#METABOLOMICS WORKBENCH sulei_20250903_235651 DATATRACK_ID:6374 STUDY_ID:ST004173 ANALYSIS_ID:AN006928 PROJECT_ID:PR002633 VERSION 1 CREATED_ON September 9, 2025, 1:12 pm #PROJECT PR:PROJECT_TITLE Metabolomics of Evolutionary Lacticaseibacillus casei (SEvo) and Wild-Type PR:PROJECT_TITLE Lacticaseibacillus casei (SAnc) PR:PROJECT_SUMMARY To compare the higher survival ability of evolutionary Lacticaseibacillus PR:PROJECT_SUMMARY casei(SEvo) than wild-type Lacticaseibacillus casei(SAnc) under hydrogen PR:PROJECT_SUMMARY peroxide (a strong oxidative stressor), we used 1.5 mM hydrogen peroxide for a PR:PROJECT_SUMMARY 2-hour stress treatment and conducted metabolomic studies based on mass PR:PROJECT_SUMMARY spectrometry technology. we analyzed the differences in metabolites among them, PR:PROJECT_SUMMARY and performed KEGG pathway enrichment analysis by investigating the abundance of PR:PROJECT_SUMMARY metabolites and the classification of metabolites, so as to explore the PR:PROJECT_SUMMARY metabolic pathways that contribute to the superiority of evolutionary strains PR:PROJECT_SUMMARY over wild-type strains under oxidative stress. PR:INSTITUTE Kunming university of science and technology PR:LAST_NAME Su PR:FIRST_NAME lei PR:ADDRESS Wujiaying Street, Chenggong District, Kunming City, Yunnan Province, Kunming, PR:ADDRESS Yunnan, 650599, China PR:EMAIL suleilei21@163.com PR:PHONE 17775175972 #STUDY ST:STUDY_TITLE Study on the metabolic pathways of evolutionary Lacticaseibacillus casei and ST:STUDY_TITLE wild-type Lacticaseibacillus casei under oxidative stress ST:STUDY_SUMMARY This study took evolutionary Lacticaseibacillus casei and wild-type ST:STUDY_SUMMARY Lacticaseibacillus casei as research objects, focusing on analyzing the ST:STUDY_SUMMARY differences in metabolic pathways between the two strains under oxidative ST:STUDY_SUMMARY stress. First, sample processing and stress induction were conducted: 1.5 mM ST:STUDY_SUMMARY hydrogen peroxide was used as the oxidative stressor, and the two strains were ST:STUDY_SUMMARY subjected to stress treatment for 2 hours respectively. After the stress ST:STUDY_SUMMARY treatment, the bacterial cells were collected by low-temperature ST:STUDY_SUMMARY centrifugation.Subsequently, ultra-high performance liquid chromatography-tandem ST:STUDY_SUMMARY mass spectrometry (UPLC-MS/MS) technology was employed for metabolite detection. ST:STUDY_SUMMARY Primary and secondary mass spectrometry data of metabolites were collected in ST:STUDY_SUMMARY positive and negative ion modes respectively, and the types of metabolites were ST:STUDY_SUMMARY initially identified by matching with metabolomics databases such as HMDB and ST:STUDY_SUMMARY METLIN.Finally, data analysis was carried out: first, partial least squares ST:STUDY_SUMMARY discriminant analysis (PLS-DA) was used for pattern recognition of the ST:STUDY_SUMMARY metabolomic data of the two strains. Metabolites with significant differences ST:STUDY_SUMMARY between groups were screened based on the criteria of VIP>1 and P<0.05. Then, ST:STUDY_SUMMARY the differential metabolites were mapped to the KEGG database, and metabolic ST:STUDY_SUMMARY pathway enrichment analysis was performed via Fisher’s Exact Test. Focus was ST:STUDY_SUMMARY placed on antioxidant, energy, and amino acid-related metabolic pathways to ST:STUDY_SUMMARY clarify the differences in metabolic pathway regulation between the two strains ST:STUDY_SUMMARY under oxidative stress, providing methodological