Wine corks decontamination using plasma activated water.

Cork taint provides off-odors and changes negatively wine composition. In fact, it is one of the most important causes of discarding bottled wine. 2,4,6-Trichloroanisole (TCA) is the most known molecule responsible of that problem. In this study, cork stoppers were artificially contaminated with a multi-pattern solution which contained different chloroanisoles and chlorophenols. Contaminated corks were immersed for 3 h in four Plasma Activated Water (PAW) generated during 1.5 min, 5 min, 15 min and 30 min. The products of OH•, NO• and NO2• with phenol were determined by HPLC for each PAW. After treating contaminated corks with PAW generated during 5 min, more than 72 % of TCA was removed and it was suggested OH• as the main reactive species decomposing TCA. Finally, other chloroanisole and chlorophenol molecules were examined after PAW treatments showing successful reductions in almost every molecule. Thus, it was presented PAW treatment as an easy solution for solving cork taint problems in wine industry.

Development and Validation of a New Method for Detecting Acetic Bacteria in Wine.

In winemaking, excessive production of acetic acid by acetic acid bacteria poses a major challenge, leading to rejection of wine by consumers. The aim of this study was to devise an economically viable and easy-to-use liquid culture medium for the preventive detection of microorganisms capable of generating acetic acid in wine. The modified medium incorporated specific nutrients that favored the growth of acetic acid bacteria and increased selectivity. Under varying conditions and with different types of wine, this medium was tested together with inoculated samples, comparing the occurrence of acetic acid and olfaction. The result was a new liquid medium based on olfactometry, designed to facilitate its use in wineries, even by untrained personnel and without the need for complex laboratory equipment. Validation was carried out on a variety of wines, determining the onset of the presence of acetic acid in the medium. This innovative culture medium provides a means to estimate the concentration of micro-organisms capable of producing acetic acid in wine. Its application in wineries facilitates proactive decision making, avoiding undesirable increases in acetic acid concentration.

Influence of the carbonic maceration winemaking method on the physicochemical, colour, aromatic and microbiological features of tempranillo red wines.

The aim of the study was to determine the differences in the microbiological, physical-chemical, aromatic and phenolic composition between two winemaking methods: carbonic maceration and the standard method of destemming and crushing. We analysed 84 commercial Rioja wines made from the Tempranillo grape variety during the 2017 vintage, 40 had been made by carbonic maceration and 44 by destemming and crushing. Despite the heterogeneity within the two groups of wines, it was possible to differentiate between them. Wines made by carbonic maceration presented higher aromatic quality due to their higher total content of esters and acetates, as well as a greater colour intensity due to a higher phenolic content and higher rates of ionization and polymerization. In addition, it was observed that the antioxidant activity, the content in coumaroyl derivatives of anthocyanins and the vitisins A and B were considerably greater in wines made by carbonic maceration.

Effect of the Sequential Inoculation of Non-Saccharomyces/Saccharomyces on the Anthocyans and Stilbenes Composition of Tempranillo Wines.

The phenolic compounds of red wines are responsible for their color, astringency, and antioxidant properties. The fermentative yeasts might be used to modulate wines in terms of their color, aroma and probably healthy properties. In this study, six non-Saccharomyces species were tested because they might enhance the properties of red Tempranillo wines from Rioja. The results confirmed that the anthocyanins and stilbenes composition of wine can be modulated with the use of a specific fermentation starter. Metschnikowia pulcherrima, Zygosaccharomyces bailii, Candida zeylanoides, and Torulaspora delbrueckii achieved the greatest improvements of the monomeric anthocyanin composition, and the latter three yeast species achieved the best results of stilbene composition when compared to S. cerevisiae and the other non-Saccharomyces yeasts. Overall, results suggested that the use of M. pulcherrima, Z. bailii, C. zeylanoides and T. delbrueckii as fermentation starters could be of great interest to achieve wines with better color and likely healthy properties.

