Non-Saccharomyces Yeasts from Organic Vineyards as Spontaneous Fermentation Agents.

Currently, non-Saccharomyces yeasts are the subject of interest, among other things, for their contribution to the aromatic complexity of wines. In this study, the characterisation of non-Saccharomyces yeasts was addressed by their isolation during spontaneous fermentations of organic Verdejo grapes, obtaining a total of 484 isolates, of which 11% were identified by molecular techniques as non-Saccharomyces yeasts. Fermentative isolates belonging to the species Hanseniaspora meyeri, Hanseniaspora osmophila, Pichia guilliermondii, Pichia kudriavzevii, Torulaspora delbrueckii, and Wickerhamomyces anomalus were analysed. Significant differences were found in the yeast populations established at the different fermentation stages. Interestingly, W. anomalus stood up as a widely distributed species in vineyards, vintages, and fermentation stages. Several of the strains studied stood out for their biotechnological potential in the production of Verdejo wine, showing the presence of relevant enzymatic activity for the release of varietal aromas and the technological improvement of the winemaking process. Three enzymatic activities were found in an important number of isolates, ß-glucosidase, protease, and ß-lyase, implicated in the positive aromatic impact on this style of white wine. In that sense, all the isolates of W. anomalus presented those activities. T. delbrueckii isolates were highlighted for their significant ß-lyase activity. In addition, T. delbrueckii was outlined because of its potential to achieve an elevated fermenting power, as well as the lack of lag phase. The results obtained highlight the importance of maintaining the microbial diversity that contributes to the production of wines with unique and distinctive characteristics of the production region.

Entrapment of Glucose Oxidase and Catalase in Silica-Calcium-Alginate Hydrogel Reduces the Release of Gluconic Acid in Must.

Glucose oxidase (GOX) and catalase (CAT) were co-immobilized in silica-calcium-alginate hydrogels to degrade must glucose. The effect of the enzyme dose (1.2-2.4 U/mL), the initial must pH (3.6-4.0), and the incubation temperature (10-20 °C) on the glucose consumption, gluconic acid concentration, pH, and color intensity of Verdejo must was studied by using a Box-Behnken experimental design and comparing free and co-immobilized enzymes. A reduction of up to 37.3 g/L of glucose was observed in co-immobilized enzyme-treated must, corresponding to a decrease in its potential alcohol strength of 2.0% vol. (v/v), while achieving a slight decrease in its pH (between 0.28 and 0.60). This slight acidification was due to a significant reduction in the estimated gluconic acid found in the must (up to 73.7%), likely due to its accumulation inside the capsules. Regarding the operational stability of immobilized enzymes, a gradual reduction in glucose consumption was observed over eight consecutive cycles. Finally, co-immobilized enzymes showed enhanced efficiency over a reaction period of 48 h, with an 87.1% higher ratio of glucose consumed per enzyme dose in the second 24 h period compared with free enzymes. These findings provide valuable insights into the performance of GOX-CAT co-immobilized to produce reduced-alcohol wines, mitigating excessive must acidification.

Silica-Calcium-Alginate Hydrogels for the Co-Immobilization of Glucose Oxidase and Catalase to Reduce the Glucose in Grape Must.

Higher temperatures due to climate change are causing greater sugar production in grapes and more alcoholic wines. The use of glucose oxidase (GOX) and catalase (CAT) in grape must is a biotechnological green strategy to produce reduced-alcohol wines. GOX and CAT were effectively co-immobilized by sol-gel entrapment in silica-calcium-alginate hydrogel capsules. The optimal co-immobilization conditions were achieved at a concentration of the colloidal silica, sodium silicate and sodium alginate of 7.38%, 0.49% and 1.51%, respectively, at pH 6.57. The formation of a porous silica-calcium-alginate structure was confirmed by environmental scanning electron microscopy and the elemental analysis of the hydrogel by X-ray spectroscopy. The immobilized GOX showed a Michaelis-Menten kinetic, while the immobilized CAT fits better to an allosteric model. Immobilization also conferred superior GOX activity at low pH and temperature. The capsules showed a good operational stability, as they could be reused for at least 8 cycles. A substantial reduction of 26.3 g/L of glucose was achieved with encapsulated enzymes, which corresponds to a decrease in potential alcoholic strength of must of about 1.5% vol. These results show that co-immobilized GOX and CAT in silica-calcium-alginate hydrogels is a promising strategy to produce reduced-alcohol wines.

