Biogenic amine production by Oenococcus oeni during malolactic fermentation of wines obtained using different strains of Saccharomyces cerevisiae

Biogenic amine production by Oenococcus oeni during malolactic fermentation of wines obtained using different strains of Saccharomyces cerevisiae

LWT - Food Science and Technology 42 (2009) 525–530 Contents lists available at ScienceDirect LWT - Food Science and Technology journal homepage: ww...

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LWT - Food Science and Technology 42 (2009) 525–530

Contents lists available at ScienceDirect

LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt

Biogenic amine production by Oenococcus oeni during malolactic fermentation of wines obtained using different strains of Saccharomyces cerevisiae Iolanda Rosi*, Francesca Nannelli, Giovanna Giovani ` di Firenze, via Donizetti 6, 50144 Firenze, Italy Dipartimento di Biotecnologie Agrarie, Universita

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 February 2008 Received in revised form 3 July 2008 Accepted 25 August 2008

Twenty-six wild Oenococcus oeni strains were investigated for their ability to form biogenic amines during malolactic fermentation in synthetic medium and in wine. Eight strains produced histamine and tyramine in screening broth at concentrations of 2.6–5.6 mg/L and 1.2–5.3 mg/L, respectively. Based on their ability to form biogenic amines, five strains were selected to inoculate three wines obtained by the fermentation of three different Saccharomyces cerevisiae strains (A, B, and C). All bacterial strains could perform malolactic fermentation for short periods in wine C, whereas only one strain performed complete malolactic fermentation in wines A and B. Two O. oeni strains (261 and 351) produced histamine and tyramine in wine C. Time-course analysis of these compounds showed that for both strains, histamine and tyramine production began at day 10 and finished on day 25, after the end of malolactic fermentation. These results indicate that the ability of O. oeni to produce histamine and tyramine is dependent on the bacterial strain and on the wine composition, which in turn depends on the yeast strain used for fermentation, and on the length of bacteria–yeast contact time after the completion of malolactic fermentation. Ó 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Key words: Histamine Tyramine Malolactic fermentation Sangiovese wine S. cerevisiae

1. Introduction Oenococcus oeni is the bacterial species most suitable for malolactic fermentation (MLF) because it can tolerate the harsh physico-chemical conditions present in wine. Therefore, O. oeni cultures have been used for direct inoculation in order to simplify the management of MLF (Henick-Kling, 1993). Historically, the selection criteria for malolactic starters have been based on classic tests of survival in wine and the extent L-malic acid consumption (Henick-Kling, Sandine, & Heatherbell, 1989). Recently, a lack of amino acid decarboxylase activity has been included in the selection criteria because the products of this enzyme, biogenic amines (BA), can cause allergic disorders if large amounts are ingested or if the natural detoxification process is inhibited or genetically deficient (Bauza et al., 1995). Even though the amounts of BA in wine are quite low in comparison to other fermented foods, the study of these compounds in wine is particularly important because ethanol can enhance their toxic effects on human metabolism by inhibiting the amine-oxidases responsible for their catabolism (Silla Santos, 1996). The role of wine lactic acid bacteria in amine biogenesis has been studied and their decarboxylating capacities vary according to

the strain and vary within a strain according to the environmental conditions (Coton, Rollan, Bertrand, & Lonvaud-Funel, 1998; Lonvaud-Funel, 2001). It has been reported that when a bacterial cell is stressed, it can activate several metabolic processes for ATP production and survival, one of which is BA production through amino acid decarboxylation (Konings, Lolkema, & Poolman, 1995; Rollan, Coton, & Lonvaud-Funel, 1995). Histamine, tyramine, and putrescine are the most significant biogenic amines present in wines where MLF has occurred (Soufleros, Barrios, & Bertrand, 1998). The decarboxylation of histidine to histamine via histidine decarboxylase (Lonvaud-Funel & Joyeux, 1994) and the production of putrescine via ornithine decarboxylase (Marcobal, de las Rivas, ˇ oz, 2004) have been demonstrated for Moreno-Arribas, & Mun single strains of O. oeni, while the main species responsible for tyramine formation in wine, via tyrosine decarboxylase, was identified as Lactobacillus brevis (Moreno-Arribas, Torlois, Joyeux, Bertrand, & Lonvaud-Funel, 2000). However, some authors (Choudhury, Hansen, Engesser, Hammes, & Holzapfel, 1990; Garai, ˜ as, Irastorza, & Moreno-Arribas, 2007; Gardini et al., 2005) Duen reported that two strains of O. oeni were able to decarboxylate tyrosine in a synthetic medium and in cider, while others (Landete,

