Sake may potentially halt the progression of Parkinson\'s disease due to its properties, yet no studies have explored its effects. This preliminary study aimed to assess the impact of sake supplementation on Parkinson\'s disease using a zebrafish model. Sixty fish were divided into six groups: control, rotenone (ROT), and groups administered rotenone along with sake at concentrations of 25, 50, 75, and 100 mg/L (25S, 50S, 75S, and 100S). After 28 days of treatment, behavioral responses and the activities of catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH), and glutathione-S-transferase (GST), as well as the expressions of TNF-α, IL-1β, and COX-2, were evaluated. The results indicated that rotenone administration significantly reduced crossing number (P = 0.001), entries in the top area (P = 0.001), and time spent in the top area (P = 0.001). It also markedly increased levels of TBARS and SH compared to the control group (P = 0.001). Rotenone significantly decreased CAT, SOD, and GSH activities while increasing GST levels. Furthermore, it upregulated the expressions of TNF-α (P = 0.001), IL-1β (P = 0.001), and COX-2 (P = 0.001). Supplementation with sake, particularly at higher doses, reversed the adverse effects of rotenone on behavioral, oxidative, and inflammatory responses. In conclusion, sake shows promise for preventing Parkinson\'s disease pending further clinical studies.

I studied at the Sleep and Circadian Neurobiology Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, from April 2018 to March 2020. At Stanford University, I mainly researched the following themes: (1) sleep basic research using mice (administering compounds with sleep or wakefulness effects to mice and examining their effects), and (2) research on circadian rhythm disorders. There are only a few institutions in the world that can conduct sleep basic research using mice, and Stanford University is a wonderful environment to immerse yourself in research, as it is home to not only psychiatrists but also neurologists and many basic researchers. In this article, I would like to review the experiments I conducted during my study abroad, using mice to verify the effects of natural compounds on wakefulness or sleep. In one study, we evaluated the effects of ginkgolides (A, B, and C) and bilobalide on arousal, locomotion, and core body temperature. The results showed that only ginkgolide B dose-dependently increased the amount of arousal and decreased the amount of NREM sleep in the physiological sleep-wake cycle of mice. In another study, we tested the sleep-inducing effects of sake yeast in mice under an acute insomnia model. We showed that sake yeast dose-dependently increased REM and non-REM sleep, decreased arousal within 6 hours after oral administration of sake yeast, and decreased locomotion and core body temperature in a new cage.

The HHP inactivation behaviors of Niigata sake yeast Saccharomyces cerevisiae strain S9arg and its aerobic respiratory-deficient mutant strains were investigated after cultivating them in a YPD media containing 2% to 15% glucose, as well as in moromi mash, in a laboratory-scale sake brewing process. The piezotolerance of strain S9arg, shown after cultivation in a YPD medium containing 2% glucose, decreased to become piezosensitive with increasing glucose concentrations in YPD media. In contrast, the piezosensitivity of a mutant strain UV1, shown after cultivation in the YPD medium containing 2% glucose, decreased to become piezotolerant with increasing glucose concentrations in the YPD medium. The intracellular ATP concentrations were analyzed for an S. cerevisiae strain with intact aerobic respiratory ability, as well as for strain UV1. The higher concentration of ATP after cultivation suggested a higher energy status and may be closely related to higher piezotolerance for the yeast strains. The decreased piezotolerance of strain S9arg observed after a laboratory-scale sake brewing test may be due to a lower energy status resulting from a high glucose concentration in moromi mash during the early period of brewing, as well as a lower aeration efficiency during the brewing process, compared with cultivation in a YPD medium containing 2% glucose.

Hineka is a type of off-flavor of sake and is attributed to the presence of several compounds, including a major one called dimethyl trisulfide (DMTS). The production of the main precursor of DMTS involves yeast methionine salvage pathway. The DMTS-producing potential (DMTS-pp) of sake brewed using the Km67 strain, a non-Kyokai sake yeast, is lower than that of sake brewed using Kyokai yeast; however, the detailed mechanism is unclear. We focused on S-adenosyl-methionine (SAM) and aimed to elucidate the mechanism that prevents DMTS production in sake brewed using the Km67 strain. We revealed that SAM is involved in DMTS production in sake, and that the conversion of SAM to the DMTS precursor occurs through an enzymatic reaction rather than a chemical reaction. Based on previous reports on ADO1 and MDE1 genes, sake brewing tests were performed using the Km67 Δmde1, Δado1, and Δmde1Δado1 strains. A comparison of the SAM content of pressed sake cakes and DMTS-pp of sake produced using the Km67 Δado1 strain showed an increase in both SAM content and DMTS-pp compared to those produced using the parent strain. However, the Km67 Δmde1Δado1 strain showed little increase in DMTS-pp compared to the Km67 Δmde1 strain, despite an increase in SAM content. These results suggest that SAM accumulation in yeast plays a role in the production of DMTS in sake through the methionine salvage pathway. Moreover, the low SAM-accumulation characteristic of the Km67 strain contributes to low DMTS production in sake.

