Alcohols, esters and heavy sulphur compounds production by pure and mixed cultures of apiculate wine yeasts

Strains of Hanseniaspora uvarum, Hanseniaspora guilliermondii and Saccharomyces cerevisiae were used as pure or mixed starter cultures in commercial medium, in order to compare their kinetic parameters and fermentation patterns. In pure and mixed cultures, yeasts presented similar ethanol yield and productivity. Pure cultures of H. uvarum and S. cerevisiae showed a specific growth rate of 0.38 h(-1); however, this value decreased when these yeasts were grown in mixed cultures with H. guilliermondii. The specific growth rate of pure cultures of H. guilliermondii was 0.41 h(-1) and was not affected by growth of other yeasts. H. guilliermondii was found to be the best producer of 2-phenylethyl acetate and 2-phenylethanol in both pure and mixed cultures. In pure cultures, H. uvarum led to the highest contents of heavy sulphur compounds, but H. guilliermondii and S. cerevisiae produced similar levels of methionol and 2-methyltetrahydrothiophen-3-one. Growth of apiculate yeasts in mixed cultures with S. cerevisiae led to amounts of 3-methylthiopropionic acid, acetic acid-3-(methylthio)propyl ester and 2-methyltetrahydrothiophen-3-one similar to those obtained in a pure culture of S. cerevisiae; however, growth of apiculate yeasts increased methionol contents of fermented media.     

Effect of pure and mixed cultures of the main wine yeast species on grape must fermentations

Mixed inoculation of non-Saccharomyces yeasts and S. cerevisiae is of interest for the wine industry for technological and sensory reasons. We have analysed how mixed inocula of the main non-Saccharomyces yeasts and S. cerevisiae affect fermentation performance, nitrogen consumption and volatile compound production in a natural Macabeo grape must. Sterile must was fermented in triplicates and under the following six conditions: three pure cultures of S. cerevisiae, Hanseniaspora uvarum and Candida zemplinina and the mixtures of H. uvarum:S. cerevisiae (90:10), C. zemplinina:S. cerevisiae (90:10) and H. uvarum:C. zemplinina:S. cerevisiae (45:45:10). The presence of non-Saccharomyces yeasts slowed down the fermentations and produced higher levels of glycerol and acetic acid. Only the pure H. uvarum fermentations were unable to finish. Mixed fermentations consumed more of the available amino acids and were more complex and thus better able to synthesise volatile compounds. However, the amount of acetic acid was well above the admissible levels and compromises the immediate application of mixed cultures.     

Saccharomyces cerevisiae and Hanseniaspora uvarum mixed starter cultures: Influence of microbial/physical interactions on wine characteristics

The growing trend in the wine industry is the revaluation of the role of non-Saccharomyces yeasts, promoting the use of these yeasts in association with Saccharomyces cerevisiae. Non-Saccharomyces yeasts contribute to improve wine complexity and organoleptic composition. However, the use of mixed starters needs to better understand the effect of the interaction between these species during alcoholic fermentation. The aim of this study is to evaluate the influence of mixed starter cultures, composed by combination of different S. cerevisiae and Hanseniaspora uvarum strains, on wine characteristics and to investigate the role of cell-to-cell contact on the metabolites produced during alcoholic fermentation. In the first step, three H. uvarum and two S. cerevisiae strains, previously selected, were tested during mixed fermentations in natural red grape must in order to evaluate yeast population dynamics during inoculated fermentation and influence of mixed starter cultures on wine quality. One selected mixed starter was tested in a double-compartment fermentor in order to compare mixed inoculations of S. cerevisiae/H. uvarum with and without physical separation. Our results revealed that physical contact between S. cerevisiae and H. uvarum affected the viability of H. uvarum strain, influencing also the metabolic behaviour of the strains. Although different researches are available on the role of cell-to-cell contact-mediated interactions on cell viability of the strains included in the mixed starter, to our knowledge, very few studies have evaluated the influence of cell-to-cell contact on the chemical characteristics of wine.     

