Koji Extract Research Articles

A Comparative Study on the Debittering of Kinnow (Citrus reticulate L.) Peels: Microbial, Chemical, and Ultrasound-Assisted Microbial Treatment

Kinnow mandarin (Citrus reticulate L.) peels are a storehouse of well-known bioactive compounds, viz., polyphenols, flavonoids, carotenoids, limonoids, and tocopherol, which exhibit an effective antioxidant capacity. However, naringin is the most predominant bitter flavanone compound found in Kinnow peels that causes their bitterness. It prohibits the effective utilization of peels in food-based products. In the present study, a novel approach for the debittering of Kinnow peels has been established to tackle this problem. A comparative evaluation of the different debittering methods (chemical, microbial, and ultrasound-assisted microbial treatments) used on Kinnow peel naringin and bioactive compounds was conducted. Among the chemical and microbial method; solid-state fermentation with A. niger led to greater extraction of naringin content (7.08 mg/g) from kinnow peels. Moreover, the numerical process optimization of ultrasound-assisted microbial debittering was performed by the Box–Behnken design (BBD) of a response surface methodology to maximize naringin hydrolysis. Among all three debittering methods, ultrasound-assisted microbial debittering led to a greater hydrolysis of naringin content and reduced processing time. The optimum conditions were ultrasound temperature (40 °C), time (30 min), and A. niger koji extract (1.45%) for the maximum extraction rate of naringin (11.91 mg/g). These debittered Kinnow peels can be utilized as raw material to develop therapeutic food products having a high phytochemical composition without any off-flavors or bitterness.

Formation of taste-active pyroglutamyl peptide ethyl esters in sake by rice koji peptidases

Formation of taste-active pyroglutamyl (pGlu) peptide ethyl esters in sake was investigated: 2 enzymes (A and B) responsible for the esterification were purified from a rice koji extract. MADLI-TOF/TOF analysis after deglycosylation identified enzyme (A) as peptidase S28 (GenBank accession number OOO13707.1) and enzyme (B) as serine-type carboxypeptidase (accession number AO090010000534). Both enzymes hydrolyzed pGlu peptides and formed ethyl esters under sake mash conditions: acidic pH (3-4) and in ethanol (5%-20% v/v) aqueous solutions. Enzyme (A) formed pGlu penta-peptide ethyl esters from pGlu undeca-peptides by a prolyl endo-type reaction. Enzyme (B) formed (pGlu) deca-peptide and its ethyl esters from pGlu undeca-peptides in an exo-type reaction. We are the first to report the enzymatic ethyl esterification reaction in the formation of pGlu peptides by rice koji peptidases.

Ougan juice debittering using ultrasound-aided enzymatic hydrolysis: Impacts on aroma and taste

The optimal sonication conditions (40 kHz, 80 W/L and 60 min) during Ougan juice debittering by Aspergillus niger koji extract were established. Enzymatic hydrolysis degrees of naringin and limonin were enhanced to 89.90% and 36.16%, and enzymatic hydrolysis time was shortened by 33%. Sonication significantly enhanced activities of α-l-rhamnosidases, β-glucosidases and limoninases from A. niger koji extract and facilitated break of CO bonds in naringin (p < 0.05). These accounted for the enhanced enzymatic hydrolysis degrees and velocities of bitter compounds. Meanwhile, sonication lowered 40%, 7% and 21%, 13%, 11%, 25% of bitter, sour tastes and green, citrus-like, floral, woody notes, but enhanced 18% and 15% of fruity and sweet notes, resulting in 38% and 33% increases in over-all taste and aroma scores. Lowered levels of bitter compounds, organic acids, green, citrus-like, floral, woody aroma compounds and enhanced levels of fruity, sweet aroma compounds caused by sonication accounted for the flavor improvements.

Cultivation temperature and length affect the antioxidant activity and total phenolic content of soybean Koji prepared with Aspergillus awamori

In this study, soybean koji was first fermented with Aspergillus awamori, a starter organism commonly used for the fermentation of sake, at 25, 30 or 35°C for a period of 1-5 days. Antioxidant activity, including DPPH radical-scavenging effect and Fe2+-chelating ability, and total phenolic content of the methanol extract of these fermented soybean koji as well as mycelial propagation of starter organism were compared. Results revealed that fermented for a same period of 3 days, the DPPH radical-scavenging effect exerted by the 30°C-koji extract was the highest showing 2.5-3.5 folds of that fermented at 25 or 35°C. The Fe2+-chelating ability exerted by the 30°C-koji extract was also found to be 3.6-3.8 times higher than those fermented at other temperatures. Furthermore, it was also noted that increasing or decreasing cultivation time from 3 days resulted in a decreased antioxidant activity of the koji extract. Cultivation temperature and length were also observed to affect the mycelial propagation and the total phenolic content of the koji extract. Maximum mycelial propagation of A. awamori was achieved after cultivation at 30°C for 3 days. Finally, the highest phenolic content noted in 30°C-koji extract was closely correlated to the highest antioxidant activity observed.

