A study into the role of L-aspartic acid on the metabolism of L-malic acid and D-glucose by Oenococcus oeni

AIMS: The purpose of this work was to study the effect of L-aspartic acid concentration on bacterial growth, D-glucose fermentation and L-malic acid consumption of Oenococcus oeni NCFB 1707. METHODS AND RESULTS: Bacterial cultures were performed in synthetic media. Bacterial growth, D-glucose fermentation and L-malic acid consumption were reduced when L-aspartic acid concentration became excessive. This inhibitory effect of high concentrations of L-aspartic acid on bacterial growth was also observed with several Oenococcus oeni strains, except O. oeni BL01. The L-aspartic acid inhibitory effect on bacterial growth could be reduced by increasing the concentration of L-glutamic acid. L-glutamic acid transport was found to be competitively inhibited by L-aspartic acid. In addition, an excessive amount of L-aspartic acid modified D-glucose metabolism, with an overproduction of acetic acid and reduced ethanol production. CONCLUSION: Since L-glutamic acid is an essential amino acid for the bacterial strain used, the L-aspartic acid inhibitory effect on bacterial growth could be linked to its involvement in an antagonistic interaction with L-glutamic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: Such antagonistic interactions between amino acids in O. oeni strains could be another explanation for the difficulties of inducing malolactic fermentation in wines.     

Effect of phenolic compounds on the co-metabolism of citric acid and sugars by Oenococcus oeni from wine

AIMS: The goal of this study was to examine the growth of Oenococcus oeni in the presence of phenolic compounds under wine conditions and to see how these compounds affect bacterial metabolism. METHODS AND RESULTS: Phenolic compounds have been added to a basal medium that simulates the composition of wine. Fifty milligrams per litre or more of phenolic compounds stimulated bacterial growth. Oenococcus oeni seemed to use citric acid and trehalose, if they were present, before glucose and fructose. Citrate was completely exhausted in three days and the yield of acetate was higher when phenolic compounds were present. CONCLUSIONS: Phenolic compounds reduced the rate of sugar consumption and enhanced citric acid consumption, increasing the yield of acetic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: This study allows a better knowledge of co-metabolism of citric acid and sugars by O. oeni in the presence of phenolic compounds of wine.     

Organic acid metabolism under different glucose concentrations of Leuconostoc oenos from wine

Leuconostoc oenos M isolated from wine did not grow in the absence of glucose and it was clearly stimulated by the presence of L-malic and citric acids in synthetic medium with different glucose concentrations. In basal medium, D-glucose and L-malic and citric acids were simultaneously consumed. L-Malic acid was metabolized at a higher rate than glucose and citric acid. When the organic acids were completely consumed only 50% of the glucose was utilized. In basal medium 1 mmol of D-lactic acid was produced per mmol of glucose consumed and the amount of ethanol formed was higher with acetate present in the medium. L-Malic acid was completely recovered as L-lactic acid, and in the presence of L-malic acid a carbon imbalance from glucose to D-lactic acid was observed. In the presence of citric acid the amount of D-lactic acid formed was directly proportional to glucose-citrate utilization and acetic acid and ethanol were produced.     

Growth, D-glucose utilization and malolactic fermentation by Leuconostoc œnos strains in 18 media deficient in one amino acid

Six Leuconostoc œnos strains were used to study the effect of the deficiency of one amino acid on growth, heterofermentative pathway and malolactic fermentation. All strains had an absolute requirement for four amino acids (isoleucine, glutamic acid, tryptophan and arginine) and needed six other amino acids (valine, methionine, cysteine, leucine, aspartic acid and histidine) for optimum growth. Each deficiency in one amino acid had a particular effect on D-glucose utilization. Overproduction and underproduction of D-lactic acid were observed. The rate of L-malic acid consumption in media deficient in one amino acid was lower than in the complete medium with all amino acids.
Although some deficiencies (glycine, phenylalanine, proline or tyrosine) had no influence on the growth, they noticeably limited the malolactic fermentation.     

Effects of yeast proteolytic activity on Oenococcus oeni and malolactic fermentation

Alcoholic fermentation of synthetic must was performed using either Saccharomyces cerevisiae or a mutant Deltapep4, which is deleted for the proteinase A gene. Fermentation with the mutant Deltapep4 resulted in 61% lower levels of free amino acids, and in 62% lower peptide concentrations at the end of alcoholic fermentation than in the control. Qualitative differences in amino acid composition were observed. Changes observed in amino acids in peptides were mainly quantitative. After alcoholic fermentation, each medium was inoculated with Oenococcus oeni. Malolactic fermentation in the medium with the Deltapep4 strain took 10 days longer than the control. This difference may have been due to a difference in the nitrogen composition of the two media. Free amino acids and amino acids in peptides were poorly consumed by O. oeni. Thus, the qualitative aspects of nitrogen composition, which depend in part on yeast metabolism, may be a determinant for the optimal growth of O. oeni in wine.     

