Oenococcus oeni allows the increase of antihypertensive and antioxidant activities in apple cider.

This study aimed to investigate the impact of the malolactic fermentation (MLF) carried out by Oenococcus oeni on antihypertensive and antioxidant activities in cider. The MLF was induced using three strains of O. oeni. The modification in phenolic compounds (PCs) and nitrogen organic compounds, antioxidant, and antihypertensive activities were determined after MLF. Among the 17 PCs analyzed caffeic acid was the most abundant compound and phloretin, (-)-epicatechin, and myricetin were detected only in malolactic ciders, however, (-)-epigallocatechin was not detected after MLF. The evaluation of nitrogen organic compounds revealed a drop in total protein concentration (from 17.58 to 14.00 mg N/L) concomitantly with a significant release of peptide nitrogen (from 0.31 to a maximum value of 0.80 mg N/L) after MLF. In addition, an extracellular proteolytic activity was evidenced in all MLF supernatants. The FRAP activity increased reaching a maximum of 120.9 µmol FeSO4/mL and the ABTS radical-scavenging activity increased until 6.8 mmol ascorbic acid/L. Moreover, the angiotensin I-converting enzyme inhibitory activity reached a maximum value of 39.8%. The MLF conducted by O. oeni in ciders enables the increase of interesting biological activities and this finding could constitute a valuable tool to add value to final product.

Metabolic behavior for a mutant Oenococcus oeni strain with high resistance to ethanol to survive under oenological multi-stress conditions.

Malolactic fermentation (MLF) positively influences the quality of the wine, and it occurs as a result of a lactic acid bacteria's metabolism, mainly of the Oenococcus oeni species. However, delays and halting of MLF are frequent problems in the wine industry. This is mainly because O. oeni's development is inhibited by different kinds of stress. Even though the sequencing of the genome of the PSU-1 strain of O. oeni, as well as other strains, has made it possible to identify genes involved in the resistance to some types of stress, all of the factors that could be involved are still unknown. With the aim of contributing to this knowledge, the random mutagenesis technique was used in this study as a strategy for genetic improvement of strains of the O. oeni species. The technique proved to be capable of generating a different and improved strain when compared to the PSU-1 strain (the parent from which it descends). Then, we evaluated the metabolic behavior of both strains in three different wines. We used synthetic MaxOeno wine (pH 3.5; 15% v/v ethanol), red wine (Cabernet Sauvignon), and white wine (Chardonnay). Furthermore, we compared the transcriptome of both strains, grown in MaxOeno synthetic wine. The specific growth rate of the E1 strain was on average 39% higher in comparison to the PSU-1 strain. Interestingly, E1 strain showed an overexpression of the OEOE_1794 gene, which encodes a UspA-like protein, which has been described as promoting growth. We observed that the E1 strain was able to convert, on average, 34% more malic acid into lactate than the PSU-1 strain, regardless of the wine being used. On the other hand, the E1 strain showed a flux rate of fructose-6-phosphate production that was 86% higher than the mannitol production rate, and the internal flux rates increase in the direction of pyruvate production. This coincides with the higher number of OEOE_1708 gene transcripts observed in the E1 strain grown in MaxOeno. This gene encodes for an enzyme fructokinase (EC 2.7.1.4) involved in the transformation of fructose to fructose-6-phosphate.

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.

Relative expression of stress-related genes during acclimation at low temperature of psychrotrophic Oenococcus oeni strains from Patagonian wine.

In the present study, we evaluated the transcriptional response of four stress-related genes in three Oenococcus oeni strains after acclimation at two different temperatures. Gene expression was analyzed at time zero and after 48 h acclimation at 18 and 21 °C. After the acclimation period cells were inoculated into sterile Pinot noir wine and MLF was followed for 25 days to investigate if different acclimation temperatures could influence cell survival and MLF performance. L-malic acid consumption, population survival, and transcriptional behavior were different upon the acclimation temperature. rmlB and hsp20 genes presented a considerable increase in their expression level when strains were acclimated at 18 °C particularly in the psychrotrophic strains UNQOe19 and UNQOe4 isolated from Patagonian Pinot noir wine in comparison with the control strain (ATCC 27310). The increase in rmlB and hsp20 expression could account for the better survival of these strains in Pinot noir in comparison with the control strain. In addition, Patagonian populations acclimated at 18 °C were able to consume a higher percentage of L-malic acid in comparison with cells acclimated at 21 °C. Our results suggest that gene expression analysis of cells acclimated at sub-optimal temperatures could benefit the selection of psychrotrophic strains aimed as starter cultures.

