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.

Preventive Roles of Rice-koji Extracts and Ergothioneine on Anxiety- and Pain-like Responses under Psychophysical Stress Conditions in Male Mice.

This study determined the effect of daily administration of Rice-koji on anxiety and nociception in mice subjected to repeated forced swim stress (FST). In a parallel experiment, it was determined whether ergothioneine (EGT) contained in Rice-koji displayed similar effects. Anxiety and nociception were assessed behaviorally using multiple procedures. c-Fos and FosB immunoreactivities were quantified to assess the effect of both treatments on neural responses in the paraventricular nucleus of the hypothalamus (PVN), nucleus raphe magnus (NRM), and lumbar spinal dorsal horn (DH). FST increased anxiety- and pain-like behaviors in the hindpaw. Rice-koji or EGT significantly prevented these behaviors after FST. In the absence of formalin, both treatments prevented decreased FosB expressions in the PVN after FST, while no effect was seen in the NRM and DH. In the presence of formalin, both treatments prevented changes in c-Fos and FosB expressions in all areas in FST mice. Further, in vitro experiments using SH-SY5Y cells were conducted. Rice-koji and EGT did not affect cell viability but changed the level of brain-derived neurotrophic factor. In conclusion, Rice-koji could reduce anxiety and pain associated with psychophysical stress, possibly mediated by the modulatory effects of EGT on neural functions in the brain.

Study of Inducing Factors on Resveratrol and Antioxidant Content in Germinated Peanuts.

When peanuts germinate, bioactive compounds such as resveratrol (RES), γ-aminobutyric acid (GABA), isoflavones, and polyphenol compounds are generated. Peanut kernels were germinated in the dark for two days, and stimuli including soaking liquid, rice koji, high-pressure processing (HPP), and ultrasonic treatment were tested for their ability to activate the defense mechanisms of peanut kernels, thus increasing their bioactive compound content. The results of this study indicate that no RES was detected in ungerminated peanuts, and only 5.58 µg/g of GABA was present, while unstimulated germinated peanuts contained 4.03 µg/g of RES and 258.83 µg/g of GABA. The RES content of the germinated peanuts increased to 13.64 µg/g after soaking in 0.2% phenylalanine solution, whereas a higher GABA content of 651.51 µg/g was observed after the peanuts were soaked in 0.2% glutamate. Soaking peanuts in 5% rice koji produced the highest RES and GABA contents (28.83 µg/g and 506.34 µg/g, respectively). Meanwhile, the RES and GABA contents of HPP-treated germinated peanuts (i.e., treated with HPP at 100 MPa for 10 min) increased to 7.66 µg/g and 497.09 µg/g, respectively, whereas those of ultrasonic-treated germinated peanuts (for 20 min) increased to 13.02 µg/g and 318.71 µg/g, respectively. After soaking peanuts in 0.5% rice koji, followed by HPP treatment at 100 MPa for 10 min, the RES and GABA contents of the germinated peanuts increased to 37.78 µg/g and 1196.98 µg/g, while the RES and GABA contents of the germinated peanuts treated with rice koji followed by ultrasonic treatment for 20 min increased to 46.53 µg/g and 974.52 µg/g, respectively. The flavonoid and polyphenol contents of the germinated peanuts also increased after exposure to various external stimuli, improving their DPPH free radical-scavenging ability and showing the good potential of germinated peanuts as functional products.

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.

Visualization of polypeptides including fragmented α-amylase in rice koji grains using mass spectrometry imaging.

The quality of rice koji greatly affects the quality of sake. To accurately evaluate the quality of rice koji, various approaches for the evaluation of rice koji are required. In this study, we directly and simultaneously visualized the distribution of polypeptides in rice koji using mass spectrometry imaging. We demonstrated four koji-specific polypeptides at m/z 4660, 6140, 8170, and 11,840 and one rice-derived polypeptide at m/z 5330. To identify the koji-specific polypeptides, extracts from rice koji were separated using tricine SDS-PAGE, and the band appeared to coincide with the polypeptide at m/z 11,840 was identified to be the N-terminal fragment of α-amylase. The polypeptide seemed to have no hydrolytic activity based on the primary structure of α-amylase. The polypeptide at m/z 11,840 seemed to coincide with the fragmented α-amylase was detected at the later stage of koji making (after 42 h). At the same period during koji making, the increasing rate of α-amylase activity decreased compared to that of glucoamylase activity, suggesting that α-amylase fragmentation possibly leads to the deceleration of the increase in α-amylase activity at the later stage of koji making. This is the first study to directly and simultaneously demonstrate the distribution of polypeptides in rice koji using mass spectrometry imaging and imply the relationship between α-amylase fragmentation and activity in rice koji.

