Isolation, identification, and tolerance analysis of yeast during the natural fermentation process of Sidamo coffee beans.

Yeast, which plays a pivotal role in the brewing, food, and medical industries, exhibits a close relationship with human beings. In this study, we isolated and purified 60 yeast strains from the natural fermentation broth of Sidamo coffee beans to screen for indigenous beneficial yeasts. Among them, 25 strains were obtained through morphological characterization on nutritional agar medium from Wallerstein Laboratory (WL), with molecular biology identifying Saccharomyces cerevisiae strain YBB-47 and the remaining 24 yeast strains identified as Pichia kudriavzevii. We investigated the fermentation performance, alcohol tolerance, SO2 tolerance, pH tolerance, sugar tolerance, temperature tolerance, ester production capacity, ethanol production capacity, H2S production capacity, and other brewing characteristics of YBB-33 and YBB-47. The results demonstrated that both strains could tolerate up to 3% alcohol by volume at a high sucrose mass concentration (400 g/L) under elevated temperature conditions (40 ℃), while also exhibiting a remarkable ability to withstand an SO2 mass concentration of 300 g/L at pH 3.2. Moreover, S. cerevisiae YBB-47 displayed a rapid gas production rate and strong ethanol productivity. whereas P. kudriavzevii YBB-33 exhibited excellent alcohol tolerance. Furthermore, this systematic classification and characterization of coffee bean yeast strains from the Sidamo region can potentially uncover additional yeasts that offer high-quality resources for industrial-scale coffee bean production.

Bioprocess optimization of interferon ß-1-a in Pichia pastoris and its improved inhibitory effect against hepatocellular carcinoma cells

Interferon-ß-1a (INF-ß-1a) has gained significant attention due to its emerging applications in the treatment of different human diseases. Therefore, many researchers have attempted to produce it in large quantities and also in a biologically active form using different expression systems. In the present study, we aimed to improve the expression level of INF-ß-1a by Pichia pastoris using optimization of culture conditions. The codon-optimized INF-ß- 1a gene was cloned into pPICZαA plasmid under the control of alcohol oxidase I (AOX1) promoter. The protein expression was induced using different concentrations of methanol at different pHs and temperatures. The biological activity of produced protein was evaluated by anti-proliferative assay. The ideal culture conditions for the expression of INF-ß-1a by P. pastoris were found to be induction with 2% methanol at pH 7.0 culture medium at 30 C which yielded a concentration of 15.5 mg/L INF-ß-1a in a shake flask. Our results indicate that differences in glycosylation pattern could result in different biological activities as INF- ß-1a produced by P. pastoris could significantly more reduce the cell viability of HepG-2 cells, a hepatocellular carcinoma cell line, than a commercially available form of this protein produced by CHO

Diversity of non-Saccharomyces yeasts of grape berry surfaces from representative Cabernet Sauvignon vineyards in Henan Province, China.

Non-Saccharomyces yeasts are important players during winemaking and may come from grapes grown in vineyards. To study the diversity of non-Saccharomyces yeasts on grape berry surfaces, 433 strains were isolated from different Cabernet Sauvignon vineyards grown in Henan Province. Our results demonstrated that these strains were classified into 16 morphotypes according to their growth morphology on Wallerstein Laboratory agar medium, and were identified as seven species from four genera-Hanseniaspora opuntiae, Hanseniaspora vineae, Hanseniaspora uvarum, Pichia occidentalis, Pichia kluyveri, Issatchenkia terricola and Saturnispora diversa-based on a series of molecular biological experiments. Hanseniaspora opuntiae was obtained from all sampling sites except Changyuan County, while Pichia kluyveri and Saturnispora diversa were only found in sites of Zhengzhou Grape Resource Garden and Minquan County, respectively. The site Minquan was home of the greatest species richness, while only one single species (Hanseniaspora opuntiae) was detected at NAPA winery from Zhengzhou or at Anyang County. Finally, this study suggested that the geographic distribution and diversity of non-Saccharomyces yeast populations on Cabernet Sauvignon grape berries were likely to be determined by a combination of grape varieties and environmental factors.

