Yeast Screen Research Articles

Modular and integrative activity reporters enhance biochemical studies in the yeast ER.

The yeast endoplasmic reticulum sequestration and screening (YESS) system is a broadly applicable platform to perform high-throughput biochemical studies of post-translational modification enzymes (PTM-enzymes). This system enables researchers to profile and engineer the activity and substrate specificity of PTM-enzymes and to discover inhibitor-resistant enzyme mutants. In this study, we expand the capabilities of YESS by transferring its functional components to integrative plasmids. The YESS integrative system yields uniform protein expression and protease activities in various configurations, allows one to integrate activity reporters at two independent loci and to split the system between integrative and centromeric plasmids. We characterize these integrative reporters with two viral proteases, Tobacco etch virus (TEVp) and 3-chymotrypsin like protease (3CLpro), in terms of coefficient of variance, signal-to-noise ratio and fold-activation. Overall, we provide a framework for chromosomal-based studies that is modular, enabling rigorous high-throughput assays of PTM-enzymes in yeast.

The Dmc1 recombinase physically interacts with and promotes the meiotic crossover functions of the Mlh1-Mlh3 endonuclease.

The accurate segregation of homologous chromosomes during the Meiosis I reductional division in most sexually reproducing eukaryotes requires crossing over between homologs. In baker's yeast approximately 80% of meiotic crossovers result from Mlh1-Mlh3 and Exo1 acting to resolve double-Holliday junction intermediates in a biased manner. Little is known about how Mlh1-Mlh3 is recruited to recombination intermediates to perform its role in crossover resolution. We performed a gene dosage screen in baker's yeast to identify novel genetic interactors with Mlh1-Mlh3. Specifically, we looked for genes whose lowered dosage reduced meiotic crossing over using sensitized mlh3 alleles that disrupt the stability of the Mlh1-Mlh3 complex and confer defects in mismatch repair but do not disrupt meiotic crossing over. To our surprise we identified genetic interactions between MLH3 and DMC1, the recombinase responsible for recombination between homologous chromosomes during meiosis. We then showed that Mlh3 physically interacts with Dmc1 in vitro and in vivo. Partial complementation of Mlh3 crossover functions was observed when MLH3 was expressed under the control of the CLB1 promoter (NDT80 regulon), suggesting that Mlh3 function can be provided late in meiotic prophase at some functional cost. A model for how Dmc1 could facilitate Mlh1-Mlh3's role in crossover resolution is presented.

Deorphanizing solute carriers in Saccharomyces cerevisiae for secondary uptake of xenobiotic compounds.

The exchange of small molecules between the cell and the environment happens through transporter proteins. Besides nutrients and native metabolic products, xenobiotic molecules are also transported, however it is not well understood which transporters are involved. In this study, by combining exo-metabolome screening in yeast with transporter characterization in Xenopus oocytes, we mapped the activity of 30 yeast transporters toward six small non-toxic substrates. Firstly, using LC-MS, we determined 385 compounds from a chemical library that were imported and exported by S. cerevisiae. Of the 385 compounds transported by yeast, we selected six compounds (viz. sn-glycero-3-phosphocholine, 2,5-furandicarboxylic acid, 2-methylpyrazine, cefadroxil, acrylic acid, 2-benzoxazolol) for characterization against 30 S. cerevisiae xenobiotic transport proteins expressed in Xenopus oocytes. The compounds were selected to represent a diverse set of chemicals with a broad interest in applied microbiology. Twenty transporters showed activity toward one or more of the compounds. The tested transporter proteins were mostly promiscuous in equilibrative transport (i.e., facilitated diffusion). The compounds 2,5-furandicarboxylic acid, 2-methylpyrazine, cefadroxil, and sn-glycero-3-phosphocholine were transported equilibratively by transporters that could transport up to three of the compounds. In contrast, the compounds acrylic acid and 2-benzoxazolol, were strictly transported by dedicated transporters. The prevalence of promiscuous equilibrative transporters of non-native substrates has significant implications for strain development in biotechnology and offers an explanation as to why transporter engineering has been a challenge in metabolic engineering. The method described here can be generally applied to study the transport of other small non-toxic molecules. The yeast transporter library is available at AddGene (ID 79999).

