Co-culture fermentation by Saccharomycopsis fibuligera and lactic acid bacteria improves bioactivity and aroma profile of wheat bran and the bran-containing Chinese steamed bread.

Co-culture fermentation with yeast and lactic acid bacteria (LAB) exhibits advantages in improving the bioactivity and flavor of wheat bran compared to single-culture fermentation, showing application potentials in bran-containing Chinese steamed bread (CSB). To explore the effects of combination of yeast and different LAB on the bioactivity and flavor of fermented wheat bran, this study analyzed the physicochemical properties, phytate degradation capacity, antioxidant activities, and aroma profile of wheat bran treated with co-culture fermentation by Saccharomycopsis fibuligera and eight different species of LAB. Further, the phenolic acid composition, antioxidant activities, texture properties, aroma profile, and sensory quality of CSB containing fermented wheat bran were evaluated. The results revealed that co-culture fermentation brought about three types of volatile characteristics for wheat bran, including ester-feature, alcohol and acid-feature, and phenol-feature, and the representative strain combinations for these characteristics were S. fibuligera with Limosilactobacillus fermentum, Pediococcus pentosaceus, and Latilactobacillus curvatus, respectively. Co-culture fermentation by S. fibuligera and L. fermentum for 36 h promoted acidification with a phytate degradation rate reaching 51.70 %, and improved the production of volatile ethyl esters with a relative content of 58.47 % in wheat bran. Wheat bran treated with co-culture fermentation by S. fibuligera and L. curvatus for 36 h had high relative content of 4-ethylguaiacol at 52.81 %, and exhibited strong antioxidant activities, with ABTS•+ and DPPH• scavenging rates at 65.87 % and 69.41 %, respectively, and ferric reducing antioxidant power (FRAP) at 37.91 µmol/g. In addition, CSB containing wheat bran treated with co-culture fermentation by S. fibuligera and L. fermentum showed a large specific volume, soft texture, and pleasant aroma, and received high sensory scores. CSB containing wheat bran treated with co-culture fermentation by S. fibuligera and L. curvatus, with high contents of 4-ethylguaiacol, 4-vinylguaiacol, ferulic acid, vanillin, syringaldehyde, and protocatechualdehyde, demonstrated strong antioxidant activities. This study is beneficial to the comprehensive utilization of wheat bran resources and provides novel insights into the enhancement of functions and quality for CSB.

Isolation of Saccharomycopsis species from plant material.

Saccharomycopsis species are natural organic sulphur auxotrophs. Their genomes do not encode genes for the uptake and assimilation of sulphate and thus these species cannot grow on media lacking e.g. methionine. Due to the similarity between sulphate and selenate, uptake and assimilation of selenate occurs through the same pathway starting from sulphate transporters encoded by the homologs of the SUL1 and SUL2 genes in S. cerevisiae. Lack of these transporters renders Saccharomycopsis species resistant to selenate levels that are toxic to other microorganisms. We used this feature to enrich environmental samples for Saccharomycopsis species. This led to the isolation of S. schoenii, S. lassenensis and a hitherto undescribed Saccharomycopsis species with limited by-catch of other yeasts, mainly belonging to Metschnikowia and Hanseniaspora. We performed growth and predation assays to characterize the potential of these new isolates as predacious yeasts. Most Saccharomycopsis species are temperature sensitive and cannot grow at 37°C; with the exception of S. lassenensis strains. Predation assays with S. schoenii and S. cerevisiae as prey indicated that predation was enhanced at 20°C compared to 30°C. We crossed an American isolate of S. schoenii with our German isolate using marker directed breeding. Viable progeny indicated that both strains are interfertile and belong to the same biological species. S. lassenensis is heterothallic, while S. schoenii and the new Saccharomycopsis isolate, for which we suggest the name S. geisenheimensis sp. nov., are homothallic.

Saccharomycopsis praedatoria sp. nov., a predacious yeast isolated from soil and rotten wood in an Amazonian rainforest biome.

