Rice Koji Research Articles

Koji amazake produced by double saccharification contains more isomaltose and modifies the gut microbiota in mice

Koji amazake, which is made from rice and rice koji (a product of Aspergillus oryzae), is a traditional Japanese beverage that has glucose as its main component. It also contains isomaltose, which has been reported to have various functionalities related to gut health. In the present study, we attempted to produce amazake with a higher concentration of isomaltose without using any additives by focusing on the saccharification step of rice koji production as a means of creating new value for amazake. Two types of rice koji that were obtained at different fermentation time points were used, and we changed the saccharification process from the usual one step of saccharification to two steps of saccharification using a different type of rice koji for each step. The amazake made by double saccharification (DSA) contained 20 times more isomaltose than the commercial amazake products. In an in vivo study, oral administration of the DSA modified the cecal microbiota in mice. Moreover, changes were seen in the abundances of several gut microorganisms, such as Anaerotignum lactatifermentans, Muribaculum intestinale, and Parabacteroides merdae. These findings indicate that our novel method may be useful for producing amazake with a high isomaltose content that may have health benefits in humans.

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

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.

Carbon stable isotopes of glucose during the degradation of rice by the koji fungus Aspergillus oryzae

Glucose, a key component of traditional Japanese fermented foods, is derived from rice starch via saccharification by hydrolytic enzymes produced by Aspergillus oryzae. The δ13C value of glucose reflects that of its rice source. However, the influence of saccharification parameters (glucose concentration, degradation temperature, and reaction time) on glucose δ13C values is unclear. Here, we investigated the influence of saccharification on the δ13C value of glucose. Our experiments showed a significant difference in the δ1³C value of glucose (−27.0 ± 0.1 ‰) obtained from saccharification compared to the ingredient rice (−27.1 ± 0.1 ‰) and remaining solid residue (−27.1 ± 0.1 ‰); however, it did not differ significantly from those of rice koji (−27.0 ± 0.1 ‰) and steamed rice (−27.1 ± 0.1 ‰), despite all values being within 0.1 ‰. Notably, glucose concentration, degradation temperature, and reaction time did not significantly affect glucose δ13C values. These findings demonstrate the remarkable preservation of glucose δ13C values. The δ13C values remain aligned with the original δ13C value of the rice, even with up to 60 % degradation during A. oryzae saccharification. This persistence of the δ13C value throughout the process offers a potential tool for authenticating the origin of rice-fermented beverages based on the δ13C value of their glucose component.

Enhancing miso production with optimized puffed-rice koji and its biological function evaluation

In order to enhance enzyme activities of koji and further improve the health benefits and nutritional value of miso, puffed rice was used to prepare koji with optimization of fermentation utilizing response surface methodology (RSM), and the optimized puffed rice koji was used to produce miso and low-salt miso. Results showed that β-glucosidase activity increased by 1.98-fold after RSM optimization. The optimized puffed rice koji also showed the highest β-amylase and protease activities (237.1 and 968.8 units/g, respectively) compared to rice koji (108.7 and 637.4 units/g, respectively). Moreover, miso made from puffed-rice koji presented a much higher polyphenol content, antioxidant activity, and aglycone conversion. These findings clarified the relationship between changes in enzyme activities in koji and the important components of miso, and also advance solid-state fermentation research and development of functional miso.

Identification and enzymatic properties of arginine decarboxylase from Aspergillus oryzae.

Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.

Rice grain structural characteristics of sake rice cultivar Hakutsurunishiki for daiginjo-shu brewing.

Hakutsurunishiki is a sake rice cultivar bred using Yamadabo (seed parent) and Wataribune 2 (pollen parent), equivalent to a Yamadanishiki sibling. This study evaluated the structural characteristics of the Hakutsurunishiki rice grain that contribute to the brewing characteristics of daiginjo-shu, via a comparison with Yamadanishiki. Hakutsurunishiki brown rice was a little heavy and had a large white core. Observing a cross-section of white rice after soaking revealed that the rice grain structure of Hakutsurunishiki was different from that of Yamadanishiki. Hakutsurunishiki white rice showed fewer voids than Yamadanishiki, promoting a slower water absorption rate. Glucose distribution in rice koji obtained by mass spectrometry imaging showed that Hakutsurunishiki rice koji, like Yamadanishiki, is tsuki-haze type, suggesting that its grain structure is suitable for making rice koji for daiginjo-shu. With these observations, we were able to clarify the structural characteristics of Hakutsurunishiki rice grain.

