A new approach for selection of Oenococcus oeni strains in order to produce malolactic starters

International Journal of Food Microbiology 105 (2005) 463 – 470 www.elsevier.com/locate/ijfoodmicro

A new approach for selection of Oenococcus oeni strains in order to produce malolactic starters Franc¸oise Coucheney a, Nicolas Desroche a, Magali Bou b, Raphae¨lle Tourdot-Mare´chal a, Laurent Dulau b, Jean Guzzo a,* a

Laboratoire de Microbiologie UMR Universite´ de Bourgogne/INRA 1232, ENSBANA, 1, Esplanade Erasme, 21 000 Dijon, France LALLEMAND SA, Laboratoire R&D Proce´de´s Toulouse, ZA Font-Grasse, 19, rue des Briquetiers, 31702 Blagnac Cedex, France

b

Received 20 July 2004; received in revised form 27 December 2004; accepted 11 April 2005

Abstract The lactic acid bacterium Oenococcus oeni, mainly responsible for malolactic fermentation (MLF), is used in new winery process as starter culture for direct inoculation. The difficulty to master MLF according to the wine led us to search a new approach to select effective O. oeni strains. Biochemical and molecular tests were performed in order to characterize three strains of O. oeni selected for malolactic starter elaboration. Malolactic and ATPase activities that appeared as a great interest in MLF were measured and the expression of a small heat shock protein Lo18 was evaluated by immunoblotting and real-time PCR. These results were correlated with the performances of strains in two red wines. Physiological and molecular characteristics of the three strains showed significant differences for the global malolactic activity on intact cell at pH 3.0 and at the level of induction of the small heat shock protein Lo18. These two parameters appeared of interest to evaluate in the ability of O. oeni strains to survive into wine after direct inoculation and to perform MLF. Indeed, a tested strain that presented the highest malolactic activity on intact cells at pH 3.0 and a high level of Lo18 induction showed a high growth rate and a high specific kinetic of malate consumption. The techniques used in this work carry out more quickly and more reliable than usual for the selection of effective strains intended for direct inoculation in wines. D 2005 Elsevier B.V. All rights reserved. Keywords: Oenococcus oeni; Malolactic starter; Malolactic and ATPase activities; Small heat shock protein

1. Introduction Processes involved in the elaboration of wines are complex and most of the time require two successive * Corresponding author. Tel.: +33 3 80 39 66 75; fax: +33 3 80 39 66 40. E-mail address: [email protected] (J. Guzzo). 0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2005.04.023

fermentations: firstly alcoholic fermentation carried out by yeasts, and secondly malolactic fermentation (MLF) by lactic acid bacteria, especially Oenococcus oeni. This second phase involves deacidification by bioconversion of l-malic acid into l-lactic acid and carbon dioxide. MLF also improves the microbiological stability and the organoleptic characteristics of wines (Kunkee, 1991).

