Shochu is traditional Japanese liquor produced from various crops and fungi Aspergillus kawachi or A. awamorii . The amount of unutilized shochu distillation remnants is increasing because of the recent prohibition of ocean dumping of these remnants. In this Article, we first describe the structures of glucosylceramides contained in shochu distillation remnants by fragment ion analysis using ESI-tandem mass spectrometry. Shochu distillation remnant produced from barley contained glucosylceramides d18:2/C16:0h, d18:2/C20:0h, d19:2/C18:1h, and d18:2/C18:0h. Koji (barley fermented with A. kawachii) contained the same glucosylceramides. Shochu distillation remnants produced from rice contained glucosylceramides d18:2/C18:0h and d19:2/C18:1h. The culture broth of A. kawachii contained glucosylceramides d19:2/C18:1h and d19:2/C18:0h. These results indicate that the glucosylceramides contained in crops and those produced by A. kawachii transfer through the processes of fermentation with yeast and distillation to the shochu distillation remnant. This information will enable utilization of shochu distillation remnants and koji as novel sources of sphingolipids.

fIn 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. In nature, microbial communities are formed through metabolic and physiochemical interactions among different microorganisms (1). The pH of cereals that have ripened and dropped from the husk to the ground is neutral to alkaline (2-4). Fermentation microbes, including Saccharomyces cerevisiae, Lactobacillus lactis, and Acetobacter aceti, efficiently utilize carbohydrates in the cereals to produce organic acids (1). As a result, the microbial communities that colonize such carbohydrate-rich cereals, as well as those found in fermented foods, reduce the pH to 4.0 to 6.0. After these microbes establish an acidic pH, they can dominate the environment because they can grow faster at acidic pH values than other microbes. Therefore, these microbes must first adapt to the alkaline environment at the initial stage of colonization on cereal; such adaptation mechanisms are also crucial for industrial fermentation.Traditional alcoholic beverages are produced via the fermentation of cereals by the yeast S. cerevisiae, which can efficiently catabolize glucose to produce ethanol. However, because S. cerevisiae is incapable of hydrolyzing starch to glucose, additional biological catalysts are often added to the fermentation reaction. For example, malt catalyzes starch hydrolysis in wheat-and barleyderived beverages such as whiskey and beer. In East and Southeast Asia, various cereals fermented by fungi are widely used as starchhydrolyzing catalysts for the production of rice-derived alcoholic beverages. These include Japanese sake (5, 6) and shochu, Korean Makgeolli, and Chinese Huangjiu, as well as various other fermented foods ...

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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 () 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. 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.

Koji, rice fermented with Aspergillus, is used for saccharification of starch contained in crops during the manufacturing of many of Japanese traditional foods and drinks. Japanese people have long eaten koji, and many beneficial substances have been reported to be contained in koji. However, there has been no report on the existence or content of galactosylceramide in koji. To address this issue, we analyzed the chemical composition of the sugar moiety of monohexosylceramide contained in koji, and elucidate that 30.3% of yellow koji is galactosylceramide, 69.7% of that is glucosylceramide, 19.2% of white koji is galactosylceramide, and 80.8% of that is glucosylceramide. This is the first report of the existence and content of galactosylceramide in koji.