Changes in microbial communities and their predictive functionalities during fermentation of toddy, an alcoholic beverage of India.

Toddy is a traditional mild-alcoholic drink of India, which is produced from fresh palm saps by natural fermentation. We studied the successional changes in bacterial and fungal communities during the natural fermentation (0 h-96 h) of toddy. During fermentation, alcohol content of the fermenting saps increased significantly from 0.6 %±0.15 to 5.6 %±0.02, pH decreased from 6.33 %±0.02-3.93 ± 0.01, volatile and titratable acidity acidity (g/100 mL) increased from 0.17 ± 0.02 (0 h) to 0.48 ± 0.02 (96 h) and 1.30 ± 0.005 (0 h) to 2.47 ± 0.005 (96 h), respectively. Total sugar content and ˚BRIX also decreased during the fermentation. Firmicutes (78.25 %) was the most abundant phylum followed by Proteobacteria (21.57 %). Leuconostoc was the most abundant genus in the early stages of fermentation. However, Lactobacillus and Gluconoacetobacter were found abundant with increase in pH during the later phases of fermentation (72 h-96 h). Ascomycota (99.02 %) was the most abundant fungal phylum. Hanseniaspora was the abundant yeast in the initial stages of fermentation, whereas the population of Saccharomyces increased significantly after 24 h of fermentation. Torulaspora, Lachancea and Starmerella showed their heterogeneous distribution throughout the fermentation. Computational analysis of metagenomes based on KEGG and MetaCyc databases showed different predictive functional profiles such as folate biosynthesis, glutathione metabolism, terpenoids biosynthesis and biosynthesis of amino acids with significant differences between the fresh palm saps and fermenting saps during toddy fermentation.

Effects of initial oxygenation on chemical and aromatic composition of wine in mixed starters of Hanseniaspora vineae and Saccharomyces cerevisiae.

The use of Saccharomyces and non-Saccharomyces yeast species as mixed starters has potential advantages over pure culture fermentation due to increased wine complexity based on modification of metabolites of oenological interest. In this work, the effects of initial oxygenation on fermentation performance, chemical and volatile composition of French Colombard wine fermented with Hanseniaspora vineae and Saccharomyces cerevisiae in sequential inoculations were investigated in 1 L flasks. Although dominated by S. cerevisiae at the middle-end of fermentation, initial aeration for 1 day boosted H. vineae populations, and allowed H. vineae to coexist longer with S. cerevisiae in mixed cultures compared to no aeration, and suppressed S. cerevisiae later in the fermentation, which resulted in extended fermentation time. More important, the major fermentation products and volatile compounds were significantly modified by aeration and different from no aeration fermentation. The wines produced by aeration of mixed fermentations were characterized with higher amounts of glycerol, lactic acid and acetate esters, and lower levels of ethanol, higher alcohol and ethyl fatty acid esters. The aeration had more potential to shape the quality of wines and diversify the aromatic characteristics relative to simple mixed inoculation, as indicated by PCA analysis. Our results suggested that the impact of early aeration on yeast physiology extends beyond the aeration phase and influences fermentation activity, chemical and aromatic compounds in the following anaerobic stage. The aeration for a short time during the cell growth stage in mixed fermentation is therefore a potential means to increase the aromatic diversity and quality of wine, possibly providing an alternative approach to meet the expectations of wine consumers for diverse aromatic qualities.

Effects of melatonin and tryptophol addition on fermentations carried out by Saccharomyces cerevisiae and non-Saccharomyces yeast species under different nitrogen conditions.