Evaulation of Effective Antagonistic Yeasts in Kalasin Province, a Biocontrol Agent against the Fungal Colletotrichum capsici Caused Anthracnose Diseases in Chili Fruits

Primary screening of epiphytic and endophytic yeasts, the majority yeast was found from rhizosphere soil (21 isolates, 31.81%), and 5 antagonistic yeasts can have inhibited the growth of mycelia of C. capsici up to 60%. Efficacy of five antagonistic yeasts were screened under plant nurseries conditions. The result found all isolates were tested and compared with control and chemical plots. The result of plant height showed that, yeast isolate KS34 had highest of plant height at 45.80 cm. For the result of branch per plant of chili plant found isolate KS37 showed highest of branch per plant at 16.60 branches and the flower of chili per plant presented that isolate KS37 had highest of flower at 18.60 flowers. The result of anthracnose disease control by antagonistic yeasts in disease incidence and category mean as plant resistance. These results found that, Isolates KS46 had a great disease incidence at 7.80% that mean this isolate prove chili plant to resistance to C. capsisci. Addition, isolate KS46 had highest of activity of chitinase enzyme at 170.72 U/ml. Isolate KS46 identified by D1/D2 domain of 26S ribosomal DNA sequencing. The sequence analysis showed maximum identity of 100% with Candida haemulonii. This assumed C. haemulonii was showed the best to control C. capsici. For confirmed this result, all isolates were determined the mode of action by studied in relationship between antagonist isolates. The action of yeast isolate could competition the nutrient with mycelia of pathogen. This mode effect to C. capsici loss nutrient caused the pathogen get stuck to braked and died later.

The Effect of Yeast Antagonist Isolated from the Fermentation of Cocoa Beans (Theobroma cacao) from Lampung, Indonesia, on the Growth of Aspergillus flavus UNJCC F-55

Mold is one of the microorganisms that causes damage to cocoa fruit because it produces mycotoxins as secondary metabolites that can decrease the crop yield. One type of pathogenic mold that destroys cocoa fruit is Aspergillus flavus. A. flavus produces aflatoxin B1 which is the most potent hepatotoxin and carcinogen to humans. Amongst the most effective ways to avoid alpha-toxin contamination in cocoa beans is to inhibit the growth of A. flavus in the beans. However, the use of pesticides and fungicides can increase the development of resistant strains of fungi and have a negative impact on the environment and human health. Therefore, we need other alternatives such as bio-control agents using antagonistic microorganisms, including yeasts. Thus, this study aims to characterize yeasts isolated from the fermented cocoa beans (Theobroma cacao) from Lampung, Indonesia, and evaluate their ability to inhibit the growth of Aspergillus flavus UNJCCF-55. The methods used were yeast screening, dual culture technique for antagonistic test, and morphological characterization. The 37ºC-growth screening gave 89 out of 98 yeast isolates. The subsequent antagonist test resulted in 13 isolates with the highest inhibition zone against A. flavus UNJCCF-55. These yeast isolates were macroscopically of smooth surface, butyrous texture, milky white color, irregular edges, and convex elevation. Microscopic observation showed that the isolates have oval cell shape, asexual reproduction of budding, and non-hyphae structure.
 Keywords: yeast antagonist, cocoa fermentation, aspergillus flavus

Isolation, screening, and application of aroma-producing yeast for red dragon fruit wine

Nineteen yeast strains were screened from the soil, dead branches, and fruit peels of a dragon fruit orchard. The yeast strain Z03M was obtained through preliminary screening, rescreening, and sensory flavor evaluation. This strain has certain alcohol-producing characteristics and good aroma-producing performance. Based on morphological and molecular identification, it was identified as Pichia kudriavzevii. The differences in flavor in dragon fruit wine from natural fermentation, inoculation with strain Z03M, commercial yeast, and mixed fermentation were comprehensive analyzed and compared via high-performance liquid chromatography, gas chromatography-mass spectrometry and gas chromatography-ion mobility spectroscopy. During the fermentation process of dragon fruit wine, We found that strain Z03M preferentially utilizes glucose. Malic acid and lactic acid are the main organic acids found in dragon fruit wine. The relative contents of volatile flavor compounds such as alcohols, aldehydes, terpenes, and heterocyclics of dragon fruit wine fermented with strain Z03M were 71.11%, 4.34%, 0.32%, and 1.78%, respectively, which were significantly higher than those of the other fermented samples. Among them, the content of 3-methyl-1-butanol was high, providing a banana aroma to the dragon fruit wine. Additionally, the wine fermented by a mixture of strain Z03M and commercial yeast had a high content of ester compounds, indicating that mixed fermentation can improve the flavor quality of dragon fruit wine. The results of this study have the potential value for the production of dragon fruit wine and for the research and development of dragon fruit products.

