Monascus-fermented Products Research Articles

Revealing the Hypoglycemic Effect of Red Yeast Rice: Perspectives from the Inhibition of α-Glucosidase and the Anti-Glycation Capability by Ankaflavin and Monascin

Red yeast rice dietary supplements have been proven to ameliorate hyperglycemia, but the mechanism was unclear. In this work, ankaflavin (AK) and monascin (MS), as typical pigments derived from red yeast rice, were found to exert noteworthy inhibitory ability against α-glucosidase, with an IC50 of 126.5 ± 2.5 and 302.6 ± 2.5 μM, respectively, compared with acarbose (IC50 = 341.3 ± 13.6 μM). They also exhibited mixed-type inhibition of α-glucosidase in vitro and caused fluorescence quenching through the static-quenching process. Molecular-docking studies indicated that AK and MS bind to amino acid residues outside the catalytic center, which induces structural changes in the enzyme, thus influencing its catalytic activity. The anti-glycation ability of Monascus-fermented products was evaluated, and they exhibited a high inhibition rate of 87.1% in fluorescent advanced glycation end-product formation at a concentration of 0.2 mg mL−1, while aminoguanidine showed a rate of 75.7% at the same concentration. These results will be significant in broadening the application scope of Monascus pigments, especially AK and MS, in treating type 2 diabetes.

Performance and mechanism of co-culture of Monascus purpureus, Lacticaseibacillus casei, and Saccharomyces cerevisiae to enhance lovastatin production and lipid-lowering effects.

To facilitate lipid-lowering effects, a lovastatin-producing microbial co-culture system (LPMCS) was constituted with a novel strain Monascus purpureus R5 in combination with Lacticaseibacillus casei S5 and Saccharomyces cerevisiae J7, which increased lovastatin production by 54.21% compared with the single strain R5. Response Surface Methodology (RSM) optimization indicated lovastatin yield peaked at 7.43mg/g with a fermentation time of 13.88 d, water content of 50.5%, and inoculum ratio of 10.27%. Meanwhile, lovastatin in LPMCS co-fermentation extracts (LFE) was qualitatively and quantitatively analyzed by Thin-Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC). Cellular experiments demonstrated that LFE exhibited no obvious cytotoxicity to L-02 cells and exhibited excellent biosafety. Most notably, high-dose LFE (100mg/L) exhibited the highest reduction of lipid accumulation, total cholesterol, and triglycerides simultaneously in oleic acid-induced L-02 cells, which decreased by 71.59%, 38.64%, and 58.85% than untreated cells, respectively. Overall, LPMCS provides a potential approach to upgrade the lipid-lowering activity of Monascus-fermented products with higher health-beneficial effects.

Adding genistein or luteolin decreased the yield of citrinin and without reducing pigments in yam solid-fermentation by Monascus.

Chinese yam fermented by Monascus, namely red mold dioscorea (RMD), has the potential of treating diseases. However, the production of citrinin limits the application of RMD. In the present study, the fermentation process of Monascus was optimized by adding genistein or luteolin to reduce citrinin yield. The results showed that citrinin in 25 g of Huai Shan yam was reduced by 48% and 72% without affecting the pigment yield by adding 0.2 g of luteolin or genistein, respectively, to a 250-mL conical flask after fermentation for 18 days at 28 °C, whereas the addition of luteolin increased the content of yellow pigment by 1.3-fold. Under optimal conditions, citrinin in 20 g of iron bar yam decreased by 55% and 74% after adding 0.2 g of luteolin or genistein. Luteolin also increased yellow pigment content by 1.2-fold. Ultra HPLC coupled to quadrupole time-of-flight mass spectrometry was used for the preliminary analysis of Monascus fermentation products. It was found that the amino acid types in RMD are similar to those in yams, but there are fewer polysaccharides and fatty acids. The results obtained in the present study showed that the addition of genistein or luteolin could reduce citrinin on the premise of increasing pigment yield, which laid a foundation for the better use of yams in Monascus fermentation. © 2023 Society of Chemical Industry.

