rossiae strains has been found as contaminant microbiota in starters preparations used in winemaking (Costantini et al., 2009) and several authors have reported the isolation of E. faecium strains from the surface of grape berries at harvest or must grapes and their ability to produce tyramine (Marcobal et al., 2004; Renouf et al., 2005). E. faecium tyramine producers had been previously isolated from other fermented products such as cheese (Linares et al., 2010). In this paper we report the identification of E. faecium strains in red wines undergoing malolactic fermentation and their ability to produce tyramine either in laboratory conditions or in microvinification trials. To our knowledge this is the first report about E. faecium viability and contribution to tyramine contents in red wine. 2. Materials and methods 2.1. Bacteria isolation and culture media Enterococcus faecium strains were isolated during a survey of LAB from two vinifications made by two different winemakers. The first vinification (Nero di Troia) showed an alcoholic content of 13.9% (v/v), pH 3.87, and residual sugar of 1.43 g l1, whereas the second (Merlot) was characterized by an alcoholic content of 12.5% (v/v), pH 3.96, and residual sugar of 2.02 g l1. Viable count and isolation were realized during vinification, according to a fixed time schedule, at the end of AF (time zero) and after 7, 14, 21, 35, 42, 50, 56 days. Strains of E. faecium were isolated on MRS media (pH 5.5), supplemented with 100 mg l1 cycloheximide (Sigma, USA) in order to prevent the growth of yeasts and other fungi. 2.2. Molecular strains identification Genomic DNA of putative E. faecium strains was isolated using the Microbial DNA extraction kit (Cabru, Milan, Italy) according to manufacturer’s procedure. The identification of the isolated strains was performed by sequencing of the 16S rRNA. The gene encoding the 16S ribosomal RNA was amplified by PCR using primers pA and pH according to Edwards et al. (1989). The amplicons were purified using GenEluteÔ PCR Clean-Up Kit (Sigma, UK) and sequenced by employing an ABI Prism 373 Stretch automated sequencer performed by Secugen (Madrid, Spain). The resulting sequences were
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compared with DNA sequences from the National Center for Biotechnology Information (NCBI) database using the standard nucleotideenucleotide homology search Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/BLAST). 2.3. Identification of tyrosine decarboxylase gene For the PCR experiment, about 100 ng of genomic DNA was added to a 50 ml PCR mixture containing 1.25 U of Taq polymerase (Qiagen, Italy) 0.2 mM each of dATP, dTTP, dGTP, dCTP, 10 mM TrisHCL, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, and 0.4 M of primers TDC/1 and TDC/2 (50 -AACTATCGTATGGATATCAACG-30 /50 -TAGTCAACCATATTGAAATCTGG-30 ) (Fernandez et al., 2004). The reaction mix was cycled through the following temperature profile: 94 C, 5 min; 94 C, 1 min, 55 C, 1 min, 72 C, 1 min for the first 15 cycles, then 12 cycles at 58 C as the annealing temperature. The PCR reaction was terminated at 72 C for 5 min. After electrophoresis gel were stained with ethidium bromide 1 mg ml1, washed for 10 min and thereafter the gel image was acquired by a VersaDoc 4000 (BIORAD). The amplified products were purified with a Quantum Prep PCR Kleen Spin Columns (Bio-Rad) and cloned in a pGEM-T easy vector (Promega) as recommended by the manufacturer, and the DNA sequencing was carried out by using an Abi 377TM DNA sequencer (Applied Biosystems, Inc). Analyses of DNA and amino acid sequences were carried out using programs accessible at the NCBI website (www.ncbi.nlm.nih.gov). The nucleotide sequences of the tyrosine decarboxylase (tdc) genes isolated from E. faecium OT23 and E. faecium OM27 strains were deposited in the GenBank database and the accession numbers FJ972172 and FJ972173 were respectively assigned. 2.4. Biogenic amine production by Enterococcus faecium Analysis of BA production was undertaken on E. faecium OT23 by reverse-phase high performance liquid chromatography (RP-HPLC) using a Waters liquid chromatograph controlled by Millennium 32 software (Waters, Milford, USA). Strain was grown in M17 medium supplemented with tyrosine, agmatine, histidine or ornithine (2 g l1) for 24 h. The cultures were centrifuged at 8000g for 10 min and the resulting supernatants filtered through a 0.2 mm Supor membrane (Pall, Ann Arbor, USA). The resulting samples were derivatized using dansyl chloride (Krause et al., 1995). Separations were performed using Waters Nova-pak C18 column (150 3.9 mm) and detection was performed at 436 nm following conditions described in Krause et al. (1995). 2.5. Microvinification assays In order to evaluate survival of E. faecium OT23 in wine, two microvinification assays were performed in 10 l of must. The two grape musts were selected on the basis of their sugar contents such as to achieve ethanol concentrations of about 10% (v/v) and 12% (v/v). Duplicate must samples were inoculated with Saccharomyces cerevisiae (var. bayanus) Maurivin™AWRI R2 (Mauri Yeast, Toowoomba, Australia) according to the producer’s instructions, and alcoholic fermentation was performed at 22 C for 16e18 days. Final experimental wines were all characterized by pH 3.8 0.2 and residual sugar content below 2.0 g l1. Thereafter, wine samples were clarified and sterilized by filtration through a 0.22 mm pore size filter. E. faecium strain OT23, was cultivated on MRS broth for 24 h, then a pre-acidic-stress treatment was realized, by inoculation on MRS broth at pH 3.5, for 16 h at 30 C. After that, the strain was inoculated in 100 ml of wine to
