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BackgroundIn recent years, researchers are interested in the discovery of active compounds from traditional remedies and natural sources, as they reveal higher therapeutic efficacies and improved toxicological profiles. Among the various traditional treatments that have been widely studied and explored for their potential therapeutic benefits, kefir, a fermented beverage, demonstrates a broad spectrum of pharmacological properties, including antioxidant, anti-inflammation, and healing activities. These health-promoting properties of kefir vary among the kefir cultures found at the different part of the world as different media and culture conditions are used for kefir maintenance and fermentation.MethodsThis study investigated the microbial composition and readily found bioactive compounds in water kefir fermented in Malaysia using 16S rRNA microbiome and UHPLC sequencing approaches. The toxicity effects of the kefir water administration in BALB/c mice were analysed based on the mice survival, body weight index, biochemistry profile, and histopathological changes. The antioxidant activities were evaluated using SOD, FRAP, and NO assays.ResultsThe 16S rRNA amplicon sequencing revealed the most abundant species found in the water kefir wasLactobacillus hilgardiifollowed byLactobacillus harbinensis,Acetobacter lovaniensis,Lactobacillus satsumensis,Acetobacter tropicalis,Lactobacillus zeae, andOenococcus oeni. The UHPLC screening showed flavonoid and phenolic acid derivatives as the most important bioactive compounds present in kefir water which has been responsible for its antioxidant activities. Subchronic toxicity study showed no toxicological signs, behavioural changes, or adverse effects by administrating 10 mL/kg/day and 2.5 mL/kg/day kefir water to the mice. Antioxidants assays demonstrated enhanced SOD and FRAP activities and reduced NO level, especially in the brain and kidney samples.ConclusionsThis study will help to intensify the knowledge on the water kefir microbial composition, available phytochemicals and its toxicological and antioxidant effects on BALB/c mice since there are very limited studies on the water kefir grain fermented in Malaysia.

Flow cytometry is a high-throughput tool for determining microbial abundance in a range of medical, environmental, and food-related samples. For wine, determining the abundance of Saccharomyces cerevisiae is well-defined and reliable. However, for the most common wine bacterium, Oenococcus oeni, using flow cytometry to determine cell concentration poses some challenges. O. oeni most often occurs in doublets or chains of varying lengths that can be greater than seven cells. This wine bacterium is also small, at 0.2-0.6 μm and may exhibit a range of morphologies including binary fission and aggregated complexes. This work demonstrates a straightforward approach to determining the suitability of flow cytometry for the chain-forming bacteria, O. oeni, and considerations when using flow cytometry for the enumeration of small microorganisms (

Wines produced from grapes cultivated in cool climate areas are characterized by high levels of organic acids. One method to correct this is malolactic fermentation (MLF). The aim of this study was to determine the effectiveness of different strains of Oenococcus oeni bacteria (Viniflora CH11, Viniflora CH16, Viniflora CH35, Viniflora Oenos, SIHA LACT Oeno) during the biological acidity reduction process. Red wine from Rondo and Regent cultivars was obtained by ethanol fermentation of the pulp, at 20 °C for 14 days. The profile of organic acids was examined with a particular focus on changes in the content of l-malic and l-lactic acids. Additionally, the impact on profile and quantity of phenolic compounds and antioxidant capacity was measured. The results showed that MLF had a positive influence on content of organic acids through the reduction of l-malic acid content with a simultaneous increase of the amount of l-lactic acid. The best effect was obtained with the CH11 and CH35 bacterial strains. The biological acidity reduction process had no significant (P > 0.05) impact on phenolic content or antioxidant capacity. However, the wine making process (ethanol fermentation, maturation) contributed to the reduction of polyphenols and in consequence lower antioxidant capacity of the final tested wines. PRACTICAL APPLICATION: The present study provides useful information on the impact of different Oenococcus oeni bacterial strains on MLF in red wines, reduction of l-malic to l-lactic acid, and stability of phenolic compounds during MLF and the maturation period. Also, this article provides information about phenolic compounds and antioxidant capacity during malolactic fermentation and maturity of red wines made from hybrids of Vitis vinifera such as Rondo and Regent cultivars.