Three yeast isolates were obtained from soil and rotting wood samples collected in an Amazonian rainforest biome in Brazil. Comparison of the intergenic spacer 5.8S region and the D1/D2 domains of the large subunit rRNA gene showed that the isolates represent a novel species of the genus Saccharomycopsis. A tree inferred from the D1/D2 sequences placed the novel species near a subclade containing Saccharomycopsis lassenensis, Saccharomycopsis fermentans, Saccharomycopsis javanensis, Saccharomycopsis babjevae, Saccharomycopsis schoenii and Saccharomycopsis oosterbeekiorum, but with low bootstrap support. In terms of sequence divergence, the novel species had the highest identity in the D1/D2 domains with Saccharomycopsis capsularis, from which it differed by 36 substitutions. In contrast, a phylogenomic analysis based on 1061 single-copy orthologs for a smaller set of Saccharomycopsis species whose whole genome sequences are available indicated that the novel species represented by strain UFMG-CM-Y6991 is phylogenetically closer to Saccharomycopsis fodiens and Saccharomycopsis sp. TF2021a (=Saccharomycopsis phalluae). The novel yeast is homothallic and produces asci with one spheroidal ascospore with an equatorial or subequatorial ledge. The name Saccharomycopsis praedatoria sp. nov. is proposed to accommodate the novel species. The holotype of Saccharomycopsis praedatoria is CBS 16589T. The MycoBank number is MB849369. S. praedatoria was able to kill cells of Saccharomyces cerevisiae by means of penetration with infection pegs, a trait common to most species of Saccharomycopsis.

Improved gene-targeting efficiency upon starvation in Saccharomycopsis.

Commonly used fungal transformation protocols rely on the use of either electroporation or the lithium acetate/single strand carrier DNA/Polyethylene glycol/heat shock method. We have used the latter method previously in establishing DNA-mediated transformation in Saccharomycopsis schoenii, a CTG-clade yeast that exhibits necrotrophic mycoparasitism. To elucidate the molecular mechanisms of predation by Saccharomycopsis we aim at gene-function analyses to identify virulence-related pathways and genes. However, in spite of a satisfactory transformation efficiency our efforts were crippled by high frequency of ectopic integration of disruption cassettes. Here, we show that overnight starvation of S. schoenii cells, while reducing the number of transformants, resulted in a substantial increase in gene-targeting via homologous recombination. To demonstrate this, we have deleted the S. schoenii CHS1, HIS3 and LEU2 genes and determined the required size of the flanking homology regions. Additionally, we complemented the S. schoenii leu2 mutant with heterologous LEU2 gene from Saccharomycopsis fermentans. To demonstrate the usefulness of our approach we also generated a S. fermentans leu2 strain, suggesting that this approach may have broader applicability.

Determining the changes in metabolites of Dendrobium officinale juice fermented with starter cultures containing Saccharomycopsis fibuligera FBKL2.8DCJS1 and Lactobacillus paracasei FBKL1.3028 through untargeted metabolomics.