Screening and characterization of potential bioethanol production yeast from tropical fruits

This study aims to separate and characterise indigenous yeast (IY) from tropical fruit waste. The techniques include isolating and characterising yeast from different kinds of fruit waste, testing yeast for ethanol and glucose tolerance, and producing bioethanol in vitro. Using a microscope and visual inspection, the yeast’s morphological identification is done. Using a spectrophotometer to measure optical density, the tolerance tests for glucose and ethanol are used to select yeast biochemically. With the Gas Chromatography-Flame Ionisation Detector (GC-FID), one can measure the amount of ethanol present. Yeast was isolated using selective media to yield six isolates: code A1 from grapes, codes NG1, NG2 from jackfruit, and codes N1, N2, and N3 from pineapple; mango produced no results. Three isolates with the codes A1, NG1, and NG2 were chosen based on test results for resistance to glucose and ethanol. The Saccharomyces cerevisae bioethanol production test yielded 6.60%, 3.30%, 4.5%, and.4.85% of ethanol for the yeast species coded A1, NG1, and NG2, respectively, in terms of ethanol. According to the study’s findings, yeast bearing the NG2 code may be used in the fermentation process to produce bioethanol.

Screening, Identification, and Fermentation Condition Optimization of a High-Yield 3-Methylthiopropanol Yeast and Its Aroma-Producing Characteristics

A high-yield 3-methylthiopropanol (3-Met) yeast Y1402 was obtained from sesame-flavored Daqu, and it was identified as Saccharomycopsis fibuligera. S. fibuligera Y1402 showed a broad range of growth temperatures and pH, as well as the maximum tolerance to glucose, NaCl, nicotine, and 3-Met at 50% (w/w), 15% (w/v), 1.2 g/L, and 18 g/L, respectively. After optimization using single-factor experiments, a Plackett–Burman design, a steepest ascent test, and a Box–Behnken design, the 3-Met yield reached 4.03 g/L by S. fibuligera Y1402 under the following optimal conditions: glucose concentration of 40 g/L, yeast extract concentration of 0.63 g/L, Tween 80 concentration of 2 g/L, L-methionine concentration of 5 g/L, liquid volume of 25 mL/250 mL, initial pH of 5.3, fermentation temperature of 32 °C, inoculum size of 0.8%, shaking speed of 210 rpm, and fermentation time of 54 h. The fermentation was scaled up to a 3 L fermenter under the optimized conditions, and the yield of 3-Met reached 0.71 g/L. Additionally, an aroma analysis revealed that the flavor substances produced by S. fibuligera Y1402 in sorghum hydrolysate medium was mainly composed of compounds with floral, sweet, creamy, roasted nut, and clove-like aromas. Therefore, S. fibuligera has great potential for application in the brewing of Baijiu and other fermented foods.

Phytosulfokine promotes fruit ripening and quality via phosphorylation of transcription factor DREB2F in tomato.

Phytosulfokine (PSK), a plant peptide hormone with a wide range of biological functions, is recognized by its receptor PHYTOSULFOKINE RECEPTOR 1 (PSKR1). Previous studies have reported that PSK plays important roles in plant growth, development, and stress responses. However, the involvement of PSK in fruit development and quality formation remains largely unknown. Here, using tomato (Solanum lycopersicum) as a research model, we show that exogenous application of PSK promotes the initiation of fruit ripening and quality formation, while these processes are delayed in pskr1 mutant fruits. Transcriptomic profiling revealed that molecular events and metabolic pathways associated with fruit ripening and quality formation are affected in pskr1 mutant lines and transcription factors are involved in PSKR1-mediated ripening. Yeast screening further identified that DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2F (DREB2F) interacts with PSKR1. Silencing of DREB2F delayed the initiation of fruit ripening and inhibited the promoting effect of PSK on fruit ripening. Moreover, the interaction between PSKR1 and DREB2F led to phosphorylation of DREB2F. PSK improved the efficiency of DREB2F phosphorylation by PSKR1 at the tyrosine-30 site, and the phosphorylation of this site increased the transcription level of potential target genes related to the ripening process and functioned in promoting fruit ripening and quality formation. These findings shed light on the involvement of PSK and its downstream signaling molecule DREB2F in controlling climacteric fruit ripening, offering insights into the regulatory mechanisms governing ripening processes in fleshy fruits.