* Corresponding author. E-mail address: [email protected]fi.it (I. Rosi). 0023-6438/$34.00 Ó 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2008.08.004

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ˇ oz, Pardo, & Ferrer, 2007; Moreno-Arribas, Polo, Jorganes, & Mun 2003) found no potential for producing biogenic amines by this species. Our aim was to screen wild strains of O. oeni for their ability to produce BA and to quantify BA production by studying their capacity to survive, carry out MLF, and produce BA in synthetic medium and in Sangiovese wines obtained by the fermentation of Sangiovese grape juice by three different Saccharomyces cerevisiae strains. 2. Materials and methods 2.1. Microorganisms Twenty-six strains of O. oeni from the malolactic bacteria collection of the Dipartimento di Biotecnologie Agrarie, University of Firenze were used in this study. These strains were isolated over 20 years from different wines subject to spontaneous MLF. The cultures were maintained at 80  C in MRS broth (Oxoid, Milano) modified by addition of 20 mL/100 mL tomato juice, 0.4 g/100 mL DL-malic acid, 0.5 g/100 mL fructose, pH 5.5, and 30 mL/100 mL glycerol. S. cerevisiae strains BM45 (A), SLO (B) (from Lallemand, Montreal, Canada), and strain BLC 83 (C) (from our wine yeast collection) were used for alcoholic fermentation of Sangiovese grape juice. 2.2. Screening for biogenic amine formation The BA-producing capabilities of the O. oeni strains were screened according to the method of Bover-Cid and Holzapfel (1999). Decarboxylase activity was re-activated by subculturing bacteria five times in modified MRS broth containing 0.1 g/100 mL of the precursor amino acids (all from Sigma, Milan, Italy) L-histidine monohydrochloride, tyrosine di-sodium salt, L-ornithine monohydrochloride, and lysine monohydrochloride, supplemented with 0.005 g/100 mL of pyridoxal-5-phosphate. Cultures were grown at 28  C to the late exponential phase, at which time duplicate aliquots were inoculated to 0.1 mL/100 mL into screening decarboxylase media with and without precursor amino acids (0.2 g/100 mL), and incubated for 15 days at 28  C. 2.3. Fermentation trials In order to replicate wine-making conditions at laboratory scale, Sangiovese grape juice was fermented with three different yeast strains, followed by the inoculation of bacterial cultures into the wines. For fermentation, Sangiovese grapes were crushed and destemmed, followed by 10 days of skin maceration. All treatments were carried out in duplicate using 25-kg lots of fruit. Sulfur dioxide (50 mg/L) was added prior to inoculation of 48 h-old yeast cultures (to 2 mL/100 mL) grown in pasteurized red grape juice (commercialized by Hero, Verona, Italy) at 25  C. The caps were mixed into the fermenting wines twice daily. The fermentation temperature was maintained at 28  C. At the end of alcoholic fermentation, the wine samples were pressed and stored in 10-L glass containers. The duplicate wine samples were combined, sterile filtered (HAWP 0.45 mm, Millipore, Bedford, MA, USA), and 80-mL aliquots were dispensed into 100-mL sterile glass bottles. Five cultures of O. oeni (139, 261, 351, 431, and 436) were chosen to induce MLF in wines. The bacterial cultures were grown in grape juice medium containing equal parts of pasteurized red grape juice and deionized water, 0.1 g/100 mL yeast extract (Oxoid, Milano; Italy), and 0.05 mL/100 mL Tween 80, at pH 4.5. The medium was sterilized at 120  C for 15 min. After 5 days of growth at 28  C, 2 mL/100 mL of each culture was centrifuged (7245  g, 150 , 4  C), the pellet was resuspended in an aliquot of each wine, and the

