• Journal of Microbiology and Biotechnology
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• v.27 no.5
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• pp.925-932
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• 2017

Changes in the metabolite profiles of Lactobacillus sakei and its growth media, based on different culture times (0, 6, 12, and 24 h), were investigated using gas chromatography-mass spectrometry (MS) and liquid chromatography-MS with partial least squares discriminant analysis, in order to understand the growth characteristics of this organism. Cell and media samples of L. sakei were significantly separated on PLS-DA score plots. Cell and media metabolites, including sugars, amino acids, and organic acids, were identified as major metabolites contributing to the difference among samples. The alteration of cell and media metabolites during cell growth was strongly associated with energy production. Glucose, fructose, carnitine, tryptophan, and malic acid in the growth media were used as primary energy sources during the initial growth stage, but after the exhaustion of these energy sources, L. sakei could utilize other sources such as trehalose, citric acid, and lysine in the cell. The change in the levels of these energy sources was inversely similar to the energy production, especially ATP. Based on these identified metabolites, the metabolomic pathway associated with energy production through lactic acid fermentation was proposed. Although further studies are required, these results suggest that MS-based metabolomic analysis might be a useful tool for understanding the growth characteristics of L. sakei, the most important bacterium associated with meat and vegetable fermentation, during growth.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.104-104
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• 2017

Sorghum (Sorghum bicolor (L.) Moench.) is one of the most important crops for human and animal nutrition. Nonetheless, sorghum has a cyanogenic glucoside compound which can be degraded into hydrogen cyanide, toxic to humans and animals even with tiny amount. In consequence, breeding materials with a low cyanide level has been a top priority in sorghum breeding programs. To fulfill our long-term goal, we are screening sorghum accessions with low cyanide level, which would be an important breeding material for food safety. We collected seeds of various sorghum accessions and analyzed relevant metabolites to find useful breeding materials of sorghum accessions containing low cyanide. Fourteen wild relatives were obtained from the University of Georgia in US, a reference accession BTx623, and three local varieties from National Agrobiodiversity Center of Rural Development Administration in Korea, and one wild species from the Wild Plant Resources Seed Bank of Korea University in Korea. Sorghum plants were grown in plastic greenhouse under natural conditions. After growing, leaf samples were harvested at different developmental stages: seedling phase, vegetative phase (right before flowering), and reproductive phase (ripening). Using collected samples, quantification analysis were performed by an HPLC system for three metabolites (dhurrin, 4-hydroxybenzaldehyde, and 4-hydroxyphenylacetic acid) in sorghum plants. Prior to metabolome analysis, specific experimental condition for HPLC system was set to be able to separate three metabolites simultaneously. Under this condition, these metabolites were quantified in each accession by HPLC system. We observed that the metabolite contents were changed differently by developmental stages and accessions. We clustered these results into five groups as patterns of their contents by developmental stages. Most of accessions showed that 4-hydroxybenzaldehyde content was very high at seedling stage and decreased rapidly at vegetative phase. Interestingly, the patterns of dhurrin content were very different among clusters. However, 4-hydroxyphenylacetic acid content was maintained at low levels by developmental stages in most accessions. The results would demonstrate how dhurrin and alternative degradation pathways are differentiated in each accession.

• Journal of Animal Science and Technology
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• v.64 no.3
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• pp.481-499
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• 2022

This study aims to determine the effects of D-methionine (D-Met) isomer and the methionine precursor 2-hydroxy-4-methylthiobutanoic acid i (HMBi) supplementation on milk protein synthesis on immortalized bovine mammary epithelial cell (MAC-T). MAC-T cells were seeded using 10-cm dishes and cultured in Dulbecco's modified Eagle's medium/F12 (DMEM/F12) basic medium. The basic medium of DMEM/F12 was replaced with the lactogenic DMEM/ F12 differentiation medium when 90% of MAC-T cells reached confluency. The best dosage at 0.6 mM of D-Met and HMBi and incubation time at 72 h were used uniformly for all treatments. Each treatment was replicated six times wherein treatments were randomly assigned in a 6-well plate. Cell, medium, and total protein were determined using a bicinchoninic acid protein assay kit. Genes, proteomics and metabolomics analyses were also done to determine the mechanism of the milk protein synthesis pathway. Data were analyzed by two-way analysis of variance (ANOVA) with supplement type and plate as fixed effects. The least significant difference test was used to evaluate the differences among treatments. The HMBi treatment group had the highest beta-casein and S6 kinase beta-1 (S6K1) mRNA gene expression levels. HMBi and D-Met treatments have higher gene expressions compared to the control group. In terms of medium protein content, HMBi had a higher medium protein quantity than the control although not significantly different from the D-Met group. HMBi supplementation stimulated the production of eukaryotic translation initiation factor 3 subunit protein essential for protein translation initiation resulting in higher medium protein synthesis in the HMBi group than in the control group. The protein pathway analysis results showed that the D-Met group stimulated fructose-galactose metabolism, glycolysis pathway, phosphoinositide 3 kinase, and pyruvate metabolism. The HMBi group stimulated the pentose phosphate and glycolysis pathways. Metabolite analysis revealed that the D-Met treatment group increased seven metabolites and decreased uridine monophosphate (UMP) production. HMBi supplementation increased the production of three metabolites and decreased UMP and N-acetyl-L-glutamate production. Taken together, D-Met and HMBi supplementation are effective in stimulating milk protein synthesis in MAC-T cells by genes, proteins, and metabolites stimulation linked to milk protein synthesis.

