Intraspecies Volatile Interactions Affect Growth Rates and Exometabolomes in Aspergillus oryzae KCCM 60345

( Digar Singh ),( Choong Hwan Lee ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.2

Volatile organic compounds (VOCs) are increasingly been recognized as the chemical mediators of mold interactions, shaping their community dynamics, growth, and metabolism. Herein, we selectively examined the time-correlated (0 D-11 D, where D = incubation days) effects of intraspecies VOC-mediated interactions (VMI) on Aspergillus oryzae KCCM 60345 (S1), following co-cultivation with partner strain A. oryzae KACC 44967 (S2), in a specially designed twin plate assembly. The comparative evaluation of S1_VMI (S1 subjected to VMI with S2) and its control (S1_Con) showed a notable disparity in their radial growth (S1_VMI < S1_Con) at 5 D, protease activity (S1_VMI > S1_Con) at 3-5 D, amylase activity (S1_VMI < S1_Con) at 3-5 D, and antioxidant levels (S1_VMI > S1_Con) at 3 D. Furthermore, we observed a distinct clustering pattern for gas chromatography-time of flight-mass spectrometry datasets from 5 D extracts of S1_VMI and S1_Con in principle component analysis (PC1: 30.85%; PC2: 10.31%) and partial least squares discriminant analysis (PLS-DA) (PLS1: 30.77; PLS2: 10.15%). Overall, 43 significantly discriminant metabolites were determined for engendering the metabolic variance based on the PLS-DA model (VIP > 0.7, p < 0.05). In general, a marked disparity in the relative abundance of amino acids (S1_VMI > S1_Con) at 5 D, organic acids (S1_VMI > S1_Con) at 5 D, and kojic acid (S1_VMI < S1_Con) at 5-7 D were observed. Examining the headspace VOCs shared between S1 and S2 in the twin plate for 5 D incubated samples, we observed the relatively higher abundance of C-8 VOCs (1-octen-3-ol, (5Z)-octa-1,5-dien-3-ol, 3-octanone, 1-octen-3-ol acetate) having known semiochemical functions. The present study potentially illuminates the effects of VMI on commercially important A. oryzae’s growth and biochemical phenotypes with subtle details of altered metabolomes.

Comparative evaluation of microbial diversity and metabolite profiles in <i>doenjang</i>, a fermented soybean paste, during the two different industrial manufacturing processes

Lee, Sunmin,Lee, Sarah,Singh, Digar,Oh, Ji Young,Jeon, Eun Jung,Ryu, Hyung SeoK,Lee, Dong Wan,Kim, Beom Seok,Lee, Choong Hwan Elsevier 2017 Food chemistry Vol.221 No.-

<P><B>Abstract</B></P> <P>Two different <I>doenjang</I> 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 <I>doenjang</I> using denaturing gradient gel electrophoresis (DGGE), exhibited that both bacteria and fungi contributed proportionally for each step in the process viz., soybean, steaming, drying, <I>meju</I> fermentation, cooling, brining, and aging. Further, correlation analysis indicated that <I>Aspergillus</I> population was linked to sugar metabolism, <I>Bacillus</I> spp. with that of fatty acids, whereas <I>Tetragenococcus</I> and <I>Zygosaccharomyces</I> were found associated with amino acids. These results suggest that the components and quality of <I>doenjang</I> are critically influenced by the microbial assortments in each process.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The mIP showed similar metabolite profiles compared to IP despite reduced time. </LI> <LI> Microbial assortments regulate differential metabolite profiles in each process. </LI> <LI> <I>Doenjang</I> quality was synchronous with microbial metabolic diversity. </LI> </UL> </P>

Bidirectional Interactions between Green Tea (GT) Polyphenols and Human Gut Bacteria

Choi Se Rin,Lee Hyunji,Singh Digar,Cho Donghyun,Chung Jin-Oh,Roh Jong-Hwa,Kim Wan-Gi,Lee Choong Hwan 한국미생물·생명공학회 2023 Journal of microbiology and biotechnology Vol.33 No.10

Green tea (GT) polyphenols undergo extensive metabolism within gastrointestinal tract (GIT), where their derivatives compounds potentially modulate the gut microbiome. This biotransformation process involves a cascade of exclusive gut microbial enzymes which chemically modify the GT polyphenols influencing both their bioactivity and bioavailability in host. Herein, we examined the in vitro interactions between 37 different human gut microbiota and the GT polyphenols. UHPLCLTQ-Orbitrap-MS/MS analysis of the culture broth extracts unravel that genera Adlercreutzia, Eggerthella and Lactiplantibacillus plantarum KACC11451 promoted C-ring opening reaction in GT catechins. In addition, L. plantarum also hydrolyzed catechin galloyl esters to produce gallic acid and pyrogallol, and also converted flavonoid glycosides to their aglycone derivatives. Biotransformation of GT polyphenols into derivative compounds enhanced their antioxidant bioactivities in culture broth extracts. Considering the effects of GT polyphenols on specific growth rates of gut bacteria, we noted that GT polyphenols and their derivate compounds inhibited most species in phylum Actinobacteria, Bacteroides, and Firmicutes except genus Lactobacillus. The present study delineates the likely mechanisms involved in the metabolism and bioavailability of GT polyphenols upon exposure to gut microbiota. Further, widening this workflow to understand the metabolism of various other dietary polyphenols can unravel their biotransformation mechanisms and associated functions in human GIT.