cultures were inoculated into the respective wines in duplicate. Wine cultures were incubated at 20  C. A bacteria-free control for each wine was included in the study. 2.4. Analyses Sampling was performed at regular intervals to monitor MLF evolution. The viable bacteria population was determined by counting cells (CFU/mL) on MRS agar plates (Oxoid, Milan, Italy) modified as above and incubated at 28  C in anaerobiosis for 7 days. The course of MLF was monitored by verifying L-malic acid consumption. Enzymatic assays (Boehringer Mannheim, Germany) were used to determine the content of L-malic, L-lactic and citric acids, glucose, and fructose. The concentration of a-amino nitrogen in the wines was determined by spectrophotometry, according to Dukes and Butzke (1998). The alcohol content, reducing sugar content, pH, free and total sulfur dioxide, and total and volatile acidity of the wines were determined according to the methods of the Office International de la Vigne et du Vin (OIV, 1990). For detecting biogenic amines (histamine, tyramine, putrescine, and cadaverine) and precursor amino acids (histidine, tyrosine, lysine, and ornithine), all culture broths and wine samples were centrifuged (7245  g, 150 , 4  C), the supernatants were filtered through a 0.2-mm membrane (Millipore, Bedford, MA, USA) and analyzed in duplicate. Quantitative detection of biogenic amines and precursor amino acids was performed by HPLC with fluorescence detection of the o-phthaldialdehyde derivatives, according to previously described methods (Pereira Monteiro & Bertrand, 1994; Pripis-Nicolau, de Revel, Marchand, Beloqui, & Bertrand, 2001) using a Perkin Elmer series 200 HPLC pump equipped with an LC-240 fluorescence detector (Perkin Elmer, Shelton, CT, USA). Turbochrom Chromatography software (Perkin Elmer, Shelton, CT, USA) was used for data storage and integration. Statistical analysis was performed using Statgraphics Plus version 4.1 1999 (Manugistic, Inc. Rockville, MD, USA).

Table 1 Histamine and tyramine content (mg/L) in the screening decarboxylase medium supplemented with 0.2 g/100 mL precursor amino acids O. oeni strain

Histamine (mg/L)

Tyramine (mg/L)

1 40 53 94 136 139 160 166 168 260 261 290 313 322 328 332 348 351 370 378 402 405 425 428 431 436 Uninoculated medium

0.4  0.06 0.4  0.07 0.4  0.08 2.6  0,12 3.4  0.08 0.8  0.06 3.9  0.05 0.7  0.04 0.4  0.06 0.8  0.05 3.7  0.10 0.5  0.07 0.6  0.04 0.5  0.06 0.6  0.05 0.5  0.04 1.2  0.06 3.8  0.09 1.2  0.07 0.7  0.04 1.2  0.07 0.7  0.05 1.3  0.06 3.6  0.07 3.4  0.08 5.6  0.09 <0.3  0.02

0.5  0.07 0.5  0.06 0.6  0.04 1.3  0.08 1.3  0.05 0.6  0.06 1.2  0.07 0.4  0.03 0.4  0.06 0.5  0.04 1.6  0.07 0.4  0.05 1.9  0.08 0.5  0.06 0.7  0.05 0.7  0.06 1.3  0.08 1.8  0.06 1.4  0.07 0.7  0.08 1.2  0.08 0.6  0.07 1.2  0.05 3.3  0.06 2.6  0.04 5.3  0.07 <0.3  0.01

Medium was inoculated to 0.1 mL/100 mL with Oenococcus oeni cultures, and cultures were incubated at 28  C for 15 days (positive strains appear in bold). Data are reported as mean values  standard deviation.

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Table 2 Chemical characterization of Sangiovese wines used for malolactic fermentation assays prior to bacterial inoculation Wine

Glucose/fructose (g/L)

Ethanol (mL/100 mL)

pH

Total aciditya (g/L)

Volatile acidityb (g/L)

Free/total SO2 (mg/L)

a-Amino nitrogen (mg/L)

L-malic

A B C

0.10/0.90 0.05/0.55 0.25/0.50

15.2 15.4 14.0

3.5 3.5 3.5

7.15 6.85 5.90

0.45 0.40 0.40

8/36 5/24 7/27

55 70 100

1.20 1.00 1.20

a b

acid (g/L)

As tartaric acid. As acetic acid.