Trabajo presentado en el 3rd Iberoamerican Congress on Biotechnology, celebrado en Braga (Portugal), del 7 al 9 de abril de 2022, Malolactic fermentation is essential for the quality of red wines and some other wine styles. This process consists in the decarboxylation of L-malic acid to L-lactic acid, increasing the pH of wine and releasing compounds with sensory impact [1]. Spontaneous malolactic fermentation is often driven by Oenococcus oeni, and commercial starters for this purpose are also often of this species. The microbial interactions during wine fermentation are a growing field of interest due to the new inoculation strategies in winemaking processes. Among other interaction mechanisms, extracellular vesicles (EVs) might play a relevant role on it. Determination of the protein cargo of the EVs is a first approach to understand how biological processes can be affected in by the EVs. In this work, the production of extracellular vesicles by O. oeni is reported for the first time. The protein content of O. oeni EVs was characterised by proteomic analysis. It shares some characteristics with the recently described protein content of Lactiplantibacillus plantarum EVs [3], a bacterial species also capable of performing malolactic fermentation of wine. EVs produced by O. oeni are similar in size to other bacterial EVs described previously, and the proteomic analysis shows an enrichment in ribosomal and integral membrane proteins, which are in accordance with other bacterial and eukaryotic EV cargo. Considering ubiquity of O. oeni in winemaking, this work will help in future studies on microbial interactions relevant for this industry. [1] Genomic variations of Oenococcus oeni strains and the potential to impact on malolactic fermentation and aroma compounds in wine, Bartowsky, E.J., Borneman, A.R., Appl. Microbiol. Biotechnol., 92., 441¿447 (2011). [2] Extracellular vesicles in food biotechnology, Morales, P., Mencher, A., Tronchoni, J., Gonzalez, R., Microb. Biotechnol., 14., 8¿11 (2021). [3] Proteomic profile of extracellular vesicles released by Lactiplantibacillus plantarum BGAN8 and their internalization by non-polarized HT29 cell line, Bajic, S.S., Cañas, M.-A., Tolinacki, M., Badia, J., Sánchez, B., Golic, N., Margolles, A., Baldomá, L., Ruas-Madiedo, P., Sci. Rep., 10., 21829 (2020)., This work was funded by the Spanish Government through grants PID2019-105159RB-I00 funded by MCIN/AEI/10.13039/501100011033 and BES-2016-077557 and PRE2020-093420 funded by MCIN/AEI/10.13039/501100011033 and by ¿ESF Investing in your future¿ (training contracts for AM and MM). JT was funded by FGCSIC by the COMFUTURO program.

Oenococcus oeni and Lactiplantibacillus plantarum are major wine-associated lactic acid bacteria that positively influence wine by carrying out malolactic fermentation. O. oeni is the most widely used commercial starter in winemaking because of its fast and efficient malate metabolism capacity under harsh wine conditions. To date, very little is known about the specific molecular mechanism underlying the differences in malate metabolism between O. oeni and L. plantarum under harsh wine conditions. Therefore, in this study, the functions of genes encoding malic enzyme (ME) and malolactic enzyme (MLE) under acid stress in O. oeni and L. plantarum, previously described to have the ability to direct malate metabolism, were comparatively verified through genetic manipulation in L. plantarum. Results showed that the MLE was the only enzyme responsible for direct malate metabolism under acid stress in O. oeni and L. plantarum. In addition, the MLEs in O. oeni and L. plantarum were positively related to acid tolerance by metabolizing malate and increasing the medium pH. Furthermore, the MLE in O. oeni exhibited significantly higher malate metabolism activity than that in L. plantarum under acid stress.

