Yeasts Inoculation Effect on Bacterial Development in Carbonic Maceration Wines Elaboration.

Carbonic maceration (CM) vinification is a very traditional method that allows saving energy without great equipment investment, obtaining high-quality wines. However, due to its particularities, CM winemaking implies a higher risk of microbial alteration. This work studies the evolution of bacterial population along carbonic maceration wines elaboration with and without yeast inoculation. In the same way, two strategies of yeast inoculation were studied: "pied de cuve" and Active Dry Yeasts (ADY) seed. For this purpose, three conditions were assayed: spontaneous fermentation (without inoculation), "pied de cuve" technology, and ADY inoculation. For each condition, two winemaking methods were compared: carbonic maceration and the standard method of destemming and crushing (DC). The bacterial evolution (lactic acid and acetic acid bacteria) was followed in different fermentation stages. Finally, the wines obtained were analysed (pH and volatile acidity). In the non-inoculated wines produced by CM, high development of the bacterial population was observed (counts of acetic acid bacteria around 4.3 log cfu/mL), and finished wines presented high values of volatile acidity (>1.5 g/L), which did not occur in the inoculated vinifications (counts of acetic acid bacteria around 1.5 log cfu/mL and 0.5 g/l of volatile acidity). Thus, the control of yeast population, as a "pied de cuve" as ADY seed, seems to be an effective tool to avoid bacterial alterations in CM vinifications.

Carbonic maceration vinification: A tool for wine alcohol reduction.

The increase in alcohol content in wine due to climate change is one of the main challenges that winemakers face today. Previous studies have indicated that carbonic maceration processing can be used to obtain a wine fraction with lower alcoholic strength. The aim of this study was to determine the effectiveness of this technique in obtaining wines with lower alcohol content. For this purpose, seven trials were carried out, studying a total of 63 wines. The physico-chemical, phenolic and aromatic composition of the wines was analysed using official methods and gas chromatography. The results showed that it is possible to obtain a fraction of carbonic maceration wine (25-35% of the total) with a reduction in alcohol content of up to almost 4% depending on the vinification conditions and the grape variety used. Therefore, this CM fraction, marketed on its own, could be a low-alcohol alternative to red wines.

Influence of the temperature and the origin of CO2 (anaerobiosis methodology) on the intracellular fermentation of wines made by carbonic maceration.

BACKGROUND: The present study aimed to determine the optimal conditions for making wines by the carbonic maceration (CM) method. In this test, we attempted to identify the causes of the higher volatile acidity of some wines vinified by this method. Accordingly, we measured the development and speed of intracellular fermentation inside the whole grapes under different vinification conditions. RESULTS: An active fermentation of the must in the tanks produced by inoculation with active dry yeasts was more efficient for the process than the addition of exogenous carbon dioxide (CO2 ). In addition, in CM vinification, the moment of devatting had a great influence on the content of acetaldehyde and acetic acid in the whole grapes. CONCLUSION: Yeast inoculation in the tanks and control of the devatting time are technological tools that could play an important role in the characteristics of the wines produced by carbonic maceration, especially with respect to acetic acid content. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Ethephon foliar application: Impact on the phenolic and technological Tempranillo grapes maturity.

In recent years, there has been increasing concern about the impact of climate change on viticulture, in particular regarding its influence on grape maturity and quality. Extensive research has shown that the rise of temperatures during grape ripening accelerates the accumulation of sugars while reducing the synthesis of phenolic and aromatic compounds. The aim of this study was to investigate whether the foliar application of the plant growth regulator ethephon might delay technological grape maturity and increase grape phenolic content, improving wine quality. For this, different concentrations of ethephon were applied during two vintages to the Tempranillo grape variety at the onset of veraison. Results showed that grape sugar accumulation was delayed in one of the two vintages, whereas the treatment favored the accumulation of phenolic compounds in both vintages. In conclusion, the application of ethephon at the onset of veraison to Tempranillo grapevines helped to couple grape phenolic and technological maturity, leading to more balanced wines, either by delaying sugar accumulation or by enhancing the synthesis of anthocyanins and phenolic compounds. PRACTICAL APPLICATION: Due to the current climate change, phenolic maturity of grapes does not match with their technological maturity, which means that harvested grapes are usually very sweet but without enough color and aroma. The foliar application of the plant growth regulator-ethephon-delays technological grape maturity and increases grape phenolic content, improving wine quality and creating wines that are more balanced.