BACKGROUND: The present study aimed to investigate the changes in volatile components and metabolites of Dendrobium officinale (D. officinale) juice fermented with starter cultures containing Saccharomycopsis fibuligera and Lactobacillus paracasei at 28 ℃ for 15 days and post-ripened at 4 ℃ for 30 days using untargeted metabolomics of liquid chromatography-mass spectrometry (LC-MS) and headspace solid-phase microextraction-gas chromatography (HS-SPME-GC-MS) before and after fermentation. RESULTS: The results showed that the alcohol contents in the S. fibuligera group before fermentation and after fermentation were 444.806 ± 10.310 µg/mL and 510.999 ± 38.431 µg/mL, respectively. Furthermore, the alcohol content in the fermentation broth group inoculated with the co-culture of L. paracasei + S. fibuligera was 504.758 ± 77.914 µg/mL, containing a significant amount of 3-Methyl-1-butanol, Linalool, Phenylethyl alcohol, and 2-Methyl-1-propanol. Moreover, the Ethyl L (-)-lactate content was higher in the co-culture of L. paracasei + S. fibuligera group (7.718 ± 6.668 µg/mL) than in the L. paracasei (2.798 ± 0.443 µg/mL) and S. fibuligera monoculture groups (0 µg/mL). The co-culture of L. paracasei + S. fibuligera significantly promoted the metabolic production of ethyl L (-)-lactate in D. officinale juice. The differential metabolites screened after fermentation mainly included alcohols, organic acids, amino acids, nucleic acids, and their derivatives. Twenty-three metabolites, including 11 types of acids, were significantly up-regulated in the ten key metabolic pathways of the co-culture group. Furthermore, the metabolic pathways, such as pentose and glucuronate interconversions, the biosynthesis of alkaloids derived from terpenoid and polyketide, and aminobenzoate degradation were significantly up-regulated in the co-culture group. These three metabolic pathways facilitate the synthesis of bioactive substances, such as terpenoids, polyketides, and phenols, and enrich the flavor composition of D. officinale juice. CONCLUSIONS: These results demonstrate that the co-culture of L. paracasei + S. fibuligera can promote the flavor harmonization of fermented products. Therefore, this study provides a theoretical basis for analyzing the flavor of D. officinale juice and the functional investigation of fermentation metabolites.

Physiological characterization and molecular identification of Saccharomycopsis fibuligera as the etiological agent of a skin lesion.

Routine laboratory methods are not effective in identifying cryptic species resulting in the underreporting of infections caused by non-Candida yeasts. This paper presents the physiological characteristics and antifungal susceptibility of Saccharomycopsis fibuligera 12-771, isolated from a tinea-like lesion. Isolate 12-771 was identified by ITS and D1/D2 analysis as S. fibuligera. The isolate presented an auxonogram profile similar to Candida utilis, as well as protease, esterase and hemolysin activity. MICs were of 0.25 â€‹µg/mL for amphotericin B, 1-2 â€‹µg/mL for echinocandins, and 16 â€‹µg/mL for fluconazole. This work represents the first record in America of S. fibuligera as an infectious agent.

Response of microbial community assembly and succession pattern to abiotic factors during the second round of light-flavor Baijiu fermentation.

The regulation of microbial communities is an important strategy for fermentation management. Dynamic microbiota during the Baijiu fermentation is shaped by a variety of abiotic factors. Therefore, this study aims to investigate the effects of the microbiota of the fermentation starter Daqu on the microbial assembly and the interaction of microbiota succession and abiotic factors during the second round (Ercha) of light-flavor Baijiu fermentation. The results revealed that Streptomyces, Bacillus, Lactobacillus, and Staphylococcus were the dominant bacterial genera in the initial fermentation, while Lactobacillus was dominant during the middle and later stages. Pichia and Saccharomycopsis were the dominant fungal genera during the whole fermentation process. A total of 54 volatile compounds were identified during the fermentation, among which 15 compounds, mainly including ethyl acetate, diethyl azelate, ethyl 2-hydroxyisocaproate, 3-furaldehyde, and ethylidene diacetate, were identified as important flavor metabolites. The SourceTracker software revealed that Daqu contributed 52.3 % of the bacterial community and 38.6 % of the fungal community to the fermentation. Ethanol, moisture, and pH were the major factors regulating the succession of dominant bacteria and fungi during the fermentation. The microbial succession and co-occurrence pattern driven by abiotic factors played a crucial role in shaping flavor profiles. These results provide guidance for controlling the fermentation process by optimizing operational parameters or bioaugmentation with specific microbes.

Development of quantitative real-time PCR and digital droplet-PCR assays for rapid and early detection of the spoilage yeasts Saccharomycopsis fibuligera and Wickerhamomyces anomalus in bread.

