Koji Mold Research Articles

Quality characteristics of Hokkaido brown bear meat sauces prepared with rice koji mold and food enzymes

Quality characteristics of Hokkaido brown bear meat sauces prepared with rice koji mold and food enzymes

Activation of Secondary Metabolism and Protease Activity Mechanisms in the Black Koji Mold Aspergillus luchuensis through Coculture with Animal Cells.

The activation of secondary metabolism plays a pivotal role in the discovery of novel natural products. We recently developed a coculture method involving actinomycetes and mouse macrophage-like cells to stimulate the production of bioactive compounds. A black koji mold, Aspergillus luchuensis IFM 61405, markedly enhanced the production of (3S,8R)-8-hydroxy-3-carboxy-2-methylenenonanoic acid (1a), (3S,8S)-8-hydroxy-3-carboxy-2-methylenenonanoic acid (1b), and (3S)-9-hydroxy-3-carboxy-2-methylenenonanoic acid (2) when coincubated with J774.1 mouse macrophage cells. The production of 1 and 2 increased by at least 3.5-fold and 2.7-fold, respectively, compared to monoculture after 7 days. A mechanistic investigation revealed that a protease from strain IFM 61405 plays a key role in enhancing the production of 1 and 2. This enhancement was not replicated in A. niger IFM 59706, a nonkoji mold, despite the presence of biosynthetic genes for 1 and 2 in A. niger IFM 59706. Furthermore, the addition of protease inhibitors suppressed the production of 1 and 2, suggesting that proteins secreted from animal cells, likely degraded by proteases secreted by strain IFM 61405, serve as precursors for 1 and 2. The results show that the strategy of coculturing koji mold with animal cells has the potential to enhance the production of natural products.

Isolation and characterization of koji mold (Aspergillus oryzae) from nature in Niigata

Koji mold (Aspergillus oryzae) is a key microorganism in brewing and fermentation in Japan. We isolated koji molds from the environment in Niigata Prefecture. Eighty-one environmental samples were placed on isolation medium made from steamed rice with wood ash and 36 Aspergillus section Flavi-like strains were obtained. Of those, 26 strains did not produce aflatoxin. We studied their morphology, sequence of ITS region, calmodulin gene, aflatoxin biosynthetic homologous gene cluster and α-amylase gene and fermentation-related enzyme activities. Furthermore, DNA-seq analysis of 14 strains from 26 non-aflatoxin producing strains were conducted and compared the three mycotoxin biosynthetic gene clusters (aflatoxin, cyclopiazonic acid, and aflatrem) and fermentation-related genes against those of reference strain A.oryzae RIB40. In some strains, gene sequences confirmed the absence of mycotoxin production, but differences in fermentation-related enzyme activities could not be explained well by amino acid substitutions. We classified the 26 isolates into 6 morphology types based on the appearance of colonies and mating types, and it was found that strains of the same morphology type had similar enzymatic profiles and gene sequences. Our results show that koji molds with various properties occur in the environment, and it will expand the possibilities of koji mold in industrial use.

Solid-state cultivation of multiple industrial strains of koji mold on different Thai unpolished rice cultivars: biotransformation of phenolic compounds and their effects on antioxidant activity.

Colored rice is abundant in polyphenols, and koji molds have potential for biotransformation. This study aimed to produce Thai-colored rice koji to study its polyphenolic biotransformation. Four industrial koji mold strains: Aspergillus oryzae 6001, A. oryzae 6020, A. sojae 7009, and A. luchuensis 8035, were cultivated on unpolished Thai-colored rice (Riceberry and Sangyod), unpolished Thai white rice (RD43), and polished Japanese white rice (Koshihikari). We discovered that koji molds grew on all the rice varieties. Methanol extracts of all rice kojis exhibited an approximately 2-fold or greater increase in total phenolic content and DPPH antioxidant activity compared to those of steamed rice. Moreover, quercetin, quercetin-3-O-glucoside, isorhamnetin-3-O-glucoside, ferulic acid, caffeic acid, protocatechuic acid, vanillic acid, (+)-catechin, and (-)-epicatechin content increased in Riceberry and Sangyod koji samples. Consequently, Aspergillus solid-state cultivation on unpolished Thai-colored rice exhibited higher functionalization than the cultivation of unpolished Thai white rice and polished Japanese white rice.

