Identification of multiple-derived peptides produced by Saccharomyces cerevisiae involved in malolactic fermentation inhibition.

An oenological strain of Saccharomyces cerevisiae was previously shown to produce a 5-10 kDa peptidic fraction responsible for the inhibition of malolactic fermentation (MLF). In the present study, we aim to further purify the anti-MLF peptides of this fraction. The yeast fermented synthetic grape juice medium was fractionated by ammonium sulfate precipitation combined with ultrafiltration. The 5-10 kDa fraction recovered at a saturation degree of 60%-80% was the only fraction that inhibited both the bacterial growth and the malate consumption in vivo. It also inhibited the malolactic enzyme activity in vitro at a pH range between 3.5 and 6.7. Therefore, it was purified by both anion and cation exchange chromatography. The eluates that inhibited the malolactic enzyme activity in vitro were migrated on Tricine SDS-PAGE and the protein bands were excised and sequenced by LC-MS/MS. The sequencing revealed nine peptides originating from eight proteins of S. cerevisiae. Two GAPDH cationic fragments of 0.9 and 1.373 kDa having a pI of 10.5 and 11 respectively, Wtm2p and Utr2p anionic fragments of 2.42 kDa with a pI of 3.5 and 4 respectively were thought to contribute the most to the MLF inhibition.

Application of directed evolution to develop ethanol tolerant Oenococcus oeni for more efficient malolactic fermentation.

Malolactic fermentation (MLF) is an important step in winemaking, which can be notoriously unreliable due to the fastidious nature of Oenococcus oeni. This study aimed to use directed evolution (DE) to produce a more robust strain of O. oeni having the ability to withstand high ethanol concentrations. DE involves an organism mutating and potentially adapting to a high stress environment over the course of extended cultivation. A continuous culture of O. oeni was established and exposed to progressively increasing ethanol content such that after approximately 330 generations, an isolate from this culture was able to complete MLF in high ethanol content medium earlier than its parent. The ethanol tolerance of a single isolate, A90, was tested to confirm the phenotype and its fermentation performance in wine. In order to investigate the genotypic differences in the evolved strain that led to the ethanol tolerance phenotype, the relative expression of a number of known stress response genes was compared between SB3 and A90. Notably, there was increase in hsp18 expression in 20% (v/v) ethanol by both strains with A90 exhibiting a higher degree of expression. This study is the first to use directed evolution for O. oeni strain improvement and confirms that this technique can be used successfully for the development of new candidate strains for the wine industry. This study also adds to the current knowledge on the genetic basis of ethanol tolerance in this bacterium.

Influence of glycosides on behavior of Oenococcus oeni in wine conditions: growth, substrates and aroma compounds.

Autochthonous Oenococcus oeni strains (MS9, MS20 and MS46) with good malolactic performance and yielding adequate diacetyl levels, were selected to investigate the effect of synthetic and grape glycosides on bacterial growth, substrate utilization and ß-glucosidase (ßGlu), α-arabinofuranosidase (αAra) and α-rhamnopyranosidase (αRha) activities in a wine-like medium containing 6% ethanol, pH 4.0 (WBM). Then, changes in the volatile compounds profile were evaluated at the end of malolactic fermentation (MLF) carried out by the MS46 strain in WBM containing 1 mg L-1 of natural glycoside. All strains grew and efficiently degraded L-malic acid in WBM where ßGlu and αAra activities were found but not αRha. In presence of a synthetic glycoside (eriodictyol 7-O-ß-rutinoside) ßGlu activity was significantly enhanced for two of the cultures tested (MS20 and MS460) while a low αRha activity was induced, presenting MS46 the better performance. Glycosides extracted from fermented grape musts under different conditions allowed maximum growths, L-malic acid utilization rates and glycosidase activities in the MS46 strain. Thus, ßGlu, αAra and αRha activities increased between 30-50 and 3-11% respectively. This indirectly correlated to significant changes in total esters and higher alcohols at the end of MLF, which increased by up to 140 and 30% respectively. Moreover, ethyl and acetate esters formed up to 100-fold than alcohols or esters degraded highlighted the main role of this microorganism in the esters synthesis. Results obtained encourage the potential use of selected indigenous O. oeni strains as a tool to enhance wine complexity through MLF, mainly on highly fruity aroma.