10 Páginas.-- 3 Tablas.-- 2 Figuras, Olives from the Sigoise, Verdale, and Sevillana cultivars were elaborated as Spanish-style table olives by four Algerian factories, and the quality and food safety of the industrial table olives have been studied by the analysis of physicochemical and microbiological parameters. Differences were observed between the treatments carried out by the different factories throughout the manufacturing process, especially during the washing stage, but no significant differences were found between the analyzed samples for the concentration of sugars and polyphenols. The final pH values reached at the end of fermentation ranged between 5.04 and 4.27, and the titratable acidity was above 0.4% for all samples. Lactic and acetic acids were produced in mean concentrations of 0.68% and 0.21% as a result of lactic acid bacteria (LAB) and yeast metabolism, respectively. However, the presence of butyric, isobutyric, and propionic acids was also detected, and was related to the growth of undesirable spoilage microorganisms, responsible for secondary fermentations. The high-throughput sequencing of bacterial DNA suggested the dominance of LAB species belonging to genera Lactiplantibacillus, Leuconostoc, Pediococcus, Oenococcus, or Enterococcus. The Enterobacteriaceae family was detected during the first days of brining and in only one sample after 120 days of fermentation. Other spoilage microorganisms were found, such as Lentilactobacillus buchneri or the Pectinatus and Acetobacter genera, capable of consuming lactic acid and these played an essential role in the onset of spoilage. The Clostridium and Enterobacter genera, producers of butyric and propionic acids, were responsible for the malodorous fermentation present in the industrial samples that were analyzed. The study concluded that the safety of the table olives analyzed was compromised by the presence of undesirable microorganisms and microbial stability was not guaranteed. The elaboration process can be improved by reducing the washing steps and the time should be reduced to avoid the loss of fermentable matter, with the goal of reaching a pH < 4.0 after the fermentation and preventing the possibility of the growth of spoilage microorganisms and foodborne pathogens., This research was supported by the Algerian Ministry of Higher Education and Scientific Research (PNE program 2019-2020), and the grant RYC2018-024752-I funded by MCIN/AEI/10.13039/501100011033 and FSE “El FSE invierte en tu futuro”.

Oenococcus oeni is the most resistant lactic acid bacteria species to the environmental stresses encountered in wine, particularly the acidity, presence of ethanol and phenolic compounds. Indigenous strains develop spontaneously following the yeast-driven alcoholic fermentation and may perform the malolactic fermentation whereby improving taste, aroma, and the microbial stability of wine. However, spontaneous fermentation is sometimes delayed, prolonged or incomplete. In order to better control its timing and quality, O. oeni strains are selected and developed to be used as malolactic starters. They are prepared under proprietary manufacturing processes to survive direct inoculation and are predominantly provided as freeze-dried preparations. In this study, we have investigated the physiological and molecular alterations occurring in O. oeni cells prepared by an industrial process that consists of preconditioning protocols and freeze-drying, and compared them to the same strain grown in a grape juice medium. We found that compared to cultured cells, the industrial production process improved survival under extreme conditions, i. e. at low pH or high tannin concentrations. In contrast, cultured cells resumed active growth more quickly and strongly than freeze-dried preparations in standard pH wines. A proteomic analysis showed that during the industrial production most non-essential metabolic processes are shut down and components of the general and the stringent stress response are upregulated. The presence of major components of the stress response facilitates protein homeostasis and physiological changes that further ensure the integrity of cells.

To successfully complete malolactic fermentation (MLF), Oenococcus oeni must overcome wine stress conditions of low pH, high ethanol, and the presence of SO2. Failure to complete MLF may result in detrimental effects to the quality and stability of the resulting wines. Research efforts to date have focused on elucidating the mechanisms and genetic features that confer the ability to withstand low pH and high ethanol concentrations on O. oeni; however, the responses to SO2 stress are less well defined. This study focused on characterizing the transcriptional response of O. oeni to SO2 challenge during cultivation in a continuous system at wine-like pH (3.5). This experimental design allowed the precise discrimination of transcriptional changes linked to SO2 stress from responses associated with growth stage and cultivation parameters. Differential gene expression analysis revealed major transcriptional changes following SO2 exposure and suggested that this compound primarily interacts with intracellular proteins, DNA, and the cell envelope of O. oeni. The molecular chaperone hsp20, which has a demonstrated function in the heat, ethanol, and acid stress response, was highly upregulated, confirming its additional role in the response of this species to SO2 stress. This work also reports the first nanopore-based complete genome assemblies for O. oeni. IMPORTANCE Malolactic fermentation is an indispensable step in the elaboration of most wines and is generally performed by Oenococcus oeni, a Gram-positive heterofermentative lactic acid bacterium species. While O. oeni is tolerant to many of the wine stresses, including low pH and high ethanol concentrations, it has high sensitivity to SO2, an antiseptic and antioxidant compound regularly used in winemaking. Understanding the physiological changes induced in O. oeni by SO2 stress is essential for the development of more robust starter cultures and methods for their use. This study describes the main transcriptional changes induced by SO2 stress in the wine bacterium O. oeni and provides foundational understanding on how this compound interacts with the cellular components and the induced protective mechanisms of this species.

