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Plant extracts including secondary metabolites have anti-inflammatory and anti-obesity activities. This study was conducted to investigate the anti-obesity properties of fermented Artemisia annua(AW) and Salicornia herbacea (GW) in vitro and in mice. The metabolite profiling of AW and GW extracts was performed using UHPLC−LTQ−Orbitrap–MS/MS, and gene expression was analyzed using real-time PCR for adipocyte difference factors. The anti-obesity effects in mice were measured using serum AST, ALT, glucose, TG, and cholesterol levels. Metabolites of the plant extracts after fermentation showed distinct differences with increasing anti-obesity active substances. The efficacy of inhibitory differentiation adipogenesis of 3T3-L1 adipocytes was better for GW than AW in a concentration-dependent manner. RT-PCR showed that the GW extract significantly reduced the expression of genes involved in adipocyte differentiation and fat accumulation (C/EBPα, PPARγ, and Fas). In C57BL/6 mice fed the HFD, the group supplemented with AW and GW showed reduced liver weight, NAS value, and fatty liver by suppressing liver fat accumulation. The GW group significantly reduced ALT, blood glucose, TG, total cholesterol, and LDL-cholesterol. This study displayed significant metabolite changes through biotransformation in vitro and the increasing anti-obesity effects of GW and AW in mice. GW may be applicable as functional additives for the prevention and treatment of obesity.

Numerous varieties of doenjang are manufactured by many food companies using different ingredients and fermentation processes, and thus, the qualities such as taste and flavor are very different. Therefore, in this study, we compared many products, specifically, 19 traditional doenjang (TD) and 17 industrial doenjang (ID). Subsequently, we performed non-targeted metabolite profiling, and multivariate statistical analysis to discover distinct metabolites in two types of doenjang. Amino acids, organic acids, isoflavone aglycones, non-DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4- one) soyasaponins, hydroxyisoflavones, and biogenic amines were relatively abundant in TD. On the contrary, contents of dipeptides, lysophospholipids, isoflavone glucosides and DDMP-conjugated soyasaponin, precursors of the above-mentioned metabolites, were comparatively higher in ID. We also observed relatively higher antioxidant, protease, and β-glucosidase activities in TD. Our results may provide valuable information on doenjang to consumers and manufacturers, which can be used while selecting and developing new products.

In this study, we investigated the altered enzymatic activities and metabolite profiles of koji fermented using varying permutations of Aspergillus oryzae and/or Bacillus amyloliquefaciens. Notably, the protease and β-glucosidase activities were manifold increased in co-inoculated (CO) koji samples (co-inoculation of A. oryzae and B. amyloliquefaciens). Furthermore, gas chromatography-mass spectrometry (GC-MS)-based metabolite profiling indicates that levels of amino acids, organic acids, sugars, sugar alcohols, fatty acids, nucleosides, and vitamins were distinctly higher in CO, SA (sequential inoculation of A. oryzae, followed by B. amyloliquefaciens), and SB (sequential inoculation of B. amyloliquefaciens, followed by A. oryzae). The multivariate principal component analysis (PCA) plot based on GC-MS datasets indicated a clustered pattern for MA and MB (koji samples inoculated either with A. oryzae or B. amyloliquefaciens) across PC2 (20.0%). In contrast, the CO, SA, and SB metabolite profiles displayed segregated patterns across PLS1 (22.2%) and PLS2 (21.1%) in the partial least square discriminant analysis (PLS-DA) model. Intriguingly, the observed disparity in the levels of primary metabolites was engendered largely by higher relative levels of sugars and sugar alcohols in MA, SA, and CO koji samples, which was commensurate with the relative amylase activities in respective samples. Collectively, the present study emphasizes the utility of integrated biochemical and metabolomic approaches for achieving the optimal permutation of fermentative inocula for industrial koji preparation.

Doenjang and ganjang are secondary fermented soybean products from meju (primary fermented product) following a complex fermentation process that separates the products into solid (doenjang) and liquid (ganjang) states. We performed a comparative study on gas chromatography mass spectrometry-(GC-MS) and liquid chromatography mass spectrometry-(LC-MS) based metabolite profiling with fungal and bacterial microbial community analysis of doenjang and ganjang during fermentation. Metabolite profiling and microbial community data showed distinct patterns, depending on the fermentation process. The relative levels of metabolic patterns were similar and most of the microorganisms produced halophilic or halotolerant microbes during the fermentation period in doenjang and ganjang. In the doenjang end products, isoflavones, soyasaponins, and amino acids were largely distributed and Debaryomyces and Staphylococcus were dominant, whereas the biogenic amine and phenylpropanoid contents were highly distributed in the ganjang end products, with higher levels of Meyerozyma and Tetragenococcus. Our results demonstrate that the quality of doenjang and ganjang is predominantly influenced by the microbiome and by metabolite changes during fermentation. Moreover, the present study provides a platform for comparing samples in different states.

