Yeast population dynamics during spontaneous fermentation of icewine and selection of indigenous Saccharomyces cerevisiae strains for the winemaking in Qilian, China.

BACKGROUND: Icewine produced in China is becoming popular, but there is only limited knowledge available on the yeast population that occurs during fermentation and also on the selection of indigenous Saccharomyces cerevisiae strains for its production. In this work, we first investigated yeast species and the evolution of yeast population in spontaneous fermentations of icewine produced in the Qilian region of China and then analyzed the biodiversity and important enological properties of S. cerevisiae isolates. RESULTS: Seven species of five genera including S. cerevisiae, S. uvarum, Torulaspora delbrueckii, Hanseniaspora uvarum, Lachancea thermotolerans, Metschnikowia aff. fructicola and H. osmophila were identified by the colony morphology on Wallerstein Laboratory Nutrient medium and sequence analysis of the 26S rRNA gene D1/D2 domain. Saccharomyces cerevisiae, H. uvarum and L. thermotolerans were the dominant species, representing almost 87% of the total yeast isolates. Microvinification with seven preselected S. cerevisiae strains were performed on Vidal. All selected strains could complete fermentations, and the enochemical parameters were within the acceptable ranges of the wine industry. W5B3 produced higher amounts of ethyl hexanoate and ethyl octanoate than other strains. R3A10 was a low volatile acid producer and the corresponding icewine presented the highest values on some odorants including ß-damascenone, 1-octen-3-ol, ethyl 2-methylbutyrate, and isoamyl alcohol. Vidal icewines fermented with R3A10, R3A16 and W5B3 were well accepted by the judges because of superior sensory quality. CONCLUSION: Three indigenous strains (R3A10, R3A16 and W5B3) could be used as starters and could potentially improve the regional character of the icewine in Qilian. © 2020 Society of Chemical Industry.

Yeast species isolated from Texas High Plains vineyards and dynamics during spontaneous fermentations of Tempranillo grapes.

Vineyards and grape musts harbor complex locally specific microbial communities, among which yeast species can be responsible of spontaneous alcoholic fermentation. Although relying on indigenous yeast can be a risk for winemaking, local yeast diversity is associated with complexity and stronger identity of the wine produced, compared to inoculated alcoholic fermentation with commercial yeast strains. In this context, the main yeast species present on grapes, leaves and soils of Tempranillo and Cabernet Sauvignon vineyards in the hot semi-arid climate of the Texas High Plains area were investigated, as well as the presence and dynamics of yeast species during spontaneous fermentations of Tempranillo grapes from the same vineyards. Molecular characterization of yeast species was performed using culture-dependent 5.8S-ITS restriction fragment length polymorphism method and sequencing. Yeast species recovered from grapes, leaves, and soils were mainly dominated by Aureobasidium pullulans, Cryptococcus species, Filobasidium species and Naganishia species, typical members of the vineyard environment. One isolate of potential enological interest, Lachancea thermotolerans, a fermenting yeast with potential in must acidification, was recovered from the vineyard environment. However, spontaneous alcoholic fermentations revealed the presence of fermenting yeast species, including Saccharomyces cerevisiae, Lachancea thermotolerans and Hanseniaspora species. The presence of the three aforementioned species is of extreme interest for winemaking in the Texas High Plains area. Indeed, Saccharomyces cerevisiae is the model for alcoholic fermentation, Hanseniaspora species have been shown to improve palatability of wines, and Lachancea thermotolerans has become of increasing interest due to its potential to acidify musts and palatability. One of the main characteristics of grapes grown in the Texas High Plains area being the lack of acidity, focusing on these three yeast species could promote the development of locally oriented started cultures for the production of wines with a stronger local identity.

Early gut mycobiota and mother-offspring transfer.

