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.

Impact of changes in wine composition produced by non-Saccharomyces on malolactic fermentation.

Non-Saccharomyces yeasts have increasingly been used in vinification recently. This is particularly true of Torulaspora delbrueckii and Metschnikowia pulcherrima, which are inoculated before S. cerevisiae, to complete a sequential alcoholic fermentation. This paper aims to study the effects of these two non-Saccharomyces yeasts on malolactic fermentation (MLF) carried out by two strains of Oenococcus oeni, under cellar conditions. Oenological parameters, and volatile and phenolic compounds were analysed in wines. The wines were tasted, and the microorganisms identified. In general, non-Saccharomyces created more MLF friendly conditions, largely because of lower concentrations of SO2 and medium chain fatty acids. The most favourable results were observed in wines inoculated with T. delbrueckii, that seemed to promote the development of O. oeni and improve MLF performance.

Assessment of ß-D-glucosidase activity and bgl gene expression of Oenococcus oeni SD-2a.

Glycosidases enhance flavor during wine-making by mediating the enzymatic release of aroma molecules. In order to better understand the aroma enhancement potential of Oenococcus oeni SD-2a, ß-D-glucosidase (ßG) activities in the culture supernatant, whole cells, and disrupted cell lysate were assessed at mid log, late log and stationary growth phase. The enzymatic activity was also compared further from cell cultures with 5 different carbon sources (glucose, cellobiose, arbutin, glucose and cellobiose, glucose and arbutin) at late log phase. Correspondingly, expression levels of 3 bgl genes, OEOE-0224, OEOE-1210, and OEOE-1569 were investigated from cell cultures of the 3 growth phases, and the 5 cell cultures with different carbon sources. Finally, the volatile aroma compounds released by O. oeni SD-2a in synthetic wines with natural glycosides were evaluated by GC-MS. Results showed ßG of O. oeni SD-2a was not extracellular enzyme, and the location of it didn't change with the change of growth phase and carbon source studied. ßG activities in the whole cells and disrupted cell lysate were similar and constant at the 3 growth phases. As for the carbon sources, ßG activities of whole cells and disrupted lysate were positively affected by cellobiose. While arbutin displayed positive and negative effect on ßG activity of whole cells and disrupted lysate, respectively. It is probably that bgl genes OEOE-0224 and OEOE-1210 were related to ßG activity of SD-2a whole cells, while OEOE-1569 was responsible for ßG activity of disrupted lysate. More kinds of volatile compounds and higher total concentration were released by SD-2a in synthetic wine compared with control. Thus, SD-2a showed a great potential for flavor enhancement under wine-like conditions. This study provides more information for further study of ßG activity from O. oeni SD-2a.

Dynamics of changes in organic acids, sugars and phenolic compounds and antioxidant activity of sea buckthorn and sea buckthorn-apple juices during malolactic fermentation.

Sea buckthorn (Hippophaë rhamnoides L.) berries have high biological value as a rich source of phenolic compounds, fatty acids and vitamins A, C, E. Due to the high organic acid content and sour taste, the fruits are rarely used in juice production. Therefore, the study aimed to determine the metabolic activity of Lactobacillus plantarum, Lactobacillus plantarum subsp. argentoratensis and Oenococcus oeni strains along with the dynamics of changes in organic acids, sugars, phenolic compounds, and antioxidant activity during 72-h fermentation of 100% sea buckthorn and mixed with apple (1:1) juices. The strongest malolactic conversion was in mixed juices (to 75.0%). The most efficient strains were L. plantarum DSM 10492, 20174 and 6872. L. plantarum strains caused an increase in flavonols and antioxidant activity of sea buckthorn-apple juices. The results can be used to select conditions and strains in industrial-scale fermentation, to produce novel sea buckthorn products and increase their consumption.

Heterologous expression of ctsR from Oenococcus oeni enhances the acid-ethanol resistance of Lactobacillus plantarum.