Wine aroma evolution throughout alcoholic fermentation sequentially inoculated with non- Saccharomyces/Saccharomyces yeasts.

Nine non-Saccharomyces yeasts belonging to 6 species (Torulaspora delbrueckii, Metschnikowia pulcherrima, Lachancea thermotolerans, Zygosaccharomyces bailii, Williopsis pratensis and Candida zeylanoides) and two mixed inoculum of T. delbrueckii and L. thermotolerans were screened for aroma formation and fermentative behaviour in sequential inoculations with Saccharomyces cerevisiae. The main differences in aromatic composition within wines were detected in the first stages of vinification between S. cerevisiae and non-Saccharomyces species, but not within the latter species. Most of the wines made with non-Saccharomyces in sequential fermentation with S. cerevisiae tended to produce higher ethanal and glycerol and lower volatile acidity than those inoculated only with S. cerevisiae. However, no significant differences were found in alcohol content. The addition of S. cerevisiae tended to standardise the wines and only those made with T. delbrueckii and L. thermotolerans, both alone and together, showed different aromatic profiles. Wines elaborated with non-Saccharomyces yeasts that quickly decreased in tanks showed characteristics similar than those made only with S. cerevisiae. Therefore, sequential inoculation of non-Saccharomyces/Saccharomyces is a useful tool to modulate wine characteristics, but only with species which remain longer in tanks. These findings can be useful to carry out selection processes within these species.

Evaluating a preventive biological control agent applied on grapevines against Botrytis cinerea and its influence on winemaking.

BACKGROUND: This research was aimed to study the influence on grape and wine quality and on the fermentation processes of the application of a preventive biological treatment against Botrytis cinerea in Tempranillo Rioja grapevines. For this purpose, a biofungicide containing Bacillus subtilis QST713 was applied twice to the vineyard. RESULTS: Results were compared with non-treated samples from the same vineyard and with samples treated with a chemical fungicide composed of fenhexamid. Data showed that general grape quality and spontaneous alcoholic fermentation performance were not influenced by either fungicide application. The chemical fungicide had a considerable influence on the clonal diversity of Saccharomyces cerevisiae species, which led to the alcoholic fermentation. Furthermore, it caused longer malolactic fermentation than with the biological fungicide and in the control. The biofungicide made malolactic fermentation 1 day shorter because the establishment of the commercial lactic acid bacteria used as a starter culture was total. After malolactic fermentation, the wines did not show significant differences in general oenological parameters. CONCLUSION: Preventive biofungicide treatment against B. cinerea did not negatively influence the quality of grapes and wines and the fermentation processes. Therefore, biological control of B. cinerea with B. subtilis applied on grapevines could be advisable in oenological terms. © 2018 Society of Chemical Industry.

Impact of Chemical and Biological Fungicides Applied to Grapevine on Grape Biofilm, Must, and Wine Microbial Diversity.

This study was aimed to measure the impact of the application of a bio-fungicide against Botrytis cinerea on the microbiota involved in the alcoholic fermentation (AF) of Tempranillo Rioja wines. For this purpose, a bio-fungicide composed of the biological control bacterium Bacillus subtilis QST713 was applied to the vineyard. The microbial diversity was analyzed from grape biofilm to wine. Impact on microbial diversity was measured employing indexes assessed with the software PAST 3.10 P.D. Results were compared to non-treated samples and to samples treated with a chemical fungicide mainly composed by fenhexamid. Overall, the impact of the biological-fungicide (bio-fungicide) on the microbial diversity assessed for grape biofilm and for musts was not remarkable. Neither of the tested fungicides enhanced the growth of any species or acted against the development of any microbial groups. The bio-fungicide had no significant impact on the wine microbiota whereas the chemical fungicide caused a reduction of microbial community richness and diversity. Although environmental threats might generate a detriment of the microbial species richness, in this study the tested bio-fungicide did not modify the structure of the microbial community. Indeed, some of the Bacillus applied at the grape surface, were detected at the end of the AF showing its resilience to the harsh environment of the winemaking; in contrast, its impact on wine quality during aging is yet unknown.