Secondary Metabolites with Antifungal Activities from Mangrove Derived Fungus Monascus purpureus WMD2424

The mold Monascus, also called red yeast rice, anka, or koji, has been used as the natural food coloring agent and food additives for more than 1000 years in Asian countries. It has also been used in Chinese herbology and traditional Chinese medicine due to its easing digestion and antiseptic effects. However, under different culture conditions, the ingredients in Monascus-fermented products may be changed. Therefore, an in-depth understanding of the ingredients, as well as the bioactivities of Monascus-derived natural products, is important. Here, through the thorough investigation into the chemical constituents of M. purpureus wmd2424, five previously undescribed compounds, monascuspurins A-E (1-5), were isolated from the EtOAc extract of mangrove-derived fungus Monascus purpureus wmd2424 cultured in RGY medium. All the constituents were confirmed via HRESIMS and 1D- and 2D-NMR spectroscopy. Their antifungal activity was also evaluated. Our results showed that four constituents (compounds 3-5) possessed mild antifungal activity against Aspergillus niger, Penicillium italicum, Candida albicans, and Saccharomyces cerevisiae. It is worth mentioning that the chemical composition of the type strain Monascus purpureus wmd2424 has never been studied.

Investigation of citrinin and monacolin K gene clusters variation among pigment producer Monascus species

The filamentous fungi Monascus spp. have been widely used in the production of food colorants. However, the presence of mycotoxin citrinin and the antihypercholestrolemia agent monacolin K in Monascus-fermented products (MFPs) has raised food safety concerns. Here we de novo-sequenced the genomes of 26 Monascus species and proposed an unprecedented classification system, consist of sections A, B and C, according to the biosynthetic gene clusters (BGCs) distribution and phylogeny results. Based on the absence of citrinin gene cluster, section B species were genetically incapable of synthesizing citrinin. A distinguished section A strain named Monascus sanguineus was believed to be a promising food-pigment-producer particularly owing to the simultaneous inactivation of citrinin and monacolin K clusters. Interestingly, gene losses within Monascus secondary metabolism gene clusters were broadly discovered, which may convey a selective advantage in nutrients and energy competition to support the strong pigment-producing ability. Overall, our sectional delimitation system will reshape the industrial strategies for this economically important fungus.

Fermentation products of the fungus Monascus spp. impairs the physiological activities of toxin-producing Vibrio cholerae

Monascus spp. are filamentous fungi used in fermented foods. They are also natural colorants and food preservatives. Certain metabolites of Monascus spp. lower cholesterol and have other health-promoting effects in humans. In the present study, we demonstrated that the fermentation products of Monascus spp. inhibited ATP synthesis and motility in toxigenic Vibrio cholerae. Single-cell tracking and rotation assays on single flagella showed that Monascus fermentation extract (MFE) significantly impaired V. cholerae swimming by disrupting flagellar rotation. A membrane potential-sensitive carbocyanine dye revealed that MFE depolarized the V. cholerae cell membrane which, in turn, lowered the membrane potential and, by extension, restricted ATP synthesis and flagellar rotation. MFE also severely hindered the motility of other pathogenic bacteria such as V. parahaemolyticus, Pseudomonas aeruginosa, Salmonella enterica Typhimurium, and Leptospira interrogans. The foregoing findings indicate that Monascus fermentation extract could potentially preventing infection caused by multiple pathogenic bacteria as the conventional prophylaxes and slow their progression and lower mortality and morbidity.

Inhibitory Effect of Water Soluble Fraction of Monascus-Fermented Rice on Lipid Accumulation in 3T3 L1 Adipocyte

Excessive lipid accumulation in the body causes people to become overweight and obese, conditions that are associated with an amplified risk of serious diseases. The fungi of genus Monascus produce various secondary metabolites such as monacolins, citrinin and fungal pigments, which are water-insoluble and have inhibitory potency for the lipid accumulation in adipocytes. However, water-soluble adipogenesis inhibitors derived from Monascus-fermented products have not yet been reported. In this study, we investigated the inhibitory activity against intracellular lipid accumulation of water-soluble fractions of Monascus-fermented red mold rice (RMR) and red mold barley (RMB) on murine 3T3-L1 cells. Water soluble fractions of ten different Monascus strains were used and the inhibitory activity of their water-soluble fractions on lipid accumulation by differentiated 3T3-L1 cells was evaluated for 8 days using oil red staining. The water-soluble fraction from Monascus pilosus NBRC4507 fermented RMR cultivated at 30C for 14 days was selected since it showed comparatively the lowest relative lipid accumulation (62±1.2%), which indicated the highest inhibitory activity of lipid accumulation in adipocytes. To study the presence of monacolin and citrinin in the water soluble fractions, thin-layer chromatography was done and the results showed that the water-soluble fractions tested were free from both monacolin and citrinin. Therefore, the present study strongly suggested that the water-soluble components, except for monacolin and citrinin, in the water soluble fraction obtained from Monascus pilosus NBRC4507-fermented rice can be used as functional food material to control overweight and obesity.

