Methionine and Glycine Stabilize Mitochondrial Activity in Sake Yeast During Ethanol Fermentation

Dodatak aminokiselina u fermentacijsku podlogu utječe na rast i aktivnost kvasca, a zadržavanje aktivnosti mitohondrija tijekom alkoholnog vrenja je kritično za aktivnost pivskog kvasca. Međutim, mehanizam djelovanja aminokiselina u fermentacijskoj podlozi i njihov utjecaj na aktivnost mitohondrija pivskog kvasca tijekom vrenja još uvijek nije poznat. U ovom radu smo utvrdili da aminokiseline u fermentacijskoj podlozi, osobito metionin i glicin, stabiliziraju aktivnost mitohondrija kvasca tijekom proizvodnje pića sake. Pomoću mutanta atg32△, koji ima narušenu aktivnost mitohondrija, istražili smo aminokiseline koje povećavaju aktivnost mitohondrija kvasca tijekom vrenja. Identificirali smo metionin i glicin kao aminokiseline koje bi mogle povećati aktivnost mitohondrija kvasca tijekom proizvodnje pića sake. Da bismo to potvrdili, izmjerili smo količinu reaktivnih oblika kisika u kvascu nakon vrenja u podlozi s metioninom i glicinom. Kvasac koji je fermentirao u podlozi s metioninom i glicinom zadržao je relativno veliku količinu reaktivnih oblika kisika u usporedbi s kvascem koji je fermentirao u podlozi bez dodatka aminokiselina. Osim toga, stanice koje su fermentirale u podlozi s dodatkom metionina imale su različit metabolom od stanica koje su fermentirale u podlozi bez dodatka aminokiseline. Rezultati pokazuju da određene aminokiseline, poput metionina i glicina, stabiliziraju aktivnost mitohondrija kvasca tijekom proizvodnje pića sake i na taj način upravljaju aktivnošću kvasca.Addition of amino acids to fermentation media affects the growth and brewing profiles of yeast. In addition, retaining mitochondrial activity during fermentation is critical for the fermentation profiles of brewer’s yeasts. However, a concrete mechanism linking amino acids in fermentation media with mitochondrial activity during fermentation of brewer’s yeasts is yet unknown. Here, we report that amino acids in fermentation media, especially methionine (Met) and glycine (Gly), stabilize mitochondrial activity during fermentation of sake yeast. By utilizing atg32Δ mutant sake yeast, which shows deteriorated mitochondrial activity, we screened candidate amino acids that strengthened the mitochondrial activity of sake yeast during fermentation. We identified Met and Gly as candidate amino acids that fortify mitochondrial activity in sake yeast during fermentation. To confirm this biochemically, we measured reactive oxygen species (ROS) levels in sake yeast fermented with Met and Gly. Yeast cells supplemented with Met and Gly retained high ROS levels relative to the non-supplemented sake yeast. Moreover, Met-supplemented cells showed a metabolome distinct from that of non-supplemented cells. These results indicate that specific amino acids such as Met and Gly stabilize the mitochondrial activity of sake yeast during fermentation and thus manipulate brewing profiles of yeast

Saccharomyces arboricola and Its Hybrids’ Propensity for Sake Production: Interspecific Hybrids Reveal Increased Fermentation Abilities and a Mosaic Metabolic Profile

The use of interspecific hybrids during the industrial fermentation process has been well established, positioning the frontier of advancement in brewing to capitalize on the potential of Saccharomyces hybridization. Interspecific yeast hybrids used in modern monoculture inoculations benefit from a wide range of volatile metabolites that broaden the organoleptic complexity. This is the first report of sake brewing by Saccharomyces arboricola and its hybrids. S. arboricola x S. cerevisiae direct-mating generated cryotolerant interspecific hybrids which increased yields of ethanol and ethyl hexanoate compared to parental strains, important flavor attributes of fine Japanese ginjo sake rice wine. Hierarchical clustering heatmapping with principal component analysis for metabolic profiling was used in finding low levels of endogenous amino/organic acids clustered S. arboricola apart from the S. cerevisiae industrial strains. In sake fermentations, hybrid strains showed a mosaic profile of parental strains, while metabolic analysis suggested S. arboricola had a lower amino acid net uptake than S. cerevisiae. Additionally, this research found an increase in ethanolic fermentation from pyruvate and increased sulfur metabolism. Together, these results suggest S. arboricola is poised for in-depth metabolomic exploration in sake fermentation

The Wnt Antagonist Frzb-1 Regulates Chondrocyte Maturation and Long Bone Development during Limb Skeletogenesis

