Malolactic Fermentation

A reversed phase liquid chromatography-DAD method is proposed for analysis of major non-flavonoid phenolic compounds in wines. The method employed a mixture of acetic acid, water and methanol as eluents and was used to evaluate the impact of malolactic fermentation in low molecular phenolic compounds. The wines analyzed underwent different treatments, like the addition of a pectolytic enzyme or lysozyme, and the way malolactic fermentation was carried out-spontaneously or with the inoculation of two different commercial lactic bacteria. The main result observed was the disappearance of hydroxycinnamoyltartaric acids and the increase of resultant free forms, regardless the way malolactic fermentation was carried out.

Use of commercial starters for malolactic fermentation (MLF) is a common practice, and outcomes of this procedure are very relevant for wine producers because aroma attributes might change according to the strain used. MLF was carried out in the Trincadeira wine variety in three batches: spontaneously and with inoculation of two different commercial starters. Wine extracts before and after MLF were obtained through liquid-liquid extraction. Gas chromatography/ion trap mass spectrometry analysis allowed the detection of 21 compounds, some of them tentatively identified through electron impact spectra comparison with a database, through interpretation of multistage mass spectrometry and chemical ionization mass spectrometry spectra and confirmation by comprehensive multidimensional gas chromatography/ time of flight-mass spectrometry (GC ¥ GC/ToF-MS). After MLF, an increase in ethyl lactate, g-butyrolactone and diethyl succinate and a drop in isoamyl acetate, ethyl octanoate, ethyl decanoate, 2,3-dimethyl-hexan-3-ol, propanol, isobutanol and 2,3-butanediol was observed. The concentrations of the 10 compounds are statistically different in wines before and after MLF, and wines after MLF show significant differences for the nine compounds.

Two autochthonous yeast strains called F-8 and F-78 (isolated and selected from the Tikveš wine-producing region) were inoculated in wine musts from Vranec and Cabernet Sauvignon grape varieties. The fermentation process and quality of the produced wines were compared to the wines produced from the same grape varieties, but with a commercial yeast strain (D-80). The fermentation process was undertaken at 23–25 °C for 16 days. The highest alcohol content was detected in Vranec and Cabernet Sauvignon wines fermented with autochthonous F-8 yeast strain. Conversely, Vranec wine fermented by the F-78 yeast strain contained the lowest alcohol content (14.11%). The low sugar amount (2–3 g/L) in both grape varieties is indicated the production of dry wines. The total amount of phenolic compounds was significantly higher in Vranec wines. Unlike the alcohol content, Vranec wine produced by commercial yeast strain D-80 indicated the highest concentration of total phenolic compounds (1450 mg/L)...

The influence of seven phenolic compounds, normally present in wine, on the growth and arginine deiminase system (ADI) of Lactobacillus hilgardii X 1 B, a wine lactic acid bacterium, was established. This system provides energy for bacterial growth and produces citrulline that reacts with ethanol forming the carcinogen ethyl carbamate (EC), found in some wines. The influence of phenolic compounds on bacterial growth was compound dependent. Growth and final pH values increased in presence of arginine. Arginine consumption decreased in presence of protocatechuic and gallic acids (31 and 17%, respectively) and increased in presence of quercetin, rutin, catechin and the caffeic and vanillic phenolic acids (between 10 and 13%, respectively). ADI enzyme activities varied in presence of phenolic compounds. Rutin, quercetin and caffeic and vanillic acids stimulated the enzyme arginine deiminase about 37-40%. Amounts of 200 mg/L gallic and protocatechuic acids inhibited the arginine deiminase enzyme between 53 and 100%, respectively. Ornithine transcarbamylase activity was not modified at all concentrations of phenolic compounds. As gallic and protocatechuic acids inhibited the arginine deiminase enzyme that produces citrulline, precursor of EC, these results are important considering the formation of toxic compounds.

Unsaturated fatty acids and sterols are essential constituents of the yeast plasma membrane. Recently, their contribution to modulating the production of yeast-derived volatile compounds has received significant attention. The objective of this study was to determine how sterol and lipid supplementation, including ergosterol, plant sterols or oleic acid, differentially influenced yeast growth as well as the production of fermentative aromas when added individually or in combinations. Oleic acid significantly altered the volatile profiles produced and lowered yeast growth. Generally, phytosterol (β-sitosterol) and ergosterol supplementation resulted in similar responses regarding the production of aromas, however, they differed in the magnitude of the response in the case of medium chain fatty acids and acetate esters synthesis. The combinations of sterols with oleic acid resulted in a response more closely associated with the oleic acid control treatment, showing lower levels of acetate ester production.