There has been a growing interest in developing co-inoculum of Oenococcus oeni and Saccharomyces cerevisiae/non-Saccharomyces for simultaneous malolactic fermentation (MLF) and alcoholic fermentation (AF) of wines. This study sought to elucidate the effects of two crucial factors (inoculation timing and paired yeast) on the fermentation performance and aroma production of Merlot wine. O. oeni used for MLF was concurrently or sequentially inoculated with two yeast cultures (i.e., single S. cerevisiae and mixed S. cerevisiae /Pichia fermentans H5Y-28) used for AF. Inoculation timing determined the overall vinification duration, and conditioned the production of principle higher alcohols, terpene and O. oeni-mediated volatiles. In contrast, paired yeast improved O. oeni viability, and showed significant effect on aromatic esters and volatile fatty acids. Possibly due to lower ethanol stress, co-inoculum allowed O. oeni to initiate MLF during AF, resulting in 45% reduction of total fermentation time. Meanwhile, O. oeni growth was stimulated by P. fermentans, with 1.7-fold of the maximum population higher than that in co-fermentation without P. fermentans. Such stimulation of O. oeni growth also occurred in sequential fermentation where P. fermentans had been replaced by S. cerevisiae. Only in sequential inoculum, P. fermentans induced high levels of 3-methylbutyl acetate, ethyl 3-methylbutanoate, ethyl hexanoate and ethyl octanoate, which may result in enhanced fresh fruity trait of wines. These findings suggested a positive effect of P. fermentans H5Y-28 on O. oeni and MLF. This work provides an alternative approach to improve wine MLF and aroma outcomes using friendly non-Saccharomyces yeast with appropriate inoculation strategy.

Fumaric acid is an additive allowed by the Codex Alimentarius and under evaluation by the International Organisation of Vine and Wine (OIV) that can be used for wine acidification but also to inhibit malolactic fermentation (MLF). The use of 300–900 mg/L of fumaric acid can inhibit MLF in red wines decreasing pH by 0.2 units or more depending on the buffer capacity. When MLF was running with populations of either 7 or 8 log CFU/mL strain alpha (Oenococcus oeni) the application of 600 mg/L of fumaric acid stopped the process for more than 50 days and cells were undetected in specific media. In triangular tastings, fumaric acid was not detected at 300–600 mg/L (p < .05). In subsequent preference tests, some tasters perceived more acidity and body. Fumaric acid is a useful technological additive to improve wine microbiological stability and freshness, also allowing reduction of SO2 levels. Trujillo San Isidro

Lactobacillus buchneri and Oenococcus oeni are two unique ethanol-tolerant Gram-positive bacteria species. Genome comparison analyses revealed that L. buchneri and O. oeni possess a pntAB locus that was absent in almost all other lactic acid bacteria (LAB) genomes. Our hypothesis is that the pntAB locus contributes to the ethanol tolerance trait of these two distinct ethanol-tolerant organisms. The pntAB locus, consisting of the pntA and pntB genes, codes for NADP(H) transhydrogenase subunits. This membrane-bound transhydrogenase catalyzes the reduction of NADP+ and is known as an important enzyme in maintaining cellular redox balance. In this study, the transhydrogenase operon from L. buchneri NRRL B-30929 and O. oeni PSU-1 were cloned and analyzed. The LbpntB shared 71.0% identity with the O. oeni (OopntB). The entire pntAB locus was expressed in Lactococcus lactis ssp . lactis IL1403 resulting in an increased tolerance to ethanol (6%), butanol (1.8%) and isopropanol (1.8%) when compared to the control strain. However, the recombinant E. coli cells carrying the entire pntAB locus did not show any improved ethanol tolerance. Independent expression of OopntB and LbpntB in recombinant E. coli BL21(DE3)pLysS host demonstrated higher tolerance to ethanol when compared with a control E. coli BL21(DE3)pLysS strain carrying pET28b vector. Ethanol tolerance comparison of E. coli strains carrying LbpntB and OopntB showed that LbpntB conferred higher ethanol tolerance (4.5%) and resulted in greater biomass, while the OopntB conferred lower ethanol tolerance (4.0%) resulted lower biomass. Therefore, the pntB gene from L. buchneri is a better choice in generating higher ethanol tolerance. This is the first study to uncover the role of pntAB locus on ethanol tolerance.

