Optimization of the ellagic acid synthesis process at the bioreactor level using non-conventional yeasts

Abstract Ellagic acid (EA) is a phenolic biomolecule. For its biosynthesis, a source of ellagitannins is required, such as strawberries and yeasts, as precursors of the tannase and ß-glucosidase enzymes responsible for hydrolysis of ellagitannins. Two experimental mixture designs were applied., varying the yeast concentration and the number of ellagitannins in the culture medium, evaluating the enzymatic activity and ellagic acid biosynthesis. Aiming to find the optimal compositions of the non-conventional yeasts assessed in the research to biosynthesize ellagic acid feasibly and efficiently using a response surface performing the statistical analysis in the StatGraphics® program for obtaining a higher yield and optimizing the ellagic acid synthesis process, the results indicate that the strains Candida parapsilosis ITM LB33 and Debaryomyces hansenii ISA 1510 have a positive effect on the synthesis of ellagic acid, since as its concentration increases in the mixture the concentration of ellagic acid in the medium also increases; on the other hand, the addition of Candida utilis ITM LB02 causes a negative effect, resulting in the compositions of 0.516876, 0.483124 and 2.58687E-9 respectively, for a treatment under the same conditions, an optimal value of ellagic acid production would be obtained. With an approximate value of 7.33036 mg/mL

Effect of freeze-dried kombucha culture on microbial composition and assessment of metabolic dynamics during fermentation.

Kombucha is a traditional fermented beverage gaining popularity around the world. So far, few studies have investigated its microbiome using next-generation DNA sequencing, whereas the correlation between the microbial community and metabolites evolution along fermentation is still unclear. In this study, we explore this correlation in a traditionally produced kombucha by evaluating its microbial community and the main metabolites produced. We also investigated the effects of starter cultures processed in three different ways (control, starter culture without liquid suspension (CSC), and a freeze-dried starter culture (FDSC)) to evaluate changes in kombucha composition, such as antioxidant activity and sensory analysis. We identified seven genera of bacteria, including Komagataeibacter, Gluconacetobacter, Gluconobacter, Acetobacter, Liquorilactobacillus, Ligilactobacillus, and Zymomonas, and three genera of yeasts, Dekkera/Brettanomyces, Hanseniaspora, and Saccharomyces. Although there were no statistically significant differences in the acceptance test in sensory analysis, different starter cultures resulted in products showing different microbial and biochemical compositions. FDSC decreased Zymomonas and Acetobacter populations, allowing for Gluconobacter predominance, whereas in the control and CSC kombuchas the first two were the predominant genera. Results suggest that the freeze-drying cultures could be implemented to standardize the process and, despite it changes the microbial community, a lower alcohol content could be obtained.

Native yeast and non-yeast fungal communities of Cabernet Sauvignon berries from two Washington State vineyards, and persistence in spontaneous fermentation.

To address a knowledge gap about the grape berry mycobiome from Washington State vineyards, next-generation sequencing of the internal transcribed spacer region (ITS1) was used to identify native yeast and fungal species on berries of cultivar 'Cabernet Sauvignon' from two vineyards at veraison and harvest in 2015 and 2016. Four hundred fifty-six different yeast amplicon sequence variants (ASV), representing 184 distinct taxa, and 2467 non-yeast fungal ASV (791 distinct taxa) were identified in this study. A set of 50 recurrent yeast taxa, including Phaeococcomyces, Vishniacozyma and Metschnikowia, were found at both locations and sampling years. These yeast species were monitored from the vineyard into laboratory-scale spontaneous fermentations. Taxa assignable to Metschnikowia and Saccharomyces persisted during fermentation, whereas Curvibasidium, which also has possible impact on biocontrol and wine quality, did not. Sulfite generally reduced yeast diversity and richness, but its effect on the abundance of specific yeasts during fermentation was negligible. Among the 106 recurring non-yeast fungal taxa, Alternaria, Cladosporium and Ulocladium were especially abundant in the vineyard. Vineyard location was the primary factor that accounted for the variation among both communities, followed by year and berry developmental stage. The Washington mycobiomes were compared to those from other parts of the world. Sixteen recurrent yeast species appeared to be unique to Washington State vineyards. This subset also contained a higher proportion of species associated with cold and extreme environments, relative to other localities. Certain yeast and non-yeast fungal species known to suppress diseases or modify wine sensory properties were present in Washington vineyards, and likely have consequences to vineyard health and wine quality.

