Koji Mold Research Articles

Production of Enzyme and Growth of Aspergillus oryzae S.on Soybean Koji

 Abstract—Soybean koji is an important ingredient for traditional fermented food in South-East Asia and East Asia. It provides large amount of enzyme from koji mold, Aspergillus oryzae S., to digest nutrients in substrates. This study aimed at production of certain enzymes in soybean koji and potential to be applied for accelerating fish sauce fermentation. Koji containing 60% soybean was used as substrate to investigate the enzyme production by A. oryzae S. The growth of this mold was enumerated by potato dextrose agar. The mycelium development of A. oryzae S. was observed by scanning electron microscope. During koji production, pH of soybean koji increased from 6.32 to 6.07. It was caused by extracellular proteins production. The highest neutral protease, alkaline protease and amylase activities were 84.38, 41.35 and 200 unit/g of dry weight, respectively. Moreover, growing of enzyme activities on soybean koji correlated with the growth of this mold. Electron micrograph showed that spores of A. oryzae S. were formed after 48 h of cultivation period. Additionally, the highest enzymes activities were also shown in this stage.

Enzyme Production and Growth of Aspergillus oryzae S. on Soybean Koji Fermentation

Soybean koji is an important ingredient for traditional fermented food in South-East Asia and East Asia. It provides large amount of enzyme from koji mold, Aspergillus oryzae S., to digest nutrients in substrates. This study was aimed at production of certain enzymes in soybean koji and potential to be applied for accelerating fish sauce fermentation. Koji containing 60% soybean was used as substrate to investigate the enzyme production by A. oryzae S. The growth of this mold was enumerated by scanning electron microscope. During koji fermentation, pH increase of soybean koji was caused by enzymes production. The highest protease and amylase activities were 84.38 and 200 unit/g of dry weight, respectively. Moreover, growing of enzyme activities on soybean koji correlated with the growth of this mold. Electron micrograph showed that spores of A. oryzae S. were formed after 48h of cultivation period. Additionally, the highest enzymes activities were also shown in this stage.

Genomic Analysis of Koji Mold Aspergillus oryzae and Investigation of Novel Peptidases by Post-genomic Approach

Aspergillus oryzae is a filamentous fungus used as a starter microorganism in the process of manufacturing various kinds of traditional fermented food. It is also used as a producer of enzymes for both food processing and pharmaceuticals, such as proteases and taka-amylase. In order to clarify the function of this microorganism in fermentation industries and utilize the new function of A. oryzae, it was subject to genomic analysis. During the analysis, the telomere of A. oryzae exhibited a novel repeated sequence distinct from other fungi. Genomic analysis also revealed that the genome of A. oryzae consists of eight chromosomes containing 12,074 genes and is larger in size than Aspergillus nidulans and Aspergillus fumigatus. A. oryzae contains sequences specific for A. oryzae for genes involved in primary and secondary metabolism, when compared with A. nidulans and A. fumigatus. Among them, the function of one of the peptidase-like gene products was investigated with a post-genomic approach, and determined as an aminopeptidase specific for acidic amino acids (aspartate and glutamate) which was first identified in filamentous fungi. It also demonstrated that the addition of cobalt ion to the growth medium greatly improved the specific activity. Using the same approach, some of the novel aminopeptidases and carboxypeptidases of A. oryzae were identified and characterized.

醤油麹を用いて製造したニギス魚醤油および蒲鉾製造ロス醤油の発酵初期に起こるタンパク質の分解について

醤油麹を用いたニギス魚醤油では，発酵開始から 7 日目以内にもろみの液化が進行し，タンパク質量が減少した。一方，蒲鉾製造ロスが主原料の醤油もろみでも，発酵開始から 1 日以内に液化が進行し，タンパク質量が減少したことから，魚醤油の発酵初期には自己消化酵素よりも麹由来のタンパク質分解酵素が強く作用することが示唆された。原料タンパク質の差異は分解産物の内訳や挙動に影響を及ぼすことが示された。すなわち，低分子のタンパク質成分の分子量分布やそれらの消長および遊離アミノ酸の総量と組成が異なることが明らかとなった。

Disruption and Overexpression of Acid Phosphatase Gene (aphA) from a Miso Koji Mold, Aspergillus oryzae KBN630, and Characterization of the Gene Product

Disruption and Overexpression of Acid Phosphatase Gene (aphA) from a Miso Koji Mold, Aspergillus oryzae KBN630, and Characterization of the Gene Product