Effect of nitrogen limitation and nature of the feed upon Oenococcus oeni metabolism and extracellular protein production

AIMS: The aim of the study was to characterize the effect of various nitrogen sources on Oenococcus oeni growth, carbon source utilization, extracellular protease activity and extracellular proteins. More generally, the goal is to understand how nitrogen-based additives might act to enhance malolactic fermentation in wine. METHODS AND RESULTS: Five yeast extracts were used. As the amino acid and nitrogen analyses revealed, they were similar in global amino acid composition, except for arginine level. Nevertheless the ratio of amino acids between free/bound, and low/high molecular weight fractions were highly different. One of the yeast extracts led to a significant protease activity in the supernatant and to a poor final biomass of the IOB84.13 strain compared to the other ones. For the IOB84.13 strain specifically, arginine addition to the arginine poor yeast extract did not restore growth. 35S-methionine-labelled extracellular proteins were separated by SDS-PAGE. Signals were detected in all media early in the growth phase and were maintained during 48 h of culture. CONCLUSIONS: A significant protease activity was detected for O. oeni supernatants during growth under nitrogen limitation but only for certain nitrogen sources. Moreover, the activity was strain dependent. Peptides (0.5-10 kDa) seemed to be more favourable for growth of wine bacteria than <0.5 kDa nitrogen sources. The extracellular protein signal patterns differed more greatly between the bacterial strains tested than between the nitrogen molecules in the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study extensively considering the role of the nitrogen source composition and level upon O. oeni growth and metabolism.     

Influence of phenolic compounds on the physiology of Oenococcus oeni from wine

This study shows that the growth of Oenococcus oeni CECT 4100 in a synthetic medium is affected by phenolic compounds in different ways, depending on their type and concentration. Generally they have no effects at low concentrations, but hydroxycinnamic acids are inhibitory at high concentrations. Malolactic fermentation was stimulated in the presence of catechin and quercetin, but increasingly delayed with increasing amounts of p-coumaric acid. Gallic acid appeared to delay or inhibit the formation of acetic acid from citric acid. This could lead to a better control of malolactic fermentation and suppress the increase in volatile acidity, which is undesirable in the wine-making process.     

Influence of phenolic acids on growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii

Aims: To determine the effect of several wine-associated, phenolic acids on the growth and viability of strains of Oenococcus oeni and Lactobacillus hilgardii. Methods and Results: Growth was monitored in ethanol-containing medium supplemented with varying concentrations of hydroxybenzoic acids (p-hydroxybenzoic, protocatechuic, gallic, vanillic and syringic acids) and hydroxycinnamic acids (p-coumaric, caffeic and ferulic acids). Progressive inactivation was monitored in ethanol-containing phosphate buffer supplemented in a similar manner to the growth experiments. Hydroxycinnamic acids proved to be more inhibitory to the growth of O. oeni than hydroxybenzoic acids. On the other hand, some acids showed a beneficial effect on growth of Lact. hilgardii. p-Coumaric acid showed the strongest inhibitory effect on growth and survival of both bacteria. Conclusions: Most phenolic acids had a negative effect on growth of O. oeni, for Lact. hilgardii this effect was only noted for p-coumaric acid. Generally, O. oeni was more sensitive to phenolic acid inactivation than Lact. hilgardii. Significance and Impact of the Study: Eight wine-derived, phenolic acids were compared for their effects on wine lactic acid bacteria. Results indicate that phenolic acids have the capacity to influence growth and survival parameters. The differences found between phenolic compounds could be related to their different chemical structures.     

Effect of Dipeptides on the Growth of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> in Synthetic Medium Deprived of Amino Acids

Oenococcus oeni has numerous amino acid requirements for growth and dipeptides could be important for its nutrition. In this paper the individual or combined effect of dipeptides on growth of O. oeni X₂L in synthetic media deficient in one or more amino acids with L-malic acid was investigated. Utilization of dipeptides, glucose, and L-malic acid was also analyzed. Dipeptides were constituted by at least one essential amino acid for growth. Dipeptides containing two essential amino acids, except leucine, had a more favorable effect than free amino acids on the growth rate. Gly-Gly was consumed to a greater extent than Leu-Leu and a rapid exodus of glycine to the extracellular medium accompanied it. The microorganism could use glycine in exchange for other essential amino acids outside the cell, favoring growth. In the presence of Leu-Leu, the increase in glucose consumption rate could be related to the additional energy required for dipeptide uptake.     