The volatile thiol 4-mercapto-4-methylpentan-2-one (4MMP) is a potent contributor to wine aroma. In grape juice, 4MMP is bound to cysteine as a non-volatile compound and requires the action of yeast during fermentation to release the aroma active thiol. A method was developed to measure 4MMP release from the precursor by headspace solid-phase microextraction and separation by gas chromatography with atomic emission detection to screen the ability of wine yeast to release 4MMP. Yeast commonly used in white wine making were grown with the precursor at two different temperatures, and the amount of 4MMP released was measured. The results demonstrate that yeast strain selection and fermentation temperature can provide an important tool to enhance or modulate the grape-derived aromas formed during wine fermentation.

The aromatic metabolites derived from yeast metabolism determine the characteristics of aroma and taste in wines, so they are considered of great industrial interest. Volatile esters represent the most important group and therefore, their presence is extremely important for the flavor profile of the wine. In this work, we use and compare two Saccharomyces cerevisiae yeast strains: P29, typical of sparkling wines resulting of second fermentation in a closed bottle; G1, a flor yeast responsible for the biological aging of Sherry wines. We aimed to analyze and compare the effect of endogenous CO2 overpressure on esters metabolism with the proteins related in these yeast strains, to understand the yeast fermentation process in sparkling wines. For this purpose, protein identification was carried out using the OFFGEL fractionator and the LTQ Orbitrap, following the detection and quantification of esters with gas chromatograph coupled to flame ionization detector (GC-FID) and stir-bar sor...

Wine is a worldwide known beverage, and even though its consumption has been associated with the reduction of heart diseases and the extent of lifespan, it also has compounds that might cause adverse effects on human health such as methanol and acetaldehyde. The aim of this study was to determine the effect of time, temperature, and pectic enzymes over wine methanol and acetaldehyde concentrations during vinification. Three temperatures (20, 30, and 35 °C) and three pectic enzyme concentrations (0, 9, and 18 mL/Kg) were tested, letting fermentation to stop due to sugar depletion. Both, metanol and acetaldehyde were quantified throughout the fermentation process. Temperature reduced metanol production, observing the lowest metanol concentration (53.543 ± 3.267 mg/100 mL of wine) at 35 °C in the absence of pectic enzyme. Acetaldehyde was not affected by these variables. Alcohol, metanol, and acetaldehyde concentrations were adjusted to mathematical models with high correlations.

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. was observed in 80% of the samples collected. Dominance of was reflected in the bacterial load (/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 . 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 , these isolates were also characterized based on technological tests such as ethanol tolerance (12 and 15%), SO resistance (0 and 80 mg l), and pH (3.1 and 3.6) and malic acid tran...

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.

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%), SO 2 (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.

Valentina Martin1, Laura Fariña1, Karina Medina1, María J. Valera1, Eduardo Boido1, Eduardo Dellacassa2*, Albert Mas3, Francisco Carrau1 Area Enología y Biotecnología de Fermentaciones, Facultad de Química, Universidad de la República, Montevideo, Uruguay e-mail: vmartin@fq.edu.uy, lfarina@fq.edu.uy, kmedina@fq.edu.uy, mariajose_valera_martinez@hotmail.com, eboido@fq.edu.uy Laboratorio de Biotecnología de Aromas, Facultad de Química, Universidad de la República, Montevideo, Uruguay e-mail: edellac@fq.edu.uy Departament Bioquímica i Biotecnologia, Facultat d'Enologia, Universitat Rovira i Virgili, Tarragona, España e-mail: albert.mas@urv.cat

Apiculate yeasts of the genus Hanseniaspora/Kloeckera are the main species present on mature grapes and play a significant role at the beginning of fermentation, producing enzymes and aroma compounds that expand the diversity of wine color and flavor. Ten species of the genus Hanseniaspora have been recovered from grapes and are associated in two groups: H. valbyensis, H. guilliermondii, H. uvarum, H. opuntiae, H. thailandica, H. meyeri, and H. clermontiae; and H. vineae, H. osmophila, and H. occidentalis. This review focuses on the application of some strains belonging to this genus in co-fermentation with Saccharomyces cerevisiae that demonstrates their positive contribution to winemaking. Some consistent results have shown more intense flavors and complex, full-bodied wines, compared with wines produced by the use of S. cerevisiae alone. Recent genetic and physiologic studies have improved the knowledge of the Hanseniaspora/Kloeckera species. Significant increases in acetyl ester...

