Glucosylceramide Changes Bacterial Metabolism and Increases Gram-Positive Bacteria through Tolerance to Secondary Bile Acids In Vitro.

Glucosylceramide is present in many foods, such as crops and fermented foods. Most glucosylceramides are not degraded or absorbed in the small intestine and pass through the large intestine. Glucosylceramide exerts versatile effects on colon tumorigenesis, skin moisture, cholesterol metabolism and improvement of intestinal microbes in vivo. However, the mechanism of action has not yet been fully elucidated. To gain insight into the effect of glucosylceramide on intestinal microbes, glucosylceramide was anaerobically incubated with the dominant intestinal microbe, Blautia coccoides, and model intestinal microbes. The metabolites of the cultured broth supplemented with glucosylceramide were significantly different from those of broth not treated with glucosylceramide. The number of Gram-positive bacteria was significantly increased upon the addition of glucosylceramide compared to that in the control. Glucosylceramide endows intestinal microbes with tolerance to secondary bile acid. These results first demonstrated that glucosylceramide plays a role in the modification of intestinal microbes.

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.

Extra copy of the mitochondrial cytochrome-c peroxidase gene confers a pyruvate-underproducing characteristic of sake yeast through respiratory metabolism.

The mechanism of pyruvate-underproduction of aneuploid sake yeast was investigated in this study. In our previous report, we revealed that an increase in chromosome XI decreases pyruvate productivity of sake yeast. In this report, we found that increased copy number of CCP1, which is located on chromosome XI and encodes cytochrome-c peroxidase, decreased the pyruvate productivity of sake yeasts. Introducing an extra copy of CCP1 activated respiratory metabolism governed by Hap4 and increased reactive oxygen species. Therefore, it was concluded that increased copy number of CCP1 on chromosome XI activated respiratory metabolism and decreased pyruvate levels in an aneuploid sake yeast. This is the first report that describes a mechanism underlying the improvement of brewery yeast by chromosomal aneuploidy.

Chemical and Bacterial Components in Sake and Sake Production Process.

Together with the worldwide Washoku (traditional Japanese foods and drinks) boom, interest in sake, a traditional Japanese alcoholic drink, is increasing around the world. There are few scientific analyses and studies on the production of sake or the final product itself. We show the diversity of bacterial contaminants during sake production and investigated the effects of different ingredients on sake (for example, amino acids). The koji mold Aspergillus oryzae converts rice starch into sugars, and then, the sake yeast Saccharomyces cerevisiae converts the sugars to ethanol. Comparative studies of the bacterial flora of different sakes have shown that various bacterial species are detected, but that there are few frequently detected bacteria. In addition, the bacterial flora does not vary much during the process of sake brewing, after the koji (steamed rice covered with koji mold) and moto (fermentation starter) are mixed, suggesting that most bacteria contaminate the sake during the process of koji and moto production. Thus, there is the possibility that the contaminating bacteria may grow due to a relationship with the koji mold and/or the sake yeast. The flavor, taste, and quality of sakes differ, even between the same brands of sakes, which may be attributed to variations in the contaminating bacteria during sake production.

Koji glycosylceramide commonly contained in Japanese traditional fermented foods alters cholesterol metabolism in obese mice.

Koji, which is manufactured by proliferating non-pathogenic fungus Aspergillus oryzae on steamed rice, is the base for Japanese traditional fermented foods. We have revealed that koji and related Japanese fermented foods and drinks such as amazake, shio-koji, unfiltered sake and miso contain abundant glycosylceramide. Here, we report that feeding of koji glycosylceramide to obese mice alters the cholesterol metabolism . Liver cholesterol was significantly decreased in obese mice fed with koji glycosylceramide. We hypothesized that their liver cholesterol was decreased because it was converted to bile acids. Consistent with the hypothesis, many bile acids were increased in the cecum and feces of obese mice fed with koji glycosylceramide. Expressions of CYP7A1 and ABCG8 involved in the metabolism of cholesterol were significantly increased in the liver of mice fed with koji glycosylceramide. Therefore, it was considered that koji glycosylceramide affects the cholesterol metabolism in obese mice.

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

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 atg32△ 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.

A new method for determining the mycelial weight of the koji-mold Aspergillus oryzae by measuring its glycosylceramide content.

At present, the quantitation of the mycelial weight of the industrially important non-pathogenic fungus Aspergillus oryzae, which is used for manufacturing koji, is performed by quantitating N-acetylglucosamine. However, since N-acetylglucosamine is a cell wall component, the extraction procedure is costly and tedious, and its quantitative performance is poor. Here, we report a novel method for the quantitation of A. oryzae mycelial weight. The amount of glycosylceramide significantly correlated with both the mycelial weight of A. oryzae and the amount of N-acetylglucosamine, an established index of the mycelial weight of A. oryzae in koji. This new method is simple and efficient and can be used in the brewing and food industries to determine the mycelial weight of A. oryzae.

