Koji Preparation Research Articles

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.

Optimizing brewing beer production using Aspergillus oryzae solid-state fermentation of sorghum koji as an adjunct

ABSTRACT This study employed solid-state fermentation (SSF) to substitute 30% barley malt with Kinmen waxy sorghum and Australian non-waxy sorghum inoculated with Aspergillus oryzae for koji preparation in the production of sorghum beer. Optimal conditions for koji preparation were determined, and various beer formulations were developed based on Kinmen sorghum koji. The optimal soaking time for Kinmen waxy and Australian non-waxy sorghum was 2.5 h at room temperature with a water absorption rate of 35%. After steaming, Kinmen sorghum and Australian sorghum exhibited blowup rates of 75.33% and 85.67%, respectively. Five beer types were fermented for 12 days using different mashing methods. Glucose content ranged from 9.93 to 17.6 g/L on the first day, decreasing to 0.03 g/L by the third day. On the 12th day, the alcohol content peaked at 3.03–4.39%, with a pH range of 4.3–5.7. Aroma analysis identified higher alcohols, such as ethanol, 3-methyl-1-butanol, and 2-phenyl ethanol, as the main contributors, with 2-phenyl ethanol imparting a solid rose flavor and ethanol providing a hop aroma and mild taste. Given the global competitiveness of the beer industry, our research emphasizes the use of Kinmen sorghum-based malt, offering unique flavors and a rich aroma, presenting significant growth potential in the market.

Additional moisture during koji preparation contributes to the pigment production of red koji (Monascus‐fermented rice) by influencing gene expression

Additional moisture in preparing red koji, Monascus-fermented rice, is a characteristic production process. To determine how additional moisture affects red koji preparation as per quality, we compared the growth of Monascus purpureus, enzyme and pigment production, and related gene expressions using our findings. We considered two kinds of red koji: one prepared with additional moisture at the middle part of the preparation and the other prepared without additional moisture. Our results showed that additional moisture did not promote the growth of M. purpureus, but it was significantly increased the pigment (red and yellow) and tended to increase the α-amylase level and saccharification power. Although adding a high amount of moisture (approximately 60% moisture content) promoted pigment production, it slightly repressed enzyme production. In contrast, adding approximately 50% moisture content promoted enzyme production. These findings showed that the additional moisture can affect the quality of red koji on the purpose. The expression of 10 pigment biosynthetic gene clusters and two glycohydrolase genes in red koji after adding moisture was analyzed through real-time qPCR. Eight genes were upregulated within 1 hr after adding water, with mppR2 being the first upregulated gene within 30min. The expression of genes as per pigment production quickly responded to additional moisture during solid-state fermentation. Moreover, acetyl-CoA, which is a starting substrate for pigment content in red koji was increased within 3 hr after adding water. This study first described the relationship between additional moisture and expression of pigment biosynthetic genes by Monascus spp. during red koji preparation.

Taro koji of Amorphophallus konjac enabling hydrolysis of konjac polysaccharides to various biotechnological interest.

Due to the indigestibility, utilization of konjac taro, Amorphophallus konjac has been limited only to the Japanese traditional konjac food. Koji preparation with konjac taro was examined to utilize konjac taro as a source of utilizable carbohydrates. Aspergillus luchuensis AKU 3302 was selected as a favorable strain for koji preparation, while Aspergillus oryzae used extensively in sake brewing industry was not so effective. Asp. luchuensis grew well over steamed konjac taro by extending hyphae with least conidia formation. Koji preparation was completed after 3-day incubation at 30°C. D-Mannose and D-glucose were the major monosaccharides found in a hydrolyzate giving the total sugar yield of 50g from 100g of dried konjac taro. An apparent extent of konjac taro hydrolysis at 55°C for 24h seemed to be completed. Since konjac taro is hydrolyzed into monosaccharides, utilization of konjac taro carbohydrates may become possible to various products of biotechnological interest.