Unraveling dynamic metabolomes underlying different maturation stages of berries harvested from Panax ginseng

Lee, Mee Youn,Seo, Han Sol,Singh, Digar,Lee, Sang Jun,Lee, Choong Hwan The Korean Society of Ginseng 2020 Journal of Ginseng Research Vol.44 No.3

Background: Ginseng berries (GBs) show temporal metabolic variations among different maturation stages, determining their organoleptic and functional properties. Methods: We analyzed metabolic variations concomitant to five different maturation stages of GBs including immature green (IG), mature green (MG), partially red (PR), fully red (FR), and overmature red (OR) using mass spectrometry (MS)-based metabolomic profiling and multivariate analyses. Results: The partial least squares discriminant analysis score plot based on gas chromatography-MS datasets highlighted metabolic disparity between preharvest (IG and MG) and harvest/postharvest (PR, FR, and OR) GB extracts along PLS1 (34.9%) with MG distinctly segregated across PLS2 (18.2%). Forty-three significantly discriminant primary metabolites were identified encompassing five developmental stages (variable importance in projection > 1.0, p < 0.05). Among them, most amino acids, organic acids, 5-C sugars, ethanolamines, purines, and palmitic acid were detected in preharvest GB extracts, whereas 6-C sugars, phenolic acid, and oleamide levels were distinctly higher during later maturation stages. Similarly, the partial least squares discriminant analysis based on liquid chromatography-MS datasets displayed preharvest and harvest/postharvest stages clustered across PLS1 (11.1 %); however, MG and PR were separated from IG, FR, and OR along PLS2 (5.6 %). Overall, 24 secondary metabolites were observed significantly discriminant (variable importance in projection > 1.0, p < 0.05), with most displaying higher relative abundance during preharvest stages excluding ginsenosides Rg1 and Re. Furthermore, we observed strong positive correlations between total flavonoid and phenolic metabolite contents in GB extracts and antioxidant activity. Conclusion: Comprehending the dynamic metabolic variations associated with GB maturation stages rationalize their optimal harvest time per se the related agroeconomic traits.

Varying Inocula Permutations (Aspergillus oryzae and Bacillus amyloliquefaciens) affect Enzyme Activities and Metabolite Levels in Koji

( Hye Jeong Gil ),( Sunmin Lee ),( Digar Singh ),( Choong Hwan Lee ) 한국미생물 · 생명공학회 2018 Journal of microbiology and biotechnology Vol.28 No.12

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.

Unraveling Metabolic Variation for Blueberry and Chokeberry Cultivars Harvested from Different Geo-Climatic Regions in Korea

Sim, Inseon,Suh, Dong Ho,Singh, Digar,Do, Seon-Gil,Moon, Kwang Hyun,Lee, Jeong Ho,Ku, Kang-Mo,Lee, Choong Hwan American Chemical Society, Books and Journals Divi 2017 Journal of agricultural and food chemistry Vol.65 No.41

<P>Temporal geo-climatic variations are presumably vital determinants of phenotypic traits and quality characteristics of berries manifested through reconfigured metabolomes. We performed an untargeted mass spectrometry (MS)-based metabolomic analysis of blueberry (<I>Vaccinium</I> spp.) and chokeberry (<I>Aronia melanocarpa</I>) sample extracts harvested from different geo-climatic regions in Korea. The multivariate statistical analysis indicated distinct metabolite compositions of berry groups based on different species and regions. The amino acids levels were relatively more abundant in chokeberry than in blueberry, while the sugar contents were comparatively higher in blueberry. However, the metabolite compositions were also dependent on geo-climatic conditions, especially latitude. Notwithstanding the cultivar types, amino acids, and sucrose were relatively more abundant in berries harvested from 35°N and 36°N geo-climatic regions, respectively, characterized by distinct duration of sunshine and rainfall patterns. The present study showed the ability of a metabolomics approach for recapitulating the significance of geo-climatic parameters for quality characterization of commercial berry types.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jafcau/2017/jafcau.2017.65.issue-41/acs.jafc.7b04065/production/images/medium/jf-2017-040656_0007.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/jf7b04065'>ACS Electronic Supporting Info</A></P>

Unraveling dynamic metabolomes underlying different maturation stages of berries harvested from Panax ginseng