Meta learning addresses noisy and under-labeled data in machine learning-guided antibody engineering

Machine learning-guided protein engineering is rapidly progressing; however, collecting high-quality, large datasets remains a bottleneck. Directed evolution and protein engineering studies often require extensive experimental processes to eliminate noise and label protein sequence-function data. Meta learning has proven effective in other fields in learning from noisy data via bi-level optimization given the availability of a small dataset with trusted labels. Here, we leverage meta learning approaches to overcome noisy and under-labeled data and expedite workflows in antibody engineering. We generate yeast display antibody mutagenesis libraries and screen them for target antigen binding followed by deep sequencing. We then create representative learning tasks, including learning from noisy training data, positive and unlabeled learning, and learning out of distribution properties. We demonstrate that meta learning has the potential to reduce experimental screening time and improve the robustness of machine learning models by training with noisy and under-labeled training data.

Ectopic expression of potato ARP1 encoding auxin-repressed protein confers salinity stress tolerance in Arabidopsis thaliana.

Salinity stress is one of the most detrimental factors affecting crop production worldwide. Genetic engineering offers a promising approach for improving agronomic traits and enhancing stress tolerance. In a previous work, several potential candidate genes were identified in potato using large-scale functional yeast screening. In this work, we characterized one of the identified genes, an auxin-repressed protein 1 (ARP1), in transgenic Arabidopsis plants. ARP1 transgenic lines were subjected to salinity stress and compared with wild-type (WT) plants. Compared to WT plants, transgenic ARP1 lines showed significant improvements in morphological parameters, such as plant height, leaves per plant, root length, and fresh weight. Additionally, biochemical and physiological analyses revealed that the transgenic ARP1 lines exhibited improved stomatal conductance, reduced electrolyte leakage, increased proline and chlorophyll accumulation, significantly enhanced malondialdehyde accumulation, and antioxidant enzyme activity. Additionally, spectral analysis revealed that transgenic ARP1 lines had increased photosynthetic capacity compared to WT plants, as indicated by various biochemical parameters and pigment indicators. Transgenic ARP1 lines also showed improved photosystem (PSII) efficiency compared to WT plants, as demonstrated by detailed chlorophyll fluorescence analyses. Moreover, both ARP1 lines showed significantly higher expression levels of SOD, CAT, and APX than the WT plants under salt stress. The highest increase in relative expression was observed with SOD (3-fold increase) as compared to their respective WT in both ARP1 lines. We conclude that potato ARP1 is a promising candidate gene for the future development of salt-tolerant crops.

Selection of Starter from Decaying Oranges and Pineapple for Bioethanol Production

Screening of yeasts for bioethanol production is an important aspect of industrial microbiology and biotechnology. Optimal yeast nutrition also requires availability of nutrients allowing for growth and ethanol production and ability to withstand the increasing concentration of the medium during fermentation. This study seeks to use appropriate methods to isolate, characterize and identify yeasts isolates with essential attributes for bioethanol production. Microbiological and Physicochemical assessment of the isolated strains was carried out on yeast maintenance media for the determination of ethanol, temperature and salt tolerance ability, growth at different pH and temperature, chloramphenicol and nalidixic acid test, determination of killer toxins production capacity of yeast, nitrate and carbon assimilation and sugar fermentation tests were carried out to select the best starter for production.
 Fifteen yeasts were isolated belonging to the genera Saccharomyces, Candida, Rhodotorula, Kluvyreomyces, Trichosporon, Pichia. S. cerevisae and K. marxianus showed efficient physico-chemical attributes. Proximate analysis of fruits juice showed a moisture content between 81.83 – 86.37%, crude protein 1.33 – 2.00%, Ash 0.73 – 0.83%, carbohydrate 11.87 – 15.67% and Ether 0.1 – 0.5%. The total sugar ranged from 1.83 – 13.17, equal mixture of oranges and pineapple juice showed a sugar content of 15 which reduced during fermentation while the alcohol content increased. Organisms were able to tolerate a percentage of 4-30% ethanol content, tolerating 15% sodium chloride which is an index of osmotolerance. At higher concentration, growth reduced, organisms produced catalase, reduced nitrate and showed a variation in utilization of different sugars.
 At the end of the screening, S. cerevisiae and K. marxianus showed the best attribute essential for bioethanol production and were chosen as starters. The ability of the selected yeast isolates to produce Killer toxins against E. coli showed a negative result. Antibiotic sensitivity test was carried out on the selected yeast isolates using chloramphenicol and nalidixic and the two organisms were resistant to the antibiotics tested.
 The ability of the organisms to grow in changing environmental conditions and ethanol tolerance are attributes essential for production while Saccharomyces cerevisiae showed the highest attribute followed by Kluveromyces marxinus. This study concluded that Saccharomyces cerevisiae can be employed as starter in the industry for the production of bioethanol and in the conversion of agricultural waste to wealth.