Studies on the cooking conditions and mechanical koji- making of black beans

Inyu, like soy sauce, is a traditionally fermented condiment in Taiwan and made by black beans. It has special properties in flavors and qualities, however, its market share is lower than soy sauce made by automatic koji-making method due to limitation in production scale and labor cost. In this study, the optimum soaking and cooking conditions were determined by response surface methodology (RSM), and the mechanically koji-making method was investigated to evaluate the feasibility of koji-making method for Inyu as compared to the traditional method. According to superimposed plots of enzyme susceptibility and enzyme activity, cooking at 132 for 23min after soaking with water at room temperature for 4 hr was the optimum cooking conditions of black beans for making Inyu. The water content and hardness of the cooked black beans were 50.7% and 61.6g, respectively. The proper water content and protein denaturation were required for growth of koji mold and protein hydrolysis by enzymes produced during koji making. The thickness of cooked black beans was related to water loss during mechanical koji-making. Water content of the koji with the 6-cm thickness was too low to be available for koji mold and only low enzyme activities were obtained. On the other hand, enzyme activities for the koji thickness at 12-18cm were higher than that from traditionally koji-making method. The activities of acid-, neutral-, alkaline-protease and amylase were 423, 543, 525, and 1127 units/g dry koji for the koji thickness at 12cm. Faster growth and the mycelium stretched to cotyledon of the black beans were observed for the koji mold by mechanical koji-making method than that of traditionally method. Therefore, koji-making through an automatic process by mechanized facilities was feasible in manufacturing of Inyu.

Breeding and identification of novel koji molds with high activity of acid protease by genome recombination between Aspergillus oryzae and Aspergillus niger

Acid protease is essential for degradation of proteins during soy sauce fermentation. To breed more suitable koji molds with high activity of acid protease, interspecific genome recombination between A. oryzae and A. niger was performed. Through stabilization with d-camphor and haploidization with benomyl, several stable fusants with higher activity of acid protease were obtained, showing different degrees of improvement in acid protease activity compared with the parental strain A. oryzae. In addition, analyses of mycelial morphology, expression profiles of extracellular proteins, esterase isoenzyme profiles, and random amplified polymorphic DNA (RAPD) were applied to identify the fusants through their phenotypic and genetic relationships. Morphology analysis of the mycelial shape of fusants indicated a phenotype intermediate between A. oryzae and A. niger. The profiles of extracellular proteins and esterase isoenzyme electrophoresis showed the occurrence of genome recombination during or after protoplast fusion. The dendrogram constructed from RAPD data revealed great heterogeneity, and genetic dissimilarity indices showed there were considerable differences between the fusants and their parental strains. This investigation suggests that genome recombination is a powerful tool for improvement of food-grade industrial strains. Furthermore, the presented strain improvement procedure will be applicable for widespread use for other industrial strains.

Extractive components and taste aspects of fermented fish pastes and bean pastes prepared using different koji molds as starters

We have evaluated four commercially available mold starters (SP-01, NY, M1, and “for kome miso”) for fermented fish pastes with the aim of determining the effect of different koji molds on the nutritional and sensory characteristics of the fermented product. Comparative studies among different fermented fish and bean pastes were also conducted. Protein contents were considerably higher in the fermented fish pastes (22.7–24.1%) than the fermented bean pastes (8.2–12.8%). Free amino acids, oligopeptides, organic acids, mineral content, and sensory scores of the fermented fish pastes were superior to those of the bean pastes in terms of nutritional aspects. Sensory evaluation of fermented fish and bean pastes clearly indicated that different koji molds used as starters had little effect on the taste of the fermented fish pastes. However, in terms of nutritional and sensory characteristics, fermented fish pastes from trash fishes were found to be both commercially valuable and a potential gain to food processing firms among the many new generic fermented food products becoming available.

Isolation of Industrial Strains of Aspergillus oryzae Lacking Ferrichrysin by Disruption of the dffA Gene

Based on studies using laboratory strains, the efficiency of gene disruption in Aspergillus oryzae, commonly known as koji mold, is low; thus, gene disruption has rarely been applied to the breeding of koji mold. To evaluate the efficiency of gene disruption in industrial strains of A. oryzae, we produced ferrichrysin biosynthesis gene (dffA) disruptants using three different industrial strains as hosts. PCR analysis of 438 pyrithiamine-resistant transformants showed dffA gene disruption efficiency of 42.9%-64.1%, which is much higher than previously reported. Analysis of the physiological characteristics of the disruptants indicated that dffA gene disruption results in hypersensitivity to hydrogen peroxide. To investigate the industrial characteristics of dffA gene disruptants, two strains were used to make rice koji and their properties were compared to those of the host strains. No differences were found between the dffA gene disruptants and the host strains, except that the disruptants did not produce ferrichrysin. Thus, this gene disruption technique is much more effective than conventional mutagenesis for A. oryzae breeding.

