Rice Koji Research Articles

Production characteristics of the antioxidant ingredient “pyranonigrin-A” using <i>shochu-koji</i> fungi

In this study, the production characteristics of pyranonigrin-A by sixteen Aspergillus strains using rice koji, barley koji, and buckwheat koji were investigated. Results showed that A. oryzae had little capacity for producing pyranonigrin-A. In contrast, it was found that A. luchuensis had a production capacity for pyranonigrin-A. In addition, it was estimated that the difference between DPPH radical-scavenging activities A. oryzae and those of A. luchuensis depended on the difference in the production of pyranonigrin-A. More pyranonigrin-A could be produced by using buckwheat for raw materials rather than rice or barley. It was clarified that the difference in the amount of pyranonigrin-A by shochu-koji fungi depended on the difference of the pyranonigrin-A production per conidial production and unit conidium.

Characterization of amylolytic enzyme overproducing mutant of Aspergillus luchuensis obtained by ion beam mutagenesis.

Aspergillus luchuensis is a kuro (black) koji fungus that has been used as a starch degrader for the awamori- and shochu-making industries in Japan. In this study, we investigated the effect of ion beam irradiation on A. luchuensis RIB2601 and obtained a high starch-degrading mutant strain U1. Strain U1 showed reduced growth rate, whereas it showed higher α-amylase, glucoamylase, and α-glucosidase activities on a mycelial mass basis than the wild type (wt) strain both on agar plates and in rice koji. In addition, strain U1 showed higher N-acetylglucosamine content in the cell wall and higher sensitivity to calcofluor white, suggesting a deficiency in cell wall composition. Interestingly, produced protein showed higher expression of acid-labile α-amylase (AmyA) and glucoamylase (GlaA) in strain U1, although real-time RT-PCR indicated no significant change in the transcription of the amyA or glaA gene. These results suggested that the high amylolytic activity of strain U1 is attributable to a high AmyA and GlaA production level, but the elevated production is not due to transcriptional regulation of the corresponding genes. Furthermore, RNA-seq analysis indicated that strain U1 shows transcriptional changes in at least 604 genes related to oxidation-reduction, transport, and glucosamine-containing compound metabolic processes, which may be involved in the deficient cell wall composition of strain U1.

Characteristic odour compounds inshochuderived from ricekoji

Shochu, a traditional Japanese distilled liquor, makes use of rice koji, which is koji mould grown on rice grain. Rice koji is an essential ingredient of Japanese liquors such as shochu, and it plays a role as a source of the enzyme to degrade starch. However, there has been no research on the effect of rice koji on the flavour of shochu. Therefore, in this study, the volatile compounds in shochu derived from rice koji were investigated. Two shochu samples were prepared to assess the contribution of rice koji to the flavour. One shochu sample was prepared from rice koji, yeast and water (rice koji-shochu). The other shochu sample was obtained from steamed rice and various enzymes instead of rice koji (enzyme-shochu), along with yeast and water. The volatile compounds were analysed by gas chromatography–mass spectrometry (GC-MS) and GC-MS/olfactometry with aroma extract dilution analysis. The results showed that enzyme-shochu had a higher flavour dilution value of dimethyl trisulphide and hexanal, whereas rice koji-shochu had a higher flavour dilution value of some ester compounds that imparted aromatic odours such as fruity. Some unknown peaks representing compounds that impart characteristic odours such as soda, potato, lavender, and tea-like were specifically detected in rice koji-shochu. The concentrations and calculated odour active values of 14 compounds were measured. These results showed that isovaleraldehyde, ethyl caprylate, ethyl caproate and ethyl 2-methylbutyrate played an important role in imparting the specific odour of rice koji-shochu. Copyright © 2016 The Institute of Brewing & Distilling

Effects of rice koji inoculated with Aspergillus luchuensis on the biochemical and sensory properties of a sailfin sandfish (Arctoscopus japonicus) fish sauce

SummaryThe effects of rice koji inoculated with Aspergillus luchuensis on the biochemical and sensory properties of fish sauce produced from sailfin sandfish (Arctoscopus japonicus) with different salt concentrations (10% and 20%) were investigated over 10 months. Fish sauces prepared from sailfin sandfish with rice koji and a low salt concentration had higher total nitrogen, amino nitrogen and total free amino acid than did those without rice koji or with a high salt concentration. Sensory evaluation of fish sauces prepared with rice koji and a low salt concentration found better taste, flavour and overall acceptance than those prepared without rice koji during fermentation (P < 0.05). The use of rice koji increased the total nitrogen, amino nitrogen and free amino acid and also enhanced the sensory qualities during fermentation. These results suggest that the use of rice koji can result in a low‐salt fish sauce with better flavour.