High Potential of Pichia kluyveri and Other Pichia Species in Wine Technology.

The surfaces of grapes are covered by different yeast species that are important in the first stages of the fermentation process. In recent years, non-Saccharomyces yeasts such as Torulaspora delbrueckii, Lachancea thermotolerans, Metschnikowia pulcherrima, and Pichia kluyveri have become popular with regard to winemaking and improved wine quality. For that reason, several manufacturers started to offer commercially available strains of these non-Saccharomyces species. P. kluyveri stands out, mainly due to its contribution to wine aroma, glycerol, ethanol yield, and killer factor. The metabolism of the yeast allows it to increase volatile molecules such as esters and varietal thiols (aroma-active compounds), which increase the quality of specific varietal wines or neutral ones. It is considered a low- or non-fermentative yeast, so subsequent inoculation of a more fermentative yeast such as Saccharomyces cerevisiae is indispensable to achieve a proper fermented alcohol. The impact of P. kluyveri is not limited to the grape wine industry; it has also been successfully employed in beer, cider, durian, and tequila fermentation, among others, acting as a promising tool in those fermentation processes. Although no Pichia species other than P. kluyveri is available in the regular market, several recent scientific studies show interesting improvements in some wine quality parameters such as aroma, polysaccharides, acid management, and color stability. This could motivate yeast manufacturers to develop products based on those species in the near future.

Improvement of Black-Odor Water by Pichia Strain GW1 under Optimized NH3-N Degradation Conditions.

In this study, a yeast strain with an outstanding NH3-N degradation ability was isolated from the sediment of a black-odor water channel in Guangdong Province, China. Based on phenotypic and phylogenetic analysis, this strain was identified as Pichia kudriavzevii GW1. The optimum conditions for NH3-N degradation by the GW1 strain were as follows: 0.3% inoculum concentration, 1.5 L/min aeration, pH 7, and a temperature of 35°C. Under optimized conditions, the GW1 strain degraded 95.5% of the NH3-N. The strain was then added to simulated black-odor water under optimal degradation conditions to investigate changes to the bacterial community over time. 16S rRNA sequencing of samples collected on days 0, 7, 14, and 21 showed that, in the presence of the GW1 strain, the relative abundances of the phyla Proteobacteria, Bacteroidetes, Chloroflexi, and Firmicutes increased in the black-odor water. In addition, the relative abundance of Propionivibrio, a known NH3-N degrading genus, increased. This study will facilitate the use of microbiological methods to repair black-odor water.

Yeast communities of primary and secondary peat swamp forests in southern Thailand.

Khanthuli peat swamp forest (PSF) is one of a few fertile peat swamp forests that remain in Thailand. It is composed of primary PSF and some areas which have been degraded to secondary PSF due to drought, wildfires and land conversion, which have resulted in a decrease in peat layers and change in the species of the plant community. In this study, diversity of yeasts in peat from both primary and secondary PSF areas of the Khanthuli PSF was determined based on culture-dependent approaches, using dilution plate and enrichment techniques. A total of 66 yeast isolates were identified by the analysis of sequence similarity of the D1/D2 region of the large subunit rRNA gene or the combined analysis of sequence of the D1/D2 region and internal transcribed spacer region and confirmed by phylogenetic analysis of the D1/D2 region to belong to 22 known yeast species and six potential new species in the genera Candida (Kurtzmaniella, Lodderomyces, Ogataea, Pichia and Yamadazyma clades), Clavispora, Cyberlindnera, Galactomyces, Hanseniaspora, Metschnikowia, Saturnispora, Schwanniomyces, Cryptotrichosporon, Pichia, Curvibasidium, Papiliotrema, Rhodotorula, and Saitozyma. The most prevalent yeasts in the primary PSF were Cyberlindnera subsufficiens and Galactomyces candidus, while Saitozyma podzolica was the most frequently found in peat from the secondary PSF. Common yeast species in both, primary and secondary PSF, were Cy. subsufficiens, G. candidus and Rhodotorula mucilaginosa.

