Succinic acid production by wine yeasts and the influence of GABA and glutamic acid.

As a consequence of alcoholic fermentation (AF) in wine, several compounds are released by yeasts, and some of them are linked to the general quality and mouthfeel perceptions in wine. However, others, such as succinic acid, act as inhibitors, mainly of malolactic fermentation. Succinic acid is produced by non-Saccharomyces and Saccharomyces yeasts during the initial stages of AF, and the presence of some amino acids such as γ-aminobutyric acid (GABA) and glutamic acid can increase the concentration of succinic acid. However, the influence of these amino acids on succinic acid production has been studied very little to date. In this work, we studied the production of succinic acid by different strains of non-Saccharomyces and Saccharomyces yeasts during AF in synthetic must, and the influence of the addition of GABA or glutamic acid or a combination of both. The results showed that succinic acid can be produced by non-Saccharomyces yeasts with values in the range of 0.2-0.4 g/L. Moreover, the addition of GABA or glutamic acid can increase the concentration of succinic acid produced by some strains to almost 100 mg/L more than the control, while other strains produce less. Consequently, higher succinic acid production by non-Saccharomyces yeast in coinoculated fermentations with S. cerevisiae strains could represent a risk of inhibiting Oenococcus oeni and therefore the MLF.

A mutant GH3 family ß-glucosidase from Oenococcus oeni exhibits superior adaptation to wine stresses and potential for improving wine aroma and phenolic profiles.

In this study, we conducted a comprehensive investigation into a GH3 family ß-glucosidase (BGL) from the wild-type strain of Oenococcus oeni and its mutated counterpart from the acid-tolerant mutant strain. Our analysis revealed the mutant BGL's remarkable capacity to adapt to wine-related stress conditions, including heightened tolerance to low pH, elevated ethanol concentrations, and metal ions. Additionally, the mutant BGL exhibited superior hydrolytic activity towards various substrates. Through de novo modeling, we identified specific amino acid mutations responsible for its resilience to low pH and high ethanol environments. In simulated wine conditions, the mutant BGL outperformed both wild-type and commercial BGLs, efficiently releasing terpene and phenolic aglycones from glycosides in wine grapes. These findings not only expand our understanding of O. oeni BGLs but also highlight their potential in enhancing wine production. The mutant BGL's enhanced adaptation to wine stress conditions opens promising avenue for improving wine quality and flavor.

Screening of Saccharomyces cerevisiae and Torulaspora delbrueckii strains in relation to their effect on malolactic fermentation.

The use of Torulaspora delbrueckii in the alcoholic fermentation (AF) of grape must is increasingly studied and used in the wine industry. In addition to the organoleptic improvement of wines, the synergy of this yeast species with the lactic acid bacterium Oenococcus oeni is an interesting field of study. In this work, 60 strain combinations were compared: 3 strains of Saccharomyces cerevisiae (Sc) and 4 strains of Torulaspora delbrueckii (Td) in sequential AF, and four strains of O. oeni (Oo) in malolactic fermentation (MLF). The objective was to describe the positive or negative relationships of these strains with the aim of finding the combination that ensures better MLF performance. In addition, a new synthetic grape must has been developed that allows the success of AF and subsequent MLF. Under these conditions, the Sc-K1 strain would be unsuitable for carrying out MLF unless there is prior inoculation with Td-Prelude, Td-Viniferm or Td-Zymaflore always with the Oo-VP41 combination. However, from all the trials performed, it appears that the combinations of sequential AF with Td-Prelude and Sc-QA23 or Sc-CLOS, followed by MLF with Oo-VP41, reflected a positive effect of T. delbrueckii compared to inoculation of Sc alone, such as a reduction in L-malic consumption time. In conclusion, the obtained results highlight the relevance of strain selection and yeast-LAB strain compatibility in wine fermentations. The study also reveals the positive effect on MLF of some T. delbrueckii strains.

Malolactic fermentation of lactic acid bacteria isolated from southern Brazilian red wine.