Meju, a type of fermented soybean paste, is used as a starter in the preparation of various Korean traditional soybean-based foods. In this study, we performed Illumina-MiSeq paired-end sequencing for microbial communities and mass spectrometry analysis for metabolite profiling to investigate the differences between 11 traditional meju products from different regions across Korea. Even though the bacterial and fungal communities showed remarkable variety, major genera including Bacillus, Enterococcus, Variovorax, Pediococcus, Weissella, and Aspergillus were detected in every sample of meju. The metabolite profile patterns of the 11 samples were clustered into two main groups: group I (M1-5) and group II (M6-11). The metabolite analysis indicated a relatively higher amino acid content in group I, while group II exhibited higher isoflavone, soyasaponin, and lysophospholipid contents. The bioactivity analysis proved that the ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) radical-scavenging activity was higher in group II and the FRAP (ferric reducing antioxidant power) activity was higher in group I. The correlation analysis revealed that the ABTS activity was isoflavonoid, lipid, and soyasaponin related, whereas the FRAP activity was amino acid and flavonoid related. These results suggest that the antioxidant activities of meju are critically influenced by the microbiome and metabolite dynamics.

Production of good Koji primarily depends upon the selection of substrate materials and fermentative microflora, which together influence the characteristic flavor and aroma. Herein, we performed comparative metabolomic analyses of volatile organic compounds (VOCs) and primary metabolites for Koji samples fermented individually with Bacillus amyloliquefaciens and Aspergillus oryzae. The VOCs and primary metabolites were analyzed using headspace solid phase microextraction (HS-SPME) followed by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). In particular, alcohols, ketones, and furans were mainly detected in Bacillus-fermented Koji (Bacillus Koji, BK), potentially due to the increased levels of lipid oxidation. A cheesy and rancid flavor was characteristic of Bacillus Koji, which is attributable to high content of typical 'off-flavor' compounds. Furthermore, the umami taste engendered by 2-methoxyphenol, (E,E)-2,4-decadienal, and glutamic acid was primarily detected in Bacillus Koji. Alternatively, malty flavor compounds (2-methylpropanal, 2-methylbutanal, 3-methylbutanal) and sweet flavor compounds (monosaccharides and maltol) were relatively abundant in Aspergillus-fermented Koji (Aspergillus Koji, AK). Hence, we argue that the VOC profile of Koji is largely determined by the rational choice of inocula, which modifies the primary metabolomes in Koji substrates, potentially shaping its volatolome as well as the aroma characteristics.

A time-correlated mass spectrometry (MS)-based metabolic profiling was performed for rice koji made using the substrates with varying degrees of milling (DOM). Overall, 67 primary and secondary metabolites were observed as significantly discriminant among different samples. Notably, a higher abundance of carbohydrate (sugars, sugar alcohols, organic acids, and phenolic acids) and lipid (fatty acids and lysophospholipids) derived metabolites with enhanced hydrolytic enzyme activities were observed for koji made with DOM of 5-7 substrates at 36 h. The antioxidant secondary metabolites (flavonoids and phenolic acid) were relatively higher in koji with DOM of 0 substrates, followed by DOM of 5DOM of 7DOM of 9 and 11 at 96 h. Hence, we conjecture that the rice substrate preprocessing between DOM of 5 and 7 was potentially optimal toward koji fermentation, with the end product being rich in distinctive organoleptic, nutritional, and functional metabolites. The study rationalizes the substrate preprocessing steps vital for commercial koji making.

Though varying metabolomes are believed to influence distinctive characteristics of different soy foods, an in-depth, comprehensive analysis of both soluble and volatile metabolites is largely unreported. The metabolite profiles of different soy products, including cheonggukjang, meju, doenjang, and raw soybean, were characterized using LC-MS (liquid chromatography–mass spectrometry), GC-MS (gas chromatography–mass spectrometry), and headspace solid-phase microextraction (HS-SPME) GC-MS. Principal component analysis (PCA) showed that the datasets for the cheonggukjang, meju, and doenjang extracts were distinguished from the non-fermented soybean across PC1, while those for cheonggukjang and doenjang were separated across PC2. Volatile organic compound (VOC) profiles were clearly distinct among doenjang and soybean, cheonggukjang, and meju samples. Notably, the relative contents of the isoflavone glycosides and DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one) conjugated soyasaponins were higher in soybean and cheonggukjang, compared to doenjang, while the isoflavone aglycones, non-DDMP conjugated soyasaponins, and amino acids were significantly higher in doenjang. Most VOCs, including the sulfur containing compounds aldehydes, esters, and furans, were relatively abundant in doenjang. However, pyrazines, 3-methylbutanoic acid, maltol, and methoxyphenol were higher in cheonggukjang, which contributed to the characteristic aroma of soy foods. We believe that this study provides the fundamental insights on soy food metabolomes, which determine their nutritional, functional, organoleptic, and aroma characteristics.