• Mass Spectrometry Letters
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• v.11 no.2
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• pp.17-24
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• 2020

Metabolomics has become an important research field with many areas of applications ranging from disease biomarker discovery to global biology systems study. A key step in metabolomics is to perform metabolome analysis to obtain quantitative information on metabolic changes among comparative samples. Mass spectrometry (MS) is widely used for highly sensitive detection of many different types of metabolites. In this review, we highlight some of the more commonly used MS techniques for metabolome analysis.

• Journal of Microbiology and Biotechnology
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• v.21 no.1
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• pp.5-13
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• 2011

In this study, seven Trichoderma species (33 strains) were classified using secondary metabolite profile-based chemotaxonomy. Secondary metabolites were analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) and multivariate statistical methods. T. longibrachiatum and T. virens were independently clustered based on both internal transcribed spacer (ITS) sequence and secondary metabolite analyses. T. harzianum formed three subclusters in the ITS-based phylogenetic tree and two subclusters in the metabolitebased dendrogram. In contrast, T. koningii and T. atroviride strains were mixed in one cluster in the phylogenetic tree, whereas T. koningii was grouped in a different subcluster from T. atroviride and T. hamatum in the chemotaxonomic tree. Partial least-squares discriminant analysis (PLS-DA) was applied to determine which metabolites were responsible for the clustering patterns observed for the different Trichoderma strains. The metabolites were hetelidic acid, sorbicillinol, trichodermanone C, giocladic acid, bisorbicillinol, and three unidentified compounds in the comparison of T. virens and T. longibrachiatum; harzianic acid, demethylharzianic acid, homoharzianic acid, and three unidentified compounds in T. harzianum I and II; and koninginin B, E, and D, and six unidentified compounds in T. koningii and T. atroviride. The results of this study demonstrate that secondary metabolite profiling-based chemotaxonomy has distinct advantages relative to ITS-based classification, since it identified new Trichoderma clusters that were not found using the latter approach.

• Journal of Microbiology and Biotechnology
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• v.30 no.6
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• pp.903-911
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• 2020

Addition of probiotics to yogurt with desired health benefits is gaining increasing attention. To further understand the effect of probiotic Lactobacillus plantarum on the quality and function of fermented milk, probiotic fermented milk (PFM) made with probiotic L. plantarum K25 and yogurt starter (L. delbrueckii ssp. bulgaricus and Streptococcus thermophilus) was compared with the control fermented milk (FM) made with only the yogurt starter. The probiotic strain was shown to survive well with a viable count of 7.1 ± 0.1 log CFU/g in the PFM sample after 21 days of storage at 4℃. The strain was shown to promote formation of volatiles such as acetoin and 2,3-butanediol with milk fragrance, and it did not cause post-acidification during refrigerated storage. Metabolomics analysis by GC-MS datasets coupled with multivariate statistical analysis showed that addition of L. plantarum K25 increased formation of over 20 metabolites detected in fermented milk, among which γ-aminobutyric acid was the most prominent. Together with several other metabolites with relatively high levels in fermented milk such as glyceric acid, malic acid, succinic acid, glycine, alanine, ribose, and 1,3-dihydroxyacetone, they might play important roles in the probiotic function of L. plantarum K25. Further assay of the bioactivity of the PFM sample showed significant (p < 0.05) increase of ACE inhibitory activity from 22.3% at day 1 to 49.3% at day 21 of the refrigerated storage. Therefore, probiotic L. plantarum K25 could be explored for potential application in functional dairy products.

• Journal of odor and indoor environment
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• v.17 no.4
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• pp.315-321
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• 2018

Phthalate is an endocrine disruptor that interferes with homeostasis and developmental regulation. It is highly toxic to the environment and is associated with various diseases of the human body. Using biological samples from 140 adult subjects, to evaluate the influencing factors which are related to contaminant concentration levels, we used correlation analysis and multiple regression analysis. Lastly, in order to analyze the health effects related to exposure to phthalates, we conducted a risk assessment by estimating acceptable daily intake exposure according to the influential factors. When we compared the concentration level according to influential factors, in general, the subjects who had engaged in home remodeling work had higher urinary phthalate metabolite concentrations levels than the subjects who had not engaged in home remodeling work. We can confirm statistically significant differences in DBP metabolites. In addition, we can confirm the concentration appeared higher in the categories such as using air freshener, sofa and foods. Through conducting a risk assessment of DEHP, BBzP, DiBP, and DnBP by using data on phthalate metabolite concentration in urine, it was found that the average concentration of all metabolites did not exceed TDI.