Identification of Rice Koji Extract Components that Increase β-Glucocerebrosidase Levels in Human Epidermal Keratinocytes.

Rice miso contains many ingredients derived from rice koji and has been a valuable source of nutrition since ancient times. We found that the consumption of rice miso led to improvements in the moisture content of cheek stratum corneum, skin viscoelasticity, and skin texture. Further, rice miso extract was found to increase the mRNA expression and activity of β-glucocerebrosidase (β-GCase), an enzyme involved in ceramide synthesis in the stratum corneum, in cultures. In this study, we identified the lipid-derived components of rice koji that increase the β-GCase activity in cultured human epidermal keratinocytes. The methanol fraction of rice koji extract induced an increase in the mRNA expression and activity of β-GCase in keratinocytes. The active fraction of rice koji was found to contain phosphatidic acid (PA) and lysophosphatidic acid (LPA). The total PA concentration in rice koji was 973.9 ng/mg dry weight, which was 17.5 times higher than that in steamed rice. Among the molecular species, PA_18:2/18:2 was the most frequently found. The total LPA concentration in rice koji was 29.6 ng/mg dry weight, and 2-LPA_18:2 was the most frequently found LPA. Since PA and LPA increase the mRNA expression and activity of β-GCase in keratinocytes, they are thought to be the active ingredients in rice koji that increase the β-GCase levels in human epidermal keratinocytes.

Untargeted metabolite profiling for koji-fermentative bioprocess unravels the effects of varying substrate types and microbial inocula

Untargeted metabolomics unraveled the effects of varying substrates (soybean, wheat, and rice) and inocula (Aspergillus oryzae and Bacillus amyloliquefaciens) on metabolite compositions of koji, a starter ingredient in various Asian fermented foods. Multivariate analyses of the hyphenated mass spectrometry datasets for different koji extracts highlighted 61 significantly discriminant primary metabolites (sugars and sugar alcohols, organic acids, amino acids, fatty acids, nucleosides, phenolic acids, and vitamins) according to varying substrates and inocula combinations. However, 59 significantly discriminant secondary metabolites were evident for koji-types with varying substrates only, viz., soybean (flavonoids, soyasaponins, and lysophospholipids), wheat (flavones and lysophospholipids), and rice (flavonoids, fatty acids derivatives, and lysophospholipids). Independently, the substrates influenced primary metabolite compositions in koji (soybean > wheat, rice). The inocula choice of A. oryzae engendered higher carbohydrates, organic acids, and lipid derivative levels commensurate with high α-amylase and β-glucosidase activities, while B. amyloliquefaciens affected higher amino acids levels, in respective koji types.

Improvement of skin conditions by ingestion of Aspergillus kawachii (Koji) extract containing 14-dehydroergosterol in a randomized, double-blind, controlled trial.

PurposeThe present study examined the effect of ingestion of Koji extract containing 14-dehydroergosterol (14-DHE), prepared from Aspergillus kawachii NBRC4308, on improvement of skin conditions among healthy volunteers.Subjects and methodsIn a randomized, double-blind, placebo-controlled, parallel-group study, 70 healthy adult women who felt that their skin was dry ingested either a placebo dietary supplement or Koji extract (200 mg/day) supplement containing 0.1% 14-DHE for 12 weeks. Throughout the treatment period and for 4 weeks afterward, objective indicators – including moisture content of the stratum corneum, trans-epidermal water loss (TEWL), and skin wrinkles – were evaluated; in addition, the subjects answered a questionnaire on their skin conditions with ratings on a visual analog scale. Statistical analysis was conducted on the basis of differences from baseline scores.ResultsCompared with the placebo group, the Koji extract group showed significantly increased forearm moisture at 4, 8, and 16 weeks (p < 0.05 on unpaired t-test). The questionnaire survey showed a marked improvement in skin conditions, particularly crow’s feet, in the Koji extract group versus the placebo group at 8 weeks (p < 0.05 by unpaired t-test). Furthermore, the Koji extract group showed a trend (p < 0.10) toward improvement in skin moisture (at 4 weeks), dryness around the eyes/mouth (at 4 weeks), and overall skin condition (at 8 weeks) versus the placebo group.ConclusionIngestion of Koji extract containing 14-DHE was demonstrated to have positive effects toward improving skin conditions – in particular, on increasing skin moisture in the stratum corneum.

