Sake Fermentation Research Articles

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.

Traditional microbial control methods used in sake brewing effectively suppress predominant bacteria emerging during production of rice koji

Rice koji is a raw material used in the production of sake; however, details regarding the microbial flora and their dynamics in rice koji during the production process are poorly understood. Clarifying these issues can contribute to proposing a method and evaluation that will improve the quality of rice koji and sake. The aim of this study was to determine the microflora in rice koji and the effectiveness of the traditional microbial control techniques used in the sake production process. We analyzed the diversity and changes in bacterial flora during rice koji production by amplicon sequencing of the 16S rRNA gene. The predominant taxon in all rice koji samples was family Staphylococcaceae. The microbial population and the changes in its distribution for five consecutive stages in rice koji production were examined by direct colony counting. Bacteria counts in all samples were below the limit of detection initially, then increased rapidly toward the final stage. The predominant bacterial colonies from all samples were yellow and were identified as Staphylococcus gallinarum through 16S rRNA gene sequence similarity. The S. gallinarum isolates exhibited faster growth in pregelatinized rice medium. Interestingly, the growth of S. gallinarum isolates was suppressed by low temperature (12 °C), ethanol concentration (≥6%) and the addition of lactic acid, which are traditional microbial control methods used during sake fermentation. Therefore, proper control of the traditional sake production process can effectively inhibit the growth of undesirable bacteria such as S. gallinarum that emerge during the production of rice koji.

Fermentation and bio-functions of sake and Chinese rice wine: Implications for 'healthy-alcoholic industry'

Traditional low-alcoholic beverages were promoted to be healthy drinks with multiple bio-functions. Research into Japanese sake and Chinese rice wine (CRW) in terms of the preparation process, bacteria diversity, authentic quality control and bio functions is of significance. Japanese sake and CRW are representative alcoholic beverages which were proven to bring health benefits. For instance, sake was found to contain bio-functional components of CRW were found to obtain antioxidant activities. In this article, the production mechanisms and their bioactive components in Japanese sake and CRW were discussed. Red yeast rice is made by fermenting the rice with Monascus. However, commercial lovastatin (a structure that similar to monacolin K) is recently reported to be illegally added to common red yeast rice to meet drug quality standards, herein, there are many safety issues of rice wine, and it is especially important to dress the authentication assessment of the CRW. A new era of CRW as a low-alcoholic healthy beverage is coming. Prospects for the future development of CRW, with an emphasis on the absorption of the successful regulation experience from Japanese sake productions which are with high reputations worldwide, are presented.

Metabolome Analysis of the Effects of Sake Lees on Adipocyte Differentiation and Lipid Accumulation

Obesity, along with hypertension and hyperlipidemia, is one of the leading factors of metabolic syndrome, which increases the risk of diabetes. However, controlling obesity is a global challenge. Sake lees, or Japanese rice wine lees, is a by-product of sake fermentation and has been consumed in Japan for a long time. Sake lees contains an abundance of amino acids, peptides, dietary fiber, and micronutrients, which make it highly nutritional. Additionally, sake lees has been reported to have multiple interesting effects when ingested and may aid in combating obesity. In this study, we investigated the effects of sake lees materials on preadipocyte differentiation and fat accumulation in preadipocyte cells (3T3-L1) and analyzed it with a metabolome analysis. We found that compared to the control group, lipid accumulation was suppressed by 80.9% when the 100 °C extract of indigestible sake lees component (ISLCs) was added to 1 mg/mL. Additionally, the metabolome analysis revealed various other differences between the control group and the group treated with ISLCs, especially in amino acids concentrations. Based on the above findings, we demonstrate that ISLCs affect the amino acid metabolic pathways, which in turn affect differentiation and lipid accumulation in adipocytes. Therefore, we suggest that sake lees may aid in combating obesity and addressing metabolic syndromes, both of which can be considered as global issues. The limitation of this research is sake lee is a general non-direct edible raw material and it is difficult to add as a regular diet.