In the present study, for the first time, high sensitive quantitative polymerase chain reaction (qPCR) and digital droplet polymerase chain reaction (ddPCR) assays were developed to detect and quantify total eumycetes with potential application in several food matrices and to specifically determine the level of contamination by Saccharomycopsis fibuligera and Wickerhamomyces anomalus cells directly in bread. Among the candidate target genes used to develop the assays, car1 gene was chosen to detect the two spoilage yeasts S. fibuligera and W. anomalus. The specificity of the PCR assays was tested using purified genomic DNA from 36 bacterial and fungal strains. The sensitivity of the assays was defined by using tenfold serial dilutions of genomic DNA starting from 106 cfu/mL to 1 cfu/mL of S. fibuligera and W. anomalus. Validation of the assays was achieved by enumeration of S. fibuligera and W. anomalus DNA copies from samples of artificially contaminated bread homogenates detecting up to 10 cfu/mL (0.06 ± 0.01 copies/µL) of W. anomalus by using ddPCR. In conclusion, the developed qPCR and ddPCR assays demonstrate strong performance in the early detection of S. fibuligera and W. anomalus in bread, representing promising tools for applying high-throughput approaches to regularly monitor bread quality.

Genetic diversity and population structure of the amylolytic yeast Saccharomycopsis fibuligera associated with Baijiu fermentation in China.

The amylolytic yeast Saccharomycopsis fibuligera is a predominant species in starters and the early fermentation stage of Chinese liquor (Baijiu). However, the genetic diversity of the species remains largely unknown. Here we sequenced the genomes of 97 S. fibuligera strains from different Chinese Baijiu companies. The genetic diversity and population structure of the strains were analyzed based on 1,133 orthologous genes and the whole genome single nucleotide polymorphisms (SNPs). Four main lineages were recognized. One lineage contains 60 Chinese strains which are exclusively homozygous with relatively small genome sizes (18.55-18.72 Mb) and low sequence diversity. The strains clustered in the other three lineages are heterozygous with larger genomes (21.85-23.72 Mb) and higher sequence diversity. The genomes of the homozygous strains showed nearly 100% coverage with the genome of the reference strain KPH12 and the sub-genome A of the hybrid strain KJJ81 at the above 98% sequence identity level. The genomes of the heterozygous strains showed nearly 80% coverage with both the sub-genome A and the whole genome of KJJ81, suggesting that the Chinese heterozygous strains are also hybrids with nearly 20% genomes from an unidentified source. Eighty-three genes were found to show significant copy number variation between different lineages. However, remarkable lineage specific variations in glucoamylase and α-amylase activities and growth profiles in different carbon sources and under different environmental conditions were not observed, though strains exhibiting relatively high glucoamylase activity were mainly found from the homozygous lineage.

Molecular characterization of the Saccharomycopsis fibuligera ATF genes, encoding alcohol acetyltransferase for volatile acetate ester formation.

Aroma ester components produced by fermenting yeast cells via alcohol acetyltransferase (AATase)-catalyzed intracellular reactions are responsible for the fruity character of fermented alcoholic beverages, such as beer and wine. Acetate esters are reportedly produced at relatively high concentrations by non-Saccharomyces species. Here, we identified 12 ATF orthologues (SfATFs) encoding putative AATases, in the diploid genome of Saccharomycopsis fibuligera KJJ81, an isolate from wheat-based Nuruk in Korea. The identified SfATF proteins (SfAtfp) display low sequence identities with S. cerevisiae Atf1p (between 13.3 and 27.0%). All SfAtfp identified, except SfAtf(A)4p and SfAtf(B)4p, contained the activation domain (HXXXD) conserved in other Atf proteins. Culture supernatant analysis using headspace gas chromatography mass spectrometry confirmed that the recombinant S. cerevisiae strains expressing SfAtf(A)2p, SfAtf(B)2p, and SfAtf(B)6p produced high levels of isoamyl and phenethyl acetates. The volatile aroma profiles generated by the SfAtf proteins were distinctive from that of S. cerevisiae Atf1p, implying difference in the substrate preference. Cellular localization analysis using GFP fusion revealed the localization of SfAtf proteins proximal to the lipid particles, consistent with the presence of amphipathic helices at their N- and C-termini. This is the first report that systematically characterizes the S. fibuligera ATF genes encoding functional AATases responsible for acetate ester formation using higher alcohols as substrate, demonstrating their biotechnological potential for volatile ester production.