BACKGROUND: The fungi in the gastrointestinal tract, the gut mycobiota, are now recognised as a significant part of the gut microbiota, and they may be important to human health. In contrast to the adult gut mycobiota, the establishment of the early gut mycobiota has never been described, and there is little knowledge about the fungal transfer from mother to offspring. METHODS: In a prospective cohort, we followed 298 pairs of healthy mothers and offspring from 36 weeks of gestation until 2 years of age (1516 samples) and explored the gut mycobiota in maternal and offspring samples. Half of the pregnant mothers were randomised into drinking probiotic milk during and after pregnancy. The probiotic bacteria included Lactobacillus rhamnosus GG (LGG), Bifidobacterium animalis subsp. lactis Bb-12 and Lactobacillus acidophilus La-5. We quantified the fungal abundance of all the samples using qPCR of the fungal internal transcribed spacer (ITS)1 segment, and we sequenced the 18S rRNA gene ITS1 region of 90 high-quantity samples using the MiSeq platform (Illumina). RESULTS: The gut mycobiota was detected in most of the mothers and the majority of the offspring. The offspring showed increased odds of having detectable faecal fungal DNA if the mother had detectable fungal DNA as well (OR = 1.54, p = 0.04). The fungal alpha diversity in the offspring gut increased from its lowest at 10 days after birth, which was the earliest sampling point. The fungal diversity and fungal species showed a succession towards the maternal mycobiota as the child aged, with Debaryomyces hansenii being the most abundant species during breast-feeding and Saccharomyces cerevisiae as the most abundant after weaning. Probiotic consumption increased the gut mycobiota abundance in pregnant mothers (p = 0.01). CONCLUSION: This study provides the first insight into the early fungal establishment and the succession of fungal species in the gut mycobiota. The results support the idea that the fungal host phenotype is transferred from mother to offspring. TRIAL REGISTRATION: Clinicaltrials.gov NCT00159523.

Unraveling microbial ecology of industrial-scale Kombucha fermentations by metabarcoding and culture-based methods.

Kombucha, historically an Asian tea-based fermented drink, has recently become trendy in Western countries. Producers claim it bears health-enhancing properties that may come from the tea or metabolites produced by its microbiome. Despite its long history of production, microbial richness and dynamics have not been fully unraveled, especially at an industrial scale. Moreover, the impact of tea type (green or black) on microbial ecology was not studied. Here, we compared microbial communities from industrial-scale black and green tea fermentations, still traditionally carried out by a microbial biofilm, using culture-dependent and metabarcoding approaches. Dominant bacterial species belonged to Acetobacteraceae and to a lesser extent Lactobacteriaceae, while the main identified yeasts corresponded to Dekkera, Hanseniaspora and Zygosaccharomyces during all fermentations. Species richness decreased over the 8-day fermentation. Among acetic acid bacteria, Gluconacetobacter europaeus, Gluconobacter oxydans, G. saccharivorans and Acetobacter peroxydans emerged as dominant species. The main lactic acid bacteria, Oenococcus oeni, was strongly associated with green tea fermentations. Tea type did not influence yeast community, with Dekkera bruxellensis, D. anomala, Zygosaccharomyces bailii and Hanseniaspora valbyensis as most dominant. This study unraveled a distinctive core microbial community which is essential for fermentation control and could lead to Kombucha quality standardization.

Yeast diversity on grapes in two German wine growing regions.

The yeast diversity on wine grapes in Germany, one of the most northern wine growing regions of the world, was investigated by means of a culture dependent approach. All yeast isolates were identified by sequence analysis of the D1/D2 domain of the 26S rDNA and the ITS region. Besides Hanseniaspora uvarum and Metschnikowia pulcherrima, which are well known to be abundant on grapes, Metschnikowia viticola, Rhodosporidium babjevae, and Curvibasidium pallidicorallinum, as well as two potentially new species related to Sporidiobolus pararoseus and Filobasidium floriforme, turned out to be typical members of the grape yeast community. We found M. viticola in about half of the grape samples in high abundance. Our data strongly suggest that M. viticola is one of the most important fermenting yeast species on grapes in the temperate climate of Germany. The frequent occurrence of Cu. pallidicorallinum and strains related to F. floriforme is a new finding. The current investigation provides information on the distribution of recently described yeast species, some of which are known from a very few strains up to now. Interestingly yeasts known for their role in the wine making process, such as Saccharomyces cerevisiae, Saccharomyces bayanus ssp. uvarum, Torulaspora delbrueckii, and Zygosaccharomyces bailii, were not found in the grape samples.

Diversity of yeast strains of the genus Hanseniaspora in the winery environment: What is their involvement in grape must fermentation?

Isolated yeast populations of Chardonnay grape must during spontaneous fermentation were compared to those isolated on grape berries and in a winery environment before the arrival of the harvest (air, floor, winery equipment) and in the air through time. Two genera of yeast, Hanseniaspora and Saccharomyces, were isolated in grape must and in the winery environment before the arrival of the harvest but not on grape berries. The genus Hanseniaspora represented 27% of isolates in the must and 35% of isolates in the winery environment. The isolates of these two species were discriminated at the strain level by Fourier transform infrared spectroscopy. The diversity of these strains observed in the winery environment (26 strains) and in must (12 strains) was considerable. 58% of the yeasts of the genus Hanseniaspora isolated in the must corresponded to strains present in the winery before the arrival of the harvest. Although the proportion and number of strains of the genus Hanseniaspora decreased during fermentation, some strains, all from the winery environment, subsisted up to 5% ethanol content. This is the first time that the implantation in grape must of populations present in the winery environment has been demonstrated for a non-Saccharomyces genus.