Live cell imaging of β-tubulin mRNA reveals spatiotemporal expression dynamics in the filamentous fungus Aspergillus oryzae

In filamentous fungi, microtubules are important for polar growth and morphological maintenance and serve as rails for intracellular trafficking. The molecular mechanisms associated with microtubules have been analyzed. However, little is known about when and where tubulin, a component of microtubules, is biosynthesized in multinuclear and multicellular filamentous fungi. In this study, we visualized microtubules based on the enhanced green fluorescence protein (EGFP)-labeled α-tubulin and β-tubulin mRNA tagged by the EGFP-mediated MS2 system in living yellow Koji mold Aspergillus oryzae cells in order to understand the spatiotemporal production mechanism of tubulin. We found that mRNA of btuA, encoding for β-tubulin, localized at dot-like structures through the apical, middle and basal regions of the hyphal cells. In addition, some btuA mRNA dots showed microtubule-dependent motor protein-like dynamics in the cells. Furthermore, it was found that btuA mRNA dots were decreased in the cytoplasm just before mitosis but increased immediately after mitosis, followed by a gradual decrease. In summary, the localization and abundance of β-tubulin mRNA is spatiotemporally regulated in living A. oryzae hyphal cells.

Clinical Perspectives of Domesticated Koji Mold - Probiotic Properties of Aspergillus oryzae: A Review Article

Clinical Perspectives of Domesticated Koji Mold - Probiotic Properties of Aspergillus oryzae: A Review Article

Koji Mold-derived Lipids Disrupt the Intracellular Redox State by Decreasing the GPx4 and Intracellular Glutathione Levels, Promoting Membrane Lipid Peroxidation, and Inducing Ferroptosis in HL-60 Cells.

In this study, we evaluated the cancer cell killing activity of koji mold-derived extracts using several solvents. The koji mold lipid extract (KML) exhibited potent cytotoxicity against a human leukemia cell line. Fractionation of the KML via silica gel chromatography revealed the presence of active components in fraction (Fr.) 6. Cytotoxic effects of Fr. 6 were inhibited by the ferroptosis inhibitors, ferrostatin-1 and SRS11-92, and the iron chelator, deferoxamine. Interestingly, ferroptosis inhibitors failed to prevent the KML-induced cell death. Fr. 6 decreased the expression of glutathione peroxidase 4 (GPx4) and increased the level of peroxidized plasma membrane lipids. Furthermore, Fr. 6 decreased the intracellular glutathione levels. Overall, our results suggest that Fr. 6 included in KML induces ferroptosis in HL-60 cells.

Shochu Koji Microstructure and Starch Structure during Preparation.

In this study, we investigated the changes in composition, microstructure, and starch molecular structure of shochu koji during preparation. We observed that the gelatinized and outer part of starch was decomposed in priority during the early and middle preparation stages. The gap between the starch granules increased with the delayed time. Finally, the koji microstructure became spongy. Shochu koji mold produced two α-amylases in different expression manners. Acid-labile α-amylase was produced in the early and middle preparation stages. Acid-stable α-amylase and saccharification power were produced in the middle and late stages. Throughout the koji preparation, reducing sugars content reached approximately 13-20 % of the total sugar content, with glucose representing over 70 % of the reducing sugars. α-Glucan fragments with C chains of degree of polymerization (DP) 4-73 were observed in the early and middle stages (<23 h), indicating the degradation of amylopectin at long B chains. In the latter stage, the amount of C chains of DP 6-30 decreased, while the longer C chains (DP 30<) did not change. These results showed that acid-labile α-amylase, acid-stable α-amylase, and saccharification enzymes including glucoamylase and α-glucosidase work preferentially on the amorphous regions of starch granules, and cooperative action of these enzymes during koji preparation contributes to the formation of the observed microstructure. Our study is the first report on the decomposition schemes of starch and the microstructure forming process in shochu koji.