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Even though MLF occurs spontaneously in wines, it starts randomly, and any delay in the starting of MLF can lead to an alteration of wine quality (Henick-Kling, 1995). Moreover, in wines with low pH, MLF remains unreliable. Therefore, recent winery practices consist in using malolactic starters for direct inoculation in wines (Nielsen et al., 1996; Maicas, 2001). However, induction of MLF by inoculation with commercially available strains of O. oeni is not always successful. The difficulty in inducing MLF in wine remains problematic because wine is a very harsh environment for bacterial growth. Nowadays, selection of strains for wine inoculation is performed by classic tests based essentially on the survival in wine and monitoring the consumption of l-malic acid (Henick-Kling et al., 1989). Knowledge of O. oeni physiology in stress conditions can be used to generate tools based on molecular and physiological approaches allowing more precise characterization of strains. Among the metabolic and enzymatic systems that could be used to this end, l-malic acid metabolism and ATPase activity are of great interest. Indeed, l-malic acid metabolism and ATPase activity together contribute to the acidophilic behaviour of O. oeni (Tourdot-Marechal et al., 1999). Both mechanisms participate in the intracellular pH (pHi) homeostasis of cells. ATPase bound to the plasma membrane extrudes protons from the cell (Koebmann et al., 2000). The role of ATPase in resistance to acid conditions was clearly demonstrated by studying ATPase deficient mutants of O. oeni (Tourdot-Marechal et al., 1999). During MLF, metabolic energy is conserved as a chemiosmotic coupling mechanism in combination with H+ consumption in the cytoplasm (Cox and Henick-Kling, 1989; Salema et al., 1996). On the other hand, when cells are under different stresses, stress proteins are synthesized. Based on the stressspecific high expression of Lo18 (Guzzo et al., 1997), this small heat shock protein (smHsp) was identified as a good marker of stress for O. oeni. No signal of Lo18 is detected in the logarithmic growth phase in normal growth conditions. The study of this protein showed that Lo18 possesses a chaperon activity in vitro and is located in part in the membrane fraction (Delmas et al., 2001). The aim of this study is to use biochemical and molecular tests to compare three strains of O. oeni selected for the elaboration of malolactic starters. The

results of these tests correlated with the performances of the strains in wines.

2. Materials and methods 2.1. Bacteria strains and rehydration conditions Three freeze-dried strains (A, B and C) of O. oeni were obtained from the collection of the laboratory of Lallemand SA (Toulouse, France) and cultivated on Lallemand medium. For the biochemical and molecular characterization, two independently freeze-dried batches were tested to confirm reproducibility of batch production for each strain. The strains were collected in stationary growth phase. Before using them, these strains were rehydrated with water containing 1 g L 1 peptone, 0.9 g L 1 NaCl at 30 8C for 45 min. Glucose (5 g L 1) was added to energize the cells before determination of their malolactic activity on intact cells. Cells were broken by ultrasonic treatment (4  45 s, at 1-min intervals in ice, at power 5) (Vibra Cell, Sonics and Materials Inc., Danbury, USA) for enzymatic measuring and with glass beads 0–50 Am in a FastPrep FP120 Instrument (6  20 s, speed 6) (ThermoSavant-BIO101, France) for Western and Northern blot analysis. 2.2. Malolactic and ATPase activity Intracellular malolactic activity was determined on cellular extract as described by Tourdot-Marechal et al. (1999). l-Malic acid was measured with a Boehringer enzymatic kit (Mannheim, Germany). This activity was expressed as Amol malate consumed per mg protein and per hour. Malolactic activity on intact cells was measured at pH 3.0 and 4.5. The protocol was the same as for intracellular malolactic activity, except that one OD600 nm unit of cells was mixed with reactional solution instead of cellular extract. This activity was expressed as Amol malate consumed per OD unit and per hour. ATPase activity associated with the plasma membrane fractions was analysed as described by TourdotMarechal et al. (1999). ATPase activity was expressed as Amol of inorganic phosphate released per mg protein and per minute.

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After cells were broken, 10 Ag of cellular proteins was loaded on 12% polyacrylamide gel in denaturing conditions (SDS-PAGE). Proteins were transferred onto a nitrocellulose membrane. Immunoblot was carried out as per Guzzo et al. (1998), with a Lo18 rabbit polyclonal antibody and peroxydase coupled to anti IgG antibody (Sigma, St. Louis, Missouri, USA).

were designated as follows: initial denaturation at 95 8C for 10 min, followed by 45 cycles of 95 8C for 15 s and 60 8C for 30 s. Fluorescence measurements were recorded during each annealing step. Four dilutions of cDNA were performed and the amplifications were duplicated. The results were calculated using the comparative critical threshold (DDCT) method in which the amount of target RNA is adjusted to a reference relative to an internal calibrated target RNA.