Food borne yeasts as DNA-bioprotective agents against model genotoxins.

Yeasts isolated from Italian beverages and foods (wine and cheeses) were identified as Saccharomyces cerevisiae and Debaryomyces hansenii by sequencing the D1/D2 domain of the 26S rRNA gene and differentiated, at strain level, by microsatellite PCR fingerprinting and RAPD-PCR. All the strains showed antioxidant activity, as demonstrated by their ability to scavenge the free radical diphenyl-1-picrylhydrazyl (DPPH). Furthermore, tested strains revealed high in vitro inhibitory activity against two model genotoxins, 4-nitroquinoline-1-oxide (4-NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as showed by short-term methods with different target cells: SOS-Chromotest with Escherichia coli PQ37 and Comet assay with HT-29 enterocytes. High inhibitory activity towards 4-NQO was associated with cell viability, while heat-inactivated cells showed a reduced antigenotoxic capability. Surprisingly, high inhibition of MNNG genotoxicity was observed even with heat-treated cells. Moreover, the strains able to inhibit the genotoxins induced some changes in the spectroscopic properties of the original compound. This result perfectly agrees with the information obtained by the two bioassays. Interestingly, strains characterized for antioxidant and antigenotoxic properties, also presented acid-bile tolerance, indicating that food autochthonous yeasts could be expected to reach gut in viable form and thus prevent genotoxin DNA damage in situ.

Direct spectroscopic (FTIR) detection of intraspecific binary contaminations in yeast cultures.

Fourier transform infrared spectroscopy has proved to be a good method to identify and characterize microorganisms. This technique has been proposed as a tool to determine the level of contamination in binary mixtures of strains belonging to different species and even to diverse kingdoms, showing a good linear relationship between spectral outputs and contamination levels. The monitoring of intraspecific contamination is a critical point in both laboratory practice and industrial monitoring, but it is challenged by the difficulty to discriminate between very similar cultures belonging to the same species. In this paper we considered binary intraspecific mixtures of strains belonging to three species (Saccharomyces cerevisiae, Debaryomyces hansenii and Rhodotorula minuta). Results showed that contaminated and pure cultures can be discriminated on the basis of their infrared spectra and that different spectral areas respond to the contamination according to the species under test. Moreover, some spectral areas change linearly with the increase of contaminants, giving the possibility of using this procedure for preliminary estimations of the contamination in addition to the even more important opportunity to indicate the presence of contaminants of the same species at low levels in fermentation cultures.

Application of whole genome amplification and quantitative PCR for detection and quantification of spoilage yeasts in orange juice.

Small cell numbers in complex food matrices and undefined PCR inhibitors often limit detection and identification of DNA species by molecular techniques. Thus in many industrial situations enrichment growths are performed. However, growth speed of different species in complex microbial mixtures in defined media is in most cases different, thus final results do not always reflect the starting situation. We tested DNA-strand displacement whole genome amplification as a possible substitute of enrichment growth. Using whole genome amplification on orange juice contaminated with Saccharomyces cerevisiae, we lowered detection level from 10(6) down to 10(2) cfu/ml. Whole genome amplification showed to be linear (R=0.992) and the relative yeast DNA copy number compared to other DNA templates did not change thus allowing quantitative estimation of initial contamination by quantitative PCR. Using melting point analysis, we were able to distinguish between the PCR products of the 5.8S-ITS region, obtained with universal primers from pure cultures of S. cerevisiae and Hanseniaspora uvarum, two major spoilage yeasts in orange juice and forming part of wine microbiota during fermentation. However, in mixed-contaminated samples, identification of both species was hampered by preferential appearance of the melting peak coinciding with H. uvarum, except when S. cerevisiae was the dominating species. Application of whole genome amplification did not prevent the preferential detection of H. uvarum. This handicap was resolved by applying an enrichment procedure up to saturation after which the melting peak of both species could clearly be identified.

The role of indigenous yeasts in traditional Irish cider fermentations.