Sterol uptake analysis in Saccharomyces and non-Saccharomyces wine yeast species.

Sterols are essential components of the yeast membrane and their synthesis requires oxygen. Yet, Saccharomyces cerevisiae has developed the ability to take up sterols from the medium under anaerobiosis. Here we investigated sterol uptake efficiency and the expression of genes related to sterol import in Saccharomyces and non-Saccharomyces wine yeast species fermenting under anaerobic conditions. The sterol uptake efficiency of 39 strains was evaluated by flow cytometry (with 25-NBD Cholesterol, a fluorescent cholesterol probe introduced in the medium) and we found an important discrepancy between Saccharomyces and non-Saccharomyces wine yeast species that we correlated to a lower final cell population and a lower fermentation rate. A high uptake of sterol was observed in the various Saccharomyces strains. Spot tests performed on 13 of these strains confirmed the differences between Saccharomyces and non-Saccharomyces strains, suggesting that the presence of the sterol uptake transporters AUS1 and PDR11 could cause these discrepancies. Indeed, we could not find any homologue to these genes in the genome of Hanseniaspora uvarum, H. guillermondii, Lachancea thermotolerans, Torulaspora delbreueckii, Metschnikowia pulcherrima, or Starmarella bacillaris species. The specialization of sterol import function for post genome-duplication species may have favored growth under anaerobiosis.

Biodiversity of yeasts isolated during spontaneous fermentation of cool climate grape musts.

Biodiversity of native yeasts, especially in winemaking, has hidden potential. In order to use the value of non-Saccharomyces strains in wine production and to minimise the possibility of its deterioration, it is necessary to thoroughly study the yeast cultures present on grape fruits and in grape must, as well as their metabolic properties. The aim of the study was to characterise the yeast microbiota found during spontaneous fermentation of grape musts obtained from grape varieties 'Rondo', 'Regent' and 'Johanniter'. Grapes from two vineyards (Srebrna Góra and Zadora) located in southern Poland were used for the research. Succession of subsequent groups of yeasts was observed during the process. Metschnikowia pulcherrima yeasts were identified both at the beginning and the end of the process. Hanseniaspora uvarum, Wickerhamomyces onychis and Torulaspora delbrueckii strains were also identified during the fermentation. Torulaspora delbrueckii and Wickerhamomyces onychis strains were identified only in grape musts obtained from grapes of the Zadora vineyard. These strains may be characteristic of this vineyard and shape the identity of wines formed in it. Our research has provided specific knowledge on the biodiversity of yeast cultures on grapes and during their spontaneous fermentation. The research results presented indicate the possibility of using native strains for fermentation of grape musts, allowing to obtain a product with favourable chemical composition and sensory profile.

Potential probiotic and food protection role of wild yeasts isolated from pistachio fruits (Pistacia vera).

BACKGROUND: The biotechnological potential of yeasts from nuts such as pistachio, not only for health applications but also for industry use, has been scarcely studied. Interest in the probiotic capability of yeasts has increased in the past years as well as their utilization as food or feed preservatives. Their capabilities as biocontrol against problematic (spoilage or toxigenic) microorganisms or as antioxidants have been revalued. As a result, both abilities would be desirable to develop a new potential probiotic microorganism which could be added to food or feed to improve their properties. RESULTS: Molecular techniques allowed the identification of a total of seven different species and 15 strains. A screening of the probiotic potential of these strains was carried out. It was found that 65% of the strains resisted the gastrointestinal conditions as well as presented a generation time of < 22 h. Additionally, some strains showed better kinetic parameters than Saccharomyces boulardii (positive control). Complementary tests were done to determine their auto-aggregation capacity, cell surface hydrophobicity, behaviour in a sequential simulated digestion, biofilm formation capability and carbon source assimilation. Finally, 67% and 13% of the studied yeasts showed biocontrol and antioxidant activities, respectively. CONCLUSIONS: Diutina rugosa 14 followed by Diutina rugosa 8 were the best wild yeast from Pistacia vera as potential probiotic and in carbon source utilization. However, Hanseniaspora guilliermondii 6 and Aureobasidium proteae 5 could be used to improve food or feed product preservation because of their notable biocontrol and antioxidant capabilities. © 2020 Society of Chemical Industry.

Yeasts Associated with Various Amazonian Native Fruits.

Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.

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 communities of secondary peat swamp forests in Thailand and their antagonistic activities against fungal pathogens cause of plant and postharvest fruit diseases.

Secondary peat swamp forest (PSF) arise by degradation of primary PSF as a result of fire and human activities. Yeasts diversity of Kuan Kreng (KK) and Rayong Botanical Garden (RBG) PSF, which are two secondary PSF in southern and in eastern Thailand, respectively, were investigated. Yeasts were isolated from soil and peat soil by the dilution plate and enrichment techniques. From six samples collected from KK PSF, 35 strains were obtained, and they were identified based on the sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene 13 species in 12 genera, and one potential new species of the genus Galactomyces were detected. Thirty-two strains were obtained from six samples collected from RBG PSF and 26 strains were identified as 13 known yeast species in 11 genera, whereas six strains were found to represent two potential new species of the genera Papiliotrema and Moesziomyces. Among yeast strains isolated from KK PSF, the number of strains in the phylum Ascomycota and Basidiomycota were equal, whereas there were slightly fewer strains in Ascomycota than in Basidiomycota among the strains obtained from RBG PSF. The yeast strains were evaluated for their antagonistic activities against fungal pathogens which cause rice diseases (Fusarium moniliforme, Helminthosporium oryzae, Rhizoctonia solani, Curvularia lunata and Pyricularia grisea) and postharvest disease of fruits (Phytophthora palmivora, Lasiodiplodia theobromae and Colletotrichum gloeosporioides). Twelve strains of seven species were found to be antagonistic yeast strains. Starmerella kuoi DMKU-SPS13-6, Hanseniaspora lindneri DMKU ESS10-9 and Piskurozyma taiwanensis DMKU-SPS12-2 capable to inhibit R. solani by 70.1-76.2%, Wickerhamomyces anomalus DMKU SPS6-1 and three Rhodotorula taiwanensis strains (DMKU SPS8-1, DMKU ESS9-3, DMKU SPS9-2) inhibited C. lunata by 69.8-71.9%, Hanseniaspora lindneri DMKU ESS10-9 and Scheffersomyces spartinae DMKU SPS9-3 inhibited P. grisea by 81.9-84.4% and four Papiliotrema laurentii strains (DMKU-SPS15-1, DMKU-ESS11-2, DMKU-ESS8-2, DMKU-ESS6-4) inhibited P. palmivora by 53.2-59.5%.

Non-tandem repeat polymorphisms at microsatellite loci in wine yeast species.

Yeast microsatellite loci consist of short tandem-repeated DNA sequences of variable length. The high mutational rate at these loci generates a remarkable repertoire of alleles, useful for strain differentiation and population genetic studies. In this work, we analyze the DNA sequences of thirteen alleles from each of ten microsatellite loci described for the yeast Starmerella bacillaris. Our results show that polymorphic variants of some informative alleles are dependent on SNPs and indels rather than on length variation at their originally defined tandem-repeated motifs. The analysis was extended to 55 previously described hypervariable microsatellite loci from a total of 26 sequenced genomes of yeast species that dominate the microbiota of spontaneously fermenting grape musts (i.e., Hanseniaspora uvarum, Saccharomyces cerevisae, Saccharomyces uvarum, and Torulaspora delbrueckii) or lead to wine spoilage (Brettanomyces bruxellensis and Meyerozyma guilliermondii). We found that allelic variants for some microsatellite loci of these yeast species are also dependent on SNPs and/or indels flanking their tandem-repeated motifs. For some loci, the number of units at their tandem repeats was found to be identical among the various characterized alleles, with allelic differences being dependent exclusively on flanking polymorphisms. Our results indicate that allele sizing of microsatellite loci using PCR, although valid for strain differentiation and population genetic studies, does not necessarily score the number of units at their tandem-repeated motifs. Sequence analysis of microsatellite loci alleles could provide relevant information for evolutionary and phylogeny studies of yeast species.

Into the wild: new yeast genomes from natural environments and new tools for their analysis.