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a final concentration of 2 106 CFU ml1 and containers were incubated at 20 C. Viable plate count analysis was realized by plating serial tenfold dilution onto MRS agar (pH 5.5) (Oxoid, UK) acidified with malic acid and added with 100 mM cycloheximide (Sigma, Germany), for 60 days, every 24 h for the first 3 days and then every week. Plates were incubated anaerobically at 30 C. Wine inoculated only with S.cerevisiae (var. bayanus) Maurivin™AWRI R2 was used as additional control. 2.6. Determination of biogenic amine in wine samples Wine samples were collected before inoculation (time 0), 30 and 60 days after E. faecium was inoculated and BA contents determined simultaneously using the method developed by Gómez-Alonso et al. (2007). A Waters 2695 series HPLC system (Waters, USA) coupled with a Waters 2996 PDA (Waters, USA) was used. The column configuration consisted of a reverse phase C18- HL column (Alltech, Alltima, 250 4.6 mm, 5 mm). The compounds analysed were identified on the basis of the aminoenone derivative retention times of the corresponding patterns (SigmaeAldrich Chemie, Steinheim, Germany) and were quantified using the internal pattern method. 3. Results 3.1. Molecular identification of Enterococcus faecium strains and isolation of the tyrosine decarboxylase (tdc) gene Enterococcus faecium strain OT23 was isolated from Nero di Troia wine sample, after 42 days from the end of alcoholic fermentation while E. faecium strain OM27 was isolated from Merlot wine sample, after 50 days from the end of alcoholic fermentation during a survey for LAB. Primary classification was based on results obtained from gram staining, cell morphology and catalase tests and subsequently confirmed by amplification and sequence analysis of their 16S rDNA genes. The nucleotide sequence of the two 16S rRNA genes, obtained from both OT23 and OM27 strains, were identical compared to those from E. faecium 16S sequences available in database, confirming that the isolated strains belonged to E. faecium species (data not shown). The presence of the gene encoding tyrosine decarboxylase (tdc), which is responsible for the decarboxylation of tyrosine to produce tyramine, was verified by PCR using a gene-specific primer pair. E. faecium strains isolated from wine were positive for the PCR reaction, rendering a unique amplification band of the expected size (890 bp) (data not shown). The DNA fragments amplified from E. faecium OT23 and OM27 strains were cloned and sequenced and similarity between these PCR products and similar tyrosine decarboxylase (tdc) sequences among Enterococcus spp. confirmed that the fragment belonged to the internal portion of the homologous genes from E. faecium (Fig. 1). 3.2. Biogenic amine production by E. faecium strains The ability of the isolated E. faecium strains to produce the biogenic amine histamine, tyramine and putrescine, was monitored by RP-HPLC analysis. Both strains were independently grown in M17 supplemented with the different possible substrates (ornithine, agmatine, histidine and tyrosine). The analysis of the chromatograms revealed that the two strains identified as E. faecium were able to produce tyramine. Indeed, a relatively strong production of tyramine with up to 70 mg l1 (OM27) and 86.8 (OT23) mg l1 was observed when tyrosine was added to the media (Fig. 2). Neither histamine or putrescine could be detected.
3.3. Survival and tyramine production of E. faecium strain OT23 in microvinification trials In order to analyse the ability of E. faecium strains to survive in wine environment, a 10 l of vinification trials were set up. E. faecium strain OT23, which has been shown to produce tyramine at the highest concentration, was chosen as pilot strain and inoculated with or without pre-acidic-stress treatment (see Materials and Methods section). Unstressed E. faecium strain was unable to survive more than five days in both 10% (v/v) and 12% (v/v) vinification trials (data not shown). On the contrary, pre-stressed strain was able to tolerate wine conditions (Fig. 3). In fact, viable cells of E. faecium OT23 were recovered in filtered wine after 60 days of microvinification trials, but different survival rates in function of the ethanol content were observed. In particular, in the trial performed in wine at 10% (v/v) ethanol, a decrease from 1.05 106 CFU ml1 to 5.87 105 CFU ml1 was detected after the first day, the second day coincides with a slight growth, after which, a decrease in survival rate was observed. From the 7th to the 21th days post-inoculation the E. faecium OT23 population remains constant and around a value of about 4.00 105 CFU ml1. After one month it diminished to 9.32 104 CFU ml1, with a further drop off noticed after 60 days. The evolution of the bacterial population, after inoculation of wine at 12% (v/v) ethanol with the OT23 strain, followed a similar profile when compared to that detected after cell inoculation at lowest ethanol content, but a minor cell growth rate was detected. In fact, viable cell count of E. faecium strain OT23 in 12% (v/v) microvinification trial starts from about 1.00 106 CFU ml1 and radically diminished down to 1 105 CFU ml1, with a 1 log loss in viability. Subsequently, we detected a slight increase of the total cell count, followed by a new reduction of the population up to 1.30 104 CFU ml1 (14th day). After 3 weeks, 1 and 2 months the number of CFU per millilitre of E. faecium in 12% (v/v) wine were 3.15 103, 8.60 102 and 5.20 102 CFU ml1, respectively. As reported in Fig. 4, at the end of alcoholic fermentation no significant amounts of tyramine was detected in all the wine analysed. In this phase of the vinification process, the level of tyramine never exceeded 0.1 mg l1 (0.07 mg l1). However, when pure culture pre-stressed E. faecium strain OT23 cells, were inoculated in sterile wine at 10% (v/v) or 12% (v/v) of ethanol, production of tyramine was observed in sterile wine at 10% (v/v) of ethanol. In particular, tyramine production (0.3 mg l1), was detectable after 60 days E. faecium OT23 strain was inoculated in sterile wine at 10% (v/v) of ethanol. The produced tyramine lead to a final content of about 4 times more than the initial concentration. No significant differences in tyramine concentration were observed when E. faecium strains was inoculated in wine with 12% (v/v) of ethanol. 4. Discussion Enterococci are natural inhabitants of the human and animal intestinal tracts, but they are also found in soil, on plants and commonly occur in large numbers in vegetables, plant material and foods, especially those of animal origin such as fermented sausages and cheeses (Giraffa, 2002; Fernández and Zúñiga, 2006). In processed meats, enterococci such as E. faecium are generally not desirable because they cause spoilage (Giraffa, 2002). In cheeses, especially those elaborated with raw milk, E. faecium can constitute one of the predominant species (Serio et al., 2007). The beneficial role of E. faecium in the development of cheese aroma has been proposed by some authors (Foulquie Moreno et al., 2006) and has led to the inclusion of E. faecium strain K77D as a starter culture in fermented dairy products by the UK Advisory Committee on Novel Foods and Processes (Sarantinopoulos et al., 2001). However,
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Fig. 1. Sequence comparison between tyrosine decarboxylase (tdc) genes of Enterococcus faecium strains OT23 and OM27 and the E. faecium gene for phenylalanine and tyrosine decarboxylase(Ef; GenBank:AJ783966.1). Identical nucleotides are indicated by asterisks.