Oenococcus oeni is a lactic acid bacterium that is widely used in wine-making to conduct malolactic fermentation (MLF). During MLF, O. oeni undergoes acid and ethanol stress that impairs its growth. In order to investigate the role that the ctsR gene plays in acid-ethanol stress, the ctsR gene from O. oeni was expressed heterologously in Lactobacillus plantarum. The transcription level of the ctsR gene and 10 additional stress response genes in L. plantarum were analyzed by RT-qPCR. Physiological assays to assess reactive oxygen species accumulation, cell membrane integrity, intracellular ATP and GSH levels, Ca2+/Mg2+-ATPase and Na+/K+-ATPase activities were also performed. Results showed that the recombinant strain WCFS1-CtsR exhibited stronger growth performance than the control strain WCFS1-Vector, and the expression of ctsR, clp and hsp genes were significantly increased under acid-ethanol stress. Furthermore, WCFS1-CtsR displayed 1.08- and 1.39-fold higher ATP and GSH concentrations, respectively, compared with the corresponding values for WCFS1-Vector under acid-ethanol stress. ROS accumulation and PI value of WCFS1-CtsR were decreased by 46.52 and 42.80%, respectively, compared with the control strain. In addition, the two ATPase activities in WCFS1-CtsR increased significantly compared with WCFS1-Vector. This is the first report demonstrating that ctsR gene enhances the acid-ethanol tolerance of L. plantarum.

Ethyl carbamate regulation and genomic expression of Saccharomyces cerevisiae during mixed-culture yellow rice wine fermentation with Lactobacillus sp.

Ethyl carbamate (EC) is a potentially carcinogenic substance present in most alcoholic beverages, especially in Chinese rice wine. Consequently, much effort has been directed at suppressing EC formation during the production of these beverages, with particular attention directed at the use of urethanase, as this enzyme can directly catalyze EC degradation. Herein, we investigated the ability of three lactic acid bacteria (Oenococcus oeni, Lactobacillus brevis, and Lactobacillus plantarum) to generate urethanase during co-cultivation with Saccharomyces cerevisiae. qPCR and transcriptomic analyses revealed that 57 genes of S. cerevisiae were significantly expressed in the presence of L. brevis, which highlighted the importance of studying urethanase-promoted EC degradation for establishing a powerful technique of EC level control. The obtained results provided deep insights into the adaptive responses of S. cerevisiae to the challenging environment of mixed-culture fermentation.

Identification of multiple-derived peptides produced by Saccharomyces cerevisiae involved in malolactic fermentation inhibition.

An oenological strain of Saccharomyces cerevisiae was previously shown to produce a 5-10 kDa peptidic fraction responsible for the inhibition of malolactic fermentation (MLF). In the present study, we aim to further purify the anti-MLF peptides of this fraction. The yeast fermented synthetic grape juice medium was fractionated by ammonium sulfate precipitation combined with ultrafiltration. The 5-10 kDa fraction recovered at a saturation degree of 60%-80% was the only fraction that inhibited both the bacterial growth and the malate consumption in vivo. It also inhibited the malolactic enzyme activity in vitro at a pH range between 3.5 and 6.7. Therefore, it was purified by both anion and cation exchange chromatography. The eluates that inhibited the malolactic enzyme activity in vitro were migrated on Tricine SDS-PAGE and the protein bands were excised and sequenced by LC-MS/MS. The sequencing revealed nine peptides originating from eight proteins of S. cerevisiae. Two GAPDH cationic fragments of 0.9 and 1.373 kDa having a pI of 10.5 and 11 respectively, Wtm2p and Utr2p anionic fragments of 2.42 kDa with a pI of 3.5 and 4 respectively were thought to contribute the most to the MLF inhibition.

Acetaldehyde released by Lactobacillus plantarum enhances accumulation of pyranoanthocyanins in wine during malolactic fermentation.

This study investigated the evolution of acetaldehyde and pyranoanthocyanins in wine during malolactic fermentation, and further evaluated the correlation between acetaldehyde and pyranoanthocyanins. Cabernet Gernischt wine after alcoholic fermentation was inoculated with four lactic acid bacteria strains. Malolactic fermentation kinetics and wine characteristics were compared. Results showed these strains exhibited different kinetics on wine malolactic fermentation. Wine with Lactobacillus plantarum had lower reducing sugar, total acid, and yellowness. Lactobacillus plantarum elevated the level of acetaldehyde in wine model medium and wine during malolactic fermentation. Malolactic fermentation using Lactobacillus plantarum significantly increased the concentration of pyranoanthocyanins, whereas O. oeni strain reduced the level of pyranoanthocyanins in wine. Polymerized anthocyanins percentage in wine was significantly enhanced after fermentation with Lactobacillus plantarum. Principal component analysis indicated that the characteristics of these strains inoculated wines after malolactic fermentation were segregated. The findings from this study could provide useful information on the wine color improvement through malolactic fermentation with suitable lactic acid bacteria strains.

