Japanese sake making using wild yeasts isolated from natural environments.

Saccharomyces cerevisiae is one of the most important microorganisms for the food industry, including Japanese sake, beer, wine, bread, and other products. For sake making, Kyokai sake yeast strains are considered one of the best sake yeast strains because these strains possess fermentation properties that are suitable for the quality of sake required. In recent years, the momentum for the development of unique sake, which is distinct from conventional sake, has grown, and there is now a demand to develop unique sake yeasts that have different sake making properties than Kyokai sake yeast strains. In this minireview, we focus on "wild yeasts," which inhabit natural environments, and introduce basic research on the wild yeasts for sake making, such as their genetic and sake fermentation aspects. Finally, we also discuss the molecular breeding of wild yeast strains for sake fermentation and the possibility for sake making using wild yeasts.

Kuratsuki bacteria and sake making.

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.

Rice grain structural characteristics of sake rice cultivar Hakutsurunishiki for daiginjo-shu brewing.

Hakutsurunishiki is a sake rice cultivar bred using Yamadabo (seed parent) and Wataribune 2 (pollen parent), equivalent to a Yamadanishiki sibling. This study evaluated the structural characteristics of the Hakutsurunishiki rice grain that contribute to the brewing characteristics of daiginjo-shu, via a comparison with Yamadanishiki. Hakutsurunishiki brown rice was a little heavy and had a large white core. Observing a cross-section of white rice after soaking revealed that the rice grain structure of Hakutsurunishiki was different from that of Yamadanishiki. Hakutsurunishiki white rice showed fewer voids than Yamadanishiki, promoting a slower water absorption rate. Glucose distribution in rice koji obtained by mass spectrometry imaging showed that Hakutsurunishiki rice koji, like Yamadanishiki, is tsuki-haze type, suggesting that its grain structure is suitable for making rice koji for daiginjo-shu. With these observations, we were able to clarify the structural characteristics of Hakutsurunishiki rice grain.

Sake yeast symbiosis with lactic acid bacteria and alcoholic fermentation.

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.

Design and rationale for an open-label, randomized, controlled pilot trial to evaluate the changes in blood uremic toxins in patients with chronic kidney disease by dietary therapy with sake lees.

BACKGROUND: Patients with chronic kidney disease (CKD) reportedly show dysbiosis, which is the imbalance of gut microbiome. Dysbiosis increases the uremic toxin level in the intestine, and uremic toxins transfer into the blood, causing CKD progression. Sake lees, a traditional Japanese fermented food, may help reduce uremic toxins by altering the gut microbiome. Additionally, D-alanine, which is present in sake lees, may have a renoprotective effect. The present pilot study aims to evaluate the effect of adding sake lees to the standard CKD dietary therapy in reducing blood uremic toxins. METHODS: This pilot study is a single-center, open-label, randomized controlled trial. Twenty-four patients with CKD will be enrolled and allocated 1:1 to the intervention and control groups. The intervention group will receive standard CKD dietary therapy with an additional intake of 50 g of sake lees per day for 8 weeks, whereas the control group will only receive standard CKD dietary therapy. The primary endpoint is the change in serum indoxyl sulfate after 8 weeks. The secondary endpoint is the plasma D-alanine and fecal microbiome changes. CONCLUSION: This pilot study provides insight into the development of a new diet focused on gut microbiome and D-amino acids in patients with CKD. CLINICAL TRIAL REGISTRATION: This protocol was approved by the Clinical Trial Review Board of Kanazawa University Hospital on October 27, 2022 (2022-001 [6139]) and available to the public on the website of the Japan Registry of Clinical Trials on November 22, 2022 (jRCT1040220095).

Significant contribution of amino acids to antioxidant capacity of Japanese rice wine (sake).