Metabolomic Profiles of Aspergillus oryzae and Bacillus amyloliquefaciens During Rice Koji Fermentation.

Rice koji, used early in the manufacturing process for many fermented foods, produces diverse metabolites and enzymes during fermentation. Using gas chromatography time-of-flight mass spectrometry (GC-TOF-MS), ultrahigh-performance liquid chromatography linear trap quadrupole ion trap tandem mass spectrometry (UHPLC-LTQ-IT-MS/MS), and multivariate analysis we generated the metabolite profiles of rice koji produced by fermentation with Aspergillus oryzae (RK_AO) or Bacillus amyloliquefaciens (RK_BA) for different durations. Two principal components of the metabolomic data distinguished the rice koji samples according to their fermenter species and fermentation time. Several enzymes secreted by the fermenter species, including α-amylase, protease, and β-glucosidase, were assayed to identify differences in expression levels. This approach revealed that carbohydrate metabolism, serine-derived amino acids, and fatty acids were associated with rice koji fermentation by A. oryzae, whereas aromatic and branched chain amino acids, flavonoids, and lysophospholipids were more typical in rice koji fermentation by B. amyloliquefaciens. Antioxidant activity was significantly higher for RK_BA than for RK_AO, as were the abundances of flavonoids, including tricin, tricin glycosides, apigenin glycosides, and chrysoeriol glycosides. In summary, we have used MS-based metabolomics and enzyme activity assays to evaluate the effects of using different microbial species and fermentation times on the nutritional profile of rice koji.

Formation of Guaiacol by Spoilage Bacteria from Vanillic Acid, a Product of Rice Koji Cultivation, in Japanese Sake Brewing.

The formation of guaiacol, a potent phenolic off-odor compound in the Japanese sake brewing process, was investigated. Eight rice koji samples were analyzed, and one contained guaiacol and 4-vinylguaiacol (4-VG) at extraordinarily high levels: 374 and 2433 μg/kg dry mass koji, respectively. All samples contained ferulic and vanillic acids at concentrations of mg/kg dry mass koji. Guaiacol forming microorganisms were isolated from four rice koji samples. They were identified as Bacillus subtilis, B. amyloliquefaciens/subtilis, and Staphylococcus gallinarum using 16S rRNA gene sequence. These spoilage bacteria convert vanillic acid to guaiacol and ferulic acid to 4-VG. However, they convert very little ferulic acid or 4-VG to guaiacol. Nine strains of koji fungi tested produced vanillic acid at the mg/kg dry mass koji level after cultivation. These results indicated that spoilage bacteria form guaiacol from vanillic acid, which is a product of koji cultivation in the sake brewing process.

Anti-obesity effects of gochujang products prepared using rice koji and soybean meju in rats.

The Korean traditional hot sauce gochujang has been reported to have biological activities. Different kinds of gochujang products were prepared based on combinations of a fungal rice koji with two kinds of bacterial soybean mejus. Diets that included gochujang products were fed to rats and anti-obesity effects were investigated. Gochujang products reduced body weight gains, epididymal fat weights, and triglyceride levels in the serum and the liver. Effects were exerted by the diet that included the non-fermented gochujang mixture, increased using a fungal rice koji, and further enhanced using a bacterial soybean meju. Dietary effects were apparently induced via inhibition of the lipogenic enzymes fatty acid synthase, malic enzyme, and lipoprotein lipase by gochujang products in epididymal adipose tissues, and inhibition of glucose-6-phosphate dehydrogenase in the liver. High levels of capsaicin and genistein in gochujang products are considered to contribute to anti-obesity effects.