Varying Inocula Permutations (Aspergillus oryzae and Bacillus amyloliquefaciens) affect Enzyme Activities and Metabolite Levels in Koji.

In this study, we investigated the altered enzymatic activities and metabolite profiles of koji fermented using varying permutations of Aspergillus oryzae and/or Bacillus amyloliquefaciens. Notably, the protease and β-glucosidase activities were manifold increased in co-inoculated (CO) koji samples (co-inoculation of A. oryzae and B. amyloliquefaciens). Furthermore, gas chromatography-mass spectrometry (GC-MS)-based metabolite profiling indicates that levels of amino acids, organic acids, sugars, sugar alcohols, fatty acids, nucleosides, and vitamins were distinctly higher in CO, SA (sequential inoculation of A. oryzae, followed by B. amyloliquefaciens), and SB (sequential inoculation of B. amyloliquefaciens, followed by A. oryzae). The multivariate principal component analysis (PCA) plot based on GC-MS datasets indicated a clustered pattern for MA and MB (koji samples inoculated either with A. oryzae or B. amyloliquefaciens) across PC2 (20.0%). In contrast, the CO, SA, and SB metabolite profiles displayed segregated patterns across PLS1 (22.2%) and PLS2 (21.1%) in the partial least square discriminant analysis (PLS-DA) model. Intriguingly, the observed disparity in the levels of primary metabolites was engendered largely by higher relative levels of sugars and sugar alcohols in MA, SA, and CO koji samples, which was commensurate with the relative amylase activities in respective samples. Collectively, the present study emphasizes the utility of integrated biochemical and metabolomic approaches for achieving the optimal permutation of fermentative inocula for industrial koji preparation.

Identification of Microbial Profile of Koji Using Single Molecule, Real-Time Sequencing Technology.

Koji is a kind of Japanese traditional fermented starter that has been used for centuries. Many fermented foods are made from koji, such as sake, miso, and soy sauce. This study used the single molecule real-time sequencing technology (SMRT) to investigate the bacterial and fungal microbiota of 3 Japanese koji samples. After SMRT analysis, a total of 39121 high-quality sequences were generated, including 14354 bacterial and 24767 fungal sequence reads. The high-quality gene sequences were assigned to 5 bacterial and 2 fungal plyla, dominated by Proteobacteria and Ascomycota, respectively. At the genus level, Ochrobactrum and Wickerhamomyces were the most abundant bacterial and fungal genera, respectively. The predominant bacterial and fungal species were Ochrobactrum lupini and Wickerhamomyces anomalus, respectively. Our study profiled the microbiota composition of 3 Japanese koji samples to the species level precision. The results may be useful for further development of traditional fermented products, especially optimization of koji preparation. Meanwhile, this study has demonstrated that SMRT is a robust tool for analyzing the microbial composition in food samples.

Effect of low frequency (20–35 kHz) airborne ultrasonication on microbiological and physicochemical properties of soybean Koji

The effect of low frequency airborne ultrasound energy on microbiological and physicochemical properties of soybean koji during preparation was investigated. Following inoculation of steam-heated soybeans with spores of Aspergillus oryzae, koji samples were treated with airborne sonication at increasing frequencies of 20, 25, 30, and 35 kHz and incubated at 30°C for 5 days. Amylase and protease activities, the number of fungal spores, bacterial growth, the moisture content, pH, and the titratable acidity were analyzed during koji preparation. All frequencies of low ultrasound treatment tested enhanced enzyme activities. After continuous ultrasonication at 30 kHz, amylase, and protease activities were significantly (p<0.05) increased by 183 and 205%, respectively, relative to corresponding control groups.