3. Results 3.1. Screening of BA formation in synthetic medium Twenty-six strains of O. oeni were tested for their ability to form histamine, tyramine, putrescine, and cadaverine in culture. Even though all of the strains were able to grow (CFU/mL ranging from 7

to 9  109) in the screening broth of Bover-Cid and Holzapfel (1999), decarboxylase activity was not detected by this qualitative procedure; the color of the culture medium of decarboxylase-positive strains should turn purple in response to a pH shift. Although no color changes were observed in the screening media, the production of BA by each O. oeni strain after 15 days of growth in the screening medium was determined by HPLC (Table 1). Amineforming ability was not widespread among the O. oeni strains investigated and only small amounts of histamine and tyramine were produced in the synthetic medium. Of the 26 strains investigated, eight were weak histamine producers, producing 2.6–5.6 mg/L of histamine. We considered only strains that produced at least 2 mg/L histamine because in certain countries this value is the recommended upper limit for histamine content in wine (Lehtonen, 1996). All of the strains that formed histamine were also weak tyramine producers, producing 1.2–5.3 mg/L of tyramine. Strain 436 was the greatest producer of both amines. No putrescine or cadaverine was produced by any strain. 3.2. Malolactic performance and biogenic amine formation Based on their capacity to produce histamine and tyramine, we selected five strains of O. oeni (four amine producers: 261, 351, 431, 436; one negative control: 139) to induce MLF and test their BA-producing behaviour in Sangiovese wines fermented by three different strains of S. cerevisiae (A, B, and C). Table 2 summarizes the characteristics of the wines at the time of bacterial inoculation. The wines differed in their ethanol, total SO2, and a-amino nitrogen content. In particular, wine A showed the lowest amino nitrogen content and the highest level of total SO2. The malolactic performance of the O. oeni strains was verified by evaluating the growth of the culturable population and the rate of L-malic acid degradation. The time-course of each O. oeni population during MLF is reported in Fig. 1. The percentage of L-malic acid consumed after 30 days of MLF is reported in Table 3. In wine C (Fig. 1), all strains maintained large culturable populations and performed complete MLF. For strains 139, 261, and 351, L-malic acid degradation began immediately after inoculation and concluded in five days; after seven days, citric acid was also completely degraded (data not shown). The remaining two strains (431 and 436) completed MLF in 19 days, but even after 30 days, citric acid was not degraded. Wines obtained with yeasts A and B (Fig. 1) were more discriminating of bacteria cultures. Strains 261 and 431 showed a progressive decrease in culturability in association with a decrease in malate degrading capacity. For strains 139 and 436,

Table 3 L-malic acid consumption (%) in wines 30 days after bacterial inoculation Oenococcus oeni strain

Fig. 1. Evolution of Oenococcus oeni viable cells (B, strain 139; , strain 261; 6, strain 351; :, strain 431; ,, strain 436) in wines A, B, and C. The mean values ( standard deviation) are shown.

139 261 351 431 436

L-malic

acid (%) in wine

A

B

C

45 10 100 (30) 10 40

60 10 100 (25) 20 50

100 100 100 100 100

Malolactic fermentation duration (in days) is reported in brackets.

(5) (5) (5) (19) (19)

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Table 4 Concentration (mg/L) of histamine (His), tyramine (Tyr), putrescine (Put), and cadaverine (Cad) in wine after 30 days of malolactic fermentation Oenococcus oeni strain

Wine A

139 261 351 431 436 Control wines

B

C

His

Tyr

Put

Cad

His

Tyr

Put

Cad

His

Tyr

Put

Cad

0.4  0.05 0.3  0.06 0.5  0.04 0.4  0.04 0.3  0.05 <0.4

0.3  0.06 0.4  0.07 0.4  0.05 0.5  0.04 0.5  0.06 <0.4

0.3  0.05 0.3  0.05 0.3  0.07 0.4  0.06 0.4  0.05 <0.4

0.4  0.06 0.3  0.07 0.3  0.06 0.4  0.05 0.3  0.05 <0.4

0.3  0.05 0.5  0.06 0.4  0.05 0.4  0.05 0.3  0.04 <0.4

0.3  0.04 0.3  0.04 0.3  0.05 0.4  0.05 0.5  0.06 <0.4

0.3  0.06 0.3  0.07 0.4  0.05 0.4  0.07 0.5  0.06 <0.4

0.3  0.04 0.4  0.05 0.3  0.07 0.5  0.05 0.3  0.06 <0.4

0.5  0.06 4.0  0.12 2.5  0.10 0.4  0.06 0.4  0.07 < 0.4

0.4  0.06 9.5  0.17 10  0.13 0.6  0.08 0.5  0.06 < 0.4

0.3  0.07 0.3  0.08 0.3  0.09 0.4  0.09 0.4  0.08 < 0.4

0.3  0.05 0.3  0.04 0.3  0.05 0.4  0.07 0.3  0.06 < 0.4

Data are reported as mean values  standard deviation.

