Consortium of selected yeasts to produce healthy soy fermented beverage: Evaluation of microbial evolution, analytical, sensorial, and functional features.

Currently, an increasing number of intolerant and vegan consumers are driving the market towards plant-based milk alternatives. Here, selected probiotic yeasts, belonging to the Candida zeylanoides, Kluyveromyces lactis and Debaryomyces hansenii species, previously characterized for their aptitude to ferment animal milk, were tested in soy milk. Trials at different fermentation times with the developed yeast consortium (Yc) coinoculated with a lactic bacterium commercial strain were carried out. Yc showed good fermentation performance, conferring distinctive analytical and aromatic properties to the resulted soy fermented beverage, a product similar to an industrial kefir. Analytical determinations did not show significant variations between the end of fermentation and cold storage (4 weeks at 4 °C), indicating full stability. Phenol amounts and antioxidant activity were significantly increased in soy fermented beverage fermented by Yc. All yeasts remained viable until the end of storage with a final concentration of approximately 8 Log CFU/ml, a value suitable for a probiotic commercial claim. Overall, the results suggest that Yc is a promising multistarter candidate for functional soy products.

Epiphytic Yeasts and Bacteria as Candidate Biocontrol Agents of Green and Blue Molds of Citrus Fruits.

Overall, 180 yeasts and bacteria isolated from the peel of citrus fruits were screened for their in vitro antagonistic activity against Penicillium digitatum and P. italicum, causative agents of green and blue mold of citrus fruits, respectively. Two yeast and three bacterial isolates were selected for their inhibitory activity on mycelium growth. Based on the phylogenetic analysis of 16S rDNA and ITS rDNA sequences, the yeast isolates were identified as Candida oleophila and Debaryomyces hansenii while the bacterial isolates were identified as Bacillus amyloliquefaciens, B. pumilus and B. subtilis. All five selected isolates significantly reduced the incidence of decay incited by P. digitatum and P. italicum on 'Valencia' orange and 'Eureka' lemon fruits. Moreover, they were effective in preventing natural infections of green and blue mold of fruits stored at 4 °C. Treatments with antagonistic yeasts and bacteria did not negatively affect the quality and shelf life of fruits. The antagonistic efficacy of the five isolates depended on multiple modes of action, including the ability to form biofilms and produce antifungal lipopeptides, lytic enzymes and volatile compounds. The selected isolates are promising as biocontrol agents of postharvest green and blue molds of citrus fruits.

The Influence of Fungicide Treatments on Mycobiota of Grapes and Its Evolution during Fermentation Evaluated by Metagenomic and Culture-Dependent Methods.

The present study evaluated the impact of organic and conventional fungicide treatments compared with untreated samples (no fungicides were used) on the grape berry yeast community of the Montepulciano variety. The yeast dynamics during the spontaneous fermentation using culture-dependent and -independent methods was also evaluated. Results showed a reduction of yeast biodiversity by conventional treatments determining a negative influence on fermenting yeasts in favor of oxidative yeasts such as Aerobasidium pullulans. Starmerella bacillaris was significantly more present in organic samples (detected by next generation sequencing (NGS)), while Hanseniaspopa uvarum was significantly less present in untreated samples (detected by the culture-dependent method). The fermenting yeasts, developed during the spontaneous fermentation, were differently present depending on the fungicide treatments used. Culture-dependent and -independent methods exhibited the same most abundant yeast species during the spontaneous fermentation but a different relative abundance. Differently, the NGS method was able to detect a greater biodiversity (lower abundant species) in comparison with the culture-dependent method. In this regard, the methodologies used gave a different picture of yeast dynamics during the fermentation process. The results indicated that the fungal treatments can influence the yeast community of grapes leading must fermentation and the final composition of wine.

Sequential Fermentation with Selected Immobilized Non-Saccharomyces Yeast for Reduction of Ethanol Content in Wine.

The average ethanol content of wine has increased over the last two decades. This increase was due to consumer preference, and also to climate change that resulted in increased grape maturity at harvest. In the present study, to reduce ethanol content in wine, a microbiological approach was investigated, using immobilized selected strains of non-Saccharomyces yeasts namely Starmerella bombicola, Metschnikowia pulcherrima, Hanseniaspora osmophila, and Hanseniaspora uvarum to start fermentation, followed by inoculation of free Saccharomyces cerevisiae cells. The immobilization procedures, determining high reaction rates, led a feasible sequential inoculation management avoiding possible contamination under actual winemaking. Under these conditions, the immobilized cells metabolized almost 50% of the sugar in 3 days, while S. cerevisiae inoculation completed all of fermentation. The S. bombicola and M. pulcherrima initial fermentations showed the best reductions in the final ethanol content (1.6 and 1.4% v/v, respectively). Resulting wines did not have any negative fermentation products with the exception of H. uvarum sequential fermentation that showed significant amount of ethyl acetate. On the other hand, there were increases in desirable compounds such as glycerol and succinic acid for S. bombicola, geraniol for M. pulcherrima and isoamyl acetate and isoamyl alcohol for H. osmophila sequential fermentations. The overall results indicated that a promising ethanol reduction could be obtained using sequential fermentation of immobilized selected non-Saccharomyces strains. In this way, a suitable timing of second inoculation and an enhancement of analytical profile of wine were obtained.