Changes in the volatile profile of Pinot noir wines caused by Patagonian Lactobacillus plantarum and Oenococcus oeni strains.

The ability of Patagonian L. plantarum and O. oeni strains to change the volatile profile of a sterile Pinot noir wine was studied through fermentation assays, at laboratory scale. Two strains of each LAB species were selected based on data regarding to their ability to survive in wine and to consume l-malic acid. Both O. oeni strains but only one L. plantarum (UNQLp 11) strain were able to remain viable, consuming l-malic acid through the fermentation assay with a concomitant increase of l-lactic acid. The volatile profile of Pinot noir wine, before and after LAB inoculation, was measured by using HS-SPME gas chromatography technique. This analysis showed that alcohols were the main volatile compounds after alcoholic fermentation and that after fermentation with the selected LAB strains, a decrease in the volatile alcohols concentration and an increase in the volatile esters concentration could be observed. The O. oeni UNQOe 73.2 strain produced the most notable change in the volatile profile, with the production of some important odorant esters at higher concentration, compared to O. oeni UNQOe 31b strain. Although, L. plantarum UNQLp 11 strain showed a better performance in the consumption of l-malic acid, this strain had a low capacity to modify the volatile compounds profile after incubation in red wine. The results found in the present work showed that different strains selected as potential malolactic starters could have different behavior when are incubated in real wine. Although L. plantarum UNQLp 11 strain showed a good consumption of l-malic acid, the O. oeni UNQOe 73.2 strain exhibited superior capacity to improve the flavor of wine due to its esterase activity that produce an increase of fruity and creamy odorants.

Influence of glycosides on behavior of Oenococcus oeni in wine conditions: growth, substrates and aroma compounds.

Autochthonous Oenococcus oeni strains (MS9, MS20 and MS46) with good malolactic performance and yielding adequate diacetyl levels, were selected to investigate the effect of synthetic and grape glycosides on bacterial growth, substrate utilization and ß-glucosidase (ßGlu), α-arabinofuranosidase (αAra) and α-rhamnopyranosidase (αRha) activities in a wine-like medium containing 6% ethanol, pH 4.0 (WBM). Then, changes in the volatile compounds profile were evaluated at the end of malolactic fermentation (MLF) carried out by the MS46 strain in WBM containing 1 mg L-1 of natural glycoside. All strains grew and efficiently degraded L-malic acid in WBM where ßGlu and αAra activities were found but not αRha. In presence of a synthetic glycoside (eriodictyol 7-O-ß-rutinoside) ßGlu activity was significantly enhanced for two of the cultures tested (MS20 and MS460) while a low αRha activity was induced, presenting MS46 the better performance. Glycosides extracted from fermented grape musts under different conditions allowed maximum growths, L-malic acid utilization rates and glycosidase activities in the MS46 strain. Thus, ßGlu, αAra and αRha activities increased between 30-50 and 3-11% respectively. This indirectly correlated to significant changes in total esters and higher alcohols at the end of MLF, which increased by up to 140 and 30% respectively. Moreover, ethyl and acetate esters formed up to 100-fold than alcohols or esters degraded highlighted the main role of this microorganism in the esters synthesis. Results obtained encourage the potential use of selected indigenous O. oeni strains as a tool to enhance wine complexity through MLF, mainly on highly fruity aroma.

Growth and metabolism of Oenococcus oeni for malolactic fermentation under pressure.