entorococci such as E. faecium, Enterococcus faecalis and Enterococcus durans are usually able to produce tyramine from tyrosine (Marcobal et al., 2004; Fernández and Zúñiga, 2006; Fernandez et al., 2007; Serio et al., 2007) and has been identified as the main responsible for tyramine production in some blue-veined cheeses elaborated with raw milk (Ladero et al., in press).
In wine, tyramine is one of the most frequent BA (0e28 mg/L) (Ancin-Azpilicueta et al., 2008) and usually produced by lactobacillus strains mainly belong to L. brevis and L. hilgardii species (Lucas et al., 2003). However, the origin of tyramine in wine may be also related to LAB rarely detected in wine. For example, several authors have already described the isolation of E. faecium strains from the
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V. Capozzi et al. / Food Microbiology 28 (2011) 434e439 100,0 90,0 80,0
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Fig. 2. Tyramine production by Enterococcus faecium OT23 and OM27 strains as revealed by RP-HPLC. Tyramine (mg/l) produced by E. faecium OT23 and OM27 strains, independently inoculated in M17 supplemented with tyrosine (2 g l1) for 24 h.
surface of grape berries at harvest (Renouf et al., 2005) or must grapes (Marcobal et al., 2004) and their ability to produce tyramine (Marcobal et al., 2004). In this paper we report the isolation and identification of E. faecium strains during malolactic fermentation of red wine in regional wineries. Viability of E. faecium analysed in microfermentation wine trials, highlights the ability of E. faecium to cope with the harsh environmental conditions found in must and wine. The behaviour of the inoculated E. faecium strain was apparently dependent from the ethanol concentration. Indeed, E. faecium strain was able to survive in wine with ethanol contents of 10% (v/v) for more than 30 days at a concentration of about 104 CFU ml1. In contrast, a rapid decline in viability was observed in wine with an ethanol concentration of 12% (v/v). However, this feature is likely influenced by other factors such as pH values. Although the E. faecium strains were isolated from wines containing more than 12% ethanol, indicating that they may survive in wine with increased alcohol content, the pH value of these wines were also significantly higher (pH 3.87 and pH 3.96) compared to pH 3.8 used for laboratory trials. The isolated E. faecium strain was able to produce tyramine in conditions that mimic wine fermentation. It is important to notice that although the amount of tyramine produced is not as high as that observed in other food products such as cheese or fermented meats, the fact that it is produced in an alcoholic beverage, a factor that is known as enhancer of the BA toxicity (Silla-Santos, 1996), could lead to provoke toxic reactions. Actually, a concentration of tyramine of 6 mg kg1 has been reported to be potentially toxic 1200000
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when ingested in combination with monoamine oxidase inhibitors (McCabe-Sellers, 1986), a value that could be lower if it is ingested in combination with alcohol. The production of tyramine was striclty dependent of the ability of E. faecium strain to survive during malolactic fermentation. Tyramine production occurred mainly between the 28th and the 60th days, after the E. faecium strain was inoculated in wine with 10% (v/v) of ethanol contents. During this time, CFU number remains constant after an initial loss of viability. This is probably consistent with the biological role of biogenic amine productions. Indeed, in prokaryotic cells the physiological role of BA synthesis mainly appears to be related to defence mechanisms used by bacteria to withstand acidic environments (Rhee et al., 2002; Lee et al., 2007). Decarboxylation increases survival under acidic stress conditions (Rhee et al., 2002) via the consumption of protons and the excretion of amines and CO2, helping to restore the internal pH (van de Guchte et al., 2002). Therefore, E. faecium strains may produce tyramine in order to survive in the acidic environment that is formed during malolactic fermentation. To our knowledge, this is the first report regarding the survival of E. faecium in wine undergoing malolactic fermentation. In addition, an in vivo contribution of E. faecium to the production of tyramine has been established. Overall, our results suggest that, at least in some wineries, E. faecium may be also considered a minor component of wine LAB microflora. In our case, contamination by E. faecium may arise from grapes, although we were unable to identify E. faecium strains on grapes used in the wineries analysed. Wineries equipments or practices may also be a font of E. faecium contamination, as well as the well water used by wineries for cleaning fermentation tanks, which are now under analyses. However, wherever contamination arises, avoiding E. faecium during malolactic fermentation may probably contribute to the reduction of tyramine in regional wines. Acknowledgments
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Fig. 4. Tyramine production by Enterococcus faecium inoculated in wine at the end of the alcoholic fermentation. Tyramine (mg/l) produced by E. faecium strain OT23 inoculated in red wine at 10% (grey bars) or 12% (white bars) of ethanol content. Tyramine was quantified by HPLC at the end of the alcoholic fermentation (a), after 28 (b) and 60 days (c) of malolactic fermentation.