2.4. Real-time PCR experiments

2.5. Composition of wine

RNAs were prepared as previously described by Jobin et al. (1997) and measured by spectrophotometry at 260 nm. RNA (5 Ag) was treated with DNase (Invitrogen, Cergy Pontoise, France) as recommended by the manufacturer. cDNA were synthesized using the thermoscript Reverse Transcription-PCR (RT-PCR) system (Invitrogen) using random hexamers as recommended by the manufacturer. The absence of chromosomal DNA contamination was checked. After dilution, 5 AL of cDNA was added to 20 AL of PCR mixture (12.5 AL of Master mix, 1 AL of each primer at 7 pM, 2.5 AL of fluoresceine at 1/10,000 and 3 AL of SYBRR Green at 1/8000) (Eurogentec, Lie`ge, Belgium). Specific cDNAs were amplified by PCR and real-time PCR with appropriate primers for the hsp18 gene. mRNAs levels were quantified by real-time 5V exonuclease PCR assay. The ldhD gene of O. oeni was chosen as an internal control gene during growth. For comparison purposes, to check the presence of intact cellular mRNAs and the uniform efficiency of each reverse transcription reaction, the ldhD cDNA level as well as the hsp18 cDNA level was quantified. Amplifications were performed on a Biorad-I-Cycler (Bio-Rad, Marnes la Coquette, France) with the SYBRR Green system. Thermal cycling conditions

Two red wines were tested: grape juice fermented in the laboratory of Lallemand SA (VT3.2) and wine obtained by the laboratory of Lallemand SA (VIR). For the elaboration of VT3.2 wine, the grape juice was fermented by a Saccharomyces cerevisiae strain (Lallemand SA) until the concentration of residual sugar was inferior to 2 g L 1. After alcoholic fermentation, the wine was clarified by centrifugation to remove yeasts. The wine was then put at 4 8C for the tartaric precipitation. Crystals were removed by filtration. Malic acid concentration and pH were adjusted to the defined values (5 g L 1 l-malic acid, pH 3.2). The VIR wine is a red Syrah wine. It was filtered after alcoholic fermentation. The physicochemical properties of the wines were analysed by the laboratory of the Station Oenologique de Champagne (France) as shown in Table 1. The wines were stored at 4 8C.

2.3. Western blot

2.6. Survival and consumption of l-malic acid in wines Twenty milliliters of each wine distributed in sterile tubes of 20 mL was inoculated with the different strains at a concentration defined in Table 2. The tubes were incubated at 18 8C, in similar growth conditions

Table 1 Composition of wines used for malolactic fermentation assays Wine VT3.2 VIR

Alcohol (% v/v)

SO2 total (mg L 1)

SO2 free (mg L 1)

pH

12.48 11.22

16 12

6 6

3.20 3.49

l-Malic acid (g L 1)

Glucose–fructose

5.0 2.8

0.1 0.4

The values were determined by the laboratory of the Station Oenologique de Champagne (France).

Acetic acid (g H2SO4 L 0.27 0.13

1

)

Acidity (g H2SO4 L 5.65 3.50

1

)

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as in a cellar. A control without inoculation was performed for each wine to verify that there were no spontaneous MLF starts. Population was determined by counting cells (CFU mL 1) spread on agar plates of MRS medium modified by Lallemand (pH 5.4) and incubated at 30 8C in anaerobiosis for 5 days. The enumeration was performed at 0, 2, 7 and 28 days after inoculation in wine. The survival rate was estimated after 48 h of incubation in the wines and it represented the cell count at 48 h divided with the cell count at inoculation. The concentration of l-malic acid was determined with a Boehringer enzymatic kit (Mannheim, Germany) at 0, 7 and 28 days after inoculation in wine. 2.7. Other procedure Protein concentration was determined using a Biorad protein assay method (Bradford, 1976) with bovine serum albumin (Euromedex, Mundolsheim, France) as a standard.