TpBGL2 codes for a Tetrapisispora phaffii killer toxin active against wine spoilage yeasts.

Tetrapisispora phaffii produces a killer toxin known as Kpkt that has extensive anti-Hanseniaspora/Kloeckera activity under winemaking conditions. Kpkt has a ß-glucanase activity and induces ultrastructural modifications in the cell wall of sensitive strains, with a higher specific cytocidal activity and a selective action towards target yeast cells. In this study, a two-step PCR-based approach was used to isolate the gene coding ß-glucanase of T. phaffii. Initially, a fragment of the open reading frame was isolated by degenerate PCR, with primers designed on the NH2 -terminal sequence of the protein and on conserved motifs of Bgl2p of Saccharomyces cerevisiae and Candida albicans. Subsequently, the entire sequence of the gene was obtained by inverse PCR. blast analyses of TpBGL2 highlight high identity with homologous genes in other yeast species, in which TpBGL2p shows no killer activity. However, gene disruption resulted in complete loss of the glucanase activity and the killer phenotype, thus confirming that TpBgl2p has a killer activity.

Grape berry yeast communities: influence of fungicide treatments.

The yeast communities colonising grape berry surfaces were evaluated for the influence of fungicide treatments in an organic vineyard (copper/sulphur-based products) and a conventional vineyard (commonly used fungicides). Analysis of yeast abundance and diversity was carried out on grape berries and juice during fermentation, using culture-dependent and -independent approaches. Yeast abundance was as generally reported for mature grapes and it was slight higher from grapes treated with conventional fungicides. Initial grape samples showed less yeast species diversity in the organic vineyard compared with the conventional one. In both vineyards, the dominant yeast were Candida zemplinina and Hanseniaspora uvarum (>50%), respectively, typical species that colonise surfaces of mature grape berries. Metschnikowia pulcherrima was widely found in the conventional samples while it was only occasionally found in organic ones. Saccharomyces cerevisiae was isolated only at the end of natural fermentation (conducted in sterile condition), with lower levels in the organic samples. S. cerevisiae strains showed less intraspecies diversity in the organic samples (two genotypes), in comparison with the conventional samples (six genotypes).

Yeast diversity in crop-growing environments in Cameroon.

In the present study, we have investigated the occurrence of yeast flora on several agricultural products coming from crop-growing environments in Cameroon, to provide better knowledge of the biodiversity of yeast flora, and to thus define the impact of this biodiversity on food products. The yeast biodiversity was investigated using traditional culture-dependent methods, along with culture-independent methods. The culture-dependent approach was carried out using both direct and enrichment procedures, to detect the broadest possible presence of yeast species. A total of 151 strains belonging to 26 different yeast species were isolated and identified using restriction pattern analysis of the internal transcribed spacer region 5.8S-ITS and sequence analysis of D1/D2 domain of 26S rRNA gene. The enrichment isolation procedures carried out in high-sugar media allowed the recognition of fermentative species such as Saccharomyces cerevisiae and Torulaspora delbrueckii, which have previously not been detected using direct isolation methodology. The results of culture-independent method using DGGE patterns and sequencing of the DNA bands revealed a lower number of yeast species when compared with the culture-dependent methodology even if the identification of several yeast species not detected by traditional microbiological procedures such as Candida tropicalis and Hanseniaspora uvarum is allowed. Thus, these multiphasic approaches to study yeast biodiversity (culture-dependent and -independent methods) have allowed us to get a more complete picture of the microbial diversity in these natural environments.

Fermentation behaviour and metabolic interactions of multistarter wine yeast fermentations.

Multistarter fermentations of Hanseniaspora uvarum, Torulaspora delbrueckii and Kluyveromyces thermotolerans together with Saccharomyces cerevisiae were studied. In grape musts with a high sugar content, mixed trials showed a fermentation behaviour and analytical profiles of wines comparable to or better than those exhibited by a pure culture of S. cerevisiae. Sequential trials of T. delbrueckii and K. thermotolerans revealed a sluggish fermentation, while those of H. uvarum exhibited an unacceptable increase in ethyl acetate content (175 ml l(-1)). A low fermentation temperature (15 degrees C) of multistarter trials of H. uvarum resulted in a stuck fermentation that was not due to a deficiency of assimilable nitrogenous compounds since lower amounts of these compounds were used. Sequential fermentation carried out by H. uvarum at 15 degrees C confirmed the high production of ethyl acetate. The persistence and level of non-Saccharomyces yeasts during multistarter fermentations under stress conditions (high ethanol content and/or low temperature) can cause stuck fermentations.