BACKGROUND: Various antioxidant components have been reported in Japanese rice wine (sake), while the contribution ratio of each component to the antioxidant capacity of whole sake has not been well understood. In this study, we evaluated the radical absorption capacity (H-ORAC and/or DPPH radical scavenging ability) and antioxidant components of commercially available sake, and estimated the major components contributing to antioxidant capacity. RESULTS: Water-soluble components with a molecular weight of less than 3000 contributed more than 90% to H-ORAC of sake by measuring the ultrafiltered fraction. The Maillard reaction seemed to have little influence on H-ORAC by browning experiments. The radical absorption capacity of sake showed a strong positive correlation with the amounts of four antioxidant amino acids (Cys, Met, Tyr, Trp) and two phenolic acids (ferulic acid and p-coumaric acid). Evaluating H-ORAC of a sake model solution composed of these antioxidants revealed that the four amino acids showed a clearly higher contribution (25.7-58.2%) than the phenolic acids. In addition, there was a slight synergistic effect when the four amino acids were mixed, whereas such effects were not seen when mixed with the phenolic acids and other sake major components (organic acids, glucose and ethanol). CONCLUSION: We concluded that the contribution of free amino acids to the radical absorption capacity is quite important in sake. These findings will provide a new perspective for antioxidant researches of sake and many fermented foods. © 2024 Society of Chemical Industry.

Protective effects of Japanese sake yeast on depressive-like behaviors, oxidative stress and inflammatory parameters in a rat model of global cerebral ischemia/reperfusion.

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.

Emergence of [GAR+ ] cells in yeast from sake brewing affects the fermentation properties.

In the traditional (kimoto) method of sake (Japanese rice wine) brewing, Saccharomyces cerevisiae yeast cells are exposed to lactate, which is produced by lactic acid bacteria in the seed mash. Lactate promotes the appearance of glucose-repression-resistant [GAR+ ] cells. Herein, we compared the resistance to glucose repression among kimoto, industrial, and laboratory yeast strains. We observed that the frequencies of the spontaneous emergence of [GAR+ ] cells among the kimoto strains were higher than those among the industrial and laboratory strains. The fermentation ability of a kimoto yeast (strain U44) was lower than that of an industrial strain (K701), as [GAR+ ] cells generally showed slower ethanol production. The addition of lactate decreased the fermentation abilities of the K701 strain by increasing the number of [GAR+ ] cells, but it did not affect those of the U44 strain. These results suggest that lactate controlled fermentation by promoting the appearance of [GAR+ ] cells in the industrial sake strains but not in the kimoto strains.

Improvement of valine and isobutanol production in sake yeast by Ala31Thr substitution in the regulatory subunit of acetohydroxy acid synthase.

The fruit-like aroma of two valine-derived volatiles, isobutanol and isobutyl acetate, has great impact on the flavour and taste of alcoholic beverages, including sake, a traditional Japanese alcoholic beverage. With the growing worldwide interest in sake, breeding of yeast strains with intracellular valine accumulation is a promising approach to meet a demand for sakes with a variety of flavour and taste by increasing the valine-derived aromas. We here isolated a valine-accumulating sake yeast mutant (K7-V7) and identified a novel amino acid substitution, Ala31Thr, on Ilv6, a regulatory subunit for acetohydroxy acid synthase. Expression of the Ala31Thr variant Ilv6 conferred valine accumulation on the laboratory yeast cells, leading to increased isobutanol production. Additionally, enzymatic analysis revealed that Ala31Thr substitution in Ilv6 decreased sensitivity to feedback inhibition by valine. This study demonstrated for the first time that an N-terminal arm conserved in the regulatory subunit of fungal acetohydroxy acid synthase is involved in the allosteric regulation by valine. Moreover, sake brewed with strain K7-V7 contained 1.5-fold higher levels of isobutanol and isobutyl acetate than sake brewed with the parental strain. Our findings will contribute to the brewing of distinctive sakes and the development of yeast strains with increased production of valine-derived compounds.

Importance and mechanisms of S-adenosylmethionine and folate accumulation in sake yeast.

Sake yeasts have a range of brewing characteristics that are particularly beneficial for sake making including high ethanol fermentability, high proliferative capacity at low temperatures, lactic acid tolerance, and high ester productivity. On the other hand, sake yeasts also accumulate a diverse range of functional components. For example, significantly greater accumulation of S-adenosylmethionine (SAM), a compound that plays important regulatory roles in a range of biological processes as a major donor of methyl groups, occurs in sake yeasts compared to other microorganisms. Significantly greater accumulation of folate, a bioactive water-soluble vitamin (vitamin B9), also occurs in sake yeasts compared to laboratory yeasts, and the methyl group on SAM is supplied by folate. Accordingly, fully characterizing 'sake yeast identity' requires detailed understanding of the mechanisms underlying both the nutritional characteristics (functional components) and the brewing characteristics in sake yeasts. Therefore, this mini-review focuses on the accumulation of SAM and folate in sake yeast including descriptions of the genes known to contribute to SAM and folate accumulation and the underlying mechanisms.