Genomic studies of yeasts from the wild have increased considerably in the past few years. This revolution has been fueled by advances in high-throughput sequencing technologies and a better understanding of yeast ecology and phylogeography, especially for biotechnologically important species. The present review aims to first introduce new bioinformatic tools available for the generation and analysis of yeast genomes. We also assess the accumulated genomic data of wild isolates of industrially relevant species, such as Saccharomyces spp., which provide unique opportunities to further investigate the domestication processes associated with the fermentation industry and opportunistic pathogenesis. The availability of genome sequences of other less conventional yeasts obtained from the wild has also increased substantially, including representatives of the phyla Ascomycota (e.g. Hanseniaspora) and Basidiomycota (e.g. Phaffia). Here, we review salient examples of both fundamental and applied research that demonstrate the importance of continuing to sequence and analyze genomes of wild yeasts.

Identification of Microorganisms Associated with the Quality Improvement of Dry-Aged Beef Through Microbiome Analysis and DNA Sequencing, and Evaluation of Their Effects on Beef Quality.

The objective of this study was to isolate and identify the microorganisms, especially yeasts and molds, related to the improvement of beef quality during dry-aging of beef through microbiome analysis, and to examine the possibility of using them as starter culture strains to improve the efficiency of dry-aging beef production. Beef sirloins were dry-aged for 28 days using different wind speeds (0, 2.5, and 5 m/s) at 1 to 3 °C and 75% relative humidity, and microbial compositions were confirmed by microbiome analysis. Mold and yeast samples were plated on potato dextrose agar supplemented with 10% tartaric acid, and the isolated colonies were identified by DNA sequencing. The isolates were subjected to microbial characterization (morphological characterization, growth condition, and enzyme activity). Microbiome analysis showed that the dominant microorganisms were molds and yeasts identified as Pilaira anomala SMFM201611 and Debaryomyces hansenii SMFM201707. Pilaira anomala SMFM201611 and D. hansenii SMFM201707 were inoculated into 24 sirloins of the lowest grade. All samples were dry-aged for 0, 14, 21, and 28 days and analyzed for microbial growth, pH, shear force, ultrastructure, and flavor compounds (free amino acids and free fatty acids). Inoculation with P. anomala SMFM201611 and D. hansenii SMFM201707 improved tenderness and cause the breakdown of myofibrils by proteolysis. Both microorganisms also produced free amino acids and fatty acids through proteolytic and lipolytic activities. These results indicate that P. anomala SMFM201611 and D. hansenii SMFM201707 isolated and identified from dry-aged beef can improve the quality of low-grade beef during dry-aging. PRACTICAL APPLICATION: During dry-aging, mold and yeast improve the quality of dry-aged beef. Pilaira anomala SMFM201611 and Debaryomyces hansenii SMFM201707 isolated from dry-aged beef can improve tenderness by breaking down myofibrils. Both microorganisms improve flavor by producing free fatty acids and amino acids, and the taste and aroma characteristics of low-grade beef may be improved during the dry-aging process.

Characterization of tolerance and multi-enzyme activities in non-Saccharomyces yeasts isolated from Vidal blanc icewine fermentation.

This work aim to study the tolerance and aroma-related enzymes activities of the non-Saccharomyces yeasts in Vidal blanc icewine from the Huanren region of China. The strains were identified by sequencing internal transcribed spacer (ITS) region and the 26S rDNA D1/D2 domain genes and all representative non-Saccharomyces yeasts were belonged to genera Metschnikowia, Hanseniaspora, Torulaspora, Candida, and Debaryomyces. A total of 28 strains were carried out for tolerance experiments and results suggested that most of them could tolerate 500 g/L glucose, 4% ethanol, 20 g/L tartaric acid, and 350 mg/L SO2 . Finally, a total of 17 strains with better tolerance were carried out for the ß-glucosidase, ß-xylosidase, and pectinase activities experiments. The results showed that Candida railenensis HC08 and the strains of Hanseniaspora genus have satisfactory multi-enzyme activities, which can be used to design mixed fermentation to produce characteristic icewine. PRACTICAL APPLICATIONS: Non-Saccharomyces yeasts produces a series of hydrolytic enzymes that are thought to have a significant contribution to the aroma complexity of wines, however, are poorly explored in icewine. In this work, most of the non-Saccharomyces yeasts screened from Chinese icewine can adapt well to the high-sugar and high-acid environment of icewine, and can secrete hydrolase. The application of these strains in mixed fermentation could provide a prospect for the production of characteristic icewine.

Diversity of yeasts in the soil adjacent to fruit trees of the Rosaceae family.