Three types of doenjang, a fermented soybean paste, were prepared by adding coriander (CR), Korean mint (KM), and peppermint (PM) and compared to the control group (CN) by studying their metabolite profiles and antioxidant activities followed by different fermentation periods (1, 30, and 150 days, respectively). The primary metabolome was analyzed by GC-TOF-MS, and 36 of metabolites were identified in four types of doenjang samples (CN, CR, KM, and PM). Samples were clustered based on the herb type and fermentation period in PCA and PLS-DA analysis. For the secondary metabolome analysis, UHPLC-Q-orbitrap-MS was used, and 26 metabolites were identified. The statistical analysis showed that the samples were clustered by herb type rather than fermentation period, and the samples containing KM and PM were located in the same group. The DPPH assay showed that PM-containing doenjang had the highest antioxidant activity. Correlation analysis indicated that organic acids such as lactic acid, malonic acid, succinic acid, uracil, vanillic acid, and quinic acid showed positive correlation with the DPPH activity. Overall, our results demonstrated that incorporating herbs in doenjang during fermentation caused significant shifts (p-value &lt, 0.05) in the doenjang metabolites and antioxidant activity. Hence, herbs could be utilized for enhancing doenjang fermentation.

The temporal metabolomes associated with Rubus coreanus (RC) vinegar production were delineated using mass spectrometry-based metabolomic analyses with multivariate studies. We observed a clustered pattern of 27 discriminant metabolites: 4 organic acids, 9 sugars and sugar derivatives, 2 alcohol and carboxylic acid, 6 esters, and 6 terpenes. The levels of organic acids and alcohols were steadily decreased throughout fermentation, whereas those of sugars and sugar derivatives, carboxylic acids, esters, and terpenes peaked at 6 days. Further, the in vivo nutraceutical potentials of RC vinegar were examined with its metabolomic implications for bone health in growing rat model. Intriguingly, the plasmatic metabolomes among treated rat groups were observed with lower levels of non-essential amino acids and lysophosphatidylcholines (LysoPCs) coupled with higher levels of fatty acids and bile acids. The plasmatic metabolomes complemented with bone morphometric and clinical parameters suggest the bone health ameliorating effects of RC vinegar.

Two different doenjang manufacturing processes, the industrial process (IP) and the modified industrial process (mIP) with specific microbial assortments, were subjected to metabolite profiling using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). The multivariate analyses indicated that both primary and secondary metabolites exhibited distinct patterns according to the fermentation processes (IP and mIP). Microbial community analysis for doenjang using denaturing gradient gel electrophoresis (DGGE), exhibited that both bacteria and fungi contributed proportionally for each step in the process viz., soybean, steaming, drying, meju fermentation, cooling, brining, and aging. Further, correlation analysis indicated that Aspergillus population was linked to sugar metabolism, Bacillus spp. with that of fatty acids, whereas Tetragenococcus and Zygosaccharomyces were found associated with amino acids. These results suggest that the components and quality of doenjang are critically influenced by the microbial assortments in each process.

Many ethnic fermented soybean products (FSPs) have long been consumed as seasoning and protein sources in East Asia. To evaluate the quality of various FSPs in East Asia, non-targeted metabolite profiling with multivariate analysis of six traditional FSPs (Natto, NT, Cheonggukjang, CG, Doenjang, DJ, Miso, MS, Doubanjiang, DB, Tianmianjiang, TM) was performed. Six FSPs could be clearly distinguished by principle component analysis (PCA) and partial least square-discriminant analysis (PLS-DA). Amino acid contents were relatively higher in NT and CG, sugar and sugar alcohol contents were relatively higher in MS and TM, isoflavone glycoside contents were relatively highest in CG, isoflavone aglycon contents were the highest in DJ, and soyasaponin contents were the highest in CG. Antioxidant activity and physicochemical properties were determined to examine the relationships between the FSPs and their antioxidant activities. We observed a negative correlation between isoflavone aglycon contents and 2,2&rsquo, azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) activity. Furthermore, the order of ABTS activity of FSPs has a positive correlation with the order of soybean content in the six FSPs. Herein it was found that primary metabolites were affected by the main ingredients and secondary metabolites were most influenced by the fermentation time, and that soybean content contributed more to antioxidant activity than fermentation time.

To investigate the effect of different acetic acid bacteria on qualities of detoxified Rhus verniciflua vinegar (DRV), different DRVs were produced by using three acetic acid bacterial strains including Acetobacter pasteurianus KACC16934 (AP), Acetobacter malorum V5-7 (AM), and Gluconoacetobacter entanii RDAF-S (GE). Little difference in pH (2.87~2.90) or titratable acidity (5.33~5.68%) was observed among different DRVs. Acetic acid yields of the AP, AM, and GE strains were 78.6%, 85.3%, and 85.9%, respectively. Organic acid content in different DRVs was in the range of 69.84~73.83 mg/mL. Oxalic acid, malic acid, succinic acid, acetic acid, and fumaric acid were detected in all DRVs. And glutamic acid, alanine, valine, leucine, tyrosine, lysine, anserine, and arginine were detected in all DRVs. Arginine and alanine were the predominant amino acids in all DRVs. PCA plots of electronic nose analysis data showed a significant differentiation of DRVs from different acetic acid bacterial strains. A sensory evaluation of all DRVs indicated that DRV fermented using AM was superior in the aspect of flavor, tasted and overall preference.