3. Results 3.1. Biochemical and molecular characterization of O. oeni strains The intracellular malolactic activity of the three strains is presented in Fig. 1A. The specific malolactic activity of strain C was the greatest with a value of 570 Amol malate h 1 mg 1 protein. Strains A and B showed lower specific malolactic activity, respectively 59% and 40%, compared to that of the strain C. Taking into account the observed standard deviation, the activity of the three strains was quite similar. In the cell, l-malic acid is decarboxylated into l-lactic acid and CO2 by the malolactic enzyme. The malate decarboxylation is an intracellular process that requires the entry of l-malate into the cell. The activity determined on intact cells (Fig. 1B) takes into account the l-malic acid uptake by cells, together with the activity of the malolactic enzyme. The malolactic activity on intact cells was tested at two pH values: 3.0 and 4.5. At these pH values, l-malate can exist both in the monoanionic form and the undissociated form. At pH

A Intracellular malolactic specific activity

Strain A

Strain B

Strain C

(µmoles malate.h-1.mg-1 protein)

233 ± 70

343 ± 77

570 ± 177

Malolactic activity on intact cells (µmol malate-1 h-1 OD units-1)

B

6 5

pH 3.0 pH 4.5

4 3 2 1 0 Strain A

Strain B

Strain C

Fig. 1. (A) Intracellular specific malolactic activity of strains A, B and C measured on cellular extract at 37 8C, pH 6.0. Vertical bars represent the standard deviation of the measurements. (B) Malolactic activity on intact cells of strains A, B and C measured at two pH values: 3.0 and 4.5. Vertical bars represent the standard deviation of the measurements. Results are based on the average of four assays for each strain by two independently freeze-dried batches.

F. Coucheney et al. / International Journal of Food Microbiology 105 (2005) 463–470

A

Strain A

Strain B Strain C Control

B

Relative level of expression

4.5, the monoanionic form predominates and is taken up via the malate permease (Tourdot-Marechal et al., 1993). On the contrary, at pH 3.0, which corresponds to the mean pH of wine, the undissociated form, which can enter by the diffusion process, is predominant (Tourdot-Marechal et al., 1993). Moreover, the rate of malate metabolism on intact cells is limited by the rate of l-malate transport (Tourdot et al., 1990). So intracellular malolactic activity determined on cellular extract (Fig. 1A) represents specific malolactic activity and malolactic activity on intact cells (Fig. 1B) informs about the malate permease activity. At pH 4.5, the three strains have about the same low activity (Fig. 1B). Nevertheless, significant differences for the global malolactic activity on intact cell at pH 3.0 appeared. Strain C is characterized by a high malolactic activity (three times higher than that of strain B). Strain A has a malolactic activity at pH 3.0 equal to 40% of the value obtained with strain C (4.4 Amol malate h 1 OD unit 1). Another mechanism plays an important role in the acid tolerance of O. oeni: membrane bound ATPase activity. This activity depends on the membrane and environmental stresses, which can alter it. The enzyme can be inhibited by SO2 and the combined effect of ethanol (Carrete et al., 2002). The strains all have about the same activity with 0.21, 0.35 and 0.29 Amol Pi min 1 mg 1 protein for strains A, B and C, respectively. Strain B has the highest ATPase activity (0.35 Amol Pi min 1 mg 1 protein) while the value of ATPase activity for strains A and C are respectively 0.21 and 0.29 Amol Pi min 1 mg 1 protein. The expression of the smHsp Lo18 was studied to compare the physiological state of the three strains. Lo18 synthesis is largely induced by multiple stresses, notably those linked to wine, due to ethanol, low pH and sulfite, stationary growth phase and heat shock in O. oeni (Guzzo et al., 1997, 1998). As shown in Fig. 2A, a signal corresponding to Lo18 was detected for each strain by immunoblotting. This result can be explained by the fact that the strains were collected at the end of growth in the stationary phase. However, the intensity of signals is different for the three strains. For strains A and B, the Lo18 signal was quite weak whereas strain C gave a strong signal. As shown in Fig. 2B, the results of RT-PCR quantification are in accordance with those obtained by immunoblotting. The induction of Lo18 is slight and the same for the