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This work was funded by the EU commission in the framework of the BIAMFOOD project (Controlling Biogenic Amines in Traditional Food Fermentations in Regional Europe - project n 211441). V. Ladero is beneficiary of I3P - CSIC contract financed by the European Social Fund.
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References Fig. 3. Viable cell count of Enterococcus faecium strain OT23 during microvinification trials at two different ethanol contents. Evolution of the bacterial population in wine after inoculation at the end of the alcoholic fermentation, in wine-like media with 10% (A) and 12% v/v (,) of ethanol, carried out by E. faecium strain OT23.
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Lee, Y.H., Kim, B.H., Kim, J.H., Yoon, W.S., Bang, S.H., Park, Y.K., 2007. CadC has a global translational effect during acid adaptation in Salmonella enterica serovar Typhimurium. J. Bacteriol. 189, 2417e2425. Linares, D. M., Martín, M.C., Ladero, V., Alvarez, M.A., Fernández, M. Biogenic amines in dairy products. Crit. Rev. Food Sci. Nutr, in press. Lonvaud-Funel, A., 2001. Biogenic amines in wines: role of lactic acid bacteria. FEMS Microbiol. Lett. 199, 9e13. Lucas, P.M., 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 Microbiol. Lett. 229, 65e71. Lucas, P.M., Wolken, W.A.M., Claisse, O., Lolkema, J.S., Lonvaud-Funel, A., 2005. Histamine-producing pathway encoded on an unstble plasmid in Lactobacillus hilgardii 0006. Appl. Environ. Microbiol. 71, 1417e1424. McCabe-Sellers, B.J., 1986. Dietary tyramine and other pressor amines in MAOI regimens: a review. J. Am. Diet. Assoc. 86, 1059e1064. Marcobal, A., de las Rivas, B., García-Moruno, E., Muñoz, R., 2004. The tyrosine decarboxylation test does not differentiate Enterococcus faecalis from Enterococcus faecium. Syst. Appl. Microbiol. 27, 423e426. Marcobal, A., Martín-Alvarez, P.J., Polo, M.C., Muñoz, R., Moreno-Arribas, M.V., 2006. Formation of biogenic amines throughout the industrial manufacture of red wine. J. Food Prot. 69, 397e404. Martín-Álvarez, P.J., Marcobal, A., Polo, C., Moreno-Arribas, M.V., 2006. Influence of technological practices on biogenic amine contents in red wines. Eur. Food Res. Technol. 222, 420e424. Moreno-Arribas, V., Lonvaud-Funel, A., 1999. Tyrosine decarboxylase activity of Lactobacillus brevis IOEB 9809 isolated from wine and Lb. brevis ATCC 367. FEMS Microbiol. Lett. 180, 55e60. Moreno-Arribas, V., Torlois, S., Joyeux, A., Bertrand, A., Lonvaud-Funel, A., 2000. Isolation, properties and behaviour of tyramine-producing lactic acid bacteria from wine. J. Appl. Microbiol. 88, 584e593. Moreno-Arribas, V., Polo, M.C., Jorganes, F., Muñoz, R., 2003. Screening of biogenic amine production by lactic acid bacteria isolated from grape must and wine. Int. J. Food Microbiol. 84, 117e123. Renouf, V., Claisse, O., Lonvaud-Funel, A., 2005. Understanding the microbial ecosystem on the grape berry surface through numeration and identification of yeast and bacteria. Aust. J. Grape Wine Res. 11, 316e327. Rhee, J.E., Rhee, J.H., Ryu, P.Y., Choi, S.H., 2002. Identification of the cadBA operon from Vibrio vulnificus and its influence on survival to acid stress. FEMS Microbiol. Lett. 208, 245e251. Sarantinopoulos, P., Andrighetto, C., Georgalaki, M.D., Rea, M.C., Lombardi, A., Cogan, T.M., Kalantzopoulos, G., Tsakalidou, E., 2001. Biochemical properties of enterococci relevant to their technological performance. Int. Dairy J. 11, 621e647. Satomi, M., Furushita, M., Oikawa, H., Yoshikawa-Takahashi, M., Yano, Y., 2008. Analysis of a 30 kbp plasmid encoding histidine decarboxylase gene in Tetragenococcus halophilus isolated from fish sauce. Int. J. Food Microbiol. 126, 202e209. Serio, A., Paparella, A., Chaves-López, C., Corsetti, A., Suzzi, G., 2007. Enterococcus populations in Pecorino Abruzzese cheese: biodiversity and safety aspects. J. Food Prot. 70, 1561e1568. Silla-Santos, M.H., 1996. Biogenic amines: their importance in foods. Int. J. Food Microbiol. 29, 213e231. van de Guchte, M., Serror, P., Chervaux, C., Smokvina, T., Ehrlich, S.D., Maguin, E., 2002. Stress responses in lactic acid bacteria. Antonie Van Leeuwenhoek 82, 187e216.
