#METABOLOMICS WORKBENCH ronghaha_20250820_221124 DATATRACK_ID:6316 STUDY_ID:ST004156 ANALYSIS_ID:AN006899 PROJECT_ID:PR002618 VERSION 1 CREATED_ON September 1, 2025, 7:26 pm #PROJECT PR:PROJECT_TITLE Influence of valeric acid fermentation on metabolites of human fecal microbial PR:PROJECT_TITLE community PR:PROJECT_SUMMARY By using an in vitro simulated intestinal fermentation system, the influence of PR:PROJECT_SUMMARY valeric acid on the metabolites of the microbial community was investigated. PR:PROJECT_SUMMARY Results: Notable differential metabolites included Isotocin, Isoleucyl-Lysine, PR:PROJECT_SUMMARY and D-glutamine. Sankey diagram analysis further demonstrated the involvement of PR:PROJECT_SUMMARY Deoxyadenosine, Xanthine, and Adenine in enriched pathways such as ABC PR:PROJECT_SUMMARY transporters, purine metabolism, and nucleotide metabolism. Comparative analysis PR:PROJECT_SUMMARY between the PMV and PS groups revealed 177 upregulated and 157 downregulated PR:PROJECT_SUMMARY metabolites. Agmatine and specific amino acids emerged as common differential PR:PROJECT_SUMMARY metabolites in both PS-vs-Control and PMV-vs-PS comparisons. The findings PR:PROJECT_SUMMARY indicate that arginine metabolism serves as a critical regulatory target of PR:PROJECT_SUMMARY valeric acid. PR:INSTITUTE Zhejiang University PR:LAST_NAME Yan PR:FIRST_NAME Fujie PR:ADDRESS Yuhangtang Road, Hangzhou, Zhejiang, 310058, China PR:EMAIL fjyan@zju.edu.cn PR:PHONE +86 15068185696 #STUDY ST:STUDY_TITLE Valerate maintains ammonia homeostasis through modulating microbial amino acid ST:STUDY_TITLE metabolism ST:STUDY_SUMMARY By using an in vitro simulated intestinal fermentation system, the influence of ST:STUDY_SUMMARY valeric acid on the metabolites of the microbial community was investigated. ST:STUDY_SUMMARY There are four groups measured in this study: (1)Control; (2) PS (polystyrene, ST:STUDY_SUMMARY 0.1 mg/mL); (3) PLV (PS+0.01 mg/mL valeric acid); (4) PMV(PS+0.1 mg/mL valeric ST:STUDY_SUMMARY acid). We conducted a comprehensive analysis of metabolic alterations in the ST:STUDY_SUMMARY fermentation broth. Partial Least Squares Discriminant Analysis (PLS-DA) ST:STUDY_SUMMARY revealed significant metabolic variations across the four experimental groups. ST:STUDY_SUMMARY Volcano plot and Venn diagram analyses identified 102 differentially expressed ST:STUDY_SUMMARY metabolites between the PS and control groups, comprising 65 upregulated and 37 ST:STUDY_SUMMARY downregulated species. Notable differential metabolites included Isotocin, ST:STUDY_SUMMARY Isoleucyl-Lysine, and D-glutamine. Sankey diagram analysis further demonstrated ST:STUDY_SUMMARY the involvement of Deoxyadenosine, Xanthine, and Adenine in enriched pathways ST:STUDY_SUMMARY such as ABC transporters, purine metabolism, and nucleotide metabolism. ST:STUDY_SUMMARY Comparative analysis between the PMV and PS groups revealed 177 upregulated and ST:STUDY_SUMMARY 157 downregulated metabolites. Agmatine and specific amino acids emerged as ST:STUDY_SUMMARY common differential metabolites in both PS-vs-Control and PMV-vs-PS comparisons. ST:STUDY_SUMMARY Notably, ammonia levels exhibited strong associations with amino acid ST:STUDY_SUMMARY metabolism, with amino acid-related metabolites constituting 34.23% of all ST:STUDY_SUMMARY differential metabolites. These included pathways such as arginine biosynthesis, ST:STUDY_SUMMARY arginine-proline metabolism, and D-amino acid synthesis. Arginine participates ST:STUDY_SUMMARY in the urea cycle to promote urea formation, which is subsequently hydrolyzed ST:STUDY_SUMMARY into ammonia via urease activity. Intriguingly, the valerate-treated group ST:STUDY_SUMMARY exhibited downregulated enzymatic activity compared to the PS group, accompanied ST:STUDY_SUMMARY by reduced levels of key metabolites including Agmatine, L-glutamic acid, ST:STUDY_SUMMARY L-ornithine, and L-aspartic acid. Collectively, these findings indicate that ST:STUDY_SUMMARY arginine metabolism serves as a critical regulatory target of valeric acid. By ST:STUDY_SUMMARY modulating gut microbiota composition, valeric acid suppresses enzymatic ST:STUDY_SUMMARY activity involved in arginine synthesis and degradation cycles, thereby ST:STUDY_SUMMARY attenuating overall pathway flux and ultimately reducing microbial ammonia ST:STUDY_SUMMARY production. ST:INSTITUTE Zhejiang University ST:DEPARTMENT Food Science and Nutrition ST:LABORATORY Key Laboratory for Exploration and High-Value Utilization of Agricultural ST:LABORATORY Products ST:LAST_NAME Yan ST:FIRST_NAME Fujie ST:ADDRESS Yuhangtang Road, Hangzhou, Zhejiang, 310058, China ST:EMAIL fjyan@zju.edu.cn ST:PHONE +86 15068185696 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS Control C-1 Genotype:Control | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_CON1.