The EHL1/2/3 genes were identified by whole-genome sequencing of Kyokai No. 7 (K7), which is a well-known representative Japanese sake yeast Saccharomyces cerevisiae. The genes are present in K7, but not in laboratory strain S288C. Although the genes were presumed to encode epoxide hydrolase based on homology analysis, their effect on cellular metabolism in sake yeast has not yet been clarified. We constructed ehl1/2/3 mutants harboring a stop codon in each gene using the haploid yeast strain H3 as the parental strain, which was derived from K701, and investigated the physiological role and effects of the EHL1/2/3 genes on sake quality. Metabolome analysis and vitamin requirement testing revealed that the EHL1/2/3 genes are partly responsible for the synthesis of pantothenate. For fermentation profiles, ethanol production by the ehl1/2/3 mutant was comparable with that of strain H3, but succinate production was decreased in the ehl1/2/3 mutant compared to strain H3 when cultured in yeast malt (YM) medium containing 10% glucose and during sake brewing. Ethyl hexanoate and isoamyl acetate levels in the ehl1/2/3 mutant strain were decreased compared to those of strain H3 during sake brewing. Thus, the EHL1/2/3 genes did not affect ethanol production but did affect the production of organic acids and aromatic components during sake brewing.

The yeast Saccharomyces cerevisiae plays a pivotal role in the production of fermented foods by converting sugars in ingredients into ethanol through alcoholic fermentation. However, how accurate is our understanding of its biological significance? Although yeast is essential to produce alcoholic beverages and bioethanol, yeast does not yield ethanol for humankind. Yeast obtains energy in the form of ATP for its own vital processes through alcoholic fermentation, which generates ethanol as a byproduct. The production of ethanol may have more significance for yeast, since many other organisms do not produce ethanol, a highly toxic substance, to obtain energy. The key to address this issue has not been found using conventional microbiology, where yeasts are isolated and cultured in pure form. This review focuses on a possible novel role of yeast alcohol fermentation, which is revealed through our recent studies of microbial interactions.

Chromosome aneuploidy is a common phenomenon in industrial yeast. Aneuploidy is considered one of the strategies to enhance the industrial properties of Saccharomyces cerevisiae strains. However, the effects of chromosomal aneuploidy on the brewing properties of sake have not been extensively studied. In this study, sake brewing was performed using a series of genome-wide segmental duplicated laboratory S. cerevisiae strains, and the effects of each segmentally duplicated region on sake brewing were investigated. We found that the duplication of specific chromosomal regions affected the production of organic acids and aromatic compounds in sake brewing. As organic acids significantly influence the taste of sake, we focused on the segmental duplication of chromosome II that alters malate levels. Sake yeast Kyokai No. 901 strains with segmental chromosome II duplication were constructed using a polymerase chain reaction-mediated chromosomal duplication method, and sake was brewed using the resultant aneuploid sake yeast strains. The results showed the possibility of developing sake yeast strains exhibiting low malate production without affecting ethanol production capacity. Our study revealed that aneuploidy in yeast alters the brewing properties; in particular, the aneuploidy of chromosome II alters malate production in sake brewing. In conclusion, aneuploidization can be a novel and useful tool to breed sake yeast strains with improved traits, possessing industrial significance.

Kuratsuki bacteria enter during the sake-making process and interact with sake yeast until their growth is attenuated by the ethanol produced by sake yeast. Due to the interaction between kuratsuki bacteria and sake yeast, the metabolism of sake yeast changes, affecting the composition of esters and organic acids and subsequently the flavor and taste of sake. We cultivated kuratsuki bacteria and sake yeast, and performed test making at sake breweries to clarify the interaction among microorganisms in the sake-making process. We aim to propose a sake-making process that controls the flavor and taste of sake by utilizing the functions of kuratsuki bacteria.

Although sake yeast mainly produces the taste of sake, sake brewery-inhabiting (kuratsuki) bacteria affect the taste of sake. Thus, kuratsuki bacteria may alter the metabolism of sake yeast through interactions between kuratsuki bacteria and sake yeast. This study aimed to confirm the effects of the combination of kuratsuki Kocuria TGY1127_2 and different sake yeast strains, AK25, K901, and K1801 on the taste of sake. Although the Brix and acidity during sake production using AK25 differed between sake with and without kuratsuki Kocuria, those using K901 and K1801 did not differ. Thus, sake yeast AK25 interacted with kuratsuki Kocuria and changed its characteristics of ethanol fermentation. In addition, the taste intensity changes, measured with a taste sensor TS-5000Z, showed that the effects of adding kuratsuki Kocuria varied among different sake yeasts. Thus, each sake yeast strain interacted with the kuratsuki bacterium and produced different metabolites, resulting in a change in the taste of sake. The findings of this study can lead to the brewing of sake using different types of kuratsuki bacteria which can affect the taste of sake.

Stroke is a serious cerebrovascular disease that causes post-stress depression and death. Stress and inflammation have pivotal roles in the induction of the disease. Several drugs and agents have been used for the treatment of disease, but their uses are faced with limitations owing to their side effects. Natural agents are more efficient for the treatment of stroke due to lower toxicity and their pharmaceutical properties. Sake yeast or Japanese rice wine is an antioxidant compound that could be used to treat stroke and post-stress depression. This study evaluates the effects of sake yeast on depressive-like behaviors, oxidative stress and inflammatory parameters in a rat model of global cerebral ischemia/reperfusion. Rats were divided into four groups, including 1) control: without bilateral common carotid artery occlusion (BCCAO) and sake supplement, 2) Ischemia group: rats induced with BCCAO and lack of therapeutic supplement, and 3 and 4) Ischemia + sake groups: rats induced with BCCAO and treated with 25 and 50 mg/kg sake yeast, respectively. Depressive-like behaviors antioxidant enzymes activities were assessed. The induction of stroke increased oxidant status, inflammatory parameters, and depressive-like behaviors, while the administration of sake could decrease inflammation, depressive-like behaviors, and oxidant status and increase antioxidant enzymes. The yeast could be used as a supplement in combination with other drugs to treat stroke.