The inhibitory effects of wine phenolics on lysozyme activity against lactic acid bacteria

The lysozyme of hen's egg white is used in winemaking to control spontaneous lactic acid bacteria (LAB). A total of eight LAB strains, isolated from grape must and wine, were used to assess the inhibitory effects of wine phenolics on lysozyme activity. The presence of phenolics, extracted from grape pomace, in growth medium reduced the mortality rate due to the lysozyme activity. This effect was especially clear in the case of strains belonging to Lactobacillus uvarum, Pediococcus parvulus and Oenococccus oeni, which are more sensitive to lysozyme than L. plantarum and L. hilgardii strains. Cell lysis assays carried out on four strains sensitive to lysozyme and Micrococcus lysodeikticus ATCC 4698, used as a reference strain, confirmed the inhibition of grape pomace phenolics on the muramidase. There was no interference from non-flavonoids, flavanols and flavonol compounds, when they were tested individually, on the lysozyme activity against the strains. Anthocyanins extracted from grape skins slightly inhibited the activity only against M. lysodeikticus. However, proanthocyanidins extracted from seed berries, strongly inhibited the lysozyme. In this extract, dimers were the predominant oligomers of flavan-3-ol. The study demonstrated that the effectiveness of lysozyme against LAB in red winemaking is related to the amount of low molecular weight proanthocyanidins that are released when the grapes are macerating.     

Ester synthesis and hydrolysis in an aqueous environment,and strain specific changes during malolactic fermentation in wine with Oenococcus oeni

Previous work has shown that Oenococcus oeni produces esterases that are capable of hydrolysing artificial substrates. Using SPME–GCMS, this study provides evidence that purified O. oeni esterases have the ability to both synthesise and hydrolyse esters. Two purified esterases (EstA2 and EstB28) synthesised ethyl butanoate and ethyl hexanoate to varying degrees. Both purified esterases hydrolysed ethyl butanoate, ethyl hexanoate and ethyl octanoate. Once this dual activity was confirmed, malolactic fermentation (MLF) trials were conducted in wine with O. oeni strains that had been previously observed to have either high or low esterase activity against artificial substrates. Strain specific differences were observed and strains with low esterase hydrolysis activity against artificial substrates had a higher level of total esters measured after MLF. The results demonstrate the impact that O. oeni has on wine aroma and relates this to the ester hydrolysis and synthesis abilities of O. oeni strains.     

Effect of phenolic acids on glucose and organic acid metabolism by lactic acid bacteria from wine

The influence of phenolic (p-coumaric, caffeic, ferulic, gallic and protocatechuic) acids on glucose and organic acid metabolism by two strains of wine lactic acid bacteria (Oenococcus oeni VF and Lactobacillus hilgardii 5) was investigated. Cultures were grown in modified MRS medium supplemented with different phenolic acids. Cellular growth was monitored and metabolite concentrations were determined by HPLC-RI. Despite the strong inhibitory effect of most tested phenolic acids on the growth of O. oeni VF, the malolactic activity of this strain was not considerably affected by these compounds. While less affected in its growth, the capacity of L. hilgardii 5 to degrade malic acid was clearly diminished. Except for gallic acid, the addition of phenolic acids delayed the metabolism of glucose and citric acid in both strains tested. It was also found that the presence of hydroxycinnamic acids (p-coumaric, caffeic and ferulic) increased the yield of lactic and acetic acid production from glucose by O. oeni VF and not by L. hilgardii 5. The results show that important oenological characteristics of wine lactic acid bacteria, such as the malolactic activity and the production of volatile organic acids, may be differently affected by the presence of phenolic acids, depending on the bacterial species or strain.     

Release of glycosidically bound flavour compounds of Chardonnay by Oenococcus oeni during malolactic fermentation

Glycosidases, produced by Oenococcus oeni strain Lalvin EQ54 during malolactic fermentation (MLF) performed in a chemically defined wine (CDW) medium, contributed to the release of volatile aglycons from their glycosylated precursors, present in a Chardonnay wine glycosidic extract. The liberation of wine volatiles during MLF was limited by the low activity of these enzymes in this strain. Six different aglycons examined were 3-hydroxydamascone, alpha-terpineol, vanillin, methyl vanillate, 4-hydroxybenzoic acid and tyrosol. Using p-nitrophenyl synthetic substrates, it was shown that O. oeni Lalvin EQ54 has β-glucosidase, and limited α-l-rhamnopyranosidase and α-l-arabinofuranosidase activities. Release of aglycons from the Chardonnay wine glycosidic extract was increased when purified α-l-rhamnopyranosidase and α-l-arabinofuranosidase were added to the CDW medium together with the malolactic bacteria culture. The results obtained confirmed that O. oeni has the necessary glycosidases for the sequential liberation of the disaccharide sugars from wine glycosides. The rate of MLF proceeded much faster in the CDW supplemented with the Chardonnay wine glycoside extract (8 days compared to 20 days), even though the bacterial growth was unaffected.     