Two Gram-stain-positive, small ellipsoidal cocci, non-motile, oxidase- and catalase-negative, and facultative anaerobic strains (UCMA15228T and UCMA17102) were isolated in France, from fermented apple juices (ciders). The 16S rRNA gene sequence was identical between the two isolates and showed 97 % similarity with respect to the closest related species Oenococcus oeni and O. kitaharae. Therefore, the two isolates were classified within the genus Oenococcus. The phylogeny based on the pheS gene sequences also confirmed the position of the new taxon. DNA–DNA hybridizations based on in silico genome-to-genome comparisons (GGDC) and Average Nucleotide Identity (ANI) values, as well as species-specific PCR, validated the novelty of the taxon. Various phenotypic characteristics such as the optimum temperature and pH for growth, the ability to metabolise sugars, the aptitude to perform the malolactic fermentation, and the resistance to ethanol and NaCl, revealed that the two strains are distinguishable from the other members of the Oenococcus genus. The combined genotypic and phenotypic data support the classification of strains UCMA15228T and UCMA17102 into a novel species of Oenococcus, for which the name O. sicerae sp. nov. is proposed. The type strain is UCMA15228T (=DSM107163T = CIRM-BIA2288T).

Based on SPME‐GC‐MS analysis, it could be found that the production of acetic acid, phenethyl acetate, and isoamyl acetate gradually increased in the apple vinegar fermentation broth with the fermentation time. Consequently, in order to systematically explore the dynamic changes of microbial diversity and metabolites in the process of apple vinegar fermentation, 16S rDNA were sequenced and analyzed in this work. The present results showed that bacterial diversity was rich and exhibited a certain variation during the dynamic fermentation process of apple vinegar. Furthermore, Lactococcus and Oenococcus were the predominant bacteria in the pre‐fermentation (alcoholic fermentation) of apple vinegar, while the dominant bacteria in the middle and late fermentation stages (acetic acid fermentation) were Lactococcus and Acetobacter. In addition, during the whole fermentation process of apple vinegar, Lactococcus was the most dominant bacteria, Oenococcus was the unique species in the stage of alcohol fermentation, and Acetobacter increased rapidly in the stage of acetic acid fermentation. In conclusion, our finding provided a theoretical basis for the processing technology of apple vinegar fermentation, and a theory evidence for the safety and health assessment of apple vinegar.

This review focuses on the considerable amount of research that has been directed towards the improvement of efficiency and reliability of malolactic fermentation (MLF), which is important in winemaking. From this large body of work, it is clear that reliable MLF is essential for process efficiency and prevention of spoilage in the final product. Impediments to successful MLF in wine, the impact of grape and wine ecology and how this may affect MLF outcome are discussed. Further focus is given to how MLF success may be enhanced, via alternative inoculation strategies, MLF progress sensing technologies and the use of different bacterial species. An update of how this information may be used to enhance and improve sensory outcomes through metabolite production during MLF and suggestions for future research priorities for the field are also provided.

Climate change is generating several problems in wine technology. One of the main ones is lack of acidity and difficulties performing malolactic fermentation to stabilize wines before bottling. Among the different available acidity management technologies, such as direct acid addition, ion exchange resins, electro-membrane treatments, or vineyard management, the microbiological option is reliable and deeply studied. The main approach is the increase in malic acid content because of the metabolism of specific Saccharomyces strains and to increase lactic acid because of the metabolism of Lachancea genus. Other non-Saccharomyces yeasts, such as Starmerella bacillaris or Candida stellata can also acidify significantly because of the production of pyruvic or succinic acid. Wine industry needs the removal of malic acid in most red wines before bottling to achieve wine stability. Oenococus oeni performs the malolactic fermentation of red wines on most conditions because of the metabolization of malic acid into lactic acid. However, modern oenology challenges such as high ethanol concentrations, high pH or low levels of malic acid have made researchers to look for other options to reduce potential risks of deviation. Other wine-related microorganisms able to de-acidify malic acid have appeared as interesting alternatives for specific difficult scenarios. Lactiplantibacillus plantarum and Schizosaccharomyces genus make up nowadays the main studied alternatives.

