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

doi:10.1016/j.ijfoodmicro.2005.04.023

International Journal of Food Microbiology

Volume 105, Issue 3, 15 December 2005, Pages 463-470

https://doi.org/10.1016/j.ijfoodmicro.2005.04.023 Get rights and content

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.

Introduction

Processes involved in the elaboration of wines are complex and most of the time require two successive 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).

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 stress-specific 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.

Section snippets

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

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 μmol 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

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 strains by analysing the intracellular

Acknowledgments

This study was supported by the Ministère de la Recherche et de l'Enseignement (France), by the Conseil Régional de Bourgogne (France) and by Lallemand SA (Toulouse, France).

References (21)

M.M. Bradford
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)
R. Carrete et al.
Inhibitory effect of sulfur dioxide and other stress compounds in wine on the ATPase activity of Oenococcus oeni
FEMS Microbiol. Lett.
(2002)
J. Guzzo et al.
Increase of sulfite tolerance in Oenococcus oeni by means of acidic adaptation
FEMS Microbiol. Lett.
(1998)
R.E. Kunkee
Some roles of malic acid in the malolactic fermentation in wine making
FEMS Microbiol. Lett.
(1991)
R. Tourdot-Marechal et al.
Acid sensitivity of neomycin-resistant mutants of Oenococcus oeni: a relationship between reduction of ATPase activity and lack of malolactic activity
FEMS Microbiol. Lett.
(1999)
Y. Vasserot et al.
Transport of glutamate in Oenococcus oeni 8403
Int. J. Food Microbiol.
(2003)
D.J. Cox et al.
Chemiosmotic energy from malolactic fermentation
J. Bacteriol.
(1989)
F. Delmas et al.
Biochemical and physiological studies of the small heat shock protein Lo18 from the lactic acid bacterium Oenococcus oeni
J. Mol. Microbiol. Biotechnol.
(2001)
I. Derre et al.
CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in gram-positive bacteria
Mol. Microbiol.
(1999)
D. Galland et al.
Absence of malolactic activity is a characteristic of H+-ATPase-deficient mutants of the lactic acid bacterium Oenococcus oeni
Appl. Environ. Microbiol.
(2003)

There are more references available in the full text version of this article.

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Oenococcus oeni is the most resistant lactic acid bacteria species to the environmental stresses encountered in wine, particularly the acidity, presence of ethanol and phenolic compounds. Indigenous strains develop spontaneously following the yeast-driven alcoholic fermentation and may perform the malolactic fermentation whereby improving taste, aroma, and the microbial stability of wine. However, spontaneous fermentation is sometimes delayed, prolonged or incomplete. In order to better control its timing and quality, O. oeni strains are selected and developed to be used as malolactic starters. They are prepared under proprietary manufacturing processes to survive direct inoculation and are predominantly provided as freeze-dried preparations. In this study, we have investigated the physiological and molecular alterations occurring in O. oeni cells prepared by an industrial process that consists of preconditioning protocols and freeze-drying, and compared them to the same strain grown in a grape juice medium. We found that compared to cultured cells, the industrial production process improved survival under extreme conditions, i. e. at low pH or high tannin concentrations. In contrast, cultured cells resumed active growth more quickly and strongly than freeze-dried preparations in standard pH wines. A proteomic analysis showed that during the industrial production most non-essential metabolic processes are shut down and components of the general and the stringent stress response are upregulated. The presence of major components of the stress response facilitates protein homeostasis and physiological changes that further ensure the integrity of cells.
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Citation Excerpt :
Also known as Lo18 or Hsp18, Hsp20 has long been described as one of the main stress-responding proteins in O. oeni with chaperone and membrane protection functions (Coucheney et al., 2005b; Guzzo et al., 1997). Indeed, it has also been proposed as a stress response marker of interest to predict MLF performances (Coucheney et al., 2005a; Olguín et al., 2010). Some DEGs relating to tRNAs were detected in the samples (Fig. 6).
Oenococcus oeni is the main agent responsible for malolactic fermentation (MLF) in wine. This usually takes place in red wines after alcoholic fermentation (AF) carried out by Saccharomyces cerevisiae. In recent years, there is an increasing interest in using non-Saccharomyces yeast, usually in combination with S. cerevisiae, to improve wine quality. Current studies report a stimulatory effect of non-Saccharomyces on MLF, generally related to a decrease in the inhibitor compounds found in wine. In this work, we followed a comparative multi-omics approach, including transcriptomic and proteomic analysis, to study the molecular adaptation of O. oeni in wines fermented with Torulaspora delbrueckii and Metschnikowia pulcherrima, two of the most frequently used non-Saccharomyces, in sequential inoculation with S. cerevisiae. We compared the results to the adaptation of O. oeni in S. cerevisiae wine to determine the main changes arising from the use of non-Saccharomyces. The duration of MLF was shortened when using non-Saccharomyces, to half the time with T. delbrueckii and to a quarter with M. pulcherrima. In this work, we observed for the first time how O. oeni responds at molecular level to the changes brought about by non-Saccharomyces. We showed a differential adaptation of O. oeni in the wines studied. In this regard, the main molecular functions affected were amino acid and carbohydrate transport and metabolism, from which peptide metabolism appeared as a key feature under wine-like conditions. We also showed that the abundance of Hsp20, a well-known stress protein, depended on the duration time. Thus, the use of non-Saccharomyces reduced the abundance of Hsp20, which could mean a less stressful wine-like condition for O. oeni.
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International Journal of Food Microbiology

Abstract

Introduction

Section snippets

Bacteria strains and rehydration conditions

Biochemical and molecular characterization of O. oeni strains

Discussion

Acknowledgments

References (21)

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

Anal. Biochem.

Inhibitory effect of sulfur dioxide and other stress compounds in wine on the ATPase activity of Oenococcus oeni

FEMS Microbiol. Lett.

Increase of sulfite tolerance in Oenococcus oeni by means of acidic adaptation

FEMS Microbiol. Lett.

Some roles of malic acid in the malolactic fermentation in wine making

FEMS Microbiol. Lett.

Acid sensitivity of neomycin-resistant mutants of Oenococcus oeni: a relationship between reduction of ATPase activity and lack of malolactic activity

FEMS Microbiol. Lett.

Transport of glutamate in Oenococcus oeni 8403

Int. J. Food Microbiol.

Chemiosmotic energy from malolactic fermentation

J. Bacteriol.

Biochemical and physiological studies of the small heat shock protein Lo18 from the lactic acid bacterium Oenococcus oeni

J. Mol. Microbiol. Biotechnol.

CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in gram-positive bacteria

Mol. Microbiol.

Absence of malolactic activity is a characteristic of H+-ATPase-deficient mutants of the lactic acid bacterium Oenococcus oeni

Appl. Environ. Microbiol.

Cited by (65)

Mixed biofilm formation by Oenococcus oeni and Saccharomyces cerevisiae: A new strategy for the wine fermentation process

The production of preconditioned freeze-dried Oenococcus oeni primes its metabolism to withstand environmental stresses encountered upon inoculation into wine

Molecular adaptation response of Oenococcus oeni in non-Saccharomyces fermented wines: A comparative multi-omics approach

Malolactic fermentation and its effects on wine quality and safety

Exopolysaccharides produced by Oenococcus oeni: From genomic and phenotypic analysis to technological valorization

Prevalence of Lentilacobacillus hilgardii over Lactiplantibacillus plantarum in Low-Temperature Spontaneous Malolactic Fermentation of a Patagonian Pinot Noir