Effect of kuratsuki Kocuria on sake's taste varies depending on the sake yeast strain used in sake brewing.

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.

Production of S-methyl-methionine using engineered Saccharomyces cerevisiae sake K6.

S-methyl-methionine (SMM), also known as vitamin U, is an important food supplement produced by various plants. In this study, we attempted to produce it in an engineered microorganism, Saccharomyces cerevisiae, by introducing an MMT gene encoding a methionine S-methyltransferase from Arabidopsis thaliana. The S. cerevisiae sake K6 strain, which is a Generally Recognized as Safe (GRAS) strain, was chosen as the host because it produces a significant amount of S-adenosylmethionine (SAM), a precursor of SMM. To increase SMM production in the host, MHT1 and SAM4 genes encoding homocysteine S-methyltransferase were knocked out to prevent SMM degradation. Additionally, MMP1, which encodes S-methyl-methionine permease, was deleted to prevent SMM from being imported into the cell. Finally, ACS2 gene encoding acetyl-CoA synthase was overexpressed, and MLS1 gene encoding malate synthase was deleted to increase SAM availability. Using the engineered strain, 1.92 g/L of SMM was produced by fed-batch fermentation. ONE-SENTENCE SUMMARY: Introducing a plant-derived MMT gene encoding methionine S-methyltransferase into engineered Saccharomyces cerevisiae sake K6 allowed microbial production of S-methyl-methionine (SMM).

High-Level Production of Isoleucine and Fusel Alcohol by Expression of the Feedback Inhibition-Insensitive Threonine Deaminase in Saccharomyces cerevisiae.

A variety of the yeast Saccharomyces cerevisiae with intracellular accumulation of isoleucine (Ile) would be a promising strain for developing a distinct kind of sake, a traditional Japanese alcoholic beverage, because Ile-derived volatile compounds have a great impact on the flavor and taste of fermented foods. In this study, we isolated an Ile-accumulating mutant (strain K9-I48) derived from a diploid sake yeast of S. cerevisiae by conventional mutagenesis. Strain K9-I48 carries a novel mutation in the ILV1 gene encoding the His480Tyr variant of threonine deaminase (TD). Interestingly, the TD activity of the His480Tyr variant was markedly insensitive to feedback inhibition by Ile, but was not upregulated by valine, leading to intracellular accumulation of Ile and extracellular overproduction of 2-methyl-1-butanol, a fusel alcohol derived from Ile, in yeast cells. The present study demonstrated for the first time that the conserved histidine residue located in a linker region between two regulatory domains is involved in allosteric regulation of TD. Moreover, sake brewed with strain K9-I48 contained 2 to 3 times more 2-methyl-1-butanol and 2-methylbutyl acetate than sake brewed with the parent strain. These findings are valuable for the engineering of TD to increase the productivity of Ile and its derived fusel alcohols. IMPORTANCE Fruit-like flavors of isoleucine-derived volatile compounds, 2-methyl-1-butanol (2MB) and its acetate ester, contribute to a variety of the flavors and tastes of alcoholic beverages. Besides its value as aroma components in foods and cosmetics, 2MB has attracted significant attention as second-generation biofuels. Threonine deaminase (TD) catalyzes the first step in isoleucine biosynthesis and its activity is subject to feedback inhibition by isoleucine. Here, we isolated an isoleucine-accumulating sake yeast mutant and identified a mutant gene encoding a novel variant of TD. The variant TD exhibited much less sensitivity to isoleucine, leading to higher production of 2MB as well as isoleucine than the wild-type TD. Furthermore, sake brewed with a mutant yeast expressing the variant TD contained more 2MB and its acetate ester than that brewed with the parent strain. These findings will contribute to the development of superior industrial yeast strains for high-level production of isoleucine and its related fusel alcohols.

Development of sake yeast breeding and analysis of genes related to its various phenotypes.