Correction to Rice Koji Extract Enhances Lipid Metabolism through Peroxisome Proliferator-Activated Receptor Alpha (PPARα) Activation in Mouse Liver.

Correction to Rice Koji Extract Enhances Lipid Metabolism through Peroxisome Proliferator-Activated Receptor Alpha (PPARα) Activation in Mouse Liver.

Extension of Chronological Life in &amp;lt;i&amp;gt;Saccharomyces cerevisiae&amp;lt;/i&amp;gt; under Ethanol Stress by Thermally Processed Rice &amp;lt;i&amp;gt;Koji&amp;lt;/i&amp;gt; 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.

Effect of black soybean koji extract on glucose utilization and adipocyte differentiation in 3T3-L1 cells.

Adipocyte differentiation and the extent of subsequent fat accumulation are closely related to the occurrence and progression of diseases such as insulin resistance and obesity. Black soybean koji (BSK) is produced by the fermentation of black soybean with Aspergilllus awamori. Previous study indicated that BSK extract has antioxidative and multifunctional bioactivities, however, the role of BSK in the regulation of energy metabolism is still unclear. We aimed to investigate the effect of glucose utilization on insulin-resistant 3T3-L1 preadipocytes and adipogenesis-related protein expression in differentiated adipocytes with BSK treatment. Cytoxicity assay revealed that BSK did not adversely affect cell viability at levels up to 200 μg/mL. The potential for glucose utilization was increased by increased glucose transporter 1 (GLUT1), GLUT4 and protein kinase B (AKT) protein expression in insulin-resistant 3T3-L1 cells in response to BSK treatment. Simultaneously, BSK inhibited lipid droplet accumulation in differentiated 3T3-L1 cells. The inhibitory effect of adipogenesis was associated with downregulated peroxisome proliferator-activated receptor γ (PPARγ) level and upregulated Acrp30 protein expression. Our results suggest that BSK extract could improve glucose uptake by modulating GLUT1 and GLUT4 expression in a 3T3-L1 insulin-resistance cell model. In addition, BSK suppressed differentiation and lipid accumulation in mature 3T3-L1 adipocytes, which may suggest its potential for food supplementation to prevent obesity and related metabolic abnormalities.

麹汁培地を用いた集積培養液から分離した酵母の性質

麹汁培地を用いた集積培養液から分離した酵母の性質

PRODUCTION OF LOVASTATIN AND SULOCHRIN BY Aspergillus terreus USING SOLID STATE FERMENTATION

Lovastatin is an anti-cholesterol agent that was produced by Aspergillus terreus using solid state fermentation (SSF). During this fermentation process, sulochrin is also produced as an unwanted co-metabolite. However, our previous result showed that sulochrin had potential as antidiabetes because it is an inhibitor agent of a-glucosidase. In this paper, we reported our observation on lovastatin and sulochrin production pattern in relation with inhibitor a-glucosidase activity during eleven days fermentation of A. terreus koji (SSF) ethyl acetate extract. Koji obtained from solid state fermentation with rice as the substrate and incubated at room temperature, sample is taken daily for eleven day (D-1 to D-11). Lovastatin and sulochrin production was measured by Liquid Chromatography- Mass Spectrometer based on their molecular weight m/z 404.5 and 332.3 respectively. The inibitory activity is measured by inhibition model of koji extract against a-glucosidase (EC 3.2.1.20) from Saccharomyces cereviceae. The results show that lovastatin production was started on the day 2 (0.04 mg/g) and achieving the optimal production on day 7 (11.46 mg/g), while sulochrin production was started on day 4 (0.60 mg/g) and keep produced until the end of fermentation period at Day 11 (3.11 mg/g). Koji extract was started to show inhibitory to a-glucosidase activity on Day 5 (IC50= 23.34 mg/mL) and keep showed activity until Day 11 (IC50=3.33 mg/mL). These results suggest that inhibitory activity of koji extract to a- glucosidase activity have relation with sulochrin biosynthesis production.

