Oenococcus Oeni Strains Research Articles

Selection of a New Highly Resistant Strain for Malolactic Fermentation under Difficult Conditions

Malolactic fermentation (MLF) is a biological process that contributes to wine quality, but it is frequently affected by various vinification conditions. Resistance to four wine-limiting factors was studied with respect to 10 Oenococcus oeni strains in order to select a suitable strain for performing reliable MLF in difficult wines. Resistance to low fermentation temperature, high SO2 and/or ethanol concentration, and low pH were assayed in laboratory tests. A pool of the most resistant strains was used in a set of laboratory MLFs. At the end of fermentation, the dominant strains were identified by RAPD-PCR. The PN4 strain was found to be dominant in the majority of cases and under the most detrimental wine conditions, and it was therefore chosen as the single-strain inoculum for the subsequent MLF trials. The effectiveness of the PN4 strain was confirmed in a series of MLFs carried out in three different countries under experimental and industrial conditions. It accomplished MLF in wines with up to 15.8% ethanol, pH as low as 3.0, 60 mg/L of free SO2, and in fermentation temperatures below 17oC. Our findings indicate that the O. oeni PN4 strain could be an effective starter, guaranteeing regular and reliable MLF fermentation.

Genetic and Physiological Characterisation of Oenococcus oeni Strains to Perform Malolactic Fermentation in Wines

Malolactic fermentation (MLF) is a process that is increasingly conducted by Oenococcus oeni industrial strains.Recently, studies of the diversity of O. oeni strains have developed some potential genetic tools to characterise theabilities of the strains. During this work, a mutation on a partial sequence of the rpoB gene and the presence ofsome genes previously established to be present in the most performing strains were tested on some strains thatare already marketed and some potential new strains. These tests were compared with a physiological test neverpreviously taken into account: the tolerance to octanoic and decanoic acid, important inhibitory compounds in wines.Our objectives were to compare the relevance of the genetic tests currently available, that of resistance to mediumchain fatty acids and the results of winemaking. Ultimately, it is clear that, as far as current knowledge is concerned,genetic tests are not yet sufficient to completely characterise the strain potential, and physiological tests thereforeare always needed. The resistance to medium chain fatty acids is an interesting point to be considered to explain thedifficulty that some strains have to resist inoculation in wine. But other criteria should also be characterised better,such as the duration of the latent phase between inoculation and the beginning of MLF, and the rate of degradationof malic acid by the different strains.

Comparative vinification assays with selected Patagonian strains of Oenococcus oeni and Lactobacillus plantarum

The performance of Patagonian Lactobacillus plantarum and Oenococcus oeni strains as malolactic starter cultures was compared. Two autochthonous strains of each species were selected, based on the presence of aroma-related genes, and inoculated in sterile wine of high ethanol content. The effects of initial inoculum size and acclimation treatment on the efficiency of malolactic fermentation (MLF) were analyzed for each strain. O. oeni strains were able to successfully conduct the MLF only when the inoculum concentration was higher than 1.108 CFU/mL and cells were acclimated in sublethal ethanol concentrations. The increase of ethanol concentration in the acclimation medium also improved the kinetics of malic acid consumption. Successful MLF with L. plantarum strains required lower inocula and no acclimation treatment. In addition, these strains showed a better profile of aroma-related genes than O. oeni. L. plantarum strains appeared to be more efficient than O. oeni strains as candidates for malolactic starter cultures to be used in Patagonian red wines.

Behaviour during Malolactic Fermentation of Three Strains of Oenococcus oeni Used as Direct Inoculation and Acclimatisation Cultures

The behaviour in malolactic fermentation (MLF) of an autochthonous strain of Oenococcus oeni, C22L9, isolated from a winery in Castilla-La Mancha (Spain), and of two other commercial strains of O. oeni, PN4 and Alpha (Lallemand Inc.), inoculated by direct inoculation (MBR®) and after a short acclimatisation phase (1-STEP®), was studied. Strain C22L9 carried out MLF slightly faster than the two other commercial strains, leading to a lower increase in volatile acidity and in 2,3-butanedione and 3-hydroxy-2-butanone concentrations, a higher lactic acid content, lower degradation of citric acid and increased degradation of ethanol. No great differences were observed in the duration of MLF, although the acclimatisation cultures were slightly faster, or in the composition of the wines when using the O. oeni strains in the form of MBR® or 1-STEP® cultures. The tasters did not detect significant differences in the wines obtained from the same strain of O. oeni in the two inoculation formats.