Mee Youn Lee,Han Sol Seo,Digar Singh,Sang Jun Lee,이충환 고려인삼학회 2020 Journal of Ginseng Research Vol.44 No.3

Background: Ginseng berries (GBs) show temporal metabolic variations among different maturationstages, determining their organoleptic and functional properties. Methods: We analyzed metabolic variations concomitant to five different maturation stages of GBsincluding immature green (IG), mature green (MG), partially red (PR), fully red (FR), and overmature red(OR) using mass spectrometry (MS)ebased metabolomic profiling and multivariate analyses. Results: The partial least squares discriminant analysis score plot based on gas chromatographyeMSdatasets highlighted metabolic disparity between preharvest (IG and MG) and harvest/postharvest (PR,FR, and OR) GB extracts along PLS1 (34.9%) with MG distinctly segregated across PLS2 (18.2%). Fortythreesignificantly discriminant primary metabolites were identified encompassing five developmentalstages (variable importance in projection > 1.0, p < 0.05). Among them, most amino acids, organic acids,5-C sugars, ethanolamines, purines, and palmitic acid were detected in preharvest GB extracts, whereas6-C sugars, phenolic acid, and oleamide levels were distinctly higher during later maturation stages. Similarly, the partial least squares discriminant analysis based on liquid chromatographyeMS datasetsdisplayed preharvest and harvest/postharvest stages clustered across PLS1 (11.1 %); however, MG and PRwere separated from IG, FR, and OR along PLS2 (5.6 %). Overall, 24 secondary metabolites were observedsignificantly discriminant (variable importance in projection > 1.0, p < 0.05), with most displayinghigher relative abundance during preharvest stages excluding ginsenosides Rg1 and Re. Furthermore, weobserved strong positive correlations between total flavonoid and phenolic metabolite contents in GBextracts and antioxidant activity. Conclusion: Comprehending the dynamic metabolic variations associated with GB maturation stagesrationalize their optimal harvest time per se the related agroeconomic traits.

Metabolomics Reveal Optimal Grain Preprocessing (Milling) toward Rice <i>Koji</i> Fermentation

Lee, Sunmin,Lee, Da Eun,Singh, Digar,Lee, Choong Hwan American Chemical Society, Books and Journals Divi 2018 Journal of agricultural and food chemistry Vol.66 No.11

<P>A time-correlated mass spectrometry (MS)-based metabolic profiling was performed for rice <I>koji</I> 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 <I>koji</I> made with DOM of 5-7 substrates at 36 h. The antioxidant secondary metabolites (flavonoids and phenolic acid) were relatively higher in <I>koji</I> with DOM of 0 substrates, followed by DOM of 5 > DOM of 7 > DOM 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 <I>koji</I> fermentation, with the end product being rich in distinctive organoleptic, nutritional, and functional metabolites. The study rationalizes the substrate preprocessing steps vital for commercial <I>koji</I> making.</P> [FIG OMISSION]</BR>

Evaluating the Headspace Volatolome, Primary Metabolites, and Aroma Characteristics of Koji Fermented with Bacillus amyloliquefaciens and Aspergillus oryzae

( Han Sol Seo ),( Sunmin Lee ),( Digar Singh ),( Min Kyung Park ),( Young-suk Kim ),( Hye Won Shin ),( Sun A Cho ),( Choong Hwan Lee ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.8

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.

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

Seo, Han Sol,Lee, Sunmin,Singh, Digar,Shin, Hye Won,Cho, Sun A,Lee, Choong Hwan Elsevier 2018 Food chemistry Vol.266 No.-

<P><B>Abstract</B></P> <P>Untargeted metabolomics unraveled the effects of varying substrates (soybean, wheat, and rice) and inocula (<I>Aspergillus oryzae</I> and <I>Bacillus amyloliquefaciens</I>) on metabolite compositions of <I>koji</I>, a starter ingredient in various Asian fermented foods. Multivariate analyses of the hyphenated mass spectrometry datasets for different <I>koji</I> 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 <I>koji</I>-types with varying substrates only, <I>viz.</I>, 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 <I>koji</I> (soybean > wheat, rice). The inocula choice of <I>A. oryzae</I> engendered higher carbohydrates, organic acids, and lipid derivative levels commensurate with high <I>α</I>-amylase and <I>β</I>-glucosidase activities, while <I>B. amyloliquefaciens</I> affected higher amino acids levels, in respective <I>koji</I> types.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Metabolomics revealed optimal substrate and inocula combination for preparing <I>koji</I>. </LI> <LI> Primary metabolites in <I>koji</I> were affected by substrates and microbial inocula. </LI> <LI> Primary metabolites except amino acids were higher in soybean substrate. </LI> <LI> Secondary metabolites varied largely according to substrate types. </LI> <LI> Flavonoids and soyasaponins abundant in soybean were higher in soybean <I>koji</I>. </LI> </UL> </P>