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Food Microbiology journal homepage: www.elsevier.com/locate/fm
Isolation and characterization of tyramine-producing Enterococcus faecium strains from red wine Vittorio Capozzi a, Victor Ladero b, Luciano Beneduce a, María Fernández b, Miguel A. Alvarez b, Bach Benoit d, Barnavon Laurent d, Francesco Grieco c, Giuseppe Spano a, * a
Dipartimento di Scienze degli Alimenti, Università di Foggia, via Napoli 25, 71100 Foggia, Italy IPLA-CSIC, Carretera de Infiesto s/n, 33300 Villaviciosa, Asturias, Spain Istituto di Scienze delle Produzioni Alimentari (ISPA), via Provinciale Lecce-Monteroni, 73100 Lecce, Italy d Inter Rhône, Scientific and Technical Services, 2260 route du grès, 84100 Orange, France b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 July 2010 Received in revised form 12 October 2010 Accepted 19 October 2010 Available online 26 October 2010
Enterococcus faecium strains were isolated from red wines undergoing malolactic fermentation and identified by comparison of their 16S rDNA gene sequences with those included in the GenEMBL Databases. The tyrosine decarboxylase gene was identified in all the strains analysed by PCR using genespecific primers and the ability to produce tyramine in a synthetic media was analysed by RP-HPLC. Survival of an E. faecium strain was also evaluated in microvinification assays using two different musts with different ethanol concentrations (10% and 12% (v/v)). Tyramine production was monitored during the vinification trials. Our results suggest that E. faecium strains isolated from wine are able to produce tyramine and tolerate wine conditions following a pre-acidic stress. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Biogenic amines Wine tdc Enterococcus faecium Tyramine
1. Introduction Biogenic amines (BA) are low molecular weight organic bases, can be formed in fermented food and beverages by bacterial metabolism via the activity of specific amino acid decarboxylases or as a spontaneous chemical reaction. The presence of BA in foods has traditionally been used as an indicator of undesired microbial activity. In the case of non fermented food products, relatively high levels of certain BA have been reported to correlate with deterioration of food products and/or their defective manufacturing. However, in fermented foods and beverages, BA-producing microorganisms can be present in the raw material or be part of the starter or secondary microbiota (Linares et al., 2010). BA are considered as a threat for human health and their toxicity has led to the general agreement that they should not be allowed to accumulate in food (Ladero et al., 2010). BA production in foods requires the availability of precursors (i.e. amino acids), the presence of microorganisms with amino acid decarboxylases, and favourable conditions for their growth and decarboxylating activity (Arena
* Corresponding author. Tel.: þ39 (0) 881 589234; fax: þ39 (0) 881 740211. E-mail address: [email protected] (G. Spano). 0740-0020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2010.10.005
et al., 2008). The most important in foods and beverages are histamine, tyramine, putrescine, cadaverine and b-phenylethylamine, which are respectively produced by the decarboxylation of histidine, tyrosine, ornithine, lysine and b-phenylalanine (LonvaudFunel, 2001; Fernandez and Zúñiga, 2006; Landete et al., 2007). However, putrescine, together with agmatine, can also derive from the arginine metabolism. Several food fermenting lactic acid bacteria (LAB) are able to produce biogenic amines and genes of diverse BA-producing pathways have been identified in LAB. Interestingly, the presence of genes seems to be rather straindependant than species-specific, suggesting that horizontal gene transfer may account for their dissemination in LAB (Lucas et al., 2005; Marcobal et al., 2006; Coton and Coton, 2009). In addition, enzymes involved in BA production can be encoded by unstable plasmids (Lucas et al., 2005; Satomi et al., 2008). The amount and type of BA formed is strongly influenced by the food composition, microbiota and by other parameters which allow bacterial growth during food processing and storage. Several BA have been identified in wine and their total concentration has been reported to range from 0.5 mg l1 to about 50 mg l1 depending on the quality and type of the wine analysed (Lonvaud-Funel, 2001; Landete et al., 2005a). Extensive research has been done to correlate BA production in wine with the presence
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of LAB involved in the winemaking process, since it is known that Lactobacillus, Leuconostoc, Pediococcus and Oenococcus spp. are implicated in biogenic amine production in wine. Leuconostoc mesenteroides has a high potential to produce tyramine or histamine in wine (Moreno-Arribas et al., 2003; Landete et al., 2005b, 2007). Oenococcus oeni is able to significantly contribute to the overall BA content of wines, mainly producing histamine, and the ability of O. oeni to produce BA varies among strains (Coton et al., 1998; Guerrini et al., 2002). Some Pediococcus parvulus strains contribute to histamine accumulation in wine (Landete et al., 2005b). Moreover, some LAB strains have the ability to simultaneously produce different BA (Coton et al., 1998; Moreno-Arribas et al., 2000; Lonvaud-Funel, 2001; Guerrini et al., 2002) suggesting that such strains might possess more than one amino acid decarboxylase activity under specific culture conditions. Different strains of Lactobacillus hilgardii, Lactobacillus buchneri, Lactobacillus brevis and Lactobacillus mali produce a variety of BA in wine (Moreno-Arribas and Lonvaud-Funel, 1999; Moreno-Arribas et al., 2000, 2003; Martín-Álvarez et al., 2006; Costantini et al., 2006; Landete et al., 2007). In addition to Oenococcus and Lactobacillus species, LAB rarely found in wine may be a potential source of BA. For example, histamine producing L. parabuchneri or L. rossiae strains has been found as contaminant microbiota in starters preparations used in winemaking (Costantini et al., 2009) and several authors have reported the isolation of E. faecium strains from the surface of grape berries at harvest or must grapes and their ability to produce tyramine (Marcobal et al., 2004; Renouf et al., 2005). E. faecium tyramine producers had been previously isolated from other fermented products such as cheese (Linares et al., 2010). In this paper we report the identification of E. faecium strains in red wines undergoing malolactic