Assessment of β-D-Glucosidase Activity from Two Typical Strains of the Lactic Acid Bacteria, Oenococcus oeni, in China

β-D-glucosidase (βG) is one of the most interesting glycosidases for the hydrolysis of glycoconjugated precursors to release active aromatic compounds in musts and wines. Oenococcus oeni strains SD-2a and 31MBR are widely used in Chinese wines to reduce the acidity. In the present study, the βG activity of the two strains was localised and partially characterised using synthetic substrate. The activity occurred in whole cells, sonication supernatants and debris, but not in the culture supernatants for both strains. Whole cells of strain SD-2a possessed greater βG activity, while strain 31MBR showed higher enzyme activity in the sonication supernatants. Strain 31MBR exhibited higher total enzyme activity than strain SD-2a. The optimum temperatures for βG from the two strains were 40oС at pH 3.5 and 50oС at pH 5.0, respectively. Ethanol at low concentrations had a positive effect on βG activity in both strains, while a wine-like pH (3.5) decreased the enzyme activity to a great extent. Whole cells of strain SD-2a showed the highest activity among all samples tested under wine-like conditions. Thus, strain SD-2a proved to have potential for aroma improvement in winemaking.

Exploitation of Simultaneous Alcoholic and Malolactic Fermentation of Incrocio Manzoni, a Traditional Italian White Wine

Malolactic fermentation (MLF) is a key feature in the production of high-quality wines. Its evolution is not always guaranteed, especially in white wine, due to certain limiting factors (low pH, sulphur dioxide, low temperature) acting against malolactic bacteria. The inoculation of grape must with bacteria is an alternative approach to the management of oenological fermentation, favouring the survival of bacteria due to the absence of ethanol or sulphur dioxide – toxic compounds made by yeasts in the first stages of winemaking. We compared the activity of two strains of Oenococcus oeni during MLF in wines made from an emerging white grape variety, native to north-eastern Italy, namely Incrocio Manzoni. Different winemaking protocols were assayed, comparing sequential or simultaneous inoculation of microbial starters. The monitoring of bacterial viability through fermentations and a comprehensive characterisation of the volatile profile of the wines were achieved by advanced analytical approaches, flow cytometry and GC-MS respectively. According to the preliminary hypothesis, the chemical composition of the grape must was characterised by high acidity, which represented a serious barrier to bacterial development. Simultaneous inoculation of the two O. oeni strains ensured a regular evolution of MLF. Some differences were highlighted, both in terms of fermentation kinetics and the aromatic profile of the wines obtained, in relation to the strain of lactic bacteria. The work provides an exhaustive overview of the opportunities and risks related to different wine fermentation approaches in order to enhance the quality of white wines made from “new” or “local” wine grapes.

Influence of Oenococcus oeni and Brettanomyces bruxellensis on Hydroxycinnamic Acids and Volatile Phenols of Aged Wine

The effect of two Oenococcus oeni strains, Viniflora® CiNe™ and Viniflora® CH11, with and without cinnamoyl esterase activity, respectively, on the contents of the hydroxycinnamic acids (HCAs) p-coumaric and ferulic acid, and their respective volatile phenols, 4-ethylphenol and 4-ethylguaiacol, was investigated over six months in Cabernet Sauvignon wines inoculated with two different Brettanomyces bruxellensis strains, CBS 73 and CBS 2499. Both CiNe™ and CH11 showed growth and malolactic fermentation in the wines. There were no clear growth interactions between B. bruxellensis and O. oeni. Furthermore, B. bruxellensis did not inhibit MLF by CiNe™ or CH11. The HCA concentrations in all wines increased until 114 days of fermentation, after which they decreased from day 114 to day 180. Wines inoculated with CiNe™ had the highest concentrations of HCAs throughout the fermentations. While CiNe™ degraded more of the tartaric ester bound forms of HCAs into free HCAs, there was no significant difference in the production of volatile phenols as compared with wines inoculated with CH11. However, there was a significant difference between wines with the two strains of B. bruxellensis in the level of volatile phenols and HCAs on day 180. Thus, the level of volatile phenols in wine depends more on strain differences of B. bruxellensis than on cinnamoyl esterase activity of O. oeni.