The objective was to isolate lactic acid bacteria (LAB) from southern Brazil's wines and investigate their potential as starter cultures for malolactic fermentation (MLF) in Merlot (ME) and Cabernet Sauvignon (CS) wines through the fermentative capacity. The LAB were isolated from CS, ME, and Pinot Noir (PN) wines in the 2016 and 2017 harvests and evaluated for morphological (color and shape of the colonies), genetic, fermentative (increase in pH, acidity reduction, preservation of anthocyanins, decarboxylation of L-malic acid, yield of L-lactic acid, and content of reduced sugars), and sensory characteristics. Four strains were identified as Oenococcus oeni [CS(16)3B1, ME(16)1A1, ME(17)26, and PN(17)65], one as Lactiplantibacillus plantarum [PN(17)75], and one as Paucilactobacillus suebicus [CS(17)5]. Isolates were evaluated in the MLF and compared to a commercial strain (O. oeni), as well as a control (without inoculation and spontaneous MLF), and standard (without MLF). CS(16)3B1 and ME(17)26 isolates finished the MLF for CS and ME wines, respectively, after 35 days, similar to the commercial strain, and CS(17)5 and ME(16)1A1 isolates ended the MLF in 45 days. In the sensory analysis, ME wines with isolated strains received better scores for flavor and overall quality than the control. Compared to the commercial strain, CS(16)3B1 isolate obtained the highest scores for buttery flavor and taste persistence. CS(17)5 isolate received the higher scores for a fruity flavor and overall quality and the lowest for a buttery flavor. The native LAB displayed MLF potential, regardless of the year and grape species from which they were isolated.

Integrative multiomics analysis of the acid stress response of Oenococcus oeni mutants at different growth stages.

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.

Assessment of the lysogenic status in the lactic acid bacterium O. oeni during the spontaneous malolactic fermentation of red wines.

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.

Protein content of the Oenococcus oeni extracellular vesicles-enriched fraction.

Malolactic fermentation is essential for the quality of red wines and some other wine styles. Spontaneous malolactic fermentation is often driven by Oenococcus oeni, and commercial starters for this purpose are also often of this species. The increasing number of microbial species and inoculation strategies in winemaking has prompted a growing interest in microbial interactions during wine fermentation. Among other interaction mechanisms, extracellular vesicles have been hypothesized to play a role in this context. Extracellular vesicles have already been described and analysed for several wine yeast species. In this work, the production of extracellular vesicles by O. oeni is reported for the first time. The protein content of these extracellular vesicles is also characterised. It shows differences and similarities with the recently described protein content of Lactiplantibacillus plantarum, a bacterial species also capable of performing malolactic fermentation of wine (and used sometimes as an alternative starter). This work further contributes to the development of the field of extracellular vesicles in food biotechnology.

Utilization of Oenococcus oeni strains to ferment grape juice: Metabolic activities and beneficial health potential.

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.

Processivity of dextransucrases synthesizing very-high-molar-mass dextran is mediated by sugar-binding pockets in domain V.

The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (∼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10-13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis.

Evaluating the Cytometric Detection and Enumeration of the Wine Bacterium, Oenococcus oeni.

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 (<0.5 µm). © 2020 International Society for Advancement of Cytometry.

Exploring the diversity of bacteriophage specific to Oenococcus oeni and Lactobacillus spp and their role in wine production.

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.

QTL mapping: an innovative method for investigating the genetic determinism of yeast-bacteria interactions in wine.

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.

Simulated lees of different yeast species modify the performance of malolactic fermentation by Oenococcus oeni in wine-like medium.

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.

Combinatorial analysis of population dynamics, metabolite levels and malolactic fermentation in Saccharomyces cerevisiae/ Lachancea thermotolerans mixed fermentations.