In several grape varieties, the dominating aryl alkyl alcohols found are the volatile group of phenylpropanoid‐related compounds, such as glycosylated benzyl and 2‐phenylethyl alcohol, which contribute to wine with floral and fruity aromas after being hydrolysed during fermentation. Saccharomyces cerevisiae is largely recognized as the main agent in grape must fermentation, but yeast strains belonging to other genera, including Hanseniaspora, are known to predominate during the first stages of alcoholic fermentation. Although non‐Saccharomyces yeast strains have a well‐recognized genetic diversity, understanding of their impact on wine flavour richness is still emerging. In this study, 11 Hansenisapora vineae strains were used to ferment a chemically defined simil‐grape fermentation medium, resembling the nutrient composition of grape juice but devoid of grape‐derived secondary metabolites. GC–MS analysis was performed to determine volatile compounds in the produced wines. Our resul...

Background and Aims: The possibility to decrease wine volatile acidity (VA) is an important aspect in wine production. This applies in particular to wines that are produced from musts with high sugar concentration, where the osmotic pressure promotes an increase in acetic acid production by Saccharomyces cerevisiae. This study aimed to identify suitable yeast strains and fermentation temperature to undertake the alcoholic fermentation of high sugar musts. Methods and Results: To lower VA during fermentation of high sugar musts, two non-Saccharomyces yeast strains, Zygotorulaspora florentina and Starmerella bacillaris, were used in multistarter fermentations with S. cerevisiae at a fermentation temperature of 14 and 20 C. The fermentation temperature influenced the yeast behaviour and the composition of the two mixed fermentations. Conclusions: Independent of fermentation temperature, the mixed fermentations with Z. florentina performed best to reduce VA. Significance of the Study: Mixed fermentations with the non-Saccharomyces yeast strains Z. florentina and S. bacillaris may represent a valuable approach for the fermentation of high sugar musts.

Aims: The purpose of this work was to study the effect of L L-aspartic acid concentration on bacterial growth, D D-glucose fermentation and L L-malic acid consumption of Oenococcus úni NCFB 1707. Methods and Results: Bacterial cultures were performed in synthetic media. Bacterial growth, D D-glucose fermentation and L L-malic acid consumption were reduced when L L-aspartic acid concentration became excessive. This inhibitory effect of high concentrations of L L-aspartic acid on bacterial growth was also observed with several Oenococcus úni strains, except O. úni BL01. The L L-aspartic acid inhibitory effect on bacterial growth could be reduced by increasing the concentration of L L-glutamic acid. L L-glutamic acid transport was found to be competitively inhibited by L L-aspartic acid. In addition, an excessive amount of L L-aspartic acid modi®ed D D-glucose metabolism, with an overproduction of acetic acid and reduced ethanol production. Conclusions: Since L L-glutamic acid is an essential amio acid for the bacterial strain used, the L L-aspartic acid inhibitory effect on bacterial growth could be linked to its involvement in an antagonistic interaction with L L-glutamic acid. Signi®cance and Impact of the Study: Such antagonistic interactions between amino acids in O. úni strains could be another explanation for the dif®culties of inducing malolactic fermentation in wines.

Lactiplantibacillus plantarum strain UNQLp 11 is a lactic acid bacterium with the potential to carry out malolactic fermentation (MLF) in red wines. Recently, the complete genome of UNQLp 11 was sequenced and this strain possesses four loci of the enzyme β-glucosidase. In order to demonstrate that these glucosidase enzymes could be functional under harsh wine conditions, we evaluated the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (p-NPG) in synthetic wine with different ethanol contents (0%, 12%, and 14% v/v) and at different pH values (3.2, 3.5, and 3.8). Then, the hydrolysis of precursor n-octyl β-D-glucopyranoside was analyzed in sterile Pinot Noir wine (containing 14.5% v/v of ethanol, at different pH values) by headspace sorptive extraction gas chromatography-mass spectrometry (HSSE-GC/MS). The hydrolysis of p-NPG showed that β-glucosidase activity is very susceptible to low pH but induced in the presence of high ethanol content. Furthermore, UNQLp 11 was able to release the glycosilated precursor n-octyl, during MLF to a greater extent than a commercial enzyme. In conclusion, UNQLp 11 could improve the aromatic profile of the wine by the release of volatile precursors during MLF.