Microbial inactivation and MLF performances of Tempranillo Rioja wines treated with PEF after alcoholic fermentation.

This study was performed with the aim of reducing the microbial communities of wines after alcoholic fermentation to improve the establishment of commercial Oenococcus oeni inoculum for developing the malolactic fermentation. Microbial community reduction was accomplished by applying Pulsed Electric Field (PEF) technology to four different wines. Overall, significant reductions in yeast population were observed. To a lesser extent, lactic acid bacteria were reduced while acetic acid bacteria were completely eliminated after the PEF treatment. In three out of the four tested wines, a decrease in the competitive pressure between microorganisms due to the detected reduction led to a general but slight shortening of the malolactic fermentation duration. In the wine with the most adverse conditions to commercial starter establishment, the shortest malolactic fermentation was reached after PEF treatment. Finally, the sensorial quality of three out of the four treated wines was considered better after the PEF treatment. Therefore, PEF technology meant an important tool for improving the malolactic fermentation performance.

Screening of enzymatic activities within different enological non-Saccharomyces yeasts.

Ninety-seven non-Saccharomyces wine yeast strains belonging to ten different genera and species (Candida spp. and Criptococcus spp.; Debaryomyces hansenii, Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia kluyveri, Sporidiobolus salmonicolor, Torulaspora delbrueckii, Williopsis pratensis and Zygosaccharomyces bailii) were screened for 13 enzymes related to wine aroma, color and clarity. Understanding the yeasts' influence in these wine characteristics provides a platform for selecting strains for their development as starter cultures and for the management of alcoholic fermentation. Most of the strains showed the presence of one or more enzymes of biotechnological interest. Our screening demonstrated several intraspecific differences within the yeast species investigated, indicating that strain selection is of great importance for their enological application, and also that some non-Saccharomyces that have not been thoroughly explored, may deserve further consideration. This research represents the first stage for selecting non-Saccharomyces strains to be used as a starter along with Saccharomyces cerevisiae to enhance some particular characteristics of wines.

Phenylalanine and urea foliar application: Effect on grape and must microbiota.

The main aim of this study was to describe the impact of foliar phenylalanine and urea application on grape and must microbial populations. The tool used to perform the ecological study was DGGE conducted with several infusions in non-enriched and enriched liquid media, as well as direct DNA extractions of grapes and musts. A total of 75 microbial species were found in the study. The alpha diversity indices of grape after both foliar nitrogen treatments did not show significant changes in comparison to the control samples, but were modified in some indices in must samples. The phenylalanine must sample was similar to the control, while foliar urea application caused significant changes in microbial diversity and population structure in comparison to the control must. Further research would be necessary to properly predict the impact on winemaking of the effects observed in this study for grape and must microbiota, especially regarding the foliar application of urea.

Genomic diversity of Oenococcus oeni populations from Castilla La Mancha and La Rioja Tempranillo red wines.

The Oenococcus oeni populations of Tempranillo wines from Castilla La Mancha and La Rioja winemaking regions were analysed from one to three years and up to ten wineries. The objective was to evaluate the genetic variability and the O. oeni population structure. For this purpose a MLST scheme based on four loci (gyrB, purK, pgm and recP genes) and PFGE with SfiI restriction enzyme were developed for later combination. The results showed an O. oeni population completely adapted to winemaking regions. A purifying selection influenced the genes evolution, especially recP that along with purK were the most interesting loci to analyse the genetic variability of the isolates. In this way linkage disequilibrium and intergenic and intragenic recombination were determined between isolates. PFGE typing with UPGMA data were not coincident with the phylograms assessed for MLST by Maximum likelihood and combination of both techniques differentiated all the isolates as strains. Those results led the research to conclude that O. oeni population from CM and LR was a panmictic population with a slight clonal evolution, so subpopulations could not be described. A broader study including more winemaking regions with different grape varieties and distinct ways of elaborating would be interesting to complete the knowledge about O. oeni populations.