Glycosylceramide modifies the flavor and metabolic characteristics of sake yeast.

In the manufacture of sake, Japanese traditional rice wine, sake yeast is fermented with koji, which is steamed rice fermented with the non-pathogenic fungus Aspergillus oryzae. During fermentation, sake yeast requires lipids, such as unsaturated fatty acids and sterols, in addition to substances provided by koji enzymes for fermentation. However, the role of sphingolipids on the brewing characteristics of sake yeast has not been studied. In this study, we revealed that glycosylceramide, one of the sphingolipids abundant in koji, affects yeast fermentation. The addition of soy, A. oryzae, and Grifola frondosa glycosylceramide conferred a similar effect on the flavor profiles of sake yeast. In particular, the addition of A. oryzae and G. frondosa glycosylceramide were very similar in terms of the decreases in ethyl caprylate and ethyl 9-decenoate. The addition of soy glycosylceramide induced metabolic changes to sake yeast such as a decrease in glucose, increases in ethanol and glycerol and changes in several amino acids and organic acids concentrations. Tricarboxylic acid (TCA) cycle, pyruvate metabolism, starch and sucrose metabolism, and glycerolipid metabolism were overrepresented in the cultures incubated with sake yeast and soy glycosylceramide. This is the first study of the effect of glycosylceramide on the flavor and metabolic profile of sake yeast.

Chromosomal Aneuploidy Improves the Brewing Characteristics of Sake Yeast.

The effect of chromosomal aneuploidy on the brewing characteristics of brewery yeasts has not been studied. Here we report that chromosomal aneuploidy in sake brewery yeast (Saccharomyces cerevisiae) leads to the development of favorable brewing characteristics. We found that pyruvate-underproducing sake yeast, which produces less off-flavor diacetyl, is aneuploid and trisomic for chromosomes XI and XIV. To confirm that this phenotype is due to aneuploidy, we obtained 45 haploids with various chromosomal additions and investigated their brewing profiles. A greater number of chromosomes correlated with a decrease in pyruvate production. Especially, sake yeast haploids with extra chromosomes in addition to chromosome XI produced less pyruvate than euploids. Mitochondrion-related metabolites and intracellular oxygen species in chromosome XI aneuploids were higher than those in euploids, and this effect was canceled in their "petite" strains, suggesting that an increase in chromosomes upregulated mitochondrial activity and decreased pyruvate levels. These findings suggested that an increase in chromosome number, including chromosome XI, in sake yeast haploids leads to pyruvate underproduction through the augmentation of mitochondrial activity. This is the first report proposing that aneuploidy in brewery yeasts improves their brewing profile.IMPORTANCE Chromosomal aneuploidy has not been evaluated in development of sake brewing yeast strains. This study shows the relationship between chromosomal aneuploidy and brewing characteristics of brewery yeast strains. High concentrations of pyruvate during sake storage give rise to α-acetolactate and, in turn, to high concentrations of diacetyl, which is considered an off-flavor. It was demonstrated that pyruvate-underproducing sake yeast is trisomic for chromosome XI and XIV. Furthermore, sake yeast haploids with extra chromosomes produced reduced levels of pyruvate and showed metabolic processes characteristic of increased mitochondrial activity. This novel discovery will enable the selection of favorable brewery yeasts by monitoring the copy numbers of specific chromosomes through a process that does not involve generation/use of genetically modified organisms.

Identification and detoxification of glycolaldehyde, an unattended bioethanol fermentation inhibitor.

Although there have been approximately 60 chemical compounds identified as potent fermentation inhibitors in lignocellulose hydrolysate, our research group recently discovered glycolaldehyde as a key fermentation inhibitor during second generation biofuel production. Accordingly, we have developed a yeast S. cerevisiae strain exhibiting tolerance to glycolaldehyde. During this glycolaldehyde study, we established novel approaches for rational engineering of inhibitor-tolerant S. cerevisiae strains, including engineering redox cofactors and engineering the SUMOylation pathway. These new technical dimensions provide a novel platform for engineering S. cerevisiae strains to overcome one of the key barriers for industrialization of lignocellulosic ethanol production. As such, this review discusses novel biochemical insight of glycolaldehyde in the context of the biofuel industry.

Erratum to: Japanese traditional dietary fungus koji Aspergillus oryzae functions as a prebiotic for Blautia coccoides through glycosylceramide: Japanese dietary fungus koji is a new prebiotic.

[This corrects the article DOI: 10.1186/s40064-016-2950-6.].