Genetically engineered Oenococcus oeni strains to highlight the impact of estA2 and estA7 esterase genes on wine ester profile

Besides deacidifying wine, Oenococcus oeni bring significant changes in the chemical composition of wine by releasing esters by the action of their own esterases. The impact of O. oeni esterases remains relatively unexplored. Four esterase genes were identified from O. oeni genome (estA2, estA7, estC, and estB). The dual objective of this study was, first to use a genetic tool enabling the expression of esterase genes in enological conditions and, second, to investigate the impact of O. oeni esterase gene expression during winemaking on wine aromatic profile. Both estA2 and estA7 genes were successfully cloned and expressed in O. oeni and recombinant strains were inoculated in Aligoté wine to initiate malolactic fermentation (MLF). Ester profile of experimental wine was established by SPME-GC-MS. EstA2 caused significant decreases in the concentrations of isoamyl acetate, ethyl hexanoate, isobutyl acetate, and hexyl acetate, by 42.7%, 23.4%, 51.5%, and 28.9%, respectively. EstA2 has preferential hydrolytic activity toward acetate esters from higher alcohols. EstA7 has synthetic activity toward hexyl acetate with a significant 22.7% increase. This study reports the first efficient expression system enabling the production of a functional protein in O. oeni in enological conditions.

Selection and characterization of Oenococcus oeni strains for use as new malolactic fermentation starter cultures

Oenococcus oeni (O. oeni) strains are critically important for winemaking. They bring microbial stabilization, de-acidification and sensory improvement through malolactic fermentation (MLF) to almost all red wines and some white wines. In this study, 23 strains of O. oeni isolated from China were evaluated for use as MLF starter cultures. Strains were characterized and screened in wine-like media with increasing selective pressure, such as low pH and high ethanol concentration. Their abilities to conduct MLF were affected significantly by ethanol concentration and pH. Eight strains were selected and subsequently evaluated by detecting the enzyme-encoding genes involved in biogenic amines synthesis and wine aroma modification. Seven well-performing strains were further assessed in newly alcohol-fermented Cabernet Sauvignon wine. The results show that strains O. oeni CS-4b and O. oeni ME-5b exhibited better performance than the commercial starter O. oeni 31MBR in wine. Therefore, they were selected as potential MLF starters suitable for wine with high ethanol concentration and low pH.

Assessment of β-d-glucosidase activity of intact cells of two Oenococcus oeni strains with synthetic and natural substrates

The β-d-glucosidase (βG) activity of intact cells of Oenococcus oeni SD-2a and 31MBR was assessed with synthetic and natural substrates, also its partial characterisation was investigated. Results showed that intact cells of both strains showed βG activity, SD-2a performing higher than 31MBR, against either synthetic or natural substrate. The high βG activity of SD-2a intact cells under wine-like temperature, pH, ethanol concentration, and glucose concentration could be of great interest for its utilisation in winemaking. SD-2a proved promising for aroma enrichment in winemaking.

Growth and consumption of L-malic acid in wine-like medium by acclimated and non-acclimated cultures of Patagonian Oenococcus oeni strains.

Five Oenococcus oeni strains, selected from spontaneous malolactic fermentation (MLF) of Patagonic Pinot noir wine, were assessed for their use as MLF starter cultures. After the individual evaluation of tolerance to some stress conditions, usually found in wine (pH, ethanol, SO2, and lysozyme), the behavior of the strains was analyzed in MLO broth with 14% ethanol and pH3.5 in order to test for the synergistic effect of high ethanol level and low pH and, finally, in a wine-like medium. Although the five strains were able to grow in MLO broth under low pH and/or high ethanol, they must be acclimated to grow in a wine-like medium. Additionally, glycosidase and tannase activities were evaluated, showing differences among the strains. The potential of the strains to ferment citrate was tested and two of the five strains showed the ability to metabolize this substrate. We did not detect the presence of genes encoding histidine, tyrosine descarboxylase, and putrescine carbamoyltransferase. All the strains tested exhibited good growth capacity and ability to consume L-malic acid in a wine-like medium after cell acclimation, and each of them showed a particular enzyme profile, which might confer different organoleptic properties to the wine.

Mixed cultures of Oenococcus oeni strains: A mathematical model to test interaction on malolactic fermentation in winemaking

The control of malolactic fermentation (MLF) by Oenococcus oeni is an essential step in winemaking process. Although selected O. œni strains are available for winemakers, the MLF is not always successful, sometimes because of indigenous presence of other strains. In this work, interaction during MLF between five strains of O. œni was studied. Experiments were performed in MRS medium modified to be closer to wine conditions (pH 3.5, 10% ethanol). Interaction in mixed cultures of 10 pairs formed by the five strains were analysed from experiments performed in a Membrane Bioreactor (MBR). Pairs were classified in three different classes among growth interaction: 1) negative reciprocal interaction of both strains (6 pairs), 2) interaction that affect negatively one strain and positively the other (3 pairs), and 3) interaction with positive effect on one strain and no effect on the other (1 pair). Thanks to a mathematical model previously established during pure cultures to link growth and malic acid consumption, effect of mixed cultures on the specific activity of cells to consume malic acid has been equally evaluated. This capacity seemed not to be affected for 4 pairs whereas it seemed activated for 6 pairs.

