1999 Biotechnology Letters by sergi maicas

349

Production of Oenococcus oeni biomass to induce malolactic fermentation in wine by control of pH and substrate addition Sergi Maicas1,2 , Pilar Gonz´alez-Cabo1,3 , Sergi Ferrer1 & Isabel Pardo1,∗ 1 Departament

de Microbiologia i Ecologia, Facultat de Biologia, Universitat de València, C/ Dr Moliner, 50, E46100 Burjassot-València, Spain Present addresses: 2 Departament de Biotecnologia, Institut d’Agroqu´ımica i Tecnologia d’Aliments (CSIC), Paterna, Spain 3 Unitat de Gen` etica. Hospital Universitari ‘La Fe’, València, Spain ∗ Author for correspondence (Fax: +34-963864372; E-mail: Isabel.Pardo@uv.es) Received 18 January 1999; Accepted 26 February 1999

Key words: biomass production, growth control, lactic acid bacteria, Oenococcus oeni, pH control

Abstract To increase the commercial production of Oenococcus oeni strains to be used for biological deacidification of wines, substrates addition and pH control have been optimized. The highest biomass yield of Oenococcus oeni (Y = 6.9 mg mmol−1 sugar) was obtained when 55 mmol glucose l−1 and 30 mmol fructose l−1 were added both to the culture medium, and the pH was controlled at 4.8. Fructose was used as carbon and energy source, but also as electron acceptor improving the ability to reoxidize NAD(P)H.

Introduction Oenococcus oeni is the major bacterial species found in wines during the malolactic fermentation (MLF), and is well adapted to the low pH and high ethanol concentration of wine (Wibowo et al. 1985, Ramos et al. 1995). The natural conditions of the malolactic conversion are unfavourable, and in recent years, winemakers have used selected strains of this microorganism to ensure a better control of the process. Inoculating the wine with a high concentration of O. oeni enhances the probability of obtaining rapid and complete MLF. The inoculation of wines or musts with optimal numbers of cells that perform the MLF requires the use of media and cultural conditions that optimize growth rate, biomass yield, and the ability to conduct the MLF in wine. Studies on the utilization of the major hexoses in wine (glucose and fructose) have been previously reported in non-pH controlled experiments (Tracey & van Rooyen 1988, Salou et al. 1994, Serpa et al. 1994). Glucose is used as the carbon and energy source by all strains of O. oeni, but it has also been shown that fructose is the most rapidly and

efficiently metabolized sugar. The use of fructose in the growth medium as an electron acceptor has usually been seen as beneficial for most strains (Salou et al. 1994). However, some exceptions have been reported (Tracey & van Rooyen 1988). A primary effect of sugar fermentation is the lowering of pH of the medium. Internal pH decreases as well and the effects inside the cell are (1) a higher consumption of energy (ATP) to pump out protons, and (2) a decrease in the cytoplasmic enzymatic activities as the internal pH decreases. When the excess of protons in the medium is neutralized by addition of the alkali, the pH remains constant outside and inside the cell and more energy can be used to increase biomass. Several authors have demonstrated the benefits of the pH control in other lactic acid bacteria cultures (Beal et al. 1989, Champagne et al. 1989), but no references have been found about its utilization to effectively produce O. oeni biomass able to develop the MLF in wine. In this paper, we describe the growth of O. oeni M42 in a basal medium supplemented with various carbohydrates, and the beneficial effect of the pH con-

350 trol to improve its commercial production. The use of this technological approach enables a high production of well-adapted biomass that can be used as an effective starter to induce the MLF in wines. Materials and methods Cultural conditions. Oenococcus oeni M42 (Pardo & Zúñiga 1992, Pardo et al. 1988), was grown on Medium for Leuconostoc oenos (MLO medium) (Caspritz & Radler 1983) without tomato juice or citrate and supplemented with glucose (55 mmol glucose l−1 ), or fructose (55 mmol fructose l−1 ), or glucose (55 mmol glucose l−1 ) plus fructose (30 mmol fructose l−1 ). pH was adjusted to 4.8 with 1 M KOH. After autoclaving, the medium was supplemented with pantothenic acid (0.01 g l−1 ). Fermenters were inoculated with 2% (v/v) washed cells harvested at the late phase of growth from a MLO preculture. Cultures were grown at 28 ◦ C stirring at 250 rev/min for 3 to 10 days. pH was monitored and, when specified, maintained at pH 4.8. Buffer assays were performed in 40 mM phosphate/20 mM tartrate buffer (HenickKling et al. 1986). For vinification assays, cultures were grown in basal MLO with glucose (55 mmol glucose l−1 ) and fructose (30 mmol fructose l−1 ) with pH control (pH 4.8). Cells were then collected and inoculated in wines at combinations of two different cell densities (about 107 and 108 cfu ml−1 ), two pH values (3.1 and 3.5), and two malic acid concentrations (3.5 and 7.0 g l−1 ). Analytical methods. Samples were collected aseptically through the fermenter sampling port, and bacterial growth was estimated by dry weight of cells and following O.D. at 600 nm against non-inoculated medium. For chemical analysis, supernatants from centrifuged samples were filtered through a C-18 cartridge and then through a 0.22 µm membrane filter. Sugars, organic acids and ethanol were quantified by HPLC as described by Frayne (1986). External standards were used to quantify the required compounds. ATP concentrations were determined as described by Firme et al. (1994). Results and discussion Effect of medium composition (sugars) on biomass yield. The fermentation of hexoses by heterofermentative lactic acid bacteria theoretically produces