Bacterial and fungal communities and their predictive functional profiles in kinema, a naturally fermented soybean food of India, Nepal and Bhutan.

Bacterial and fungal communities in kinema, a naturally fermented soybean food of the Eastern Himalayan regions of India, Nepal and Bhutan were profiled by high-throughout sequence analysis. Firmicutes (78.4%) was the most abundant phylum in kinema, followed by Proteobacteria (14.76%) and other phyla. Twenty seven species of Bacillus were detected, among which Bacillus subtilis (28.70%) was the most abundant bacterium, followed by B. licheniformis, B. thermoamylovorans, B. cereus, Ignatzschineria larvae, Corynebacterium casei, B. sonorensis, Proteus vulgaris, Brevibacillus borstelensis, Thermoactinomyces vulgaris, Lactobacillus fermentum and Ignatzschineria indica. Ascomycota was the most abundant fungal phylum in kinema. Wallemia canadensis, Penicillium spp., Aspergillus spp., Exobasidium spp., Arthrocladium spp., Aspergillus penicillioides, Mortierella spp., Rhizopus arrhizus and Mucor circinelloides, were major moulds, and Pichia sporocuriosa, Trichosporon spp., Saccharomycopsis malanga and Rhodotorula cycloclastica were abundant yeasts in kinema. We detected 277 species of bacteria among which, 99.09% were culturable and 0.91% were unculturable; and 80 fungal species among which, 33.72% were culturable and 66.28% were unculturable. Several unique bacterial genera to each country were observed, whereas no unique fungal genus was observed in kinema. Maximum coverage of sequencing depth was observed in all samples. Based on PCA plot, close relation was observed between samples of India and Nepal, whereas samples of Bhutan was clearly distinctive. Predictive functional features of bacterial and fungi related to metabolisms were inferred by the KEGG Orthology and MetaCyc databases, respectively.

Arabinoxylo- and Arabino-Oligosaccharides-Specific α-L-Arabinofuranosidase GH51 Isozymes from the Amylolytic Yeast Saccharomycopsis fibuligera.

Two genes encoding probable α-L-arabinofuranosidase (E.C. 3.2.1.55) isozymes (ABFs) with 92.3% amino acid sequence identity, ABF51A and ABF51B, were found from chromosomes 3 and 5 of Saccharomycopsis fibuligera KJJ81, an amylolytic yeast isolated from Korean wheat-based nuruk, respectively. Each open reading frame consists of 1,551 nucleotides and encodes a protein of 517 amino acids with the molecular mass of approximately 59 kDa. These isozymes share approximately 49% amino acid sequence identity with eukaryotic ABFs from filamentous fungi. The corresponding genes were cloned, functionally expressed, and purified from Escherichia coli. SfABF51A and SfABF51B showed the highest activities on p-nitrophenyl arabinofuranoside at 40~45°C and pH 7.0 in sodium phosphate buffer and at 50°C and pH 6.0 in sodium acetate buffer, respectively. These exo-acting enzymes belonging to the glycoside hydrolase (GH) family 51 could hydrolyze arabinoxylo-oligosaccharides (AXOS) and arabino-oligosaccharides (AOS) to produce only L-arabinose, whereas they could hardly degrade any polymeric substrates including arabinans and arabinoxylans. The detailed product analyses revealed that both SfABF51 isozymes can catalyze the versatile hydrolysis of α-(1,2)-and α-(1,3)-L-arabinofuranosidic linkages of AXOS, and α-(1,2)-, α-(1,3)-, and α-(1,5)-linkages of linear and branched AOS. On the contrary, they have much lower activity against the α-(1,2)-and α-(1,3)-double-substituted substrates than the single-substituted ones. These hydrolases could potentially play important roles in the degradation and utilization of hemicellulosic biomass by S. fibuligera.