Metabolite Profiling and Anti-Aging Activity of Rice Koji Fermented with Aspergillus oryzae and Aspergillus cristatus: A Comparative Study.

Rice koji, used as a starter for maximizing fermentation benefits, produces versatile end products depending on the inoculum microbes used. Here, we performed metabolite profiling to compare rice koji fermented with two important filamentous fungus, Aspergillus oryzae and A. cristatus, during 8 days. The multivariate analyses showed distinct patterns of primary and secondary metabolites in the two kojis. The rice koji fermented with A. oryzae (RAO) showed increased α-glucosidase activity and higher contents of sugar derivatives than the one fermented with A. cristatus (RAC). RAC showed enhanced ß-glucosidase activity and increased contents of flavonoids and lysophospholipids, compared to RAO. Overall, at the final fermentation stage (8 days), the antioxidant activities and anti-aging effects were higher in RAC than in RAO, corresponding to the increased metabolites such as flavonoids and auroglaucin derivatives in RAC. This comparative metabolomic approach can be applied in production optimization and quality control analyses of koji products.

Red rice koji extract alleviates hyperglycemia by increasing glucose uptake and glucose transporter type 4 levels in skeletal muscle in two diabetic mouse models.

BACKGROUND: Red rice koji (RRK), prepared by growing Monascus species on steamed rice, has been reported to lower blood glucose levels in diabetic animal models. However, the action mechanism is not yet completely understood. OBJECTIVE: The objective of this study was to examine the mechanism underlying the hypoglycemic action of RRK extract in two diabetic animal models: the insulin-deficiency mice, where the insulin deficiency was induced by streptozotocin (STZ), and insulin-resistance mice, where the insulin resistance was induced by a high-fat diet (HFD). DESIGN: Low (12.5 mg/kg body weight [BW]) and high (50.0 mg/kg BW) doses of RRK extract were orally administered to the mice for 10 successive days (0.25 mL/day/mouse). The protein expression levels of glucose transporter type 4 (GLUT4) in the skeletal muscle and glucose transporter type 2 (GLUT2) in the liver were measured. Blood glucose (BG) levels of STZ-treated mice in insulin tolerance test (ITT) and BG and insulin levels of HFD-fed mice in intraperitoneal glucose tolerance test (IPGTT) were investigated. RESULTS: In the STZ-treated mice, oral administration of RRK extract lowered BG levels and food intake but increased plasma 1,5-anhydroglucitol level. Moreover, the RRK extract lowered the BG levels of STZ-treated mice as measured by ITT. In the HFD-fed mice, we confirmed that the orally administered RRK extract lowered the BG and the homeostasis model assessment index for insulin resistance. Furthermore, the RRK extract lowered the BG and insulin levels of HFD-fed mice in IPGTT. Regarding the protein levels of GLUT, the orally administered RRK extract increased the GLUT4 level in the skeletal muscle; however, the RRK extract did not alter the GLUT2 level in the liver of either the STZ-treated or the HFD-fed mice. DISCUSSION: Our study demonstrates that RRK extract can improve impaired glucose tolerance in mouse models of diabetes by enhancing GLUT4 expression in skeletal muscle. CONCLUSION: These results suggest that RRK extract could potentially be a functional food for the treatment of diabetes mellitus.

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.

Amazake made from sake cake and rice koji suppresses sebum content in differentiated hamster sebocytes and improves skin properties in humans.