despite a culturable cell density of about 106 CFU/mL, L-malate degradation was rather slow (Table 3). For strain 351 in wines A and B, 25 and 30 days, respectively, were necessary to complete MLF. It is possible that the interactions between yeasts and bacteria was related to the nitrogen composition (Table 2). The different a-amino nitrogen content of the three wines at the end of alcoholic fermentation indicated that the three yeast strains used amino acids as sources of nitrogen to differing extents. In order to verify whether the bacterial strains investigated had produced BA in wines, we measured BA levels 30 days after the induction of MLF (Table 4). Wines were checked for the presence of histamine, tyramine, putrescine, and cadaverine before bacterial inoculation, and the levels were lower than 0.4 mg/L for each amine. Thirty days after inoculation, the levels of BA in wines B and A were unchanged, while wine C inoculated with strains 261 and 351 showed increased concentrations of tyramine (10 mg/L) and histamine (2.5–4 mg/L). As reported in Table 5, wines were also characterized for histidine, tyrosine, ornithine, and lysine concentration before inoculation and after 30 days of MLF. Thirty days after the induction of MLF, the levels of precursor amino acid in wines A and B were similar to those found in the control wines whereas in wine C inoculated with strains 261 and 351, the concentration of histidine and tyrosine was one-half of that found in control wine. To determine when bacterial strains 261 and 351 began BA production, we monitored the evolution of histamine and tyramine formation and the growth of culturable cells during and after MLF (Figs. 2 and 3). Both O. oeni strains started to produce tyramine and histamine ten days after MLF completion (day 15), when the bacterial population was decreasing but still at 105–106 CFU/mL. Histamine and tyramine production continued to increase up to day 30 at 2–4 mg/L and 10 mg/L, respectively. By day 65, the production of these compounds had ceased and the bacterial populations showed further decreases in culturability. 4. Discussion Even if MLF is in general a desired process for some wines, the metabolic activity of some strains of O. oeni may give rise to

undesirable compounds, such as biogenic amines. The decarboxylase reaction, which induces an increase in pH and leads to the formation of ATP, can stimulate bacterial survival in difficult conditions. Although the decarboxylation reaction does not yield metabolic energy directly, the free energy of the decarboxylase reaction can be converted to metabolic energy indirectly through an Hþ pump mechanism. Lolkema, Poolman, and Konings (1995) found that an electrogenic precursor/product exchange, as in histidine/ histamine exchange, could contribute to the generation of a proton motive force that consists of both a membrane potential and a pH gradient, similarly to primary proton pumps. Histamine, tyramine, and putrescine are the most significant amines present in wines and previous studies have indicated that their concentrations may increase during MLF, depending on the bacterial strain and on some wine-making factors, such as grape variety, vintage, ageing of wines on lees, and grape skin maceration (Landete, Ferrer, & Pardo, 2005; Marques, Leitao, & San Romao, 2008; Martin-Alvarez, Marcobal, Polo, & Moreno-Arribas, 2006; Soufleros et al., 1998). No evidence of amino acid decarboxylase activity in O. oeni strains was detected in the screening broth; the little BA that was formed (about 10 mg/L) was not sufficient to induce a rise in the pH and a subsequent color change in the media. This result is not surprising; previous reports have acknowledged that this qualitative method for detecting BA is valid only when the level of BA in the medium exceeds 100 mg/L (Landete et al., 2005). HPLC-mediated quantitative detection of BA in the supernatants of cultures grown in decarboxylase broth allowed us to identify histamineand tyramine-producing strains (Table 1). In agreement with previous reports (Landete, Ferrer, & Pardo, 2007), no putrescine producers were found in any of the O. oeni strains investigated. Since wines are complex environments characterized by factors that influence the growth and the amino acid decarboxylase activity of lactic acid bacteria, we studied the amine-forming behavior of five O. oeni strains during MLF in wines obtained by fermenting grape juice with three different strains of S. cerevisiae (Table 2). The strain of S. cerevisiae used to produce the wine is known to influence the survival, development (Fig. 1), and metabolic activity of the O. oeni present in the wine (Tables 3–5).