Malolactic fermentation is a biological deacidification process of wine, characterized by the transformation of l-malic acid to l-lactic acid and CO2 . Oenococcus oeni is able to perform malolactic fermentation and to survive under wine harsh conditions, representing great interest for wine industry. The aim of this work was to evaluate the effect of high pressure on the metabolism of O. oeni growing in culture media, regarding malolactic fermentation, sugars metabolism and bacterial growth. A pressure stress of 50 MPa during 8 h did not result in significant modifications in bacterial metabolism. In contrast, a stress of 100 MPa during 8 h resulted in lower amounts of l-lactic acid, while higher amounts of d-lactic acid were also registered, indicating changes in bacterial metabolism. A pressure stress of 0·5 MPa during 300 h resulted in complete inactivation of O. oeni, but malolactic fermentation was still observed at some extent, showing that malolactic enzyme was not completely inactivated at these conditions. It was concluded that high pressure causes modification of O. oeni metabolism, and possibly in enzyme activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that high pressure affects the viability and metabolism of Oenococcus oeni on a culture medium, depending on the pressure intensity and holding time applied. These effects were particularly noteworthy on malolactic fermentation. After high pressure (HP)-stress of 100 MPa for 8 h, modifications in the activity of malolactic enzyme were detected, possibly due to a change in specificity. After a HP-stress of 300 MPa for 0·5 h, malolactic enzyme showed some residual activity, although O. oeni was completely inactivated. This study provides relevant information about the impact of high pressure on malolactic fermentation, opening interesting possibilities to the improvement of biocatalytic processes.

Diversity of the microbiota involved in wine and organic apple cider submerged vinegar production as revealed by DHPLC analysis and next-generation sequencing.

Unfiltered vinegar samples collected from three oxidation cycles of the submerged industrial production of each, red wine and organic apple cider vinegars, were sampled in a Slovene vinegar producing company. The samples were systematically collected from the beginning to the end of an oxidation cycle and used for culture-independent microbial analyses carried out by denaturing high pressure liquid chromatography (DHPLC) and Illumina MiSeq sequencing of 16S rRNA gene variable regions. Both approaches showed a very homogeneous bacterial structure during wine vinegar production but more heterogeneous during organic apple cider vinegar production. In all wine vinegar samples Komagataeibacter oboediens (formerly Gluconacetobacter oboediens) was a predominating species. In apple cider vinegar the acetic acid and lactic acid bacteria were two major groups of bacteria. The acetic acid bacterial consortium was composed of Acetobacter and Komagataeibacter with the Komagataeibacter genus outcompeting the Acetobacter in all apple cider vinegar samples at the end of oxidation cycle. Among the lactic acid bacterial consortium two dominating genera were identified, Lactobacillus and Oenococcus, with Oenococcus prevailing with increasing concentration of acetic acid in vinegars. Unexpectedly, a minor genus of the acetic acid bacterial consortium in organic apple cider vinegar was Gluconobacter, suggesting a possible development of the Gluconobacter population with a tolerance against ethanol and acetic acid. Among the accompanying bacteria of the wine vinegar, the genus Rhodococcus was detected, but it decreased substantially by the end of oxidation cycles.

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 pH 3.5 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.

Effectiveness of chitosan against wine-related microorganisms.

The antimicrobial action of chitosan against wine related microorganisms, including Lactobacillus plantarum, Saccharomyces cerevisiae, Oeonococcus oeni, Lactobacillus hilgardii, Brettanomyces bruxellensis, Hanseniaspora uvarum and Zygosaccharomyces bailii was examined in laboratory media. In order to assess the potential applicability of chitosan as a microbial control agent for wine, the effect of chitosan, applied individually and/or in combination with sulphur dioxide (SO2), on the growth of microorganisms involved in various stages of winemaking and on the fermentative performance of S. cerevisiae was investigated. Of the seven wine-related microorganisms studied, S. cerevisiae exhibited the strongest resistance to antimicrobial action of chitosan in laboratory media with a minimum inhibitory concentration (MIC) greater than 2 g/L. L. hilgardii, O. oeni and B. bruxellensis were the most susceptible to chitosan since they were completely inactivated by chitosan at 0.2 g/L. The MIC of chitosan for L. plantarum, H. uvarum and Z. bailii was 2, 0.4 and 0.4 g/L, respectively. In wine experiments, it was found that chitosan had a retarding effect on alcoholic fermentation without significantly altering the viability and the fermentative performance of S. cerevisiae. With regard to non-Saccharomyces yeasts (H. uvarum and Z. bailii) involved in winemaking, the early deaths of these yeasts in mixed cultures with S. cerevisiae were not probably due to the antimicrobial action of chitosan but rather due to ethanol produced by the yeasts. The complex interactions between chitosan and wine ingredients as well as microbial interactions during wine fermentation considerably affect the efficacy of chitosan. It was concluded that chitosan was worthy of further investigation as an alternative or complementary preservative to SO2 in wine industry.