fermentation and their ability to produce tyramine either in laboratory conditions or in microvinification trials. To our knowledge this is the first report about E. faecium viability and contribution to tyramine contents in red wine. 2. Materials and methods 2.1. Bacteria isolation and culture media Enterococcus faecium strains were isolated during a survey of LAB from two vinifications made by two different winemakers. The first vinification (Nero di Troia) showed an alcoholic content of 13.9% (v/v), pH 3.87, and residual sugar of 1.43 g l1, whereas the second (Merlot) was characterized by an alcoholic content of 12.5% (v/v), pH 3.96, and residual sugar of 2.02 g l1. Viable count and isolation were realized during vinification, according to a fixed time schedule, at the end of AF (time zero) and after 7, 14, 21, 35, 42, 50, 56 days. Strains of E. faecium were isolated on MRS media (pH 5.5), supplemented with 100 mg l1 cycloheximide (Sigma, USA) in order to prevent the growth of yeasts and other fungi. 2.2. Molecular strains identification Genomic DNA of putative E. faecium strains was isolated using the Microbial DNA extraction kit (Cabru, Milan, Italy) according to manufacturer’s procedure. The identification of the isolated strains was performed by sequencing of the 16S rRNA. The gene encoding the 16S ribosomal RNA was amplified by PCR using primers pA and pH according to Edwards et al. (1989). The amplicons were purified using GenEluteÔ PCR Clean-Up Kit (Sigma, UK) and sequenced by employing an ABI Prism 373 Stretch automated sequencer performed by Secugen (Madrid, Spain). The resulting sequences were
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compared with DNA sequences from the National Center for Biotechnology Information (NCBI) database using the standard nucleotideenucleotide homology search Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/BLAST). 2.3. Identification of tyrosine decarboxylase gene For the PCR experiment, about 100 ng of genomic DNA was added to a 50 ml PCR mixture containing 1.25 U of Taq polymerase (Qiagen, Italy) 0.2 mM each of dATP, dTTP, dGTP, dCTP, 10 mM TrisHCL, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, and 0.4 M of primers TDC/1 and TDC/2 (50 -AACTATCGTATGGATATCAACG-30 /50 -TAGTCAACCATATTGAAATCTGG-30 ) (Fernandez et al., 2004). The reaction mix was cycled through the following temperature profile: 94 C, 5 min; 94 C, 1 min, 55 C, 1 min, 72 C, 1 min for the first 15 cycles, then 12 cycles at 58 C as the annealing temperature. The PCR reaction was terminated at 72 C for 5 min. After electrophoresis gel were stained with ethidium bromide 1 mg ml1, washed for 10 min and thereafter the gel image was acquired by a VersaDoc 4000 (BIORAD). The amplified products were purified with a Quantum Prep PCR Kleen Spin Columns (Bio-Rad) and cloned in a pGEM-T easy vector (Promega) as recommended by the manufacturer, and the DNA sequencing was carried out by using an Abi 377TM DNA sequencer (Applied Biosystems, Inc). Analyses of DNA and amino acid sequences were carried out using programs accessible at the NCBI website (www.ncbi.nlm.nih.gov). The nucleotide sequences of the tyrosine decarboxylase (tdc) genes isolated from E. faecium OT23 and E. faecium OM27 strains were deposited in the GenBank database and the accession numbers FJ972172 and FJ972173 were respectively assigned. 2.4. Biogenic amine production by Enterococcus faecium Analysis of BA production was undertaken on E. faecium OT23 by reverse-phase high performance liquid chromatography (RP-HPLC) using a Waters liquid chromatograph controlled by Millennium 32 software (Waters, Milford, USA). Strain was grown in M17 medium supplemented with tyrosine, agmatine, histidine or ornithine (2 g l1) for 24 h. The cultures were centrifuged at 8000g for 10 min and the resulting supernatants filtered through a 0.2 mm Supor membrane (Pall, Ann Arbor, USA). The resulting samples were derivatized using dansyl chloride (Krause et al., 1995). Separations were performed using Waters Nova-pak C18 column (150 3.9 mm) and detection was performed at 436 nm following conditions described in Krause et al. (1995). 2.5. Microvinification assays In order to evaluate survival of E. faecium OT23 in wine, two microvinification assays were performed in 10 l of must. The two grape musts were selected on the basis of their sugar contents such as to achieve ethanol concentrations of about 10% (v/v) and 12% (v/v). Duplicate must samples were inoculated with Saccharomyces cerevisiae (var. bayanus) Maurivin™AWRI R2 (Mauri Yeast, Toowoomba, Australia) according to the producer’s instructions, and alcoholic fermentation was performed at 22 C for 16e18 days. Final experimental wines were all characterized by pH 3.8 0.2 and residual sugar content below 2.0 g l1. Thereafter, wine samples were clarified and sterilized by filtration through a 0.22 mm pore size filter. E. faecium strain OT23, was cultivated on MRS broth for 24 h, then a pre-acidic-stress treatment was realized, by inoculation on MRS broth at pH 3.5, for 16 h at 30 C. After that, the strain was inoculated in 100 ml of wine to
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a final concentration of 2 106 CFU ml1 and containers were incubated at 20 C. Viable plate count analysis was realized by plating serial tenfold dilution onto MRS agar (pH 5.5) (Oxoid, UK) acidified with malic acid and added with 100 mM cycloheximide (Sigma, Germany), for 60 days, every 24 h for the first 3 days and then every week. Plates were incubated anaerobically at 30 C. Wine inoculated only with S.cerevisiae (var. bayanus) Maurivin™AWRI R2 was used as additional control. 2.6. Determination of biogenic amine in wine samples Wine samples were collected before inoculation (time 0), 30 and 60 days after E. faecium was inoculated and BA contents determined simultaneously using the method developed by Gómez-Alonso et al. (2007). A Waters 2695 series HPLC system (Waters, USA) coupled with a Waters 2996 PDA (Waters, USA) was used. The column configuration consisted of a reverse phase C18- HL column (Alltech, Alltima, 250 4.6 mm, 5 mm). The compounds analysed were identified on the basis of the aminoenone derivative retention times of the corresponding patterns (SigmaeAldrich Chemie, Steinheim, Germany) and were quantified using the internal pattern method. 