A novel method for beef potentiator preparation and identification of its characteristic aroma compounds

Beef potentiator (BP) is the most popular savoury flavour and regarded as the soul of the modern food industry. In this work, BP was prepared by a novel method with Aspergillus oryzae and Aspergillus niger (BPSF). Three other BPs prepared using commercial enzymes (Protamex, Flavourzyme and papain; BPCEs) were used as controls to investigate its aroma characteristics and related compounds. Sensory evaluation showed that BPSF possessed more favourable and distinctive sauce-like, meat-like, roast and alcoholic attributes when compared with BPCEs. Significantly higher contents (peak areas) and proportions of pyrazines, pyrroles, sulfurous compounds and alcohols in BPSF were responsible for its sensory characteristics, and most of these aroma compounds were derived from microbial metabolism during beef koji preparation and the Maillard reaction. BP prepared by synergistic fermentation with A. oryzae and A. niger is a potential alternative for BP preparation.

Metabolomics-Based Optimal Koji Fermentation for Tyrosinase Inhibition Supplemented with Astragalus Radix

The present study was focused on improving the quality of rice koji by fermentation with a selected Aspergillus oryzae strain and a plant Astragalus radix. A. oryzae KCCM 60345 was used as main inoculant and the Astragalus radix was added as supplement in rice koji preparation. LC-MS based metabolite analysis and tyrosinase inhibitory activities were studied for different time periods. A. oryzae KCCM 60345 fermented rice koji supplemented with Astragalus showed higher tyrosinase inhibition activity at 4 d of fermentation and metabolite analysis with PCA and PLS-DA indicated differences in kojic acid, calycosin-7-O-β-D-glucoside, ononin, calycosin, and formononetin as compared with other forms of rice koji fermentation. By correlation analysis between metabolites and tyrosinase inhibitory activity, calycosin and kojic acid were identified as major tyrosinase inhibitors. Based on these results, we concluded that A. oryzae KCCM 60345 supplemented with Astragalus radix is useful for whitening effects, and we identified optimal conditions for rice koji preparation.

Optimization for rice koji preparation using aspergillus oryzae CJCM-4 isolated from a korean traditional meju

To obtain a useful mold strain in rice-doenjang production, enzyme activities were investigated in rice and wheat koji prepared with 13 different strains isolated from traditional Korean meju. The CJCM-4 strain, which had been selected as a superior strain for the production of protease, was identified as Aspergillus oryzae by the gene sequence of the ITS-5.8S rDNA region as well as aflatoxin production capacity. The enzyme activities of koji prepared with CJCM-4 according to the polishing degree of rice bran were examined. The enzyme activities of koji prepared with 70% milling degree of rice bran were higher than those of other kojis. Protease activity showed its highest level on day 3 at 30°C. Glucoamylase activity showed its highest level on day 2 at 30°C. The optimum enzyme production of koji was determined at different conditions such as incubation time, temperature, moisture, and inorganic salt content. The optimum ratio of water added to the rice for the production of enzymes was 35% on the dry basis of the rice, and the optimum temperature was 25°C. When inorganic salts were added to increase enzyme activity, the combined addition of 0.15% CaCO3 and 0.05% NaH2PO4 resulted in the highest enzyme activities.

Identification of 2,4,6-Trichloroanisole (TCA) causing a musty/muddy off-flavor in sake and its production in rice koji and moromi mash

2,4,6-Trichloroanisole (TCA), which has been identified as the main component responsible for the cork taint in wine, was detected in sake samples having a musty/muddy off-flavor by stir bar sorptive extraction (SBSE). We confirmed that TCA is one of the components causing this off-flavor in sake, as in other alcoholic beverages, from a sensory analysis showing the correlation between TCA concentration and the intensity of the musty/muddy off-flavor. We investigated the route of TCA production in the rice koji preparation process and in the moromi mash process for sake brewing. We found that TCA is produced mainly by the biomethylation of 2,4,6-trichlorophenol (TCP) by rice koji in brewing and that TCP originates from the wooden tools used in preparing rice koji.