The effects of grape must fermentation conditions on volatile alcohols and esters formed by Saccharomyces cerevisiae

Changes in aroma compounds synthesised from grape must during fermentation carried out by Saccharomyces cerevisiae, in semi-aerobic, anaerobic, short aeration conditions and after adding ergosterol and oleic acid to the must were studied. The biosynthesis of these aroma compounds was strongly dependent on the fermentation conditions and on the growth of the yeast. Ethanol, isoamyl alcohols, isobutyl alcohol, phenethyl alcohol and isoamyl, butyl and hexyl acetates were produced in greater concentrations in semiaerobic conditions, mainly during cellular growth. 1-Butanol and 1-pentanol were produced in greater levels in anaerobic conditions, when cellular growth was lower. Ergosterol and oleic acid added to the musts generally increased the levels of the aroma compounds in wine compared to those obtained in anaerobic conditions. © 1997 SCI.     

Monitoring a mixed starter of Hanseniaspora vineae-Saccharomyces cerevisiae in natural must: impact on 2-phenylethyl acetate production

The effect of simultaneous or sequential inoculation of Hanseniaspora vineae CECT 1471 and Saccharomyces cerevisiae T73 in non-sterile must on 2-phenylethyl acetate production has been examined. In both treatments tested, no significant differences in Saccharomyces yeast growth were found, whereas non-Saccharomyces yeast growth was significantly different during all days of fermentation. Independently of the type of inoculation, S. cerevisiae was the predominant species from day 3 till the end of the fermentation. The dynamics of indigenous and inoculated yeast populations showed H. vineae to be the predominant non-Saccharomyces species at the beginning of fermentation in sequentially inoculated wines, whereas the simultaneous inoculation of S. cerevisiae did not permit any non-Saccharomyces species to become predominant. Differences found in non-Saccharomyces yeast growth in both fermentations influenced the analytical profiles of final wines and specifically 2-phenylethyl acetate concentration which was two-fold increased in sequentially inoculated wines in comparison to those co-inoculated. In conclusion we have shown that H. vineae inoculated as part of a sequential mixed starter is able to compete with native yeasts present in non-sterile must and modify the wine aroma profile.     

Glucose metabolism,enzymic analysis and product formation in chemostat culture of Hanseniaspora uvarum

The physiology of Hanseniaspora uvarum K₅ was studied in glucose-limited chemostat cultures and upon glucose pulse. Up to a dilution rate of 0·28 h^?1, glucose was completely metabolized in biomass and CO₂. Above this value, increase in the dilution rate was accompanied by sequential production of metabolites (glycerol, acetate and ethanol) and decrease in cell yield. Similar results were observed upon glucose pulse. From the enzyme activities (pyruvate dehydrogenase, pyruvate decarboxylase, NAD and NADP-dependent acetaldehyde dehydrogenases, acetyl coenzyme A synthetase and alcohol dehydrogenase) and substrate affinities, the following conclusions were drawn with respect to product formation of cells: (1) pyruvate was preferentially metabolized via pyruvate dehydrogenase, when biomass and CO₂ were the only products formed; (2) acetaldehyde formed by pyruvate decarboxylase was preferentially oxidized in acetate by NADP-dependent aldehyde dehydrogenase; acetate accumulation results from insufficient activity of acetyl-CoA synthetase required for the complete oxidation of acetate; (3) acetaldehyde was oxidized in ethanol by alcohol dehydrogenase, in addition to acetate production.     

Multi-enzyme production by pure and mixed cultures of Saccharomyces and non-Saccharomyces yeasts during wine fermentation

Saccharomyces and non-Saccharomyces yeasts release enzymes that are able to transform neutral compounds of grape berries into active aromatic compounds, a process that enhances the sensory attributes of wines. So far, there exists only little information about enzymatic activity in mixed cultures of Saccharomyces and non-Saccharomyces during grape must fermentations. The aim of the present work was to determine the ability of yeasts to produce extracellular enzymes of enological relevance (β-glucosidases, pectinases, proteases, amylases or xylanases) in pure and mixed Saccharomyces/non-Saccharomyces cultures during fermentation. Pure and mixed cultures of Saccharomyces cerevisiae BSc562, Hanseniaspora vinae BHv438 and Torulaspora delbrueckii BTd259 were assayed: 1% S. cerevisiae/99% H. vinae, 10% S. cerevisiae/90% H. vinae, 1% S. cerevisiae/99% T. delbrueckii and 10% S. cerevisiae/90% T. delbrueckii. Microvinifications were carried out with fresh must without pressing from Vitis vinifera L. c.v. Pedro Jiménez, an autochthonous variety from Argentina. Non-Saccharomyces species survived during 15-18days (BTd259) or until the end of the fermentation (BHv438) and influenced enzymatic profiles of mixed cultures. The results suggest that high concentrations of sugars did not affect enzymatic activity. β-Glucosidase and pectinase activities seemed to be adversely affected by an increase in ethanol: activity diminished with increasing fermentation time. Throughout the fermentation, Saccharomyces and non-Saccharomyces isolates assayed produced a broad range of enzymes of enological interest that catalyze hydrolysis of polymers present in grape juice. Vinifications carried out by a pure or mixed culture of BTd259 (99% of T. delbrueckii) showed the highest production of all enzymes assayed except for β-glucosidase. In mixed cultures, S. cerevisiae outgrew H. vinae, and T. delbrueckii was only detected until halfway the fermentation process. Nevertheless, their secreted enzymes could be detected throughout the fermentation process. Our results may contribute to a better understanding of the microbial interactions and the influence of some enzymes on vinification environments.     