Alterations in d-amino acid concentrations and microbial community structures during the fermentation of red and white wines

Alterations in d-amino acid concentrations and microbial community structures during the fermentation of red and white wines were analyzed to clarify the relationship between the occurrence of d-amino acids and the actions of fermentative microorganisms. Relatives of Saccharomyces cerevisiae and Oenococcus oeni were detected in red wine samples, and relatives of S. cerevisiae, O. oeni, and Gluconacetobacter saccharivorans were detected in white wine samples. The S. cerevisiae relatives were detected throughout the fermentation process, whereas the O. oeni relatives were detected at the late stage of fermentation in both the red and white wine samples. The G. saccharivorans relative was detected in the early stage of fermentation. The amino acid analysis showed that d-alanine, d-glutamic acid, and d-lysine were present in both the red and white wine samples. The concentrations of these d-amino acids increased as the fermentation continued, especially from the malolactic fermentation stage to the end of the fermentation processes. These increases seem to be linked to the presence of O. oeni relatives.     

Release of wine monoterpenes from natural precursors by glycosidases from Oenococcus oeni

It is now well established that wine-related lactic acid bacteria (LAB), especially Oenococcus oeni, possess glycosidase activities that positively contribute to wine aroma through the hydrolysis of grape-derived aroma precursors. In our recent studies, we have identified and characterised several LAB glycosidases with potential in these terms. Here, we report that both a glucosidase and an arabinosidase from O. oeni can release high amounts of monoterpenes from natural substrates under optimal conditions, indicating that these intracellular enzymes might play a significant role in the hydrolysis of aroma precursors during malolactic fermentation. The enzymes from O. oeni exhibited broad substrate specificities (release of both primary/tertiary terpene alcohols) and were even active in grape juice. Further, a sensory panel clearly preferred enzyme-treated Riesling wines over the controls and affirmed that the glycosidases from O. oeni could improve the typical Riesling aroma.     

Inactivation of Lactobacillus brevis in Beer Utilizing a Combination of High‐Pressure Homogenization and Lysozyme Treatment

Inactivation of Lactobacillus brevis in beer by high‐pressure homogenization (HPH) and lysozyme addition was evaluated. The minimum inhibitory concentration of lysozyme against L. brevis was determined and found to be 100 mg.L^?1. The effects of the homogenization process on lysozyme antimicrobial activity and muramidase activity, and the microbial reduction promoted by HPH, and by HPH associated with lysozyme, were evaluated. A significant reduction in lysozyme muramidase activity was observed under 250 MPa, however, no reduction in antimicrobial activity was observed after homogenization up to 300 MPa. The HPH at 100, 140 and 150 MPa promoted 1, 3 and 6 decimal reductions in L. brevis microbial counts, respectively. The HPH and lysozyme association had an additive effect on microbial inactivation, immediately after homogenization, and the lysozyme remained active during 10 days of storage, increasing the inactivation of L. brevis up to a 6 decimal reduction. Therefore, the application of lysozyme with HPH has the potential to reduce the level of pressure required for beer processing, improving the economic costs when utilizing HPH.     

Pre-fermentative replacement of sulphur dioxide by lysozyme and oenological tannins: Effect on the formation and evolution of volatile compounds during the bottle storage of white wines

In this work, the effects on the volatile profile of the pre-fermentative substitution of SO₂ with lysozyme and oenological tannins were studied in white wines. At the same time, in order to understand the changes of volatile compounds in SO₂-free wines, the evolution of volatiles was evaluated over 1 year of storage in bottles. For this purpose, a number of laboratory scale fermentations of SauvignonBlanc musts were carried out and the effects of three variables (SO₂, lysozyme and oenological tannins) were investigated by means of GC–MS analysis. Results showed that the replacement of SO₂ with lysozyme and oenological tannins influenced the volatile composition of wines at the end of the alcoholic fermentation. Wines fermented with SO₂ showed higher total alcohol amounts, while the presence of oenological tannins augmented the level of esters. The presence of SO₂ influenced also the alcohols and esters profiles of wines during bottle storage. Moreover, the presence of oenological tannins displayed a positive role in maintaining the amounts of esters over certain levels in wine stored for 1 year, likely due to their oxygen scavenging ability. By contrast, acids were less affected by the investigated adjuvants both at the end of the alcoholic fermentation and during the storage time.     