Yeasts are common constituents of different types of soil. Their diversity depends on the season, the type and depth of the soil, the plant species, and the locality. In this study, diversity of yeasts isolated from the soil adjacent to five fruit trees (apple, appricot, peach, pear, and plum) in two localities (in Slovakia) in four sampling periods was examined. Our results demonstrated differences in the species richness and evenness among the yeast populations, which inhabited the soil beneath individual fruit tree species in both localities. Altogether, 32 ascomycetous and 27 basidiomycetous yeast species were discovered. The highest species richness was found in the soil adjacent to the apricot trees. Galactomyces candidum, Metschnikowia pulcherrima, Hanseniaspora uvarum, Schwanniomyces capriottii, and Tausonia pullulans, as well as the genus Apiotrichum, were present in soil samples in all samplings. Two species of the genus Holtermanniella (H. festucosa and H. takashimae) were exclusively isolated during Sampling IV in April. Cyberlindnera spp., Clavispora reshetovae, S. capriottii, and Trichosporon asahii were found only in one of two localities. Ascomycetous yeasts were present more frequently than their basidiomycetous counterparts in the three samplings (one in June and two in October); they formed from 65.6% to 70.8% of the total yeast population, whereas basidiomycetous yeasts prevailed in the April sampling (61.2%).

Yeast-like fungi and yeasts in withered grape carposphere: Characterization of Aureobasidium pullulans population and species diversity.

Yeast-like fungi and yeasts residing on carposphere of withered grapes for Italian passito wine production have been scarcely investigated. In the present study, isolates from single berries, both sound and damaged, of Nosiola, Corvina and Garganega varieties were analyzed at the end of the withering process. Great variation of cell concentration among single berries was observed. In sound berries, yeast-like fungi were significantly more frequent than yeasts. Species identification of isolates was carried out by BLAST comparative analysis on gene databases and phylogenetic approach. All yeast-like fungi isolates belonged to Aureobasidium pullulans. They displayed different culture and physiological characteristics and inhibitory capacity against phytopathogenic fungi. Moreover, PCR profile analysis revealed high genotypic similarity among these strains. A total of 35 species were recognized among yeast isolates. Ascomycetes prevailed over basidiomycetes. To the best of our knowledge, Naganishia onofrii and Rhodosporidiobolus odoratus were identified for the first time among yeasts isolated from grapes, must or wine. Hanseniaspora uvarum and Starmerella bacillaris were the most frequent species. Most species were found only in one grape variety (nine in Nosiola, 10 in Corvina and five in Garganega). The sanitary state of withered grapes could have an important impact on the structure of these epiphytic populations.

Population and oenological characteristics of non-Saccharomyces yeasts associated with grapes of Northwestern Argentina.

Yeasts population associated with grapes from Northwest Argentina, a region with a significant vine-growing increase over the past years, was evaluated. Ten species of non-Saccharomyces yeasts were identified from four grape varieties (Malbec, Merlot, Syrah and Torrontes) being Hanseniaspora uvarum the dominant species. Typing of isolates revealed genetic variability within Hanseniaspora genus and also high variability was observed according to their oenological characteristics. Based on the oenological properties, the most adequate strains as starter cultures were H. uvarum HuT7, HuMe15, HuS16, H. vineae HvT-mc1 and Metschnikowia pulcherrima MpT2/MpT3. These selected yeasts exhibited moderate resistance to SO2, reduced values of volatile acidity, null or low production of H2S, high levels of enzymes related to aroma and did not produce killer toxins. Further studies using mixed cultures of these non-Saccharomyces strains and S. cerevisiae are needed to validate the contribution of selected indigenous yeasts on wine organoleptic characteristics.

Microbiology of high-sugar must fermentation by novel yeasts from the chihuahuan desert.

This work reports important results of aromatic profiles produced by native yeasts isolated from desert-grown wine grapes from the south of Chihuahua, México. These grapes stand very high temperatures during the ripening season, developing high sugar concentration and high pH. Yeast species found in grapes were identified by polymerase chain reaction and sequence analysis of the 5.8S internal transcribed spacer ribosomal RNA region. Aureobasidium namibiae, Sporobolomyces johnsonii, Candida apicola, Hanseniaspora uvarum, Candida thaimueangensis, Hanseniaspora opuntiae were identified. All of them can grow at glucose concentration of 35% (w/v) and 100 ppm of SO2, and produce low volatile acidity (0.2-1.0 g acetic acid/L). Volatile organic compounds analysis showed that C. thaimueangensis and one strain of C. apicola produce high levels of esters, and Hanseniaspora species produces high levels of higher alcohols and carbonyl compounds. The results of this study contribute to the knowledge about yeast communities associated with desert-grown winegrape yeasts.