Generation of Large Chromosomal Deletions in Koji Molds Aspergillus oryzae and Aspergillus sojae via a Loop-Out Recombination

We established a technique for efficiently generating large chromosomal deletions in the koji molds Aspergillus oryzae and A. sojae by using a ku70-deficient strain and a bidirectional marker. The approach allowed deletion of 200-kb and 100-kb sections of A. oryzae and A. sojae, respectively. The deleted regions contained putative aflatoxin biosynthetic gene clusters. The large genomic deletions generated by a loop-out deletion method (resolution-type recombination) enabled us to construct multiple deletions in the koji molds by marker recycling. No additional sequence remained in the resultant deletion strains, a feature of considerable value for breeding of food-grade microorganisms. Frequencies of chromosomal deletions tended to decrease in proportion to the length of the deletion range. Deletion efficiency was also affected by the location of the deleted region. Further, comparative genome hybridization analysis showed that no unintended deletion or chromosomal rearrangement occurred in the deletion strain. Strains with large deletions that were previously extremely laborious to construct in the wild-type ku70(+) strain due to the low frequency of homologous recombination were efficiently obtained from Delta ku70 strains in this study. The technique described here may be broadly applicable for the genomic engineering and molecular breeding of filamentous fungi.

Genomics of Aspergillus oryzae: learning from the history of Koji mold and exploration of its future.

At a time when the notion of microorganisms did not exist, our ancestors empirically established methods for the production of various fermentation foods: miso (bean curd seasoning) and shoyu (soy sauce), both of which have been widely used and are essential for Japanese cooking, and sake, a magical alcoholic drink consumed at a variety of ritual occasions, are typical examples. A filamentous fungus, Aspergillus oryzae, is the key organism in the production of all these traditional foods, and its solid-state cultivation (SSC) has been confirmed to be the secret for the high productivity of secretory hydrolases vital for the fermentation process. Indeed, our genome comparison and transcriptome analysis uncovered mechanisms for effective degradation of raw materials in SSC: the extracellular hydrolase genes that have been found only in the A. oryzae genome but not in A. fumigatus are highly induced during SSC but not in liquid cultivation. Also, the temperature reduction process empirically adopted in the traditional soy-sauce fermentation processes has been found to be important to keep strong expression of the A. oryzae-specific extracellular hydrolases. One of the prominent potentials of A. oryzae is that it has been successfully applied to effective degradation of biodegradable plastic. Both cutinase, responsible for the degradation of plastic, and hydrophobin, which recruits cutinase on the hydrophobic surface to enhance degradation, have been discovered in A. oryzae. Genomic analysis in concert with traditional knowledge and technology will continue to be powerful tools in the future exploration of A. oryzae.

Traditional healthful fermented products of Japan

A variety of fermentation products, such as foods containing probiotic bacteria, black rice vinegar (kurosu), soy sauce (shoyu), soybean-barley paste (miso), natto and tempeh, are sold in food stores in Japan. These fermented food products are produced by traditional methods that exploit mixed cultures of various non-toxic microorganisms. These microorganisms include lactic acid bacteria, acetic acid bacteria, sake yeast, koji molds and natto bacteria. Many traditional fermented foods have been studied and their effects on metabolism and/or immune system have been demonstrated in animal and/or human cells. This review summarizes the scientific basis for the effects of these traditional food products, which are currently produced commercially in Japan.

Characterization of yeast cell surface displayed Aspergillus oryzae β-glucosidase 1 high hydrolytic activity for soybean isoflavone

The yeast Saccharomyces cerevisiae GRI-117-UK was transformed to either display or secrete β-glucosidase 1 (BGL1) from the koji mold, Aspergillus oryzae. The β-glucosidase activity of BGL1-displaying yeast strains reached 405.9 U/g dry cell mass after 72 h of cultivation in YPD medium. The optimal pH and temperature for BGL1 displayed on the cell surfaces of the yeast were 5.0 and 55 °C, while the optima for BGL1 secreted by the yeast were 4.0 and 55 °C. The displayed BGL1 was stable at higher pH compared with the secreted BGL1. In addition, the thermostability of BGL1 was improved by displaying the enzyme on the yeast cell surfaces. In addition, the displayed and secreted forms of BGL1 had similar substrate specificity. β-Glucosidase hydrolyzes daidzin and genistin, which are the glycoside forms of soybean isoflavones, to the aglycones. Isoflavone aglycones were efficiently produced by BGL1-displaying yeast from an isoflavone mixture; at optimal temperature and pH the rate of aglycone production was at least 15.8 g/(l h). After 144 h of reaction, almost isoflavones were converted to its aglycone by BGL1-displaying yeast. The results of the present study demonstrate that BGL1-displaying yeast strains are effective whole cell biocatalysts of isoflavone aglycone production.