Effectiveness of chitosan against wine-related microorganisms.

The antimicrobial action of chitosan against wine related microorganisms, including Lactobacillus plantarum, Saccharomyces cerevisiae, Oeonococcus oeni, Lactobacillus hilgardii, Brettanomyces bruxellensis, Hanseniaspora uvarum and Zygosaccharomyces bailii was examined in laboratory media. In order to assess the potential applicability of chitosan as a microbial control agent for wine, the effect of chitosan, applied individually and/or in combination with sulphur dioxide (SO2), on the growth of microorganisms involved in various stages of winemaking and on the fermentative performance of S. cerevisiae was investigated. Of the seven wine-related microorganisms studied, S. cerevisiae exhibited the strongest resistance to antimicrobial action of chitosan in laboratory media with a minimum inhibitory concentration (MIC) greater than 2 g/L. L. hilgardii, O. oeni and B. bruxellensis were the most susceptible to chitosan since they were completely inactivated by chitosan at 0.2 g/L. The MIC of chitosan for L. plantarum, H. uvarum and Z. bailii was 2, 0.4 and 0.4 g/L, respectively. In wine experiments, it was found that chitosan had a retarding effect on alcoholic fermentation without significantly altering the viability and the fermentative performance of S. cerevisiae. With regard to non-Saccharomyces yeasts (H. uvarum and Z. bailii) involved in winemaking, the early deaths of these yeasts in mixed cultures with S. cerevisiae were not probably due to the antimicrobial action of chitosan but rather due to ethanol produced by the yeasts. The complex interactions between chitosan and wine ingredients as well as microbial interactions during wine fermentation considerably affect the efficacy of chitosan. It was concluded that chitosan was worthy of further investigation as an alternative or complementary preservative to SO2 in wine industry.

Transcriptomic and proteomic analysis of Oenococcus oeni PSU-1 response to ethanol shock.

The correct development of malolactic fermentation depends on the capacity of Oenococcus oeni to survive under harsh wine conditions. The presence of ethanol is one of the most stressful factors affecting O. oeni performance. In this study, the effect of ethanol addition (12% vol/vol) on O. oeni PSU-1 has been evaluated using a transcriptomic and proteomic approach. Transcriptomic analysis revealed that the main functional categories of the genes affected by ethanol were metabolite transport and cell wall and membrane biogenesis. It was also observed that some genes were over-expressed in response to ethanol stress (for example, the heat shock protein Hsp20 and a dipeptidase). Proteomic analysis showed that several proteins are affected by the presence of ethanol. Functions related to protein synthesis and stability are the main target of ethanol damage. In some cases the decrease in protein concentration could be due to the relocation of cytosolic proteins in the membrane, as a protective mechanism. The omic approach used to study the response of O. oeni to ethanol highlights the importance of the cell membrane in the global stress response and opens the door to future studies on this issue.

Genetic diversity of Oenoccoccus oeni isolated from wines treated with phenolic extracts as antimicrobial agents.

Molecular techniques have been applied to study the evolution of wine-associated lactic acid bacteria from red wines produced in the absence and presence of antimicrobial phenolic extracts, eucalyptus leaves and almond skins, and to genetically characterize representative Oenococcus oeni strains. Monitoring microbial populations by PCR-DGGE targeting the rpoB gene revealed that O. oeni was, as expected, the species responsible for malolactic fermentation (MLF). Representative strains from both extract-treated and not-treated wines were isolated and all were identified as O. oeni species, by 16S rRNA sequencing. Typing of isolated O. oeni strains based on the mutation of the rpoB gene suggested a more favorable adaptation of L strains (n = 63) than H strains (n = 3) to MLF. Moreover, PFGE analysis of the isolated O. oeni strains revealed 27 different genetic profiles, which indicates a rich biodiversity of indigenous O. oeni species in the winery. Finally, a higher number of genetic markers were shown in the genome of strains from control wines than strains from wines elaborated with phenolic extracts. These results provide a basis for further investigation of the molecular and evolutionary mechanisms leading to the prevalence of O. oeni in wines treated with polyphenols as inhibitor compounds.