467

4 3 2 1 0 Strain A

Strain B

Strain C

Fig. 2. (A) Immunoblotting analysis of Lo18 in three strains of O. oeni. Each crude extract was separated on a 12% SDS gel and revealed with rabbit polyclonal Lo18 antibodies. (B) Relative level of the hsp18 gene expression determined by real-time RT-PCR Quantitative. The relative levels of expression were calculated with the comparative critical threshold (DDCT) method in relation to the lowest expression of hsp18. The ldhD gene was used as internal control.

strains A and B. On the other hand, in strain C the hsp18 transcription is four times higher. 3.2. Survival in wines and catabolism of l-malic acid Fig. 3 shows the viability measured at 48 hours after direct inoculation with freeze-dried strains. The VT3.2 wine appeared more discriminating with the following characteristics: it was more acidic (pH 3.2) and had a higher concentration of ethanol. The poor survival rate in VT3.2 wine observed with strains A and B, respectively 35% and 10%, can be explained by these physico-chemical conditions, and notably the low pH. Nevertheless, strain C showed a 65% survival rate, but with a high variability. Despite a high number of viable cells, strain C was unable to perform MLF after 28 days in VT3.2 wine. Indeed, the population decreased during 7 days, and then a growth was observed with a final population at 1.05  106 CFU. mL 1 (data not shown). It is known that the malate consumption in wine depends on the number of viable cells and the metabolic activities of the individual cells. For strain A, after 2 days, the population stayed constant during the experiment. The population of strain B decreased during 28 days (data not shown).

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100

VT3.2 Wine

VIR Wine

90

Survival rate (%)

80 70 60 50 40 30 20 10 0 Strain A

Strain B

Strain C

Fig. 3. Influence of wine on survival rate of three strains after two days of incubation. The survival rate was calculated by dividing the cell count at 48 h with the cell count at inoculation. Vertical bars represent the standard deviation of the two independent measurements.

In VIR wine, the survival rate was superior to 65% for all strains and the behaviour of strains was very similar. Consequently the kinetics of MLF were compared for all of the strains during growth in VIR wine (Table 2). In absence of inoculation, no spontaneously start was observed (data not shown). The VIR wine was inoculated with a large number of bacteria to obtain after inoculation, an initial population of around 2  106 CFU mL 1. We can notice that, for unknown reasons, the population count of strain C was systematically inferior to this level of inoculation. As shown in Table 2, both strains with the highest initial cell density (A and B) differed in both their final population and the number of days to perform MLF. MLF was the fastest and completed in 14 days for the strain A which also had the highest final population. In contrast, the strain B, which started with the highest number of viable cells, had the lowest final population and completed MLF in 28 days. Following the initial decrease in viable cell number, strain C seemed to regain its viability because the strain reached an identical final popula-

tion to strains A and B within 28 days. Moreover, strain C showed a high capacity to perform MLF. The determination of l-malic consumption in relation to the biomass confirmed this result with a value of 7.16  10 5 Ag l malic acid day 1 CFU 1 for strain C. The survival rate for this strain was identical in both wines. Strain C thus appears to be the strain the most capable of surviving in bdifficultQ wine after direct inoculation.

4. Discussion MLF and ATPase play a central role in the acidophilic behaviour of O. oeni and recently a link between the two enzymatic systems has been demonstrated (Galland et al., 2003). Comparison of the intracellular malolactic activities of strains A, B and C has revealed no significant difference. The same conclusion can be deduced from the results obtained from the comparison of ATPase activities. Consequently, it is difficult to distinguish between these

Table 2 Behaviour of three strains of Oenococcus oeni in VIR wine and capacity to perform malolactic fermentation Strain A a

1

Initial population (CFU mL ) Final populationa (CFU ml 1) Days to complete MLF Specific kinetics of l-Malic acid consumptionb ( 10 5 Ag l-malic acid day 1 CFU 1)

Strain B 5

4

8.63  10 F 7.64  10 1.62  108 F 4.95  106 14 2.65 F 0.32

Strain C 6

5

1.28  10 F 1.44  10 8.97  107 F 8.01  106 28 1.40 F 0.18

The data shown are mean values of duplicate measurements. a Initial and final populations were respectively counted at 0 and 28 days after inoculation. b Specific kinetics of l-malic acid consumption was calculated after seven days of incubation.

