Kazunori Takamine

Characterization of glucoamylase and α-amylase from Monascus anka: Enhanced production of α-amylase in red koji

To enhance glucoamylase and α-amylase production from Monascus anka, we investigated the influence of different culture conditions on enzyme production and purified and characterized these enzymes. The effect of different raw materials was investigated by using solid-state plates of raw materials such as barley and non-waxy or waxy rice. The barley plate was the most suitable for mycelial growth, but glucoamylase and α-amylase production per growth area did not differ according to the raw material. Investigation of the effect of temperature showed that incubation at 37 °C promoted maximal cell growth, while incubation at 25 °C and at 40 °C resulted in enhanced α-amylase and glucoamylase production, respectively. Characterization of the purified enzymes revealed that α-amylase was unstable at acidic pH and less resistant to heat (stable at < 40 °C) than glucoamylase. When these culture conditions were applied to enzyme production in red koji, reducing the temperature from 35 °C to 25 °C for 48 h in the late stages of growth resulted in higher glucoamylase and α-amylase production (1.4 and 18 times, respectively) with a concomitant increase in protein stability.

Rice koji reduced body weight gain, fat accumulation, and blood glucose level in high-fat diet-induced obese mice

Rice koji is considered a readily accessible functional food that may have health-promoting effects. We investigated whether white, yellow, and red koji have the anti-obesity effect in C57BL/6J mice fed a high-fat diet (HFD), which is a model for obesity. Mice were fed HFD containing 10% (w/w) of rice koji powder or steamed rice for 4 weeks. Weight gain, epididymal white adipose tissue, and total adipose tissue weight were significantly lower in all rice koji groups than in the HFD-rice group after 4 weeks. Feed efficiency was significantly reduced in the yellow koji group. Blood glucose levels were significantly lower in the white and red koji groups with HOMA-R and leptin levels being reduced in the white koji group. White and red koji increased glucose uptake and GLUT4 protein expression in L6 myotube cells. These results showed that all rice koji have the anti-obesity or anti-diabetes effects although the mechanisms may differ depending on the type of rice koji consumed.

Development of a heat-processing method for koji to enhance its antioxidant activity

We developed a heat-processing method to enhance the antioxidant activity of koji. The superoxide anion scavenging activity (SOSA) and oxygen radical absorbance capacity (ORAC) of heat-processed koji (HP-koji) at 55 °C for 7 days were 4.9 times and 4.2 times, respectively, those of unheated koji. These results showed that heat processing effectively enhances the antioxidant activity of koji. Analysis of the antioxidant activities of koji subjected to a range of temperatures (45-75 °C) revealed that the SOSA is enhanced by heating at higher temperatures, which might be catalyzed by Maillard reaction, whereas the ORAC was enhanced by heating at lower temperatures, which might be catalyzed by an enzymatic reaction. Assuming these enhancements in antioxidant activities are contributed by both Maillard and enzyme reactions, we hypothesized that the antioxidant activity of HP-koji could be more effectively amplified by heating at a higher temperature after the progression of the enzymatic reaction at a moderate temperature. Therefore, we evaluated the effect of heating of koji in a stepwise manner, first at 55 °C for 2 days and then at 75 °C for 5days. The antioxidant activities of stepwise-heated HP-koji were higher than those of koji heated at either 55 °C or 75 °C. The SOSA and ORAC of stepwise-heated HP-koji were 94 times and 6 times, respectively, those of unheated koji. This result suggests that enzymatic reaction followed by Maillard reaction can effectively enhance the antioxidant activity of HP-koji. Thus, we developed a novel heat-processing method to enhance the antioxidant activity of koji.

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.