During wine fermentation, yeasts produce metabolites that are known growth regulators. The relationship between certain higher alcohols derived from aromatic amino acid metabolism and yeast signalling has previously been reported. In the present work, tryptophol (TrpOH) or melatonin (MEL), which are putative growth regulators, were added to alcoholic fermentations. Fermentations were performed with three different inocula, combining Saccharomyces cerevisiae and four non-Saccharomyces yeast species, under two nitrogen conditions. The combinations tested were: (i) only S. cerevisiae; (ii) the mixture of four non-Saccharomyces species; and (iii) the combination of all five species together. The results revealed that the TrpOH and MEL addition caused changes in fermentation kinetics, viability and species distribution during fermentation, but it was dependent on the nitrogen present in the media and the composition of the inocula. Low nitrogen condition seemed to favour the presence of non-Saccharomyces species until mid-fermentation, although at the end of fermentation the imposition of Saccharomyces was higher in this condition. The presence of high concentrations of TrpOH resulted in limited growth and a delay in fermentation, noticeably significant in fermentations performed with S. cerevisiae inocula. These effects were reversed by the presence of non-Saccharomyces yeast in the medium. Low TrpOH concentration allowed faster fermentation with mixed non-Saccharomyces and Saccharomyces inocula. Moreover, in the absence of S. cerevisiae, a low concentration of TrpOH increased the presence of Torulaspora delbrueckii during fermentation with high nitrogen availability but not under low nitrogen conditions, when the population of S. bacillaris was higher than that in the control. The effects of MEL were particularly evident at the beginning and end of the process, primarily favouring the growth of non-Saccharomyces strains, especially the first hours after inoculation.

Microbial cell-free extracts as sources of enzyme activities to be used for enhancement flavor development of ewe milk cheese.

Freeze-dried cell-free extracts (CFE) from Lactobacillus casei LC01, Weissella cibaria 1XF5, Hafnia alvei Moller ATCC 51815, and Debaryomyces hansenii LCF-558 were used as sources of enzyme activities for conditioning the ripening of ewe milk cheese. Compared with control cheese (CC), CFE did not affect the gross composition and the growth of the main microbial groups of the cheeses. As shown through urea-PAGE electrophoresis of the pH 4.6-soluble nitrogen fraction and the analysis of free AA, the secondary proteolysis of the cheeses with CFE added was markedly differed from that of the CC. Compared with CC, several enzyme activities were higher in the water-soluble extracts from cheeses made with CFE. In agreement, the levels of 49 volatile compounds significantly differentiated CC from the cheeses made with CFE. The level of some alcohols, ketones, sulfur compounds, and furans were the lowest in the CC, whereas most aldehydes were the highest. Each CFE seemed to affect a specific class of chemical compounds (e.g., the CFE from H. alvei ATCC 51815 mainly influenced the synthesis of sulfur compounds). Apart from the microbial source used, the cheeses with the addition of CFE showed higher score for acceptability than the control cheese. Cheese ripening was accelerated or conditioned using CFE as sources of tailored enzyme activities.

Heavy sulphur compounds, higher alcohols and esters production profile of Hanseniaspora uvarum and Hanseniaspora guilliermondii grown as pure and mixed cultures in grape must.

Hanseniaspora guilliermondii and Hanseniaspora uvarum were tested in grape must fermentations as pure and mixed starter cultures with Saccharomyces cerevisiae. In pure cultures, the specific growth rates found were 0.29 h(-1) for H. uvarum, 0.23 h(-1) for H. guilliermondii and 0.18 h(-1) for S. cerevisiae. No significant differences were observed between these values and those obtained in mixed cultures. Results presented in this work show that growth of apiculate yeasts during the first days of fermentation enhances the production of desirable compounds, such as esters, and may not have a negative influence on the production of higher alcohols and undesirable heavy sulphur compounds. Growth of apiculate yeasts reduced the total content of higher alcohols in wines, when compared to those produced by a pure culture of S. cerevisiae. Furthermore, the highest levels of 2-phenylethyl acetate were obtained when H. guilliermondii was inoculated in grape musts, whereas H. uvarum increased the isoamyl acetate content of wines. Apiculate yeasts produced high amounts of ethyl acetate; however, the level of this compound decreased in mixed cultures of apiculate yeasts and S. cerevisiae. When S. cerevisiae was used as a starter culture, wines showed higher concentrations of glycerol, 2-phenylethanol and ethyl hexanoate. In mixed cultures of apiculate yeasts and S. cerevisiae, wines presented amounts of methionol, acetic acid-3-(methylthio)propyl ester, 4-(methylthio)-1-butanol, 2-mercaptoethanol and cis-2-methyltetrahydro-thiophen-3-ol similar to those produced by a pure culture of S. cerevisiae. An increase in the amounts of 3-(ethylthio)-1-propanol, trans-2-methyltetrahydro-thiophen-3-ol and 3-mercapto-1-propanol was obtained in wines produced from mixed cultures with H. guilliermondii.

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.