Growth and exopolysaccharide (EPS) production by Oenococcus oeni I4 and structural characterization of their EPSs

AIMS: To study the influence of medium constituents on growth, and exopolysaccharide (EPS) production by a strain of Oenococcus oeni. The structure of one of the EPSs has also been characterized. METHODS AND RESULTS: EPS concentration was estimated by the phenol/sulfuric acid method. After purification and fractionation of crude EPSs, the sugar composition was determined by GLC-MS of the TMS methyl glycosides. The major polysaccharide is 2-substituted-(1-3)-beta-D-glucan. This structure was determined by methylation analysis and conventional (1)H- and (13)C-nuclear magnetic resonance spectroscopy. In addition, O. oeni synthesized two heteropolysaccharides, although a lesser proportion, constituted by galactose and glucose, and one of them also showed rhamnose. The sugar source has a clear influence on growth and EPS synthesis, and EPS production was not enhanced by adding ethanol or increasing the nitrogen source. EPS biosynthesis starts in the exponential growth phase, and continued during the stationary growth phase. CONCLUSIONS: Higher EPS yields were obtained on cultures grown on glucose + fructose. O. oeni produces a beta-glucan, as the predominant EPS, and it is also able to produce two heteropolysaccharides. Significance and Impact of the Study: This work provides a better understanding of EPS synthesis by O. oeni and shows the first EPS structure described for this species.     

Oligopeptide assimilation and transport by Oenococcus oeni

Aims: Oenococcus oeni is a slow‐growing wine bacterium with a low growth yield. It thrives better on complex nitrogen sources than on free amino‐acid medium. We aimed to characterize the oligopeptide use of this micro‐organism. Methods and Results: Several peptides of two to eight amino‐acid residues were able to provide essential amino acids. The disappearance of various peptides from extracellular medium was assessed with whole cells. Initial rates of utilization varied with the peptide, and free amino acids were released into the medium. Conclusions: Oenococcus oeni was able to transport the oligopeptides with two to five amino‐acid residues tested and to hydrolyse them further. Significance and Impact of the Study: This study has clear implications for the relationship between wine nitrogen composition and the ability of O. oeni to cope with its environment.     

Functional divergence in the genus Oenococcus as predicted by genome sequencing of the newly-described species, Oenococcus kitaharae

Oenococcus kitaharae is only the second member of the genus Oenococcus to be identified and is the closest relative of the industrially important wine bacterium Oenococcus oeni. To provide insight into this new species, the genome of the type strain of O. kitaharae, DSM 17330, was sequenced. Comparison of the sequenced genomes of both species show that the genome of O. kitaharae DSM 17330 contains many genes with predicted functions in cellular defence (bacteriocins, antimicrobials, restriction-modification systems and a CRISPR locus) which are lacking in O. oeni. The two genomes also appear to differentially encode several metabolic pathways associated with amino acid biosynthesis and carbohydrate utilization and which have direct phenotypic consequences. This would indicate that the two species have evolved different survival techniques to suit their particular environmental niches. O. oeni has adapted to survive in the harsh, but predictable, environment of wine that provides very few competitive species. However O. kitaharae appears to have adapted to a growth environment in which biological competition provides a significant selective pressure by accumulating biological defence molecules, such as bacteriocins and restriction-modification systems, throughout its genome.     

The effect of amino acid composition of serum-free medium on DNA synthesis in primary hepatocyte cultures in the presence of epidermal growth factor

Summary The presence of optimal nutritional elements in cell culture medium is very important in studies of cultured cells. For this reason, several researchers have experimented with adding or increasing the concentration of one or more amino acids to the medium they were using to determine “essential” amino acids and optimal concentrations. We studied how leaving out one amino acid at a time from Dulbecco’s modified Eagle’s medium would affect epidermal growth factor-induced DNA synthesis in primary hepatocytes of the rat. Our “modified” DMEM contained only eight amino acids: arginine, cysteine, isoleucine, leucine, lysine, phenylalanine, tryptophan, and valine. Proline was found to be an essential amino acid in normal DMEM but not in the modified DMEM, and some other amino acids reduced DNA synthesis in this medium. This study showed that perhaps no single amino acid such as proline can be called “essential,” but rather an optimal balance of amino acids is required for each major function of each cell type cultured.     