Comparison of taste characteristics between koji mold–ripened cheese and Camembert cheese using an electronic tongue system

Koji mold, classified in the genus Aspergillus, is used to produce traditional Japanese fermented foods such as miso, soy sauce, and sake. In recent years, the application of koji mold to cheese ripening has attracted attention, and cheese surface-ripened with koji mold (koji cheese) has been studied. In this study, to evaluate the taste characteristics of koji cheese, an electronic tongue system was employed to measure the taste values of cheese samples ripened using 5 strains of koji mold in comparison with commercial Camembert cheese. All koji cheese samples exhibited lower sourness and greater bitterness, astringency, saltiness, and umami richness than the Camembert cheese samples. The intensity of each taste characteristic differed depending on the koji mold strain. These results indicate that koji cheese has a different taste value than conventional mold-ripened cheese. Furthermore, the results also indicate that various taste characteristics can be achieved by selecting different koji molds.

Are Current Aspergillus sojae Strains Originated from a Native Aflatoxigenic Aspergillus Species Population Also Present in California?

Aspergillus sojae has long been considered a domesticated strain of Aspergillus parasiticus. This study delineated relationships among the two species and an Aspergillus PWE36 isolate. Of 25 examined clustered aflatoxin genes of PWE36, 20 gene sequences were identical to those of A. sojae, but all had variations to those of A. parasiticus. Additionally, PWE36 developmental genes of conidiation and sclerotial formation, overall, shared higher degrees of nucleotide sequence identity with A. sojae genes than with A. parasiticus genes. Examination of defective cyclopiazonic acid gene clusters revealed that the PWE36 deletion pattern was identical only to those of A. sojae. Using A. sojae SMF134 genome sequence as a reference, visualization of locally collinear blocks indicated that PWE36 shared higher genome sequence homologies with A. sojae than with A. parasiticus. Phylogenetic inference based on genome-wide single nucleotide polymorphisms (SNPs) and total SNP counts showed that A. sojae strains formed a monophyletic clade and were clonal. Two (Argentinian and Ugandan) A. parasiticus isolates but not including an Ethiopian isolate formed a monophyletic clade, which showed that A. parasiticus population is genetically diverse and distant to A. sojae. PWE36 and A. sojae shared a most recent common ancestor (MRCA). The estimated divergence time for PWE36 and A. sojae was about 0.4 mya. Unlike Aspergillus oryzae, another koji mold that includes genetically diverse populations, the findings that current A. sojae strains formed a monophyletic group and shared the MRCA with PWE36 allow A. sojae to be continuously treated as a species for food safety reasons.

An efficient Agrobacterium-mediated system based on the pyrG auxotrophic marker for recombinant expression in the filamentous fungus Penicillium rubens