AbstractThe Wnt antagonist Frzb-1 is expressed during limb skeletogenesis, but its roles in this complex multistep process are not fully understood. To address this issue, we determined Frzb-1 gene expression patterns during chick long bone development and carried out gain- and loss-of-function studies by misexpression of Frzb-1, Wnt-8 (a known Frzb-1 target), or different forms of the intracellular Wnt mediator LEF-1 in developing limbs and cultured chondrocytes. Frzb-1 expression was quite strong in mesenchymal prechondrogenic condensations and then characterized epiphyseal articular chondrocytes and prehypertrophic chondrocytes in growth plates. Virally driven Frzb-1 misexpression caused shortening of skeletal elements, joint fusion, and delayed chondrocyte maturation, with consequent inhibition of matrix mineralization, metalloprotease expression, and marrow/bone formation. In good agreement, misexpression of Frzb-1 or a dominant-negative form of LEF-1 in cultured chondrocytes maintained the cells at an immature stage. Instead, misexpression of Wnt-8 or a constitutively active LEF-1 strongly promoted chondrocyte maturation, hypertrophy, and calcification. Immunostaining revealed that the distribution of endogenous Wnt mediator β-catenin changes dramatically in vivo and in vitro, from largely cytoplasmic in immature proliferating and prehypertrophic chondrocytes to nuclear in hypertrophic mineralizing chondrocytes. Misexpression of Frzb-1 prevented β-catenin nuclear relocalization in chondrocytes in vivo or in vitro. The data demonstrate that Frzb-1 exerts a strong influence on limb skeletogenesis and is a powerful and direct modulator of chondrocyte maturation, phenotype, and function. Phases of skeletogenesis, such as terminal chondrocyte maturation and joint formation, appear to be particularly dependent on Wnt signaling and thus very sensitive to Frzb-1 antagonistic action

Medical Application of Substances Derived from Non-Pathogenic Fungi Aspergillus oryzae and A. luchuensis-Containing Koji

Although most fungi cause pathogenicity toward human beings, dynasties of the East Asian region have domesticated and utilized specific fungi for medical applications. The Japanese dynasty and nation have domesticated and utilized koji fermented with non-pathogenic fungus Aspergillus oryzae for more than 1300 years. Recent research has elucidated that koji contains medicinal substances such as Taka-diastase, acid protease, koji glycosylceramide, kojic acid, oligosaccharides, ethyl-α-d-glucoside, ferulic acid, ergothioneine, pyroglutamyl leucine, pyranonigrin A, resistant proteins, deferriferrichrysin, polyamines, Bifidobacterium-stimulating peptides, angiotensin I-converting enzyme inhibitor peptides, 14-dehydroergosterol, beta-glucan, biotin, and citric acid. This review introduces potential medical applications of such medicinal substances to hyperlipidemia, diabetes, hypertension, cardiovascular and cognitive diseases, chronic inflammation, epidermal permeability barrier disruption, coronavirus disease 2019 (COVID-19), and anti-cancer therapy

Residual mitochondrial transmembrane potential decreases unsaturated fatty acid level in sake yeast during alcoholic fermentation

Oxygen, a key nutrient in alcoholic fermentation, is rapidly depleted during this process. Several pathways of oxygen utilization have been reported in the yeast Saccharomyces cerevisiae during alcoholic fermentation, namely synthesis of unsaturated fatty acid, sterols and heme, and the mitochondrial electron transport chain. However, the interaction between these pathways has not been investigated. In this study, we showed that the major proportion of unsaturated fatty acids of ester-linked lipids in sake fermentation mash is derived from the sake yeast rather than from rice or koji (rice fermented with Aspergillus). Additionally, during alcoholic fermentation, inhibition of the residual mitochondrial activity of sake yeast increases the levels of unsaturated fatty acids of ester-linked lipids. These findings indicate that the residual activity of the mitochondrial electron transport chain reduces molecular oxygen levels and decreases the synthesis of unsaturated fatty acids, thereby increasing the synthesis of estery flavors by sake yeast. This is the first report of a novel link between residual mitochondrial transmembrane potential and the synthesis of unsaturated fatty acids by the brewery yeast during alcoholic fermentation

A Temperature-Sensitive dcw1 Mutant of Saccharomyces cerevisiae Is Cell Cycle Arrested with Small Buds Which Have Aberrant Cell Walls