We performed mass spectrometry (MS)-based metabolite profiling of Cheonggukjang (CGJ) and three types of garlic (normal, sprouted, and fermented) added CGJ (G-CGJ). Metabolite profiling suggested that the major distinguishing factor between the four types of CGJ lay in whether garlic was added, although different metabolic states were observed in G-CGJ. Among the discriminant metabolites between CGJ and G-CGJ, the levels of four amino acids such as l -valine, l -isoleucine, l -leucine, and glycine were decreased in G-CGJ compared to CGJ because garlic inhibited the protease activity of Bacillus subtilis during fermentation. In addition, the relative contents of l -cysteine, s -allyl-cysteine, s -allyl-cysteine sulfoxide, soyasaponin βg, and soyasaponin γg, which showed positive correlation with antioxidant activities, were high in G-CGJ. These results suggest that MS-based metabolite profiling could be a useful tool for understanding the metabolic differences of fermented foods according to the additives, and their relationship with antioxidant activity.

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In this study, vinegar was produced using urushiol-free fermented Rhus verniciflua extract to create a lacquer with added value. The effect of manufacturing conditions on the quality of vinegar using detoxified R. verniciflua extract for fermentation was investigated. The acidity of the vinegar for inoculations with various liquid starter contents was 4.8~4.9%, and it was similar among all treatment groups. The acidity of vinegar was higher when the initial alcohol content was high. The acetic acid yields were 82.8%, 84.4%, 77.7%, and 69.5%, and the maximum yield was observed when the initial alcohol content was 6%. For acetic acid fermentation using different amounts of detoxified R. verniciflua extracts, the acidity of the vinegar with the extract after fermentation was 5.3~5.9%. However, the acidity of vinegar without the extract was 5.5%. The intensity of the brown color was high for vinegar without the extract. Hunter’s L values were high for vinegar with an extract content of 2%. Acetic acid (53.3~65.8 mg/mL) was the predominant acid. Arginine (190.3~333.3 μg/mL), proline (125.6~290.8 μg/mL), alanine (126.1~270.9 μg/mL), and glutamic acid (159.0~262.4 μg/mL) were the predominant amino acids in detoxified R. verniciflua vinegar.

건국대학교 생명공학과For the selection of a starter for vinegar, six strains of acetic-acid bacteria were isolated from traditional Korean vinegar fermented through the static method. These strains were investigated for their acetic-acid fermentation and identification characteristics. The 16S rRNA sequences of six strains were identified as Acetobacter pasteurianus, A. malorum, Gluconacetobacter entanii, Ga. intermedius, and Ga. xylinus respectively. The overoxidation of acetic acid, acetic-acid and pH tolerances, and acetic-acid production of these strains were investigated. None seemed to have been overoxidized. The Gluconacetobacter genus showed acetic-acid tolerance. Among the acetic-acid bacteria, A. malorum V5-7 exhibited the highest pH tolerance. The Ga. intermedius V11-5 and Ga. xylinus V8-1 strains produced colloids that exopolysaccharides of fiber. The acetic-acid production by isolated acetic-acid bacteria and type strain was a achieved at a shaking culture at 30for 5 days. A. malorum V5-7, A. pasteurianus Gam2, and Ga. intermedius V11-5 exhibited the highest acetic acid production. The study results indicate that appropriate strains of acetic-acid bacteria improved the thraditional Korean vinegar fermented through the static method. Key words vinegar, starter, acetic acid bacteria, 16S rRNA gene, static fermentation

In this study, a comprehensive metabolite profile analysis of doenjang, a fermented soybean paste, at various steps of its industrial 5-step production process was conducted, by combining gas and liquid chromatography-mass spectrometry techniques with multivariate analysis. From the partial least squares discriminant analysis of primary and secondary metabolites, the patterns were clearly distinguishable between the various processing steps (step 1: steaming, step 2: drying, step 3: meju fermentation, step 4: brining, step 5: doenjang aging). Of the primary metabolites, most of the monosaccharides, amino acids, and fatty acids increased in steps 3-5. Isoflavone and soyasaponin derivatives were major secondary metabolites identified during the processing of doenjang. Isoflavone glycosides gradually decreased after step 1, while isoflavone aglycones distinctly increased in steps 4-5. Soyasaponins generally decreased during processing after step 2. Increased isoflavone aglycones, such as daidzein, glycitein, and genistein, were observed in steps 4-5 showed the strongest positive correlation with doenjang's antioxidant potential and total phenolic content.

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 (LysoPC's) 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.

This study aimed to investigate the biotransformation of soybean isoflavones to hydroxyisoflavones, and the primary and secondary metabolite change during Aspergillus oryzae KACC40247-mediated fermentation by gas chromatography–time of flight-mass spectrometry and LC-MS with multivariate analysis. The mass spectrometric analysis revealed that acetylglycosides and glycosides decreased during the first 12 h of fermentation, while the aglycones increased up to that time point. This was followed by a decrease in aglycone levels due to the formation of hydroxyisoflavones. The hydroxyflavones, 8-hydroxydaidzein, hydroxygenistein, and hydroxyglycitein, resulting from the biotransformation of the corresponding aglycones, increased up to 24 h, and then subsequently decreased. During fermentation, the levels of monosaccharides, aspartic acid, pyroglutamic acid, gamma-aminobutyric acid, and organic acids gradually decreased, whereas the levels of threonine, serine, and glycine increased. Hydroxyisoflavone was more strongly correlated with antioxidant activity than the other metabolites. Our results suggest that biotransformation has the potential to improve the nutritional properties of soy-based food.