Yeast diversity investigation of Vitis davidii Föex during spontaneous fermentations using culture-dependent and high-throughput sequencing approaches.

Vitis davidii Föex is widely planted in South China as a potential wine making grape. In this study, yeast communities during the spontaneous fermentations of two varieties of Vitis davidii Föex in Guizhou, China were investigated. Hanseniaspora uvarum, Pichia terricola, Saccharomyces cerevisiae/S. mikatae, and Schizosaccharomyces japonicus were the four main yeast species detected by culture-dependent and high-throughput sequencing approaches. However, the composition of minor yeast species was quite different as revealed by the two approaches. Ten yeast species including Debaryomyces hansenii, Rhodotorula mucilaginosa, Sporidiobolus paraoseus, Starmerella bacillaris, Zygoascus meyerae, etc. were detected by culture-dependent approaches, whereas the other five species were found by high-throughput sequencing analysis. S. japonicus was widely found during spontaneous fermentations and its concentrations were mostly higher than that of S. cerevisiae. The difference in grape varieties and fermentation conditions contributed to yeast diversity. The results of this study provide basic information on indigenous yeast diversity from Vitis davidii Föex in Guizhou, which would help to exploit the potential of non-Saccharomyces yeast species with good oenological attributes.

Identification of Novel Endophytic Yeast Strains from Tangerine Peel.

Seven endophytic yeast strains were isolated from tangerine peel (Citrus reticulata Blanco) and genotyped through clustering with D1/D2 and ITS1-5.8S-ITS2 sequences from GenBank. Phenotypic characteristics were obtained through commercial kits and through assisted species identification. Indole-3-acetic acid (IAA) production by the yeast strains was assessed using Salkowski reagent and High-Performance Liquid chromatography (HPLC). The growth-promoting effects of the yeast were evaluated using the 'ragdoll' method. CRYb1, CRYb2 and CRYb7 isolates were identified as the closest species Hanseniaspora opuntiae. CRYb3 was identified as Pichia kluyveri. CRYb4, CRYb5 and CRYb6 were identified as Meyerozyma guilliermondii. CRYb1, CRYb5, CRYb6 and CRYb7 were found to be capable of IAA production. The most promising yeast strains now require further evaluation for their ability to promote plant growth in vitro and in vivo. These data increase our knowledge of the distribution and biological properties of endophytic yeast. This is important information that will be required to fully harness the growth-promoting properties of yeast strains.

Identifying yeasts using surface enhanced Raman spectroscopy.

The molecular fingerprints of yeasts Saccharomyces cerevisiae, Dekkera bruxellensis, and Wickerhamomyces anomalus (former name Pichia anomala) have been examined using surface-enhanced Raman spectroscopy (SERS) and helium ion microscopy (HIM). The SERS spectra obtained from cell cultures (lysate and non-treated cells) distinguish between these very closely related fungal species. Highly SERS active silver nano-particles suitable for detecting complex biomolecules were fabricated using a simple synthesis route. The yeast samples mixed with aggregated Ag nanoparticles yielded highly enhanced and reproducible Raman signal owing to the high density of the hot spots at the junctions of two or more Ag nanoparticles and enabled to differentiate the three species based on their unique features (spectral fingerprint). We also collected SERS spectra of the three yeast species in beer medium to demonstrate the potential of the method for industrial application. These findings demonstrate the great potential of SERS for detection and identification of fungi species based on the biochemical compositions, even in a chemically complex sample.

Impact of seasonality and environmental conditions on yeast diversity from camel's milk collected in Algeria.