Malolactic fermentation is a complex process that involves many reactions aside from the decarboxylation of L-malic acid. But we still have only glimpses of that complexity. It is not clear if the phenolic composition and colour are affected by malolactic fermentation and, if so, to what extent. So, the aims of this study were: 1) to evaluate the behaviour of native Patagonian strains of Oenococcus oeni and Lactobacillus plantarum in two wine varieties, and 2) to analyse the effect of malolactic fermentation on the colour of these wines. Our results show that the survival of bacteria and L-malic acid decarboxylation is different depending on the lactic acid bacteria strain employed and the wine variety. In addition, we found that O. oeni can survive in wine even when L-malic acid is not being consumed. We found some correlations between MLF and colour-related parameters in Pinot noir but not for Merlot. In fact, some of the colour-related parameters measured in Merlot (total polyphenolic index, colour intensity, hue, as well as the CIELAB parameters) were affected even when L-malic acid was not being consumed.

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.

The aim of this work was to evaluate the changes due to acclimation to ethanol on the fatty acid composition of three oenological Lactobacillus plantarum strains and their effect on the resistance to ethanol and malic acid consumption (MAC). Lactobacillus plantarum UNQLp 133, UNQLp 65.3 and UNQLp 155 were acclimated in the presence of 6 or 10% v/v ethanol, for 48 h at 28°C. Lipids were extracted to obtain fatty acid methyl esters and analysed by gas chromatography interfaced with mass spectroscopy. The influence of change in fatty acid composition on the viability and MAC in synthetic wine was analysed by determining the Pearson correlation coefficient. Acclimated strains showed a significant change in the fatty composition with regard to the nonacclimated strains. Adaptation to ethanol led to a decrease in the unsaturated/saturated ratio, mainly resulting from an increase in the contribution of short-length fatty acid C12:0 and a decrease of C18:1. The content of C12:0 was related ...

Malolactic fermentation is a vital red wine-making process to enhance the sensory quality. The objective of this study is to elucidate the starter cultures' role in modifying phenolic compounds, biogenic amines, and volatile compounds after red wine malolactic fermentation. We initiated the malolactic fermentation in Cabernet Gernishct wine by using two Oenococcus oeni and two Lactobacillus plantarum strains. Results showed that after malolactic fermentation, wines experienced a content decrease of total flavanols and total flavonols, accompanied by the accumulation of phenolic acids. The Lactobacillus plantarum strains, compared to Oenococcus oeni, exhibited a prevention against the accumulation of biogenic amines. The malolactic fermentation increased the total esters and modified the aromatic features compared to the unfermented wine. The Lactobacillus plantarum strains retained more aromas than the Oenococcus oeni strains did. Principal component analysis revealed that different strains could distinctly alter the wine characteristics being investigated in this study. These indicated that Lactobacillus plantarum could serve as a better alternative starter for conducting red wine malolactic fermentation.

The effect of malolactic fermentation (MLF) on the flavour quality of cider was examined. Leuconostoc mesenteroides subsp. mesenteroides Z25 was used to start MLF taking place at 25°C for 12 days after the completion of alcoholic fermentation (AF) by Saccharomyces cerevisiae. Strain Z25 showed good activity in starting MLF of cider with 10% alcoholic concentration. The content of malic acid, whose high concentration gives negative organoleptic characteristics to the cider, dropped significantly from 4.0 g/l to 0.25 g/l via MLF. The concentration of lactic acid increased significantly from 0.99 g/l to 3.50 g/l, contributing to volatile acidity. The acetic acid content of the ciders was 0.74 g/l. Among 51 volatile compounds detected by GC-MS, higher alcohols, esters, and carbonyl compounds were formed in ciders through MLF. The total concentration of aromatic substances doubled compared to the controls. The occurrence of MLF started by strain Z25 enabled the cider containing more vola...

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.

Biogenic amines (BA) in wine represent a toxicological risk for the health of the consumer, with several trade implications. In this study 26 strains of Lactobacillus plantarum were analyzed for their ability to degrade BA commonly found during wine fermentation. Two strains of L. plantarum were selected in reason of their ability to degrade putrescine and tyramine. The degradation was assessed in vitro, both in presence of the BA and in presence of the specific chemical precursor and of producer bacteria. The two L. plantarum biotypes were found capable to work synergically. In addition, the survival in wine-like medium and the aptitude to degrade malic acid after alcoholic fermentation of the selected L. plantarum strains was analyzed. Our results suggest the potential application of wine L. plantarum strains to design malolactic starter cultures able to degrade BA in wine.