Selective Wine Aroma Enhancement through Enzyme Hydrolysis of Glycosidic Precursors.

Selective enhancement of wine aroma was achieved using a broad spectrum of exogenous glycosidases. Eight different enzyme preparations were added to Verdejo wine, resulting in an increase in the levels of varietal volatile compounds compared to the control wine after 15 days of treatment. The enzyme preparations studied were robust under winemaking conditions (sulfur dioxide, reducing sugars, and alcohol content), and no inhibition of ß-glucosidase activity was observed. Significant differences were detected in four individual terpenes (α-terpineol, terpinen-4-ol, α-pinene, and citronellal) and benzyl alcohol in all the treated wines compared to the control wine, contributing to the final wine to varying degrees. In addition, a significant increase in the other aromatic compounds was observed, which showed different patterns depending on the enzyme preparation that was tested. The principal component analysis of the data revealed the possibility of modulating the different aromatic profiles of the final wines depending on the enzyme preparation used. Taking these results into account, enhancement of the floral, balsamic, and/or fruity notes of wines is possible by using a suitable commercial enzyme preparation.

Screening and Enzymatic Evaluation of Saccharomyces cerevisiae Populations from Spontaneous Fermentation of Organic Verdejo Wines.

Microbial populations in spontaneous winemaking contribute to the distinctiveness and quality of the wines. In this study, molecular methods were applied to 484 isolated yeasts to survey the diversity of the Saccharomyces cerevisiae population in spontaneous fermentations of organic Verdejo grapes. Identification was carried out at strain level for samples from different vineyards correct.and stages of the winemaking process over the course of two vintages, establishing 54 different strains. The number of isolates belonging to each strain was not homogeneous, as two predominant strains represented more than half of the isolates independent of vineyard or vintage. Regarding the richness and abundance, differences among the stages of fermentation were confirmed, finding the highest diversity values in racked must and in the end of fermentation stages. Dissimilarity in S. cerevisiae communities was found among vineyards and vintages, distinguishing representative groups of isolates for each of the populations analysed. These results highlight the effect of vineyard and vintage on yeast communities as well as the presence of singular strains in populations of yeasts. Oenologically relevant enzymatic activities, ß-lyase, protease and ß-glucanase, were detected in 83.9%, 96.8% and 38.7% of the isolates, respectively, which may be of interest for potential future studies.

Volatile composition and sensory properties of wines from vineyards affected by iron chlorosis.

Recent studies have shown that mild to moderate iron chlorosis can have positive effects on grape quality potential, including volatile profile. The main objective of this work was to investigate, for the first time, how moderate iron stress in grapevines affects the presence of volatile organic compounds (VOCs) in wines. The study was carried out during 2018-2019 seasons, in 20 Tempranillo vineyard subzones with different degree of iron deficiency, located in Ribera del Duero (North-Central Spain). The results showed that moderate iron stress increased in wines the concentrations of VOCs associated with floral notes, such as 2-phenylacetaldehyde, 2-phenylethanol and 2-phenylethyl acetate, while reducing the presence of C6-alcohols, responsible for green-herbaceous aroma. A favourable reduction of pH and a betterment of parameters related to colour were detected in wines from iron deficient subzones. Chlorosis incidence was associated to improvements in wine sensory attributes as layer intensity, black fruit and aroma intensity.

Relationships between chlorophyll content of vine leaves, predawn leaf water potential at veraison, and chemical and sensory attributes of wine.