Phenolic compounds and antioxidant properties of different grape cultivars grown in China

Skins and seeds of 18 grape cultivars belonging to Oriental and North American Vitis Species/hybrids, and Vitis vinifera were analysed for health beneficial properties. Four phenolic compound parameters (total phenols, flavonoids, flavan-3-ols and anthocyanins) and three antioxidant property parameters (DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging, ABTS [2,2-azino-di-(3-ethylbenzothialozine-sulphonic acid)] radical scavenging and FRAP (ferric reducing antioxidant power)) were measured. Principal component analysis (PCA) was used for this evaluation and results showed that both phenolic compounds and antioxidant properties in the seeds and skins varied among the cultivars investigated. V. vinifera “Cabernet Sauvignon” had the highest values of phenolic compounds and antioxidant properties in seeds followed by Muscadines, while the lowest appeared in the Oriental Vitis species. As expected, these values of the Euro-Asian or Euro-American hybrids fell between the parents. However, far less variation of these values was observed in the skins among different grape cultivars investigated. Interestingly, even the total phenolic contents in the berries of two cultivars are similar, distributions of phenolic compounds in seeds and skins varied greatly among them. Additionally, significant correlations among different antioxidant assays in both seeds and skins were observed. These antioxidant properties were also found highly correlated to the main phenolic compounds.     

野生山葡萄酒杂醇油含量偏高的原因及对策

根据野生山葡萄（毛葡萄）的原料特点及工艺过程，找出导致野生葡萄酒杂醇油含量偏高的原因，并制定相庆的对策，有效地解决了酒后“上头”问题。     

Temperature effect on the biological activity of Bifidobacterium longum CRL 849 and Lactobacillus fermentum CRL 251 in pure and mixed cultures grown in soymilk

The influence of temperature on the growth and biological activity of two probiotic strains (Bifidobacterium longum CRL 849 and Lactobacillus fermentum CRL 251) as pure and mixed cultures in soymilk (SM) were evaluated. Maximum growth was observed at 37°C in both mixed and pure cultures. In a product prepared with the mixed culture (1:1) at 37°C, the amount of lactic acid produced was approximately 55 mmol l⁻¹ after 24 h with a slow production rate (2.8 mmol l⁻¹ h⁻¹); the formation of acetic acid was higher with respect to pure cultures (82.01 mmol l⁻¹ after 24 h), and final pH (24 h) was 5.0. About 85% of the total amount of sugars in SM was reduced, mainly sucrose. Stachyose was reduced (71%) after 4 h of incubation. Maximum activity of alpha-galactosidase (alpha-gal) (13.2 U ml⁻¹) was observed after 6 h. At 37°C the bifidobacterium strain was viable in mixed culture throughout the period assayed. At lower (30°C) or higher (42°C) temperatures, mixed culture showed slower growth and lower acid production in SM but the alpha-gal activity was stimulated at 30°C.     

Anthocyanin profile in berry skins and fermenting must/wine, as affected by grape ripeness level of Vitis vinifera cv. Shiraz/R99