This work aimed to construct a versatile, effective, and food-grade Agrobacterium tumefaciens-mediated transformation (ATMT) system for recombinant expression in the filamentous fungus Penicillium rubens (also known as Pencillium chrysogenum). In this study, the wild-type P. chrysogenum VTCC 31172 strain was re-classified as P. rubens by a multilocus sequencing analysis. Further, the pyrG gene required for uridine/uracil biosynthesis was successfully deleted in the VTCC 31172 strain by homologous recombination to generate a stable uridine/uracil auxotrophic mutant (ΔpyrG). The growth of the P. rubens ΔpyrG strain could be restored by uridine/uracil supplementation, and a new ATMT system based on the uridine/uracil auxotrophic mechanism was established for this strain. The optimal ATMT efficiency could reach 1750 transformants for 106 spores (equivalent to 0.18%). In addition, supplementation of uridine/uracil at the concentrations of 0.005-0.02% during the co-cultivation process significantly promoted transformation efficiency. Especially, we demonstrated that the pyrG marker and the amyB promoter from the koji mold Aspergillus oryzae were fully functional in P. rubens ΔpyrG. Expression of the DsRed reporter gene under the regulation of the A. oryzae amyB promoter lighted up the mycelium of P. rubens with a robust red signal under fluorescence microscopy. Furthermore, genomic integration of multiple copies of the Aspergillus fumigatus phyA gene under the control of the amyB promoter significantly enhanced phytase activity in P. rubens. The ATMT system developed in our work provides a safe genetic platform for producing recombinant products in P. rubens without using drug resistance markers.

Improvement in salt-tolerance of Aspergillus oryzae γ-glutamyl transpeptidase via protein chimerization with Aspergillus sydowii homolog

γ-Glutamyl transpeptidase is one of the key enzymes involved in glutamate production during high-salt fermentation of soy sauce and miso by koji mold, Aspergillus oryzae. However, the activity of γ-glutamyl transpeptidase from A. oryzae (AOggtA) is markedly reduced in the presence of NaCl, thus classifying it as a non-salt-tolerant enzyme. In contrast, the homologous protein from the xerophilic mold, A. sydowii (ASggtA) maintains its activity under high-salt conditions. Therefore, in this study, a chimeric enzyme, ASAOggtA, was designed and engineered to improve salt-tolerance in AOggtA by swapping the N-terminal region, based on sequence and structure comparisons between salt-tolerant ASggtA and non-salt-tolerant AOggtA. The parental AOggtA and ASggtA and their chimera, ASAOggtA, were heterologously expressed in A. oryzae and purified. The chimeric enzyme inherited the superior activity and stability from each of the two parent enzymes. ASAOggtA showed > 2-fold greater tolerance than AOggtA in the presence of 18% NaCl. In addition, the chimera showed a broader range of pH stability and greater thermostability than ASggtA. AOggtA and ASAOggtA were sy over the range pH 3.0 to pH 10.5. Thermal stability was found to be in the order AOggtA (57.5 °C, t1/2 = 32.5 min) > ASAOggtA (55 °C, t1/2 = 20.5 min) > ASggtA (50 °C, t1/2 = 12.5 min). The catalytic and structural characteristics indicated that non-salt-tolerant AOggtA would not undergo irreversible structural changes in the presence of NaCl, but rather a temporary conformational change, which might result in reducing the substrate binding and catalytic activity, on the basis of kinetic properties. In addition, the chimeric enzyme showed hydrolytic activity toward L-glutamine that was as high as that of AOggtA. The newly-designed chimeric ASAOggtA might have potential applications in high-salt fermentation, such as miso and shoyu, to increase the content of the umami-flavor amino acid, L-glutamate.

Random mutagenesis of koji mold (Aspergillus oryzae) to enhance the catalytic efficiency and thermostability of glucoamylase

Random mutagenesis of koji mold (Aspergillus oryzae) to enhance the catalytic efficiency and thermostability of glucoamylase

Phenotypic, Genomic, and Transcriptomic Comparison of Industrial Aspergillus oryzae Used in Chinese and Japanese Soy Sauce: Analysis of Key Proteolytic Enzymes Produced by Koji Molds.