[Investigation of microbial community involved in fermentation of Massa Medicata Fermentata].

This study aimed to clarify the microbial diversity, dominant species and the change of community structures in the fermentation of Liushenqu(Massa Medicata Fermentata), and explore the material foundation of its pharmacodynamics effect. On the basis of standardizing the fermentation process, Massa Medicata Fermentata was prepared by screening and optimizing the recipes and the standard formula issued by the Ministry. The community structure and growth process of fungi and bacteria in the samples at five time points(0, 17, 41, 48, 65 h) in the fermentation process of Massa Medicata Fermentata were analyzed by using isolation and culture of eight different media and high-throughput DNA sequencing technology. The results indicated that the samples of the two recipes pre-sented high microbial diversity at the initial fermentation stage, with Aspergillus spp. as the dominant species. As the fermentation process goes forward, Saccharomycopsis fibuligera and Rhizopus oryzae soon became dominant species from 17 h after fermentation commencement point to the fermentation end, while the other species were inhibited at a lower level from 17 h. The species diversity of bacteria in the initial fermentation samples was also high, and Enterobacter was the dominant species. Enterobacter cloacae, Pediococcus pentosaceus and Cronobacter sakazakii became dominant bacterial species 17 h after fermentation commencement, while the species diversity was decreased. Our results will be a scientific basis for promoting the fermentation process of Massa Medicata Fermentata by using pure microbial cultures.

Effects of Saccharomycopsis fibuligera and Saccharomyces cerevisiae inoculation on small fermentation starters in Sichuan-style Xiaoqu liquor.

Xiaoqu liquor is a type of distilled spirit in China prepared on a small scale from a small solid starter culture. Although this liquor is popular in southwestern China, it can have a dull taste, limiting its market. To improve the flavour profile of Xiaoqu liquor, we selected two functional yeast strains (Saccharomycopsis fibuligera and Saccharomyces cerevisiae) from Zaopei (fermented grain) of Baijiu liquor and used them for Xiaoqu liquor fermentation. Compared with traditional Xiaoqu (Starter), bioaugmentation inoculation increased the glucoamylase and acidic protease activities and the ethanol synthesis rate, while decreasing the acidity of the Zaopei (fermented grains) in the early stage of fermentation. By the end of the fermentation process, the alcohol and ester content had also increased by 42.5% and 11.8%, respective, and that of aldehydes and ketones, and heterocyclic compounds decreased by 73.7% and 77.1%, respectively. Traditional isolation and high-throughput sequencing were employed to analyse the microorganisms in the Zaopei. Bioaugmentation inoculation increased the microbial diversity of Xiaoqu liquor during the fermentation process. The dominant fungus during fermentation using the two types of starter cultures was S. cerevisiae, whereas the dominant bacteria was Pseudomonas, followed by Bacillus, Weissella, Lactobacillus, and Bacteroides. Principal component analysis of the bacterial community structure and flavour substances in the Zaopei produced using the two strains revealed that there were few differences between the two liquors and that inoculation with functional yeasts may not change the flavour substances in Xiaoqu liquor. However, correlation analysis showed that Escherichia Shigella, Terrisporobacter, Bacillus, Clostridium, and Prevotellaceae are the main microorganisms in the Xiaoqu liquor fermentation process. These results lay the foundation to improve the quality of Xiaoqu liquor.

In Vitro Studies on Therapeutic Potential of Probiotic Yeasts Isolated from Various Sources.