Studies on growth and metabolism of Oenococcus oeni on sugars and sugar mixtures

AIMS: To study the effect of sugars and sugar mixtures on the growth kinetics of Oenococcus oeni NCIMB 11648 in batch culture with the aim of producing a high cell productivity system for starter cultures. METHODS AND RESULTS: The growth of O. oeni was investigated on single sugars (glucose, fructose or sucrose) and their mixtures (glucose-fructose, glucose-sucrose or fructose-sucrose). Better growth was obtained on sugar mixtures compared with growth on a single sugar. The production system of O. oeni biomass was investigated in batch culture with or without pH control with respect to kinetics, specific growth rate and biomass yield. The effect of pH and substrate concentration on fermentation balances and ATP yield were determined. The optimal growth of O. oeni was achieved on the glucose-fructose mixture (9 g l(-1), 1 : 1) at pH 4.5 and 25 degrees C with pH control, with highest cell volumetric productivity (7.9 mg cell l(-1) h(-1)), biomass yield (0.041 g cell g(-1) sugar) and specific growth rate (0.066 h(-1)). CONCLUSIONS: The limitations to the growth of O. oeni were pH and inhibition by end product resulting in poor utilization of the medium with low cell yields. The cell productivity of the system can be improved by the appropriate use of mixed sugar growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study uniquely showed that appropriate sugar mixtures with the correct environmental conditions can significantly improve the productivity of O. oeni cultures.     

Lactobacillus casei metabolic potential to utilize citrate as an energy source in ripening cheese: a bioinformatics approach

AIMS: To identify potential pathways for citrate catabolism by Lactobacillus casei under conditions similar to ripening cheese. METHODS AND RESULTS: A putative citric acid cycle (PCAC) for Lact. casei was generated utilizing the genome sequence, and metabolic flux analyses. Although it was possible to construct a unique PCAC for Lact. casei, its full functionality was unknown. Therefore, the Lact. casei PCAC was evaluated utilizing end-product analyses of citric acid catabolism during growth in modified chemically defined media (mCDM), and Cheddar cheese extract (CCE). Results suggest that under energy source excess and limitation in mCDM this micro-organism produces mainly L-lactic acid and acetic acid, respectively. Both organic acids were produced in CCE. Additional end products include D-lactic acid, acetoin, formic acid, ethanol, and diacetyl. Production of succinic acid, malic acid, and butanendiol was not observed. CONCLUSIONS: Under conditions similar to those present in ripening cheese, citric acid is converted to acetic acid, L/D-lactic acid, acetoin, diacetyl, ethanol, and formic acid. The PCAC suggests that conversion of the citric acid-derived pyruvic acid into acetic acid, instead of lactic acid, may yield two ATPs per molecule of citric acid. Functionality of the PCAC reductive route was not observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This research describes a unique PCAC for Lact. casei. Additionally, it describes the citric acid catabolism end product by this nonstarter lactic acid bacteria during growth, and under conditions similar to those present in ripening cheese. It provides insights on pathways preferably utilized to derive energy in the presence of limiting carbohydrates by this micro-organism.     

Vanillin production from simple phenols by wine-associated lactic acid bacteria

AIMS: The ability of lactic acid bacteria (LAB) to metabolize certain phenolic precursors to vanillin was investigated. METHODS AND RESULTS: Gas chromatography-mass spectrometry (GC-MS) or HPLC was used to evaluate the biosynthesis of vanillin from simple phenolic precursors. LAB were not able to form vanillin from eugenol, isoeugenol or vanillic acid. However Oenococcus oeni or Lactobacillus sp. could convert ferulic acid to vanillin, but in low yield. Only Lactobacillus sp. or Pediococcus sp. strains were able to produce significant quantities of 4-vinylguaiacol from ferulic acid. Moreover, LAB reduced vanillin to the corresponding vanillyl alcohol. CONCLUSIONS: The transformation of phenolic compounds tested by LAB could not explain the concentrations of vanillin observed during LAB growth in contact with wood. SIGNIFICANCE AND IMPACT OF THE STUDY: Important details of the role of LAB in the conversion of phenolic compounds to vanillin have been elucidated. These findings contribute to the understanding of malolactic fermentation in the production of aroma compounds.     

Interactions between Saccharomyces cerevisiae and malolactic bacteria: preliminary characterization of a yeast proteinaceous compound(s) active against Oenococcus oeni

AIMS: To investigate the occurrence and extent of Saccharomyces cerevisiae and Oenococcus oeni interactions. METHODS AND RESULTS: Interactions between S. cerevisiae and O. oeni were investigated by double-layer and well-plate assays showing the occurrence of specific interactions for each yeast-malolactic bacteria (MLB) coupling. Heat and protease treatments of synthetic grape juice fermented by the S. cerevisiae strain F63 indicated that the inhibitory activity exerted by this yeast on O. oeni is due to a proteinaceous factor(s) which exerts either bacteriostatic or bactericidal effect depending on concentration and affects malolactic fermentation in natural grape juice and wine. CONCLUSIONS: A proteinaceous factor(s) produced by a S. cerevisiae wine strain able to inhibit O. oeni growth and malic acid fermentation was characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: The individuation, characterization and exploitation of yeast proteinaceous factor(s) exerting inhibitory activity on MLB may offer new opportunities for the management of malolactic fermentation.