Metabolomic differences between green tea (GT) and Aspergillus oryzae -fermented green tea (FGT) were investigated using Liquid Chromatography–Electrospray Ionization-Ion Trap–Tandem Mass Spectrometry (LC-ESI-IT-MS/MS). To identify the metabolomic differences, principal component analysis (PCA) and orthogonal projection to latent structures discriminate analysis (OPLS-DA) of GT and FGT were performed. A total of 17 metabolites differed between GT and FGT. The major flavonoid compounds of GT, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), decreased during fermentation, while the levels of phenolic compounds, such as gallic acid, 3- p -coumaroylquinic acid, and other flavonoid metabolites like gallocatechin, epicatechin, and epigallocatechin, increased significantly during fermentation. Notably, caffeine degradation was also observed during fermentation of GT by A. oryzae . Although the total flavonoid content of FGT decreased, the antioxidant activity of FGT increased significantly due to increased levels of other identified and unidentified metabolites. These results show that fermentation-dependent metabolomic changes have the potential to increase the bioactivity of GT.

As a nontargeted metabolomics approach, we investigated changes in the plasma metabolite levels in a mouse model of obesity induced by a high-fat diet and fermented soybean product diet. We analyzed the plasma samples by using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). In the present study, the animals were divided into four groups according to the diet type; normal fat diet control group (ND), high-fat diet control group (HD), high-fat diet plus 30% cooked soybean power (HD + S), and high-fat diet plus 30% 72-h fermented Cheonggukjang powder (HD + CGJ). To examine the changes in plasma metabolite levels because of high-fat diet feeding, total cholesterol and triglyceride levels were measured. Total cholesterol and triglyceride levels were lower in the HD + S and HD + CGJ groups than in the ND group. According to partial least-squares discriminant analysis (PLS-DA), major metabolites contributing to the discrimination between each group were assigned as lipid metabolites in plasma, e.g., lyso-phosphatidylcholines and phosphatidylcholines. Therefore, diets containing soy-based food products, which are rich sources of isoflavonoids, might be helpful for controlling the lipid metabolism under high-fat diet conditions.

The microbial community and the metabolites of barley nuruk were studied to determine the time-dependent correlation between the fermentation of microbes and metabolites. Samples were analyzed by a polyphasic approach based on culture-dependent, culture-independent (PCR-DGGE and qPCR analysis), and metabolite analysis using GC-MS. Barley nuruk consists of varying amounts of bacteria, yeasts, and molds. The PCR-DGGE results showed that only one phylotype, Aspergillus oryzae, was predominant throughout fermentation, reaching a maximum on day 9. The bacterial load was higher on day 6 of fermentation, and then gradually decreased because of increased fungal activity. The shift in fungal and bacterial diversity observed by DGGE was further confirmed by qPCR analysis. In addition, microbes closely related to Pantoea agglomerans and Saccharomycopsis fibuligera appeared to play key roles in the fermentation of barley nuruk. GC-MS analysis combined with multivariate analysis, including PCA, PLS-DA, and OPLS-DA, showed fermentation time-dependent metabolite patterns. A total of 21 metabolites, including organic acids, amino acids, sugars, and sugar alcohols, were identified. In particular, glycerol, malic acid, fructose, glucose, sucrose, and maltose were produced at the early fermentation stages (0-6 d), whereas glutamine, aspartic acid, glutamic acid, mannitol, and xylitol were produced during the latter stages of fermentation (9-18 d). Mixed culture fermentation was found throughout the natural fermentation of barley nuruk starter. Most likely, A. oryzae had a major role in saccharification, along with other mixed cultures.

Bacterial blight, an important and potentially destructive bacterial disease in rice caused by Xanthomonas oryzae pv. oryzae (Xoo), has recently developed resistance to the available antibiotics. In this study, mass spectrometry (MS)-based metabolite profiling and multivariate analysis were employed to investigate the correlation between time-dependent metabolite changes and antimicrobial activities against Xoo over the course of Phomopsis longicolla S1B4 fermentation. Metabolites were clearly differentiated based on fermentation time into phase 1 (days 4-8) and phase 2 (days 10-20) in the principal component analysis (PCA) plot. The multivariate statistical analysis showed that the metabolites contributing significantly for phases 1 and 2 were deacetylphomoxanthone B, monodeacetylphomoxanthone B, fusaristatin A, and dicerandrols A, B, and C as identified by liquid chromatography-mass spectrometry (LC-MS), and dimethylglycine, isobutyric acid, pyruvic acid, ribofuranose, galactofuranose, fructose, arabinose, hexitol, myristic acid, and propylstearic acid were identified by gas chromatography-mass spectrometry (GC-MS)-based metabolite profiling. The most significantly different secondary metabolites, especially deacetylphomoxanthone B, monodeacetylphomoxanthone B, and dicerandrol A, B and C, were positively correlated with antibacterial activity against Xoo during fermentation.

