2024 Research results

Background
The quantity and composition of rice proteins play a crucial role in determining taste quality of sake, Japanese rice wine. However, the spatial distribution of proteins within rice grains, especially in endosperm tissue, and the differences between rice varieties remain unclear.
Results
Here, we analyzed the crude protein contents and composition ratios of table (Nipponbare and Koshihikari) and genuine sake rice varieties (Yamadanishiki, Gohyakumangoku, Dewasansan, Dewanosato, and Yumenokaori) to elucidate their spatial distribution within the Japonica rice grain endosperm. Seven sake rice varieties were polished over five harvest years using a brewer’s rice-polishing machine. We obtained fractions at 90%–70% (the outermost endosperm fraction), 70%–50%, 50%–30%, and 30%–0% (the central region of the endosperm fraction). Yamadanishiki and Dewanosato exhibited considerably lower crude protein contents than the other cultivars. After applying SDS-PAGE, the protein composition, comprising glutelin/total protein (G/TP), prolamin/TP (P/TP), and G/P ratios of these fractions was determined. In white rice (at a 90% rice-polishing ratio), the average ratio of the major protein composition was G/TP 41%, P/TP 21%, and G/P ratios of 1.97. Gohyakumangoku and Yamadanishiki had higher G/TP ratio, while Dewanosato had a lower value. Despite having lower crude protein contents, Yamadanishiki and Dewanosato exhibited significantly varying G/TP ratios. The G/TP ratio markedly varied among rice varieties, particularly in the rice grains’ central region. The 50%–30% fraction had the highest P/TP ratio among all tested rice varieties, suggesting spatial differences in P/TP within rice grains. Koshihikari had the lowest P/TP ratio. In addition, the 50%–30% fraction had the lowest G/P ratio among all tested rice varieties, with Gohyakumangoku having the highest G/P ratio. Dewanosato had the lowest G/P value, and this value significantly differed from that of Yamadanishiki in the 30%–0% fraction.
Conclusions
We found substantial differences in protein composition within distinct spatial regions of rice grains, and larger differences among rice varieties were observed in the rice grain's central region.

In discussions on the compatibility of alcoholic beverages with food, certain types of alcohol consumed with fish dishes may impart a fishy odor. However, sake has been reported to effectively suppress this odor, enhanciing its compatibility with food. This study conducted sensory evaluations, confirming that when beer was paired with dried squid (Surume), a fish-derived metallic odor was noticeable. In contrast, when sake was combined with Surume, the metallic odor was effectively suppressed, as confirmed by sensory assessments. The contributing substance to this metallic odor was identified as the previously reported (Z)-1,5-octadien-3-one. A quantitative analysis of (Z)-1,5-octadien-3-one at levels below 1 ng/L (ppt) in alcoholic beverages was conducted. A highly sensitive and streamlined quantitative method was established, combining SBSE extraction, PFBOA derivatization, NCI ionization, and GC/MS/MS analysis. Measurement of (Z)-1,5-octadien-3-one production when mixing sake, beer, and Surume revealed significantly lower levels in sake, with an average generation rate 7.1 times lower than that in beer. This study demonstrates -- for the first time from a component perspective -- the inhibition of the ‛metallic odor', considered a factor contributing to the fishy odor in sake, providing insights into the ‛compatibility with food' at a component level.

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.

