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.

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.

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.

Molecular tools for the analysis of the microbiota involved in malolactic fermentation: from microbial diversity to selection of lactic acid bacteria of enological interest.

Winemaking is a complex process involving two successive fermentations: alcoholic fermentation, by yeasts, and malolactic fermentation (MLF), by lactic acid bacteria (LAB). During MLF, LAB can contribute positively to wine flavor through decarboxylation of malic acid with acidity reduction and other numerous enzymatic reactions. However, some microorganisms can have a negative impact on the quality of the wine through processes such as biogenic amine production. For these reasons, monitoring the bacterial community profiles during MLF can predict and control the quality of the final product. In addition, the selection of LAB from a wine-producing area is necessary for the formulation of native malolactic starter cultures well adapted to local winemaking practices and able to enhance the regional wine typicality. In this sense, molecular biology techniques are fundamental tools to decipher the native microbiome involved in MLF and to select bacterial strains with potential to function as starter cultures, given their enological and technological characteristics. In this context, this work reviews the different molecular tools (both culture-dependent and -independent) that can be applied to the study of MLF, either in bacterial isolates or in the microbial community of wine, and of its dynamics during the process.

Whey permeate as a substrate for the production of freeze-dried Lactiplantibacillus plantarum to be used as a malolactic starter culture.

The aim of this work was to obtain freeze-dried biomass of the native Patagonian Lactiplantibacillus plantarum strain UNQLp 11 from a whey permeate (WP)-based medium and compare it with the growth in commercial MRS broth medium. Survival and activity of the freeze-dried Lb. plantarum strain were investigated after inoculation in wine as a starter culture for malolactic fermentation (MLF). The effect of storage and rehydration condition of the dried bacteria and the nutrient supplementation of wine were also studied. The freeze-dried cultures from WP and those grown in MRS showed similar survival results. Rehydration in MRS broth for 24 h and the addition of a rehydration medium to wine as nutrient supplementation improved the survival under wine harsh conditions and guaranteed the success of MLF. Storage at 4 °C under vacuum was the best option, maintaining high cell viability for at least 56 days, with malic acid consumption higher than 90% after 7 days of inoculation in a wine-like medium. These results represent a significant advance for sustainable production of dried malolactic starter cultures in an environmentally friendly process, which is low cost and easy to apply in winemaking under harsh physicochemical conditions.

Lactobacillus plantarum as a malolactic starter culture in winemaking: a new (old) player?

Malolactic fermentation (MLF) is a process in winemaking responsible for the conversion of L-malic acid to L-lactic acid and CO2, which reduces the total acidity, improves the biological stability, and modifies the aroma profile of wine. MLF takes place during or after alcoholic fermentation and is carried out by one or more species of lactic acid bacteria (LAB), which are either present in grapes and cellars or inoculated with malolactic starters during the winemaking process. Although the main bacterium among LAB used in commercial starter cultures for MLF has traditionally been Oenococcus oeni, in the last decade, Lactobacillus plantarum has also been reported as a malolactic starter, and many works have shown that this species can survive and even grow under harsh conditions of wine (i.e., high ethanol content and low pH values). Furthermore, it has been proved that some strains of L. plantarum are able to conduct MLF just as efficiently as O. oeni. In addition, L. plantarum exhibits a more diverse enzymatic profile than O. oeni, which could play an important role in the modification of the wine aroma profile. This enzymatic diversity allows obtaining several starter cultures composed of different L. plantarum biotypes, which could result in distinctive wines. In this context, this review focuses on showing the relevance of L. plantarum as a MLF starter culture in winemaking.

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.

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.

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.

Pre-alcoholic fermentation acidification of red grape must using Lactobacillus plantarum.

Red grape musts from overripe grapes are characterised by high pH and sugar concentration. Corrections with organic acids are commonly used to secure the alcoholic fermentation and improve the organoleptic characteristics of the wine. In this study we test an alternative biological acidification method using the ability of Lactobacillus plantarum to produce high concentrations of lactic acid. The time course of sugars, organic acids and pH were measured. Available sugars were consumed by L. plantarum producing up to 8.3 g L(-1) of lactic acid. Lactic acid changed the pH from 3.9 to 3.4 after 14 days post-inoculation without yielding a relevant concentration of acetic acid (0.34 g L(-1)).

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.