Starmerella bacillaris is a non-Saccharomyces yeast proposed for must fermentation together with Saccharomyces cerevisiae because of its high glycerol and moderate volatile acidity production. Furthermore, it was demonstrated that the same S. bacillaris strains that possess interesting technological properties exhibited antifungal activity against Botrytis cinerea, suggesting the release of this yeast in the vineyard. To obtain a positive effect during the following winemaking process, the maintenance of suitable concentrations of S. bacillaris is essential. Therefore, to obtain information on the survival of S. bacillaris, a small-scale field trial was performed. One week before the harvest, two different concentrations of S. bacillaris (106 and 107 cells/mL) were sprayed on Pinot grigio bunches, and the strain concentration was monitored by means of qPCR during the subsequent fermentation process. In addition, the combined effect of different winemaking techniques was evaluated, i...

The aromatic metabolites derived from yeast metabolism determine the characteristics of aroma and taste in wines, so they are considered of great industrial interest. Volatile esters represent the most important group and therefore, their presence is extremely important for the flavor profile of the wine. In this work, we use and compare two Saccharomyces cerevisiae yeast strains: P29, typical of sparkling wines resulting of second fermentation in a closed bottle; G1, a flor yeast responsible for the biological aging of Sherry wines. We aimed to analyze and compare the effect of endogenous CO 2 overpressure on esters metabolism with the proteins related in these yeast strains, to understand the yeast fermentation process in sparkling wines. For this purpose, protein identification was carried out using the OFFGEL fractionator and the LTQ Orbitrap, following the detection and quantification of esters with gas chromatograph coupled to flame ionization detector (GC-FID) and stir-bar sorptive extraction, followed by thermal desorption and gas chromatography-mass spectrometry (SBSE-TD-GC-MS). Six acetate esters, fourteen ethyl esters, and five proteins involved in esters metabolism were identified. Moreover, significant correlations were established between esters and proteins. Both strains showed similar behavior. According to these results, the use of this flor yeast may be proposed for the sparkling wine production and enhance the diversity and the typicity of sparkling wine yeasts.

Nowadays, grape berries mature earlier due to climate change. Higher sugar contents are observed, whereas lower ones in organic acids, leading to increased pH values. Those biochemical changes have direct consequences on the balance and quality of wines made from such grapes. Chemical acidification, commonly used to compensate for the lack of acidity in musts or wines, includes tartaric, malic, lactic, and citric acids. Fumaric acid (FA), naturally present in the grape berries at low concentrations and already authorized in the member states of the OIV to inhibit malolactic fermentation in wines, seems to be a promising alternative to those acids to lower the pH. However, the evolution of FA levels added at bottling and its impact on wine quality during its conservation have not yet been studied. Therefore, the aim of the present study was to develop and validate a simple method using liquid chromatography coupled with a diode array detector, which can be used routinely for the determination of the added fumaric acid in wines. The proposed and validated method uses a ProntoSIL C-18 analytical column and an isocratic elution with water acidified at 0.1% formic acid. The run time, including column cleaning with acetonitrile and re-equilibration, was 40 minutes.

Nowadays, grape berries mature earlier due to climate change. Higher sugar contents are observed, whereas lower ones in organic acids, leading to increased pH values. Those biochemical changes have direct consequences on the balance and quality of wines made from such grapes. Chemical acidification, commonly used to compensate for the lack of acidity in musts or wines, includes tartaric, malic, lactic, and citric acids. Fumaric acid (FA), naturally present in the grape berries at low concentrations and already authorized in the member states of the OIV to inhibit malolactic fermentation in wines, seems to be a promising alternative to those acids to lower the pH. However, the evolution of FA levels added at bottling and its impact on wine quality during its conservation have not yet been studied. Therefore, the aim of the present study was to develop and validate a simple method using liquid chromatography coupled with a diode array detector, which can be used routinely for the dete...