Developing a novel selection method for alcoholic fermentation starters by exploring wine yeast microbiota from Greece.

The selection of native yeast for alcoholic fermentation in wine focuses on ensuring the success of the process and promoting the quality of the final product. The purpose of this study was firstly to create a large collection of new yeast isolates and categorize them based on their oenological potential. Additionally, the geographical distribution of the most dominant species, Saccharomyces cerevisiae, was further explored. Towards this direction, fourteen spontaneously fermented wines from different regions of Greece were collected for yeast typing. The yeast isolates were subjected in molecular analyses and identification at species level. RAPD (Random Amplified Polymorphic DNA) genomic fingerprinting with the oligo-nucleotide primer M13 was used, combined with Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. All yeast isolates were scrutinized for their sensitivity to killer toxin, production of non-desirable metabolites such as acetic acid and H2S, β-glucosidase production and resistance to the antimicrobial agent; SO2. In parallel, S. cerevisiae isolates were typed at strain level by interdelta - PCR genomic fingerprinting. S. cerevisiae strains were examined for their fermentative capacity in laboratory scale fermentation on pasteurized grape must. Glucose and fructose consumption was monitored daily and at the final point a free sorting task was conducted to categorize the samples according to their organoleptic profile. According to our results, among the 190 isolates, S. cerevisiae was the most dominant species while some less common non-Saccharomyces species such as Trigonopsis californica, Priceomyces carsonii, Zygosaccharomyces bailii, Brettanomyces bruxellensis and Pichia manshurica were identified in minor abundancies. According to phenotypic typing, most isolates were neutral to killer toxin test and exhibited low acetic acid production. Hierarchical Cluster Analysis revealed the presence of four yeast groups based on phenotypic fingerprinting. Strain level typing reported 20 different S. cerevisiae strains from which 65% indicated fermentative capacity and led to dry wines. Sensory evaluation results clearly discriminated the produced wines and consequently, the proposed yeast categorization was confirmed. A novel approach that employs biostatistical tools for a rapid screening and classification of indigenous wine yeasts with oenological potential, allowing a more efficient preliminary selection or rejection of isolates is proposed.

Fission Yeast TORC1 Promotes Cell Proliferation through Sfp1, a Transcription Factor Involved in Ribosome Biogenesis.

Target of rapamycin complex 1 (TORC1) is activated in response to nutrient availability and growth factors, promoting cellular anabolism and proliferation. To explore the mechanism of TORC1-mediated proliferation control, we performed a genetic screen in fission yeast and identified Sfp1, a zinc-finger transcription factor, as a multicopy suppressor of temperature-sensitive TORC1 mutants. Our observations suggest that TORC1 phosphorylates Sfp1 and protects Sfp1 from proteasomal degradation. Transcription analysis revealed that Sfp1 positively regulates genes involved in ribosome production together with two additional transcription factors, Ifh1/Crf1 and Fhl1. Ifh1 physically interacts with Fhl1, and the nuclear localization of Ifh1 is regulated in response to nutrient levels in a manner dependent on TORC1 and Sfp1. Taken together, our data suggest that the transcriptional regulation of the genes involved in ribosome biosynthesis by Sfp1, Ifh1, and Fhl1 is one of the key pathways through which nutrient-activated TORC1 promotes cell proliferation.