Development of fermented adzuki bean pastes using rice koji and their physicochemical properties

Development of fermented adzuki bean pastes using rice koji and their physicochemical properties

Traditional microbial control methods used in sake brewing effectively suppress predominant bacteria emerging during production of rice koji

Rice koji is a raw material used in the production of sake; however, details regarding the microbial flora and their dynamics in rice koji during the production process are poorly understood. Clarifying these issues can contribute to proposing a method and evaluation that will improve the quality of rice koji and sake. The aim of this study was to determine the microflora in rice koji and the effectiveness of the traditional microbial control techniques used in the sake production process. We analyzed the diversity and changes in bacterial flora during rice koji production by amplicon sequencing of the 16S rRNA gene. The predominant taxon in all rice koji samples was family Staphylococcaceae. The microbial population and the changes in its distribution for five consecutive stages in rice koji production were examined by direct colony counting. Bacteria counts in all samples were below the limit of detection initially, then increased rapidly toward the final stage. The predominant bacterial colonies from all samples were yellow and were identified as Staphylococcus gallinarum through 16S rRNA gene sequence similarity. The S. gallinarum isolates exhibited faster growth in pregelatinized rice medium. Interestingly, the growth of S. gallinarum isolates was suppressed by low temperature (12 °C), ethanol concentration (≥6%) and the addition of lactic acid, which are traditional microbial control methods used during sake fermentation. Therefore, proper control of the traditional sake production process can effectively inhibit the growth of undesirable bacteria such as S. gallinarum that emerge during the production of rice koji.

Carbon stable isotopic compositions of glucose and ethanol in sake after simultaneous saccharification and fermentation processes.

The carbon stable isotopic composition of glucose is transferred to that of ethanol during the simultaneous saccharification and fermentation processes of sake production. However, there is limited information regarding carbon isotope discrimination between the ingredient rice and the sake components. Our fermentation experiments show that the carbon stable isotopic composition of rice is intermediate between those of glucose and ethanol in sake and do not differ significantly from those of rice koji and sake lees. Carbon isotope discrimination from rice to ethanol and from glucose to ethanol is 0.9±0.1‰ (mean±standard deviation, n=18) and 1.9±0.2‰, respectively. This is approximately half of the isotope discrimination observed in grape wine due to the saccharification process during sake manufacture. Carbon isotope discrimination from ingredient rice to sake components provides valuable insights for the manufacturing process and the authentication of sake.

Expression of heterochromatin protein 1 affects citric acid production in Aspergillus luchuensis mut. kawachii

A putative methyltransferase, LaeA, controls citric acid production through epigenetic regulation of the citrate exporter gene, cexA, in the white koji fungus Aspergillus luchuensis mut. kawachii. In this study, we investigated the role of another epigenetic regulator, heterochromatin protein 1, HepA, in citric acid production. The ΔhepA strain exhibited reduced citric acid production in liquid culture, although to a lesser extent compared to the ΔlaeA strain. In addition, the ΔlaeA ΔhepA strain showed citric acid production similar to the ΔlaeA strain, indicating that HepA plays a role in citric acid production, albeit with a less-significant regulatory effect than LaeA. RNA-seq analysis revealed that the transcriptomic profiles of the ΔhepA and ΔlaeA strains were similar, and the expression level of cexA was reduced in both strains. These findings suggest that the genes regulated by HepA are similar to those regulated by LaeA in A.luchuensis mut. kawachii. However, the reductions in citric acid production and cexA expression observed in the disruptants were mitigated in rice koji, a solid-state culture. Thus, the mechanism by which citric acid production is regulated differs between liquid and solid cultivation. Further investigation is thus needed to understand the regulatory mechanism in koji.