During this study, we characterized the seasonality's impact and environmental conditions on the yeast diversity from raw camel's milk collected in Algeria. The yeast counts were estimated to 3.55 × 102 CFU mL-1, with a maximum of 6.3 × 102 CFU mL-1. The yeasts were categorized phenotypically by API 20C AUX, MALDI-TOF and genetically by sequencing 26S rDNA and ITS1-5.8S-ITS2. The rDNA sequencing approaches revealed 12 species including unusual ones such as Trichosporon asahii, Pichia fermentans, Millerozyma farinosa, Pichia galeiformis, Candida tartarivorans and Pichia manshurica. The most dominant species were T. asahii (23%), P. fermentans (19%) and Rhodotorula mucilaginosa (14%). The high occurrence and large diversity were registered in samples collected during the autumn season, in the semi-arid and arid highlands regions with 0.66 × 103 CFU mL-1 and 0.51 × 103 CFU mL-1, respectively. Interestingly, T. asahii, R. mucilaginosa, P. fermentans, C. parapsilosis and C. zeylanoides were detected during both spring and autumn.

Development of air-blast dried non-Saccharomyces yeast starter for improving quality of Korean persimmon wine and apple cider.

A total of 512 yeasts, including 422 non-Saccharomyces yeasts, were isolated from various fruits including apple, aronia, Muscat Bailey A grapes, and persimmon. These were used to prepare persimmon wine and apple cider starters that produced high levels of aromatic compounds, which contribute to high-quality fermented products. Environmental tolerance testing with 20% glucose and 8% EtOH, alongside a sniffing test, led to the selection of Wickerhamomyces anomalus (Synonym Pichia anomala) SJ20, Meyerozyma caribbica (Synonym Pichia caribbica) YP1, Pichia kluyveri CD34, Hanseniaspora uvarum SJ69 (for persimmon wine), W. anomalus CS7-16 (for apple cider), and Starmerella bacillaris (Synonym Candida zemplinina) CD80 (for both wines) as wine starters. These strains had high environmental stress tolerance and the highest sniffing test scores. Persimmon wine and apple cider were fermented using these strains in single- or mixed-culture with S. cerevisiae W-3 to determine the improved effect on wine aroma. In accordance with the results of volatile ester compounds and sensory evaluation, W. anomalus SJ20, H. uvarum SJ69, and W. anomalus CS7-16 had an excellent potential as persimmon wine and apple cider starters. Moreover, other strains also showed a good potential for a distinctive persimmon wine and apple cider because of the different compositions of the various volatile ester compounds. Six types of sugars (fructose, glucose, maltose, sucrose, raffinose, sucrose, and trehalose), four types of rehydration solutions (distilled water, 1× phosphate buffered saline, 0.85% NaCl, and 1% peptone water), and two types of antioxidants (l-ascorbic acid and glutathione) were examined to improve the survival rate of air-blast dried non-Saccharomyces yeast cells. Optimal sugar and rehydration conditions for each strain were validated, and scanning electron microscopy showed that each cell was surrounded by protectants, including sugar, skim milk, and lactomil. Storability assessment of air-blast dried yeast cells maintained at 4 °C for two months indicated that at least one condition in each strain had a higher survival rate than the control, regardless of the concentration or type of antioxidant treatment, except for M. caribbica YP1. These results suggest that antioxidant treatment contributes to maintaining the viability of air-blast dried cells in hostile environments.

Screening of new yeast Pichia manchurica for arabitol production.

Arabitol has several applications in food and pharmaceutical industries as a natural sweetener, dental caries inhibitor, and texturing agent. Newly isolated yeast strains from seawater, sugarcane plantation soil samples, and Zygosaccharomyces rouxii 2635 from MTCC were tested for arabitol production. The yield of arabitol was found to be higher in seawater isolate (24.6 g L-1 ) compared to two soil isolates (22.5 g L-1 ) and Z. rouxii (19.4 g L-1 ). Based on ITS 26S rDNA sequence analysis, the seawater isolate was identified as Pichia manchurica. In the present study, the effect of different substrates, trace elements, nitrogen sources, pH, and temperature on arabitol production was examined. Three different carbon sources viz. glucose, arabinose, and galactose were studied. Glucose was determined to be the best substrate for arabitol production (27.6 g L-1 ) followed by arabinose (13.7 g L-1 ) and galactose (7.7 g L-1 ). Maximum production of arabitol was observed at pH 6.0 (34.7 g L-1 ). In addition, arabitol production was high (35.7 g L-1 ) at temperature of 30 °C. Among the different concentrations of ammonium sulfate tested (3, 4.5, 6, 7.5, and 9 g L-1 ) concentration of 6 g L-1 resulted in higher arabitol Individual metal ions had no effect on arabitol production by this strain as compared to control. Results obtained in this study identify ways for improved arabitol production with natural isolates using microbial processes.