The release of anthocyanin compounds from berry skins into the wine is affected by several viticulture and oenological practices. Ripeness level seems to play a significant role. The study aimed to evaluate the extraction kinetics of individual anthocyanins from grape skins into wine under controlled conditions of small-scale vinification of Shiraz grapes, harvested at three ripeness levels (23, 25 and 28°Brix). The anthocyanin profile of intact berry skins, fermenting musts/wines and crushed skins during the period between crushing and pressing was analysed by HPLC. Ripeness level greatly impacted on the skin?/?juice ratio (ranging from 169?± 4.5?g?L^?1 at 23°B, 185?±?7.2?g?L^?1 at 25°B and 256?±?10.7?g?L^?1 at 28°B) and on grape anthocyanin concentration (ranging from 0.80?±?0.13?g?kg^?1 at 23°B, 0.78?±?0.07?g?kg^?1 at 25°B and 1.21?±?0.13?g?kg^?1 at 28°B) and profile (%) at harvest. In addition, it affected the rate and amount of individual anthocyanin extraction from skins into wine and their transformation during fermentation, consistent with the relevant chemical group classification (3-glucoside, 3-acetyl-glucoside, 3-p-coumaroyl-glucoside). At pressing, the anthocyanin concentration of the three wines was similar (319?mg?L^?1, on average), but the anthocyanin profile was different, particularly the ratio of 3-glucoside?/?3-p-coumaroyl-glucoside derivatives, which was on average 3.55 at 23°B and 25°B and 2.6 at 28°B. Viticultural choices, such as to harvest earlier or later, influencing the chemical and physical composition of the berries, may influence the extraction kinetics of individual anthocyanins and their destiny in the fermenting must, offering to winemakers different basic wines suitable for the production of different wine products. These findings are valuable to improve viticultural and oenological practices for Shiraz wine production, allowing the improvement of wine quality and the purposeful creation of different styles of wine.     

不同有孢汉逊酵母与酿酒酵母混合发酵对威代尔冰葡萄酒香气的影响

为了研究菌株和接种方式对冰葡萄酒非挥发性产物和挥发性香气成分的影响,选用2株自筛非酿酒酵母——仙人掌有孢汉逊酵母(Hanseniaspora opuntiae)和葡萄汁有孢汉逊酵母(Hanseniaspora uvarum),分别与商业酿酒酵母DV10混合发酵。结果表明:H.uvarum与DV10同时接种混合发酵可以显著降低乙酸产量(降低26.90%);H.opuntiae与DV10同时接种混合发酵促进了酯类和萜烯类物质的合成,总量分别增产88.61%和21.40%,其中乙酸苯乙酯含量提高14.60倍,β-大马士酮增产8.85%。除此之外,感官品评结果显示H.opuntiae与DV10混合发酵可显著增强花果香和黄油味。综合分析,认为H.opuntiae更适合应用于冰葡萄酒发酵,为冰酒混合发酵的进一步研究提供了数据基础和理论依据。     

Effect of temperature and pH on the formation of higher alcohols,fatty acids and esters in the malt whisky fermentation

The levels of higher alcohols, fatty acids and esters in small-scale whisky fermentations which were carried out at different temperatures and initial pH values were investigated. Neither the total higher alcohol content nor the relative abundance of different alcohols was affected by varying the temperature between 20 and 30°C. However, the level of octanoic acid, decanoic acid, ethyl hexanoate and ethyl octanoate were depressed at higher temperatures. The highest level of acetate esters were observed at 25 and 30°C. Altering the initial pH of the wort had little effect on the level of higher alcohols. However, increasing the pH from 4·0 to 7·0 resulted in an increase in the level of octanoic, decanoic and dodecanoic acids. Maximum levels of acetate esters of higher alcohols were obtained when initial pH values of between 5·0 and 6·0 were used.     

Chitosan elicits mono-glucosylated stilbene production and release in fed-batch bioreactor cultures of grape cells

The present paper reports the study of the optimal conditions for grape (Vitis vinifera cv. Barbera) cell suspensions in batch and fed-batch bioreactor cultures, in order to specifically improve the production of mono-glucosylated stilbenes, which are resveratrol derivatives. These compounds are physiologically as active as free resveratrol in cardio- and chemoprotection, but are more stable and bioavailable after ingestion through diet. In fed-batch conditions the production of mono-glucosides was considerably increased together with that of free resveratrol. For the first time, an elicitor (chitosan) was tested in a bioreactor system, demonstrating its efficacy in inducing the production of stilbenes. The bioreactor culture conditions also allowed the accumulation of other polyphenols, such as catechins. The vast majority of the produced compounds was released into the culture media, which represents a relevant advantage for the recovery of specific molecules or of polyphenol-enriched mixtures. These results represent a further step toward the employment of grape cells in fed-batch cultures, as a promising alternative to whole plant extraction for the industrial production of plant polyphenols, considering the necessity for developing sustainable processes.