The metabolite profile of meju during fermentation was analyzed using mass spectrometry techniques, including GC-MS and LC-MS, and the bacterial diversity was characterized. The relative proportions of bacterial strains indicated that lactic acid bacteria, such as Enterococcus faecium and Leuconostoc lactis, were the dominant species. In partial least-squares discriminate analysis (PLS-DA), the componential changes, which depended on fermentation, proceeded gradually in both the GC-MS and LC-MS data sets. During fermentation, lactic acid, amino acids, monosaccharides, sugar alcohols, and isoflavonoid aglycones (daidzein and genistein) increased, whereas citric acid, glucosides, and disaccharides decreased. MS-based metabolite profiling and bacterial diversity characterization of meju demonstrated the changes in metabolites according to the fermentation period and provided a better understanding of the correlation between metabolites and bacterial diversity.

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.

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.

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.

We used ultra-performance-liquid-chromatography with quadrupole-time-of-flight mass spectrometry to study the changes in metabolites in the mixture of Cudrania tricuspidata, Lonicera caerulea, and soybean (CLM) during fermentation. Additionally, the antiobesity effects of CLM and fermented-CLM (FCLM) were studied based on the analysis of plasma from high-fat diet (HFD)-fed mice. The levels of cyanidin and the glycosides of luteolin, quercetin, and cyanidin derived from L. caerulea were decreased, whereas the levels of luteolin and quercetin were increased during fermentation. Isoflavone glycosides and soyasaponins originating from the soybean were decreased, whereas their aglycones such as daidzein, glycitein, and genistein were increased. As for prenylated flavonoids from C. tricuspidata, these metabolites were decreased at the early stage of fermentation, and were increased at end of the fermentation. In terms of the functional food product, various metabolites derived from diverse natural products in CLM had complementary effects and demonstrated higher antioxidant and pancreatic lipase inhibition activities after fermentation; these activities were closely related to flavonoid aglycones including genistein, daidzein, glycitein, luteolin, and quercetin. In an in vivo experiment, several clinical parameters affected by HFD were improved by the administration of either CLM or FCLM, but there was a difference in the antiobesity effects. The levels of lysoPCs with C20:4, C16:0, and C22:6 were significantly attenuated by CLM administration, while the attenuated levels of lysoPCs with C20:4 and C18:2 were significantly restored by FCLM administration. These metabolites may explain the above-mentioned differences in antiobesity effects. Although only the changes in plasma lysophospholipids could not fully explain antiobesity effects between non-fermented and fermented plant mixtures from our results, we suggest that metabolomics approach could provide a way to reveal the metabolite alterations in the complex fermentation process and understand the differences or changes in bioactivity according to fermentation.

Metabolic changes in fast-fermented bean paste (cheonggukjang) as a function of fermentation time were observed in inoculated Bacillus strains using gas chromatography time-of-flight mass spectrometry (GC-TOF-MS)- and capillary electrophoresis TOF-MS (CE-TOF-MS)-based metabolomics techniques. From the combined GC-MS and CE-MS analysis of fermented cheonggukjang samples, 123 metabolites were recovered (55% by GC-MS and 45% by CE-MS). Multivariate statistical analysis of fermented cheonggukjang samples showed that the separation of metabolites was influenced by the fermentation period (range, 0-72 h) and not by strain. When comparing the metabolites of fermented cheonggukjang with the metabolic pathways, uracil and thymine contents showed a rapid 20-fold increase after 24 h fermentation up to the end of fermentation. Xanthine and adenine levels increased slightly from 24 to 48 h fermentation and then decreased slightly at the end of fermentation. Hypoxanthine and guanine levels also increased remarkably during fermentation. Purine metabolism differed according to the microorganism used for cheonggukjang fermentation. Most intermediates in nucleoside biosynthesis were detected by CE-TOF-MS and were related to amino acid metabolism.

In this study, the metabolite profiles of three Aspergillus strains during rice koji fermentation were compared. In the partial least squares discriminant analysis-based gas chromatography-mass spectrometry data sets, the metabolite patterns of A. oryzae (KCCM 60345) were clearly distinguished from A. kawachii (KCCM 60552) and only marginal differences were observed for A. oryzae (KCCM 60551) fermentation. In the 2 days fermentation samples, the overall metabolite levels of A. oryzae (KCCM 60345) were similar to the A. oryzae (KCCM 60551) levels and lower than the A. kawachii (KCCM 60552) levels. In addition, we identified discriminators that were mainly contributing tyrosinase inhibition (kojic acid) and antioxidant activities (pyranonigrin A) in A. oryzae (KCCM 60345) and A. kawachii (KCCM 60552) inoculated rice koji, respectively. In this study, we demonstrated that the optimal inoculant Aspergillus strains and fermentation time for functional rice koji could be determined through a metabolomics approach with bioactivity correlations.