Agrochemicals: Effect on genetic resistance in yeasts colonizing winter wheat kernels.

Crop protection agents are widely used in modern agriculture and exert direct effects on non-target microorganisms such as yeasts. Yeasts abundantly colonize wheat grain and affect its chemical composition. They can also limit pathogen growth. This study evaluated the sensitivity of yeast communities colonizing winter wheat kernels to benzimidazole, strobilurin, triazole and morpholine fungicides, trinexapac-ethyl, a commercial mixture of o-nitrophenol+p-nitrophenol+5-nitroguaiacol, and chitosan applied during the growing season of winter wheat and in vitro in a diffusion test. A molecular identification analysis of yeasts isolated from winter wheat kernels was performed, and nucleotide polymorphisms in the CYTb gene (G143A) conferring resistance to strobilurin fungicides in yeast cells were identified. The size of yeast communities increased during grain storage, and the total counts of endophytic yeasts were significantly (85%) reduced following intensive fungicide treatment (fenpropimorph, a commercial mixture of pyraclostrobin, epoxiconazole and thiophanate-methyl). This study demonstrated that agrochemical residues in wheat grain can drive selection of yeast communities for reduced sensitivity to xenobiotics. A mutation in the CYTb gene (G143A) was observed in all analyzed isolates of the following azoxystrobin-resistant species: Aureobasidium pullulans, Debaryomyces hansenii, Candida albicans and C. sake. Agrochemicals tested in vitro were divided into four classes of toxicity to yeasts: (1) tebuconazole and a commercial mixture of flusilazole and carbendazim - most toxic to yeasts; (2) fenpropimorph and a commercial mixture of pyraclostrobin and epoxyconazole; (3) propiconazole, chitosan, thiophanate-methyl and a commercial mixture of o-nitrophenol, p-nitrophenol and 5-nitroguaiacol; (4) trinexapac-ethyl and azoxystrobin - least toxic to yeasts. It was found that agrochemicals can have an adverse effect on yeast abundance and the composition of yeast communities, mostly due to differences in fungicide resistance between yeast species, including the clinically significant C. albicans.

MALDI-TOF MS Supplementary database for species identification employing the yeast diversity encountered on southern Brazil grapes.

The study of grape microflora is of interest when autochthonous yeasts, which are related to typical wine characteristics, are intended to be used in winemaking. The election of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) as the first method for yeast identification was based on its accuracy and rapidity compared to alternative laboratory protocols for identification. The aims of this study are to consolidate the MALDI-TOF MS Supplementary database for environmental yeasts already constructed, to expand it through the addition of standard spectra of not included yeast species, and to discuss the grape microflora encountered in Southern Brazil. A total of 358 strains, isolated from grape berries, were submitted to protein profiling employing Biotyper and Supplementary database. Molecular biology techniques were used as alternatives to identify 6.4% of strains not promptly designated by protein profiling. These strains corresponded to the species Candida californica, Zygoascus meyerae, Candida akabanensis, Candida azyma, and Hanseniaspora vineae. The MALDI-TOF MS spectra of the identified species were added to Supplementary database. The presented results strengthen the need for further expansion of the mass spectra database to broaden its microbiological application.

Yeast species diversity in apple juice for cider production evidenced by culture-based method.

Identification of yeasts isolated from apple juices of two cider houses (one located in a plain area and one in an alpine area) was carried out by culture-based method. Wallerstein Laboratory Nutrient Agar was used as medium for isolation and preliminary yeasts identification. A total of 20 species of yeasts belonging to ten different genera were identified using both BLAST algorithm for pairwise sequence comparison and phylogenetic approaches. A wide variety of non-Saccharomyces species was found. Interestingly, Candida railenensis, Candida cylindracea, Hanseniaspora meyeri, Hanseniaspora pseudoguilliermondii, and Metschnikowia sinensis were recovered for the first time in the yeast community of an apple environment. Phylogenetic analysis revealed a better resolution in identifying Metschnikowia and Moesziomyces isolates than comparative analysis using the GenBank or YeastIP gene databases. This study provides important data on yeast microbiota of apple juice and evidenced differences between two geographical cider production areas in terms of species composition.