Sake is a traditional Japanese alcoholic beverage made from rice and water, fermented by the filamentous fungi Aspergillus oryzae and the yeast Saccharomyces cerevisiae. Yeast strains, also called sake yeasts, with high alcohol yield and the ability to produce desired flavor compounds in the sake, have been isolated from the environment for more than a century. Furthermore, numerous methods to breed sake yeasts without genetic modification have been developed. The objectives of breeding include increasing the efficiency of production, improving the aroma and taste, enhancing safety, imparting functional properties, and altering the appearance of sake. With the recent development of molecular biology, the suitable sake brewing characteristics in sake yeasts, and the causes of acquisition of additional phenotypes in bred yeasts have been elucidated genetically. This mini-review summarizes the history and lineage of sake yeasts, their genetic characteristics, the major breeding methods used, and molecular biological analysis of the acquired strains. The data in this review on the metabolic mechanisms of sake yeasts and their genetic profiles will enable the development of future strains with superior phenotypes.

Isolation and analysis of a sake yeast mutant with phenylalanine accumulation.

Sake is a traditional Japanese alcoholic beverage brewed by the yeast Saccharomyces cerevisiae. Since the consumption and connoisseurship of sake has spread around the world, the development of new sake yeast strains to meet the demand for unique sakes has been promoted. Phenylalanine is an essential amino acid that is used to produce proteins and important signaling molecules involved in feelings of pleasure. In addition, phenylalanine is a precursor of 2-phenylethanol, a high-value aromatic alcohol with a rose-like flavor. As such, adjusting the quantitative balance between phenylalanine and 2-phenylethanol may introduce value-added qualities to sake. Here, we isolated a sake yeast mutant (strain K9-F39) with phenylalanine accumulation and found a missense mutation on the ARO80 gene encoding the His309Gln variant of the transcriptional activator Aro80p involved in the biosynthesis of 2-phenylethanol from phenylalanine. We speculated that mutation of ARO80 would decrease transcriptional activity and suppress the phenylalanine catabolism, resulting in an increase of intracellular phenylalanine. Indeed, sake brewed with strain K9-F39 contained 60% increase in phenylalanine, but only 10% less 2-phenylethanol than sake brewed with the parent strain. Use of the ARO80 mutant in sake brewing may be promising for the production of distinctive new sake varieties. ONE-SENTENCE SUMMARY: The ARO80 mutant is appropriate for controlling the content of phenylalanine and 2-phenylethanol.

Effect of sake lees on cheese components in cheese ripened by Aspergillus oryzae and lactic acid bacteria.

More than 2,000 varieties of cheese currently exist in the world, and cheese manufacture continues to flourish. To develop the cheese ripening process, additional ingredients are used during cheese production. In this study, the effect of sake lees as an additional ingredient on the fermentation of cheese using Aspergillus oryzae (koji mold), known as koji cheese, was investigated. Aspergillus oryzae is used in the fermentation of Japanese traditional foods, such as sake and soy sauce, given its strong enzymatic activities, as well as in cheese production (i.e., koji cheese). Sake lees, a by-product of the fermentation of rice with A. oryzae and yeasts in the sake brewing process, contains various metabolites, such as amino acids. Here, supplementation with sake lees enhanced the activities of lactic acid bacteria and affected the color of the cheese. Metabolome analysis revealed that sake lees altered the balance of carbohydrates and fatty acids in the cheese. Remarkably, supplementation with sake lees enhanced the production of umami-enhancing γ-glutamyl (kokumi-active) peptides. This study suggests that a new type of cheese can be produced using A. oryzae and sake lees, and information on the synergistic effects of A. oryzae and sake lees will aid the development of cheese production.

Effects of rice fermented extracts, "Sake Lees", on the functional activity of odontoblast-like cells (KN-3 cells).

This study was designed to investigate the effects of Sake Lees extracts (SLE, Sake Kasu) on the functional activity of odontoblastic cells and tooth pulp of the rats. For in vitro studies, a rat clonal odontoblast-like cell line, KN-3 cells were cultured. SLE significantly decreased KN-3 cell proliferation, but showed no significant cytotoxicity. SLE effects on several protein productions of KN-3 cells were compared with PBS. SLE and PBS increased alkaline phosphatase (ALP), dentin sialoprotein (DSP), and osterix in a day-course dependent manner, while SLE increased the induction of ALP on day 9-21 and DSP on day 15-21. SLE also increased Runx2 expression on day 3 and 9 compared to PBS. Alizarin Red stainings revealed that SLE showed a subtle increase in mineralization of KN-3 cells on day 15 and 21. A histological investigation was conducted to assess if SLE induced reparative dentin formation after direct capping at the exposed tooth pulp in rats, suggesting that SLE could increase the reparative dentin formation more than PBS. These findings suggest that Sake Lees could have functional roles in the alterations of odontoblastic activity, which might influence the physiology of the tooth pulp.