대두를 이용한 Lovastatin 대량생산용 Seed Culture의 제조기술

Lovastatin (Mevinolin, Monacolin K) is a well-known drug for the therapy of hypercholesterolemia. It is an important fungal secondary metabolite as it inhibits 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) which catalyzes a major rate-limiting step in the biosynthesis of cholesterol. Both soybeans and black soybeans with Aspergillus terreus ATCC 20542 mutant were used as the seed culture for the mass production of lovastatin. The production of lovastatin in soybean seed culture of Asp. terreus was twofold compared to that of black soybean seed culture. The effect of two different vessels (petri dish and Erlenmeyer flask) on lovastatin production was also studied. The production of lovastatin on petri dish was tenfold to that of Erlenmeyer flask. Furthermore, the most lovastatin production on rice bran was achieved when the soybean seed culture was treated by heat shock at 30 o C for 1 hour, representing 82% of HMG-CoA reductase inhibition in the koji extract. We estimated that the heat treated soybean seed culture could be a new method for the mass production of lovastatin.

Production of Bacteriocin by Staphylococcus sp. NPSI 38 in Koji Extract Medium with Rice Protein Hydrolyzate and Its Growth-inhibitory Activity against Hiochi-bacteria

Enhanced production of bacteriocin by Staphylococcus sp. NPSI 38 (NPSI 38) using koji extract medium was investigated. The bacteriocin produced by NPSI 38 in MRS medium was found to be effective for inhibiting the growth of Lactobacillus hilgardii NBRC 15886T, one of the representative hiochi-bacteria. In cultivations using test tubes without pH control, meat extract and Polypepton were effective for bacteriocin production by NPSI 38 with 10%(v/v) koji extract medium. When the koji extract medium supplemented with hydrolyzate of a rice protein preparation was used instead of meat extract and Polypepton, NPSI 38 produced 160 U/ml of bacteriocin in the cultivation with pH control, which was almost as high as that (156 U/ml) observed in cultivation using MRS medium with pH control. When the cells of L. hilgardii NBRC 15886T collected at logarithmic growth and stationary phases were inoculated into fresh modified MRS medium with 11 U/ml of the bacteriocin, negligible cell growth was observed, irrespective of different growth phases of cells inoculated. Little or no increase in cell concentrations in the media containing bacteriocin showed that the action of the bacteriocin produced by NPSI 38 was bacteriostatic against the hiochi-bacterium.

Suppression on the mutagenicity of 4-nitroquinoline- N-oxide by the methanol extracts of soybean koji prepared with various filamentous fungi

In this study, solid fermentation of soybean with various GRAS filamentous fungi including Aspergillus sojae BCRC 30103, Aspergillus oryzae BCRC 30222, Aspergillus awamori, Actinomucor taiwanesis and Rhizopus sp. was performed to prepare various soybean kojis. Toxicity, mutagenicity and suppression on the mutagenesis induced by a direct mutagen, 4-nitroquinoline- N-oxide (4-NQO) on Salmonella typhimurium TA 100, by the various methanol extracts of the prepared soybean koji and unfermented soybean were determined and compared. Results revealed that methanol extracts of unfermented soybean and kojis show no toxicity and mutagenic activity within the dose levels examined on test organism. On the other hand, antimutagenic activity against 4-NQO was observed with the extract of unfermented soybean. Furthermore, fermentation, regardless of the starter organism employed, resulted in an enhanced antimutagenic effect on the mutagenesis of 4-NQO by the extracts of the soybean koji. Across the dose range (0.625–5.0 mg/plate) tested, a dose-dependent antimutagenic activity was observed. Antimutagenic activities of the koji extracts varied with starter organism, with A. awamori-prepared koji extract exhibiting the highest rate of suppression on the mutagenicity of 4-NQO. Further study with A. awamori also revealed that fermentation temperature affected the antimutagenic activity of the prepared koji extract. In general, the extract of the A. awamori-soybean koji prepared at 30 °C showed a higher antimutagenic activity than those prepared at 25 or 35 °C.

Enzymatic Production of Caffeic Acid by Koji from Plant Resources Containing Caffeoylquinic Acid Derivatives

The effect of a koji (Aspergillus awamori mut.) extract on the caffeoylquinic acid derivatives purified from sweetpotato (Ipomoea batatas L.) leaves was examined to develop the mass production of caffeic acid. A koji extract hydrolyzed the caffeoylquinic acid derivatives, chlorogenic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid and 3,4,5-tri-O-caffeoylquinic acid, to caffeic acid. Furthermore, the koji extract also converted the major polyphenolic components from sweetpotato, burdock (Arctium lappa L.), and mugwort (Artemisia indica var. maximowiczii) leaves to caffeic acid. These results suggest that the production of caffeic acid from plant resources containing caffeoylquinic acid derivatives is possible.