Table 5 Concentration (mg/L) of histidine, tyrosine, ornithine, and lysine in wine after 30 days of malolactic fermentation Oenococcus oeni strain

139 261 351 431 436 Control wines

Wine A

B

C

Histidine

Tyrosine

Ornithine

Lysine

Histidine

Tyrosine

Ornithine

Lysine

Histidine

Tyrosine

Ornithine

Lysine

15  0.08 20  0.15 15  0.18 20  0.07 15  0.08 20  0.04

25  0.06 30  0.12 25  0.15 30  0.08 30  0.05 30  0.03

100  0.14 105  0.18 100  0.15 105  0.06 100  0.07 110  0.05

130  0.16 130  0.08 130  0.09 135  0.06 130  0.05 140  0.06

30  0.15 30  0.08 30  0.07 30  0.10 30  0.06 35  0.04

45  0.07 50  0.15 45  0.08 50  0.08 50  0.07 55  0.05

115  0.10 120  0.07 115  0.09 120  0.07 115  0.09 125  0.03

205  0.09 205  0.15 200  0.07 205  0.08 200  0.09 210  0.06

45  0.08 25  0.18 25  0.15 45  0.09 40  0.06 50  0.06

70  0.07 50  0.15 45  0.13 70  0.09 70  0.05 95  0.05

230  0.09 225  0.18 225  0.12 230  0.14 230  0.08 235  0.04

330  0.08 325  0.07 325  0.05 330  0.07 320  0.06 340  0.06

Data are reported as mean values  standard deviation.

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decarboxylase broth did not form BA in wine, not even in the wines in which MLF was successful. In fact, for these strains, amino acid decarboxylase activity was enhanced neither by the lack of malic acid nor by the higher concentration of amino acid precursors present in wine C. Characterization of the histidine decarboxylase activity of O. oeni IOEB 9204 showed that citric acid has an inhibitory effect on histidine decarboxylase activity (Lonvaud-Funel & Joyeux, 1994). Since strains 431 and 436 did not utilize citric acid during MLF, it is possible that the presence of this acid inhibited the expression and activity of amino acid decarboxylases.

Fig. 2. Evolution of viability (B) and histamine (His, M) and tyramine (Tyr, ) content in wine C during and after malolactic fermentation (MLF) by Oenococcus oeni strain 261. The mean values ( standard deviation) are shown.

Our results confirmed that interaction between yeast and bacteria in wine is important for the success of MLF (Alexandre, Costello, Remize, Guzzo, & Guilloux-Benatier, 2004). In the ‘‘easier’’ wine, C (lowest alcohol content and highest level of a-amino nitrogen), all strains were able to perform MLF, whereas only one strain (strain 351) was able to maintain a high degree of culturability and metabolic activity under difficult physico-chemical and nutritional conditions (as in wines A and B). In any case, all strains performed MLF when the population did not exceed the inoculum concentration of 106 CFU/mL and was in a ‘‘maintenance mode’’, as previously reported by Arnink and Henick-Kling (2005). After 30 days, wine C inoculated with O. oeni strains 261 and 351 showed an increase in histamine and tyramine concentration (Table 4). Strains 261 and 351 performed MLF quickly in wine C, completely consuming the L-malic acid by five days after inoculation. After 10 days when malic and citric acid had been metabolized the bacteria started to convert histidine to histamine and tyrosine to tyramine resulting in 3–4 mg/L histamine and 10 mg/L of tyramine. In accordance with other reports (Coton, Rollan, & LonvaudFunel, 1998; Konings et al., 1997), we propose that the lack of fermentable substrate (L-malic acid) and the low concentration of a primary energy source (carbohydrates) can activate histidine and tyrosine decarboxylation for metabolic energy generation. Moreover, our results showed that elevated levels of histamine and tyramine corresponded with a decrease in histidine and tyrosine (Table 5), confirming that BA formation is greater when the concentration of amino acid precursors is higher (Lonvaud-Funel & Joyeux, 1994; Moreno-Arribas et al., 2000), although this association was not linear. In fact not all amino acids would be decarboxylated, some also would be integrated into the bacterial cells. Regarding the other bacterial strains investigated, strain 139 (negative control) did not form BA in wines or in decarboxylase broth. On the contrary, strains 431 and 436 that produced BA in

Fig. 3. Evolution of viability (B) and histamine (His, M) and tyramine (Tyr, ) content in wine C during and after malolactic fermentation (MLF) by Oenococcus oeni strain 351. The mean values ( standard deviation) are shown.