Cell surface damage and morphological changes in Oenococcus oeni after freeze-drying and incubation in synthetic wine.

The aim of the present study was to evaluate the effects of freeze-drying in the presence of trehalose as a cryoprotectant, followed by incubation in synthetic wine, on surface damage, viability and l-malic acid consumption of the oenological strain Oenococcus oeni UNQOe 73.2. After freeze-drying, no significant differences were observed in the number of viable cells (for both acclimated and non-acclimated cultures) respect to the fresh culture. In contrast, loss of viability was observed after wine incubation for 24 h, being acclimated freeze-dried cells the best conditions for this. After the preservation process, small changes in cell morphology were observed by Atomic Force Microscopy (AFM). The Zeta potential and AFM showed that 24 h of wine incubation was enough to induce several cell surface modifications. Plate count data allowed us to establish that surface damage is an important factor for loss of viability, regardless of the acclimation treatment. Although the number of surviving O. oeni cells decreased dramatically after incubation in synthetic wine for 15 days, the consumption of l-malic acid was higher than 70%, with freeze-dried cells showing a better performance than fresh cultures. These results demonstrate that O. oeni freeze-dried cultures could be applied to direct wine inoculation, to conduct malolactic fermentation, maintaining its technological properties and reducing the time and costs of the winemaking process.

Oenococcus oeni in Chilean Red Wines: Technological and Genomic Characterization.

The presence and load of species of LAB at the end of the malolactic fermentation (MLF) were investigated in 16 wineries from the different Chilean valleys (Limarí, Casablanca, Maipo, Rapel, and Maule Valleys) during 2012 and 2013, using PCR-RFLP and qPCR. Oenococcus oeni was observed in 80% of the samples collected. Dominance of O. oeni was reflected in the bacterial load (O. oeni/total bacteria) measured by qPCR, corresponding to >85% in most of the samples. A total of 178 LAB isolates were identified after sequencing molecular markers, 95 of them corresponded to O. oeni. Further genetic analyses were performed using MLST (7 genes) including 10 commercial strains; the results indicated that commercial strains were grouped together, while autochthonous strains distributed among different genetic clusters. To pre-select some autochthonous O. oeni, these isolates were also characterized based on technological tests such as ethanol tolerance (12 and 15%), SO2 resistance (0 and 80 mg l-1), and pH (3.1 and 3.6) and malic acid transformation (1.5 and 4 g l-1). For comparison purposes, commercial strain VP41 was also tested. Based on their technological performance, only 3 isolates were selected for further examination (genome analysis) and they were able to reduce malic acid concentration, to grow at low pH 3.1, 15% ethanol and 80 mg l-1 SO2. The genome analyses of three selected isolates were examined and compared to PSU-1 and VP41 strains to study their potential contribution to the organoleptic properties of the final product. The presence and homology of genes potentially related to aromatic profile were compared among those strains. The results indicated high conservation of malolactic enzyme (>99%) and the absence of some genes related to odor such as phenolic acid decarboxylase, in autochthonous strains. Genomic analysis also revealed that these strains shared 470 genes with VP41 and PSU-1 and that autochthonous strains harbor an interesting number of unique genes (>21). Altogether these results reveal the presence of local strains distinguishable from commercial strains at the genetic/genomic level and also having genomic traits that enforce their potential use as starter cultures.

Mapping the Physiological Response of Oenococcus oeni to Ethanol Stress Using an Extended Genome-Scale Metabolic Model.