Antimicrobial activity of pediocin PA-1 against Oenococcus oeni and other wine bacteria.

Pediocin PA-1 is an antimicrobial peptide produced by lactic acid bacteria (LAB) that has been sufficiently well characterised to be used in food industry as a biopreservative. Sulphur dioxide is the traditional antimicrobial agent used during the winemaking process to control bacterial growth and wine spoilage. In this study, we describe the effect of pediocin PA-1 alone and in combination with sulphur dioxide and ethanol on the growth of a collection of 53 oenological LAB, 18 acetic acid bacteria and 16 yeast strains; in addition, production of pediocin PA-1 by Pediococcus acidilactici J347-29 in presence of ethanol and grape must is also reported. Inhibitory concentrations (IC) and minimal bactericide concentrations of pediocin PA-1 were determined against LAB, and revealed a bacteriostatic effect. Oenococcus oeni resulted more sensitive to pediocin PA-1 (IC(50) = 19 ng/ml) than the other LAB species (IC(50) = 312 ng/ml). Cooperative inhibitory effects of pediocin PA-1 and either sulphur dioxide or ethanol were observed on LAB growth. Moreover, the pediocin PA-1 producing P. acidilactici strain J347-29 was able to grow and produce the bacteriocin in presence of ethanol (up to 4% ethanol in the fermentation broth) and grape must (up to 80%), which indicated that pediocin PA-1 can be considered as a potential biopreservative in winemaking.

Genetic and physiological studies on Oenococcus oeni PsuI in response to ethanol stress.

Malolactate fermentation (MLF), which is known to decreases total acidity and improves the stability and quality of cider is conducted by Oenococcus oeni; the principal microorganism responsible for MLF under stress conditions. Understanding O. oeni physiology in stress conditions can be used to generate tools based on molecular and physiological approaches allowing more precise characterization of strains. Regarding intracellular protein, the results showed an increase in the levels of amino acids under ethanol stress. To study the expressed genes under ethanol stress, one gene were sequenced. An outer-membrane lipoprotein carrier protein precursor, Lo1A was expressed under ethanol stress conditions. Scanning electron microscopy was used to study the effect of ethanol stress on cell morphology. SEM revealed aggregation of bacterial cells as the level of ethanol increases in culture medium in comparison to controls.

[Storage of Oenococcus oeni in liquid nitrogen].

OBJECTIVE: To develop cryopreservation for Oenococcus oeni. METHODS: We investigated the influence of the age and concentration of the strain in the protective medium, rate of freezing, temperature of thawing and the protective medium on the number of viable cells after cryopreservation by colony counting. RESULTS: We got the highest number of viable cells after cryopreservation by collecting cells in the early stationary growth phase, keeping their concentration in the protective medium at 10(9) CFU/mL, transferring cells into liquid nitrogen directly and thawing in a water bath at 37 degrees C. The ratio of viable cells was above 99% for 21 of the Oenococcus oeni after storing in liquid nitrogen for 6 months. CONCLUSION: We The storage of Oenococcus oeni in liquid nitrogen is convenient for strains preserved in culture collections.

Wine Phenolic Compounds Differently Affect the Host-Killing Activity of Two Lytic Bacteriophages Infecting the Lactic Acid Bacterium Oenococcus oeni.

To provide insights into phage-host interactions during winemaking, we assessed whether phenolic compounds modulate the phage predation of Oenococcus oeni. Centrifugal partition chromatography was used to fractionate the phenolic compounds of a model red wine. The ability of lytic oenophage OE33PA to kill its host was reduced in the presence of two collected fractions in which we identified five compounds. Three, namely, quercetin, myricetin and p-coumaric acid, significantly reduced the phage predation of O. oeni when provided as individual pure molecules, as also did other structurally related compounds such as cinnamic acid. Their presence was correlated with a reduced adsorption rate of phage OE33PA on its host. Strikingly, none of the identified compounds affected the killing activity of the distantly related lytic phage Vinitor162. OE33PA and Vinitor162 were shown to exhibit different entry mechanisms to penetrate into bacterial cells. We propose that ligand-receptor interactions that mediate phage adsorption to the cell surface are diverse in O. oeni and are subject to differential interference by phenolic compounds. Their presence did not induce any modifications in the cell surface as visualized by TEM. Interestingly, docking analyses suggest that quercetin and cinnamic acid may interact with the tail of OE33PA and compete with host recognition.