Potential of endophytic yeasts as biocontrol of Phytophthora capsici, causative agent of foot rot disease on black pepper

Abstract. Safitri D, Wiyono S, Purbajanti ED. 2023. Potential of endophytic yeasts as biocontrol of Phytophthora capsici, causative agent of foot rot disease on black pepper. Biodiversitas 24: 5847-5853. Phytophthora capsici is the main pathogen of foot rot disease of black pepper. This disease contributes to a significant decrease in black pepper productivity. Conventional disease management strategies usually require the use of chemical pesticides, which can have harmful environmental and human health consequences. Introducing biological agents such as endophytic yeasts for controlling foot rot disease has minimal effect on human health and is sustainable. One technique to control foot rot disease is to use biological agents such as endophytic yeasts. The aim of this research was to find yeast against Phytophthora capsici. The methods used included (i) study development and identification of endophytic yeast and (ii) in vitro and in vivo antagonism screening of yeast against P. capsici. The results showed that 49 endophytic yeasts were isolated from the leaves, fruits and roots of three black pepper lines. Five isolates had selected based on the highest antibiosis in antagonism test by in vitro: END6, END21, END66, END87, and END14. Isolate END87 had a mycelium inhibition rate of 64.55%, and a low level of inhibition was found in isolate 21, which had a mycelium size of 42.81%. The molecular characterization based on the sequence partial of NL1 and NL4. Moreover, 5 isolates of endophytic yeast (END6, END21, END66, END87, and END14) were identified as Rhodotorula mucilaginosa, Hannaella oryzae, Cryptococcus tephrensis.

Genome mining yields putative disease-associated ROMK variants with distinct defects.

Bartter syndrome is a group of rare genetic disorders that compromise kidney function by impairing electrolyte reabsorption. Left untreated, the resulting hyponatremia, hypokalemia, and dehydration can be fatal, and there is currently no cure. Bartter syndrome type II specifically arises from mutations in KCNJ1, which encodes the renal outer medullary potassium channel, ROMK. Over 40 Bartter syndrome-associated mutations in KCNJ1 have been identified, yet their molecular defects are mostly uncharacterized. Nevertheless, a subset of disease-linked mutations compromise ROMK folding in the endoplasmic reticulum (ER), which in turn results in premature degradation via the ER associated degradation (ERAD) pathway. To identify uncharacterized human variants that might similarly lead to premature degradation and thus disease, we mined three genomic databases. First, phenotypic data in the UK Biobank were analyzed using a recently developed computational platform to identify individuals carrying KCNJ1 variants with clinical features consistent with Bartter syndrome type II. In parallel, we examined genomic data in both the NIH TOPMed and ClinVar databases with the aid of Rhapsody, a verified computational algorithm that predicts mutation pathogenicity and disease severity. Subsequent phenotypic studies using a yeast screen to assess ROMK function-and analyses of ROMK biogenesis in yeast and human cells-identified four previously uncharacterized mutations. Among these, one mutation uncovered from the two parallel approaches (G228E) destabilized ROMK and targeted it for ERAD, resulting in reduced cell surface expression. Another mutation (T300R) was ERAD-resistant, but defects in channel activity were apparent based on two-electrode voltage clamp measurements in X. laevis oocytes. Together, our results outline a new computational and experimental pipeline that can be applied to identify disease-associated alleles linked to a range of other potassium channels, and further our understanding of the ROMK structure-function relationship that may aid future therapeutic strategies to advance precision medicine.

Critical assessment of the endocrine potential of Linalool and Linalyl acetate: proactive testing strategy assessing estrogenic and androgenic activity of Lavender oil main components.

The acyclic linear monoterpenes Linalool (Lin) and Linalyl acetate (LinAc) occur in nature as major constituents of various essential oils such as lavender oils. A potential endocrine activity of these compounds was discussed in literature including premature thelarche and prepubertal gynecomastia due to lavender product use. This study aims to follow-up on these critical findings reported by testing Lin and LinAc in several studies in line with current guidance and regulatory framework. No relevant anti-/ER and AR-mediated activity was observed in recombinant yeast cell-based screening tests and guideline reporter gene in vitro assays in mammalian cells. Findings in the screening test suggested an anti-androgenic activity, which could not be confirmed in the respective mammalian cell guideline assay. Mechanistic guideline in vivo studies (Uterotrophic and Hershberger assays) with Lin did not show significant dose related changes in estrogen or androgen sensitive organ weights and a guideline reproductive toxicity screening study did not reveal evident effects on sex steroid hormone sensitive organ weights, associated histopathological findings and altered sperm parameters. Estrous cycling and mating/fertility indices were not affected and no evident Lin-related steroid hormone dependent effects were found in the offspring. Overall, the initial concerns from literature were not confirmed. Findings in the yeast screening test were aberrant from follow-up guideline in vitro and in vivo studies, which underlines the need to apply careful interpretation of single in vitro test results to support a respective line of evidence and to establish a biologically plausible link to an adverse outcome.