原料米水浸漬液へのモノグリセリド添加が麹の性質に及ぼす影響

We investigated the effect of monoglyceride (MG) addition on qualities of koji. Koji was prepared with MG (0.00-0.20% of rice) addition during rice soaking. We examined cell mass, enzyme activity, and quality evaluation on the basis of the analytical data obtained from the koji. The results were as follows: Cell mass and various enzyme activities of koji were increased by MG addition. Quality evaluation of koji with MG addition was higher than koji without MG. These results suggest that MG addition to the soaking water in the koji preparation process might improve the quality of koji.

ナイシン生産性乳酸菌を利用した米麹及び米味噌の製造方法

[in Japanese]

Isoflavone transformation during soybean koji preparation and subsequent miso fermentation supplemented with ethanol and NaCl.

Soybeans were soaked with water for 4 h, steam-cooked, inoculated with the conidia of Aspergillus oryzae, and incubated for 3 days for koji preparation. The koji was then mixed with water-soaked and steam-cooked soybeans (1:2, w/w), ground into paste, and supplemented with 15% ethanol and 12.5% NaCl or 3% ethanol and 6% NaCl for miso fermentation at 30 degrees C. Daidzin, genistin, daidzein, and genistein contents were extracted from the lyophilized and pulverized soybean powder or from the miso homogenate by a developed one-tube procedure and analyzed with an HPLC. After water soaking, daidzein and genistein contents increased markedly, whereas daidzin and genistin contents decreased. Further increases of daidzein and genistein contents and decreases of daidzin and genistin contents were observed after koji mold growth. During fermentation, fungal and lactic acid bacterial (LAB) growth in the miso products was inhibited, whereas soluble protein contents increased much more rapidly in the low-salt miso products supplemented with 3% ethanol and 6% NaCl than the other products. When the 4- and 8-week-fermented miso products were cooked with tofu for sensory evaluation, flavor ratings of the low-salt products were higher than that of a popular commercial product. In both products, the most daidzins and genistins were hydrolyzed after 4 weeks of fermentation. The hydrolytic enzymes contributing to isoflavone transformation originated from soybeans after water soaking and from koji with mold growth. It was of merit that the low-salt fermented products were fairly acceptable in flavor rating and rich in daidzein and genistein contents after 4 weeks of fermentation.

A modified chemical procedure for rapid determination of glucosamine and its application for estimation of mold growth in peanut kernels and koji.

One-step hydrolysis of chitin to release glucosamine for quantitation was achieved by combining a chitin-containing sample (10-200 mg of sample size) in a test tube with 1 mL of 10 M HCl followed by vacuum treatment for 10 min, incubation at 28 degrees C for 30 min, replenishment with 3 mL of deionized water, nitrogen flushing, screw capping, and heat treatment at 140 degrees C for 60 min. A phosphate buffer solution (pH 12.5, 0.2 M) was effective in pH stabilization and enhancing colorimetric determination of glucosamine content. When the modified procedure was applied to analyze glucosamine content in the mycelia of various molds, glucosamine content varied mainly depending on mold species. In estimations of mold growth of the uninoculated peanut kernels incubated under a humidified condition for 5 weeks, cooked rice and soybean inoculated with conidia of Aspergillus oryzae for koji preparation, logarithms of the internal mold populations and glucosamine contents both increased with increases of incubation time. The modified procedure provided a rapid and reliable estimation of mold growth in various substrates.

青ヶ島の芋焼酎製造用粉状麹の製麹特性と糸状菌相粉状麹に関する研究 (第1報)

Sake has been produced from ancient times using Mothi-koji (as cake) in China and Southeast Asia, and Bara-koji (as grain) in Japan.On the other hand, Kona-koji, which differs from these koji, is found in Aogashima. This uses crushed or milled grain with the raw material. There are seven factories in Aogashima, we investigated one of them. We investigated the production of Kona-koji at the factory in Aogashima, and studied the form, microflora and characteristics of Kona-koji used during the preparation of koji. As the koji is made from roasted and milled barley without the addition of a koji starter (Tane-koji), it took a week to prepare the koji. Nettopteris antiqua was used the during preparation of koji. Pink spore and yellow spores were found in the material after 24 hours. The yellow spores grew rapidly in the fast 4days, and the koji was finished after 7 days. Yellow spore were present at 2.7×1011 colonies per gram? koji and black spore were present at 3.1×1010 colonies per gram koji. Therefore, the dominant koji mold were the yellow spores. The molds of 23 strains were isolated from the material during the preparation and completion of the koji. As a result of classification, we found 21 strains of Aspergillus oryzae group and 2 strains of A. niger group. Therefore, the koji mainly consisted of molds belonging to the Aspergillus oryzae with a small number of Aspergillus niger strains present.