The role of carbon and nitrogen sources in the production of bioactive compounds in Monascus fermentation products: a mini review

Angkak is one of the Monascus fermented products with rice as a substrate. In addition, Chinese yam, potatoes, soybean, ginseng and onions can be used as a substrate for fermentation products by Monascus sp. It was known that the fermented product by Monascus sp. produces several bioactive compounds that have antihyperlipidemic activity. These bioactive compounds are monacolin-K and pigment compounds (monascin and ankaflavin). Each of these compounds has different cholesterol inhibitory activity. The production of these bioactive compounds is strongly influenced by the nutritional composition of the fermentation media. The addition of the right carbon and nitrogen sources can accelerate the production of bioactive compounds by Monascus sp.

Investigations into Chemical Components from Monascus purpureus with Photoprotective and Anti-Melanogenic Activities.

Monascus species are asexually or sexually reproduced homothallic fungi that can produce a red colorant, specifically the so-called red yeast rice or Anka, which is used as a food ingredient in Asia. Traditional experiences of using Monascus for treating indigestion, enhancing blood circulation, and health remedies motivate us to investigate and repurpose Monascus-fermented products. Here, two new 5H-cyclopenta[c]pyridine type azaphilones, 5S,6S-monaspurpyridine A (1) and 5R,6R-monaspurpyridine A (2), two new xanthonoids, monasxanthones A and B (3 and 4), one new naphthalenone, monasnaphthalenone (5), and one new azaphilone, monapurpurin (6), along with two known compounds were isolated from the 70% EtOH extract of a citrinin-free domesticated strain M. purpureus BCRC 38110. The phytochemical properties of the xanthonoid and naphthalenone components were first identified from Monascus sp. differently from the representative ingredients of polyketide-derived azaphilones. UVB-induced cell viability loss and reactive oxygen species (ROS) overproduction in human keratinocytes were attenuated by monascuspirolide B (7) and ergosterol peroxide (8), indicating their photoprotective potentials. Ergosterol peroxide (8) decreased the melanin contents and tyrosinase activities of mouse melanocytes, depending on the concentration, suggesting their anti-melanogenic effects. In conclusion, six new and two known compounds were isolated from M. purpureus BCRC 38110, and two of them exhibited dermal protective activities. The results revealed the novel potential of M. purpureus for developing natural cosmeceutics against skin photoaging.

The characteristics of fermented purple sweet potato (Ipomoea batatas) and black rice (Oryza sativa) using UV-irradiated Monascus purpureus

Abstract. Novita I, Oedjijono, Asnani A. 2021. The characteristics of fermented purple sweet potato (Ipomoea batatas) and black rice (Oryza sativa) using UV-irradiated Monascus purpureus. Biodiversitas 22: 684-690. This research aimed to produce Monascus fermented product (MFP) with purple sweet potato (Ipomoea batatas (L.) Lam.) and black rice (Oryza sativa L.) using UV-irradiated Monascus purpureus Went and evaluated the characteristic of its antibacterial activity against Salmonella typhi. M. purpureus was irradiated with UV at l254 nm for 0, 2, 3, and 4 min. The solid-state fermentation process was carried out for 7, 14, and 21 days. The pigments were measured at l390 nm for yellow and l500 nm for red. The ethanol extracts of MFP were analyzed for their antibacterial activity against S. typhi using the Kirby–Bauer method. The results showed that the highest yield of MFP was obtained from MFP–black rice (51.88%) that used UV-irradiated M. purpureus for 2 min and fermentation for 21 days. The highest absorbance value of MFP–purple sweet potato was obtained from UV-irradiated M. purpureus for 3 min, whereas the highest absorbance value of MFP–black rice was obtained with UV-irradiated M. purpureus for 2 min. Ethanol extracts of both MFP3–purple sweet potato and MFP2–black rice showed antibacterial activities against S. typhi with minimum inhibitory concentration values of 0.2 and 0.15 g/mL, respectively. Thin-layer chromatography analysis of the ethanol extract from MFP2–black rice revealed the presence of bioactive saponin and flavonoid. These findings suggest that UV-irradiated M. purpureus was able to use both purple sweet potato and black rice substrates to produce MFP with antibacterial activity against S. typhi.