Nitrogen fertilization of rice plants before flowering affects sake fermentation and quality

AbstractBackground and ObjectiveNitrogen fertilizer applied as top‐dressing to rice plants before flowering affects the yield and quality of rice grains. However, little is known about the effects of varying dosages of nitrogen at this stage on the brewing properties of sake using the rice grains. To explore this topic, we cultivated the sake rice cultivar “Koshitanrei” with low and high levels of nitrogen (equivalent to 10 kg and 30 kg N per hectare, respectively) applied 1 week before flowering and then conducted a small‐scale sake brewing test using the harvested rice.FindingsSake made from rice from the low‐N treatment was fermented more efficiently. This yielded a higher volume of sake with a higher alcohol concentration and decreased amino acid concentrations.ConclusionsThe use of rice from the low‐N treatment enhanced alcohol fermentation, resulting in increased alcohol yield and decreased concentrations of amino acids and related compounds.Significance and NoveltyThe amount of nitrogen applied to rice plants before flowering affects the fermentation properties of rice grains in sake brewing, and this can alter the quality and sensory properties of sake even though the nitrogen content of rice grains is unaffected.

Metagenomics analyses of microbial dynamics associated with putative flavor development in mash fermentation of sake

Sake is a traditional alcoholic beverage in Japan. To examine the impact of co-fermentation on microbial composition, fermentation with and without pineapple was conducted. The dynamics of microbial communities during fermentation were investigated using the metagenomics of 16S rRNA and internal transcribed spacers. In regular (RS) and co-fermented (CS) sakes, the fungal microbiota was dominated by Saccharomyces. Hierarchical clustering and non-metric multidimensional scaling identified two phases of bacterial succession. Compared with the dividing point between phases in RS, the transition in CS was delayed by five days. In both experiments, Leuconostoc dominated the premature stage and was replaced by Erwinia in the maturation stage; the alcohol tolerators of Staphylococcus and Bacillus were persistent. In RS, bacterial succession was characterized by a dramatic decrease in Pseudomonas in the maturation stage, whereas it occurred earlier in the premature stage in CS. Nineteen genera were exclusively found in CS, suggesting that co-fermentation may nurture diverse bacteria that enrich the sake flavor. Analysis of functional pathways by PICRUSt2 revealed that sugar alcohol degradation was fundamental to fermentation, whereas flavor-rich CS was attributable to the activation of l-arginine biosynthesis and amine and polyamine degradation. Aerococcus was positively correlated with enzymes involved in phenylethyl metabolism.

Deep Ocean Water Concentrate Changes Physicochemical Characteristics, the Profile of Volatile Components and Consumer Acceptance for Taiwanese Rice Shochu.

To study the effects of deep-ocean water concentrate (DOWC) on sake quality, Taichung No. 10 indica rice (Oryza sativa subsp. indica) and Tainan No. 11 japonica rice (O. sativa subsp. japonica) were used as raw materials, and basic physicochemical property parameters in shochu were analyzed differentially. Sake fermentation mash analysis results revealed that DOWC addition did not significantly affect the basic physicochemical properties during sake brewing, but it significantly reduced citric acid and malic acid contents in Taichung No. 10 indica rice sake sample by 52–66% and 73–93%, respectively. DOWC addition significantly increased citric acid content in Tainan No. 11 japonica rice sake sample by 32–202%. Rice shochu analysis results revealed that DOWC addition significantly increased isoamyl acetate, ethyl hexanoate, and ethyl octanoate contents in shochu made from japonica rice and indica rice, respectively. The results indicate that rice variety directly affects the types of volatile compounds in rice shochu. Principal component analysis and sensory evaluation results revealed that DOWC addition affected the composition of volatile compounds in the two types of rice shochu and resulted in differences in flavor evaluation. DOWC addition affects yeast metabolites and directly changes the volatile compound composition and flavor of rice shochu.