青ヶ島の芋焼酎製造用粉状麹の主要糸状菌の由来源粉状麹に関する研究 (第2報)

Kona-koji was prepared without the addition of a koji starter (Tanc-koji) for the production of Imo-shochu. The spores of molds carried in the air affected growth during the preparation of koji. So, we isolated the spores of molds from the vicinity of the places used for the preparation of koji and production of shochu.As shown by the results, a 103 order of colonies per gram·koji of A. oryzae were isolated from kojimuro solids, shochu distillery solids and from the leaves of N. antiqua that are used to wrap the koji A. oryzae spores were also isolated from the air of the koji-muro and shochu distillery. In addition spores of A. niger and other molds were also isolated.We measured the enzyme activity of these strains and investigated their physiological characteristics. Their acidity was higher than that of the standard strains. The characteristics of these strains agreed with those of strains isolated from kona-koji. Therefore, we found that spores of Aspergillus sp. were mixed with the raw material for koji from the leaves of N. antiqua or airborne spores, and these spores grew during the preparation of koji.Further more, spores of other mold contaminating koji were isolated from the leaves of N. antiqua, but they were not detected in the koji. We found that their mold were selected against as the temperature of koji rose to 40C during its preparation.

米麹からの尿素の抽出と製麹中における尿素の経時的変化

We have investigated on conditions for extraction of urea from rice-koji to determine urea content in koji.The most appropriate condition for extraction of urea is as follows: rice-koji is solubilized at 55°C by a saccharifying enzyme (e.g.Gluc S etc.) following heat inactivation of enzymes of koji, and then the amount of urea is determined by the enzymatic assay. Alternatively, the condition under which urea is extracted with distilled water at room temperatureis sufficient for comparing urea contents in various koji samples.When we estimated urea content in rice-koji during koji preparation under these conditions, most of urea contained in rice was consumed by koji mold (Aspergillus oryzae) for its germination in early stage and thereafter the amount of urea in koji increased as koji mold showed vigorous mycelial growth.

Preparation of Koji from corn hulls for alcoholic fermentation without cooking

Corn hulls, the outer peel covering the corn grain, were used for preparation of koji, which was applied to alcoholic fermentation without cooking of raw strachy materials. Corn hull koji had lower saccharifying power, α-amylase, CMCase and xylanase than wheat bran koji, but higher protease and pectinase activities, Its alcoholic fermentations of cassava starch and sweet potato were also superior to those of wheat bran koji: corn hull koji gave 10.3% (v/v) of alcohol with 93% yield from 20 g of cassava starch, while wheat bran koji gave 9.4% (v/v) of alcohol with 90.4% yeild; and corn hull koji gave 9.1% (v/v) of alcohol with 92.6% yielded from 50 g of sweet potato, while wheatbran koji gave 8.1 (v/v) of alcohol with 88.6% yield.

The production of aroma by Aspergillus oryzae during the preparation of soy sauce koji

The rôle of koji in the preparation of soy sauce has been a subject of controversy. A study of the volatiles produced during koji preparation and a comparison with uninoculated bean-flour mixture shows that the growth and sporulation of Aspergillus oryzae is related to the production of volatiles. The volatiles present at the mycelial stage are different from the volatiles of sporulated koji. The ether extracts of the acidic homogenates were different from those of the neutral and alkaline homogenates.