The present study investigates the therapeutic properties of probiotic yeasts viz. Yarrowia lipolytica VIT-MN01, Kluyveromyces lactis VIT-MN02, Lipomyces starkeyi VIT-MN03, Saccharomycopsis fibuligera VIT-MN04 and Brettanomyces custersianus VIT-MN05. The antimutagenic activity of probiotic yeasts against the mutagens viz. Benzo[a]pyrene (B[a]P), and Sodium azide (SA) was tested. S. fibuligera VIT-MN04 showed highest antimutagenicity (75%). Binding ability on the mutagen acridine orange (AO) was tested and L. starkeyi VIT-MN03 was able to bind AO effectively (88%). The probiotic yeasts were treated with the genotoxins viz. 4-Nitroquinoline 1-Oxide (NQO) and Methylnitronitrosoguanidine (MNNG). The prominent changes in UV shift confirmed the reduction in genotoxic activity of S. fibuligera VIT-MN04 and L. starkeyi VIT-MN03, respectively. Significant viability of probiotic yeasts was noted after being exposed to mutagens and genotoxins. The adhesion capacity and anticancer activity were also assessed using Caco-2 and IEC-6 cell lines. Adhesion ability was found to be more in IEC-6 cells and remarkable antiproliferative activity was noted in Caco-2 cells compared to normal cells. Further, antagonistic activity of probiotic yeasts was investigated against S. typhimurium which was found to be more in S. fibuligera VIT-MN04 and L. starkeyi VIT-MN03. The inhibition of α-glucosidase and α-amylase activity confirmed the antidiabetic activity of probiotic yeasts. Antioxidant activity was also tested using standard assays. Therefore, based on the results, it can be concluded that probiotic yeasts can serve as potential therapeutic agents for the prevention and treatment of colon cancer, type 2 diabetes and gastrointestinal infections.

Saccharomycopsis oxydans sp. nov., a new non-fermentative member in the genus Saccharomycopsis isolated from a traditional dairy product of Iran.

A total of 21 yeast isolates were recovered as part of a research project on biodiversity of yeasts in traditional dairy products in Alborz province, Iran. Standard protocols were used to carry out phenotypic, biochemical, physiological characterization and the phylogenetic analysis of combined the D1/D2 domain of the large ribosomal subunit (26S or LSU) and ITS region sequences. Five strains represented a potential new ascomycetous yeast species. Ascospore formation was not observed in these strains, and they did not ferment the examined carbon sources. Phylogenetic analysis placed these isolates in a well-supported sub-clade in the genus Saccharomycopsis. Here, we describe this novel yeast as Saccharomycopsis oxydans sp. nov.

Nanoencapsulation of Saccharomycopsis fibuligera VIT-MN04 using electrospinning technique for easy gastrointestinal transit.

In this study, probiotic yeast Saccharomycopsis fibuligera (S. fibuligera) VIT-MN04 was encapsulated with wheat bran fibre (WBF) and exopolysaccharide (EPS) along with 5% polyvinylpyrrolidone (PVP) using electrospinning technique for easy gastrointestinal transit (GIT). The electrospinning materials viz. WBF (10%), EPS (15%), PVP (5%) and electrospinning parameters viz. applied voltage (10 kV) and tip to collector distance (15 cm) were optimised using response surface methodology to produce fine nanofibres to achieve maximum encapsulation efficiency (100%) and GIT tolerance (97%). The probiotic yeast was successfully encapsulated in nanofibre and investigated for potential properties. The survival of encapsulated S. fibuligera VIT-MN04 was increased compared to the free cells during in vitro digestion. In addition, encapsulated yeast cells retained their viability during storage at 4°C for 56 days. The nanofibres were characterised using scanning electron microscopy, atomic force microscopy, X-ray diffraction, thermogravimetric analysis, zeta potential analysis and Fourier transform infrared spectroscopy followed by gas chromatography-mass spectrometry and nuclear magnetic resonance analysis. This work provides an efficient approach for encapsulation of probiotic yeast with the nanofibres which can also broaden the application of the prebiotic like WBF providing an idea for the efficient preparation of functional synbiotic supplements in the food industry.