Cultivation temperature and length affect the antioxidant activity and total phenolic content of soybean Koji prepared with Aspergillus awamori

In this study, soybean koji was first fermented with Aspergillus awamori, a starter organism commonly used for the fermentation of sake, at 25, 30 or 35°C for a period of 1-5 days. Antioxidant activity, including DPPH radical-scavenging effect and Fe2+-chelating ability, and total phenolic content of the methanol extract of these fermented soybean koji as well as mycelial propagation of starter organism were compared. Results revealed that fermented for a same period of 3 days, the DPPH radical-scavenging effect exerted by the 30°C-koji extract was the highest showing 2.5-3.5 folds of that fermented at 25 or 35°C. The Fe2+-chelating ability exerted by the 30°C-koji extract was also found to be 3.6-3.8 times higher than those fermented at other temperatures. Furthermore, it was also noted that increasing or decreasing cultivation time from 3 days resulted in a decreased antioxidant activity of the koji extract. Cultivation temperature and length were also observed to affect the mycelial propagation and the total phenolic content of the koji extract. Maximum mycelial propagation of A. awamori was achieved after cultivation at 30°C for 3 days. Finally, the highest phenolic content noted in 30°C-koji extract was closely correlated to the highest antioxidant activity observed.

Profiling of taste-related compounds during the fermentation of Japanese sake brewed with or without a traditional seed mash (kimoto)

Japanese sake production involves three processes: rice koji fermentation, seed mash fermentation, and main mash fermentation. Traditional seed mash (kimoto) production utilizes natural lactic acid produced by lactic acid bacteria for pure cultures of only sake yeast, preventing the growth of wild yeast and other unwanted bacteria. Recently, because kimoto production requires substantial time and labor, sake yeast mass-cultured in usual liquid medium has been used as a seed mash alternative. Sake quality is highly similar to that of kimoto, suggesting that they share similar component profiles. However, comparative component analyses of sake brewed with kimoto and sake brewed with cultured yeast are lacking. In this study, a time-course analysis of hydrophilic compounds in the main mash brewed with kimoto and with cultured yeast as well as a sensory evaluation of the products were performed. As a result, differences in various compounds and in umami taste level between sake brewed with kimoto and cultured yeast were detected. This is the first comparative analysis of changes in the component profile during sake main mash brewing using kimoto seed mash and cultured sake yeast; our results clarify the effects of kimoto seed mash on main mash brewing and sake quality.

Characteristic analysis of the fermentation and sporulation properties of the traditional sake yeast strain Hiroshima no.6.

General sake yeasts (e.g., Kyokai no.7, K7) show high fermentation ability and low sporulation frequency. Former is related to stress-response defect due to the loss-of-function of MSN4 and RIM15. Later is mainly caused by low IME1 expression, leading to difficulty in breeding and genetic analysis. Sake yeast Hiroshima no.6 (H6), which had been applied for sake fermentation, has sporulation ability. However, its detailed properties have not been unveiled. Here we present that the fermentation ability of H6 is suitable for sake brewing, and the precursor of dimethyl trisulfide in sake from H6 is low. MSN4 but not RIM15 of H6 has the same mutation as K7. Our phylogenetic analysis indicated that H6 is closely related to the K7 group. Unlike K7, H6 showed normal sporulation frequency in a partially RIM15-dependent manner, and IME1 in H6 was expressed. H6 possesses excellent properties as a partner strain for breeding by crossing.

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.

Regulatory mechanisms for amylolytic gene expression in the koji mold Aspergillus oryzae.

The koji mold Aspergillus oryzae has been used in traditional Japanese food and beverage fermentation for over a thousand years. Amylolytic enzymes are important in sake fermentation, wherein production is induced by starch or malto-oligosaccharides. This inducible production requires at least two transcription activators, AmyR and MalR. Among amylolytic enzymes, glucoamylase GlaB is produced exclusively in solid-state culture and plays a critical role in sake fermentation owing to its contribution to glucose generation from starch. A recent study demonstrated that glaB gene expression is regulated by a novel transcription factor, FlbC, in addition to AmyR in solid-state culture. Amylolytic enzyme production is generally repressed by glucose due to carbon catabolite repression (CCR), which is mediated by the transcription factor CreA. Modifying CCR machinery, including CreA, can improve amylolytic enzyme production. This review focuses on the role of transcription factors in regulating A. oryzae amylolytic gene expression.

Histone deacetylases in sake yeast affect fermentation characteristics.