2.22  105 F 6.36  104 1.07  108 F 7.35  107 28 7.42 F 2.34

F. Coucheney et al. / International Journal of Food Microbiology 105 (2005) 463–470

strains by analysing the intracellular enzymatic activities alone. In contrast, analysis of the malolactic activity on intact cells was more discriminating. In this case, clear differences between the strains were observed. According to Tourdot et al. (1990), the intracellular activity of decarboxylation is rate-limited by l-malic acid uptake, suggesting that the transport of l-malic acid would be the main limiting step for MLF in wine. The three strains had a greater activity at pH 3.0 than at pH 4.5. However, diffusion of the undissociated form of l-malic acid inside the cell increases with pH close to 3.0 (more than 50% of total l-malic acid uptake) (Tourdot-Marechal et al., 1993). Strain C had high malolactic activity at pH 3.0; this result suggests that strain C adapts better to acidic conditions than the two other strains, which are only slightly affected by changes in pH. Nevertheless, strain B presented slight permease activity, as did strain A, in these conditions of measurement, which suggests behaviour less dependant of pH. The results obtained on intact cells showed different metabolic capacities for the different strains. Consequently, analysis of malolactic activity on intact cells reflecting the permease activity seems to be an interesting tool for the selection of strains. With regard to the expression of Lo18, strain C presented the highest level of induction. We have previously observed that the level of hsp18 expression was higher in the strain C than the other tested strains for optimal growth conditions in laboratory (data not shown). The regulation of hsp18, the gene encoding Lo18, is not yet completely understood. In the promoter region of hsp18, a DNA sequence similar to the target of the repressor CtsR has been described (Derre et al., 1999). Thus, a negative regulation by the repressor CtsR is suggested for this gene in O. oeni. No information about the role of CtsR in the stationary growth phase is available at this time. Previously studies (Guzzo et al., 1994) showed that O. oeni cells exhibit increased synthesis of stress proteins (notably Lo18) and simultaneously acquire a greater ability to survive in wine and to perform MLF. According to these biochemical and molecular data, strain C presents the best criteria for direct inoculation into an acidic wine. Microvinifications showed that strain C is able to survive in both wines, even the difficult wine VT3.2. However, like strains A and B, strain C was not able to grow in this

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wine. One can suppose that a nitrogen nutrient deficiency in the VT3.2 wine prevents the growth of strains, or that this lack of growth is the result of the inability of O. oeni to use some of these nutrients (Vasserot et al., 2003). The microvinifications in VIR wine showed the specificity of one strain for one kind of wine. Thus, strain A appeared to be the best strain to perform MLF in VIR wine because the MLF was completed in 14 days. In contrast, strain C, that presented the most acidophilic behaviour and the highest induction of Lo18, took twice as long to complete MLF (28 days). Despite a particularly low level of inoculation, strain C presented a high biomass production and high specific kinetics of malate consumption. According to these data, strain C seems to be the strain with the best capacities to perform MLF in a bdifficultQ wine. This work presents a study about the characterization of strains with molecular and physiological tools in order to select malolactic starters. The techniques presented can be carried out more quickly and more reliably than usual for the selection and production of effective strains intended for direct inoculation into wine. However, the tests used in this work to characterize strains are not exhaustive and improvement of physiological knowledge of O. oeni will allow other tools to emerge in order to master the characterization of malolactic starters.

Acknowledgments This study was supported by the Ministe`re de la Recherche et de l’Enseignement (France), by the Conseil Re´gional de Bourgogne (France) and by Lallemand SA (Toulouse, France).

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