Pichia nanzhaoensis sp. nov. and Pichia paraexigua f.a., sp. nov., two yeast species isolated from rotting wood.

Four yeast strains were isolated from rotting wood samples collected in the Baotianman Nature Reserve in Henan Province, Central China. On the basis of sequence analysis of the D1/D2 domains of the large subunit rRNA gene and the internal transcribed spacer regions, they were suggested to be two novel species of the genus Pichia. Pichia nanzhaoensis sp. nov. produces one to four spherical ascospores per ascus, and is most closely related to Candida pseudolambica. Pichia paraexigua f.a., sp. nov. is a sister taxa to Pichia exigua, but the formation of ascospores was not observed on various sporulation media. P. nanzhaoensis sp. nov. can weakly assimilate inulin, whereas P. paraexigua sp. nov. can weakly assimilate d-glucosamine. The type strain of Pichia nanzhaoensis is NYNU 178136T (=CICC 33279T=CBS 15346T) and the type strain of Pichia paraexigua is NYNU 178135T (=CICC 33278T=CBS 15237T).

Population genomics shows no distinction between pathogenic Candida krusei and environmental Pichia kudriavzevii: One species, four names.

We investigated genomic diversity of a yeast species that is both an opportunistic pathogen and an important industrial yeast. Under the name Candida krusei, it is responsible for about 2% of yeast infections caused by Candida species in humans. Bloodstream infections with C. krusei are problematic because most isolates are fluconazole-resistant. Under the names Pichia kudriavzevii, Issatchenkia orientalis and Candida glycerinogenes, the same yeast, including genetically modified strains, is used for industrial-scale production of glycerol and succinate. It is also used to make some fermented foods. Here, we sequenced the type strains of C. krusei (CBS573T) and P. kudriavzevii (CBS5147T), as well as 30 other clinical and environmental isolates. Our results show conclusively that they are the same species, with collinear genomes 99.6% identical in DNA sequence. Phylogenetic analysis of SNPs does not segregate clinical and environmental isolates into separate clades, suggesting that C. krusei infections are frequently acquired from the environment. Reduced resistance of strains to fluconazole correlates with the presence of one gene instead of two at the ABC11-ABC1 tandem locus. Most isolates are diploid, but one-quarter are triploid. Loss of heterozygosity is common, including at the mating-type locus. Our PacBio/Illumina assembly of the 10.8 Mb CBS573T genome is resolved into 5 complete chromosomes, and was annotated using RNAseq support. Each of the 5 centromeres is a 35 kb gene desert containing a large inverted repeat. This species is a member of the genus Pichia and family Pichiaceae (the methylotrophic yeasts clade), and so is only distantly related to other pathogenic Candida species.

Low-pH production of d-lactic acid using newly isolated acid tolerant yeast Pichia kudriavzevii NG7.

Lactic acid is a platform chemical for the sustainable production of various materials. To develop a robust yeast platform for low-pH production of d-lactic acid (LA), an acid-tolerant yeast strain was isolated from grape skins and named Pichia kudriavzevii NG7 by ribosomal RNA sequencing. This strain could grow at pH 2.0 and 50°C. For the commercial application of P. kudriavzevii NG7 as a lactic acid producer, the ethanol fermentation pathway was redirected to lactic acid by replacing the pyruvate decarboxylase 1 gene (PDC1) with the d-lactate dehydrogenase gene (d-LDH) derived from Lactobacillus plantarum. To enhance lactic acid tolerance, this engineered strain was adapted to high lactic acid concentrations, and a new transcriptional regulator, PAR1, responsible for acid tolerance, was identified by whole-genome resequencing. The final engineered strain produced 135 g/L and 154 g/L of d-LA with productivity over 3.66 g/L/hr at pH 3.6 and 4.16 g/L/hr at pH 4.7, respectively.