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_CON1.mzML SUBJECT_SAMPLE_FACTORS Control C-2 Genotype:Control | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_CON2.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_CON2.mzML SUBJECT_SAMPLE_FACTORS Control C-3 Genotype:Control | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_CON3.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_CON3.mzML SUBJECT_SAMPLE_FACTORS Control C-4 Genotype:Control | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_CON4.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_CON4.mzML SUBJECT_SAMPLE_FACTORS Control C-5 Genotype:Control | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_CON5.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_CON5.mzML SUBJECT_SAMPLE_FACTORS Control C-6 Genotype:Control | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_CON6.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_CON6.mzML SUBJECT_SAMPLE_FACTORS PS PS-1 Genotype:Model | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PS1.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PS1.mzML SUBJECT_SAMPLE_FACTORS PS PS-2 Genotype:Model | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PS2.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PS2.mzML SUBJECT_SAMPLE_FACTORS PS PS-3 Genotype:Model | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PS3.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PS3.mzML SUBJECT_SAMPLE_FACTORS PS PS-4 Genotype:Model | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PS4.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PS4.mzML SUBJECT_SAMPLE_FACTORS PS PS-5 Genotype:Model | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PS5.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PS5.mzML SUBJECT_SAMPLE_FACTORS PS PS-6 Genotype:Model | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PS6.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PS6.mzML SUBJECT_SAMPLE_FACTORS LV LV-1 Genotype:Intervention-1 | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PLV1.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PLV1.mzML SUBJECT_SAMPLE_FACTORS LV LV-2 Genotype:Intervention-1 | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PLV2.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PLV2.mzML SUBJECT_SAMPLE_FACTORS LV LV-3 Genotype:Intervention-1 | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PLV3.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PLV3.mzML SUBJECT_SAMPLE_FACTORS LV LV-4 Genotype:Intervention-1 | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PLV4.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PLV4.mzML SUBJECT_SAMPLE_FACTORS LV LV-5 Genotype:Intervention-1 | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PLV5.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PLV5.mzML SUBJECT_SAMPLE_FACTORS LV LV-6 Genotype:Intervention-1 | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PLV6.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PLV6.mzML SUBJECT_SAMPLE_FACTORS MV MV-1 Genotype:Intervention-2 | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PMV1.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PMV1.mzML SUBJECT_SAMPLE_FACTORS MV MV-2 Genotype:Intervention-2 | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PMV2.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PMV2.mzML SUBJECT_SAMPLE_FACTORS MV MV-3 Genotype:Intervention-2 | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PMV3.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PMV3.mzML SUBJECT_SAMPLE_FACTORS MV MV-4 Genotype:Intervention-2 | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PMV4.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PMV4.mzML SUBJECT_SAMPLE_FACTORS MV MV-5 Genotype:Intervention-2 | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PMV5.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PMV5.mzML SUBJECT_SAMPLE_FACTORS MV MV-6 Genotype:Intervention-2 | Sample source:Supernatant of fecal fermentation broth RAW_FILE_NAME(Raw file name)=NEG_He10FJ_PMV6.mzML; RAW_FILE_NAME(Raw file name)=POS_He10FJ_PMV6.mzML #COLLECTION CO:COLLECTION_SUMMARY Fresh fecal samples were collected from three healthy volunteers and immediately CO:COLLECTION_SUMMARY transferred to a sterile laminar flow hood. A 10 g aliquot of feces was CO:COLLECTION_SUMMARY homogenized in 80 mL of sterile phosphate-buffered saline (PBS) using a sterile CO:COLLECTION_SUMMARY glass rod, followed by filtration through triple-layered sterile gauze. Side-arm CO:COLLECTION_SUMMARY test tubes were sealed at the lower port, and 27 mL of sterilized intestinal CO:COLLECTION_SUMMARY fermentation medium and 3 mL of fecal filtrate were aseptically introduced into CO:COLLECTION_SUMMARY each tube. The mixture