A mathematical model of the link between growth and L-malic acid consumption for five strains of Oenococcus oeni.

In winemaking, after the alcoholic fermentation of red wines and some white wines, L-malic acid must be converted into L-lactic acid to reduce the acidity. This malolactic fermentation (MLF) is usually carried out by the lactic acid bacteria Oenococcus oeni. Depending on the level of process control, selected O. oeni is inoculated or the natural microbiota of the cellar is used. This study considers the link between growth and MLF for five strains of O. oeni species. The kinetics of growth and L-malic acid consumption were followed in modified MRS medium (20 °C, pH 3.5, and 10 % ethanol) in anaerobic conditions. A large variability was found among the strains for both their growth and their consumption of L-malic acid. There was no direct link between biomass productivities and consumption of L-malic acid among strains but there was a link of proportionality between the specific growth of a strain and its specific consumption of L-malic acid. Experiments with and without malic acid clearly demonstrated that malic acid consumption improved the growth of strains. This link was quantified by a mathematical model comparing the intrinsic malic acid consumption capacity of the strains.

Effect of physicochemical factors on glycerol production by simultaneous cultures of wine micro-organisms using the response surface method.

AIM: To evaluate the effect of temperature, pH and SO2 on growth and glycerol production improvement by Saccharomyces cerevisiae mc2, Kloeckera apiculata mF and Oenococcus oeni X2L using the response surface method (RSM). METHODS AND RESULTS: Multifactorial design of cultures with physicochemical factors variations was performed. The micro-organisms grew in all cultures conditions. Overall, after 6 days yeasts prevailed, especially S. cerevisiae (10(9) CFU ml(-1)), while O. oeni reached 10(7) CFU ml(-1). At initial fixed pH 5·5, metabolic behaviour of cultures showed a temperature-dependent response. Total malate consumption occurred at 26°C, 50 mg l(-1) SO2. Glucose and pentoses utilization was highly modified when varying SO2. Ethanol showed negative interaction with temperature-SO2 relationship. At low SO2, glycerol and acetate production increased when temperature enhanced. Predictive results of RSM indicate that 26°C, 60·24 mg l(-1) SO2 and pH 5·5 were the optimal conditions for glycerol and organic acids synthesis compatible with wine quality. CONCLUSIONS: We propose a predictive condition to improve the performance of mixed cultures for must fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: To optimize the culture conditions to design mixed starters containing autochthonous yeasts and O. oeni strains for winemaking and to obtain products with high glycerol content, low acidity and maintenance of regional characteristics.

Simultaneous and successive inoculations of yeasts and lactic acid bacteria on the fermentation of an unsulfited Tannat grape must.

Interactions between yeasts and lactic acid bacteria are strain specific, and their outcome is expected to change in simultaneous alcoholic--malolactic fermentations from the pattern observed in successive fermentations. One Oenococcus oeni strain Lalvin VP41™ was inoculated with two Saccharomyces cerevisiae strains either simultaneously, three days after the yeast inoculation, or when alcoholic fermentation was close to finish. Early bacterial inoculations with each yeast strain allowed for the growth of the bacterial populations, and the length of malolactic fermentation was reduced to six days. Alcoholic fermentation by Lalvin ICV D80® yeast strain left the highest residual sugar, suggesting a negative effect of the bacterial growth and malolactic activity on its performance. In sequential inoculations the bacterial populations did not show actual growth with either yeast strain. In this strategy, both yeast strains finished the alcoholic fermentations, and malolactic fermentations took longer to finish. Lalvin ICV D80® allowed for higher viability and activity of the bacterial strain than Fermicru UY4® under the three inoculation strategies. This was beneficial for the sequential completion of both fermentations, but negatively affected the completion of alcoholic fermentation by Lalvin ICV D80® in the early bacteria additions. Conversely, Fermicru UY4®, which was rather inhibitory towards the bacteria, favored the timely completion of both fermentations simultaneously. As bacteria in early inoculations with low or no SO2 addition can be expected to multiply and interact with fermenting yeasts, not only are the yeast-bacterium strains combination and time point of the inoculation to be considered, but also the amount of bacteria inoculated.