The noncovalent conjugations of human serum albumin (HSA) with MS/AK and the effect on anti-oxidant capacity as well as anti-glycation activity of Monascus yellow pigments.

Monascin (MS) and ankaflavin (AK), as typical yellow lipid-soluble pigments identified from Monascus-fermented products, have been confirmed to possess diverse biological activities such as anti-oxidation, reversing diabetes, and anti-atherosclerosis, and have received increasing attention in recent years. Certainly Monascus-fermented product with a high content of MS/AK is also a concern. The current work explored interactions between MS/AK and human serum albumin (HSA) as well as their influence on the anti-oxidant properties of MS/AK. Moreover, the anti-glycation potential of Monascus-fermented products rich in MS and AK (denoted as Mps) was assessed. The results showed that the fluorescence emission of HSA was quenched by MS/AK through a static quenching mechanism, and MS-HSA and AK-HSA complexes were mainly formed by van der Waals forces and hydrophobic interactions, but AK showed a higher binding affinity than MS. Although the DPPH radical-scavenging abilities of MS-HSA and AK-HSA complexes declined, Mps significantly reduced the formation of fructosamine, α-dicarbonyl compounds and advanced glycation end products (AGEs) in the in vitro glycation model (HSA-glucose). Notably, approximately 80% of fluorescent-AGEs were suppressed by Mps at a concentration of 0.95 mg mL-1, while aminoguanidine (AG, a reference standard) caused only 65% decrease at the same concentration. Although radical scavenging and metal chelating activities could justify the observed anti-glycation activity of Mps, in-depth research on the structures of other functional compounds present in Mps except MS/AK and reaction mechanisms should be performed. Overall, the present study proved that Mps would be promising sources of food-based anti-glycation agents because of their superior inhibitory effect on AGEs.

Assessment of the thermal and oxidative stability of Antrodia salmonea extract in dripping pills via DSC and TAM air analyses

Lovastatin, also known as monacolin K, is the major active component in red yeast rice. It is the principal active substance in Monascus fermentation products and a statin (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) that inhibits rate-limiting enzymes in the production of cholesterol. Lovastatin is effective in reducing cholesterol levels in humans and is a lipid-lowering drug. Herein, lovastatin was produced from Antrodia salmonea by a liquid-state fermentation process. The A. salmonea extract contained 431 μg g–1 (d.b) lovastatin. A. salmonea extract with high lovastatin content is insoluble, resulting in poor bioavailability, which is problematic. In this study, we utilized a dripping pill delivery system to improve the absorption of lovastatin. Comparisons of the oxidative and thermal stability properties of the lovastatin medicine, A. salmonea extract and dripping pill were conducted by using high-performance liquid chromatography (HPLC) analyses, differential scanning calorimetry (DSC) tests and isothermal microcalorimetry (TAM Air) measurements. We also investigated the thermal degradation of the A. salmonea extract dripping pill under various atmospheric environments to determine the proper storage conditions, including the onset time, total reaction time and heat power of the exothermic reaction, by isothermal storage experimental analyses. The analysis of the oxidative and thermal stability properties suggested that the A. salmonea extract had a higher lovastatin content than the lovastatin medicine, and the results demonstrated the superiority of the dripping pills. Overall, the results can be applied to determine the suitable conditions for the production, storage, heat treatment and food processing of A. salmonea extract dripping pills, and the results will be beneficial to food and pharmaceutical applications.

Modulation of Proinflammatory Cytokines by Red Mold Dioscorea Ethanol Extract in Radioactive Cobalt-60 Exposure

Monascus-fermented products offer valuable therapeutic benefits and have been extensively used in East Asia. Secondary polyketide metabolites ofMonascus, including monacolin K (MK), ankaflavin (AK) and monascin (MS), have been reported to have antioxidation, antiinflammation and tumor metastasis suppression effects. This study investigated the attenuation of radiation-induced proinflammatory cytokines overexpression by ethanol extract of red mold dioscorea (RMDE). We used the B16-F0 melanoma cell line to establish an animal radiotherapy model and serum proinflammatory cytokines (i.e. TNF-a, IL-1b and IL-6) were analyzed. RAW 264.7 cells were pre-treated with different pure compounds [AK, MS and citrinin (CT)] of RMDE and irradiation of 2 Gy cobalt-60 g-radiation. The animal experiment results showed that oral administration of RMDE increased feed intake in C57BL/6 mice. Serum proinflammatory cytokine levels (i.e. TNF-α, IL-1β and IL-6) and profibrotic cytokine (TGF-b) were significantly reduced in the RMDE group compared to control group (p ＜0.01). For in vitro cell model, we evaluated the antiinflammative effects of AK, MS and CT in irradiated RAW 264.7 cells. Both AK and MS significantly reduced radiation-induced overexpression of proinflammatory cytokines and profibrotic cytokines. We have shown for the first time that Monascus-fermented products can attenuate ionizing radiation-induced inflammatory response. AK and MS are the main components of RMDE responsible for this effect.