• Journal of Ginseng Research
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• v.45 no.2
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• pp.334-343
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• 2021

Background: Gut microbiota mainly function in the biotransformation of primary ginsenosides into bioactive metabolites. Herein, we investigated the effects of three prebiotic fibers by targeting gut microbiota on the metabolism of ginsenoside Rb1 in vivo. Methods: Sprague Dawley rats were administered with ginsenoside Rb1 after a two-week prebiotic intervention of fructooligosaccharide, galactooligosaccharide, and fibersol-2, respectively. Pharmacokinetic analysis of ginsenoside Rb1 and its metabolites was performed, whilst the microbial composition and metabolic function of gut microbiota were examined by 16S rRNA gene amplicon and metagenomic shotgun sequencing. Results: The results showed that peak plasma concentration and area under concentration time curve of ginsenoside Rb1 and its intermediate metabolites, ginsenoside Rd, F2, and compound K (CK), in the prebiotic intervention groups were increased at various degrees compared with those in the control group. Gut microbiota dramatically responded to the prebiotic treatment at both taxonomical and functional levels. The abundance of Prevotella, which possesses potential function to hydrolyze ginsenoside Rb1 into CK, was significantly elevated in the three prebiotic groups (P < 0.05). The gut metagenomic analysis also revealed the functional gene enrichment for terpenoid/polyketide metabolism, glycolysis, gluconeogenesis, propanoate metabolism, etc. Conclusion: These findings imply that prebiotics may selectively promote the proliferation of certain bacterial stains with glycoside hydrolysis capacity, thereby, subsequently improving the biotransformation and bioavailability of primary ginsenosides in vivo.

• Microbiology and Biotechnology Letters
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• v.51 no.2
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• pp.147-156
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• 2023

Tetragenococcus halophilus CY54, an isolate from jeotgal, grows best in media with 5% NaCl and can grow at 18% and higher salt concentration. Three different doenjang samples were prepared with multiple starters including T. halophilus CY54. TBZA doenjang was prepared with T. halophilus, Bacillus subtilis, Zygosaccharomyces rouxii and Aspergillus oryzae. BZA doenjang was prepared with the same 3 starters except T. halophilus. KACC doenjang was prepared with a single starter, B. subtilis KACC16750. During 16 weeks of fermentation at 25℃, the viable counts were maintained in the range of 7-8 log CFU/g in all 3 samples. As fermentation progressed, pH decreased and titratable acidity (TA) gradually increased. Crude protein contents decreased slightly. TBZA doenjang showed higher amino-type nitrogen (ANN) and volatile basic nitrogen (VBN) contents, and KACC doenjang showed higher ammonia-type nitrogen (AMN) content. TBZA doenjang showed higher fibrinolytic and protease activity than other doenjang samples. Metabolites analyses by GC/MS showed that doenjang samples were separated from each other by partial least squares-discriminant analysis (PLS-DA) analysis. Seventeen major metabolites involved in the differences between samples were identified and they included organic acids, amino acids, sugars, fatty acids and alcohols. TBZA doenjang showed higher contents for most metabolites responsible for flavor and taste of fermented foods including doenjang. These results showed that T. halophilus could be useful as a starter for doenjang and can improve the product quality by accelerating the fermentation processes.

• Journal of Microbiology and Biotechnology
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• v.33 no.10
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• pp.1351-1360
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• 2023

Endocrine-disrupting chemicals (EDCs) are compounds that disturb hormonal homeostasis by binding to receptors. EDCs are metabolized through hepatic enzymes, causing altered transcriptional activities of hormone receptors, and thus necessitating the exploration of the potential endocrine-disrupting activities of EDC-derived metabolites. Accordingly, we have developed an integrative workflow for evaluating the post-metabolic activity of potential hazardous compounds. The system facilitates the identification of metabolites that exert hormonal disruption through the integrative application of an MS/MS similarity network and predictive biotransformation based on known hepatic enzymatic reactions. As proof-of-concept, the transcriptional activities of 13 chemicals were evaluated by applying the in vitro metabolic module (S9 fraction). Identified among the tested chemicals were three thyroid hormone receptor (THR) agonistic compounds that showed increased transcriptional activities after phase I+II reactions (T3, 309.1 ± 17.3%; DITPA, 30.7 ± 1.8%; GC-1, 160.6 ± 8.6% to the corresponding parents). The metabolic profiles of these three compounds showed common biotransformation patterns, particularly in the phase II reactions (glucuronide conjugation, sulfation, GSH conjugation, and amino acid conjugation). Data-dependent exploration based on molecular network analysis of T3 profiles revealed that lipids and lipid-like molecules were the most enriched biotransformants. The subsequent subnetwork analysis proposed 14 additional features, including T4 in addition to 9 metabolized compounds that were annotated by prediction system based on possible hepatic enzymatic reaction. The other 10 THR agonistic negative compounds showed unique biotransformation patterns according to structural commonality, which corresponded to previous in vivo studies. Our evaluation system demonstrated highly predictive and accurate performance in determining the potential thyroid-disrupting activity of EDC-derived metabolites and for proposing novel biotransformants.