Extension of Chronological Life in <i>Saccharomyces cerevisiae</i> under Ethanol Stress by Thermally Processed Rice <i>Koji</i> Extracts

The effect of thermally processed rice koji extracts on survival of yeast Saccharomyces cerevisiae was examined in comparison with non-heated koji extract. In chronological life span (CLS) tests on high-sugar fermentation, the survivals of the yeast cells grown with heated koji extracts were higher than non-heated koji extract. Heat treatment of the extracts by autoclaving led to a loss in most of amino acids due to the Maillard reaction, although histidine contents slightly increased. In glucose-arginine mixtures, arginine was partly converted to histidine by autoclaving and the addition of histidine prolonged the CLS of yeast cells. The yeast cells grown with the non-heated extracts were more resistant to oxidative stress whereas the antioxidant activities were lower than those of the heated extracts. The yeast cells grown with the heated extracts were more tolerant to ethanol and had a higher reduction capacity in the late stationary phase when the cells were incubated in the presence of ethanol. Maillard reaction products elevated the levels of reactive oxygen species to yeast cells grown under ethanol stress in the late stationary phase. These results suggest that thermally processed koji extracts can act as a protectant against ethanol stress during the late stationary phase of yeast growth and extend the CLS due to the increase of histidine contents by autoclaving.

Amelioration of Lead-induced Toxicity in Blood, Liver and Kidney Tissues of Male Wistar Rats by Fermented Ofada Rice

The protective effects of ‘ofada’ rice koji (ORK, fermented ofada rice) and ascorbic acid (AA) against lead (Pb)-induced toxicity in the blood, liver and kidney tissues of male Wistar rats was investigated. The animals were divided into four treatment groups (A – D, n = 5). Groups B, C and D were intoxified by intra-peritoneal injection of 75 mg lead acetate/kg body weight. Groups C and D only had their feed mixed with ORK and AA, respectively. The results showed no significant difference in % packed cell volume (PCV) and Pb concentrations. Feeding with ORK and AA significantly decreased alanine aminotransferase activities (36.50 ± 3.54 and 34.02 ± 0.05 UL-1 respectively) compared to Pb-only treated group (85.50 ± 3.25 UL-1). The ferric reducing antioxidant power (FRAP) for organs increased significantly following intake of feeds mixed with ORK and AA; increases in FRAP was higher for ORK-treated group possibly due to increased total flavonoids concentration following fermentation. Furthermore, Pb-induced high plasma creatinine levels decreased upon treatment with feeds mixed with ORK and ascorbic acid. These findings strongly indicated that feed supplementation with ORK by 45% may be more effective at ameliorating the effects of Pb-induced toxicity in tissues compared to supplementation with AA by 2%.