Amazake is a traditional Japanese health drink. Here, we examined the effects of amazake on skin in cells and humans. Treatment with sake cake or rice koji suppressed intracellular lipid accumulation in differentiated hamster sebocytes, likely through the reduced expression of peroxisome proliferator-activated receptor-gamma (PPARγ) mRNA. In double-blind, placebo-controlled trial, seventeen Japanese women ingested either amazake or placebo for 4 weeks. Ingestion of the amazake decreased the sebum content compared to the placebo. The questionnaires showed improvements in "face color," "dark circles under the eyes," "glossy hair," and "waking up well", only in the amazake. In accordance with the questionnaires, additional analysis revealed the change in the L* values under the eyes was statistically increased in the amazake compared to the placebo. These results indicate that amazake may decrease sebum content in cells and humans and increase the L* values under the eyes, with some additional beneficial effects in humans.

Gochujang prepared using rice and wheat koji partially alleviates high-fat diet-induced obesity in rats.

The aim of this study was to compare whether gochujang products prepared using giant embryo rice koji (rice gochujang, RG) and wheat koji (wheat gochujang, WG) have anti-obesity effects on rats fed a high-fat diet (HFD), who served as a model for obesity. The nutritional composition of RG and WG including proximate constituents, amino acid and fatty acid compositions were investigated. Consequently, the secondary fermented metabolites were analyzed in RG and WG by ultrahigh-performance liquid chromatography and mass spectrometry. Rats were fed a HFD containing 10% RG powder (HFD-RG) or 10% WG powder (HFD-WG) for 8 weeks. Body weight gain, weights of liver, epididymal, retroperitoneal, perirenal, and total white fat pads, and levels of serum triglyceride (TG), total cholesterol, low-density lipoprotein cholesterol, and leptin were lower in all gochujang groups than in the HFD group. Furthermore, RG and WG treatment decreased the hepatic TG content and lipid accumulation and significantly reduced the size of epididymal adipocytes. These effects are probably mediated through inhibition of hepatic fatty acid synthase, acetyl CoA carboxylase, malic enzyme, and adipose tissue lipoprotein lipase activities. The anti-obesity effect was slightly greater in the HFD-RG group than in the HFD-WG group. This effect may be attributed to secondary metabolites, such as capsaicin, genistein, daidzein, soyasaponin, and lysophosphatidylcholines, contained in gochujang prepared using giant embryo rice or wheat 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.

Superinfection of hepatitis A virus in hepatocytes infected with hepatitis B virus.

Hepatitis A virus (HAV) infection is a major cause of acute hepatitis including acute liver failure. Hepatitis B infection (HBV) occurs worldwide, with the highest rates in Asian and African countries, and there are several reports that HAV infection may have a more severe clinical course in patients with chronic HBV infection. We previously demonstrated that Japanese miso extracts have inhibitory effects on HAV replication. In the present study, we examined the replication of HAV and HBV in a hepatocyte superinfection model and the inhibitory effects of Japanese miso extracts on both viruses. According to the results, HAV infection inhibited HBV replication in superinfected hepatocytes, and Japanese rice-koji miso extracts had inhibitory effects on HAV replication. Our findings provide useful information for clinicians in managing HAV infection in patients with chronic HBV infection.

Inhibitory effect of Japanese rice-koji miso extracts on hepatitis A virus replication in association with the elevation of glucose-regulated protein 78 expression.

Hepatitis A virus (HAV) infection is one of the major causes of acute hepatitis and acute liver failure in developing and developed countries. Although effective vaccines for HAV infection are available, outbreaks of HAV infection still cause deaths, even in developed countries. One approach to control HAV infection is prevention through diet, which can inhibit HAV propagation and replication. Glucose-regulated protein 78 (GRP78) is a member of the heat shock protein 70 family of molecular chaperone required for endoplasmic reticulum stress and stress-induced autophagy. We previously showed that the elevation of GRP78 expression inhibits HAV replication. It has been reported that Japanese miso extracts, which was made from rice-koji, enhance GRP78 expression. In the present study, we used human hepatoma Huh7 cells and human hepatocyte PXB cells to examine the efficacy of Japanese miso extracts as antiviral agents against HAV. Japanese miso extracts enhanced GRP78 expression and inhibited HAV replication in human hepatocytes. Together, these results demonstrate that Japanese miso extracts may partly modulate GRP78 expression and additively or synergistically work as antivirals against HAV infection. Japanese miso extracts can be used as effective dietary supplements for severe hepatitis A.

