Impact of malolactic fermentation on the volatile composition of Turkish Kalecik karası red wines

The effects of spontaneous malolactic fermentation (MLF) and a malolactic starter culture (Oenococcus oeni) on the volatile compounds of Kalecik karas? red wines were investigated. The volatile compounds were extracted using liquid–liquid extraction and quantified by GC–MS–flame ionisation detection. Starter cultures of O. oeni had a positive effect in shortening MLF and producing lower volatile acidity. After MLF, the total volatile compounds increased in both the inoculated wines and the spontaneously fermented wine. Ethyl lactate, diethyl succinate and γ‐butyrolactone increased upon inoculation with cultures of O. oeni strains PN4 and VP41. MLF increased the diacetyl concentration in all Kalecik karas? wines. Strain dependent differences were found in the level of diacetyl, with O. oeni PN4 producing more diacetyl than O. oeni VP41. After MLF, the concentrations of acetoin, 4‐ethylguaicol and 4‐ethylphenol increased in spontaneously fermented wine compared with wines inoculated with O. oeni. Accordingly, the use O. oeni PN4 is recommended for a controlled MLF to produce better quality Kalecik karas? wines. © 2018 The Institute of Brewing & Distilling     

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.     

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.     

Molecular analysis of Oenococcus oeni population dynamics and the effect of aeration and temperature during alcoholic fermentation on malolactic fermentation

The effects of aeration and temperature during alcoholic fermentation (AF) on spontaneous and inoculated malolactic fermentation (MLF) of wine have been analysed by following the population dynamics of Oenococcus oeni strains with multiplex random amplified polymorphic DNA‐polymerase chain reaction. It has enabled us to follow precisely the proportion of different autochthonous strains and the starter strain through fermentations. Lack of aeration led to delays in AF, which meant that autochthonous lactic acid bacteria could develop earlier and prevented the starter strain from developing correctly. Temperature was not found to lead to any differences. Two strains were isolated in the same spontaneous MLF, suggesting that, in some cases, multiple strains might be responsible for the degradation of malic acid in wine. It can be concluded that delays in the AF can negatively affect the control of MLF and that this can be studied by following the development of the different strains of O. oeni.     

Characterization and amino acid metabolism performances of indigenous Oenococcus oeni isolated from Chinese wines

Oenococcus oeni is a multiple physical stress-tolerant lactic acid bacterium that plays an important role in wine making. It is often added as a starter culture to carry out malolactic fermentation (MLF). In this study, a total of 22 out of 127 lactic acid bacteria, isolated from Chinese wines undergoing MLF, were identified as O. oeni by species-specific PCR and 16S rRNA sequencing. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis showed that all strains could be typed under these conditions, and three main groups were determined by cluster analysis, which showed intraspecific homology higher than 69 %. Eight strains, representative of SE-AFLP clusters, were tested for malolactic activity. Significant differences were observed among strains with regard to the amount of malic acid consumed. Seventeen amino acids in different wines that were inoculated by 4 O. oeni strains, respectively, were analyzed before and after MLF. The results indicated that the amino acid metabolism of the 4 strains was significantly different between each strain.     

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.     

Evidence of mixed wild populations of Oenococcus oeni strains during wine spontaneous malolactic fermentations

Two hundred and four bacterial isolates from Rioja red wines undergoing spontaneous malolactic fermentation (MLF) were studied. Bacterial species was determined both by microbiological identification methods and by specific PCR analysis. Oenococcus oeni was shown to be the predominant species (98.5% of total isolates). Pulsed field gel electrophoresis (PFGE) of chromosomal DNA digested with SfiI was used to differentiate individual strains of O. oeni. A wide variety of restriction digest patterns were detected, which indicated a rich biodiversity of indigenous strains. Most fermentations (37 out of 41) showed from 2 to 6 clones growing in the same tank. Five O. oeni strains were the most frequently found, appearing in more than three of the 13 studied wineries, and most times in combination with other less frequently found strains. PFGE was shown to be a suitable method for strain differentiation, for monitoring individual strains and determining which strains actually survive and carry out MLF. A high genotypic heterogeneity of wild O. oeni strains was demonstrated and 90% of the studied wines showed mixed populations of O. oeni strains during MLF.     

Improvement of volatile composition of wines by controlled addition of malolactic bacteria

The effect of malolactic fermentation (MLF) on the volatile composition of red wines was studied by inoculation with selected lactic acid bacteria. Four wines were inoculated with different Oenococcus oeni (syn. Leuconostoc oenos) strains, the major malolactic species found in wines, and one was inoculated with a Lactobacillus sp. strain. A non inoculated wine was also analyzed to act as a control. Malolactic fermentation and evolution of non volatile compounds were followed by HPLC and after the depletion of the malic acid present in wine the volatile compounds were extracted and analyzed by gas chromatography with flame ionization and mass spectrometry. Wines which had undergone the MLF showed a significant increment in total higher alcohols, esters and acids that are important in the sensory properties and quality of wine.     