The outcome of co- or sequential inoculation of Lachancea thermotolerans in winemaking remains unpredictable due to a lack of integrated data regarding the impact of grape juice composition on L. thermotolerans fermentation behaviour. Here, we investigate the impact of nitrogen composition on fermentation characteristics and aroma compound production in grape juice sequentially inoculated with commercial L. thermotolerans and S. cerevisiae strains. Subsequently, all treatments were subjected to malolactic fermentation (MLF) using two commercial strains of Oenococcus oeni. Addition of amino acids led to faster growth for S. cerevisiae fermentations, compared to the nitrogen-equivalent addition of diammonium phosphate (DAP). L. thermotolerans persistence in the mixed fermentations was significantly higher following DAP addition, with higher glycerol and lactic acid production. Interestingly, the lower total Nitrogen content in DAP-treated musts compared to other treatments did not alter the subsequent growth of S. cerevisiae. MLF was more similar between musts fermented with L. thermotolerans, regardless of nutrient regime, whereas significant differences in MLF completion times were observed for different nitrogen treatments in S. cerevisiae fermentations. Collectively, the data present an integrated view of the impact of nitrogen treatment on multispecies co-inoculation (growth kinetics and aromatic outcomes) and the downstream impact on MLF.

Influences of acid and ethanol stresses on Oenococcus oeni SD-2a and its proteomic and transcriptional responses.

BACKGROUND: During winemaking, malolactic fermentation (MLF) is usually induced by Oenococcus oeni owing to its high resistance to wine stress factors. To ensure a controlled and efficient MLF process, starter cultures are inoculated in wine. In previous studies, O. oeni strains with sub-lethal acid or ethanol stresses showed higher freeze-drying vitality and better MLF performance. To explore the mechanisms involved, influences of acid and ethanol stresses on O. oeni SD-2a were investigated in this study to gain a better understanding of the cross-protection responses. RESULTS: The results showed that acid and ethanol stresses both caused damage to cell membranes and decreased cellular adenosine triphosphate concentration. At the same time, acid stress increased the uptake of glutathione, while ethanol stress led to cell depolarization. The results of comparative proteomic analysis highlighted that heat shock protein was induced with almost all acid and ethanol stresses. In addition, the expression of stress-relevant genes (hsp20, clpP, trxA, ctsR, recO, usp) increased greatly with ethanol and acid stress treatments. Finally, the viability of O. oeni was improved with acid and ethanol pretreatments after freeze-drying. CONCLUSIONS: This study demonstrated that acid and ethanol stresses had mixed influences on O. oeni SD-2a. Some physiological and molecular changes would contribute to a more stress-tolerant state of O. oeni, thereby improving the viability of lyophilized cells. © 2020 Society of Chemical Industry.

The Expansion of Inosine at the Wobble Position of tRNAs, and Its Role in the Evolution of Proteomes.

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.

Identification and Characterization of the Small Heat Shock Protein Hsp20 from Oenococcus oeni SD-2a.

Oenococcus oeni can exert its function in hostile wine conditions during the malolactic fermentation process. Therefore, it is an important microbial resource for exploring resistance genes. Hsp20 is an important small heat shock protein from O. oeni. The conserved consensus motif "A-x-x-x-x-G-x-L" of Hsp20 announced its role as a member of the small heat shock protein family. The hsp20 gene from O. oeni SD-2a was cloned to create the recombinant plasmid pTriEx-Hsp20. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and the Hsp20 protein was induced by isopropyl-ß-D-thiogalactoside (IPTG). The hsp20 gene from O. oeni SD-2a was successfully expressed, and a 20-kDa fusion protein was identified by SDS-PAGE. The purified Hsp20 protein was obtained using Ni-affinity chromatography. Additionally, BL21(DE3)/Hsp20 and BL21(DE3)/Ctrl were treated at high temperatures of 42 and 52 °C, at pH values of 2.0-12.0, under oxidative shock with 0.1% (v/v) and 0.2% (v/v) H2O2, and under an osmotic shock of 430 and 860 mM NaCl to compare the effects of heterologous expression of the Hsp20 protein from O. oeni SD-2a for stress resistance. Notably, Hsp20 overexpression showed enhanced resistance than the control strain did when confronted with different elevated stress conditions. The results demonstrated heterologous expression of the hsp20 gene from O. oeni SD-2a significantly improved the resistance of the host E. coli bacteria against stress conditions.