support for analyzing the ST:STUDY_SUMMARY stress resistance mechanism of the evolutionary strain. ST:INSTITUTE Kunming university of science and technology ST:LAST_NAME Su ST:FIRST_NAME lei ST:ADDRESS Wujiaying Street, Chenggong District, Kunming City, Yunnan Province, Kunming, ST:ADDRESS Yunnan, 650599, China ST:EMAIL suleilei21@163.com ST:PHONE 17775175972 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Lacticaseibacillus casei SU:TAXONOMY_ID 1582 #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 - SAnc_1 Sample source:bacterial cells | Bacterial strain:WT RAW_FILE_NAME_NEG=SAnc_1.raw; RAW_FILE_NAME_POS=SAnc_p1.raw SUBJECT_SAMPLE_FACTORS - SAnc_2 Sample source:bacterial cells | Bacterial strain:WT RAW_FILE_NAME_NEG=SAnc_2.raw; RAW_FILE_NAME_POS=SAnc_p2.raw SUBJECT_SAMPLE_FACTORS - SAnc_3 Sample source:bacterial cells | Bacterial strain:WT RAW_FILE_NAME_NEG=SAnc_3.raw; RAW_FILE_NAME_POS=SAnc_p3.raw SUBJECT_SAMPLE_FACTORS - SAnc_4 Sample source:bacterial cells | Bacterial strain:WT RAW_FILE_NAME_NEG=SAnc_4.raw; RAW_FILE_NAME_POS=SAnc_p4.raw SUBJECT_SAMPLE_FACTORS - SAnc_5 Sample source:bacterial cells | Bacterial strain:WT RAW_FILE_NAME_NEG=SAnc_5.raw; RAW_FILE_NAME_POS=SAnc_p5.raw SUBJECT_SAMPLE_FACTORS - SAnc_6 Sample source:bacterial cells | Bacterial strain:WT RAW_FILE_NAME_NEG=SAnc_6.raw; RAW_FILE_NAME_POS=SAnc_p6.raw SUBJECT_SAMPLE_FACTORS - SEvo_1 Sample source:bacterial cells | Bacterial strain:Evo RAW_FILE_NAME_NEG=SEvo_1.raw; RAW_FILE_NAME_POS=SEvo_p1.raw SUBJECT_SAMPLE_FACTORS - SEvo_2 Sample source:bacterial cells | Bacterial strain:Evo RAW_FILE_NAME_NEG=SEvo_2.raw; RAW_FILE_NAME_POS=SEvo_p2.raw SUBJECT_SAMPLE_FACTORS - SEvo_3 Sample source:bacterial cells | Bacterial strain:Evo RAW_FILE_NAME_NEG=SEvo_3.raw; RAW_FILE_NAME_POS=SEvo_p3.raw SUBJECT_SAMPLE_FACTORS - SEvo_4 Sample source:bacterial cells | Bacterial strain:Evo RAW_FILE_NAME_NEG=SEvo_4.raw; RAW_FILE_NAME_POS=SEvo_p4.raw SUBJECT_SAMPLE_FACTORS - SEvo_5 Sample source:bacterial cells | Bacterial strain:Evo RAW_FILE_NAME_NEG=SEvo_5.raw; RAW_FILE_NAME_POS=SEvo_p5.raw SUBJECT_SAMPLE_FACTORS - SEvo_6 Sample source:bacterial cells | Bacterial strain:Evo RAW_FILE_NAME_NEG=SEvo_6.raw; RAW_FILE_NAME_POS=SEvo_p6.raw #COLLECTION CO:COLLECTION_SUMMARY We obtained an evolved strain of Lacticaseibacillus casei (formerly CO:COLLECTION_SUMMARY Lactobacillus casei) from the wild-type Lacticaseibacillus casei via aerobic CO:COLLECTION_SUMMARY stress. Both the wild-type and evolved Lacticaseibacillus casei strains were CO:COLLECTION_SUMMARY cultured overnight in MRS medium for approximately 16 hours. Subsequently, CO:COLLECTION_SUMMARY hydrogen peroxide was added to adjust its concentration to 1.5 mM, and the CO:COLLECTION_SUMMARY mixture was incubated in a constant-temperature incubator at 37°C for 2 hours. CO:COLLECTION_SUMMARY After that, the bacterial cells were collected by centrifugation at 4°C (3,000 CO:COLLECTION_SUMMARY rpm for 8 minutes). The collected cells were washed three times with distilled CO:COLLECTION_SUMMARY water, followed by another centrifugation step at 4°C (3,000 rpm for 8 CO:COLLECTION_SUMMARY minutes). The final bacterial cells were placed in 1.5 mL centrifuge tubes, with CO:COLLECTION_SUMMARY the weight of each sample required to be at least 80 mg. These samples were then CO:COLLECTION_SUMMARY transported at 4°C for subsequent testing. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY Sample type: bacterial cells; Collection time: 2 hours post-treatment; TR:TREATMENT_SUMMARY Collection tools and conditions: sterile centrifuge tubes were used, and samples TR:TREATMENT_SUMMARY were transported under refrigeration at 4°C; Collection volume: 80 TR:TREATMENT_SUMMARY mg.Intervention subjects: two bacterial strains, namely evolved bacteria and TR:TREATMENT_SUMMARY wild-type bacteria. The overnight cultures of evolved bacteria and wild-type TR:TREATMENT_SUMMARY bacteria were centrifuged, then resuspended in 1.5 mM hydrogen peroxide, and TR:TREATMENT_SUMMARY incubated for 2 hours.After being treated with 1.5 mM hydrogen peroxide for 2 TR:TREATMENT_SUMMARY hours, the evolved bacteria and wild-type bacteria were washed three times with TR:TREATMENT_SUMMARY distilled water. Subsequently, bacterial cells were collected via centrifugation TR:TREATMENT_SUMMARY at 4°C (3,000 rpm for 8 minutes), and finally placed in 1.5 mL centrifuge tubes TR:TREATMENT_SUMMARY for subsequent detection. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY After being treated with 1.5 mM hydrogen peroxide for 2 hours, the evolved SP:SAMPLEPREP_SUMMARY bacteria and wild-type bacteria were washed three times with distilled water. SP:SAMPLEPREP_SUMMARY Subsequently, bacterial cells were collected via centrifugation at 4°C (3,000 SP:SAMPLEPREP_SUMMARY rpm for 8 minutes), and finally placed in 1.5 mL centrifuge tubes for subsequent SP:SAMPLEPREP_SUMMARY detection. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Ultra-High Performance Liquid Chromatography (UPLC) with Reverse Phase (RP) CH:CHROMATOGRAPHY_SUMMARY chromatography (based on the use of ACQUITY UPLC HSS T3 column, a typical CH:CHROMATOGRAPHY_SUMMARY reverse-phase chromatographic column, and mobile phases with water and CH:CHROMATOGRAPHY_SUMMARY acetonitrile, consistent with RP separation principles) CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 99.9% acetonitrile/0.1% formic acid CH:FLOW_GRADIENT 0.0 min 5% B; 1.0 min 5% B; 4.7 min 95% B; 6 min 95% B; 6.1 min 5% B; 8.5 min 5% CH:FLOW_GRADIENT B CH:FLOW_RATE 0.4mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Exploris 120 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Spray voltage –3.0 kV; other parameters (sheath/auxiliary gas, temps, scan MS:MS_COMMENTS range, resolution, AGC, Max IT, collision energy, dynamic exclusion) are the MS:MS_COMMENTS same as positive mode. Raw data (*.raw) directly imported into MS-DIAL; peaks MS:MS_COMMENTS absent in >50% QC samples filtered; missing values imputed (gap filling); MS:MS_COMMENTS normalization applied. Suitable for deprotonated metabolites. Thermo Xcalibur MS:MS_COMMENTS (v4.7) for DDA acquisition. MS-DIAL (v4.9.221218) for feature extraction and MS:MS_COMMENTS annotation; databases: PSNGM, mzCloud, LIPID MAPS, HMDB, MoNA, NIST_2020_MSMS, MS:MS_COMMENTS AI-predicted MS/MS. Matching tolerance: MS1 ±0.01 m/z, MS2 ±0.05 m/z; features MS:MS_COMMENTS with score ≥70 accepted. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS intensity MS_METABOLITE_DATA_START Samples SAnc_1 SAnc_2 SAnc_3 SAnc_4 SAnc_5 SAnc_6 SEvo_1 SEvo_2 SEvo_3 SEvo_4 SEvo_5 SEvo_6 Factors Sample source:bacterial cells | Bacterial strain:WT Sample source:bacterial cells | Bacterial strain:WT Sample source:bacterial cells | Bacterial strain:WT Sample source:bacterial cells | Bacterial strain:WT Sample source:bacterial cells | Bacterial strain:WT Sample source:bacterial cells | Bacterial strain:WT Sample source:bacterial cells | Bacterial strain:Evo Sample source:bacterial cells | Bacterial strain:Evo Sample source:bacterial cells | Bacterial strain:Evo Sample source:bacterial cells | Bacterial strain:Evo Sample source:bacterial cells | Bacterial strain:Evo Sample source:bacterial cells | Bacterial strain:Evo Provitamin C 30250352.45 40290392.14 38465016.91 33616997.07 28455681.23 33884535.53 35804187.46 34892927.23 28906364.36 29394475.24 35951143.67 43811857.07 Glutathione sulfonate 31469383.82 22765458.13 42613674.23 51328366.45 23039332.68 27343776.26 8514347.004 47351308.94 48085926.54 28443676.47 40149654 50291787.46 Thioglycolic acid 6562277.029 6320477.08 5715312.947 6465992.604 3813692.909 4542063.152 5051247.592 7878265.456 6978393.621 6571574.959 6834208.607 5914011.557 alpha,beta-Dihydroresveratrol 28218806.1 28053683.09 29273958.91 26726533.14 26126233.61 27682172.67 29873984.88 29323175.44 30278718.83 29293338.68 29481304.17 28870683.92 Bergapten 4510876.213 9675787.105 4428783.629 9798864.201 6789798.1 3698844.258 5419357.651 11167341.08 12098910.72 5496798.919 10545582 10482633.06 Pantothenate 18476791.86 22596325.81 20358864.49 19585857.76 20755177.98 22590972.71 25141151.31 22735729.39 22566451.82 19939732.63 21965024.57 21256439.52 Methoxsalen 161173869.8 153957479.7 153269958.7 151795833.3 154014264.8 157602512.4 144040219.9 147584589.7 157119657.3 154593009.8 147958939.8 154679679.9 Lunularic acid 77142774.12 75240516.93 76815517.78 71396781.25 73115582.32 73765537.28 78872548.33 77168295.12 80771936.39 76935512.98 79047421.28 73767556.9 Pantetheine 6412398.698 6208807.476 6699375.074 6294556.508 6382199.224 6685516.335 6714379.215 6367896.131 6928972.436 6174724.216 6564936.613 6811959.987 Sphingosine-1-phosphate 4236075.119 794349.8485 3260172.518 3684367.717 814538.9692 794323.773 351505.6087 609316.1767 346798.8109 244102.0655 202068.387 320847.4445 Sodium pyruvate 478474.0291 27361462.65 421799.9685 481984.3231 428109.7608 463316.7404 460538.9041 414269.9789 348134.6596 445674.3694 454294.5683 425417.0905 Pyruvate 55777496.66 49058347.23 57671126.3 54501306.45 49496300.53 44366869.79 56489158.22 55853438.89 58438803.95 59344944.85 52638211.93 55791474.95 Lactate 5809801.176 5569097.668 5626034.832 4790101.913 3676889.023 5633370.411 4864854.145 5065453.171 4257875.379 4316819.299 4234439.454 4125168.327 Malic 102755741.3 107583577.6 111925159.6 112938018.6 111265744.3 103707781.3 103361617.9 104326671.5 108306283.9 103255835.8 105258976.5 112582874.8 Citric 15349832.76 16305189.27 16075368.03 14445986.22 14221155.41 15824085.15 18198506.62 16595634.03 17785296.59 13285688.69 17038941.95 15825224.58 Succinic 565447910.7 530606435.8 590897844.4 531014276.9 538730191.1 538229382.2 531118872.4 531638456.6 536428979.5 506505234.1 583344307.4 522000305.6 Indolelactate 5809801.176 5569097.668 5626034.832 4790101.913 3676889.023 5633370.411 4864854.145 5065453.171 4257875.379 4316819.299 4234439.454 4125168.327 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quantified m/z KEGG ID Provitamin C 113.02443 C03342 Glutathione sulfonate 354.06113 - Thioglycolic acid 91.00373 - alpha,beta-Dihydroresveratrol 229.08302 - Bergapten 215.03279 C01557 Pantothenate 218.10345 C00864 Methoxsalen 215.03285 C01864 Lunularic acid 257.07785 C10268 Pantetheine 277.11942 C00831 Sphingosine-1-phosphate 378.24142 C06124 Sodium pyruvate 87.01119 - Pyruvate 87.00873 C00022 Lactate 204.06676 C02043 Malic 133.01427 C00149 Citric 205.03546 - Succinic 117.01932 C00042 Indolelactate 204.06676 C02043 METABOLITES_END #END