Background: The study conducted benchmarking research, assessed and identified suitable factors for the efficient benchmarking of Karnataka wines. As the Grape Processing Industry Board is being formed by the Wine Board of Karnataka (Wine Policy '2007'), which may have control of grape quality, standards formulation, label approval and market promotion, the study focused on recommending strategies for quality grape-growing, wine-making for label approvals. Methods: The study adopted the Quality Function Deployment (QFD) tool to build the House of Quality to benchmark Karnataka wines with the new world wines. Result: The findings demonstrate that the informative label was given high importance by customers during wine purchases. The wine industry to meet this requirement should focus more, on the factor-crushing method of grapes. Karnataka wineries are inferior to wine from other New World regions when it comes to other aspects such as flavour, colour, brand name, grape variety, meal pairings, alcohol content, wine label designand label information.

The aim of this research was to investigate the efficiency of microwaves (MW) for wine microbiological stabilization and thereby assess the potential application of a Weibullian mathematical model in wine industry. The study focused on (i) the influence of treatment time (come-up time and temperature maintenance) and temperature on a simple wine-like matrix, as well as (ii) the specific resistance of yeast and bacteria selected according to their representativeness in red wines (A. aceti ATCC_15973, S. cerevisiae FX10, B. bruxellensis CRBO_L0619 and AWRI_1499), and finally (iii) the potential usefulness of a new mathematical model adapted to heat treatment to evaluate strain sensitivity. This research aimed to show that microwave treatment decreases the main wine-associated microorganism populations and that the reduction in viable counts mainly depends on the MW treatment temperature. Complete microbiological stabilisation was obtained under a temperature of 50 °C a very short trea...

Vitis vinifera L. cv Graciano is often used as a blending partner of Tempranillo based wines because it is considered to contribute significantly to the quality. The aim of this study is to discriminate between Tempranillo and Graciano monovarietal wines, and those made by the incorporation of 20% of Graciano variety in two different stages (at the end of malolactic fermentation and mixing the two grape varieties in the pre-fermentative maceration stage) of the winemaking process of the Tempranillo variety. To achieve this, supervised and unsupervised pattern recognition tools were applied to the data obtained in the study of the detailed polyphenolic composition, colour and other oenological parameters (143 variables). Patterns related to stages in the winemaking and ageing process, different wines and vintages can be observed using principal component analyses. Furthermore, linear discriminant analysis has been applied in order to characterise the wine samples. From the 143 variables, flavan-3-ols have exerted a profound influence on wine differentiation.

Non-wine yeasts could enhance the aroma and organoleptic profile of wines. However, compared to wine strains, they have specific intolerances to winemaking conditions. To solve this problem, we generated intra-and interspecific hybrids using a non-GMO technique (rare-mating) in which non-wine strains of S. uvarum, S. kudriavzevii and S. cerevisiae species were crossed with a wine S. cerevisiae yeast. The hybrid that inherited the wine yeast mitochondrial showed better fermentation capacities, whereas hybrids carrying the non-wine strain mitotype reduced ethanol levels and increased glycerol, 2,3-butanediol and organic acid production. Moreover, all the hybrids produced several fruity and floral aromas compared to the wine yeast: bphenylethyl acetate, isobutyl acetate, c-octalactone, ethyl cinnamate in both varietal wines. Sc 3 Sk crosses produced three-to sixfold higher polyfunctional mercaptans, 4-mercapto-4-methylpentan-2-one (4MMP) and 3-mercaptohexanol (3MH). We proposed that the exceptional 3MH release observed in an S. cerevisiae 3 S. kudriavzevii hybrid was due to the cleavage of the non-volatile glutathione precursor (Glt-3MH) to detoxify the cell from the presence of methylglyoxal, a compound related to the high glycerol yield reached by this hybrid. In conclusion, hybrid generation allows us to obtain aromatically improved yeasts concerning their wine parent. In addition, they reduced ethanol and increased organic acids yields, which counteracts climate change effect on grapes.

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La vinificación de varietales tintos patagónicos ocurre en abril y mayo, con bajas temperaturas ambientales que inhiben el progreso de la fermentación maloláctica (FML), por ser un factor de estrés para las bacterias lácticas (BAL) que conducen el proceso. Las bodegas deben calentar los tanques de fermentación, incrementando los costos de producción. El desarrollo de cultivos malolácticos nativos, tolerantes a bajas temperaturas, constituye una herramienta estratégica para resolver el problema y potenciar el carácter regional de los vinos. El objetivo del trabajo consistió en obtener cepas enológicas patagónicas de BAL, capaces de sobrevivir y consumir ácido Lmálico en vinificaciones a bajas temperaturas (4 y 10 oC). Los aislamientos psicrotolerantes procedieron de vino Pinot noir (bodega comercial de General Roca, Río Negro). Su identificación se realizó por secuenciación del gen 16S rRNA y la similitud genética se analizó por RAPD-PCR con el primer M13. Como primer criterio de sel...