Production of melatonin and other tryptophan derivatives by Oenococcus oeni under winery and laboratory scale.

Malolactic fermentation (MLF) in Valtellina Superiore DOCG red wine was monitored in 4 cellars and the final products were analysed to determine the content of melatonin (MEL) and other tryptophan (TRP) derivatives, including tryptophan ethyl ester (TEE) and MEL isomers (MISs), and to isolate predominant O. oeni strains. MEL and TEE significantly increased in wines after MLF from two cellars out of four. Six strains were isolated during the MLF of red wines and under laboratory scale, in rich and synthetic wine cultural media, together with other four O. oeni strains able to trigger the MLF. Results showed that the presence of stressful growth factors, like ethanol and acid pH, has a pivotal role in triggering the release of TEE by oenococci. Indeed, all the strains became capable to produce also MEL and MISs, together with TEE. under harsh growth conditions, as in a synthetic wine medium. The production of these compounds was strain-dependent and a maximum amount of 0.0078 ±â€¯0.0023 ngT/mL (UMB472) and 619.85 ±â€¯196.16 ngT/mL (UMB436) of MEL and TEE was obtained, respectively. In particular, different MISs were detected under oenological and laboratory scale suggesting that other factors (i.e. technological and/or physico-chemical) could affect the synthesis of TRP derivatives.

The metagenome-assembled genome of Candidatus Oenococcus aquikefiri from water kefir represents the species Oenococcus sicerae.

Recently, a metagenomic study of a water kefir fermentation ecosystem enabled the reconstruction of a metagenome-assembled genome (MAG) of an Oenococcus species that was different from the three species of this genus known so far. Therefore, the name Candidatus Oenococcus aquikefiri was proposed for this novel Oenococcus species. In the meantime, however, a fourth member of the genus, Oenococcus sicerae, isolated from French cider, was reported. The comparison of its genome sequence with the Candidatus O. aquikefiri MAG showed an average nucleotide identity (ANI) value of 98.53%. In addition, the 16S rRNA and pheS genes of the two species were 99.4% and 99.9% identical, respectively. As the presence of O. sicerae in a water kefir metagenome was also revealed by metagenomic recruitment plotting, it can be stated that Candidatus O. aquikefiri and O. sicerae belong to the same species. Intraspecies variations include the presence or absence of a citrate lyase operon and components of various phosphotransferase (PTS) transport systems.

Effect of Saccharomyces cerevisiae, Torulaspora delbrueckii and malolactic fermentation on fermentation kinetics and sensory property of black raspberry wines.

Alcoholic fermentation (AF) and malolactic fermentation (MLF) both have significant influence on the production of black raspberry wine. In this study, three microbes associated with AF and MLF including S. cerevisiae, T. delbrueckii and O. oeni were used to investigate their combined effect on basic compositional, volatile and sensory property of black raspberry wine, and four fermentation trials including single S. cerevisiae inoculation plus spontaneous MLF (BSU) and controlled MLF with O. oeni (BSO), sequential culture of T. delbrueckii and S. cerevisiae plus spontaneous MLF (BTSU) and controlled MLF (BTSO) were tested and compared. Fermentation results showed MLF in BSU, BSO and BTSO were successful, with respective period of 40, 25 and 23 days, whereas a stuck MLF occurred in BTSU. Volatile compounds were determined by HS-GC-IMS method, with a total of 45 aromas identified. BTSO was distinguished by a significant higher signal intensity of many fruity esters and a lower production of several alcohols and terpenes, which was in agreement with its perception result of strong 'fruity' and slight note of 'solvent' and 'herbaceous' during quantitative descriptive analysis. On the contrary, BSU was found to reinforce the synthesis of most detected volatiles, resulting in the enhancement of both beneficial and off-flavour compounds, therefore scoring lower in the 'global aroma' descriptor. Principal component analysis showed BSU and BSO were similar in the volatile composition, whereas BTSO was quite different. Overall, BTSO had greater potential to be used in the production of black raspberry wine.