The potential of the newly isolated thermotolerant yeast Pichia kudriavzevii RZ8-1 for high-temperature ethanol production

Abstract High potential, thermotolerant, ethanol-producing yeasts were successfully isolated in this study. Based on molecular identification and phylogenetic analysis, the isolated thermotolerant yeasts were clustered in the genera of Pichia kudriavzevii, Candida tropicalis, Candida orthopsilosis, Candida glabrata and Kodamea ohmeri. A comparative study of ethanol production using 160 g/L glucose as a substrate revealed several yeast strains that could produce high ethanol concentrations at high temperatures. When sugarcane bagasse (SCB) hydrolysate containing 85 g/L glucose was used as a substrate, the yeast strain designated P. kudriavzevii RZ8-1 exhibited the highest ethanol concentrations of 35.51 g/L and 33.84 g/L at 37 °C and 40 °C, respectively. It also exhibited multi-stress tolerance, such as heat, ethanol and acetic acid tolerance. During ethanol fermentation at high temperature (42 °C), genes encoding heat shock proteins (ssq1 and hsp90), alcohol dehydrogenases (adh1, adh2, adh3 and adh4) and glyceraldehyde-3-phosphate dehydrogenase (tdh2) were up-regulated, suggesting that these genes might play a crucial role in the thermotolerance ability of P. kudriavzevii RZ8-1 under heat stress. These findings suggest that the growth and ethanol fermentation activities of this organism under heat stress were restricted to the expression of genes involved not only in heat shock response but also in the ethanol production pathway.

Description of Komagataella mondaviorum sp. nov., a new sibling species of Komagataella (Pichia) pastoris.

Five methylotrophic strains (UCDFST 71-1024T, UCDFST 54-11.16, UCDFST 54-11.141, UCDFST 68-967.1 and UCDFST 74-1030) from the Phaff Yeast Culture Collection (University of California Davis, USA) that were originally designated as Pichia pastoris were found to represent a novel Komagataella species. Strains of Komagataella mondaviorum sp. nov. UCDFST 71-1024T(type strain) = CBS 15017, UCDFST 54-11.16, UCDFST 54-11.141, UCDFST 68-967.1, and UCDFST 74-1030 were isolated in USA, respectively, from cottonwood tree Populus deltoides in 1971 (Davis, CA), slime flux of Quercus sp. in 1954 (CA), exudate of black oak Q. kelloggii in 1954 (Central Sierra Nevada. CA), dry frass from Salix sp. in 1968 (Soleduck Road, Olympic National Park, WA) and from flux of hackberry tree Celtis sp. in 1974 (CA). The new species was differentiated from Komagataella kurtzmanii, Komagataella pastoris, Komagataella phaffii, Komagataella populi, Komagataella pseudopastoris and Komagataella ulmi by divergence in gene sequences for D1/D2 LSU rRNA, ITS1-5.8S-ITS2, RNA polymerase subunit I and translation elongation factor-1α. Komagataella mondaviorum sp. nov. is registered in MycoBank under MB 821789.

Candida infanticola and Candida spencermartinsiae yeasts: Possible emerging species in cancer patients.

