Hye Won Shin

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.

Metabolomics reveal that amino acids are the main contributors to antioxidant activity in wheat and rice gochujangs (Korean fermented red pepper paste)

Metabolite profiling of gochujangs (Korean fermented red pepper paste) produced from different kinds of raw materials (WG, wheat gochujang; RG, rice gochujang) was performed using gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). Using principal component analysis (PCA), gochujang samples were differentiated based on the differences in starchy source and secondary materials used in their production. The content of amino acids and sugars was higher in WG, whereas the content of flavonoids, soyasaponins, and γ-aminobutyric acid (GABA) was higher in RG. The content of flavonoids and soyasaponins was related to the higher soybean koji content of RG. The level of antioxidant activity in WG was higher than that in RG and showed a strong positive correlation with amino acid content and a negative correlation with flavonoid content. Although the Trolox equivalent antioxidant capacity of flavonoids was only 10-fold higher than that of amino acids in an antioxidant activity assay, the total amino acid content was as much as several hundred fold higher than total flavonoid content in gochujang. This result suggests that the amino acids could be a main contributor to the antioxidant capacity in gochujang.

Time-resolved comparative metabolomes for Koji fermentation with brown-, white-, and giant embryo-rice

A time-resolved non-targeted metabolomic study towards evaluating the effects of three rice substrate types viz., white rice (WR), giant embryo rice (GER), and brown rice (BR), on Koji fermentation was performed. Notwithstanding the relatively higher proportions of metabolites in unfermented BR (BR 0>WR 0, GER 0), the relative levels of metabolic repertoire in BR Koji were marginally enhanced during 36h fermentation except those for phenolic acids, fatty acids, and vitamins. The WR Koji showed higher levels of lysophospholipids (LysoPCs) and protease-released amino acids (WR 36>GER 36>BR 36). The higher β-glucosidase activity in GER Koji effected the increased levels of sugars and flavonoid aglycons complementing its higher antioxidant activity (GER Koji>BR Koji∼WR Koji). The present study holistically underpins the dynamic metabolomes and enzymatic states during rice Koji fermentation with varying substrate types. The present study finds applications in optimization of commercial Koji production.

Effect of fermented soybean products intake on the overall immune safety and function in mice

Various functional activities have been reported for the fermented soybean products doenjang (DJ) and cheonggukjang (CGJ), although no systemic investigations of their immune functions have been conducted to date. We examined the effects of an experimental diet of DJ, CGJ, or a mixture of unfermented raw material for 4 weeks on overall immunity and immune safety in mice. No significant alterations were observed in peripheral or splenic immune cells among groups. Enhanced splenic natural killer cell activity was observed in the DJ and CGJ groups compared with the plain diet group. T helper type-1 (Th1)-mediated immune responses were enhanced in the DJ and CGJ groups with an upregulated production ratio of IFN-γ vs. IL-4 and IgG2a vs. IgG1 in stimulated splenic T and B cells, respectively. Resistance to Listeria monocytogenes infection was observed in the DJ and CGJ groups. Overall, the results of this study suggest that DJ and CGJ intake consolidates humoral and cellular immunity to Th1 responses.

Process specific differential metabolomes for industrial gochujang types (pepper paste) manufactured using white rice, brown rice, and wheat

The metabolic perplexes for gochujang (GCJ) fermentative bioprocess, a traditional Korean pepper paste, has largely remain equivocal for preparative conditions and raw material (RM) additives exacerbating its commercial standardization. Herein, we outlined a differential non-targeted metabolite profiling for three GCJ (white rice-WR; brown rice-BR; wheat-WT) under varying processing steps (P1 - fermentation; P2 - meju addition; P3 - ripening; and P4 - red pepper addition). We correlated the process specific metabolomes with corresponding physicochemical factors, enzymatic phenotypes, and bioactivities for GCJ-types. The P1 was characterized by a uniform increase in the levels of RM-derived lysoPCs. In contrast, P2 was observed with proportionally higher levels of meju-released isoflavones and soyasaponins in WR-GCJ, followed by BR and WT-GCJ. The P3 involved a cumulative increase in primary metabolites in all GCJ samples except lower organic acid contents in WT-GCJ. The pepper derived flavonoids and alkaloids were selectively increased while P4 in all GCJ-types.

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.