도정도에 따른 쌀의 침지 특성 및 쌀입국의 발효 특성

본 논문의 목적은 청주의 원료인 쌀의 도정도가 고두밥 및 쌀입국 발효주에 미치는 영향을 조사하는데 있다. 연구방법으로는 쌀도정에 따른 수분흡수량 및 입국특성 등을 분석하였고 연구기간은 8개월 소요되었다. 도정을 많이 할수록 도정미의 단백질 성분구성은 지속적으로 감소되었으며 지질 및 회분 성분은 20% 도정시까지는 급격히 감소하다 그 이상 도정시는 서서히 감소하였다. 도정미의 침지시 도정도가 높을수록 수분흡수 속도가 빨랐으며 최대 수분흡수율도 높았고, 침지 초기에는 수온이 높을 때 수분흡수가 빨랐으나 일정시간 경과후에는 수온이 낮을 때 수분흡수가 지속적으로 이루어져 최대흡수율도 높아졌다. 입국 발효 술덧의 알코올 함량은 도정도10% 입국시료가 16.3-16.9%(v/v)으로 다른 도정도 입국시료의 17.1-17.5%(v/v)인 것에 비해 낮은 알코올함량을 보였고 침지시간은 영향이 없었다. pH는 도정도 10% 입국이 다소 높았으며 그 외엔 유사하였다. 총산함량은 도정도 10%가 0.71-0.76%로 다른 도정도보다 낮은 결과를 보였고 도정도 30%인 시료가 0.93-0.99%로 가장 높게 나타났다. 결론적으로 쌀의 도정도는 쌀의 물리화학적 특성 및 고두밥 상태에 영향을 미치는 것으로 나타났다. We investigated the effects of milling degrees of rice on characteristics of the steamed rice and fermentation of rice koji as a raw material of cheongju. It was studied for 8 months about ratio of water absorption and koji charateristics according to milling degrees. The The more increasing rice milling, the less the protein contents of milled rice was continuously decreased and the lipid and ash contents were sharply decreased to until the milling degrees 20%, and then gradually decreased. When steeping the milled rice, higher milling degrees raised rapid water asorption and higher maximum water asorption, lower water temperature raised gradual water absorption and higher maximum absorption. The longer steeping time, the steamed rice milling degrees 40% showed significantly lower hardness and springness than others. Alcohol contents of the fermented mash with rice koji milling degrees 10% was lower with 16.3-16.9%(v/v) than other sample's 17.1-17.5% (v/v). The total acid contents showed that the milling degrees 10% indicated the lowest content(0.71-0.76%) and the milling degrees 30% indicated highest content(0.93-0.99%). Within the same milling degrees, As the steeping time increases, the total acid contents imcrease. In conclusion, the milling degrees influenced on physicochemical chatacteristics of rice and steamed rice condition.

Formation of ethyl ferulate from feruloylated oligosaccharide by transesterification of rice koji enzyme under sake mash conditions

Formation of ethyl ferulate (EF) and ferulic acid (FA) under sake mash conditions was studied using feruloylated oligosaccharide (FO), prepared from rice grains, as the substrate for rice koji enzyme. EF and FA were produced from FO over six times faster than from alkyl ferulates however, under the same ethanol concentration, only small differences were observed between the EF/FA ratios when either FO or methyl ferulate were used as substrates. Esterification and hydrolysis of FO or methyl ferulate showed similar pH dependencies and similar EF/FA ratios for each substrate in all of the pH ranges tested. Ethanol concentration clearly affected the EF/FA ratio; the ratio increased as ethanol concentration increased. Formation of EF and FA in the sake mash simulated rice digest was accelerated by addition of exogenous FO. These results indicated that supply of FO to sake mash is a crucial step for EF and FA formation, and ethanol is an influencing factor in the EF/FA ratio. The rice koji enzyme reaction suggested that EF and FA are formed through a common feruloylated enzyme intermediate complex by transesterification or hydrolysis, and these reactions occur competitively.

쌀 및 쌀 전분을 활용한 약주의 품질특성

Yakju, a Korean traditional alcoholic beverage, is made from rice and Nuruk. In this study, the fermentation characteristics of Yakju was investigated using rice and rice starch. Ingredients was classified into raw material (rice, rice starch) and starter (enzyme supplements, modified Nuruk, traditional Nuruk, and yellow rice koji) for fermentation. The crude protein content of rice, rice starch, and starter were determined as follows (%): rice 6.69, rice starch 0.44, enzyme supplements 7.84, modified Nuruk 15.29, traditional Nuruk 14.28, and yellow rice koji 7.28. The alcohol content of rice with traditional Nuruk (20.13±0.12%) was higher than other Yakju. The concentration of organic acids of rice starch-based Yakju (389.83~538.34 mg%) was higher than that of rice Yakju (259.27~357.70 mg%). The concentration of nitrogen compound of rice Yakju (498.38~5976.93 ppm) was higher than that of rice starch-based Yakju (600.43~4463.79 ppm). In line with these findings, further studies will be necessary for the quality analysis of the rice, rice starch and fermented starter (enzyme supplements, modified Nuruk, traditional Nuruk and yellow rice koji).