Protective role of glutathione addition against wine-related stress in Oenococcus oeni

Oenococcus oeni is the main species responsible for the malolactic fermentation (MLF) of wine due to its ability to survive in this environment. Some wine-related stress factors, such as ethanol and low pH, may alter the cell redox balance of O. oeni. For the first time, the ability to uptake glutathione (GSH), an almost universal tripeptide with antioxidant properties, has been associated to the improvement of stress response in O. oeni. Despite the inability of O. oeni to synthesize GSH, this bacterium can capture it from the media. The ability of 30 O. oeni strains to uptake GSH was assessed in this study. Although all of the strains tested were able to import GSH, substantial variability among them was detected. To assess the physiological function of GSH, three strains with different GSH-import capacities were selected. Significant changes in membrane fatty acids composition were observed due to GSH addition. The most relevant was the increase of cyclopropane fatty acids in cell membrane, in both the exponential and the stationary phases. Cells grown with GSH showed an improved survival against ethanol shock (14% v/v). GSH addition also increased biomass production during the adaptation to wine stress conditions (pH 4, pH 3.4 and 6% ethanol). The results suggest that GSH enrichment could improve the resistance to stress to O. oeni, which could be useful for the adaptation of MLF starter cultures.     

Effect of lysozyme on "flor" velum yeasts in the biological aging of sherry wines

Biological aging is a key step in the production of Sherry wine classified as “fine”. During this stage, a film of yeast referred to as “flor velum” covers the surface of the wine and substantially alters its characteristics. Other microorganisms may coexist with flor yeasts, such as lactic acid bacteria and non-Saccharomyces yeasts, whose growth may be favored under certain conditions, causing organoleptic deviations and deterioration of the wine. To prevent the development of lactic bacteria, lysozyme usage has been introduced. Lysozyme is a hydrolytic enzyme with muramidase activity that can lyse gram-positive bacteria; its use in winemaking was approved by the OIV in 1997 (resolution OENO 10/97). Thus far, the use of lysozyme during the production of Sherry wines is not widespread despite its effectiveness in controlling lactic acid bacteria. However, there have been no studies on the effect of lysozyme on flor velum. The aim of this study was to determine the influence of lysozyme on yeast growth and the formation, development and metabolism of flor velum during the biological aging process of Sherry wine. The results indicate that lysozyme does not affect the flor yeast during the fermentative stage or biofilm stage. However, if yeast inoculation is carried out under submerged culture conditions during biological aging, low doses of lysozyme (≥12.5 g/hL) affect cell multiplication and the membrane hydrophobicity of the yeast, inhibiting their aggregation and flotation and the subsequent development of flor velum. Thus, the yeast inoculation protocol and the methodology used for the addition of lysozyme influence velum development, its metabolism and the wine characteristics.     

Lactobacillus: the Next Generation of Malolactic Fermentation Starter Cultures��an Overview

Malolactic fermentation (MLF) is a secondary wine fermentation conducted by lactic acid bacteria (LAB). This fermentation is important in winemaking as it deacidifies the wine, it contributes to microbial stability and lastly it contributes to wine aroma through the production of metabolites. Oenococcus oeni is the main species used in commercially available starter culture currently, but research has indicated that different Lactobacillus species also partake in MLF and this has shifted the focus in the MLF field to evaluate the potential of lactobacilli as starter cultures for the future. There are 17 different species of Lactobacillus associated with winemaking either being associated with the grapes/beginning of alcoholic fermentation or the MLF and wine. Lactobacillus associated with wine is mainly facultative or obligatory heterofermentative and can withstand the harsh wine conditions such as high ethanol levels, low pH and temperatures and sulphur dioxide. Wine lactobacilli contain the malolactic enzyme encoding gene, but sequence homology shows that it clusters separate from O. oeni. Lactobacillus also possesses more enzyme encoding genes compared to O. oeni, important for the production of wine aroma compounds such as glycosidase, protease, esterase, phenolic acid decarboxylase and citrate lyase. Another characteristic associated with wine lactobacilli is the production of bacteriocins, especially plantaricins which would enable them to combat spoilage LAB. All these characteristics, together with their ability to conduct MLF just as efficiently as O. oeni, make them suitable for a new generation of MLF starter cultures.     