was thoroughly vortexed, and anaerobic conditions were CO:COLLECTION_SUMMARY established by purging the headspace with nitrogen gas for 5 min. Tubes were CO:COLLECTION_SUMMARY incubated at 37 ℃ in a shaking incubator (180 rpm) for 24 h to stabilize the CO:COLLECTION_SUMMARY microbial community. Following stabilization, indicated concentrations of CO:COLLECTION_SUMMARY PS/valeric acid were introduced into the fermentation broth. The anaerobic CO:COLLECTION_SUMMARY environment was re-established by nitrogen gas purging (5 min), and tubes were CO:COLLECTION_SUMMARY returned to the 37 ℃ shaking incubator for continued fermentation. CO:COLLECTION_SUMMARY Fermentation broth samples (3 mL) were collected at 24-hour intervals over a CO:COLLECTION_SUMMARY 72-hour period. The supernatant obtained after centrifugation of the CO:COLLECTION_SUMMARY fermentation broth was used for metabolomics determination. CO:SAMPLE_TYPE Fecal fermentation broth #TREATMENT TR:TREATMENT_SUMMARY Healthy individuals' feces were selected for in vitro fermentation. One day TR:TREATMENT_SUMMARY later, polystyrene microplastics (PS, 0.1 mg/mL)and different concentrations of TR:TREATMENT_SUMMARY valeric acid (PLV, 0.01 mg/mL; PMV, 0.1 mg/mL) were added to the fermentation TR:TREATMENT_SUMMARY liquid. Three days later, the fermentation supernatant was collected for TR:TREATMENT_SUMMARY metabolomics analysis. There are four groups: Control, PS, PLV, PMV. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 100 μL liquid sample was added to a 1.5 mL centrifuge tube with 800 μL SP:SAMPLEPREP_SUMMARY solution (acetonitrile: methanol = 1:1(v:v)) containing four internal standards SP:SAMPLEPREP_SUMMARY (0.02 mg/mL L-2-chlorophenylalanine, etc.) to extract metabolites. The samples SP:SAMPLEPREP_SUMMARY were mixed by vortex for 30 s and low-temperature sonicated for 30 min (5°C, 40 SP:SAMPLEPREP_SUMMARY KHz)。The samples were placed at -20°C for 30 min to precipitate the proteins. SP:SAMPLEPREP_SUMMARY Then the samples were centrifuged for 15 min (4°C, 13000 g). The supernatant SP:SAMPLEPREP_SUMMARY was removed and blown dry under nitrogen. The sample was then re-solubilized SP:SAMPLEPREP_SUMMARY with 100 µL solution (acetonitrile: water = 1:1) and extracted by SP:SAMPLEPREP_SUMMARY low-temperature ultrasonication for 5 min (5°C, 40 KHz), followed by SP:SAMPLEPREP_SUMMARY centrifugation at 13000 g and 4°C for 10 min.The supernatant was transferred to SP:SAMPLEPREP_SUMMARY sample vials for LC-MS/MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Solvent A: 0.1% formic acid in water:acetonitrile (2:98, v/v); Solvent B: 0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid in acetonitrile CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:SOLVENT_A 2% water/98% acetonitrile; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0min, 100% A; 3min, 80% A; 4.5min, 65% A; 15.5min, 15% A; 16min, 3% A; 18min, 3% CH:FLOW_GRADIENT A; 18.1min, 100% A; 21min, 100% A CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The UHPLC system was coupled to a UHPLC-Q Exactive HF-X system Mass Spectrometer MS:MS_COMMENTS equipped with an electrospray ionization (ESI) source operating in positive mode MS:MS_COMMENTS and negative mode. The optimal conditions were set as followed: source MS:MS_COMMENTS temperature at 400℃ ; sheath gas flow rate at 40 arb; Aux gas flow rate at 10 MS:MS_COMMENTS arb; ion-spray voltage floating (ISVF) at -2800V in negative mode and 3500V in MS:MS_COMMENTS positive mode, respectively; Normalized collision energy , 20-40-60V rolling for MS:MS_COMMENTS MS/MS. Data acquisition was performed with the Data Dependent Acquisition (DDA) MS:MS_COMMENTS mode. The detection was carried out over a mass range of 70-1050 m/z. The MS:MS_COMMENTS pretreatment of LC/MS raw data was performed by Progenesis QI (Waters MS:MS_COMMENTS Corporation， Milford, USA) software, and a three-dimensional data matrix in MS:MS_COMMENTS CSV format was exported. The information in this three-dimensional matrix MS:MS_COMMENTS included: sample information, metabolite name and mass spectral response MS:MS_COMMENTS intensity. Internal standard peaks, as well as any known false positive peaks MS:MS_COMMENTS (including noise, column bleed, and derivatized reagent peaks), were removed MS:MS_COMMENTS from the data matrix, deredundant and peak pooled. At the same time, the MS:MS_COMMENTS metabolites were identified by searching database, and the main databases were MS:MS_COMMENTS the HMDB (http://www.hmdb.ca/), Metlin ( https://metlin.scripps.edu/) and the MS:MS_COMMENTS self-compiled Majorbio Database (MJDB) of Majorbio Biotechnology Co., Ltd. MS:MS_COMMENTS (Shanghai, China) MS:MS_RESULTS_FILE ST004156_AN006899_Results.txt UNITS:peak intensity Has m/z:Yes Has RT:No RT units:No RT data #END