Isolation, screening and identification of ethanol producing yeasts from Ethiopian fermented beverages

The growing demand for renewable energy sources such as bioethanol is facing a lack of efficient ethanologenic microbes. This study aimed to isolate and screen ethanologenic yeasts from Ethiopian fermented beverages. A progressive screening and selection approach was employed. Selected isolates were evaluated for bioethanol production using banana peel waste as substrate. A total of 102 isolates were obtained. Sixteen isolates were selected based on their tolerance to stress conditions and carbohydrate fermentation and assimilation capacity. Most found moderately tolerant to 10 %, but slightly tolerant at 15 and 20 % (v/v) ethanol concentration. They yield 15.3 to 20.1 g/L and 9.1 ± 0.6 to 12.9 ± 1.3 g/L ethanol from 2 % (w/v) glucose and 80 g/L banana peel, respectively. Molecular characterization identified them as Saccharomyces cerevisiae strains. Results demonstrate insight about their potential role in the ethanol industry. Optimization of the fermentation conditions is recommended.

Screening and Identification of Potential Indigenous Yeasts Isolated During Fermentation of Wine Coffee

Wine coffee is a fermented coffee product that involves yeast as the fermentative agent which has potency as probiotics. This study aimed to determine the potency of yeast isolated from wine coffee fermentation and to identify the yeast species with the best probiotic properties. This study comprised three main steps: coffee fermentation, yeast isolation, and probiotic characterization. A series of probiotic tests were carried out, including resistance tests at low pH (pH 2, 3, & 4) and bile salts (0.5% & 2%), antimicrobial activity tests, antibiotic resistance tests, hemolytic activity tests, and species identification based on the ITS rDNA sequence. The data obtained were analyzed using One-way ANOVA (p≤0.05) and continued with the Tukey test. A total of 25 yeast isolates were isolated and purified. Nine isolates (A2, B1, B3, C3, D4, D5, E2, E3 & E5) had the highest tolerance to pH 2 and 2% bile salts with survival rates were more than 100% and 90%, respectively. Nine isolates were resistant to all tested antibiotics, and only isolate A2 exhibited a pathogenic characteristic (β-hemolysis). Three isolates (B3, E3 & E5) could inhibit the five indicator pathogens, with the highest inhibitory activity shown by isolating E3 against Bacillus cereus by 68 AU/mL. The isolate E3 was selected as the best yeast with probiotic properties identified as Pichia kudriavzevii with 100% similarities towards strain iwate20191107.

Isolation and identification of gastric acid-tolerant yeast from tapai

Lactobacilli and Bifidobacteria are the most extensively employed bacterial strains in marketable probiotic supplements. However, another probiotic was recently developed from yeast screening based on tolerance against gastric acid. This research aimed to isolate yeasts from traditional Indonesian fermented food (tapai). Screening of probiotic yeasts was based on their survival in gastric acid of pH 2.0. Yeast strains were cultured in malt extract agar, and their phenotype and genotype characteristics were identified. Phenotype characteristics were based on yeast cells’ colony, microscopy, and physiology. Meanwhile, genotype characteristics were determined using the PCR-fingerprinting technique to identify the sequence homology compared to the GenBank database and the phylogenetic tree construction. The result showed that SUL and SM isolates have the highest survival on artificial gastric acid of pH 2.0. The SUL isolate from tapai brand “Sumber Madu” has a morphologically wrinkled colony, no pseudo mycelium, white surface colony, and round cell shape. In contrast, the SM isolate from tapai brand “Sari Madu” has a thin wide colony with no pseudo mycelium, turbid white surface, and oval cell shape. After 2 h incubation on gastric acid, SUL and SM isolates grew up to 6.20±0.35 CFU/mL (survival yeast of 82.71%) and 5.75±0.45 CFU/mL (survival yeast of 79.74%), respectively. The SUL isolate was identified as Kodamaea ohmeri, while the SM isolate was identified as Pichia kudriavzevii.