Activity of ethanol-stressed Oenococcus oeni cells: a flow cytometric approach.

AIMS: To study the effect of ethanol on Oenococcus oeni activity at the single cell level. METHODS AND RESULTS: The active extrusion of the fluorescent probe carboxy fluorescein (cF) was used to assess the metabolic activity of ethanol-stressed O. oeni cells. Subsequent flow cytometric analysis revealed that O. oeni cells extrude the accumulated cF upon energizing with l-malic acid. However, O. oeni cells exposed to 12% (v/v) ethanol for 1 h showed a decreased capacity for active extrusion of cF. Moreover, two subpopulations could be distinguished, one of which being able to extrude cF and the other one remaining cF fluorescent. Growing cells in the presence of 8% (v/v) ethanol resulted in robust cells that maintained the capacity to actively extrude cF after being exposed to 12% (v/v) ethanol, which in turn correlated with the high levels of ATP observed in these ethanol stressed, malolactic fermentation (MLF) performing cells. CONCLUSION: From our results, it becomes evident that active extrusion of cF can be used to assess malolactic activity in O. oeni. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides information for the development of a rapid method to assess the malolactic activity of individual O. oeni cells performing MLF during wine production.

Microbial inactivation and MLF performances of Tempranillo Rioja wines treated with PEF after alcoholic fermentation.

This study was performed with the aim of reducing the microbial communities of wines after alcoholic fermentation to improve the establishment of commercial Oenococcus oeni inoculum for developing the malolactic fermentation. Microbial community reduction was accomplished by applying Pulsed Electric Field (PEF) technology to four different wines. Overall, significant reductions in yeast population were observed. To a lesser extent, lactic acid bacteria were reduced while acetic acid bacteria were completely eliminated after the PEF treatment. In three out of the four tested wines, a decrease in the competitive pressure between microorganisms due to the detected reduction led to a general but slight shortening of the malolactic fermentation duration. In the wine with the most adverse conditions to commercial starter establishment, the shortest malolactic fermentation was reached after PEF treatment. Finally, the sensorial quality of three out of the four treated wines was considered better after the PEF treatment. Therefore, PEF technology meant an important tool for improving the malolactic fermentation performance.

Impact of inhibitory peptides released by Saccharomyces cerevisiae BDX on the malolactic fermentation performed by Oenococcus oeni Vitilactic F.

A previous study has shown that the malolactic fermentation (MLF) was inhibited during sequential fermentations performed with the pair Saccharomyces cerevisiae BDX/Oenococcus oeni Vitilactic F in synthetic grape juices. A yeast peptidic fraction with an apparent MW of 5-10kDa was involved in the inhibition. In the present study, the MLF was also inhibited in Cabernet Sauvignon and Syrah wines. The inhibition due to the peptidic fraction was maintained despite high phenolic contents. Kinetic studies showed that the peptidic fraction was gradually released during the alcoholic fermentation (AF). Its highest anti-MLF effect was reached when isolated from late stages of the AF stationary phase. The peptidic fraction was tested in vitro on cell-free bacterial cytosolic extracts containing the malolactic enzyme in a pH range between 3.5 and 6.7. Results showed that it was able to directly inhibit the malolactic enzyme activity with an increasing inhibitory kinetic correlated to the AF time at which it was collected.

Comparative study of the inhibitory effects of wine polyphenols on the growth of enological lactic acid bacteria.