Glycerol production by Oenococcus oeni during sequential and simultaneous cultures with wine yeast strains.

Growth and fermentation patterns of Saccharomyces cerevisiae, Kloeckera apiculata, and Oenococcus oeni strains cultured in grape juice medium were studied. In pure, sequential and simultaneous cultures, the strains reached the stationary growth phase between 2 and 3 days. Pure and mixed K. apiculata and S. cerevisiae cultures used mainly glucose, producing ethanol, organic acids, and 4.0 and 0.1 mM glycerol, respectively. In sequential cultures, O. oeni achieved about 1 log unit at 3 days using mainly fructose and L-malic acid. Highest sugars consumption was detected in K. apiculata supernatants, lactic acid being the major end-product. 8.0 mM glycerol was found in 6-day culture supernatants. In simultaneous cultures, total sugars and L-malic acid were used at 3 days and 98% of ethanol and glycerol were detected. This study represents the first report of the population dynamics and metabolic behavior of yeasts and O. oeni in sequential and simultaneous cultures and contributes to the selection of indigenous strains to design starter cultures for winemaking, also considering the inclusion of K. apiculata. The sequential inoculation of yeasts and O. oeni would enhance glycerol production, which confers desirable organoleptic characteristics to wines, while organic acids levels would not affect their sensory profile.

Influence of controlled inoculation of malolactic fermentation on the sensory properties of industrial cider.

Given the lack of research in the traditional cider making field when compared to the efforts devoted to winemaking, this work focused on the effects of controlled inoculation of the malolactic fermentation (MLF) on the sensory properties of cider. MLF develops spontaneously in cider making at industrial level. In this work, industrial cider samples were inoculated with selected indigenous Oenococcus oeni strains and the benefits on the aroma and flavour in cider production compared to non-inoculated ciders were evaluated. Randomly amplified polymorphic DNA PCR was used to monitor strain colonization ability, outnumbering the indigenous microbiota, after completion of the alcoholic fermentation at industrial scale (20,000 l). Aroma-active compounds of experimentally inoculated ciders were analysed by HPLC and GC-MS, and sensory profiles were determined by fractioning aroma extracts using reversed-phase HPLC. Principal component analysis allowed the identification of relationships and differences among ciders with or without inoculation, including several highly appreciated commercial ones obtained under spontaneous conditions. Under controlled inoculation conditions, not only could MLF be shortened by half but, interestingly, enhancement of aroma complexity and flavour resulted in ciders enriched with a higher fruity note. In addition, important aromatic groups analysed here had not been previously described, thus affording deeper knowledge on aroma characterization of apple cider.

Simultaneous and successive inoculations of yeasts and lactic acid bacteria on the fermentation of an unsulfited Tannat grape must

Interactions between yeasts and lactic acid bacteria are strain specific, and their outcome is expected to change in simultaneous alcoholic -malolactic fermentations from the pattern observed in successive fermentations. One Oenococcus oeni strain Lalvin VP41TM was inoculated with two Saccharomyces cerevisiae strains either simultaneously, three days after the yeast inoculation, or when alcoholic fermentation was close to finish. Early bacterial inoculations with each yeast strain allowed for the growth of the bacterial populations, and the length of malolactic fermentation was reduced to six days. Alcoholic fermentation by Lalvin ICV D80® yeast strain left the highest residual sugar, suggesting a negative effect of the bacterial growth and malolactic activity on its performance. In sequential inoculations the bacterial populations did not show actual growth with either yeast strain. In this strategy, both yeast strains finished the alcoholic fermentations, and malolactic fermentations took longer to finish. Lalvin ICV D80® allowed for higher viability and activity of the bacterial strain than Fermicru UY4® under the three inoculation strategies. This was beneficial for the sequential completion of both fermentations, but negatively affected the completion of alcoholic fermentation by Lalvin ICV D80® in the early bacteria additions. Conversely, Fermicru UY4®, which was rather inhibitory towards the bacteria, favored the timely completion of both fermentations simultaneously. As bacteria in early inoculations with low or no SO2 addition can be expected to multiply and interact with fermenting yeasts, not only are the yeast-bacterium strains combination and time point of the inoculation to be considered, but also the amount of bacteria inoculated.