Aspergillus oryzae, which generates numerous enzymes for the breakdown of raw materials, is an essential koji mold in soy sauce production. For better soy sauce productivity and flavor quality, China and Japan have developed their own industrial A. oryzae strains at distinct evolutionary branches for use in soy sauce production for decades. However, systematic comparison between the two national industrial strains has been poorly conducted, and thus we have not been able to generate adequate knowledge, especially regarding what are the key hydrolytic enzymes produced by A. oryzae during koji production. This study sequenced and assembled three high-quality genome sequences of industrial A. oryzae originating from China and Japan. Based on the genome sequences, a phylogenetic tree analysis was performed and revealed the evolutional distances between the two national industrial koji molds. Meanwhile, a comparative phenotypic analysis revealed that the two national industrial strains differed in growth and catalytic characteristics, particularly in proteolytic enzyme activities. To investigate the molecular mechanism underlying the phenotypic difference, we conducted systematic comparative genome and transcriptome investigations. We found minor differences in the quantity and diversity of proteolytic enzyme genes between Chinese and Japanese koji molds, while the protease secretion ratio and transcriptional level were dissimilar. We identified 58 potential important enzymes associated with high protein breakdown efficiency during industrial koji fermentation by combining comparative phenotypic and transcriptome data. More research is required to confirm the function of these putative key hydrolytic enzymes. IMPORTANCE Aspergillus oryzae is widely used as an industrial koji mold for soy sauce brewing due to its powerful raw material decomposition capability. Although various proteases in A. oryzae have been identified, it remains a challenge to find essential enzymes involved in soy sauce production. Generally, the industrial A. oryzae used in soy sauce brewing has excellent proteolytic activity. Based on this, we analyzed key proteolytic enzymes according to a comparison of the genome and transcriptome between three industrial strains. This study found little difference in gene numbers and mutations of proteolytic enzymes between three industrial A. oryzae strains. However, variations in protease secretion ratio and transcriptome were discovered between industrial strains. Based on that, we generated 58 candidate key proteolytic enzymes. This work comprehensively analyzed three industrial koji molds, revealing genome development under separate artificial domestication and helping in the study of key proteolytic enzymes during soy sauce production.

Aspergillus oryzae Fermentation Extract Alleviates Inflammation in Mycoplasma pneumoniae Pneumonia.

The filamentous fungus Aspergillus oryzae, also known as koji mold, has been used for centuries in the production of fermented foods in East Asia. A. oryzae fermentation can produce enzymes and metabolites with various bioactivities. In this study, we investigated whether A. oryzae fermentation extract (AOFE) has any effect on Mycoplasma pneumoniae (Mp) pneumonia. We performed solid-state fermentation of A. oryzae and obtained the ethanol extract. AOFE was analyzed by HPLC, and the major component was identified to be kojic acid. In vitro, AOFE suppressed Mp growth and invasion into A549 lung epithelial cells as determined by the gentamicin protection assay. AOFE treatment also suppressed Mp-stimulated production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 at mRNA and protein levels in murine MH-S alveolar macrophages. In a mouse model of Mp pneumonia, Mp infection induced a marked pulmonary infiltration of neutrophils, which was significantly reduced in mice pre-treated orally with AOFE. AOFE administration also suppressed the production of proinflammatory cytokines and chemokines in the lungs. Collectively, our results show that AOFE has the potential to be developed into a preventive/therapeutic agent for Mp pneumonia.

Effect of kuratsuki Kocuria on sake brewing in different koji conditions.

Koji is made using steamed rice and a koji mold, which plays an essential role in sake brewing. We challenge to build a new sake brewing method using the kuratsuki bacteria that have inhabited each sake brewery. In this paper, effects of the kuratsuki Kocuria strain TGY1127_2 were estimated on sake brewing in different koji conditions. Sake was produced by incubation of a mixture solution of koji, water, and sake yeast (strain K1401) with and without the kuratsuki Kocuria TGY1127_2. The effects of the kuratsuki Kocuria on the taste of the sake differed among different koji. The kuratsuki Kocuria led to an increase in ethanol concentration. Additionally, the sugar content (Brix) and acidity of the sake increased in proportion to the amount of koji. These results strongly suggest that the kuratsuki Kocuria does not adversely affect the fermentation activity of the sake yeast. Thus, the kuratsuki Kocuria had different effects on the taste of sake among different koji but the fermentation activity of the sake yeast was maintained.