Methyl jasmonate foliar application to Tempranillo vineyard improved grape and wine phenolic content.

The importance of phenolic compounds for grape and wine quality has drawn attention to studying different practices with the aim of increasing their content. Cluster application of elicitors is a viticultural practice that has shown promising results in recent years. However, cluster application requires a previous defoliation, which is time-consuming and expensive. In the present study, methyl jasmonate was foliar applied to Tempranillo grapevines in order to study its effect on grape and wine phenolic composition. Methyl jasmonate foliar application increased anthocyanin and stilbene content in both grape and wine, besides enhancing wine flavonol content. This treatment induced the synthesis of 3-O-glucosides of petunidin and peonidin and trans-p-coumaroyl derivatives of cyanidin and peonidin. For stilbenes, trans-piceid content was considerably increased in both grape and wine. The results obtained suggest that methyl jasmonate foliar application could be a simple and accessible practice to enhance grape and wine quality.

Molecular analysis of Oenococcus oeni and the relationships among and between commercial and autochthonous strains.

The presence and distribution of genotypes from malolactic starter cultures between the autochthonous microbiota in fermenting Rioja wines have been studied in this paper. The commercial cultures characterization allowed to identify different species and common pulsed field gel electrophoresis and randomly amplified polymorphic DNA genotypes in several brands of Oenococcus oeni starter cultures. Four indistinguishable genotypes were found between the commercial and the autochthonous O. oeni strains. These four genotypes appeared during different vintages in seven of the 10 sampled wineries despite bacterial starter cultures had never been used. Therefore, the detection of commercial LAB genotypes indistinguishable from the autochthonous ones involved their removal from a selection process. These genotypes represented the 17.7% of autochthonous O. oeni isolated in this region during three consecutive years. This fact demonstrated that performing similar studies as previous selection criteria is advisable to avoid large work and the proposal of one commercialized bacteria.

Oenococcus oeni strain typification by combination of Multilocus Sequence Typing and Pulsed Field Gel Electrophoresis analysis.

Oenococcus oeni is usually the main lactic acid bacteria (LAB) responsible for conducting malolactic fermentation (MLF) in wines. Pulsed Field Gel Electrophoresis (PFGE) is one of the most common methods used to identify different genotypes among the wine LAB populations. Although PFGE is a powerful typing tool, it is time-consuming and its results are not easily exchangeable between laboratories so typing methods such as Multilocus Sequence Typing (MLST) have been developed. In this study, thirty O. oeni isolates from Rioja Tempranillo wines were characterized performing SfiI and ApaI PFGE and MLST with eight housekeeping genes. Using the latter technique, six new alleles have been described for five genes. PFGE was slightly more efficient than MLST because of the number of genotypes and of the index of diversity (ID) that each technique discriminated. This has been the first time that PFGE and MLST results have been combined to shape a unique dendrogram. Thus, the combination of results from both typing methods allowed the discrimination of twenty-two PFGE-ST genotypes showing the highest ID of these research (0.947). According to these results, the future application of the combination of PFGE and MLST results could be successful for reliable O. oeni strain typification.

Pulsed electric field treatment enhanced stilbene content in Graciano, Tempranillo and Grenache grape varieties.

The purpose of this paper was to study the effect of pulsed electric fields (PEF) on the stilbene content of three grape varieties. For this purpose, four different PEF treatments were applied using a continuous system over three varieties, Graciano, Tempranillo and Grenache, destemmed and crushed. In addition, the influence of PEF on their physicochemical composition was studied. PEF treatments did not affect the pH or total acidity of Graciano, however, musts from Tempranillo and Grenache had higher pH values and lower total acidity. In the three varieties, all treatments resulted in an increase of potassium content, deeper colour intensity and total polyphenol index and lower tonality, more pronounced in the treatments with higher time and energy. The stilbene content of the must significantly increased with respect to the control. This increase depended on the variety and the treatment applied. Tempranillo increased up 200% the total stilbene concentration, Grenache 60% and Graciano 50%. For the three varieties, the treatment with the highest time and energy was the most effective on the total stilbene extraction. These results indicate that PEF could be a suitable technology for obtaining musts with deeper colour and higher phenolic content, including resveratrol and piceid.

