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.

Visualization of dipeptidyl peptidase B enzymatic reaction in rice koji using mass spectrometry imaging.

Enzyme histochemistry via mass spectrometry imaging (MSI) has garnered attention as a straightforward approach for visualizing enzymatic reactions. While several studies in the medical and physiological fields have shown its promising application potential, its applicability to agricultural or food studies has not yet been demonstrated. Rice koji, known as an enzyme source for various fermented products, is a suitable model for demonstrating the applicability of this method to food-related materials. In this study, the enzymatic reaction of dipeptidyl peptidase B (DppB) in rice koji was visualized using MSI for the first time. The method was optimized and applied to investigate the effects of rice variety, polishing ratio, and cultivation time on the location of the DppB reaction. The DppB enzymatic reaction was found to occur in different locations in each of the two rice varieties, Yamadanishiki and Hakutsurunishiki. The polishing ratio also affected the distribution of the DppB enzymatic reactions. Furthermore, a time-course investigation of rice koji cultivation revealed that while the location of the reaction was largely associated with mycelial penetration, the structure and features of the rice grain may also affect the location of the enzymatic reaction. In summary, these results demonstrate the applicability of enzyme histochemistry by MSI to food-related materials.

Metabolic Visualization Reveals the Distinct Distribution of Sugars and Amino Acids in Rice Koji.

The compounds inside rice koji have been thoroughly investigated as an essential material in making many food-related products, including sake. However, these studies focused only on quantitative aspects, leaving features that can still be uncovered if seen from a new perspective. Visualization of the metabolites inside rice koji may as well be the new angle needed to retrieve more information regarding rice koji making. Here we utilized mass spectrometry imaging (MSI) to visualize the distribution of sugars, sugar alcohols, and amino acids inside rice koji. Imaging results revealed that several sugars alcohols and amino acids were shown to have characteristic distribution near the edges or surface of rice koji. Furthermore, the distribution appears to be correlated with the different structure of rice koji. This study is the first report of using MSI to visualize sugars, sugar alcohols, and amino acids in rice koji.

Mapping haze-komi on rice koji grains using ß-glucuronidase expressing Aspergillus oryzae and mass spectrometry imaging.

In sake brewing, the quality of rice koji is evaluated by experienced sake brewers based on visual inspection of the haze, which is defined by the extent of fungal hyphae spread on/into the rice grains. There is an increasing interest in understanding the factors that affect the quality of rice koji, which is dependent on its making process. Several studies have focused on the degree of mycelial penetration (haze-komi) and enzyme production during rice koji production. However, there are limited analytical methods available to monitor hyphal growth on a solid surface. Here we used a ß-glucuronidase (GUS)-expressing strain of Aspergillus oryzae to visualize and map the fungal growth on rice koji grains. Observation of indigo color revealed that A. oryzae hyphae penetrated the steamed rice grain in the early stage (24 h) of rice koji-making before spreading on the surface during the later stages. Additionally, hyphae penetrated along the endosperm cells and penetrated the cells to form the sou-haze. Furthermore, mass spectrometry imaging (MSI) of glucose demonstrated that the area of mycelial penetration is directly correlated with the spread of glucose during fermentation. This is the first report on utilizing new tools such as GUS-body-mapping of A. oryzae and MSI to monitor fungal growth during rice koji making.

Analyzing the relationship between the inorganic element profile of sake dilution water and dimethyl trisulfide formation using multi-element profiling.

Dimethyl trisulfide (DMTS) is the main component of hineka, an off-flavor generated in sake during storage. Genshu, or undiluted sake, is usually diluted with water during warimizu, the process of adjusting the alcohol content of sake. In this study, we evaluated how the inorganic element composition of sake dilution water affects the DMTS-producing potential of the sake (DMTS-pp, determined as the DMTS concentration in sake stored at 70°C for 1 week after dilution) using inductively coupled plasma-mass spectrometry (ICP-MS). Partial least squares (PLS) regression analysis was conducted with the ICP-MS data as the explanatory variable and DMTS-pp as the response variable, and the selection of inorganic elements for the construction of the PLS model was performed using variable importance in projection scores. The findings confirmed that some of the compounds containing the inorganic elements extracted from the PLS regression analysis contribute to DMTS-pp.

Changes in transcription and metabolism during the early stage of replicative cellular senescence in budding yeast.

Age-related damage accumulates and a variety of biological activities and functions deteriorate in senescent cells. However, little is known about when cellular aging behaviors begin and what cellular aging processes change. Previous research demonstrated age-related mRNA changes in budding yeast by the 18th to 20th generation, which is the average replicative lifespan of yeast (i.e. about half of the population is dead by this time point). Here, we performed transcriptional and metabolic profiling for yeast at early stages of senescence (4th, 7th, and 11th generation), that is, for populations in which most cells are still alive. Transcriptional profiles showed up- and down-regulation for ∼20% of the genes profiled after the first four generations, few further changes by the 7th generation, and an additional 12% of the genes were up- and down-regulated after 11 generations. Pathway analysis revealed that these 11th generation cells had accumulated transcripts coding for enzymes involved in sugar metabolism, the TCA cycle, and amino acid degradation and showed decreased levels of mRNAs coding for enzymes involved in amino acid biosynthetic pathways. These observations were consistent with the metabolomic profiles of aging cells: an accumulation of pyruvic acid and TCA cycle intermediates and depletion of most amino acids, especially branched-chain amino acids. Stationary phase-induced genes were highly expressed after 11 generations even though the growth medium contained adequate levels of nutrients, indicating deterioration of the nutrient sensing and/or signaling pathways by the 11th generation. These changes are presumably early indications of replicative senescence.

Novel strategy for non-targeted isotope-assisted metabolomics by means of metabolic turnover and multivariate analysis.

Isotope-labeling is a useful technique for understanding cellular metabolism. Recent advances in metabolomics have extended the capability of isotope-assisted studies to reveal global metabolism. For instance, isotope-assisted metabolomics technology has enabled the mapping of a global metabolic network, estimation of flux at branch points of metabolic pathways, and assignment of elemental formulas to unknown metabolites. Furthermore, some data processing tools have been developed to apply these techniques to a non-targeted approach, which plays an important role in revealing unknown or unexpected metabolism. However, data collection and integration strategies for non-targeted isotope-assisted metabolomics have not been established. Therefore, a systematic approach is proposed to elucidate metabolic dynamics without targeting pathways by means of time-resolved isotope tracking, i.e., "metabolic turnover analysis", as well as multivariate analysis. We applied this approach to study the metabolic dynamics in amino acid perturbation of Saccharomyces cerevisiae. In metabolic turnover analysis, 69 peaks including 35 unidentified peaks were investigated. Multivariate analysis of metabolic turnover successfully detected a pathway known to be inhibited by amino acid perturbation. In addition, our strategy enabled identification of unknown peaks putatively related to the perturbation.