AIMS: To study the role of the indigenous yeast flora in traditional Irish cider fermentations. METHODS AND RESULTS: Wallerstein laboratory nutrient agar supplemented with biotin, ferric ammonium citrate, calcium carbonate and ethanol was employed together with PCR-restriction fragment length polymorphism analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene in the identification of indigenous yeasts at the species level, from traditional Irish cider fermentations. By combining the molecular approach and the presumptive media it was possible to distinguish between a large number of yeast species, and to track them within cider fermentations. The Irish cider fermentation process can be divided into three sequential phases based on the predominant yeast type present. Kloeckera/Hanseniaspora uvarum type yeasts predominate in the initial 'fruit yeast phase'. Thereafter Saccharomyces cerevisiae type yeast dominate in the 'fermentation phase', where the alcoholic fermentation takes place. Finally the 'maturation phase' which follows, is dominated by Dekkera and Brettanomyces type yeasts. H. uvarum type yeast were found to have originated from the fruit. Brettanomyces type yeast could be traced back to the press house, and also to the fruit. The press house was identified as having high levels of S. cerevisiae type yeast. A strong link was noted between the temperature profile of the cider fermentations, which ranged from 22 to 35 degrees C and the yeast strain population dynamics. CONCLUSIONS: Many different indigenous yeast species were identified. The mycology of Irish cider fermentations appears to be very similar to that which has previously been reported in the wine industry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the role of indigenous yeast species in 'Natural' Irish cider fermentations.

Analysis of yeast populations during alcoholic fermentation: a six year follow-up study.

Wine yeasts were isolated from fermenting Garnatxa and Xarel.lo musts fermented in a newly built and operated winery between 1995 and 2000. The species of non-Saccharomyces yeasts and the Saccharomyces cerevisiae strains were identified by ribosomal DNA and mitochondrial DNA RFLP analysis respectively. Non-Saccharomyces yeasts, particularly Hanseniaspora uvarum and Candida stellata, dominated the first stages of fermentation. However Saccharomyces cerevisiae was present at the beginning of the fermentation and was the main yeast in the musts in one vintage (1999). In all the cases, S. cerevisiae took over the process in the middle and final stages of fermentation. The analysis of the S. cerevisiae strains showed that indigenous strains competed with commercial strains inoculated in other fermentation tanks of the cellar. The continuous use of commercial yeasts reduced the diversity and importance of the indigenous S. cerevisiae strains.

Fourier-transform infrared microspectroscopy, a novel and rapid tool for identification of yeasts.

Fourier-transform infrared (FT-IR) microspectroscopy was used in this study to identify yeasts. Cells were grown to microcolonies of 70 to 250 micro m in diameter and transferred from the agar plate by replica stamping to an IR-transparent ZnSe carrier. IR spectra of the replicas on the carrier were recorded using an IR microscope coupled to an IR spectrometer, and identification was performed by comparison to reference spectra. The method was tested by using small model libraries comprising reference spectra of 45 strains from 9 genera and 13 species, recorded with both FT-IR microspectroscopy and FT-IR macrospectroscopy. The results show that identification by FT-IR microspectroscopy is equivalent to that achieved by FT-IR macrospectroscopy but the time-consuming isolation of the organisms prior to identification is not necessary. Therefore, this method also provides a rapid tool to analyze mixed populations. Furthermore, identification of 21 Debaryomyces hansenii and 9 Saccharomyces cerevisiae strains resulted in 92% correct identification at the strain level for S. cerevisiae and 91% for D. hansenii, which demonstrates that the resolution power of FT-IR microspectroscopy may also be used for yeast typing at the strain level.

Optimisation of methodology for enumeration of xerophilic yeasts from foods.

Xerophilic yeasts grow in intermediate moisture foods (aw, 0.65-0.85) such as sugar syrups, fruit concentrates, jams and brines. Non-osmophilic yeasts are enumerated by diluting in 0.1% peptone and then plated onto media such as malt extract or glucose yeast extract agar. In the presence of moulds the yeasts are enumerated in dichloran rose bengal chloramphenicol agar (DRBC). These procedures were demonstrated to be unsatisfactory for the enumeration of xerophilic yeasts in low aw foods. Investigations using pure cultures of xerophilic yeasts as well as naturally contaminated apple juice concentrates and glacé cherries have shown that a reduced aw diluent, in particular 30% w/w glycerol in combination with tryptone 10% glucose yeast extract agar (TGY) optimises the recovery of the yeasts, especially sublethally injured cells. The inclusion of sodium chloride in either the diluents or the culture media was not necessary to optimise the recovery of D. hansenii growing in 20% sodium chloride broths.