The metabolomics is the studies on metabolites, and by-products of the chemical reactions that continuously go on in every biological system. The combined mass spectrometry approaches used in this study may be useful in understanding the overall fermentation metabolism in the various microbes.

Liquid chromatography mass spectrometry and multivariate analysis were employed to investigate the correlation between fermentation time-dependent metabolite changes in cheonggukjang, a traditional fermented soybean product, and changes in its antioxidant activity over 72 h. The metabolite patterns were clearly distinguished not by strains but by fermentation time, into patterns I (0-12 h), II (12-24 h), and III (24-72 h), which appeared as distinct clusters on principal component analysis. The compounds that significantly contributed to patterns I, II, and III were soyasaponins, isoflavonoid derivatives, and isoflavonoid aglycons respectively. Partial least square analysis for metabolite to antioxidant effects showed correlations between the ferric reducing/antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay during 24-36 h, and 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) test and total phenol content (TPC) during 36-72 h. Compared with the strong negative correlations of glucosylated-isoflavonoids with DPPH, ABTS and TPC during fermentation, the isoflavonoid aglycon displayed strong positive correlations with these compounds during fermentation.

Methanol extracts of Cheonggukjang, fermented for various times, were evaluated for their free-radical-scavenging and tyrosinase-inhibitory activities and the underlying mechanisms were elucidated. The free-radical-scavenging activity was highest for Cheonggukjang extracts fermented for 40 h and this decreased as the fermentation time shortened. The tyrosinase-inhibition activity increased with the length of fermentation. While levels of total phenolic compounds were highest in the 40-h-fermented Cheonggukjang samples, the total flavonoid content was lower compared to samples fermented for shorter times. Therefore, the antioxidative and tyrosinase-inhibition activities exhibited by methanol-extracted Cheonggukjang samples may be attributable to the contents of total phenolic compounds.

Carnobacterium jeotgali strain MS3(T) was isolated from traditionally fermented Korean shrimp produced with bay salt. The bacterium belongs to the family Carnobacteriaceae, produces lactic acid and contains gene clusters involved in the production of lactate, butyrate, aromatic compounds and exopolysaccharides. Carnobacterium jeotgali strain MS3(T) was characterized through extensive comparison of the virulence potential, genomic relatedness and sequence similarities of its genome with the genomes of other Carnobacteria and lactic acid bacteria. In addition, links between predicted functions of genes and phenotypic characteristics, such as antibiotic resistance and lactate and butyrate production, were extensively evaluated. Genomic and phenotypic analyses of strain MS3(T) revealed promising features, including minimal virulence genes and lactate production, which make this bacterium a desirable candidate for exploitation by the fermented food industry.

A Gram-variable, motile, endospore-forming, halotolerant bacillus, strain JG-30T, was isolated from the traditional Korean fermented seafood jeotgal, and was subjected to a polyphasic taxonomic study. This organism grew optimally at 37 °C and in the presence of 0–1 % (w/v) NaCl. 16S rRNA gene sequence analysis showed that strain JG-30T forms a distinct phylogenetic lineage within the evolutionary radiation encompassed by the genus Bacillus. Strain JG-30T was characterized chemotaxonomically as having cell-wall peptidoglycan based on meso-diaminopimelic acid, MK-7 as the predominant menaquinone and iso-C15 : 0 and iso-C14 : 0 as the major fatty acids. The DNA G+C content was 45 mol%. Strain JG-30T exhibited levels of 16S rRNA gene sequence similarity of less than 95·7 % to Bacillus species with validly published names. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain JG-30T (=KCTC 3880T=DSM 16189T) was classified within the genus Bacillus as a novel species, for which the name Bacillus cibi sp. nov. is proposed.

New melanin synthesis inhibitors, melanocins A, B and C, were isolated from the fermentation broth and mycelium extract of Eupenicillium shearii F80695. Melanocin A, an isocyanide compound, inhibited mushroom tyrosinase and melanin biosynthesis of B16 melanoma cells with IC50 value of 9.0 nM and MIC value of 0.9 microM, respectively. Melanocin A also inhibited growth of Streptomyces bikiniensis. While, the structurally very related but non-isocyanide compounds melanocins B and C did not show inhibitory activity in these assays. Melanocins A, B and C showed potent antioxidant activity with scavenging activity of DPPH radical and superoxide anion radical.

Metabolite profiles of seven commercial vinegars and two traditional vinegars were performed by gas chromatography time-of-flight mass spectrometry with multivariate statistical analysis. During alcohol fermentation, yeast, nuruk, and koji were used as sugars for nutrients and as fermentation substrates. Commercial and traditional vinegars were significantly separated in the principal component analysis and orthogonal partial least square discriminant analysis. Six sugars and sugar alcohols, three organic acids, and two other components were selected as different metabolites. Target analysis by ultra-performance liquid chromatography quadruple-time-of-flight mass spectrometry and liquid chromatographyion trap-mass spectrometry/mass spectrometry were used to detect several metabolites having antioxidant activity, such as cyanidin-3-xylosylrutinoside, cyanidin-3-rutinoside, and quercetin, which were mainly detected in Rural Korean Black raspberry vinegar (RKB). These metabolites contributed to the highest antioxidant activity measured in RKB among the nine vinegars. This study revealed that MS-based metabolite profiling was useful in helping to understand the metabolite differences between commercial and traditional vinegars and to evaluate the association between active compounds of vinegar and antioxidant activity.