equimolecular amounts of lactate, carbon dioxide, and acetate or ethanol (Kandler 1983). However, modifications in culture conditions or substrate composition may result in variable stoichiometric data (Ragout et al. 1994). When glucose was used as sole substrate, growth was poor. Under these conditions, sugar degradation was not detected and the pH was dropped by less than 0.1 units. Fructose was completely metabolized after ten days when it was the only carbon source in the medium (Figure 1a). The degradation was accomplished through two alternative pathways: fifty seven percent of the fructose was reduced to mannitol to reoxidize NAD(P)H, while the reminder was degraded by the heterolactic pathway, essentially yielding lactate and acetate, i.e. 1.4 mmol ATP/mmol of sugar (Table 1). Additional redox power, supplied by fructose reduction, allowed the production of six times more biomass, molar growth yield Y = 5.61 mg mmol−1 of sugar, than did a glucose based medium. Small amounts of ethanol and glycerol were also detected, showing the non effective contribution of their production pathways to NAD(P)H regeneration (Tables 1 and 2). Maximum biomass levels, Y = 1.8 mg mmol−1 sugar, were achieved during the co-metabolism of glucose and fructose. In a batch culture without pH control, eight times more biomass (180 µg mmol−1 sugar) occurred than with glucose alone (data not shown). Fructose was almost completely converted to mannitol while only 37% of glucose was metabolized (Table 2). The simultaneous consumption of both substrates led to increased relative yields of lactate, acetate and ethanol, compared to yields in the reference culture (i.e. fructose alone) (Table 1). After complete degradation of fructose, O. oeni was not able to metabolize the remaining glucose, which was due to a lack of oxidized cofactors. During a co-fermentation of glucose and fructose, O. oeni may use glucose by the heterolactic pathway, generating energy (1.84 mmol ATP mmol−1 of sugar) and biomass (Y = 6.89 mg/mmol of sugar), while fructose can be reduced to mannitol by mannitol dehydrogenase. This provides cells of additional oxidized redox power to that obtained from glucose culture alone (Salou et al. 1994) and explains the improved results when both sugars were present. pH changes in the media were coupled with growth of O. oeni M42. When pH was not controlled it decreased to 4.1 (fructose alone) and 4.0 (cofermentation glucose and fructose) and halted hexoses metabolism. These results indicate the benefits to operate a fermenter under pH control to improve the production of O. oeni starter biomass.

351

Fig. 1. Growth of Oenococcus oeni and carbohydrate fermentation kinetics in a culture with fructose alone or glucose plus fructose. (a) no pH control; (b) under pH control. Dotted lines, dry weight; , fructose alone; , glucose-fructose mixture. Lines, substrate concentrations; , fructose alone; , fructose (in a glucose-fructose mixture); , glucose (in a glucose-fructose mixture).

Effect of pH control on biomass yield. As described earlier, growth was poor in glucose-based media, and the pH decreased less than 0.1 units from the initial value (4.8). Therefore, we did not perform a pHcontrolled batch with glucose alone, given that the pH control activation threshold was over this value. In a fructose based system, the results achieved when the pH was not controlled (decrease of 0.7 units) and in the pH controlled assay were similar (Figures 1a, b). All the fructose in the medium was metabolized in ten days with or without pH control. Under pH control, increased lactate and acetate yield was detected when compared to those in a non pH controlled assay (Table 2). Our results suggest that in O. oeni M42 the enzymes involved in lactic acid production may be partially inhibited by a low pH. This has been previously described by Ramos et al. (1995), who showed that O. oeni lactate dehydrogenase activity decreased at a reduced pH. Acetate production was also affected by pH but to a lesser extent. To elucidate the effect of external pH in the lactate/acetate ratio we assayed the degradation of fructose in tartrate phosphate buffer at different initial values (ranging from 3.0 to 5.0). As the initial pH decreased, the lactate/acetate ratio dropped from 0.81 to 0.68. These values, obtained in a buffered system, were similar to those in a synthetic medium, supporting the decrease of lactate

dehydrogenase activity as pH decreased. Acetate production was also affected, but at a lesser extent. The increase in production of lactate (20%) in a growth medium when pH was controlled yielded 1.2-folds ATP and consequently a 10% increase of biomass, up to 160 µg/mmol of sugar. However, generation times were both about 8.7 h−1 . These results are significatively better (2–3 folds) than those previously reported (Beelman et al. 1980, Naouri et al. 1989). Co-fermentation of glucose and fructose revealed greater pH dependence. In the incubation time, 30% more glucose was degraded under pH control, yielding higher quantities of final products. This led to an increase in acetate production (ATP linked), producing 38% more biomass (220 µg mmol−1 of sugar). These differences were possible because of the increased metabolic activity of the cells when pH was controlled (Ragout et al. 1994). While fructose was completely metabolized in less than 30 h under pH control, it took more than 50 h in its absence (Figures 1a, b). The kinetics of mannitol production was coupled to fructose consumption under both conditions. Glucose degradation, which is fructose dependent, was also influenced by pH control (Figures 1a, b). In any case, remaining quantities of glucose were detected after stopping the experimentation due to the restriction in oxidized cofactors (Table 2).