麹汁培地を用いて花から分離した酵母の清酒醸造試験

We used natural flowers to isolate yeast strains which have the characteristics of sake yeast, using a medium containing yeastcidin produced by Aspergillus oryzae No.G. Although a synthetic culture medium was employed in previous papers, a koji extract culture medium was applied this time.As a result, Saccharomyces species was isolated from the flower of 48 items among 241 samples collected in Japan. Furthermore, results of the examination of physiological character showed 16 strains were a non-forming sake yeast type. These strains were used in sake brewing tests on a small scale. Analysis of the aroma of these sake tested the strains. The AB-2 strain, which shows fresh organic functions with features to the flavor MR-4, and the KS-3 strains were obtained. In fragrance, the AB-2 strain produced a high concentration of ethyl caproate on a par with the ND-4 strain. The MR-4 strain produced isoamyl acetate higher than the BK-1 strain. The KS-3 strain contained ethyl caproate and isoamyl acetate at a sufficient balance like the NI-2 strain.When the test brewing sake using these three strains of organic acid composition was analyzed, the rate of malic acid to succinic acid (MA/SA ratio) was high. However, the total acidity of the test brewing sake had low values. Moreover, these three strains had a strong fermentation ability in sake mash.

Inhibitory effect of red koji extracts on mushroom tyrosinase.

Red koji has been recognized as a cholesterol-lowering diet supplement because of it contains fungi metabolites, monacolins, which reduce cholesterol synthesis by inhibiting HMG-CoA reductase. In this study, water extracts of red koji were loaded onto a C(18) cartridge, and the acetonitrile eluate was collected as test fraction. Red koji water extracts and its C(18) cartridge acetonitrile eluent had total phenols concentrations of 5.57 and 1.89 mg/g of red koji and condensed tannins concentrations of 2.71 and 1.20 mg/g of red koji, respectively. Both exhibited an antioxidant activity and an inhibitory activity to mushroom tyrosinase. The higher antioxidant activity of the red koji acetonitrile eluent was due to the existence of a high percentage of condensed tannins. The results from the kinetic study for inhibition of mushroom tyrosinase by red koji extracts showed that the compounds in the extracts competitively inhibited the oxidation of tyrosine catalyzed by mushroom tyrosinase with an ID(50) of 5.57 mg/mL.

Yeast-immobilized SPV device for koji quality control in sake brewing process

The malted rice, koji, is an indispensable material for the brewing of sake. It saccharifies rice starch and supplies vitamins for the yeast in sake brewing. Since the quality of sake depends strongly on the quality of koji, quality control of koji is very important in the brewing. There are some methods to measure the activity of enzymes and the quantity of vitamins with the quality of koji. None of these methods, however, directly relate to the yeast metabolism. We constructed a sensor system to monitor the yeast metabolism in sake brewing by use of immobilized Saccharomyces cerevisiae and a Surface PhotoVoltage device (SPV). In this system, S. cerevisiae K701 and K9, designed for use in sake brewing by the Brewing Society of Japan, were employed as immobilized microbe. The pH change due to the production of organic acids in sake brewing is measured using the SPV. A linear relationship was observed between decrease in the photocurrent (the metabolism response) and the concentration to less than 60 mM of glucose ( r=0.990). Then we measured the koji extract and observed the difference of response between K701 and K9 which corresponded to the productivity of acidic substances by batch test.

A novel method for measurement of α-amylase activity in rice koji by capillary isoelectric focusing

A capillary isoelectric focusing (CIEF) method has been developed for the analysis of α-amylase activity in rice koji. The standards of α-amylase and rice koji extracts were successfully separated by CIEF in a large pH gradient. The α-amylase from A. oryzae was found to have pI of 3.7, as determined from the migration times of six standard proteins. A simple method has been developed for the measurement of α-amylase activity in rice koji, and the results were compared with those obtained by the official method. The coefficient of variation (C.V.) of the α-amylase assay was less than 0.5% in both intra-day and inter-day experiments. This CIEF method can be applied to the routine quality control of α-amylase activity in rice koji for the brewing process in sake production.