The study aimed to investigate the effects of Rubus coreanus (RC) vinegar supplementation on anti-obesity in high-fat-diet-induced obesity rat. The RC vinegar was processed starting with 25°Brix of sugar concentration and was alcohol fermented at temperature of 30℃ for 10 days. The conditions for the acetic acid fermentation were 14 days of stationary fermentation at 30℃, with 6% alcohol. A total 52 4-wk old male Sprague-Dawley rats were fed a normal chow diet or a high-fat diet (HFD) for 12 weeks to induce obesity and then were randomized into normal control group (NC), HFD + distilled water group (OB-DW), HFD + 1% Acetic acid group (OB-AA) for control, HFD + 1% RC vinegar group (OB-RC), HFD + 15mg/d caffeine group (OB-CAF). After 2 months, body weights, organ and fat weights, adipocyte size were analyzed. OB-CAF rats showed most decreased body weight about 15% and OB-AA and OB-RC also showed decreased body weight about 6% compared to the obese rats. The adipocyte size were decreased in OB rats treated w...

New melanin synthesis inhibitors (MR566A and B) and six related known isocyanocyclopentenes were isolated from the fermentation broth of Trichoderma harzianum. The IC50 values of MR566A and B against mushroom tyrosinase were 1.72 and 47 microM, respectively. They inhibited melanin biosynthesis in B16 melanoma cells with MIC values of 0.1 and 2.2 microM, respectively. Also isolated from the same culture extract of T. harzianum was a new oxazole (MR93B), which showed no inhibitory activity against mushroom tyrosinase at a concentration of 1,000 microg/ml.

Aspergillus oryzae KACC 40247 was selected from among 60 fungal strains as an effective 7,8,4'-trihydroxyisoflavone (8-hydroxydaidzein)-producing fungus. The optimal culture conditions for production by this strain in a 7-L fermentor were found to be 30 °C, pH 6, and 300 rpm. Under these conditions, A. oryzae KACC 40247 produced 62 mg/L of 8-hydroxydaidzein from soybean extract in 30 h, with a productivity of 2.1 mg/L/h. These are the highest production and productivity for 8-hydroxydaidzein ever reported. To increase production, several concentrations of daidzin and of daidzein as precursor were added at several culture times. The optimal addition time and concentration for daidzin were 12 h and 1,248 mg/L, and those for daidzein were 12 h and 254 mg/L respectively. Maximum production and productivity for 8-hydroxydaidzein with the addition of daidzein were 95 mg/L and 3.2 mg/L/h respectively, and those with the addition of daidzin were 160 mg/L and 4.4 mg/L/h respectively.

Identification of a new phomoxanthone antibiotic from Phomopsis longicolla and its antimicrobial correlation with other metabolites during fermentation

To identify the major factors contributing to the quality of commercial gochujang (fermented red pepper paste), metabolites were profiled by mass spectrometry. In principal component analysis, cereal type (wheat, brown rice, and white rice) and species of hot pepper (Capsicum annuum, C. annuum cv. Chung-yang, and C. frutescens) affected clustering patterns. Relative amino acid and citric acid levels were significantly higher in wheat gochujang than in rice gochujang. Sucrose, linoleic acid, oleic acid, and lysophospholipid levels were high in brown-rice gochujang, whereas glucose, maltose, and γ-aminobutyric acid levels were high in white-rice gochujang. The relative capsaicinoid and luteolin derivative contents in gochujang were affected by the hot pepper species used. Gochujang containing C. annuum cv. Chung-yang and C. frutescens showed high capsaicinoid levels. The luteolin derivative level was high in gochujang containing C. frutescens. These metabolite variations in commercial gochujang may be related to different physicochemical phenotypes and antioxidant activity.

The present study was focused on improving the quality of rice koji by fermentation with a selected Aspergillus oryzae strain and a plant Astragalus radix. A. oryzae KCCM 60345 was used as main inoculant and the Astragalus radix was added as supplement in rice koji preparation. LC-MS based metabolite analysis and tyrosinase inhibitory activities were studied for different time periods. A. oryzae KCCM 60345 fermented rice koji supplemented with Astragalus showed higher tyrosinase inhibition activity at 4 d of fermentation and metabolite analysis with PCA and PLS-DA indicated differences in kojic acid, calycosin-7-O-β-D-glucoside, ononin, calycosin, and formononetin as compared with other forms of rice koji fermentation. By correlation analysis between metabolites and tyrosinase inhibitory activity, calycosin and kojic acid were identified as major tyrosinase inhibitors. Based on these results, we concluded that A. oryzae KCCM 60345 supplemented with Astragalus radix is useful for whitening effects, and we identified optimal conditions for rice koji preparation.