5. Conclusion The detection method using a synthetic nutrient medium with high amino acid content and relying on a pH change to indicate the formation of biogenic amines is not sufficient for analyzing strains of O. oeni for their potential to produce biogenic amines. The ability of O. oeni to produce biogenic amines, in particular histamine and tyramine, has been shown to depend on the screening method used, on the bacterial strain, on the wine composition (which in turn depends on the yeast strain inoculated), and on the length of bacteria–yeast contact time after MLF completion. This latter aspect is of practical importance, confirming the necessity to separate the bacterial biomass from the wine immediately following the end of MLF to avoid a loss of quality, not only in sensorial terms but also for health reasons. The results of this study represent another tool for interpreting the compatibility between yeast and bacteria, with regard to not only the success or failure of MLF but also to healthier wine. References Alexandre, H., Costello, P. J., Remize, F., Guzzo, J., & Guilloux-Benatier, M. (2004). Saccharomyces cerevisiae–Oenococcus oeni interactions in wine: current knowledge and perspectives. International Journal of Food Microbiology, 93, 141–154. Arnink, K., & Henick-Kling, T. (2005). Influence of Saccharomyces cerevisiae and Oenococcus oeni strains on successful malolactic conversion in wine. American Journal of Enology and Viticulture, 56, 228–237. Bauza, T., Blaise, A. P., Teissedre, L., Cabanis, J. C., Kanny, G., Moneret-Vautrin, D. A., et al. (1995). Les amines biogenes du vin. Me´tabolism et toxicite´. Bullettin de l’ O.I.V., 68, 42–67. Bover-Cid, S., & Holzapfel, W. H. (1999). Improved screening procedure for biogenic amine production by lactic acid bacteria. International Journal of Food Microbiology, 53, 33–41. Choudhury, N., Hansen, W., Engesser, D., Hammes, W. P., & Holzapfel, W. H. (1990). Formation of histamine and tyramine by lactic acid bacteria in decarboxylase assay medium. Letters in Applied Microbiology, 11, 278–281. Coton, E., Rollan, G. C., Bertrand, A., & Lonvaud-Funel, A. (1998). Histamine producing lactic acid bacteria: early detection, frequency and distribution. American Journal of Enology and Viticulture, 49, 199–204. Coton, E., Rollan, G. C., & Lonvaud-Funel, A. (1998). Histidine decarboxylase of Leuconostoc oenos 9204: purification, kinetic properties, cloning and nucleotide sequence of the hdc gene. Journal of Applied Microbiology, 84, 143–151. Dukes, B. C., & Butzke, C. E. (1998). Rapid determination of primary amino acids in grape juice using an o-Phthaldialdehyde/N-acetyl-L-cysteine spectrophotometric assay. American Journal of Enology and Viticulture, 49, 125–134. ˜ as, M. T., Irastorza, A., & Moreno-Arribas, M. V. (2007). Biogenic Garai, G., Duen amine production by lactic acid bacteria isolated from cider. Letters in Applied Microbiology, 45, 473–478. Gardini, F., Zaccarelli, A., Belletti, N., Faustini, F., Cavazza, A., Martuscelli, M., et al. (2005). Factors influencing biogenic amines production by a strain of Oenococcus oeni in a model system. Food Control, 16, 609–616. Henick-Kling, T. (1993). Malolactic fermentation. In G. H. Fleet (Ed.), Wine microbiology and biotechnology (pp. 289–326). Chur, Switzerland: Harward Academic Publisher. Henick-Kling, T., Sandine, W. E., & Heatherbell, D. A. (1989). Evaluation of malolactic bacteria isolated from Oregon wines. Applied Environmental Microbiology, 55, 2010–2016. Konings, W. N., Lolkema, J. S., Bolhvis, M., van Heen, H. W., Poolman, B., & Driessen, A. J. (1997). The role of transport processes in survival of lactic acid bacteria. Antonie van Leeuwenhoek, 71, 117–128. Konings, W. N., Lolkema, J. S., & Poolman, B. (1995). The generation of metabolic energy by solute transport. Archives of Microbiology, 164, 235–242. Landete, J. M., Ferrer, S., & Pardo, I. (2005). Which lactic acid bacteria are responsible for histamine production in wine? Journal of Applied Microbiology, 99, 580–586. Landete, J. M., Ferrer, S., & Pardo, I. (2007). Biogenic amine production by lactic acid bacteria, acetic acid bacteria and yeast isolated fromwine. Food Control,18,1569–1574.

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