3. Results 3.1. Molecular identification of Enterococcus faecium strains and isolation of the tyrosine decarboxylase (tdc) gene Enterococcus faecium strain OT23 was isolated from Nero di Troia wine sample, after 42 days from the end of alcoholic fermentation while E. faecium strain OM27 was isolated from Merlot wine sample, after 50 days from the end of alcoholic fermentation during a survey for LAB. Primary classification was based on results obtained from gram staining, cell morphology and catalase tests and subsequently confirmed by amplification and sequence analysis of their 16S rDNA genes. The nucleotide sequence of the two 16S rRNA genes, obtained from both OT23 and OM27 strains, were identical compared to those from E. faecium 16S sequences available in database, confirming that the isolated strains belonged to E. faecium species (data not shown). The presence of the gene encoding tyrosine decarboxylase (tdc), which is responsible for the decarboxylation of tyrosine to produce tyramine, was verified by PCR using a gene-specific primer pair. E. faecium strains isolated from wine were positive for the PCR reaction, rendering a unique amplification band of the expected size (890 bp) (data not shown). The DNA fragments amplified from E. faecium OT23 and OM27 strains were cloned and sequenced and similarity between these PCR products and similar tyrosine decarboxylase (tdc) sequences among Enterococcus spp. confirmed that the fragment belonged to the internal portion of the homologous genes from E. faecium (Fig. 1). 3.2. Biogenic amine production by E. faecium strains The ability of the isolated E. faecium strains to produce the biogenic amine histamine, tyramine and putrescine, was monitored by RP-HPLC analysis. Both strains were independently grown in M17 supplemented with the different possible substrates (ornithine, agmatine, histidine and tyrosine). The analysis of the chromatograms revealed that the two strains identified as E. faecium were able to produce tyramine. Indeed, a relatively strong production of tyramine with up to 70 mg l1 (OM27) and 86.8 (OT23) mg l1 was observed when tyrosine was added to the media (Fig. 2). Neither histamine or putrescine could be detected.
3.3. Survival and tyramine production of E. faecium strain OT23 in microvinification trials In order to analyse the ability of E. faecium strains to survive in wine environment, a 10 l of vinification trials were set up. E. faecium strain OT23, which has been shown to produce tyramine at the highest concentration, was chosen as pilot strain and inoculated with or without pre-acidic-stress treatment (see Materials and Methods section). Unstressed E. faecium strain was unable to survive more than five days in both 10% (v/v) and 12% (v/v) vinification trials (data not shown). On the contrary, pre-stressed strain was able to tolerate wine conditions (Fig. 3). In fact, viable cells of E. faecium OT23 were recovered in filtered wine after 60 days of microvinification trials, but different survival rates in function of the ethanol content were observed. In particular, in the trial performed in wine at 10% (v/v) ethanol, a decrease from 1.05 106 CFU ml1 to 5.87 105 CFU ml1 was detected after the first day, the second day coincides with a slight growth, after which, a decrease in survival rate was observed. From the 7th to the 21th days post-inoculation the E. faecium OT23 population remains constant and around a value of about 4.00 105 CFU ml1. After one month it diminished to 9.32 104 CFU ml1, with a further drop off noticed after 60 days. The evolution of the bacterial population, after inoculation of wine at 12% (v/v) ethanol with the OT23 strain, followed a similar profile when compared to that detected after cell inoculation at lowest ethanol content, but a minor cell growth rate was detected. In fact, viable cell count of E. faecium strain OT23 in 12% (v/v) microvinification trial starts from about 1.00 106 CFU ml1 and radically diminished down to 1 105 CFU ml1, with a 1 log loss in viability. Subsequently, we detected a slight increase of the total cell count, followed by a new reduction of the population up to 1.30 104 CFU ml1 (14th day). After 3 weeks, 1 and 2 months the number of CFU per millilitre of E. faecium in 12% (v/v) wine were 3.15 103, 8.60 102 and 5.20 102 CFU ml1, respectively. As reported in Fig. 4, at the end of alcoholic fermentation no significant amounts of tyramine was detected in all the wine analysed. In this phase of the vinification process, the level of tyramine never exceeded 0.1 mg l1 (0.07 mg l1). However, when pure culture pre-stressed E. faecium strain OT23 cells, were inoculated in sterile wine at 10% (v/v) or 12% (v/v) of ethanol, production of tyramine was observed in sterile wine at 10% (v/v) of ethanol. In particular, tyramine production (0.3 mg l1), was detectable after 60 days E. faecium OT23 strain was inoculated in sterile wine at 10% (v/v) of ethanol. The produced tyramine lead to a final content of about 4 times more than the initial concentration. No significant differences in tyramine concentration were observed when E. faecium strains was inoculated in wine with 12% (v/v) of ethanol. 4. Discussion Enterococci are natural inhabitants of the human and animal intestinal tracts, but they are also found in soil, on plants and commonly occur in large numbers in vegetables, plant material and foods, especially those of animal origin such as fermented sausages and cheeses (Giraffa, 2002; Fernández and Zúñiga, 2006). In processed meats, enterococci such as E. faecium are generally not desirable because they cause spoilage (Giraffa, 2002). In cheeses, especially those elaborated with raw milk, E. faecium can constitute one of the predominant species (Serio et al., 2007). The beneficial role of E. faecium in the development of cheese aroma has been proposed by some authors (Foulquie Moreno et al., 2006) and has led to the inclusion of E. faecium strain K77D as a starter culture in fermented dairy products by the UK Advisory Committee on Novel Foods and Processes (Sarantinopoulos et al., 2001). However,
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Fig. 1. Sequence comparison between tyrosine decarboxylase (tdc) genes of Enterococcus faecium strains OT23 and OM27 and the E. faecium gene for phenylalanine and tyrosine decarboxylase(Ef; GenBank:AJ783966.1). Identical nucleotides are indicated by asterisks.
entorococci such as E. faecium, Enterococcus faecalis and Enterococcus durans are usually able to produce tyramine from tyrosine (Marcobal et al., 2004; Fernández and Zúñiga, 2006; Fernandez et al., 2007; Serio et al., 2007) and has been identified as the main responsible for tyramine production in some blue-veined cheeses elaborated with raw milk (Ladero et al., in press).