Key volatile compounds in red koji-shochu, a Monascus-fermented product, and their formation steps during fermentation

Red koji, which refers the solid culture grown koji mold on the steamed rice, is one of the ingredients of Asian fermented foods including the Japanese spirit shochu. This study was aimed at elucidating the characteristic flavor and key volatile compounds of red koji-shochu as well as the mechanism of their formation. Sensory evaluation showed that red koji-shochu has the distinctive flavors cheese, sour, milky, and oily. Fifteen key volatile compounds of red koji-shochu were identified by gas chromatography-mass spectrometry and high-performance liquid chromatography, and by comparison between red koji-shochu and white koji-shochu, as another typical shochu. The mash analysis revealed that ketone compounds and short-chain acids derive from red koji. Furthermore, although other key compounds were produced by yeast, it is highly likely that their concentrations were affected directly or indirectly by the high activities of protease and lipase in red koji.

Acute effects of traditional Japanese alcohol beverages on blood glucose and polysomnography levels in healthy subjects

Background. Alcohol consumption is a lifestyle factor associated with type 2 diabetes. This relationship is reportedly different depending on the type of alcohol beverage. The purpose of this study was to examine the acute effects of traditional Japanese alcohol beverages on biochemical parameters, physical and emotional state, and sleep patterns. Methods. Six healthy subjects (three men and three women; age, 28.8 ± 9.5 years; body mass index, 21.4 ± 1.6 kg/m2) consumed three different types of alcohol beverages (beer, shochu, and sake, each with 40 g ethanol) or mineral water with dinner on different days in the hospital. Blood samples were collected before and 1, 2, and 12 h after drinking each beverage, and assessments of physical and emotional state were administered at the same time. In addition, sleep patterns and brain waves were examined using polysomnography. Results. Blood glucose levels at 1 h and the 12-h area under the curve (AUC) value after drinking shochu were significantly lower than that with water and beer. The 12-h blood insulin AUC value after drinking shochu was significantly lower than that with beer. Blood glucose × insulin level at 1 h and the 2-h blood glucose × insulin AUC value with shochu were significantly lower than that with beer. The insulinogenic indexes at 2 h with beer and sake, but not shochu, were significantly higher than that with water. The visual analogue scale scores of physical and emotional state showed that the tipsiness levels with beer, shochu, and sake at 1 h were significantly higher than that with water. These tipsiness levels were maintained at 2 h. The polysomnography showed that the rapid eye movement (REM) sleep latency with shochu and sake were shorter than that with water and beer. Conclusions. Acute consumption of alcohol beverages with a meal resulted in different responses in postprandial glucose and insulin levels as well as REM sleep latency. Alcohol beverage type should be taken into consideration for people with impaired glucose tolerance.

Ethnic Fermented Foods and Alcoholic Beverages of Japan

Japanese traditional fermented foods are centerpieces of taste of Washoku cuisine which was added to UNESCO’s Intangible Cultural Heritage list in 2013. Miso is fermented soybean paste. It is used for every day’s miso soup and as a seasoning in many kinds of cooking. Shoyu – soy sauce – is a liquid-type seasoning made of soybeans, wheat, koji, and salt water. Natto is soybeans fermented with Bacillus, with sticky but tasty γ-polyglutamic acid. Su is a traditional vinegar of cereal or fruit. Rice vinegar which is usually made directly from rice and rice koji is the most popular in Japan. Tsukemono is pickled vegetables, of which the traditional one is fermented with lactic acid bacteria. Tsukemono is usually served daily as a side dish with boiled rice. A variety of fermented seafoods, salted type (gyoshyo, shiokara, kusaya), pickled type (nare-zushi, nuka-zuke), and molded type (fushi) are also developed. The last one “fushi” is an indispensable ingredient in Japanese cuisine. Two kinds of alcoholic beverages, famous sake (rice wine) and shochu (spirits), are also important gifts of fermentation. The outline, including information of microbes, preparation methods and functionality and health benefits, of these traditional fermented foods and beverages popular in Japan are introduced.