Dynamics of lactic acid bacteria populations in Rioja wines by PCR-DGGE, comparison with culture-dependent methods.

Lactic acid bacteria populations of red wine samples from industrial fermentations, including two different vinification methods were studied. For this investigation, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis was employed to supplement previous results that were obtained by culture-dependent methods. PCR-DGGE was aimed to study two targeted genes, 16S ribosomal DNA (rDNA) and rpoB, and the results were useful to evaluate the microbial populations in wine samples. Moreover, an improvement of a detection limit determined so far for DGGE analysis was obtained with the method described in this study, what made possible to identify lactic acid bacteria populations below 10(1) colony-forming unit/mL. The species Oenococcus oeni was the most frequently detected bacterium, but identifications close to species Oenococcus kitaharae and Lactococcus lactis that are not often found in wine were firstly identified in samples of this research. PCR-DGGE allowed to detect 9 out of 11 lactic acid bacteria species identified in this study (nine by PCR-16S rDNA/DGGE and four by PCR-rpoB/DGGE), while five species were detected using the modified de Man, Rogosa and Sharpe agar. Therefore, the two methods were demonstrated to be complementary. This finding suggests that analysis of the lactic acid bacteria population structure in wine should be carried out using both culture-dependent and culture-independent techniques with more than one primer pair.

Ecology of indigenous lactic acid bacteria along different winemaking processes of Tempranillo red wine from La Rioja (Spain).

Ecology of the lactic acid bacteria (LAB) during alcoholic fermentation (AF) and spontaneous malolactic fermentation (MLF) of Tempranillo wines from four wineries of La Rioja has been studied analyzing the influence of the winemaking method, processing conditions, and geographical origin. Five different LAB species were isolated during AF, while, during MLF, only Oenococcus oeni was detected. Although the clonal diversity of O. oeni strains was moderate, mixed populations were observed, becoming at least one strain with distinct PFGE profile the main responsible for MLF. Neither the winemaking method nor the cellar situation was correlated with the LAB diversity. However, processing conditions influenced the total number of isolates and the percentage of each isolated species and strains. The winemaking method could cause that genotypes found in semicarbonic maceration did not appear in other wineries. Four genotypes of O. oeni were isolated in more than one of the rest wineries. These four together with other dominant strains might be included in a future selection process.

Dynamics of indigenous lactic acid bacteria populations in wine fermentations from La Rioja (Spain) during three vintages.

Diversity of lactic acid bacteria (LAB) species has been analyzed for three consecutive years (2006, 2007, and 2008) during alcoholic and malolactic fermentations of Tempranillo wine in a winery at La Rioja. The results showed differences in malolactic fermentation duration, and in both diversity of LAB species and diversity of Oenococcus oeni genotypes. O. oeni was shown to be the predominant species (73% of total isolates). Monitoring the different strains of O. oeni using pulsed-field gel electrophoresis of chromosomal DNA digested with SfiI and ApaI allowed detection of a total of 37 distinct genotypes, most of them comprised at least two isolates. Six appeared in more than one vintage, one of them being present in the three studied years. Moreover, four genotypes were indistinct of the strains isolated from the air of this same winery in 2007 vintage. The frequency of participation of each genotype varied from year to year, thus dominant genotypes at one year were minority or not present at another year. This suggests that distinct indigenous O. oeni strains are better adapted to the different winery conditions every year. Predominant genotypes that appeared in more than one vintage and lead to quality wines with low histamine contents could be considered as interesting for selecting of new malolactic starter cultures.