탄소원으로서 입국을 이용한 유산균 발효

Recently, several health benefits of rice wine, makgeolli, were known due to the interest on the traditional Korean liquor and the researches on the rice wine are increasing. Organic acids produced during the process of rice wine fermentation play important roles in the taste and flavor. In this study, we have examined the optimal conditions for lactic acid production in rice koji as a carbon source. Skim milk was also used as a supplementary ingredient for the optimization of lactic acid fermentation. Bacterial growth of Lactobacillus sakei was monitored under this condition. The pH, acidity of the culture and the ethanol tolerance of this bacterium were also tested. Through these experiments, we were able to optimize the growth condition of lactic acid bacteria by the addition of skim milk. This was also able to affect the change of pH, acidity, sugar concentration and alcohol tolerance, which might contribute to the improvement of the quality of rice wine. The optimal condition for the growth was 2 days with 10% (w/v) of skim milk concentration. With these results, it was confirmed that rice koji was an effective carbon source for the growth of lactic acid bacteria.

Quality characteristics of distilled alcohols prepared with different fermenting agents

This study was performed to investigate the quality characteristics of distilled spirits made with different fermenting agents in copper distiller. The highest distillate yield was observed in the mashing made with fermenting agent A. The distillate made with fermenting agent A showed also significantly the highest level of ester and higher-alcohol concentration. In addition, the distilled spirit made with fermenting agent A indicated significantly higher preferences in appearance, smell, taste, quality, and overall acceptability than those of other agents. However, the aging in bottle for 6 months had no significant differences in sensory improvement in distilled spirits regardless in kind of fermenting agents. This study emphasizes the importance of rice koji as fermenting agent in distilled spirit prepared with rice and using with multistage distiller made of copper to improve aroma substances and yield ratio of distillate.

Rice koji reduced body weight gain, fat accumulation, and blood glucose level in high-fat diet-induced obese mice.

Rice koji is considered a readily accessible functional food that may have health-promoting effects. We investigated whether white, yellow, and red koji have the anti-obesity effect in C57BL/6J mice fed a high-fat diet (HFD), which is a model for obesity. Mice were fed HFD containing 10% (w/w) of rice koji powder or steamed rice for 4 weeks. Weight gain, epididymal white adipose tissue, and total adipose tissue weight were significantly lower in all rice koji groups than in the HFD-rice group after 4 weeks. Feed efficiency was significantly reduced in the yellow koji group. Blood glucose levels were significantly lower in the white and red koji groups with HOMA-R and leptin levels being reduced in the white koji group. White and red koji increased glucose uptake and GLUT4 protein expression in L6 myotube cells. These results showed that all rice koji have the anti-obesity or anti-diabetes effects although the mechanisms may differ depending on the type of rice koji consumed.

Factors affecting phenolic acid liberation from rice grains in the sake brewing process

Phenolic acid (ferulic and p-coumaric acid) liberation from rice grains was examined using rice samples containing phenolic acid at different levels, using two sake mash simulated digestion tests to elucidate influencing factors. Phenolic acid levels in a digest made from steamed rice using dialyzed rice koji enzymes were smaller than levels in a rice koji self-digest. Differences in phenolic acid levels among rice samples in the rice koji self-digest were larger than levels in a digest of steamed rice. In the rice koji self-digest, phenolic acid levels in the ingredient rice grains or in the formed digest related to feruloylesterase (FE) activity in the rice koji. Addition of exogenous FE to rice koji self-digestion increased phenolic acid levels, while addition of xylanase (Xyl) showed weak effects. A concerted effect of FE and Xyl was not clearly observed. Addition of ferulic acid to koji made from α-rice grains raised FE activity, but it did not increase the activity of other enzymes. A similar phenomenon was observed in an agar plate culture of koji mold. These results indicated that ferulic acid levels in ingredient rice grains correlate with FE activities of koji, as a resulut, they affect the phenolic acid levels in sake mash.