Identification and Localization of β-D-Glucosidase from Two Typical Oenococcus oeni Strains.

β-D-glucosidase (βG) gene from Oenococcus oeni SD-2a and 31MBR was cloned, sequenced and analyzed, also intracellular βG of the two strains was further localized. The results showed that βG gene of the two strains was in high homology (> 99%) to reported βG gene, con-firming both strains possess βG activity at the molecular level. Intracellular βG of SD-2a is a mainly soluble protein, existing mostly in the cytoplasm and to some extent in the periplasm. While for 31MBR, intracellular βG is mainly insoluble protein existing in the cytoplasmic membrane. This study provides basic information for further study of the metabolic mechanism of βG from O. oeni SD-2a and 31MBR.

Application and validation of autochthonous Lactobacillus plantarum starter cultures for controlled malolactic fermentation and its influence on the aromatic profile of cherry wines

Lactic acid bacteria (LAB) used for malolactic fermentation (MLF) has a great effect on the production and quality of cherry wines. The present study used an autochthonous Lb. plantarum strain of SGJ-24 which was isolated from spontaneous MLF cherry wines and selected by its best MLF performance and tolerance, to investigate its effect on the kinetic of vinification and on chemical and volatile characteristics of Rainer and May Duck cherry wines, in comparison with a commercial Oenococcus oeni strain of 31 MBR. Monitoring of MLF was carried out by measuring cell viability and malic acid metabolism, and results showed that for both cherry varieties, SGJ-24 can significantly minimize MLF duration. After fermentation, wine samples were chemically characterized and analyzed for volatile profiles. Results demonstrated that no negative impact on the analytical parameters has been found, and a general increase of volatile esters and terpenes was observed when SGJ-24 was involved. Sensory analysis revealed that the global aromatic intensity was enhanced by the introduction of SGJ-24. All these data suggested that the application of Lb. plantarum strain of SGJ-24 as a worthwhile alternative LAB species for Rainer and May Duck cherry winemaking.

Simultaneous inoculation of yeasts and lactic acid bacteria: Effects on fermentation dynamics and chemical composition of Negroamaro wine

Traditional vinification process is undertaken with the inoculation of the lactic acid bacteria (LAB) at the end of alcoholic fermentation (AF) to induce malolactic fermentation (MLF). MLF is an important phase during winemaking and the LAB co-inoculation with yeast starter represents a promising approach to enhance the quality and safety of wine. In this investigation we have studied: i) the effect of timing of LAB inoculation on the vinification dynamics and chemical features of Negroamaro wines; ii) the interactions between two commercial yeast and two commercial Oenococcus oeni strains. The fermentations dynamics were monitored by microbial counts, quantifying l-malic acid concentration and analyzing the volatile compounds contents in the obtained wines. Our results indicate that simultaneous yeasts/bacteria inoculation at the beginning of vinification reduces the processes duration and simultaneously lowers of volatile acidity. Wine obtained after co-inoculum showed a profile dominated by red and ripe fruits notes associated to esters and to buttery and creamy notes linked to diethyl succinate and ethyl lactate. Furthermore, compatibility specification between commercial yeasts and LAB strains were observed, suggesting the importance of the assessment of microbial-compatibility before their utilization in large-scale vinification.

Genome Sequences of Three Oenococcus oeni Strains Isolated from Maipo Valley, Chile.

Oenococcus oeni is part of the microbial terroir involved in wine production. Here, we present three genome sequences of O. oeni strains isolated from spontaneous malolactic fermentation of cultivar Cabernet Sauvignon Maipo Valley, Chile.

Arginine deiminase pathway genes and arginine degradation variability in Oenococcus oeni strains.