Identification of Rice Koji Extract Components that Increase ß-Glucocerebrosidase Levels in Human Epidermal Keratinocytes.

Rice miso contains many ingredients derived from rice koji and has been a valuable source of nutrition since ancient times. We found that the consumption of rice miso led to improvements in the moisture content of cheek stratum corneum, skin viscoelasticity, and skin texture. Further, rice miso extract was found to increase the mRNA expression and activity of β-glucocerebrosidase (β-GCase), an enzyme involved in ceramide synthesis in the stratum corneum, in cultures. In this study, we identified the lipid-derived components of rice koji that increase the β-GCase activity in cultured human epidermal keratinocytes. The methanol fraction of rice koji extract induced an increase in the mRNA expression and activity of β-GCase in keratinocytes. The active fraction of rice koji was found to contain phosphatidic acid (PA) and lysophosphatidic acid (LPA). The total PA concentration in rice koji was 973.9 ng/mg dry weight, which was 17.5 times higher than that in steamed rice. Among the molecular species, PA_18:2/18:2 was the most frequently found. The total LPA concentration in rice koji was 29.6 ng/mg dry weight, and 2-LPA_18:2 was the most frequently found LPA. Since PA and LPA increase the mRNA expression and activity of β-GCase in keratinocytes, they are thought to be the active ingredients in rice koji that increase the β-GCase levels in human epidermal keratinocytes.

Metabolomics Reveal Optimal Grain Preprocessing (Milling) toward Rice Koji Fermentation.

A time-correlated mass spectrometry (MS)-based metabolic profiling was performed for rice koji made using the substrates with varying degrees of milling (DOM). Overall, 67 primary and secondary metabolites were observed as significantly discriminant among different samples. Notably, a higher abundance of carbohydrate (sugars, sugar alcohols, organic acids, and phenolic acids) and lipid (fatty acids and lysophospholipids) derived metabolites with enhanced hydrolytic enzyme activities were observed for koji made with DOM of 5-7 substrates at 36 h. The antioxidant secondary metabolites (flavonoids and phenolic acid) were relatively higher in koji with DOM of 0 substrates, followed by DOM of 5 > DOM of 7 > DOM of 9 and 11 at 96 h. Hence, we conjecture that the rice substrate preprocessing between DOM of 5 and 7 was potentially optimal toward koji fermentation, with the end product being rich in distinctive organoleptic, nutritional, and functional metabolites. The study rationalizes the substrate preprocessing steps vital for commercial koji making.

Time-resolved comparative metabolomes for Koji fermentation with brown-, white-, and giant embryo-rice.

A time-resolved non-targeted metabolomic study towards evaluating the effects of three rice substrate types viz., white rice (WR), giant embryo rice (GER), and brown rice (BR), on Koji fermentation was performed. Notwithstanding the relatively higher proportions of metabolites in unfermented BR (BR 0>WR 0, GER 0), the relative levels of metabolic repertoire in BR Koji were marginally enhanced during 36h fermentation except those for phenolic acids, fatty acids, and vitamins. The WR Koji showed higher levels of lysophospholipids (LysoPC's) and protease-released amino acids (WR 36>GER 36>BR 36). The higher ß-glucosidase activity in GER Koji effected the increased levels of sugars and flavonoid aglycons complementing its higher antioxidant activity (GER Koji>BR Koji∼WR Koji). The present study holistically underpins the dynamic metabolomes and enzymatic states during rice Koji fermentation with varying substrate types. The present study finds applications in optimization of commercial Koji production.

Metabolomic Profiles of Aspergillus oryzae and Bacillus amyloliquefaciens During Rice Koji Fermentation.

