令和7年度 研究成果

OKUDA Masaki, JOYO Midori, TAKAHASHI Kei, and MUKAI Nobuhiko

Background and Objectives: The facile low‐cost evaluation of rice starch properties may help assess the suitability of rice for sake production and other applications. Herein, the starch gelatinization properties of raw rice and enzymatic digestibility of steamed rice were simply evaluated by observing the swelling of raw rice grains in urea solutions.
Findings: Polished rice grains were soaked in urea solutions of varying concentrations to determine the concentration at which the rice grains started swelling or completely absorbed the solution. The swelling‐onset concentration was strongly positively correlated with the DSC‐determined gelatinization temperature and rapid visco analysis–determined pasting temperature of raw rice and negatively correlated with the enzymatic digestibility of steamed rice (R² = 0.81–0.94) while being only slightly affected by the rice polishing ratio and moisture content.
Conclusions: The starch gelatinization properties of raw rice and enzymatic digestibility of steamed rice could be simply evaluated using the urea swelling test.
Significance and Novelty: The developed simple method does not require apparatus and flouring and provides results that are not influenced by rice moisture content and polishing ratio, thus holding promise for analyzing rice used in sake production and starch‐containing food materials.

OKUDA Masaki, JOYO Midori, TAKAHASHI Kei, and MUKAI Nobuhiko

Background and Objectives: The physicochemical properties of cereal starch affect food quality and processing properties, for example, the properties of rice starch influence the suitability of rice for sake (traditional Japanese alcoholic beverage) production. This study reports a convenient method for evaluating the starch gelatinization and retrogradation properties of rice grains and enzymatic digestibility of steamed rice to determine the suitability of japonica rice for sake brewing.
Findings: The swelling properties of polished rice grains were observed during the heating of nonfloured (whole‐grain) samples. The swelling‐onset temperature and swelling ratio were significantly correlated with the amylopectin side‐chain structure of rice starch, starch gelatinization/retrogradation properties, and enzymatic digestibility of steamed rice under sake production–like conditions. Swelling‐onset temperatures at a swelling ratio of 1.2 were measured for 31 samples with a polishing ratio of 70%, and the strength of the corresponding correlations (R² = 0.79–0.93) were comparable with those observed for gelatinization and pasting temperatures (determined by DSC and rapid visco analysis, respectively).
Conclusions: The starch properties and enzymatic digestibility of multiple steamed rice samples could be rapidly evaluated without flouring.
Significance and Novelty: The method facilitates the evaluation of rice used for sake brewing and analysis of starch properties in a wide range of food industries.

KANAI Muneyoshi, SHIBATA Tomoko, ZHOU Yan, HAYASHI Risa, FUKUBA Ikuko, KOCHI Takayuki, TERAMOTO Satoko, SHIMOI Hitoshi, TAKAHASHI Hidekazu, and AKAO Takeshi

Saccharomyces cerevisiae, a unicellular eukaryotic microorganism, includes various strains used in alcoholic beverage production, like sake, shochu/awamori, and wine yeasts. Despite being the same “Saccharomyces cerevisiae”, each strain has unique genes and mutations that make them suitable for specific production processes. We focused on sake yeast, Saccharomyces cerevisiae, suitable for sake making. To identify genes and mutations contributing to sake yeast’s characteristics more efficiently, we improved the quantitative trait loci (QTL) analysis system. This genetic statistical method used spore-separating haploid strains (F1 segregant haploids) from crossing sake yeast and laboratory yeast haploid strains. We increased the number of F1 segregant haploids for QTL analysis from 100 to 400 and set DNA markers uniformly across the genome (approximately 12 Mbp) at 5,267 locations using single nucleotide polymorphisms (SNPs) spaced about 3 kb apart. Additionally, a small-scale sake making test using 400 F1 segregant haploids and QTL analysis of ethanol concentration in sake sample identified the PBS2 gene and its causative mutation (amino acid substitution at position 545). The PBS2 gene was also implicated in producing organic acids (fumaric, succinic, and malic acids) and inorganic acids (phosphoric acid) for sake. These findings validated the improved QTL analysis system as effective genes screening method.