Analysis of the effect of rice wine koji on the fermentation quality of rice wine based on high-depth sequencing

Rice wine koji is made from “ChengQu” as the parent species and is prepared by enriching microorganisms in the environment, which plays a decisive role in the formation of quality rice wine. This study explored the microbiome of 18 rice wine koji collected from Xiaogan, Fangxian, Hubei, Dazhou, and Sichuan and analyzed the sensory quality and microbiome of rice wine made with rice koji as a fermentation agent. Although our results showed that there were large differences in microbial communities and functions in rice wine koji, samples from the same region had a higher similarity, and the closer the distance, the higher the similarity. Five suspected new species (Lactococcus sp., Weissella sp., Lactobacillus sp., and Acetobacter sp.) were discovered by ultra-high-depth metagenomic sequencing; their functional maps and phase correlation analysis showed that they contributed positively to the aroma of rice wine. The sensory quality and microbial community structure of rice wine fermented with different rice wine koji have obvious differences, consistent with the structural difference of microorganisms in rice wine koji in different regions. These findings indicate that rice wine koji directly determines the structure of microorganisms in rice wine and directly or indirectly affects the sensory quality of rice wine.

A study on the volatile compounds in Japanese alcoholic beverage (Awamori) produced from Thai rice varieties

Awamori is a famous alcoholic beverage brewed in the Southern part of Japan. Thai rice is well known and there are many varieties of rice including white rice and pigmented rice. This research aimed to study the properties and volatile compounds of Awamori produced from five Thai rice varieties including KDML105, Chainat, RD6, brown KDML105, and riceberry rice. Rice koji was prepared by inoculating the steamed rice with tane-koji from Aspergillus awamori at 0.2% by weight. Then, water and Saccharomyces cerevisiae were added and alcoholic fermentations were performed at around 2 weeks. The production of alcohol and pH changes during the liquid fermentation were monitored, and volatile compounds of the Awamori were determined by gas chromatography-mass spectrophotometry (GC-MS). The highest soluble solid and alcohol content were observed in the fermentation of KDML 105. The alcohol content after 2 weeks of fermentation ranged from 14.5-18 %, with the pH around 4.02-4.5. The same volatile compounds, ethyl alcohol, isobutyl alcohol, isoamyl alcohol, phenylethyl alcohol, isoamyl acetate, ethyl laurate, ethyl linoleate, with the flavor descriptions as alcohol, wine-like, mushroom-like, floral, rose, fruity, banana, sweet, ice-cream, mild fatty, oily, and honey-like odors were detected in all samples from Thai rice while KDML105 presented greater diversity of volatile compounds than the other Thai rice.

Solid-state fungal fermentation transforms low-quality plant-based foods into products with improved protein quality

To meet global protein demand sustainably in the future, we have to move to alternative, non-animal sources. A problem of many plant-derived foods is their low protein quality compared to animal-based proteins, in particular their deficiency in lysine. To improve the protein quality, plant proteins could be converted to fungal proteins using solid-state fungal fermentation (SSFF). In this project Rhizopus microsporus var. oligosporus and Aspergillus oryzae were used to ferment barley and rice, producing tempeh and koji, respectively. SSFF yielded products with 6–13% (dry weight basis) fungal biomass. Protein quality was defined by the parameters indispensable amino acid index (IAAI) and protein digestibility corrected amino acid score (PDCAAS). SSFF improved both parameters in all samples. Lysine was the limiting amino acid in unfermented barley and rice, with PDCAAS of 0.54 and 0.57, respectively. SSFF increased the PDCAAS of lysine by approximately 30% in barley koji, barley tempeh and rice koji and 10% in rice tempeh. These results demonstrate the use of SSFF to improve the protein quality of staple foods. Thereby, this fermentation method can aid in meeting protein demands sustainably in the future through plant-based diets.

Nutritional Improvement of Germinated Riceberry Rice (Oryza sativa) Cultivated with Pleurotus ostreatus Mycelium