The modification of adenosine to inosine at the first position of transfer RNA (tRNA) anticodons (I34) is widespread among bacteria and eukaryotes. In bacteria, the modification is found in tRNAArg and is catalyzed by tRNA adenosine deaminase A, a homodimeric enzyme. In eukaryotes, I34 is introduced in up to eight different tRNAs by the heterodimeric adenosine deaminase acting on tRNA. This substrate expansion significantly influenced the evolution of eukaryotic genomes in terms of codon usage and tRNA gene composition. However, the selective advantages driving this process remain unclear. Here, we have studied the evolution of I34, tRNA adenosine deaminase A, adenosine deaminase acting on tRNA, and their relevant codons in a large set of bacterial and eukaryotic species. We show that a functional expansion of I34 to tRNAs other than tRNAArg also occurred within bacteria, in a process likely initiated by the emergence of unmodified A34-containing tRNAs. In eukaryotes, we report on a large variability in the use of I34 in protists, in contrast to a more uniform presence in fungi, plans, and animals. Our data support that the eukaryotic expansion of I34-tRNAs was driven by the improvement brought by these tRNAs to the synthesis of proteins highly enriched in certain amino acids., This work was supported by the Spanish Ministry of Economy and Competitiveness (grant numbers BES2013-064551 to [A.R.-Y.], and BIO2015-64572 to [L.R.d.P.]).

The widespread existence of bacteriophage has been of great interest to the biological research community and ongoing investigations continue to explore their diversity and role. They have also attracted attention and in-depth research in connection to fermented food processing, in particular from the dairy and wine industries. Bacteriophage, mostly oenophage, may in fact be a 'double edged sword' for winemakers: whilst they have been implicated as a causal agent of difficulties with malolactic fermentation (although not proven), they are also beginning to be considered as alternatives to using sulphur dioxide to prevent wine spoilage. Investigation and characterisation of oenophage of Oenococcus oeni, the main species used in winemaking, are still limited compared to lactococcal bacteriophage of Lactococcus lactis and Lactiplantibacillus plantarum (formally Lactobacillus plantarum), the drivers of most fermented dairy products. Interestingly, these strains are also being used or considered for use in winemaking. In this review, the genetic diversity and life cycle of phage, together with the debate on the consequent impact of phage predation in wine, and potential control strategies are discussed. KEY POINTS: • Bacteriophage detected in wine are diverse. • Many lysogenic bacteriophage are found in wine bacteria. • Phage impact on winemaking can depend on the stage of the winemaking process. • Bacteriophage as potential antimicrobial agents against spoilage organisms.

This study aimed to investigate the behavior of Oenococcus oeni MS9 and MS46 strains in sterile grape juice (SGJ, pH 4.0) incubated at 30 °C, in terms of growth and glucose, organic acids and total phenolic compounds utilization. In addition, their antimicrobial activity and the changes in antioxidant properties of fermented juice with selected strain were evaluated. Both strains grew without lag period by ~1.40 log CFU/mL at 12 days with maximum growth rates of about 0.019 h−1. After this time the MS9 and MS46 strains counts declined by 0.6 log units and remained unchanged respectively. O. oeni MS46 was evaluated in SGJ for low inoculum size (~104 CFU/mL). In this condition it also grew without lag period by 3.11 ± 0.01 log CFU/mL with a μmax of 0.05 h−1. Glucose and L-malic and citric acids were simultaneously utilized but at different rates and extents, yielding mainly lactic acid with concomitant pH reduction. Acetic acid ranged between 11 and 19 mmol/L. Total phenolic compounds significantly decreased in fermented SGJ with strain MS9 but not MS46. In this last condition, the antioxidant activity increased by 21%. In addition, both O. oeni strains showed antibacterial properties against Escherichia coli 700, Salmonella Typhimurium and Listeria monocytogenes. O. oeni strains, especially MS46, with the ability to growth in SGJ, high malolactic potential and adequate sugars and organic acids profiles from the sensorial viewpoint may be used to ferment grape juice with safer and healthier properties than fresh juice.