Yeast histone deacetylases (HDAC) affect the production of alcoholic beverages. In this study, we evaluated the sake fermentation characteristics when using HDAC gene-disrupted yeast strain Kyokai No. 701. Flavor components of the sake product were significantly changed. RPD3 or HDA1 disruption increased twofold the amount of isoamyl acetate, and isoamyl alcohol levels also increased in the rpd3Δ strain. To determine the contribution of Rpd3L and Rpd3S complexes to sake characteristics, a gene responsible for Rpd3L and/or Rpd3S formation was also disrupted. Disruption of DEP1 or SDS3 that is an essential component of Rpd3L led to increased isoamyl alcohol production similar to that of the rpd3Δ strain, but the efficiency of isoamyl alcohol esterification was not affected. In addition, Rpd3 and Hda1may regulate the responsiveness to oxygen in isoamyl acetate production. We conclude that HDAC genes regulate the production of flavor components during sake fermentation. Abbreviations: HDAC: Histone deacetylase; HAT: histone acetyltransferase; K701: sake yeast Kyokai No. 701; PCR: polymerase chain reaction; HPLC: high performance liquid chromatography; E/A: Ester/Alcohol; BCAA: branched chain-amino acid; Atf: alcohol acetyltransferase.

History, lineage and phenotypic differentiation of sake yeast.

Sake yeast was first isolated as a single yeast strain, Saccharomyces sake, during the Meiji era. Yeast strains suitable for sake fermentation were subsequently isolated from sake brewers and distributed as Kyokai yeast strains. Sake yeast strains that produce characteristic flavors have been bred in response to various market demands and individual preferences. Interestingly, both genetic and morphological studies have indicated that sake yeast used during the Meiji era differs from new sake yeasts derived from Kyokai Strain No. 7 lineage. Here, we discuss the history of sake yeast breeding, from the discovery of sake yeast to the present day, to highlight the achievements of great Japanese scientists and engineers.

Metabolite profiling of the fermentation process of "yamahai-ginjo-shikomi" Japanese sake.

Sake is a traditional Japanese alcoholic beverage prepared by multiple parallel fermentation of rice. The fermentation process of “yamahai-ginjo-shikomi” sake is mainly performed by three microbes, Aspergillus oryzae, Saccharomyces cerevisiae, and Lactobacilli; the levels of various metabolites fluctuate during the fermentation of sake. For evaluation of the fermentation process, we monitored the concentration of moderate-sized molecules (m/z: 200–1000) dynamically changed during the fermentation process of “yamahai-ginjo-shikomi” Japanese sake. This analysis revealed that six compounds were the main factors with characteristic differences in the fermentation process. Among the six compounds, four were leucine- or isoleucine-containing peptides and the remaining two were predicted to be small molecules. Quantification of these compounds revealed that their quantities changed during the month of fermentation process. Our metabolomic approach revealed the dynamic changes observed in moderate-sized molecules during the fermentation process of sake, and the factors found in this analysis will be candidate molecules that indicate the progress of “yamahai-ginjo-shikomi” sake fermentation.

Bioethanol Production from Brewers Spent Grains Using a Fungal Consolidated Bioprocessing (CBP) Approach

Production of bioethanol from brewers spent grains (BSG) using consolidated bioprocessing (CBP) is reported. Each CBP system consists of a primary filamentous fungal species, which secretes the enzymes required to deconstruct biomass, paired with a secondary yeast species to ferment liberated sugars to ethanol. Interestingly, although several pairings of fungi were investigated, the sake fermentation system (A. oryzae and S. cerevisiae NCYC479) was found to yield the highest concentrations of ethanol (37 g/L of ethanol within 10 days). On this basis, 1 t of BSG (dry weight) would yield 94 kg of ethanol using 36 hL of water in the process. QRT-PCR analysis of selected carbohydrate degrading (CAZy) genes expressed by A. oryzae in the BSG sake system showed that hemicellulose was deconstructed first, followed by cellulose. One drawback of the CBP approach is lower ethanol productivity rates; however, it requires low energy and water inputs, and hence is worthy of further investigation and optimisation.

RIM15 antagonistic pleiotropy is responsible for differences in fermentation and stress response kinetics in budding yeast.