Trace amounts of the carcinogenic ethyl carbamate can appear in wine as a result of a reaction between ethanol and citrulline, which is produced from arginine degradation by some bacteria used in winemaking. In this study, arginine deiminase (ADI) pathway genes were evaluated in 44 Oenococcus oeni strains from wines originating from several locations in order to establish the relationship between the ability of a strain to degrade arginine and the presence of related genes. To detect the presence of arc genes of the ADI pathway in O. oeni, pairs of primers were designed to amplify arcA, arcB, arcC and arcD1 sequences. All strains contained these four genes. The same primers were used to confirm the organization of these genes in an arcABCD1 operon. Nevertheless, considerable variability in the ability to degrade arginine among these O. oeni strains was observed. Therefore, despite the presence of the arc genes in all strains, the expression patterns of individual genes must be strain dependent and influenced by the different wine conditions. Additionally, the presence of arc genes was also determined in the 57 sequenced strains of O. oeni available in GenBank, and the complete operon was found in 83% of strains derived from wine. The other strains were found to lack the arcB, arcC and arcD genes, but all contained sequences homologous to arcA, and some of them had also ADI activity.

Assessment of the genetic polymorphism and physiological characterization of indigenous Oenococcus oeni strains isolated from Aglianico del Vulture red wine.

The aim of this study was a reliable intra-species discrimination and strain biodiversity in Oenococcus oeni populations of two different Aglianico wineries by molecular, biochemical, and physiological characterization. Pulsed field gel electrophoresis (PFGE) analysis revealed a high polymorphism related to the origin (winery) of strains, while differential display PCR (DD-PCR) allowed a further discrimination of strains from the same winery. Moreover, the heterogeneity of these natural populations was investigated by capillary electrophoresis and enzymatic assays. A variability related to a different surface charge distribution was observed among strains, linked to their origin. Malolactic activity study evidenced strain-specific differences in malic acid degradation, and then, only the presence of L(-)-malic acid in the medium induced the mle gene. This study provided evidences on the importance of intra-species biodiversity of malolactic bacterial populations in wine ecosystems, as each wine possess peculiar winemaking conditions and physical-chemical properties which make specific the bacterial survival and growth. This study highlighted a great biodiversity among O. oeni strains that can be also winery specific. Such biodiversity within a certain winery and winemaking area is important for selecting malolactic starters, and strain-specific trait identification is especially important to match individual strains to specific industrial process.

Together is better. Experience of simultaneous fermentation of yeast and bacteria as a possible strategy to prevent stuck fermentation in difficult wines

In the field of winemaking, malolactic fermentation is a key aspect in obtaining high quality wines. Unfortunately, in some oenological contexts effective evolution does not take place because of the occurrence of certain limiting factors for malolactic bacteria in wine. Simultaneous alcoholic and malolactic fermentation in grape must is a promising alternative that promotes the survival of bacteria, due to the absence of certain limiting factors such as ethanol or other toxic substances produced by yeasts in the native grape must. The risk of wine depreciation due to the spoilage activity of malolactic bacteria can be reduced by using selected strains of Oenococcus oeni, with proven behaviour in terms of malolactic fermentation occurring in the presence of sugars. In this work we compared the activity of a strain of Oenococcus oeni in malolactic fermentation of a Chardonnay grape must, using different winemaking protocols characterised by sequential or simultaneous inoculums of microbial starters. The results are discussed both in terms of fermentative behaviour and the quality of the wines obtained, with careful analysis of the main chemical parameters of the wines and of 47 different volatile compounds, giving an exhaustive overview of the opportunities and the risks related to different wine fermentation strategies.

Together is better. Experience of simultaneous fermentation of yeast and bacteria as a possible strategy to prevent stuck fermentation in difficult wines

In the field of winemaking, malolactic fermentation is a key aspect in obtaining high quality wines. Unfortunately, in some oenological contexts effective evolution does not take place because of the occurrence of certain limiting factors for malolactic bacteria in wine. Simultaneous alcoholic and malolactic fermentation in grape must is a promising alternative that promotes the survival of bacteria, due to the absence of certain limiting factors such as ethanol or other toxic substances produced by yeasts in the native grape must. The risk of wine depreciation due to the spoilage activity of malolactic bacteria can be reduced by using selected strains of Oenococcus oeni, with proven behaviour in terms of malolactic fermentation occurring in the presence of sugars. In this work we compared the activity of a strain of Oenococcus oeni in malolactic fermentation of a Chardonnay grape must, using different winemaking protocols characterised by sequential or simultaneous inoculums of microbial starters. The results are discussed both in terms of fermentative behaviour and the quality of the wines obtained, with careful analysis of the main chemical parameters of the wines and of 47 different volatile compounds, giving an exhaustive overview of the opportunities and the risks related to different wine fermentation strategies.