BACKGROUND: Water deficit and iron deficiency (iron chlorosis) are common environmental stresses that affect grapevine production in the Mediterranean area. Studies on the impact of both stresses, when they act simultaneously, are rare. The main objective of this investigation was to evaluate the combined effects of the incidence of iron chlorosis and the vine water status on quality of Tempranillo wine. For this, 20 non-irrigated vineyard subzones (10 m × 10 m each), from non-affected to moderately affected by iron chlorosis, were monitored in the Ribera del Duero area (north-central Spain) during two consecutive seasons. RESULTS: Factorial analyses of variance were performed to study the effects of predawn leaf water potential and foliar chlorophyll content, both measured at veraison, on chemical and sensory characteristics of wine. With an impact much greater than water status, the incidence of iron stress decreased pH of the wine and enhanced sensory attributes as tonality, layer intensity, flavour intensity, and persistence in the mouth. There were increases in red colour, astringency, and persistence of the wine associated with chlorosis, although they might be restricted in water-deficit conditions. CONCLUSION: The results have demonstrated that mild to moderate iron stress can have positive effects on chemical and sensory attributes of Tempranillo wine. Measurements of foliar chlorophyll content at veraison could be very useful to map quality potential in rainfed vineyards affected by iron deficiency. © 2020 Society of Chemical Industry.

Evaluating the influence of simultaneous inoculation of SiO2-alginate encapsulated bacteria and yeasts on volatiles, amino acids, biogenic amines and sensory profile of red wine with lysozyme addition.

The influence of the timing of inoculation (sequential and simultaneous alcoholic fermentation (AF)/malolactic fermentation (MLF)) on the chemical and sensory properties of red wines was studied. The impact of the encapsulation of Oenococcus oeni into SiO2-alginate hydrogel (Si-ALG) and the addition of lysozyme in wines inoculated with encapsulated bacteria were also analysed. There was a significant influence of the timing of inoculation on the volatile composition of the wines just as on the amino acid and biogenic amine content. The wines produced by simultaneous AF/MLF showed the highest contents of some volatile compounds, such as ethyl esters and terpenes, as well as amino acids and tyramine. Bacterial encapsulation affected the volatile and amino acid profile of the wines, while the biogenic amine composition was not modified. The chemical composition of the wines was not altered by the presence of lysozyme. A trained panel did not perceive substantial differences between treatments.

Effect of stressful malolactic fermentation conditions on the operational and chemical stability of silica-alginate encapsulated Oenococcus oeni.

Oenococcus oeni was encapsulated into inter-penetrated polymer networks of silica-alginate (SiO2-ALG). Fourier transform infrared spectroscopy analysis proved the presence and the polycondensation of the siliceous material used in SiO2-ALG capsules. Environmental scanning electron microscopy showed that the structure of SiO2-ALG biocapsules was rougher than in alginate (ALG) biocapsules. The behaviour of SiO2-ALG biocapsules was evaluated at pH 3.0-3.6 and alcohol degrees of 12-15%. Repeated-batch malolactic fermentations (MLF) demonstrated that SiO2-ALG biocapsules can be reused efficiently for five times in either low-pH or high-ethanol wines, while free bacteria only can be used once under the most favourable MLF conditions. The inclusion of siliceous materials into ALG hydrogel improved the stability of the biocapsules, reducing their shrinking and achieving an excellent integrity under winemaking conditions. These results proved the possibility of industrial application of SiO2-ALG biocapsules in winemaking.

Highly Efficient Malolactic Fermentation of Red Wine Using Encapsulated Bacteria in a Robust Biocomposite of Silica-Alginate.

Bacteria encapsulation to develop malolactic fermentation emerges as a biotechnological strategy that provides significant advantages over the use of free cells. Two encapsulation methods have been proposed embedding Oenococcus oeni, (i) interpenetrated polymer networks of silica and Ca-alginate and (ii) Ca-alginate capsules coated with hydrolyzed 3-aminopropyltriethoxysilane (hAPTES). On the basis of our results, only the first method was suitable for bacteria encapsulation. The optimized silica-alginate capsules exhibited a negligible bacteria release and an increase of 328% and 65% in L-malic acid consumption and mechanical robustness, respectively, compared to untreated alginate capsules. Moreover, studies of capsule stability at different pH and ethanol concentrations in water solutions and in wine indicated a better behavior of silica-alginate capsules than untreated ones. The inclusion of silicates and colloidal silica in alginate capsules containing O. oeni improved markedly their capacity to deplete the levels of L-malic acid in red wines and their mechanical robustness and stability.

Research progress in coating techniques of alginate gel polymer for cell encapsulation.