Glucose, a key component of traditional Japanese fermented foods, is derived from rice starch via saccharification by hydrolytic enzymes produced by Aspergillus oryzae. The δ¹³C value of glucose reflects that of its rice source. However, the influence of saccharification parameters (glucose concentration, degradation temperature, and reaction time) on glucose δ¹³C values is unclear. Here, we investigated the influence of saccharification on the δ¹³C value of glucose. Our experiments showed a significant difference in the δ¹³C value of glucose (-27.0 ± 0.1 ‰) obtained from saccharification compared to the ingredient rice (-27.1 ± 0.1 ‰) and remaining solid residue (-27.1 ± 0.1 ‰); however, it did not differ significantly from those of rice koji (-27.0 ± 0.1 ‰) and steamed rice (-27.1 ± 0.1 ‰), despite all values being within 0.1 ‰. Notably, glucose concentration, degradation temperature, and reaction time did not significantly affect glucose δ¹³C values. These findings demonstrate the remarkable preservation of glucose δ¹³C values. The δ¹³C values remain aligned with the original δ¹³C value of the rice, even with up to 60 % degradation during A. oryzae saccharification. This persistence of the δ¹³C value throughout the process offers a potential tool for authenticating the origin of rice-fermented beverages based on the δ¹³C value of their glucose component.

A large number of recombinant plasmids for the yeast Saccharomyces cerevisiae have been constructed and accumulated over the past four decades. It is desirable to apply the recombinant plasmid resources to Saccharomyces sensu stricto species group, which contains an increasing number of natural isolate and industrial strains. The application to the group encounters a difficulty. Natural isolates and industrial strains are exclusively prototrophic and polyploid, whereas direct application of most conventional plasmid resources imposes a prerequisite in host yeast strains of an auxotrophic mutation (i.e., leu2) that is rescued by a selection gene (e.g., LEU2) on the recombinant plasmids. To solve the difficulty, we aimed to generate leu2leu2 mutants from yeast strains belonging to the yeast Saccharomyces sensu stricto species group by DNA editing. First, we modified an all-in-one type CRISPR-Cas9 plasmid pML104 by adding an antibiotic-resistance gene and designing guide sequences to target the LEU2 gene and to enable wide application in this yeast group. Then, the resulting CRISPR-Cas9 plasmids were exploited to seven strains belonging to five species of the group, including natural isolate, industrial, and allopolyploid strains. Colonies having the designed mutations in the gene appeared successfully by introducing the plasmids and assisting oligonucleotides to the strains. Most of the plasmids and resultant leu2leu2-mutants produced in this study will be deposited in several repository organizations.

KEY POINTS:

• All-in-one type CRISPR-Cas9 plasmids targeting LEU2 gene were designed for broad application to Saccharomyces sensu stricto group species strains.
• Application of the plasmids generated leu2 mutants from strains including natural isolates, industrial, and allopolyploid strains.
• The easy conversion to leu2 mutants permits free access to recombinant plasmids having a LEU2 gene.

Why was the work done:
Elevated levels of isovaleraldehyde (3-methylbutanal) in sake gives rise to an unfavourable aroma of ‘mureka’ or ‘stuffy smell. The concentration of isovaleraldehyde is typically higher in unpasteurised than pasteurised sake. Controlling the concentration of isovaleraldehyde in unpasteurised sake remains a major challenge for quality control. As existing methods for the quantification of isovaleraldehyde in sake require specialised sample preparation, there is a need for a simple and precise method.

How was the work done:
High-performance liquid chromatography with fluorescence detection and post-column derivatisation (HPLC-PCD-FLD) for determining the isovaleraldehyde content in sake has been developed with optimisation of the separation of peaks and derivatisation of aldehyde compounds. The new method was compared with the established method of headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS).

What are the main findings:
The limit of quantification of the new method was 87 µg/L, and accordingly, the HPLC-PCD-FLD method could determine the concentration of isovaleraldehyde in sake below the reported threshold level. The precision of the HPLC-PCD-FLD method for the analysis of sake containing isovaleraldehyde (〉 threshold level) either matched or was superior to the HS-SPME-GC-MS method.

Why is the work important:
The new approach requires only particle removal for sample preparation, with a rapid analysis time (〉1 h per sample), and requires a smaller sample volume (≈ 100 µL) than the alternative method (10 mL). These improvements contribute to a simpler and more efficient workflow for routine analysis of isovaleraldehyde in the quality control of sake.