Background Acid stress is one of the most important environmental stresses that adversely affect the growth of lactic acid bacteria (LAB), such as Oenococcus oeni which was isolated from grape-berries and mainly used in wine fermentation. The aim of this paper is to comprehensively characterize the mechanisms of acid stress regulation in O. oeni and to provide a viable theoretical basis for breed and improvement of existing LAB. Method First, six O. oeni mutants with acid-sensitive (strains b2, a1, c2) and acid-tolerant (strains b1, a3, c1) phenotypes were screened from three wild-type O. oeni, and then their genome (sequencing), transcriptome and metabolome (LC-MS/MS) were examined. Results A total of 459 genes were identified with one or more intragenic single nucleotide polymorphisms (SNPs) in these mutants, and were extensively involved in metabolism and cellular functions with a high mutation rates in purine (46%) and pyrimidine (48%) metabolic pathways. There were 210 mutated genes that cause significant changes in expression levels. In addition, 446 differentially accumulated metabolites were detected, and they were consistently detected at relatively high levels in the acid-tolerant O. oeni mutant. The levels of intracellular differentially expressed genes and differential metabolites changed with increasing culture time. Conclusion The integrative pathways analysis showed that the intracellular response associated with acid regulation differed significantly between acid-sensitive and acid-tolerant O. oeni mutants, and also changed at different growth stages.

The two most commonly used wine microorganisms, Saccharomyces cerevisiae yeast and Oenococcus oeni bacteria, are responsible for completion of alcoholic and malolactic fermentation (MLF), respectively. For successful co-inoculation, S. cerevisiae and O. oeni must be able to complete fermentation; however, this relies on compatibility between yeast and bacterial strains. For the first time, quantitative trait loci (QTL) analysis was used to elucidate whether S. cerevisiae genetic makeup can play a role in the ability of O. oeni to complete MLF. Assessment of 67 progeny from a hybrid S. cerevisiae strain (SBxGN), co-inoculated with a single O. oeni strain, SB3, revealed a major QTL linked to MLF completion by O. oeni. This QTL encompassed a well-known translocation, XV-t-XVI, that results in increased SSU1 expression and is functionally linked with numerous phenotypes including lag phase duration and sulphite export and production. A reciprocal hemizygosity assay was performed to elucidate the effect of the gene SSU1 in the SBxGN background. Our results revealed a strong effect of SSU1 haploinsufficiency on O. oeni's ability to complete malolactic fermentation during co-inoculation and pave the way for the implementation of QTL mapping projects for deciphering the genetic bases of microbial interactions. KEY POINTS: • For the first time, QTL analysis has been used to study yeast-bacteria interactions. • A QTL encompassing a translocation, XV-t-XVI, was linked to MLF outcomes. • S. cerevisiae SSU1 haploinsufficiency positively impacted MLF by O. oeni.

The use of non-Saccharomyces yeast together with S. cerevisiae in winemaking is a current trend. Apart from the organoleptic modulation of the wine, the composition of the resulting yeast lees is different and may thus impact malolactic fermentation (MLF). Yeasts of Saccharomyces cerevisiae, Torulaspora delbrueckii and Metschnikowia pulcherrima were inactivated and added to a synthetic wine. Three different strains of Oenococcus oeni were inoculated and MLF was monitored. Non-Saccharomyces lees, especially from some strains of T. delbrueckii, showed higher compatibility with some O. oeni strains, with a shorter MLF and a maintained bacterial cell viability. The supplementation of lees increased nitrogen compounds available by O. oeni. A lower mannoprotein consumption was related with longer MLF. Amino acid assimilation by O. oeni was strain specific. There may be many other compounds regulating these yeast lees-O. oeni interactions apart from the well-known mannoproteins and amino acids. This is the first study of MLF with different O. oeni strains in the presence of S. cerevisiae and non-Saccharomyces yeast lees to report a strain-specific interaction between them.

After alcoholic fermentation, most wines undergo malolactic fermentation (MLF), driven by the lactic acid bacterium Oenococcus oeni, which improves their organoleptic properties and microbiological stability. Prophages were recently shown to be notably diverse and widely disseminated in O. oeni genomes. Such in silico predictions confirmed previous cultivation-based approaches which showed frequent lysis of strains upon treatment with the inducing agent mitomycin C. Both strategies used to assess lysogeny in the species were so far applied to a number of strains collected from distinct countries, wineries, cepages and fermentation processes. Results may not therefore be representative of the lysogenic population in natural communities driving the MLF during winemaking. Here we report the prevalence of lysogeny during winemaking in three wineries in the Bordeaux area. The dominant LAB population was collected in 11 red wines upon completion of MLF. Using VNTR and prophage typing analyses, our data confirm the presence of lysogens in the population driving the spontaneous MLF in all tested wines, although lysogeny rates varied across wineries. Higher prevalence of lysogeny was associated to a reduced diversity in VNTR profiles, the dominance of a few prophage-types and presence of some bacterial genetic backgrounds that were particularly prone to lysogenization.