TAKAHASHI Masayuki, MORIKAWA Kana, and AKAO Takeshi

Lactobacillus acetotolerans and Fructilactobacillus fructivorans are responsible for the spoilage of Japanese sake, and sake brewers want to control these bacteria to prevent product-quality deterioration. Due to these bacteria's slow growth, a long time must pass before a product's spoilage can be identified by a culture-dependent method; a method enabling the rapid detection of spoilage is thus desired for maintaining sake's quality and reducing processing times. Herein, we developed a novel, simple method for determining the ori copy and ter copy numbers of both bacterial species by a single multiplex digital polymerase chain reaction (PCR). Our investigation about the ori and ter copy numbers of L. acetotolerans RIB9124 strain revealed that (i) the ter-region copy number was highly correlated with the colony-forming units, and (ii) the ori:ter ratio during log phase (mean: 1.36) was higher than that during non-log phase (mean: 1.16). The relationship between the ori:ter ratio and the bacterial doubling time was parabola-shaped, not linear. We constructed a model for estimating the two species' doubling times based on the ori:ter ratio and then created a generalized linear model with a probit link function using the ori:ter ratio, the doubling time predicted by the ori:ter ratio, and the ter copy number as parameters. The model was able to predict whether sake inoculated with either species would spoil within 4 months (spoilage-prediction error rate validated with 45 samples: 15.6 %). In light of these results, we suggest some criteria for judging the risk of Japanese sake spoiling within 4 months.

AKAMATSU Fumikazu, OSAFUNE Yukio, FUJITA Akiko, BOERZHIJIN Surina, TAKAHASHI Masayuki, and Atsuko ISOGAI

Oxygen stable isotope analysis is a well-established technique for determining the geographical origin and authenticity of beverages. However, volatile organic compounds (VOCs) present in some beverages can hinder accurate analysis. This study presents a straightforward method to overcome this challenge. Granular activated carbon co-incubated with the beverage sample during CO₂-H₂O equilibration effectively removes VOCs across a range of beverages tested. This includes propan-1-ol, 2-methylpropan-1-ol, ethyl acetate, 3-methylbutan-1-ol, 3-methylbutyl acetate, and ethyl hexanoate, achieving an average reduction of 77%. Consequently, this method minimizes background noise interferences, preserving δ¹⁸O values of water in a wide range of beverages, such as sake, sparkling sake, wine, beer, cider, awamori, brandy, shochu, whisky, and fruit juices. Notably, grain-derived alcoholic beverages like sake and beer exhibit more negative δ¹⁸O values compared to fruit-derived alcoholic beverages such as wine and cider, reflecting the distinct source waters and production processes employed. This universally applicable approach minimizes VOC interferences during oxygen stable isotope analysis of aroma-rich beverages. Its inherent simplicity and cost-effectiveness facilitate seamless integration into existing analytical workflows, leading to enhanced efficiency and productivity. This practical solution has potential for brand protection and authenticity verificationwithin the beverage industry.

GONG Yifeng, Norapat Klinkaewboonwong, HAYASHI Risa, ZHOU Yan, NISHIDA Ikuhisa, SAITO Rei, GOSHIMA Tetsuya, NISHI Tomoyuki, WATANABE Daisuke, HIRATA Dai, AKAO Takeshi, and OHYA Yoshikazu

Isoamyl acetate and ethyl caproate are the primary aroma compounds responsible for the fruity fragrance characteristic of Ginjo sake. Simultaneous high-level production of both compounds is crucial to achieving a balanced aroma and complex flavor. Isoamyl acetate is predominantly produced by hda1∆/hda1∆ and LEU4(G516S)/LEU4(G516S), while ethyl caproate is produced in high quantities by FAS2(G1250S)/FAS2(G1250S). In this study, to maximize the production of both aroma compounds, genome editing was employed to generate sake yeast strains combining these mutations. After small-scale fermentation tests were conducted to evaluate the production of aroma compounds, we found that the isoamyl acetate–enhancing effect ofhda1∆ /hda1∆ was almost completely masked by FAS2(G1250S)/FAS2(G1250S). In contrast, the effects of LEU4(G516S)/LEU4(G516S) were not entirely masked by FAS2(G1250S)/FAS2(G1250S), resulting in 2.4- and 5.4-fold greater production of isoamyl acetate and ethyl caproate, respectively. This study highlights the utility of genome editing in the combinatorial breeding of sake yeast.