Japanese traditional dietary fungus koji Aspergillus oryzae functions as a prebiotic for Blautia coccoides through glycosylceramide: Japanese dietary fungus koji is a new prebiotic.

BACKGROUND: The Japanese traditional cuisine, Washoku, considered to be responsible for increased longevity among the Japanese, comprises various foods fermented with the non-pathogenic fungus Aspergillus oryzae (koji). We have recently revealed that koji contains an abundant amount of glycosylceramide. Intestinal microbes have significant effect on health. However, the effects of koji glycosylceramide on intestinal microbes have not been studied. MATERIALS AND METHODS: Glycosylceramide was extracted and purified from koji. C57BL/6N mice were fed a diet containing 1 % purified koji glycosylceramide for 1 week. Nutritional parameters and faecal lipid constituents were analyzed. The intestinal microbial flora of mice on this diet was investigated. RESULTS: Ingested koji glycosylceramide was neither digested by intestinal enzymes nor was it detected in the faeces, suggesting that koji glycosylceramide was digested by the intestinal microbial flora. Intestinal microbial flora that digested koji glycosylceramide had an increased ratio of Blautia coccoides. Stimulation of B. coccoides growth by pure koji glycosylceramide was confirmed in vitro. CONCLUSIONS: Koji functions as a prebiotic for B. coccoides through glycosylceramide. Since there are many reports of the effects of B. coccoides on health, an increase in intestinal B. coccoides by koji glycosylceramide might be the connection between Japanese cuisine, intestinal microbial flora, and longevity.

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.

Glucosylceramide Contained in Koji Mold-Cultured Cereal Confers Membrane and Flavor Modification and Stress Tolerance to Saccharomyces cerevisiae during Coculture Fermentation.

In nature, different microorganisms create communities through their physiochemical and metabolic interactions. Many fermenting microbes, such as yeasts, lactic acid bacteria, and acetic acid bacteria, secrete acidic substances and grow faster at acidic pH values. However, on the surface of cereals, the pH is neutral to alkaline. Therefore, in order to grow on cereals, microbes must adapt to the alkaline environment at the initial stage of colonization; such adaptations are also crucial for industrial fermentation. Here, we show that the yeast Saccharomyces cerevisiae, which is incapable of synthesizing glucosylceramide (GlcCer), adapted to alkaline conditions after exposure to GlcCer from koji cereal cultured with Aspergillus kawachii. We also show that various species of GlcCer derived from different plants and fungi similarly conferred alkali tolerance to yeast. Although exogenous ceramide also enhanced the alkali tolerance of yeast, no discernible degradation of GlcCer to ceramide was observed in the yeast culture, suggesting that exogenous GlcCer itself exerted the activity. Exogenous GlcCer also increased ethanol tolerance and modified the flavor profile of the yeast cells by altering the membrane properties. These results indicate that GlcCer from A. kawachii modifies the physiology of the yeast S. cerevisiae and demonstrate a new mechanism for cooperation between microbes in food fermentation.

SUMO expression shortens the lag phase of Saccharomyces cerevisiae yeast growth caused by complex interactive effects of major mixed fermentation inhibitors found in hot-compressed water-treated lignocellulosic hydrolysate.

The complex inhibitory effects of inhibitors present in lignocellulose hydrolysate suppress the ethanol fermentation of Saccharomyces cerevisiae. Although the interactive inhibitory effects play important roles in the actual hydrolysate, few studies have investigated glycolaldehyde, the key inhibitor of hot-compressed water-treated lignocellulose hydrolysate. Given this challenge, we investigated the interactive effects of mixed fermentation inhibitors, including glycolaldehyde. First, we confirmed that glycolaldehyde was the most potent inhibitor in the hydrolysate and exerted interactive inhibitory effects in combination with major inhibitors. Next, through genome-wide analysis and megavariate data modeling, we identified SUMOylation as a novel potential mechanism to overcome the combinational inhibitory effects of fermentation inhibitors. Indeed, overall SUMOylation was increased and Pgk1, which produces an ATP molecule in glycolysis by substrate-level phosphorylation, was SUMOylated and degraded in response to glycolaldehyde. Augmenting the SUMO-dependent ubiquitin system in the ADH1-expressing strain significantly shortened the lag phase of growth, released cells from G2/M arrest, and improved energy status and glucose uptake in the inhibitor-containing medium. In summary, our study was the first to establish SUMOylation as a novel platform for regulating the lag phase caused by complex fermentation inhibitors.

Variations in mitochondrial membrane potential correlate with malic acid production by natural isolates of Saccharomyces cerevisiae sake strains.