This paper reports a comparative study of the inhibitory potential of 18 phenolic compounds, including hydroxybenzoic acids and their derivatives, hydroxycinnamic acids, phenolic alcohols and other related compounds, stilbenes, flavan-3-ols and flavonols, on different lactic acid bacteria (LAB) strains of the species Oenococcus oeni, Lactobacillus hilgardii and Pediococcus pentosaceus isolated from wine. In general, flavonols and stilbenes showed the greatest inhibitory effects (lowest IC50 values) on the growth of the strains tested (0.160-0.854 for flavonols and 0.307-0.855 g/L for stilbenes). Hydroxycinnamic acids (IC50 > 0.470 g/L) and hydroxybenzoic acids and esters (IC50 >1 g/L) exhibited medium inhibitory effect, and phenolic alcohols (IC50 > 2 g/L) and flavanol-3-ols (negligible effect) showed the lowest effect on the growth of the LAB strains studied. In comparison to the antimicrobial additives used in winemaking, IC50 values of most phenolic compounds were higher than those of potassium metabisulphite for O. oeni strains (e.g., around 4-fold higher for quercetin than for potassium metabisulphite), but lower for L. hilgardii and P. pentosaceus strains (e.g., around 2-fold lower for quercetin). Lysozyme IC50 values were negligible for L. hilgardii and P. pentosaceus, and were higher than those corresponding to most of the phenolic compounds tested for O. oeni strains, indicating that lysozyme was less toxic for LAB than the phenolic compounds in wine. Scanning electron microscopy confirmed damage of the cell membrane integrity as a consequence of the incubation with antimicrobial agents. These results contribute to the understanding of the inhibitory action of wine phenolics on the progress of malolactic fermentation, and also to the development of new alternatives to the use of sulphites in enology.

Pesticides' influence on wine fermentation.

Wine quality strongly depends on the grape quality. To obtain high-quality wines, it is necessary to process healthy grapes at the correct ripeness stage and for this reason the farmer has to be especially careful in the prevention of parasite attacks on the grapevine. The most common fungal diseases affecting grape quality are downy and powdery mildew (Plasmopara viticola and Uncinula necator), and gray mold (Botrytis cinerea). On the other hand, the most dangerous insects are the grape moth (Lobesia botrana), vine mealybug (Planococcus ficus), and the citrus mealybug (Planococcus citri). Farmers fight grape diseases and insects applying pesticides that can be found at harvest time on grapes. The persistence of pesticides depends on the chemical characteristic of the active ingredients as well as on photodegradation, thermodegradation, codistillation, and enzymatic degradation. The pesticide residues on grapes can be transferred to the must and this can influence the selection and development of yeast strains. Moreover, yeasts can also influence the levels of the pesticides in the wine by reducing or adsorbing them on lees. During the fermentative process, yeasts can cause the disappearance of pesticide residues by degradation or absorption at the end of the fermentation when yeasts are deposited as lees. In this chapter, we reviewed the effect of commonly used herbicides, insecticides, and fungicides on yeasts. We also studied the effect of alcoholic and malolactic fermentation on pesticide residues.

Cell membrane damage induced by phenolic acids on wine lactic acid bacteria.

The aim of this work was to investigate the effect of phenolic acids on cell membrane permeability of lactic acid bacteria from wine. Several phenolic acids were tested for their effects on the cell membrane of Oenococcus oeni and Lactobacillus hilgardii by measuring potassium and phosphate efflux, proton influx and by assessing culture viability employing a fluorescence technique based on membrane integrity. The experimental results indicate that hydroxycinnamic acids (p-coumaric, caffeic and ferulic acids) induce greater ion leakages and higher proton influx than hydroxybenzoic acids (p-hydroxibenzoic, protocatechuic, gallic, vanillic, and syringic acids). Among the hydroxycinnamic acids, p-coumaric acid showed the strongest effect. Moreover, the exposure of cells to phenolic acids caused a significant decrease in cell culture viability, as measured by the fluorescence assay, in both tested strains. The results agree with previous results obtained in growth experiments with the same strains. Generally, phenolic acids increased the cell membrane permeability in lactic acid bacteria from wine. The different effects of phenolic acids on membrane permeability could be related to differences in their structure and lipophilic character.