Rice koji, used early in the manufacturing process for many fermented foods, produces diverse metabolites and enzymes during fermentation. Using gas chromatography time-of-flight mass spectrometry (GC-TOF-MS), ultrahigh-performance liquid chromatography linear trap quadrupole ion trap tandem mass spectrometry (UHPLC-LTQ-IT-MS/MS), and multivariate analysis we generated the metabolite profiles of rice koji produced by fermentation with Aspergillus oryzae (RK_AO) or Bacillus amyloliquefaciens (RK_BA) for different durations. Two principal components of the metabolomic data distinguished the rice koji samples according to their fermenter species and fermentation time. Several enzymes secreted by the fermenter species, including α-amylase, protease, and ß-glucosidase, were assayed to identify differences in expression levels. This approach revealed that carbohydrate metabolism, serine-derived amino acids, and fatty acids were associated with rice koji fermentation by A. oryzae, whereas aromatic and branched chain amino acids, flavonoids, and lysophospholipids were more typical in rice koji fermentation by B. amyloliquefaciens. Antioxidant activity was significantly higher for RK_BA than for RK_AO, as were the abundances of flavonoids, including tricin, tricin glycosides, apigenin glycosides, and chrysoeriol glycosides. In summary, we have used MS-based metabolomics and enzyme activity assays to evaluate the effects of using different microbial species and fermentation times on the nutritional profile of rice koji.

Anti-obesity effects of gochujang products prepared using rice koji and soybean meju in rats.

The Korean traditional hot sauce gochujang has been reported to have biological activities. Different kinds of gochujang products were prepared based on combinations of a fungal rice koji with two kinds of bacterial soybean mejus. Diets that included gochujang products were fed to rats and anti-obesity effects were investigated. Gochujang products reduced body weight gains, epididymal fat weights, and triglyceride levels in the serum and the liver. Effects were exerted by the diet that included the non-fermented gochujang mixture, increased using a fungal rice koji, and further enhanced using a bacterial soybean meju. Dietary effects were apparently induced via inhibition of the lipogenic enzymes fatty acid synthase, malic enzyme, and lipoprotein lipase by gochujang products in epididymal adipose tissues, and inhibition of glucose-6-phosphate dehydrogenase in the liver. High levels of capsaicin and genistein in gochujang products are considered to contribute to anti-obesity effects.

Antioxidants in heat-processed koji and the production mechanisms.

We previously developed antioxidative heat-processed (HP)-koji via two-step heating (55 °C/2days → 75 °C/3 days) of white-koji. In this study, we isolated antioxidants in HP-koji and investigated their formation mechanisms. The antioxidants were identified to be 5-hydroxymethyl furfural (HMF) and 5-(α-D-glucopyranosyloxymethyl)-2-furfural (GMF) based on nuclear magnetic resonance spectral analysis. HMF and GMF were not present in intact koji, but were formed by heating at 75 °C. As production of these antioxidants was more effective by two-step heating than by constant heating at 55 °C or 75 °C, we presumed that the antioxidant precursors are derived enzymatically at 55°C and that the antioxidants are formed subsequently by thermal reaction at 75 °C. The heating assay of saccharide solutions revealed glucose and isomaltose as HMF and GMF precursors, respectively, and thus the novel finding of GMF formation from isomaltose. Finally, HMF and GMF were effectively formed by two-step heating from glucose and isomaltose present in koji.

Effects of Aspergillus species inoculation and their enzymatic activities on the formation of volatile components in fermented soybean paste (doenjang).

The volatile components of rice-koji doenjang produced in association with various Aspergillus species (A. oryzae, A. sojae, and A. kawachii) during 8 weeks of fermentation were compared using gas chromatography-mass spectrometry analysis and multivariate statistical analysis. In addition, the activities of diverse enzymes (α-amylase, protease, lipase, and esterase) were determined to investigate their effects on the formation of volatile compounds. Regardless of the fungi types, carbonyls including 4-methylheptan-2-one, heptan-2-one, (E)-hept-2-enal, and hexanal were found to contribute mainly to early phase fermentation, whereas the contents of ethyl esters of long-chained fatty acids were considerably enhanced in the latter stage of fermentation. With the exception of α-amylase, the activities of the studied enzymes generally increased as fermentation proceeded, and there were significant differences in enzymes activities between the species of fungi. The lipase activity was strongly correlated with the formation of long-chain fatty acid esters, which could be related to the distinctive organoleptic properties of rice-koji doenjang.