The effect of ethanol on the metabolism of Oenococcus oeni, the bacterium responsible for the malolactic fermentation (MLF) of wine, is still scarcely understood. Here, we characterized the global metabolic response in O. oeni PSU-1 to increasing ethanol contents, ranging from 0 to 12% (v/v). We first optimized a wine-like, defined culture medium, MaxOeno, to allow sufficient bacterial growth to be able to quantitate different metabolites in batch cultures of O. oeni. Then, taking advantage of the recently reconstructed genome-scale metabolic model iSM454 for O. oeni PSU-1 and the resulting experimental data, we determined the redistribution of intracellular metabolic fluxes, under the different ethanol conditions. Four growth phases were clearly identified during the batch cultivation of O. oeni PSU-1 strain, according to the temporal consumption of malic and citric acids, sugar and amino acids uptake, and biosynthesis rates of metabolic products - biomass, erythritol, mannitol and acetic acid, among others. We showed that, under increasing ethanol conditions, O. oeni favors anabolic reactions related with cell maintenance, as the requirements of NAD(P)+ and ATP increased with ethanol content. Specifically, cultures containing 9 and 12% ethanol required 10 and 17 times more NGAM (non-growth associated maintenance ATP) during phase I, respectively, than cultures without ethanol. MLF and citric acid consumption are vital at high ethanol concentrations, as they are the main source for proton extrusion, allowing higher ATP production by F0F1-ATPase, the main route of ATP synthesis under these conditions. Mannitol and erythritol synthesis are the main sources of NAD(P)+, countervailing for 51-57% of its usage, as predicted by the model. Finally, cysteine shows the fastest specific consumption rate among the amino acids, confirming its key role for bacterial survival under ethanol stress. As a whole, this study provides a global insight into how ethanol content exerts a differential physiological response in O. oeni PSU-1 strain. It will help to design better strategies of nutrient addition to achieve a successful MLF of wine.

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.

Genome-Scale Reconstruction of the Metabolic Network in Oenococcus oeni to Assess Wine Malolactic Fermentation.

Oenococcus oeni is the main responsible agent for malolactic fermentation in wine, an unpredictable and erratic process in winemaking. To address this, we have constructed and exhaustively curated the first genome-scale metabolic model of Oenococcus oeni, comprising 660 reactions, 536 metabolites and 454 genes. In silico experiments revealed that nutritional requirements are predicted with an accuracy of 93%, while 14 amino acids were found to be essential for the growth of this bacterial species. When the model was applied to determine the non-growth associated maintenance, results showed that O. oeni grown at 12% ethanol concentration spent 30 times more ATP to stay alive than in the absence of ethanol. Most of this ATP is employed for extruding protons outside of the cell. A positive relationship was also found between specific consumption rates of fructose, amino acids, oxygen, and malic acid and the specific production rates of erythritol, lactate, and acetate, according to the ethanol content of the medium. The metabolic model reconstructed here represents a unique tool to predict the successful completion of wine malolactic fermentation carried out either by different strains of Oenococcus oeni, as well as at any particular physico-chemical composition of wine. It will also allow the development of consortium metabolic models that could be applied to winemaking to simulate and understand the interactions between O. oeni and other microorganisms that share this ecological niche.

Distribution of Native Lactic Acid Bacteria in Wineries of Queretaro, Mexico and Their Resistance to Wine-Like Conditions.

Native lactic acid bacteria (LAB) are capable of growing during winemaking, thereby strongly affecting wine quality. The species of LAB present in musts, wines during malolactic fermentation (MLF), and barrels/filters were investigated in wineries from the emerging wine region of Queretaro, México using multiplex PCR and culture. The resistance to wine-like conditions (WLC): ethanol (10, 12, and 13%), SO2 (30 mg⋅l-1), and low pH (3.5) of native LAB strains was also studied. Five species were detected within 61 samples obtained: Oenococcus oeni, Lactobacillus plantarum, Pediococcus parvulus, Lactobacillus hilgardi, and Lactobacillus brevis. Four species (excepting L. brevis) were found in must; O. oeni and P. parvulus were ubiquitous in wine and L. plantarum and L. brevis were mainly present at the initial stage of MLF, while L. hilgardii was mostly detected at the advanced stage. Furthermore, some species detected in barrel/filter, prove them to be hazardous reservoirs. From 822 LAB isolates, only 119 resisted WLC with 10% ethanol; the number of strains able to grow in WLC with 13% ethanol decreased approximately by 50%, O. oeni being the most versatile species with 65% of resistant isolates, while Lactobacillus spp. and P. parvulus were the most strongly affected, especially those recovered from barrel/filter, with less than 10% of resistant isolates. This study evidences the presence of local strains able to be used as starter cultures, and also enabled the assessment of the risks derived from the presence of spoilage LAB strains resistant to WLC.

The rapid identification of lactic acid bacteria present in Chilean winemaking processes using culture-independent analysis.

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 16S ribosomal RNA (rRNA) genes was developed to identify lactic acid bacteria (LAB) that are commonly present in winemaking processes (Oenococcus, Pediococcus, Lactobacillus, and Leuconostoc). This culture-independent approach revealed the presence of Oenococcus in the spontaneous malolactic fermentation in industrial Chilean wines.

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.