El objetivo de esta tesis ha sido abordar un estudio global, tanto a nivel transcripcional como funcional, de los genes y proteinas de O. oeni involucrados en su adaptacion a las condiciones del vino y al desarrollo de la FML. A partir del estudio transcripcional hemos observado una relacion entre el nivel de expresion de ciertos genes y las condiciones particulares del cultivo. Tambien hemos estudiado por primera vez en vino tinto, la expresion de un gen que codifica para la enzima β-glucosidasa. Adicionalmente, hemos encontrado que diversas proteinas se ven afectadas luego del choque con 12% etanol. Estas estan relacionadas con la biosintesis y estabilidad proteica y a la biosintesis de la pared celular. Estos resultados contribuyen a una mejor compresion de los mecanismos relacionados con la respuesta fisiologica de O. oeni durante la adaptacion a las condiciones del vino y proveen informacion para la caracterizacion de cepas.

Malolactic fermentation is a complex process that involves many reactions aside from the decarboxylation of L-malic acid. But we still have only glimpses of that complexity. It is not clear if the phenolic composition and colour are affected by malolactic fermentation and, if so, to what extent. So, the aims of this study were: 1) to evaluate the behaviour of native Patagonian strains of Oenococcus oeni and Lactobacillus plantarum in two wine varieties, and 2) to analyse the effect of malolactic fermentation on the colour of these wines. Our results show that the survival of bacteria and L-malic acid decarboxylation is different depending on the lactic acid bacteria strain employed and the wine variety. In addition, we found that O. oeni can survive in wine even when L-malic acid is not being consumed. We found some correlations between MLF and colour-related parameters in Pinot noir but not for Merlot. In fact, some of the colour-related parameters measured in Merlot (total polyphenolic index, colour intensity, hue, as well as the CIELAB parameters) were affected even when L-malic acid was not being consumed.

Spontaneous malolactic fermentation (MLF) in the North Patagonian region of Argentina may have to bear low environmental temperatures, an important stress factor for the lactic acid bacteria (LAB) that conduct this process. Thus, inducing MLF by inoculation of a native starter culture able to lead this biological process at low temperatures is encouraged because it would allow preserving the regional character of the wine. The goal of this work was to obtain autochthonous Oenococcus oeni strains able to consume L-malic acid when MLF takes place at temperatures lower than 15 °C. Among the psychrotrophic isolates obtained from a Pinot noir wine, sixteen O. oeni strains were identified by sequencing the 16S rRNA gene and typing by RAPD PCR, allowing the differentiation of five genotypic groups. After the evaluation of the viability and metabolic performance of the strains in sterile wine at low temperatures, strains UNQOe4 and UNQOe19 were selected to evaluate their behavior in a non-sterile wine at 4 and 10 °C. The strains selected showed good implantation capacity and were able to successfully conduct MLF, suggesting that their use as starter cultures is suitable to improve this biological process in a wine region where environmental temperatures can be low.

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 UNQOe19 is a native strain isolated from a Patagonian pinot noir wine undergoing spontaneous malolactic fermentation. Here, we present the 1.83-Mb genome sequence of O. oeni UNQOe19, the first fully assembled genome sequence of a psychrotrophic strain from an Argentinean wine.

The correct development of malolactic fermentation depends on the capacity of Oenococcus oeni to survive under harsh wine conditions. The presence of ethanol is one of the most stressful factors affecting O. oeni performance. In this study, the effect of ethanol addition (12% vol/vol) on O. oeni PSU-1 has been evaluated using a transcriptomic and proteomic approach. Transcriptomic analysis revealed that the main functional categories of the genes affected by ethanol were metabolite transport and cell wall and membrane biogenesis. It was also observed that some genes were overexpressed in response to ethanol stress (for example, the heat shock protein Hsp20 and a dipeptidase). Proteomic analysis showed that several proteins are affected by the presence of ethanol. Functions related to protein synthesis and stability are the main target of ethanol damage. In some cases the decrease in protein concentration could be due to the relocation of cytosolic proteins in the membrane, as a protective mechanism. The omic approach used to study the response of O. oeni to ethanol highlights the importance of the cell membrane in the global stress response and opens the door to future studies on this issue.