Different natural yeast populations have faced dissimilar selective pressures due to the heterogeneous fermentation substrates available around the world; this increases the genetic and phenotypic diversity in Saccharomyces cerevisiae In this context, we expect prominent differences between isolates when exposed to a particular condition, such as wine or sake musts. To better comprehend the mechanisms underlying niche adaptation between two S. cerevisiae isolates obtained from wine and sake fermentation processes, we evaluated fermentative and fungicide resistance phenotypes and identify the molecular origin of such adaptive variation. Multiple regions were associated with fermentation rate under different nitrogen conditions and fungicide resistance, with a single QTL co-localizing in all traits. Analysis around this region identified RIM15 as the causative locus driving fungicide sensitivity, together with efficient nitrogen utilization and glycerol production in the wine strain. A null RIM15 variant confers a greater fermentation rate through the utilization of available glucose instead of its storage. However, this variant has a detrimental effect on fungicide resistance since complex sugars are not synthesized and transported into the membrane. Together, our results reveal the antagonist pleiotropic nature of a RIM15 null variant, positively affecting a series of fermentation related phenotypes, but apparently detrimental in the wild.

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.

Novel 4-methyl-2-oxopentanoate reductase involved in synthesis of the Japanese sake flavor, ethyl leucate.

Ethyl-2-hydroxy-4-methylpentanoate (ethyl leucate) contributes to a fruity flavor in Japanese sake. The mold Aspergillus oryzae synthesizes leucate from leucine and then the yeast Saccharomyces cerevisiae produces ethyl leucate from leucate during sake fermentation. Here, we investigated the enzyme involved in leucate synthesis by A. oryzae. The A. oryzae gene/cDNA encoding the enzyme involved in leucate synthesis was identified and expressed in E. coli and A. oryzae host cells. The purified recombinant enzyme belonged to a D-isomer-specific 2-hydroxyacid dehydrogenase family and it NADPH- or NADH-dependently reduced 4-methyl-2-oxopentanate (MOA), a possible intermediate in leucine synthesis, to D-leucate with a preference for NADPH. Thus, we designated this novel enzyme as MOA reductase A (MorA). Furthermore, an A. oryzae strain overexpressing morA produced 125-fold more leucate than the wild-type strain KBN8243. The strain overexpressing MorA produced 6.3-fold more ethyl leucate in the sake than the wild-type strain. These findings suggest that the strain overexpressing morA would help to ferment high-quality sake with an excellent flavor. This is the first study to identify the MOA reductase responsible for producing D-leucate in fungi.

Inhibitory Role of Greatwall-Like Protein Kinase Rim15p in Alcoholic Fermentation via Upregulating the UDP-Glucose Synthesis Pathway in Saccharomyces cerevisiae.

The high fermentation rate of Saccharomyces cerevisiae sake yeast strains is attributable to a loss-of-function mutation in the RIM15 gene, which encodes a Greatwall-family protein kinase that is conserved among eukaryotes. In the present study, we performed intracellular metabolic profiling analysis and revealed that deletion of the RIM15 gene in a laboratory strain impaired glucose-anabolic pathways through the synthesis of UDP-glucose (UDPG). Although Rim15p is required for the synthesis of trehalose and glycogen from UDPG upon entry of cells into the quiescent state, we found that Rim15p is also essential for the accumulation of cell wall β-glucans, which are also anabolic products of UDPG. Furthermore, the impairment of UDPG or 1,3-β-glucan synthesis contributed to an increase in the fermentation rate. Transcriptional induction of PGM2 (phosphoglucomutase) and UGP1 (UDPG pyrophosphorylase) was impaired in Rim15p-deficient cells in the early stage of fermentation. These findings demonstrate that the decreased anabolism of glucose into UDPG and 1,3-β-glucan triggered by a defect in the Rim15p-mediated upregulation of PGM2 and UGP1 redirects the glucose flux into glycolysis. Consistent with this, sake yeast strains with defective Rim15p exhibited impaired expression of PGM2 and UGP1 and decreased levels of β-glucans, trehalose, and glycogen during sake fermentation. We also identified a sake yeast-specific mutation in the glycogen synthesis-associated glycogenin gene GLG2, supporting the conclusion that the glucose-anabolic pathway is impaired in sake yeast. These findings demonstrate that downregulation of the UDPG synthesis pathway is a key mechanism accelerating alcoholic fermentation in industrially utilized S. cerevisiae sake strains.