Analysis of Ferulic and p‐Coumaric Acids in Japanese Rice for Sake Brewing

ABSTRACTFerulic and p‐coumaric acids were analyzed in 50 rice (Oryza sativa L.) samples from 32 cultivars harvested in Japan. In brown rice, ferulic and p‐coumaric acid levels ranged from 309 to 607 mg/kg and from 49 to 100 mg/kg, respectively. In 70% polished rice, ferulic and p‐coumaric acid levels ranged from 27 to 103 mg/kg and from 0.4 to 3.5 mg/kg, respectively. Ratios of average phenolic acid levels in the 70% polished rice to the brown rice were 13.9% for ferulic acid and 1.9% for p‐coumaric acid. The ferulic acid level was highly correlated between brown and 70% polished rice (R = 0.815; P < 0.01), but there was no clear correlation for p‐coumaric acid. Phenolic acid levels in the 70% polished rice did not show any clear correlations between the analytical index measurements for sake brewing suitability (weight of 1,000 grains, water absorption, digestibility, crude protein, and potassium content). Phenolic acid levels in the 70% polished rice directly affected levels in the rice koji enzyme digest. The results indicated that phenolic acid levels in sake were affected by the levels in ingredient rice grains, which may then influence the sensory quality of sake.

쌀과 미입국의 배합비율에 따른 쌀 당화액의 품질특성

The effects of Aspergillus oryzae rice koji (AO) and Asp. kawachii rice koji (AK) as enzyme preparations, on the quality characteristics of rice mash were invested in this study. The amount of AORM (Asp. oryzae rice mash) and AKRM (Asp. kawachii rice mash) were 50, 100, 200% (w/w) based on 100 g of rice. Firstly, in the titer measurement result on the α-amylase and glucoamylase activities of AO and AK. On the other hand, the acid protease activity has values of 31.56 unit for AO and 849.17 unit for AK. The sugar solid of the AORM and AKRM groups significantly increased as the rice koji ratio on rice was higher, which were shown with values as high as 17.63∼20.53 and 17.51∼19.28, respectively. Glucose and maltose were detected for free sugar of AORM. Only glucose was found in AKRM. Citric acid, malic acid, and lactic acid were detected as the organic acid of KORM; oxalic acid, citric acid, and succinic acid were detected for AKRM, and the content increased as the rice koji ratio on rice increased (P<0.05). From the above result, rice koji with useful mold is expected to be used broadly in foods by looking at the fact that it has starch degradation ability and organic acid producibility.

과일과 전통발효제로부터 추출한 단백질분해 조효소의 비교 특성

본 연구에서는 과일(무화과, 키위, 파인애플)에서 추출한 단백질분해 조효소와 전통발효제(개량누룩, 전통누룩, 메주, 입국)에서 추출한 단백질분해 조효소의 특징을 비교하였다. 전통발효제에서 추출한 단백질분해 조효소는 과일에서 추출한 단백질분해조효소보다 높은 온도(70°C), 높은 염 내성(1~3%), 그리고 낮은 pH(3~6)에서 활성을 보였다. 같은 조건에서 활성을 비교하였을 때, 바이오 누룩>입국>전통누룩>무화과>파인애플>메주>키위 순으로 활성이 높았다. 이러한 결과는 과일에서 추출한 단백질분해 조효소보다 전통발효제에서 추출한 단백질분해 조효소의 활성이 우수하고 고온, 산성, 그리고 염의 조건에서도 활성이 저하되지 않으므로, 소시지나 치즈 제조 등의 다양한 식품산업에 이용될 수 있는 가능성을 보여준다.

Formation of ethyl ferulate by rice koji enzyme in sake and mirin mash conditions

Formation mechanism of ethyl ferulate (EF) in sake and mirin mash conditions was investigated to understand EF level control in the manufacturing process. Rice koji formed EF from ferulic acid (FA) and ethanol and decomposed EF to FA. This did not occur in sake yeast and chemical esterification was rare. Esterification of FA and hydrolysis of EF by rice koji might be due to feruloyl esterase(s). The rice koji enzyme showed normal Michaelis-Menten kinetics for FA in ethyl esterification and for EF in hydrolysis, but not for ethanol in the esterification reaction. Substrate specificity of the rice koji enzyme for hydroxycinnamic acids suggested that the main enzyme involved might be similar to type A feruloyl esterase. We studied the rice koji enzyme properties, short-term digestion of steamed rice grains with exogenous ethanol and small scale mirin making with pH adjustment. Our results suggested differences in the esterification and hydrolysis properties of the enzyme, in particular, different pH dependencies and different behaviors under high ethanol conditions; these factors might cause the differing EF levels in sake and mirin mashes.