Research on the relationship between mitochondrial membrane potential and fermentation profile is being intensely pursued because of the potential for developing advanced fermentation technologies. In the present study, we isolated naturally occurring strains of yeast from sake mash that produce high levels of malic acid and demonstrate that variations in mitochondrial membrane potential correlate with malic acid production. To define the underlying biochemical mechanism, we determined the activities of enzymes required for malic acid synthesis and found that pyruvate carboxylase and malate dehydrogenase activities in strains that produce high levels of malic acid were elevated compared with the standard sake strain K901. These results inspired us to hypothesize that decreased mitochondrial membrane potential was responsible for increased malic acid synthesis, and we present data supporting this hypothesis. Thus, the mitochondrial membrane potential of high malic acid producers was lower compared with standard strains. We conclude that mitochondrial membrane potential correlates with malic acid production.

Quantitation and structural determination of glucosylceramides contained in sake lees.

Sake lees are solid parts filtered from the mash of sake, the traditional rice wine of Japan, which is brewed with Aspergillus oryzae and Saccharomyces cerevisiae. The moisture-holding activity of sake lees has long been recognized in Japan. However, the constituent responsible for this activity has not been elucidated. In this study, we first determined the structure of the glucosylceramides contained in sake lees. The glucosylceramides contained in sake lees were N-2'-hydroxyoctadecanoyl-l-O-ß-D-glucopyranosyl-9-methyl-4,8-sphingadienine (d19:2/C18:0h), N-2'-hydroxyoctadecanoyl-l-O-ß-D-glucopyranosyl-4,8-sphingadienine (d18:2/C18:0h), N-2'-hydroxyicosanoyl-l-O-ß-D-glucopyranosyl-4,8-sphingadienine (d18:2/C20:0h) and N-2'-hydroxyicosanoyl-l-O-ß-D-glucopyranosyl-4,8-sphingadienine (d18:2/C22:0h), which corresponded to those of A. oryzae and rice. The glucosylceramide produced by A. oryzae constituted the most abundant species (43% of the total glucosylceramide) in the sake lees. These results will be of value in the utilization of sake lees for cosmetics and functional foods.

Mitochondrial metabolism and stress response of yeast: Applications in fermentation technologies.

Mitochondria are sites of oxidative respiration. During sake brewing, sake yeasts are exposed to long periods of hypoxia; the structure, role, and metabolism of mitochondria of sake yeasts have not been studied in detail. It was first elucidated that the mitochondrial structure of sake yeast transforms from filamentous to dotted structure during sake brewing, which affects malate metabolism. Based on the information of yeast mitochondria during sake brewing, practical technologies have been developed; (i) breeding pyruvate-underproducing sake yeast by the isolation of a mutant resistant to an inhibitor of mitochondrial pyruvate transport; and (ii) modifying malate and succinate production by manipulating mitochondrial activity. During the bread-making process, baker's yeast cells are exposed to a variety of baking-associated stresses, such as freeze-thaw, air-drying, and high sucrose concentrations. These treatments induce oxidative stress generating reactive oxygen species due to mitochondrial damage. A novel metabolism of proline and arginine catalyzed by N-acetyltransferase Mpr1 in the mitochondria eventually leads to synthesis of nitric oxide, which confers oxidative stress tolerance on yeast cells. The enhancement of proline and arginine metabolism could be promising for breeding novel baker's yeast strains that are tolerant to multiple baking-associated stresses. These new and practical methods provide approaches to improve the processes in the field of industrial fermentation technologies.

Improved sake metabolic profile during fermentation due to increased mitochondrial pyruvate dissimilation.

Although the decrease in pyruvate secretion by brewer's yeasts during fermentation has long been desired in the alcohol beverage industry, rather little is known about the regulation of pyruvate accumulation. In former studies, we developed a pyruvate under-secreting sake yeast by isolating a strain (TCR7) tolerant to ethyl α-transcyanocinnamate, an inhibitor of pyruvate transport into mitochondria. To obtain insights into pyruvate metabolism, in this study, we investigated the mitochondrial activity of TCR7 by oxigraphy and (13) C-metabolic flux analysis during aerobic growth. While mitochondrial pyruvate oxidation was higher, glycerol production was decreased in TCR7 compared with the reference. These results indicate that mitochondrial activity is elevated in the TCR7 strain with the consequence of decreased pyruvate accumulation. Surprisingly, mitochondrial activity is much higher in the sake yeast compared with CEN.PK 113-7D, the reference strain in metabolic engineering. When shifted from aerobic to anaerobic conditions, sake yeast retains a branched mitochondrial structure for a longer time than laboratory strains. The regulation of mitochondrial activity can become a completely novel approach to manipulate the metabolic profile during fermentation of brewer's yeasts.