Light Represses Conidiation in Koji MoldAspergillus oryzae

In the filamentous fungus Aspergillus oryzae, there has been no report on photoreaction. Here we investigated the effect of light in A. oryzae and found that conidiation was repressed by white light. This reaction is contrary to that of other Aspergilli, which show abundant conidiation under light. Moreover, red light also caused reduced conidiation. Genome sequencing of A. oryzae indicated the existence of homologs of some light-related genes in other filamentous fungi. To approach the molecular mechanism of this photoresponse, the effect of red light on the expression level of several genes putatively responsible for conidiation or photoperception, i.e., brlA, a gene known to be required for conidiation, AofphA, the putative homolog of the A. nidulans phytochrome gene fphA, and AoveA, the putative homolog of the negative regulator gene in conidiation in A. nidulans, was examined. These three genes showed no significant response to red light at the transcriptional level. The results indicate that A. oryzae perceives and responds to red light in a manner independent of the transcriptional regulation of these genes.

Changes in extractive components and microbial proliferation during fermentation of fish sauce from underutilized fish species and quality of final products

To make use of underutilized fish species and produce high-quality condiments substitutable for those of mammalian origin, fish sauces were produced on a small industrial scale from three fish species, the flyingfish Cypselurus agoo agoo, the small dolphinfish Coryphaena hippurus, and the deepsea smelt Glossanodon semifasciatus, using salt and koji mold. During 180 days of fermentation, the pH decreased to approximately 4.5 and total nitrogen and free amino acids increased to approximately 2 g/100 mL and 6000 mg/100 mL, respectively. Halophilic and extreme halophilic bacterial counts dramatically increased from 106 to 108 colony forming units/mL between days 14 and 30 and decreased the reafter. In the final products, the pH and salt concentration decreased to between soy sauce and a Vietnamese fish sauce, Nuoc mam. The total amino acid and organic acid contents were almost the same as those of Nuoc mam and soy sauce. Sensory evaluation found these products to have lower smell, saltiness, and bitterness, and higher sweetness and umami taste than Nuoc mam. During fermentation, the histamine content increased in one or two of three fermentation tanks for each fish species. As a result, the histamine content of the final products was higher than that in Nuoc mam.

Gene Silencing by RNA Interference in the Koji MoldAspergillus oryzae

We found the orthologous genes required for RNA interference (RNAi) in the Aspergillus oryzae genome database, and constructed a set of tools for gene silencing using RNAi in A. oryzae. This system utilizes compatible restriction enzyme sites so that only a single target gene fragment is required to create the hairpin RNA cassette. For ease of handling, we also separated the construction of the hairpin RNA cassette for the target gene from its subsequent introduction into the expression vector. Using the brlA gene as a target for RNAi, we detected decreased mRNA levels and a delayed conidiation phenotype in the transformants. Furthermore, even though A. oryzae possesses three copies of the alpha-amylase gene, a single copy of an alpha-amylase RNAi construct was sufficient to downregulate the mRNA levels and decrease the enzymatic activity to 10% of control levels. Gene silencing by RNAi should provide a powerful genetic tool for post-genomic studies of the industrially important fungus A. oryzae.

Study on the Koji Mold Producing Substrate-Possible Application as a Food Supplement

In the production of soy sauce, the koji making stage during which koji mold is cultivated, is the key process for producing a high quality final product. Koji mold and contaminated bacteria act as antagonists as the koji making stage proceeds. As a result, the usable moisture content was reduced and an antibacterial substance was produced. Both of these effects were observed during our experiments. We confirmed the presence of antibacterial substances in koji. We purified the two main components and identified their chemical structures precisely. Furthermore, we examined the minimum inhibitory concentrations (MIC) of these substrates using selected bacteria, yeasts and molds. Out of these microorganisms, antibacterial substances in koji showed the strongest antibacterial activity toward Legionella erythra.

本格焼酎製造におけるソバの原料特性に関する研究 (第1報)

Koji making was carried out using buckwheat grits showed no growth of koji mold on the surfaceof the buckwheat testa. The explanation for this was that the growth of the mold was inhibited by a defensive mechanism against the mold by the plant and the structure of buckwheat testa. To investigate this, confirmation of the inhibitor and defensive mechanisms of the cuticular layer and lignin to themold was performed. These experiments proved that the buckwheat testa did not have an inhibiting, defensive mechanism in the cuticular layer and lignin to the mold. Furthermore, the surface structure of the buckwheat testa was determined by scanning electron microscope analysis. The result showed that the absence of a gap in the surface structure of the buckwheat testa, in contrast to the surface structure of other raw materials such as rice and barley; the structure was not attacked by the enzymes from the koji mold. From these results, it was suggested that the growth of the mold was inhibited due to these complex structures of the buckwheat testa.

醤油麹菌のエキソ-1, 5-α-L-アラビナナーゼの精製と性質の応用

[in Japanese]

胞子形成能の低い麹菌株の簡便な胞子形成法

胞子形成能の低い麹菌株の簡便な胞子形成法