Mitochondrial citrate transporters CtpA and YhmA are involved in lysine biosynthesis in the white koji fungus, Aspergillus luchuensis mut. kawachii

Aspergillus luchuensis mut. kawachii produces a large amount of citric acid during the process of fermenting shochu, a traditional Japanese distilled spirit. In this study, we characterized A. kawachii CtpA and YhmA, which are homologous to the yeast Saccharomyces cerevisiae mitochondrial citrate transporters Ctp1 and Yhm2, respectively. CtpA and YhmA were purified from A. kawachii and reconstituted into liposomes. The proteoliposomes exhibited only counter-exchange transport activity; CtpA transported citrate using counter substrates especially for cis-aconitate and malate, whereas YhmA transported citrate using a wider variety of counter substrates, including citrate, 2-oxoglutarate, malate, cis-aconitate, and succinate. Disruption of ctpA and yhmA caused deficient hyphal growth and conidia formation with reduced mycelial weight–normalized citrate production. Because we could not obtain a ΔctpA ΔyhmA strain, we constructed a ctpA-S conditional expression strain in the ΔyhmA background using the Tet-On promoter system. Knockdown of ctpA-S in ΔyhmA resulted in a severe growth defect on minimal medium, indicating that double disruption of ctpA and yhmA leads to synthetic lethality; however, we subsequently found that the severe growth defect was relieved by addition of lysine. Our results indicate that CtpA and YhmA are mitochondrial citrate transporters involved in citric acid production and that transport of citrate from mitochondria to the cytosol plays an important role in lysine biogenesis in A. kawachii. IMPORTANCE Citrate transport is believed to play a significant role in citrate production by filamentous fungi; however, details of the process remain unclear. This study characterized two citrate transporters from Aspergillus luchuensis mut. kawachii. Biochemical and gene disruption analyses showed that CtpA and YhmA are mitochondrial citrate transporters required for normal hyphal growth, conidia formation, and citric acid production. In addition, this study provided insights into the links between citrate transport and lysine biosynthesis. The characteristics of fungal citrate transporters elucidated in this study will help expand our understanding of the citrate production mechanism and facilitate the development and optimization of industrial organic acid fermentation processes.

Glycosylceramide modifies the flavor and metabolic characteristics of sake yeast

In the manufacture of sake, Japanese traditional rice wine, sake yeast is fermented with koji, which is steamed rice fermented with the non-pathogenic fungus Aspergillus oryzae. During fermentation, sake yeast requires lipids, such as unsaturated fatty acids and sterols, in addition to substances provided by koji enzymes for fermentation. However, the role of sphingolipids on the brewing characteristics of sake yeast has not been studied. In this study, we revealed that glycosylceramide, one of the sphingolipids abundant in koji, affects yeast fermentation. The addition of soy, A. oryzae, and Grifola frondosa glycosylceramide conferred a similar effect on the flavor profiles of sake yeast. In particular, the addition of A. oryzae and G. frondosa glycosylceramide were very similar in terms of the decreases in ethyl caprylate and ethyl 9-decenoate. The addition of soy glycosylceramide induced metabolic changes to sake yeast such as a decrease in glucose, increases in ethanol and glycerol and changes in several amino acids and organic acids concentrations. Tricarboxylic acid (TCA) cycle, pyruvate metabolism, starch and sucrose metabolism, and glycerolipid metabolism were overrepresented in the cultures incubated with sake yeast and soy glycosylceramide. This is the first study of the effect of glycosylceramide on the flavor and metabolic profile of sake yeast.

Pex16 is involved in peroxisome and Woronin body formation in the white koji fungus, Aspergillus luchuensis mut. kawachii

We characterized Pex16 in Aspergillus luchuensis mut. kawachii to examine the role of peroxisomes on citric acid production during the shochu-fermentation process. Rice koji made using a Δpex16 strain exhibited no significant change in citric acid accumulation but a 1.4-fold increase in formic acid production. Microscopic observation of mRFP-SKL (a peroxisome protein marker) showed that pex16 disruption decreased the number of dot-like structures per hyphal cell to 5% of the control. Pex16-GFP exclusively co-localized with mRFP-SKL throughout the hyphae including the very close position to the septal pore. Moreover, the Δpex16 strain was hypersensitive to calcofluor white, which appeared to induce bursting of the hyphal tip and translocation of mRFP-SKL signals to the septal pore. These results indicate that Pex16 does not play a role in citric acid accumulation but is significantly involved in peroxisome and Woronin body formation in Aspergillus kawachii.