Screening and identification of Monascus strains with high-yield monacolin K and undetectable citrinin by integration of HPLC analysis and pksCT and ctnA genes amplification.

Red yeast rice (RYR), produced by inoculating Monascus strains to steamed rice, contains many kinds of physiologically bioactive compounds, among which monacolin K can be used as an antihypercholesterolaemic agent. However, RYR can be polluted by the mycotoxin citrinin, which has nephrotoxic and hepatotoxic activities. To avoid the risk of citrinin contamination in Monascus fermented products, it is important to screen for Monascus strains that produce no or low citrinin. Five autochthonous Monascus strains with high-yield monacolin K and undetectable citrinin were obtained using high-performance liquid chromatography (HPLC). All five strains were identified as Monascus ruber based on Genealogical Concordance Phylogenetic Species Recognition criteria. Polymerase chain reaction revealed that citrinin polyketide synthase (pksCT) gene was found in these strains, but transcriptional regulator (ctnA) was not found. Five strains are potential strains for producing high-quality RYR. The distribution of the pksCT gene was not restricted to Monascus purpureus and Monascus sanguineus, and M. ruber strains were diverse in pksCT and ctnA genes. The integration of citrinin HPLC analysis and pksCT and ctnA genes amplification could provide a complementary approach in valuable Monascus strains screening.

Molecular insight on the binding of monascin to bovine serum albumin (BSA) and its effect on antioxidant characteristics of monascin

Monascin (MS) is a yellow lipid-soluble azaphilonoid pigment identified from Monascus-fermented products with promising biological activities. This work studied interactions between MS and bovine serum albumin (BSA) as well as their influences on the antioxidant activity of MS. Experimental results demonstrated that the fluorescence emission of BSA was quenched by MS via static quenching mechanism and the formed BSA-MS complex was mainly maintained by hydrophobic and hydrogen bond interactions. Meanwhile, the probable binding pocket of MS located near site I of BSA and the corresponding conformational and structural alterations of BSA were determined. Furthermore, the molecular modeling approach was performed to understand the visual representation of binding mode between BSA and MS. It was noticeable that the BSA-MS complex exhibited reduced DPPH radical-scavenging ability, which might be attributed to the restraining effect of BSA on the relevant reaction pathways involved in antioxidation by MS.

Monascus spp. used in wheat kernel solid-state fermentations: growth, extrolite production and citrinin cytotoxicity

Monascus fermentation products can be consumed as food or feed supplements or used as food colouring or flavouring agents. In this study, qPCR and Q-TOF LC/MS methods were developed to monitorMonascus ruber andMonascus purpureus growth and extrolite (lovastatin, mevastatin, as well as the regulated mycotoxin, citrinin (CIT)) production, respectively. Wheat kernels were inoculated with one strain of each species during a solid-state fermentation followed over 63 days. Different growth and extrolite production patterns were clearly observed for the 2 tested strains. After 63 days, high lovastatin levels (up to 0.5 mg/g) were reached forM. ruber wheat fermented kernels whileM. purpureus only yielded 0.1 mg/g of lovastatin at best, suggesting thatM. ruber may be a better candidate for lovastatin production in a wheat-based model. Mevastatin levels were low and stable for both species. However,M. ruber fermented wheat kernels also contained the highest CIT content, up to 4.2 μg/g, i.e. at levels above the regulation threshold set by the European Union. CIT toxicity was then determinedin vitro on bovine kidney cells, showing an IC10 of 6.10-4 M. At maximum concentrations encountered during solid-state fermentation, CIT toxicity was very low under chronic exposure.