Investigation of microbial community involved in fermentation of Massa Medicata Fermentata / 中国中药杂志

This study aimed to clarify the microbial diversity, dominant species and the change of community structures in the fermentation of Liushenqu(Massa Medicata Fermentata), and explore the material foundation of its pharmacodynamics effect. On the basis of standardizing the fermentation process, Massa Medicata Fermentata was prepared by screening and optimizing the recipes and the standard formula issued by the Ministry. The community structure and growth process of fungi and bacteria in the samples at five time points(0, 17, 41, 48, 65 h) in the fermentation process of Massa Medicata Fermentata were analyzed by using isolation and culture of eight different media and high-throughput DNA sequencing technology. The results indicated that the samples of the two recipes pre-sented high microbial diversity at the initial fermentation stage, with Aspergillus spp. as the dominant species. As the fermentation process goes forward, Saccharomycopsis fibuligera and Rhizopus oryzae soon became dominant species from 17 h after fermentation commencement point to the fermentation end, while the other species were inhibited at a lower level from 17 h. The species diversity of bacteria in the initial fermentation samples was also high, and Enterobacter was the dominant species. Enterobacter cloacae, Pediococcus pentosaceus and Cronobacter sakazakii became dominant bacterial species 17 h after fermentation commencement, while the species diversity was decreased. Our results will be a scientific basis for promoting the fermentation process of Massa Medicata Fermentata by using pure microbial cultures.

A script for initiating molecular biology studies with non-conventional yeasts based on Saccharomycopsis schoenii.

Non-conventional yeasts (NCYs), i.e. all yeasts other than Saccharomyces cerevisiae, are emerging as novel production strains and gain more and more attention to exploit their unique properties. Yet, these yeasts can hardly compete against the advanced methodology and genetic tool kit available for exploiting and engineering S. cerevisiae. Currently, for many NCYs one has to start from scratch to initiate molecular genetic manipulations, which is often time consuming and not straight-forward. More so because utilization of S. cerevisiae tools based on short-flank mediated homologous recombination or plasmid biology are not readily applicable in NCYs. Here we present a script with discrete steps that will lead to the development of a basic and expandable molecular toolkit for ascomycetous NCYs and will allow genetic engineering of novel platform strains. For toolkit development the highly efficient in vivo recombination efficiency of S. cerevisiae is utilized in the generation and initial testing of tools. The basic toolkit includes promoters, reporter genes, selectable markers based on dominant antibiotic resistance genes and the generation of long-flanking homology disruption cassettes. The advantage of having pretested molecular tools that function in a heterologous host facilitate NCY strain manipulations. We demonstrate the usefulness of this script on Saccharomycopsis schoenii, a predator yeast with useful properties in fermentation and fungal biocontrol.

Multi-omics characterization of the necrotrophic mycoparasite Saccharomycopsis schoenii.

Pathogenic yeasts and fungi are an increasing global healthcare burden, but discovery of novel antifungal agents is slow. The mycoparasitic yeast Saccharomycopsis schoenii was recently demonstrated to be able to kill the emerging multi-drug resistant yeast pathogen Candida auris. However, the molecular mechanisms involved in the predatory activity of S. schoenii have not been explored. To this end, we de novo sequenced, assembled and annotated a draft genome of S. schoenii. Using proteomics, we confirmed that Saccharomycopsis yeasts have reassigned the CTG codon and translate CTG into serine instead of leucine. Further, we confirmed an absence of all genes from the sulfate assimilation pathway in the genome of S. schoenii, and detected the expansion of several gene families, including aspartic proteases. Using Saccharomyces cerevisiae as a model prey cell, we honed in on the timing and nutritional conditions under which S. schoenii kills prey cells. We found that a general nutrition limitation, not a specific methionine deficiency, triggered predatory activity. Nevertheless, by means of genome-wide transcriptome analysis we observed dramatic responses to methionine deprivation, which were alleviated when S. cerevisiae was available as prey, and therefore postulate that S. schoenii acquired methionine from its prey cells. During predation, both proteomic and transcriptomic analyses revealed that S. schoenii highly upregulated and translated aspartic protease genes, probably used to break down prey cell walls. With these fundamental insights into the predatory behavior of S. schoenii, we open up for further exploitation of this yeast as a biocontrol yeast and/or source for novel antifungal agents.