Cell encapsulation is used as a biotechnology tool to solve the technological problems derived from handling and application of cells in a great range of fields. This involves immobilization of the cells within a polymeric gel that permits the preservation of their metabolic activity. Alginate is widely established as the most suitable polymer for cell encapsulation. However, alginate gel capsules suffer several disadvantages because of their lack of mechanical and chemical stability. This review summarizes results of recent advances in coating techniques that include ionic and covalent cross-linking between alginate and coating materials for cell encapsulation as a strategy to solve the disadvantages mentioned before. Throughout this review, physicochemical properties of coated-alginate capsules and the effect of coating process on metabolic activity and viability of immobilized cells have been specially discussed.

Immobilization of Oenococcus oeni in lentikats® to develop malolactic fermentation in wines.

Entrapment of Oenococcus oeni into a polymeric matrix based on polyvinyl alcohol (PVA) (Lentikats®) was successfully used to get a better development of malolactic fermentation (MLF) in wine. The incubation of immobilized cells in a nutrient medium before starting the MLF, did not improve the degradation of malic acid. In only one day, 100% of conversion of malic acid was achieved using a high concentration of immobilized cells (0.35 g gel/ml of wine with a cell-loading of 0.25 mg cells/mg of gel). While a low concentration of 0.21 g gel/ml of wine (cell-loading of 0.25 mg cells/mg of gel) needed 3 days to get a reduction of 40%. The entrapped cells could be reused through six cycles (runs of 3 days), retaining 75% of efficacy for the conversion of malic acid into lactic acid. The immobilized cells in PVA hydrogels gave better performance than free cells because of the increase of the alcohol toleration. Consequently, the inhibitory effect of ethanol for developing MLF could be reduced using immobilized cells into PVA hydrogels.

Proteolysis and texture of hard ewes' milk cheese during ripening as affected by somatic cell counts.

Ewes' milk samples with low (<500,000 ml(-1)), medium (1,000,000-1,500,000 ml(-1)) and high (> 2,500,000 ml(-1)) somatic cell counts (SCC) were used to manufacture hard ewes' cheese using the Zamorano cheese manufacturing protocol. Cheeses that had been ripened for 1, 2 and 3 months were used to obtain isoelectric ovine casein that was analysed by capillary electrophoresis. The texture of the cheeses during ripening was determined instrumentally using the Warner-Bratzler maximum shear force and assessed for sensory qualities by consumers using hedonic tests. The study revealed that the pH value and the lactose content of the milk were affected by high SCC and that the coagulation properties were dependent on the somatic cell content. The protein and moisture contents of the cheeses were unaffected by SCC but a significant increase of pH with ripening time were observed in high-SCC cheeses. The results also pointed to a significant increase in proteolysis related to SCC levels, showing that intact casein, both alphas1 and beta-casein, decreased as the SCC of milk increased, and that the proteolytic fragments, mainly I-alphas1, increased with SCC levels. Significant differences in texture were found among the samples, the cheeses made with high levels of SCC being significantly less compact at each ripening time. The differences in texture were detected by the consumers, who reported defects in cheeses made with high levels of SCC. Indeed, high SCC cheeses were significantly less well accepted.

Analysis of European and north american maize inbred and hybrid lines by monolithic and perfusion reversed-phase high-performance chromatography and multivariate analysis.

Monolithic and perfusion RP-HPLC methods have been employed for the separation of maize proteins from several European and North American inbred and hybrid lines in analysis times close to 4 min for the perfusion column and close to 8 min for the monolithic column. A study of the repeatability of the protein extraction conditions and of the perfusion and monolithic RP-HPLC methods was performed, indicating low values of variance for the relative peak areas and for the retention times. On the other hand, a low inter-kernel variability of these chromatographic parameters was also found, confirming the possibility of a variety identification and classification of maize inbred and hybrid lines by using a RP-HPLC analysis of the maize proteins. A multivariate analysis of the chromatographic data was carried out in order to characterize and identify the inbred and hybrid maize lines. Cluster analysis of the data showed how far or closely related were the maize lines. Principal component analysis showed that protein chromatographic data had enough information to distinguish between the different groups of maize lines. Finally, a linear discriminant analysis enabled the correct classification of the inbred and hybrid lines according to their geographical origin showing the best percentage of cases correctly classified for monolithic RP-HPLC.