The correct performance of wine malolactic fermentation (MLF) depends on the metabolic characteristics of the Oenococcus oeni strain/s responsible for this process. This study characterizes four O. oeni strains, which behave differently in terms of malolactic performance, the citric acid use related to acetic acid production, and stress adaptation. Metabolic evolution and its associated enzymatic activities were studied and the transcriptional response of the genes related to MLF, citrate metabolism and stress response was compared among strains. A higher initial expression of both the malolactic enzyme and the encoding gene mleA may be related to faster MLF. The initial transcriptional levels of citrate lyase (citE) proved indicative of early citrate consumption. Moreover, the strains that performed best in wine-like conditions presented a much higher relative expression of several stress responsive genes, particularly hsp18, clpP, ctsR and rmlB. Finally, an interstrain comparison of the transcriptional levels of selected genes at different times during MLF proved a useful tool in characterizing strains based on their metabolic behaviour.

The consumption of citrate by the malolactic bacterium Oenococcus oeni changes the aromatic profile of wines due to the production of volatile compounds such as diacetyl and acetic acid. In this study, the expression of genes related to citrate utilization in the O. oeni strain PSU-1 was investigated to further understand the role of this metabolic pathway in the adaptation to wine environment and its impact on organoleptic qualities. Different conditions of ethanol content (0% and 10%) and pH (3.5 and 4.0) were assayed to evaluate the transcriptional response to both these stress factors. In the presence of ethanol, metabolic and transcriptional behavior was different than the observed when ethanol was absent. The expression of citrate pathway genes was mainly affected by ethanol, while pH showed a lower effect. Among the studied genes, citE, ackA and alsD were the genes revealing a distinctive transcriptional response. The differences observed in gene expression were in correlation with the different content of end products such as acetic acid and diacetyl. The increment of gene expression observed in the presence of ethanol at low pH suggests the participation of citrate metabolism in the response to stress conditions.

Some phenolic acids can either inhibit or stimulate the growth of Oenococcus oeni and other lactic acid bacteria (LAB) in wine. It has been observed that some LAB metabolism could have an influence on wine colour. In this article, some of the relationships between LAB, malolactic fermentation (MLF) and phenolic compounds are summarised; these relationships are important for the selection of LAB to make starter cultures and are of interest for wineries in terms of its effect on wine colour.

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 SO 2 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.

) is a biological process that contributes to wine quality,but it is frequently affected by various vinification conditions. Resistance to four wine-limiting factors was studied with respect to 10 Oenococcus oeni strains in order to select a suitable strain for performing reliable MLF in difficult wines. Resistance to low fermentation temperature, high SO2 and/or ethanol concentration, and low pH were assayed in laboratory tests. A pool of the most resistant strains was used in a set of laboratory MLFs. At the end of fermentation, the dominant strains were identified by RAPD-PCR. The PN4 strain was found to be dominant in the majority of cases and under the most detrimental wine conditions, and it was therefore chosen as the single-strain inoculum for the subsequent MLF trials, The effectiveness of the PN4 strain was confirmed in a series of MLFs carried out in three different countries under experimental and industrial conditions. It accomplished MLF in wines with up to 15.8% ethanol, pH as low as 3.0,60 mg/L of free SO2, and in fermentation temperatures below 17°C. Our findings indicate that the 0. oeni PN4 strain could be an effective starter, guaranteeing regular and reliable MLF fermentation.

The exploitation of organo-silica immobilized lactic acid bacteria (LAB) to perform malolactic fermentation (MLF) in wine is described. The immobilization of a large amount of Oenococcus oeni cell culture was achieved by a two-step process in an original pilot plant. Cells are entrapped in Ca-alginate microbeads, coated with an organo-silica membrane obtained by two treatments: the first a sol suspension of tetraetoxysilane, the second using methyltriethoxysilane in gas phase. The resulting material improves the physico-chemical features of alginate, avoids cell leakage during fermentation, and protects the cells from antimicrobial compounds. In MLFs carried out at the microvinification scale, the activity of immobilized cells did not differ from that of free cells, and no differences were found in the chemical composition of the wines obtained. The use of immobilized bacteria allowed: (1) simultaneous alcoholic and malolactic fermentations in must inoculated with free yeast and immobilized bacteria; (2) the sequential MLF of three wine lots with the same biomass of immobilized bacteria; (3) the achievement of MLF in a wine with lysozyme added to suppress wild LAB and their potential spoilage.