Genetic typification by pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) of wild <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Oenococcus oeni</Emphasis> wine strains

Genetic typification of 120 bacterial isolates of Lactobacillus plantarum and Oenococcus oeni from different Rioja musts and wines was performed by numerical analysis of pulsed-field gel electrophoresis (PFGE) patterns with endonuclease SfiI, and 46 of them were also studied by randomly amplified polymorphic DNA (RAPD)-PCR. A comparative study of both typification methods applied to L. plantarum and O. oeni oenological strains was performed. Bacterial species was determined both by biochemical identification methods and by specific PCR analysis. A wide variety of restriction digest patterns were detected by PFGE among L. plantarum strains (36 unrelated patterns and one closely related pattern, out of 48 isolates), as well as among O. oeni strains (18 unrelated patterns out of 72 isolates). PFGE was shown to be a suitable method for strain differentiation and to determine which strains are present in wine fermentations, with a discriminatory power to type L. plantarum and O. oeni strains higher than that of RAPD-PCR.     

The chemical and sensorial effects of lysozyme addition to red and white wines over six months' cellar storage

Lysozyme is an enzyme with muramidase activity which can lyse Gram-positive bacteria, including wine lactic acid bacteria. This enzyme provides a practical method for delaying or preventing the growth of Oenococcus oeni and consequently the onset of malolactic fermentation during the vinification of red and white wines. This paper reports the impact of lysozyme on the chemical and sensorial properties of commercially vinified red (Cabernet Sauvignon and Shiraz) and white (Riesling) wines. The addition of lysozyme to these wines had little or no effect on the content of alcohol, free and total sulfur dioxide and titratable acidity, and pH value. The lysozyme retained 75–80% activity in the Riesling wine after six months, however no detectable activity remained in the Cabernet Sauvignon and Shiraz wines after two days. Upon addition of lysozyme to both of the red wines, a rapid initial decrease (up to 17%) in red wine colour density and phenolic content occurred in association with the formation of a light precipitate. The reduction in red wine colour was also noted by the sensory panel. When added to the Riesling wine, lysozyme did not cause an increase in browning over the six month storage time, but did induce heat instability (haze), suggesting that white wines may require protein stabilisation following treatment with lysozyme. Sensory assessment by triangle difference testing revealed that, during the six month storage period, treatment with lysozyme did not cause important changes to either the aroma or palate of the red and white wines tested.     

Influence of pH and ethanol on malolactic fermentation and volatile aroma compound composition in white wines

The present study investigated the influences of pH and ethanol on malolactic fermentation (MLF) and the volatile aroma profile of the subsequent white wines from Riesling and Chardonnay inoculated with two different Oenococcus oeni strains. In all cases MLF was induced after completion of alcoholic fermentation (AF). Partial MLF occurred under low pH 3.2 and high alcohol (118.3 g/L) conditions. In the cases with complete MLF, the time required for each strain varied from 13 to 61 days and was dependent on bacterial culture, cultivar and wine parameter. Chemical properties of each wine were determined after AF, complete and partial MLF. The wines showed significant differences in total higher alcohols, esters and acids that are important for the sensory profile and quality of wine. This work demonstrated that the wine matrix as well as the pH and alcohol concentration affects MLF and the final volatile aroma profile. Results indicate that changes in volatile aroma composition are not necessarily related to complete MLF and that partial MLF already has distinct influences on the wine aroma profile of white wines.     

Malolactic Fermentation and Secondary Metabolite Production by Oenoccocus oeni Strains in Low pH Wines

Abstract: Different wine varieties, including some with low pH, were studied to determine the ability to grow and produce secondary metabolites of a previously selected autochthonous Oenococcus oeni strain (C22L9), compared with a commercial strain. Monitoring of malolactic fermentation (MLF) was carried out by microbiological and chemical analysis of wines. The concentration of some major volatile compounds and biogenic amines in wines before and after malolactic fermentation was also determined. The results showed major differences in MLF duration both between wines and strains, although the differences between strains were slight for most of the analyzed compounds. Statistically significant differences in citric acid degradation were found in all wine varieties and it was confirmed that O. oeni C22L9 is a poor degrader of citric acid; this means that MLF can be prolonged without the risk of producing high concentrations of diacetyl and acetoin. Sensory analysis of wines after MLF showed similar characteristics in wines from both strains. This study thus shows that O. oeni C22L9 possesses even better sensory and fermentation properties than the commercial strain and can be used in wines with different characteristics, which makes it highly valuable for industrial use. Practical Application: The increasingly use of grape varieties of low pH in winemaking and the higher alcohol content of wines, as a consequence of the climatic change, make interesting the study of the behavior during MLF of O. oeni strains in order to determine their ability to grow, when growth conditions are not optimal, and to produce secondary metabolites. A comparative study was conducted using an autochthonous O. oeni strain (C22L9) and a commercial O. oeni strain and 4 wine varieties.     