Opportunistic infections due to Candida species occur frequently in intensive care settings. We investigated the prevalence of Candida species among 65 clinical specimens obtained from 200 cancer patients by phenotypic and molecular (ITS sequencing and AFLP) methods. Among the 65 yeast isolates, Candida albicans was the most commonly isolated species (n = 34, 52.3%), whereas other Candida species comprised 47.7% (n = 31) and consisted of Candida glabrata (n = 14, 21.5%), Candida tropicalis (n = 5, 7.7%) and uncommon Candida species (n = 12, 18.5%) such as Candida pelliculosa (n = 3, 4.6%), Pichia kudriavzevii (= Candida krusei, n = 2, 3.1%), Candida orthopsilosis (n = 2, 3.1%), Candida parapsilosis (n = 1, 1.5%), Candida infanticola (n = 2, 3.1%), Candida spencermartinsiae (n = 1, 1.5%), and Kluyveromyces marxianus (=Candida kefyr, n = 1, 1.5%). Candida infanticola and Candida spencermartinsiae were recovered from oral lesions of cancer patients. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) easily confirmed these isolates as less common Candida isolates (4.6%). The in vitro antifungal susceptibilities of C. spencermartinsiae and the two strains of C. infanticola were determined according to CLSI guidelines (M27-A3). MIC results among these isolates showed they were susceptible to isavuconazole, posaconazole and voriconazole, however, fluconazole and caspofungin had high MIC values. These Candida species that may occur more commonly in infections remain unnoticed using commonly used phenotypical methods in routine microbiology laboratories. MALDI-TOF MS proved to be a more fast and robust diagnostic technique for identification of the yeasts isolated from different clinical specimens of cancer patients.

The potential of the newly isolated thermotolerant yeast Pichia kudriavzevii RZ8-1 for high-temperature ethanol production.

High potential, thermotolerant, ethanol-producing yeasts were successfully isolated in this study. Based on molecular identification and phylogenetic analysis, the isolated thermotolerant yeasts were clustered in the genera of Pichia kudriavzevii, Candida tropicalis, Candida orthopsilosis, Candida glabrata and Kodamea ohmeri. A comparative study of ethanol production using 160g/L glucose as a substrate revealed several yeast strains that could produce high ethanol concentrations at high temperatures. When sugarcane bagasse (SCB) hydrolysate containing 85g/L glucose was used as a substrate, the yeast strain designated P. kudriavzevii RZ8-1 exhibited the highest ethanol concentrations of 35.51g/L and 33.84g/L at 37°C and 40°C, respectively. It also exhibited multi-stress tolerance, such as heat, ethanol and acetic acid tolerance. During ethanol fermentation at high temperature (42°C), genes encoding heat shock proteins (ssq1 and hsp90), alcohol dehydrogenases (adh1, adh2, adh3 and adh4) and glyceraldehyde-3-phosphate dehydrogenase (tdh2) were up-regulated, suggesting that these genes might play a crucial role in the thermotolerance ability of P. kudriavzevii RZ8-1 under heat stress. These findings suggest that the growth and ethanol fermentation activities of this organism under heat stress were restricted to the expression of genes involved not only in heat shock response but also in the ethanol production pathway.

Potential characterization of yeasts isolated from Kazak artisanal cheese to produce flavoring compounds.

Cheese is a typical handcrafted fermented food in Kazak minority from the Uighur Autonomy Region in China and Central Asia. Among the microbial community that is responsible for Kazak cheese fermentation, yeasts play important role in flavor formation during ripening. To develop ripening cultures, we isolated 123 yeasts from 25 cheese products in Kazak, and identified 87 isolates by the D1/D2 domain of the large subunit rRNA gene sequence. Pichia kudriavzevii was the dominant yeast in Kazak cheese, followed by Kluyveromyces marxianus and Kluyveromyces lactis. Of these, the ability to exhibit enzyme of dominant isolates and contribution to the typical flavor of cheeses was assessed. Enzyme producing yeast strains were inoculated in Hazak cheese-like medium and volatile compounds were identified by head space solid phase micro extraction coupled to gas chromatography and mass spectroscopy. Pichia kudriavzevii N-X displayed the strongest extracellular proteolytic and activity on skim milk agar and produced a range of aroma compounds (ethanol, ethyl acetate, 3-methylbutanol, and acetic acid) for Kazak cheese flavor, could be explored as ripening cultures in commercial production of Kazak cheeses.