In wine, tyramine is one of the most frequent BA (0e28 mg/L) (Ancin-Azpilicueta et al., 2008) and usually produced by lactobacillus strains mainly belong to L. brevis and L. hilgardii species (Lucas et al., 2003). However, the origin of tyramine in wine may be also related to LAB rarely detected in wine. For example, several authors have already described the isolation of E. faecium strains from the
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Tyramine (mg/l)
70,0 60,0 50,0 40,0 30,0 20,0 10,0 0,0
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Fig. 2. Tyramine production by Enterococcus faecium OT23 and OM27 strains as revealed by RP-HPLC. Tyramine (mg/l) produced by E. faecium OT23 and OM27 strains, independently inoculated in M17 supplemented with tyrosine (2 g l1) for 24 h.
surface of grape berries at harvest (Renouf et al., 2005) or must grapes (Marcobal et al., 2004) and their ability to produce tyramine (Marcobal et al., 2004). In this paper we report the isolation and identification of E. faecium strains during malolactic fermentation of red wine in regional wineries. Viability of E. faecium analysed in microfermentation wine trials, highlights the ability of E. faecium to cope with the harsh environmental conditions found in must and wine. The behaviour of the inoculated E. faecium strain was apparently dependent from the ethanol concentration. Indeed, E. faecium strain was able to survive in wine with ethanol contents of 10% (v/v) for more than 30 days at a concentration of about 104 CFU ml1. In contrast, a rapid decline in viability was observed in wine with an ethanol concentration of 12% (v/v). However, this feature is likely influenced by other factors such as pH values. Although the E. faecium strains were isolated from wines containing more than 12% ethanol, indicating that they may survive in wine with increased alcohol content, the pH value of these wines were also significantly higher (pH 3.87 and pH 3.96) compared to pH 3.8 used for laboratory trials. The isolated E. faecium strain was able to produce tyramine in conditions that mimic wine fermentation. It is important to notice that although the amount of tyramine produced is not as high as that observed in other food products such as cheese or fermented meats, the fact that it is produced in an alcoholic beverage, a factor that is known as enhancer of the BA toxicity (Silla-Santos, 1996), could lead to provoke toxic reactions. Actually, a concentration of tyramine of 6 mg kg1 has been reported to be potentially toxic 1200000
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when ingested in combination with monoamine oxidase inhibitors (McCabe-Sellers, 1986), a value that could be lower if it is ingested in combination with alcohol. The production of tyramine was striclty dependent of the ability of E. faecium strain to survive during malolactic fermentation. Tyramine production occurred mainly between the 28th and the 60th days, after the E. faecium strain was inoculated in wine with 10% (v/v) of ethanol contents. During this time, CFU number remains constant after an initial loss of viability. This is probably consistent with the biological role of biogenic amine productions. Indeed, in prokaryotic cells the physiological role of BA synthesis mainly appears to be related to defence mechanisms used by bacteria to withstand acidic environments (Rhee et al., 2002; Lee et al., 2007). Decarboxylation increases survival under acidic stress conditions (Rhee et al., 2002) via the consumption of protons and the excretion of amines and CO2, helping to restore the internal pH (van de Guchte et al., 2002). Therefore, E. faecium strains may produce tyramine in order to survive in the acidic environment that is formed during malolactic fermentation. To our knowledge, this is the first report regarding the survival of E. faecium in wine undergoing malolactic fermentation. In addition, an in vivo contribution of E. faecium to the production of tyramine has been established. Overall, our results suggest that, at least in some wineries, E. faecium may be also considered a minor component of wine LAB microflora. In our case, contamination by E. faecium may arise from grapes, although we were unable to identify E. faecium strains on grapes used in the wineries analysed. Wineries equipments or practices may also be a font of E. faecium contamination, as well as the well water used by wineries for cleaning fermentation tanks, which are now under analyses. However, wherever contamination arises, avoiding E. faecium during malolactic fermentation may probably contribute to the reduction of tyramine in regional wines. Acknowledgments
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Fig. 4. Tyramine production by Enterococcus faecium inoculated in wine at the end of the alcoholic fermentation. Tyramine (mg/l) produced by E. faecium strain OT23 inoculated in red wine at 10% (grey bars) or 12% (white bars) of ethanol content. Tyramine was quantified by HPLC at the end of the alcoholic fermentation (a), after 28 (b) and 60 days (c) of malolactic fermentation.
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This work was funded by the EU commission in the framework of the BIAMFOOD project (Controlling Biogenic Amines in Traditional Food Fermentations in Regional Europe - project n 211441). V. Ladero is beneficiary of I3P - CSIC contract financed by the European Social Fund.
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References Fig. 3. Viable cell count of Enterococcus faecium strain OT23 during microvinification trials at two different ethanol contents. Evolution of the bacterial population in wine after inoculation at the end of the alcoholic fermentation, in wine-like media with 10% (A) and 12% v/v (,) of ethanol, carried out by E. faecium strain OT23.
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