352 Table 1. Influence of sugar substrate and pH control on stoichiometrya of final products in growth of Oenococcus oeni M42. T = 28 ◦ C; pH0 = 4.8; stirring = 250 rev/min.

Fructose. Without pH control Fructose. With pH controlc Glucose + Fructose. Without pH control Glucose + Fructose. With pH controlc

Substrate (mmol l−1 ) Hexoseb

Products (mmol l−1 ) Lactate Acetate Ethanol

Glycerol

Fructose converted to mannitol (%)

1 1 1 1

0.65 0.72 0.81 0.95

0.06 0.06 0.02 0.07

57 56 91 99

0.74 0.80 1.07 1.13

0.04 0.04 0.31 0.56

a Results are means of determinations of duplicate cultures. b Hexoses represents total degraded glucose plus degraded fructose not converted to mannitol. c pH was controlled by addition of 5 M KOH and 5 M HCl.

Table 2. Effect of pH control and medium composition on substrate utilization and fermentation of Oenococcus oeni M42 in batch culture. T = 28 ◦ C; pH0 = 4.8; stirring = 250 rev/min. Medium A: basal MLO with 55 mmol fructose l−1 ; medium B: basal MLO with glucose (55 mmol glucose ml−1 ) plus fructose (30 mmol fructose ml−1 ).

Glucose (residual) Fructose (residual) Lactate Acetate Ethanol Glycerol Mannitol

Final concentration (mmol l−1 )a Medium A (after 10 days) No pH control pH controlb

Medium B (after 3 days) No pH control pH controlb

0.0 1.4 15.1 17.5 0.9 1.4 30.5

34.4 0.5 18.7 24.9 7.3 0.5 26.8

0.0 0.4 17.1 18.5 0.9 1.5 30.8

28.1 0.2 25.6 30.6 15.1 1.9 29.6

a Results are means of determinations of duplicate cultures. b pH was controlled by addition of 5 M KOH and 5 M HCl.

Table 3. Malolactic fermentation in red wine following inoculation with Oenococcus oeni M42 after 3 weeks of incubation Viable bacteria in wine (cfu m−1 )

Malic acid concentration (g l−1 ) Initial Final

Malic acid degradation (%) after 3 weeks

pH Initial

Final

1.2 × 107 1.1 × 107 1.2 × 107 1.3 × 107 8.2 × 107 9.4 × 107 1.1 × 108 9.6 × 107

3.5 7.0 3.5 7.0 3.5 7.0 3.5 7.0

34 16 71 40 77 44 97 86

3.10 3.10 3.50 3.50 3.10 3.10 3.50 3.50

3.18 3.12 3.57 3.57 3.20 3.17 3.64 3.74

2.3 5.9 1.0 4.2 0.8 3.9 0.1 1.0

353 Vinification assays. To determine the usefulness of the starter cultures produced under optimal conditions, several vinifications have been conducted, and two malic acid concentrations were tested (3.5 and 7.0 g l−1 ) (Table 3). The rate of malolactic activity was directly related to the number of bacteria added, and when 1 × 108 cfu ml−1 were inoculated, the MLF was generally completed in 1–3 days. MLF was developed in red wines within 2–3 weeks when inoculating about 107 cfu ml−1 . The vinifications were assayed at various pH values to assess the produced biomass to degrade the malic acid present in wine. Best results were observed, as expected, at pH 3.5. However, good degradation taxes were obtained at pH 3.1 by using 1 × 108 cfu ml−1 . Thus, the inhibitory effect of low pH (Bousbouras & Kunkee 1971, Davis et al. 1986) can be overcome by using high numbers of cells. Reductions of about 60–90% of the initial concentrations of malic acid are here described (Table 3). These results offer the possibility for developing the malic acid degradation under unfavourable conditions (low pH and high quantities of malic acid). Very few approaches have been successfully reported to solve these adverse situations for an effective MLF in wine (Davis et al. 1986). The data obtained in this study have shown that differences in the sugar composition of the culture medium influences growth kinetics and biomass yields of O. oeni M42. We suggest the utilization of two combined sugar substrates (glucose and fructose) in culture media for O. oeni. This co-fermentation permits production of up to eight times more biomass than with the fermentation of one sugar alone (glucose). The pH control, which avoids the acidification of the culture medium, also gave a 38% increase in biomass production. The utilization of this biomass to perform the MLF under several experimental conditions produced improved results.

Acknowledgements This work has been partially supported by grants from the Comisión Interministerial de Ciencia y Tecnología (ALI93-0246) and by a grant from the M.E.C. (Spanish Government) to S.M.

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