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.

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.

Evaluation of lysozyme to control vinification process and histamine production in Rioja wines.

Lysozyme and the reduction of metabisulfite addition to red wine were evaluated during a winemaking process and after malolactic fermentation (MLF). Treatment, with lysozyme, of the must from Tempranillo grapes and at the end of alcoholic fermentation (AF) caused the 100% implantation of the inoculated bacterial strain and shortened the duration of MLF by 7 days. At the end of the MLF, wines treated with lysozyme showed lower volatile acidity, color intensity, and biogenic amine content. The differences in color intensity disappeared during wine stabilization. The lysozyme addition after MLF led to lower histamine concentrations in wines. These phenomena occurred irrespective of the lactic acid bacteria (LAB) proliferation control and of the Oenococcus oeni dominant strain identified at this period. The results of this study show the significance of preventive use of lysozyme in vinification of red wine to maintain low histamine levels and ensure a successful implantation of inoculated Oenococcus oeni starters.

The occurrence of fungi, yeasts and bacteria in the air of a Spanish winery during vintage.

This research studies the presence of microorganisms of enological interest (yeasts, bacteria and molds) and their evolution in the air of a wine cellar. The samples were taken throughout the winemaking campaign (September-December) in a winery of the D.O.Ca. Rioja, Spain. They were collected using an airIDEAL atmosphere sampler from Biomerieux. For the isolation, specific selective media were used for each group of microorganisms. The results obtained indicate that the presence in the winery air of the various different microorganisms studied is directly related to the winemaking processes that are taking place in the winery. Thus, the number of molds present decreases once grapes have ceased to be brought into the winery. The maximum number of yeasts in the air is found when all the vats in the cellar are fermenting, while the lactic bacteria are not detected until the first malolactic fermentation begins. The species of yeasts and molds identified are also related to the winemaking processes. The coincidence of strains of Saccharomyces cerevisiae among those present in the vats during alcoholic fermentation and those isolated from the air, confirms the role of the latter as a transmitter of microorganisms.

Analysis of yeast population during spontaneous alcoholic fermentation: effect of the age of the cellar and the practice of inoculation.

An ecological study of spontaneous fermentation in red wine carried out in two wineries located in the same village of the D.O.Ca. Rioja appellation (Spain). These two wineries were very different: one was very old and the other commenced its operations with the 2000 vintage. The study was conducted over seven consecutive years (1997-2003); the first 3 years in the old winery; and the other 4 years in the new one. Another difference between the wineries was the use of commercial strains: no starters were ever used in the old winery and two known strains (A and B) were employed only in a white must vat in the new winery. The number of different Saccharomyces cerevisiae strains detected for each vintage and the frequency of their appearance varied between the old winery and the new winery. There was no one clearly dominant clone in the fermentations studied in the old winery, whereas in the vinifications monitored in the new winery, a clear majority strain was detected. The same S. cerevisiae clone took over the spontaneous fermentation of all the vintages studied in this new winery. Analysis of the mtDNA restriction pattern of this strain revealed that it was identical to one of the commercial strain used (starter A), despite the fact that the fermentation studied was spontaneous. Microbial analysis conducted on winery equipment and winery surfaces prior to the 2003 vintage revealed that starter A had become a resident of the winery with a percentage of 50% among all S. cerevisiae. The presence of non-Saccharomyces yeasts in the early stages of vinification also differed between the old and new wineries.