Dcw1p and Dfg5p in Saccharomyces cerevisiae are homologous proteins that were previously shown to be involved in cell wall biogenesis and to be essential for growth. Dcw1p was found to be a glycosylphosphatidylinositol-anchored membrane protein. To investigate the roles of these proteins in cell wall biogenesis and cell growth, we constructed mutant alleles of DCW1 by random mutagenesis, introduced them into a Δdcw1 Δdfg5 background, and isolated a temperature-sensitive mutant, DC61 (dcw1-3 Δdfg5). When DC61 cells were incubated at 37°C, most cells had small buds, with areas less than 20% of those of the mother cells. This result indicates that DC61 cells arrest growth with small buds at 37°C. At 37°C, fewer DC61 cells had 1N DNA content and most of them still had a single nucleus located apart from the bud neck. In addition, in DC61 cells incubated at 37°C, bipolar spindles were not formed. These results indicate that DC61 cells, when incubated at 37°C, are cell cycle arrested after DNA replication and prior to the separation of spindle pole bodies. The small buds of DC61 accumulated chitin in the bud cortex, and some of them were lysed, which indicates that they had aberrant cell walls. A temperature-sensitive dfg5 mutant, DF66 (Δdcw1 dfg5-29), showed similar phenotypes. DCW1 and DFG5 mRNA levels peaked in the G(1) and S phases, respectively. These results indicate that Dcw1p and Dfg5p are involved in bud formation through their involvement in biogenesis of the bud cell wall

Effect of Fermented Rice Drink “Amazake” on Patients with Nonalcoholic Fatty Liver Disease and Periodontal Disease: A Pilot Study

The worldwide increase in nonalcoholic fatty liver disease (NAFLD) is a major public health problem. Obesity and diabetes are risk factors for NAFLD and the development of liver fibrosis is a risk factor for liver cancer. Periodontal disease bacteria can also exacerbate NAFLD. We previously reported that amazake, a traditional Japanese fermented food, improves the quality of life (QOL) of patients with liver cirrhosis. In this study, we investigated the effect of amazake intake on NAFLD patients with periodontal disease. Ten patients (mean age: 57.1 ± 19.2 years) consumed 100 g of amazake daily for 60 days. On days 0 and 60, their body mass index (BMI), body fat percentage, serum biochemical parameters, periodontal disease bacteria in saliva, and ten visual analog scales (VASs), namely, sense of abdomen distension, edema, fatigue, muscle cramps, loss of appetite, taste disorder, constipation, diarrhea, depression, and sleep disorder, were measured. For periodontal bacteria, the numbers of six types of bacteria in saliva (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Fusobacterium necleatum) and P. gingivalis-specific fimA genotype were determined. After 60 days of amazake consumption, eosinophils (p p p p p p < 0.05) had significantly decreased. All subjective symptoms improved after amazake intake. No change was observed in the periodontal bacteria. In conclusion, amazake significantly decreased TNFα and improved the QOL of the patients with NAFLD and periodontitis. However, caution should be exercised because amazake, which is manufactured using techniques that lead to concentrations of glucose from the saccharification of rice starch, may worsen glucose metabolism in NAFLD patients. Amazake may be an effective food for improving the symptoms of a fatty liver if energy intake is regulated

Ethanol-induced death in yeast exhibits features of apoptosis mediated by mitochondrial fission pathway

AbstractCell death in yeast (Saccharomyces cerevisiae) involves several apoptotic processes. Here, we report the first evidence of the following processes, which are also characteristic of apoptosis, in ethanol-induced cell death in yeast: chromatin condensation and fragmentation, DNA cleavage, and a requirement for de novo protein synthesis. Mitochondrial fission protein, Fis1, appears to mediate ethanol-induced apoptosis and ethanol-induced mitochondrial fragmentation. However, mitochondrial fragmentation in response to elevated ethanol levels was not correlated with cell death. Further, in the presence of ethanol, generation of reactive oxygen species was elevated in mutant fis1Δ cells. Our characterization of ethanol-induced cell death in yeast as being Fis1-mediated apoptosis is likely to pave the way to overcoming limitations in large-scale fermentation processes, such as those employed in the production of alcoholic beverages and ethanol-based biofuels

Induction of Chromosomal Aneuploids from Brewery Shochu Yeast with Novel Brewery Characteristics

The first development method of brewery shochu yeast focusing on chromosomal aneuploidy is reported in this study. Euploidy diploid shochu yeast S-3 was treated with a microtubule inhibitor, nocodazole, for the purpose of inducing aneuploidy. Next, 2,3,5-triphenyl tetrazolium chloride (TTC) staining and the growth rate were investigated to select aneuploids. Aneuploids were selected at a frequency of 8.2 × 10−4, which was significantly higher than that of the control group, mainly at chromosomes I, II, III, IX, XII, XIII, and XVI. The acquired aneuploids were evaluated for their metabolic and brewing characteristics. A hierarchical cluster analysis based on endogenous metabolite data discriminated euploid S-3 and aneuploids. In addition, principal-component analysis of the constituents of the broth brewed with the strains discriminated between euploid S-3 and aneuploids. Sensory evaluation of the broth brewed with euploid S-3 and aneuploids showed that it tended to differ in aroma and taste. Specific ethanol production rates of the aneuploids were not deteriorated. The method of this selection made it possible to efficiently obtain aneuploids with various brewing characteristics from brewer’s yeast, which do not correspond to genetically modified organisms. This novel breeding method focusing on chromosomal aneuploidy will facilitate the development of novel shochu yeast strains