An overview of the bioactivity of monacolin K / lovastatin

Monacolin K (MK) is the principal active substance in Monascus-fermentation products (e.g. red yeast rice). MK is effective in reducing cholesterol levels in humans and has been widely used as a lipid-lowering drug. The mechanism for this is through a high degree of competitive inhibition of the rate-limiting enzyme HMG-CoA reductase (HMGR) in the cholesterol synthesis pathway. In addition to lowering blood lipid levels, MK also prevents colon cancer, acute myeloid leukemia and neurological disorders such as Parkinson's disease and type I neurofibromatosis. The aim of this manuscript is to comprehensively review the progress in the study of the biological activity of MK and its imechanism of action in reducing blood lipid concentration, prevention of cancer and its neuroprotective, anti-inflammatory and antibacterial properties. This review provides a reference for future applications of MK in functional foods and medicine.

Screening and identification of Monacus strain with high TMP production and statistical optimization of its culture medium composition and liquid state fermentation conditions using response surface methodology (RSM)

ABSTRACTTeramethylpyrazine (TMP) is a pyrazine alkaloid with a variety of pharmacological effects and widely used in clinical practice. In this study, Monascus strain M-3 with high TMP and low citrinin productions was screened from red yeast rice collected from different areas of Fujian province in China. Monascus strain M-3 was further identified through molecular biological methods based on the sequencing of different gene regions. Results showed that primer set of β-tubulin-F/β-tubulin-R targeting the β-tubulin gene was more suitable for the identification of Monascus strain M-3. Based on comparative analysis of the results obtained by different primer sets, Monacus strain M-3 was finally identified as Monascus purpureus. Then, the composition of culture medium and liquid state fermentation conditions of Monacus strain M-3 were optimized by single-factor experiment and central composite design of response surface methodology, with the purpose of optimizing the TMP productions. The optimum composition of culture medium and liquid state fermentation conditions are as follows: starch 5.1%, peptone 3.51%, Mg2SO4 0.1%, NaNO3 0.2%, KH2PO4 0.25%, fermentation temperature of 28.23 °C, fermentation time of 9 d, shaker speed of 180 r/min, inoculum size of 7.21% and loading volume of 100 mL/250 mL triangular flask. Under the optimum composition of culture medium and liquid state fermentation conditions, the TMP production of Monacus strain M-3 can reach 13.49 µg/mL. This study is expected to provide a new approach of TMP production through biosynthesis by Monascus strain, and promote the further development of functional foods or medicines from Monascus fermentation products.

The Prevention of Alzheimer's Disease and Parkinson’s Disease by Monascus purpureus NTU 568-Fermented Compounds

Alzheimer's disease (AD) and Parkinson’s disease (PD), which are senile neurodegenerative diseases, have become more common in recent year. An important cause for AD is currently the deposition of amyloid β (Aβ) in the brain, leading to severe oxidative stress and inflammation, as well as neuronal death and memory damage in AD patients. PD is a chronic degeneration of the central nervous system that mainly affects the motor system. Its symptoms usually appear gradually, and are most obviously tremors, body stiffness, slowed movement, and gait abnormality during early stage; though the mechanism for the death of dopaminergic neurons is not yet completely understood, oxidative stress has been shown to be a very important factor in triggering the development of PD in many human and animal studies. As has been revealed in past research, fermented products of Monascus and their extracts can effectively improve the course of AD and PD, possessing significant preventative and alleviating effects. The main effectors are confirmed to be monascin and ankaflavin for AD treatment, and dimerumic acid and deferricoprogen for PD treatment. This review, therefore, focuses on the analysis and comprehensive discussion of these four active components, for an overall understanding of their role in the AD and PD improvement function of Monascus fermented products.

Key volatile compounds in red koji-shochu, a Monascus-fermented product, and their formation steps during fermentation.

Red koji, which refers the solid culture grown koji mold on the steamed rice, is one of the ingredients of Asian fermented foods including the Japanese spirit shochu. This study was aimed at elucidating the characteristic flavor and key volatile compounds of red koji-shochu as well as the mechanism of their formation. Sensory evaluation showed that red koji-shochu has the distinctive flavors cheese, sour, milky, and oily. Fifteen key volatile compounds of red koji-shochu were identified by gas chromatography-mass spectrometry and high-performance liquid chromatography, and by comparison between red koji-shochu and white koji-shochu, as another typical shochu. The mash analysis revealed that ketone compounds and short-chain acids derive from red koji. Furthermore, although other key compounds were produced by yeast, it is highly likely that their concentrations were affected directly or indirectly by the high activities of protease and lipase in red koji.