Riceberry rice is a whole grain rice variety, which is rich in vitamin E, anthocyanin, phenolic acids, flavonoids, phytosterol antioxidants and fiber. The nutritional of Riceberry rice could be further improved by germination process, with this process the g-aminobutyric acid (GABA) in rice samples is increased. Furthermore, solid stage fermentation (SSF) of rice and grains with fungi have been employed in the food industry such as rice fermented with Aspergillus oryzae for rice koji production, or soybean fermented with Rhizopus oryzae to produce Tempeh. This fermentation step is not only for the food preservation but also improve the nutritional in foods. In this study, the germination and fermentation with Pleurotus ostreatus mycelium can improve the nutritional content present in Riceberry rice. The amounts of GABA, anthocyanin, and bioactive substances contained in Riceberry rice (RR), germinated Riceberry rice (GR) and germinated Riceberry rice with P. ostreatus mycelium (MR) were compared. During germination, the g-aminobutyric acid (GABA) content of RR increased by 71 and 73 % when further fermented with P. ostreatus mycelium. The highest level of GABA was found in the MR (38.58±0.29 mg/100 g) treatment. Furthermore, an untargeted metabolomics approach using Liquid Chromatograph Quadrupole Time-of-Flight Mass Spectrometer; LC-QTOF, revealed an abundance of various amino acids such as valine, alanine, histidine, methionine, L-glutamic acid as well as essential fatty acids such as steric acid and linoleic acid in germinated Riceberry rice with P. ostreatus mycelium samples. The results from this study could lead to nutritional improvement of Riceberry rice through fermentation with P. ostreatus mycelium, which could be applied in further rice flour applications and the product could be recognized as functional food. HIGHLIGHTS The GABA content in Riceberry rice increased by 71 % during germination and 73 % when germinated Riceberry rice was further fermented with ostreatus mycelium Amino acids, fatty acids, and phenolic compounds differed significantly among Riceberry rice (RR), germinated Riceberry rice (GR) and germinated Riceberry rice with ostreatus mycelium (MR) Contents of different amino acids in Riceberry rice were significantly increased when the germinated Riceberry rice was fermented with ostreatus mycelium Essential fatty acids such as Stearic acid and Linoleic acid were the highest in the germinated Riceberry rice with ostreatus mycelium sample GRAPHICAL ABSTRACT

Microbial ecology of Australian commercial rice koji and soybean miso

AbstractBackgroundRice koji and miso are growing in popularity globally. This is because of both the unique sensory properties of the foods, and the belief that fermented foods may be beneficial for our health. This study aimed to increase the collective understanding of the physiochemical properties and microorganisms found in rice koji and miso.ResultsThe microbial communities within rice koji and soybean miso were analyzed using physiochemical, microbiological and metagenomic methods. Metagenomic analysis found Bacillus and Weizmannia were the dominant bacterial genera in rice koji samples, and Tetragenococcus, Staphylococcus and Bacillus predominant in the miso samples. Aspergillus was the predominant fungal genera in all three rice koji samples, and in five of the seven miso samples. Debaryomyces was the dominant genera in one miso sample, while no fungal microorganisms were identified in the other miso sample. The physiochemical analysis found rice koji water activity (Aw) between 0.90 and 0.91 and pH from 5.3 to 5.6. The miso samples showed Aw ranging from 0.73 to 0.88, pH between 4.2 and 5.3, and salt concentration from 6.4% to 15.5%.ConclusionThe predominant genera detected and physiochemical properties in each sample varied according to supplier, length of fermentation and ratio of ingredients used. Details of the microorganisms identified in varieties of unpasteurised rice koji and miso can be used to assist producers with food safety processes, manipulate the flavor of the product and enable accurate labeling of the foods.

Food safety considerations in the production of traditional fermented products: Japanese rice koji and miso

AbstractWhile established in Asia, rice koji and miso are fermented foods that are becoming more popular in western countries. They have been shown to contain a variety of microorganisms, consisting of bacteria, yeasts, and fungal species. Many contemporary miso varieties are not pasteurized as consumers are looking for more natural products, and/or have the desire to consume fermented foods containing live microorganisms. While correctly prepared fermented foods are rarely associated with food safety outbreaks, incidences have been recorded. On these occasions, pathogenic, or spoilage microorganisms were introduced into the products from external sources such as the raw material or the processing environment. Consequently, hygiene and fermentation conditions need to be carefully monitored to ensure food safety. Furthermore, many of the production steps during koji and miso manufacture do not fit into contemporary food safety guidelines for foods. Although pH is a required food safety hurdle for fermented foods, this does not apply to nonacidic foods such as koji or miso. This review focuses on control of microbial pathogens and discusses the processes of miso fermentation, and how fermentation of rice koji and miso fits with current food safety hurdles in western countries.