Effects of Milk Amazake on Skin Elasticity, Hydration, and Transepidermal Water Loss: An 8-Week Double-Blind, Randomized, Controlled Trial.

Amazakes made from rice and koji mold are rich in nutrients, such as groups of vitamin B, minerals, essential amino acids, and oligosaccharides, and can improve skin moisturization. However, there are few reports on milk amazake, made from milk and koji mold. Therefore, in this double-blind, randomized controlled trial, we investigate the effect of milk amazake on skin function. Healthy women and men (n = 40) were randomly allocated to the milk amazake or placebo group. The test beverage was consumed once daily for 8 weeks. Skin elasticity, hydration, and transepidermal water loss (TEWL) were measured at baseline and at weeks 4 and 8, and all subjects completed the trial. Skin elasticity (R2 and R5) at 8 weeks was significantly increased in the milk amazake group compared with baseline. In addition, changes in R5 in the milk amazake group were significantly higher than those in the placebo group. Conversely, TEWL, an evaluation item of skin moisturizing function at 8 weeks, was significantly decreased in the active group compared with baseline. In conclusion, milk amazake may be useful as a functional food for improving skin function.

Volatile and soluble metabolite profiles in surface-ripened cheeses with Aspergillus oryzae and Aspergillus sojae

To determine the impact of traditional koji molds on chemical characteristics of soft-type natural cheese, novel surface mold-ripened cheeses with Aspergillus oryzae and Aspergillus sojae were studied by non-targeted metabolite profiling. Comprehensive water-soluble and volatile metabolite profiles of koji cheese were evaluated among five Aspergillus strains and other mold-ripened cheeses. Time-course changes in the metabolite profiles and degrading enzyme activities were also compared with those of an industrial Penicillium candidum starter culture. Koji cheeses differed from Camembert, Brie, and blue cheeses in higher lactic acid, amino acid, and acetoin levels and lower methyl ketone and volatile fatty acid levels. Time-course analysis revealed the associations of rapid accumulations of glutamic, aspartic, and 3-methylbutanoic acids and 3-methylbutanal with higher proteolytic activity, and methyl ketone and fatty acid derivative suppressions with lower lipolytic activity. Ethyl butanoate, diacetyl, and malic acid also characterized koji cheeses as strain-dependent metabolites. This study highlighted the key compositional difference derived from cheese ripening with Aspergillus strains. The findings could help quality improvements of koji cheese product.

Effect of sake lees on cheese components in cheese ripened by Aspergillus oryzae and lactic acid bacteria

More than 2,000 varieties of cheese currently exist in the world, and cheese manufacture continues to flourish. To develop the cheese ripening process, additional ingredients are used during cheese production. In this study, the effect of sake lees as an additional ingredient on the fermentation of cheese using Aspergillus oryzae (koji mold), known as koji cheese, was investigated. Aspergillus oryzae is used in the fermentation of Japanese traditional foods, such as sake and soy sauce, given its strong enzymatic activities, as well as in cheese production (i.e., koji cheese). Sake lees, a by-product of the fermentation of rice with A. oryzae and yeasts in the sake brewing process, contains various metabolites, such as amino acids. Here, supplementation with sake lees enhanced the activities of lactic acid bacteria and affected the color of the cheese. Metabolome analysis revealed that sake lees altered the balance of carbohydrates and fatty acids in the cheese. Remarkably, supplementation with sake lees enhanced the production of umami-enhancing γ-glutamyl (kokumi-active) peptides. This study suggests that a new type of cheese can be produced using A. oryzae and sake lees, and information on the synergistic effects of A. oryzae and sake lees will aid the development of cheese production.

麹菌 －細胞生物学から酒造りまで－

麹菌 －細胞生物学から酒造りまで－