Oenococcus oeni and malolactic fermentation – moving into the molecular arena

Malolactic fermentation (MLF) is the bacterial‐driven decarboxylation of L‐malic acid to L‐lactic acid and carbon dioxide, and brings about deacidification, flavour modifications and microbial stability of wine. Pasteur first described the presence of ‘bacteria’ in wine nearly one‐hundred‐and‐fifty years ago and the subsequent elucidation of the bacterial‐driven malolactic reaction was described about fifty years later. Over the following years the occurrence of MLF became apparent in wines worldwide, and eventually Oenococcus oeni was identified as the principal organism involved in the process. O. oeni is remarkable in its ability to tolerate the nutritionally poor and challenging, harsh wine environment; however, it can be a difficult and sometimes unreliable organism to work with in the winery. A greater knowledge of its biology would undoubtedly facilitate the development of strains and practices, with improved performance outcomes. We already know a considerable amount about the biochemistry and physiology of O. oeni, and ironically, although we know little about its genetics, its genome has been sequenced. With this groundwork in place and molecular biology techniques at our disposal we are poised to increase our knowledge and understanding of this organism enormously.     

The influence of malolactic fermentation and Oenococcus oeni strain on glycosidic aroma precursors and related volatile compounds of red wine

Free and glycosidically bound volatile compounds of red wine were measured after malolactic fermentation (MLF) with four different commercial starter cultures of Oenococcus oeni. MLF resulted in a significant decrease in the concentration of total glycosides, expressed as phenol‐free glycosyl glucose. Gas chromatographic analyses of wine enzyme hydrolysates showed that the extent of hydrolysis of glycosides during MLF was dependent on both bacterial strain and chemical structure of the substrate. The highest decrease was observed for glycosidic precursors of primary terpene alcohols. Glycoside‐related aroma compounds such as linalool, farnesol, and β‐damascenone were increased after MLF with all the bacterial strains tested. Two of the strains were also able to release significant amounts of vinylphenols during MLF. Copyright © 2006 Society of Chemical Industry     

RNA fingerprinting analysis of Oenococcus oeni strains under wine conditions

Oenococcus oeni is a lactic acid bacterium of economic interest used in winemaking. This bacterium is the preferred species for malolactic fermentation (MLF) due its adaptability to the chemically harsh wine environment. MLF enhances the organoleptic properties and ensures deacidification of wines.     

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.     

Effects of Inhibitory Environmental Factors on Growth of Oenococcus oeni CCSYU2068 for Malolactic Fermentation of Cider Production

The effects of several inhibitory factors (sulfur dioxide, pH and ethanol) on the growth of lactic acid bacteria and the subsequent malolactic fermentation (MLF) were studied by inoculation of different culture strains of Oenococcus oeni, the major lactic acid bacteria (LAB) in cider production. After comparing their organoleptic properties, three strains of Oenococcus oeni were selected from indigenous and commercial sources and their inhibitory effects on cell growth and MLF examined. The malolactic bacteria expressed variations in tolerance to the environmental conditions of pH, sulfur and ethanol concentration. Isolated from an indigenous cider production facility, O. oeni L4 had a better capacity with constant growth even when the concentration of SO₂ was 50 ppm, ethanol 10% (v/v) and pH 3.0. O. oeni L4 showed better properties for metabolizing the major acids: malic, lactic and acetic acid. The decomposition mean rate of malic acid was as high as 228.52 mg/L per day with a low acetic acid concentration of 101.78 mg/L under the stress conditions of cider production.     

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.     

Indigenous lactic acid bacteria communities in alcoholic and malolactic fermentations of Tempranillo wines elaborated in ten wineries of La Rioja (Spain)

Indigenous lactic acid bacteria (LAB) communities have been analyzed for three years (2006, 2007 and 2008) during alcoholic (AF) and malolactic (MLF) fermentations of Tempranillo wines in ten wineries of La Rioja. The results showed that analytical composition of wines and physical–chemical conditions of elaboration influenced the LAB populations, the MLF duration and the percentage of each isolated species and strains. The highest diversity of LAB species was observed during AF in all the wineries. Oenococcus oeni was present in all studied stages of the fermentation process, being the predominant species at final AF stage. The study of 925 isolates of O. oeni by Pulsed Field Gel Electrophoresis (PFGE) allowed the detection of a total of 112 distinct genotypes. Most fermentation stages of both AF and MLF showed mixed O. oeni strain populations, so that there were different genotypes able to share their ecological niche or tank in spontaneous MLF. The frequency of participation of each genotype varied either from year to year or from winery to winery. Otherwise, seven genotypes were detected in the three studied years and in at least three out of the ten studied wineries, being four of them also present in the three studied subzones of this region. These results suggest the existence of an endemic microbiota in this region, the adaptation of indigenous O. oeni strains to the winery conditions every year and the interest of selecting predominant genotypes in order to preserve the biodiversity and peculiarity of these wines.     

Oenococcus oeni,a species born and moulded in wine: a critical review of the stress impacts of wine and the physiological responses

Oenococcus oeni is a lactic acid bacterium highly adapted to the stressful environment of wine, and is widely used as an industrial starter culture to conduct malolactic fermentation (MLF), the conversion of L‐malate to L‐lactate. Some cold‐climate wine regions still produce wines which prove too stressful for the application of a starter culture, resulting in sluggish or stuck MLFs which compromise the quality and stability of the wine. The literature on the three primary stress factors, low pH, ethanol and SO₂, is reviewed as well as their toxic mechanisms; possible interactions between the stress factors are also discussed. In particular, the toxic mechanisms of SO₂, an extremely inhibitory compound, are poorly understood. in vitro studies as well as in vivo studies in other prokaryotes point to numerous mechanisms which could have widespread impacts on the cell envelope, proteins and DNA in O. oeni. We also review the responses of O. oeni to these stress factors and offer a simplified model of events which cultures inoculated into particularly stressful wines may experience. Although significant advances have been made in our understanding of the stress responses in recent years, many fundamental aspects still require study.     

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.     

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.