Changes in volatile aroma profiles of soybean, rice, and wheatkojiduring fermentation using stir bar sorptive extraction followed by gas chromatography‐mass spectrometry and descriptive sensory analysis

AbstractDoenjang, a fermented soybean paste, is a traditional food ingredient in Korea. While the flavor ofdoenjangis heavily dependent onkoji, studies on the aroma characteristics ofkojiremain limited. This study aimed to determine the changes in the aroma profiles of soybeankoji(SBK), ricekoji(RK), and wheatkoji(WK). Aroma analysis was performed using stir bar sorptive extraction followed by gas chromatography‐mass spectrometry (GC‐MS) and descriptive sensory analysis. In SBK, bean‐associated aroma was strongly developed due to the lipid‐derived compounds, whereas in RK, sweet aromas were relatively strong at 72 h due to the sweet‐smelling ester compounds. In WK, the nutty cracker attribute was significantly strengthened during fermentation due to the high concentration of aldehydes. The results showed that the aroma profile ofkojiwas influenced by main ingredient, and this finding is useful in improving aroma development with the use ofkojiin the fermented food industry.Practical ApplicationKojiis one of the key ingredients utilized in commercializeddoenjangmaking process in Korea, in order to accelerate the fermentation process. In this study, influences of raw materials ofkojiin the aroma development ofdoenjangwere investigated using descriptive sensory analysis and instrumental flavor analysis using GC‐MS. Raw material‐driven aroma compounds were documented in differentkojisamples, in which influences the aroma characteristics ofdoenjangmade withkojimade with different raw materials. Finding from this study can aiddoenjangmanufacturers for versatile aroma development of commercializeddoenjang.

Study of Inducing Factors on Resveratrol and Antioxidant Content in Germinated Peanuts.

When peanuts germinate, bioactive compounds such as resveratrol (RES), γ-aminobutyric acid (GABA), isoflavones, and polyphenol compounds are generated. Peanut kernels were germinated in the dark for two days, and stimuli including soaking liquid, rice koji, high-pressure processing (HPP), and ultrasonic treatment were tested for their ability to activate the defense mechanisms of peanut kernels, thus increasing their bioactive compound content. The results of this study indicate that no RES was detected in ungerminated peanuts, and only 5.58 μg/g of GABA was present, while unstimulated germinated peanuts contained 4.03 µg/g of RES and 258.83 μg/g of GABA. The RES content of the germinated peanuts increased to 13.64 μg/g after soaking in 0.2% phenylalanine solution, whereas a higher GABA content of 651.51 μg/g was observed after the peanuts were soaked in 0.2% glutamate. Soaking peanuts in 5% rice koji produced the highest RES and GABA contents (28.83 µg/g and 506.34 μg/g, respectively). Meanwhile, the RES and GABA contents of HPP-treated germinated peanuts (i.e., treated with HPP at 100 MPa for 10 min) increased to 7.66 μg/g and 497.09 μg/g, respectively, whereas those of ultrasonic-treated germinated peanuts (for 20 min) increased to 13.02 μg/g and 318.71 μg/g, respectively. After soaking peanuts in 0.5% rice koji, followed by HPP treatment at 100 MPa for 10 min, the RES and GABA contents of the germinated peanuts increased to 37.78 μg/g and 1196.98 μg/g, while the RES and GABA contents of the germinated peanuts treated with rice koji followed by ultrasonic treatment for 20 min increased to 46.53 μg/g and 974.52 μg/g, respectively. The flavonoid and polyphenol contents of the germinated peanuts also increased after exposure to various external stimuli, improving their DPPH free radical-scavenging ability and showing the good potential of germinated peanuts as functional products.

ホスファターゼ低生産麹菌株KBN8048を用いた非加熱だし入り米味噌のイノシン酸分解特性

非加熱だし入り米味噌を製造するための一助として, ホスファターゼ低生産麹菌株の米味噌への適用性を検討した. ホスファターゼ低生産麹菌株KBN8048 (A. oryzae) または変異株 (KBN8048-m671及びKBN8048-m671-mm558) は, 味噌醸造用一般菌株である丸福EM-2号と比べて, 米麹の酸性ホスファターゼ活性がリン酸ナトリウムの併用によって減少した. また, 30℃で60日間温醸した米味噌に0.2 %のイノシン酸を添加してイノシン酸残存率が約50 %にまで低下する期間を品質保持期限とすると, 4℃保存した場合は味噌醸造用一般菌株では1週間で, ホスファターゼ低生産麹菌株または変異株では7週間であり, その優位性が明らかになった. 30℃保存でも, ホスファターゼ低生産麹菌株または変異株は味噌醸造用一般菌株よりも高いイノシン酸分解抑制が認められた. したがって, ホスファターゼ低生産麹菌株または変異株を使用した米味噌は, 非加熱であってもイノシン酸の分解抑制についての有効性が実証され, 非加熱だし入り米味噌の開発に向けた新たな知見が示された.