Biogenic amine production by Oenococcus oeni during malolactic fermentation of wines obtained using different strains of Saccharomyces cerevisiae

Twenty-six wild Oenococcus oeni strains were investigated for their ability to form biogenic amines during malolactic fermentation in synthetic medium and in wine. Eight strains produced histamine and tyramine in screening broth at concentrations of 2.6-5.6 mg/L and 1.2-5.3 mg/L, respectively. Based on their ability to form biogenic amines, five strains were selected to inoculate three wines obtained by the fermentation of three different Saccharomyces cerevisiae strains (A, B, and C). All bacterial strains could perform malolactic fermentation for short periods in wine C, whereas only one strain performed complete malolactic fermentation in wines A and B. Two O. oeni strains (261 and 351) produced histamine and tyramine in wine C. Time-course analysis of these compounds showed that for both strains, histamine and tyramine production began at day 10 and finished on day 25, after the end of malolactic fermentation. These results indicate that the ability of O. oeni to produce histamine and tyramine is dependent on the bacterial strain and on the wine composition, which in turn depends on the yeast strain used for fermentation, and on the length of bacteria-yeast contact time after the completion of malolactic fermentation.     

Study of the oenological characteristicsand enzymatic activities of wine yeasts

Seventy-four wine strains of theSaccharomycesgenus were tested to study their oenological characteristics and enzymatic activities. These strains were isolated from spontaneous microbiota taken from cellars in the Valdepeñas region (Spain) and identified by contour homogeneous electric field (CHEF) technique. When Cluster Analysis was applied to oenological properties, only SH₂production, tolerance to 12⁰of ethanol and synthesis of killer toxin contributed to the distribution of the strains in the different statistical groups. All the strains possessed esterase activity and some also showed protease and pectinase activities. None of them hadβ-D-glucosidase activity.     

Comparison of the effects of high pressure homogenization and high pressure processing on the enzyme activity and antimicrobial profile of lysozyme

The effects of high pressure homogenization (HPH, < 190 MPa) and high pressure processing (HPP, < 600 MPa) on hen egg white lysozyme muramidase and antimicrobial activities were assessed. The results showed enzyme activation under mild process conditions (< 120 MPa for HPH and < 400 MPa for HPP, both at 20 °C) and mostly for activity measured at non-optimum pH and temperature. When processes were carried out at 50 °C, lower activation were observed (< 18% for HPH and < 13% for HPP), possibly indicating that processes at 50 °C delivered enough energy to promote undesirable unfolding on lysozyme. HPH induced a greater increase in muramidase activity (29%) than HPP (17%), but this not reflected the antimicrobial performance of the processed lysozyme, since only HPP reduced the minimum inhibitory concentration of the lysozyme against Bacillus cereus (50%) and Geobacillus stearothermophilus (66%). The results highlighted that each process changed differently the lysozyme muramidase and antimicrobial activity.Industrial relevanceHPP and HPH are generally described as technologies able to increase the activity of several enzymes and are suggested as tools to improve the performance of commercial enzymes. The results showed that although HPP and HPH were able to increase the muramidase activity of lysozyme, improvement of the antibacterial performance was only observed for samples processed by HPP. Therefore HPP was highlighted as the better pressure process to physically modify lysozyme.     

Assessment of the genetic polymorphism and biogenic amine production of indigenous Oenococcus oeni strains isolated from Greek red wines

In the warm climate country of Greece malolactic fermentation (MLF) has received limited attention. Molecular techniques and High Performance Liquid Chromatography (HPLC) were used to study the genetic polymorphism of autochthonous lactic acid bacteria developing towards the end of spontaneous MLF of Greek red wines and for the assessment of their potential to produce harmful biogenic amines. This research revealed that native Oenococcus oeni isolates are very much adapted to specific winery conditions since the majority of spontaneous MLF were driven mostly or exclusively by a single strain of O. oeni. Native O. oeni strains showed only limited dispersion since cluster analysis uncovered only few common genotypes among indigenous isolates from different wineries. The genotype of a frequently used malolactic starter was more than often detected among autochthonous isolates without nevertheless compromising the biodiversity of natural microflora residing in wineries but rather becoming a part of it. For the majority of the wine samples studied, MLF implementation and storage in bottles resulted in negligible changes on the levels of the BA histamine, tyramine, phenylethylamine, cadaverine as well as of ethylamine, methylamine, isobutylamine. We provide evidence that autochthonous O. oeni isolates can only contribute to putrescine accumulation in Greek wines but still the specific trait behaves as strain-specific with a limited dispersion.