Koji amazake Maintains Water Content in the Left Cheek Skin of Healthy Adults: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group, Comparative Trial.

Purpose: Improvement in water content and skin barrier function on human skin is believed to be induced by koji amazake, a non-alcoholic beverage derived from rice fermented by Aspergillus oryzae (A. oryzae). In order to scientifically identify the effects of koji amazake on human skin, we performed a randomized, double-blind, placebo-controlled, parallel-group comparative trial and quantified the content of glucosylceramide (GlcCer) which would be responsible for the effects. Participants and Methods: Healthy adults concerned with their skin dryness were divided into koji amazake (N = 30) or placebo group (N = 30). During this test, the test beverages were ingested at 118 g/day. Their water content and trans-epidermal water loss (TEWL) were measured at 0 week (baseline) and 8 weeks. The content of GlcCer in test beverages was quantified by HPLC-ELSD. Results: In comparison with the placebo group, the water content in the left cheek of individuals in the koji amazake group was maintained for 8 weeks. In addition, changes in water content from the baseline to 8 weeks differed significantly between the koji amazake (0.19) and placebo groups (-3.98). Unexpectedly, there was no significant difference in the TEWL between koji amazake and placebo group. We analyzed GlcCer in both koji amazake and placebo beverages, which were found to contain 1.35 ± 0.11 and 0.30 ± 0.07 mg/118 g, respectively. The amount of GlcCer in koji amazake was approximately equal to the dosage of plant-derived GlcCer which has the ability to improve water content and TEWL in humans. Conclusion: Present study has shown that intake of koji amazake contributes to maintain the water content only on the left cheek. The content of GlcCer derived from koji amazake was adequate for maintenance of the water content compared to previous reports. Therefore, it was concluded that GlcCer in koji amazake acts as a functional ingredient.

Intake of Koji Amazake Improves Defecation Frequency in Healthy Adults.

Reportedly, the intake of koji amazake, a beverage made from steamed rice fermented by Aspergillus oryzae, improves defecation frequency. However, its functional ingredients and mechanism of action remain unclear. To compare the effects of koji amazake and a placebo beverage on defecation frequency and to identify the functional ingredients and mechanism of action, a randomized, placebo-controlled, double-blind parallel-group comparative trial was performed on two groups. The koji amazake had 302 ± 15.5 mg/118 g of A. oryzae cells, which was not in the placebo. Compared with the placebo group, the koji amazake group showed a significant increase in weekly defecation frequency at 2 weeks (5.09 days vs. 4.14 days), 3 weeks (5.41 days vs. 4.18 days), and 4 weeks (5.09 days vs. 3.95 days), along with an increase in the weekly fecal weight at 4 weeks (724 g vs. 501 g). The intake of koji amazake did not induce significant intergroup differences in the fecal SCFA concentration, whereas it significantly decreased the relative abundance of Blautia and significantly increased that of Bacteroides at 3 weeks. Therefore, koji amazake intake improved defecation frequency, and A. oryzae cells played potentially important roles as functional ingredients.

Effect of temperature on saccharification and oligosaccharide production efficiency in koji amazake.

Koji amazake, prepared from rice koji, is a traditional Japanese sweet beverage. The main source of sweetness is glucose derived from rice starch following digestion by enzymes of Aspergillus oryzae during saccharification. The temperature of this process was empirically determined as 45°C-60°C, but no studies have systematically investigated the effect of temperature on saccharification efficiency. We addressed this in the present study by evaluating saccharification efficiency at various temperatures. We found that glucose content was the highest at 50°C (100%) and was reduced at temperatures of 40°C (66.4%), 60°C (91.9%), and 70°C (76.6%). We previously reported that 12 types of oligosaccharides are present in koji amazake; the levels of eight of these, namely nigerose, kojibiose, trehalose, isomaltose, gentiobiose, raffinose, panose, and isomaltotriose, were the highest at 50°C-60°C, whereas sophorose production was maximal at 70°C. Based on these findings, we initially performed saccharification at 50°C and then switched the temperature to 70°C. The maximum amount of each saccharide including sophorose that was produced was close to the values obtained at these two temperatures. Thus, oligosaccharide composition of koji amazake is dependent on saccharification temperature. These findings provide useful information for improving the consumer appeal of koji amazake by enhancing oligosaccharide content.

The impact of a single-nucleotide mutation of bgl2 on cellulase induction in a Trichoderma reesei mutant.

BACKGROUND: The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that ß-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular ß-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose. RESULTS: To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked ß-1,4, ß-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7. CONCLUSION: We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

Conversion of L-galactono-1,4-lactone to L-ascorbate is regulated by the photosynthetic electron transport chain in Arabidopsis.

In this study we focused on the effects of light irradiation and the addition of L-galactono-1,4-lactone (L-GalL) on the conversion of exogenous L-GalL to L-ascorbate (AsA) and the total AsA pool size in detached leaves of Arabidopsis plants and transgenic plants expressing the rat L-gulono-1,4-lactone oxidase gene. Increases in the total AsA level in L-GalL-treated leaves depended entirely on light irradiation. Treatment with an inhibitor of photosynthetic electron transport together with L-GalL reduced the increase in total AsA under light. Light, particularly the redox state of photosynthetic electron transport, appeared to play an important role in the regulation of the conversion of L-GalL to AsA in the mitochondria, reflecting the cellular level of AsA in plants.

Light regulation of ascorbate biosynthesis is dependent on the photosynthetic electron transport chain but independent of sugars in Arabidopsis.

It has been known that leaves exposed to high light contain more L-ascorbic acid (AsA) than those in the shade. However, the mechanism of the light regulation of the AsA pool size in plants is largely unknown. In this work, the relationship between gene expression levels related to AsA biosynthesis and photosynthesis have been studied. When 2-week-old Arabidopsis plants grown under a 16 h daily photoperiod were moved into the dark, the AsA level in the leaves was decreased by 91% in 72 h, whereas it increased by 171% in the leaves of plants exposed to continuous light during the same period. Among the several enzymes of the AsA biosynthesis pathway, the transcript levels of GDP-D-mannose pyrophosphorylase, L-galactose 1-P phosphatase, L-galactono-1,4-lactone dehydrogenase, and the VTC2 gene were down-regulated in the dark. Treatment with inhibitors of photosynthesis, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and atrazine, arrested a rise in the AsA pool size accompanying the decrease in the transcript levels of the genes of the above enzyme in the leaves. When the plants were transferred to a medium containing 0.5% (w/v) sucrose, the photosynthesis activities and the leaf AsA levels were lowered even under exposure to light compared with those in plants on the medium without sucrose. In contrast, the AsA level in leaves of the sugar-insensitive Arabidopsis mutant abi4/sun6 was unaffected by external sucrose. No significant difference in the expression profiles for AsA biosynthesis enzymes was observed between the wild-type and mutant plants by sucrose feeding. The results suggest that photosynthetic electron transport of chloroplasts is closely related to AsA pool size regulation in leaves.

Functional characterization of D-galacturonic acid reductase, a key enzyme of the ascorbate biosynthesis pathway, from Euglena gracilis.

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38-39 kDa, as judged by SDS-PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5+/-4.5 microM and uronic acids, such as D-galacturonic acid (Km=3.79+/-0.5 mM) and D-glucuronic acid (Km=4.67+/-0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP(+). The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H(2)O(2), suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.