[A CASE OF BRONCHIAL ASTHMA CAUSED BY EXPOSURE TO ASPERGILLUS ORYZAE IN A JAPANESE RICE WINE BREWERY WORKER].

A-55-year-old man who have been working in a Sake (Japanese rice wine) brewer for 27 years, came to the outpatient clinic because cough, dyspnea, and wheeze gradually worsen. These symptoms occurred immediately after exposure to Aspergillus oryzae in the brewing process since age 43. A dust mask was required to reduce these symptoms, but that work was interrupted by exacerbation of these symptoms. These symptoms disappeared when he was away from the on-site work. The SMART therapy using combined inhaler of budesonide (ICS) with formoterol (LABA) was effective to reduce these symptoms. In serological test total IgE antibody and Aspergillus specific IgE antibodies increased, whereas Aspergillus precipitating antibody and Asp f 1 (a major allergen of Aspergillus fumigatus) specific IgE antibody were negative. Eosinophilia in peripheral blood was not observed, and FeNO was not increased. Values of peak expiratory flow was reduced by 20.8% after exposure to Aspergillus oryzae in that work. Lung function test including reversibility test was intact, but FEV1 was fluctuated up to 400mL (15.9%) in the clinical course. Based on these variable clinical manifestations, laboratory data, and lung function test findings, this case was diagnosed as adult-onset atopic (Aspergillus-sensitized) bronchial asthma without allergic bronchopulmonary aspergillosis. Involvement of eosinophilic inflammation is unknown. Allergen may be considered to be Aspergillus oryzae, because these symptoms do not occur in any environment without exposure to Aspergillus oryzae. This patient is the first case of occupational asthma related to Aspergillus oryzae in a Japanese rise wine brewer.

Changes in Bacterial and Chemical Components and Growth Prediction for Lactobacillus sakei during Kimoto-Style Fermentation Starter Preparation in Sake Brewing: a Comprehensive Analysis.

Kimoto-style seed mash is a traditional preparation method for sake that takes advantage of spontaneous lactic acid fermentation before the growth of yeast. Lactic acid helps decrease the pH in seed mash and control the growth of unfavorable microorganisms. In this study, we carried out a comprehensive analysis of the change in the bacterial community and chemical composition during the lactic acid fermentation stage in kimoto-style seed mash preparation. The bacterial transitions were diverse at five sake breweries, but they exhibited three patterns. Lactobacillus sakei was the dominant species in the later stage of lactic acid fermentation in all sake breweries. This species was found to be the most important bacterium for the accumulation of lactic acid, because its average production rate of lactic acid in seed mash reached 4.44 × 10-11 mg cell-1 h-1, which is 10 times higher than those of other species. As a result of specific growth rate analysis, it was revealed that the growth rate of L. sakei was influenced by the strain, pH, and temperature. The effects of pH and temperature were explained by the square root model, and the result indicates that the strains isolated in this study were incapable of growth below pH 3.9. The growth curve predicted using the growth model fit the actual cell density in two out of five sake breweries; however, our model did not work well for the remaining three sake breweries, and we presume that the error was caused by the strain or an unknown factor.IMPORTANCE It is important to produce lactic acid in kimoto-style seed mash; however, the bacterial transition is different depending on the sake brewery. The reason why there are diverse bacterial transitions during kimoto-style seed mash preparation for each sake brewery is unclear so far, and it causes difficulty in starting kimoto-style seed mash. Our findings indicate that the changes in pH caused by lactic acid bacteria grown prior to L. sakei in seed mash influence the growth of L. sakei and are related to the diversity of the bacterial transition. This study uses comprehensive analytical methods to reveal that there is a diversity of bacterial transition and chemical compositions in kimoto-style seed mash depending on the sake brewery and to explain the differences in bacterial transition depending on the characteristics of L. sakei.

Determination of proteinaceous free amino acids by gas chromatography.

A method was developed for determination of proteinaceous free amino acids by gas chromatography-mass spectrometry (GC-MS). The guanidino group of arginine in amino acid samples was modified with 1,2-cyclohexanedione at room temperature under basic conditions, and all amino acids were directly derivatized with isobutyl chloroformate. The amino acid derivatives formed were analyzed by GC-MS. The method developed was applied successfully for the determination of amino acids in the Japanese alcoholic beverage, sake.