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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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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 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.

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.

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. [ABSTRACT FROM AUTHOR]

Abstract The effects of pulsed electric field (PEF) technology on the microbiota of wines aging in oak barrels without additional chemical preservatives were studied. Two differently aged wines without sensory deviations, naturally having Brettanomyces/Dekkera, were PEF-treated. Results showed that the PEF treatment in continuous flow inactivated the most abundant spoilage microbial population of each wine. However, the recovery of some microorganisms after treatment was also observed. Therefore, to determine PEF as an alternative to the use of SO 2 , additional studied are needed with a wider sampling set. Highlights • Pulsed electric fields inactivated Brettanomyces/Dekkera in long-term aged wine. • Pulsed electric fields inactivated lactic acid bacteria in short-term aged wines. • Spoilage microorganisms were recovered throughout the study. • Volatile compounds were significantly lower in long-term aged wine. • Pulsed electric fields applied during ageing could be useful. [ABSTRACT FROM AUTHOR]

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 (biofungicide) 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. [ABSTRACT FROM AUTHOR]

Barrel aging is crucial for the production of high-quality wines, with barrel reuse playing a key role in this process. Therefore, disinfecting and cleaning barrels are vital to prevent health and safety issues, being B. bruxellensis one of the most extended problems. In this study, naturally contaminated oak barrels with B. bruxellensis were immersed during 3 h in four Plasma Activated Water (PAW) generated for 1.5 min, 5 min, 15 min and 30 min. The presence of secondary radicals (OH•, NO•, NO 2 •) was observed after HPLC and spectrometry analysis. The results suggested that those reactive species played an important role in the inactivation of B. bruxellensis. PAW_5 (generated during 5 min), which achieved a reduction of 3.49 ± 0.83 logarithmic units in B. bruxellensis population, was chosen as the best one in terms of economic and time consumption. Thus, an ecofriendly, sustainable and inexpensive solution was presented to inactivate B. bruxellensis from wine barrels. [Display omitted] • Oak barrels naturally contaminated with B. bruxellensis were disinfected using PAW. • Immersion in PAW_5 during 3 h achieved 3.49 log reduction of B. bruxellensis. • OH·, NO·, NO 2 · played a key role in inactivating B. bruxellensis in wood samples. • PAW could be a promising method to disinfect and clean oak wood barrels. [ABSTRACT FROM AUTHOR]

Four different treatments of pulsed electric field (PEF) technology were tested in a continuous-flow system for inactivating 25 species of yeast, lactic acid bacteria and acetic acid bacteria associated with wines. Overall, the reached inactivation varied from 0.64 to 4.94 log units. Curiously, it was determined that the application of higher specific energy did not provide significant declination of yeast population, although significantly different inactivations were determined for bacteria. The inactivation of bacteria highly depended on each species and results corroborated different sensitivity of two Oenococcus oeni strains what could be related with their genetic characteristics. [ABSTRACT FROM AUTHOR]

Abstract: This study presents a novel microwave technology application which investigates the ability of a new equipment prototype, based on high frequency microwaves, to reduce microbial populations in oak wine barrels. American and French oak staves from highly contaminated barrels were treated. It was demonstrated that microwave treatment was able to significantly decrease the main wine associated microorganism populations from the first 8 mm of oak surfaces. However, the reduction in viable counts depended on the studied microorganism. Thus, counts were reduced by 36%–38% for total yeast, from 35% to 67% for Brettanomyces and around 91%–100% for lactic acid bacteria and acetic acid bacteria, using a very short treatment time (3 min). Nowadays, there is not a convenient method for decontaminating barrels and microwave technology may be a more efficient treatment than currently available methods. Findings from this research suggested that microwave would be beneficial for wine industry and environment by increasing barrel functionality, reducing frequency of replacement, improving microbiological control of oak wood, as well as minimizing of preservative use. [Copyright &y& Elsevier]

Abstract: Indigenous lactic acid bacteria (LAB) communities have been analyzed for three years (2006, 2007 and 2008) during alcoholic (AF) and malolactic (MLF) fermentations of Tempranillo wines in ten wineries of La Rioja. The results showed that analytical composition of wines and physical–chemical conditions of elaboration influenced the LAB populations, the MLF duration and the percentage of each isolated species and strains. The highest diversity of LAB species was observed during AF in all the wineries. Oenococcus oeni was present in all studied stages of the fermentation process, being the predominant species at final AF stage. The study of 925 isolates of O. oeni by Pulsed Field Gel Electrophoresis (PFGE) allowed the detection of a total of 112 distinct genotypes. Most fermentation stages of both AF and MLF showed mixed O. oeni strain populations, so that there were different genotypes able to share their ecological niche or tank in spontaneous MLF. The frequency of participation of each genotype varied either from year to year or from winery to winery. Otherwise, seven genotypes were detected in the three studied years and in at least three out of the ten studied wineries, being four of them also present in the three studied subzones of this region. These results suggest the existence of an endemic microbiota in this region, the adaptation of indigenous O. oeni strains to the winery conditions every year and the interest of selecting predominant genotypes in order to preserve the biodiversity and peculiarity of these wines. [Copyright &y& Elsevier]

Abstract: The aim of this work was to study the effect of pH on biogenic amine formation during the elaboration and conservation (7 months) of Tempranillo wines. Grapes at two pHs (3.4 and 3.7) were vinified to achieve this purpose. After alcoholic fermentation the wines were either inoculated with a commercial malolactic starter or not inoculated. Identification and clonal distribution of lactic acid bacteria and amino acid concentration were determined in an attempt to explain the biogenic amine generation. The results showed that the pH of the must influenced the clonal distribution of the Oenococcus oeni strains which conducted the malolactic fermentation and also the concentration of amino acids in the wines after alcoholic fermentation. These aspects could account for the higher biogenic amine formation in wines with the lowest pH during malolactic fermentation. In these wines, inoculation with a malolactic starter was favourable since it produced a lower biogenic amine concentration after malolactic fermentation. Furthermore, the pH also influenced the concentration of amino acids after malolactic fermentation and the lactic acid bacteria distribution during the conservation of the wines. These aspects allow explaining the greater formation of histamine, tyramine and putrescine in the wines with the lowest pH vinified via lactic acid bacteria inoculation after seven months of conservation. [Copyright &y& Elsevier]