#METABOLOMICS WORKBENCH sulei_20250903_235651 DATATRACK_ID:6374 STUDY_ID:ST004173 ANALYSIS_ID:AN006927 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 POSITIVE MS:MS_COMMENTS Spray voltage +3.5 kV; sheath gas 40 arb; auxiliary gas 10 arb (N₂); capillary MS:MS_COMMENTS temp 320 ℃; auxiliary gas temp 300 ℃; MS1 full scan, m/z 70–1000, MS:MS_COMMENTS resolution 60,000 FWHM, AGC target standard, Max IT 100 ms; MS2 top-4 DDA, HCD MS:MS_COMMENTS CE 30%, dynamic exclusion 4 s, resolution 15,000 FWHM, AGC target standard, Max MS:MS_COMMENTS IT Auto. Raw data (*.raw) directly imported into MS-DIAL; peaks absent in >50% MS:MS_COMMENTS QC samples filtered; missing values imputed (gap filling); normalization MS:MS_COMMENTS applied. Suitable for protonated metabolites. Thermo Xcalibur (v4.7) for DDA MS:MS_COMMENTS acquisition. MS-DIAL (v4.9.221218) for feature extraction and annotation; MS:MS_COMMENTS databases: PSNGM, mzCloud, LIPID MAPS, HMDB, MoNA, NIST_2020_MSMS, AI-predicted MS:MS_COMMENTS MS/MS. Matching tolerance: MS1 ±0.01 m/z, MS2 ±0.05 m/z; features with score MS:MS_COMMENTS ≥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 Xanthine 12458985.28 12788792.49 11856662.97 11594870.55 11239571.46 11541780.75 15463951.1 16741315.21 17601001.37 15578631.04 16850435.94 16268585.95 Ser-Val 28179529.57 29080714.23 30292270.95 29012473.96 28196155.06 28669658.69 39085951.97 37031320.89 35547189.43 35145584.18 34978456.72 36809676.01 D-Valyl-L-threonine 48076301.5 49300106.71 52832911.69 51906359.66 51789525.17 50678208.69 64876737.09 61264566.52 64452722.16 64600589.48 59371007.89 60562701.35 (1R,3S,4S,5R,7R)-4-(3-hydroxybutyl)-5-methyl-10-methylidene-8-oxatricyclo[5.3.0.0l,]decan-9-one 8604470.812 8632947.874 9404353.575 8240048.414 8731923.487 9196476.024 12904185.45 11525052.97 12279725.81 10865850.44 11776799.46 11727325.58 5-hydroxy-2,2,6,6-tetramethyl-4-{2-methyl-1-[2,4,6-trihydroxy-3-(2-methylpropanoyl)phenyl]propyl}cyclohex-4-ene-1,3-dione 7422662.574 6005210.019 6211015.353 5740635.475 6472139.344 7781125.196 3746051.326 3982701.87 3392662.963 3431988.442 3549427.141 3453635.318 Ethyl 2-naphthoate 20521735.87 20752682.43 19610774.88 19597483.16 19715253.65 19263662.29 22220641.71 22382637.58 23107332.86 23900499.03 22245153.07 23104709.8 L-Isoleucyl-L-serine 14186843.27 14787357.38 17392365.9 18025775.41 20525645.71 16375253.78 31855401.97 29086605.77 38976735.81 41140060.43 30909138.21 28706783.18 Ala-Leu 8325682.418 8353077.063 8628906.345 8916425.785 8261952.488 9093527.264 11679786.67 10618364.15 11701073.67 10349880.62 11987316.42 10783675.33 L-alanyl-D-glutamic acid 16187420.48 16756388.96 18414176.4 16702340.65 16681994.1 16463386.24 21515529.38 20166921.38 19932002.4 19245541.8 20674360.4 20417797.18 Lauryldimethylamine oxide 45675630.98 41655194.24 48873529.5 49185004.37 44794756.32 48998692.87 34970839.13 39631412.03 33281705.79 33343488.83 34258211.67 33653802.2 Ile-Glu 86383042.02 91879028.13 111605101.9 103733781.6 106301649.7 98225645.57 148574463.4 135966274 131906633.6 138251591.3 140697543.9 122157007 Ile-Ile 26637126.04 28357615.57 34599712.07 32409889.74 32502080.84 32182175.41 43433747.56 41106255.68 45200738.99 45458778.87 44903759.76 41264505.44 Leu-Glu 114415535.2 122467440.3 143990924.7 143000486.7 149189663.8 134436366.2 195067536.6 181133987.8 192244870.7 202611832.9 171419417 180800151.3 L-Isoleucyl-L-alanine 60271627.57 69800481.99 77830968.59 82147078.11 82182127.15 66695233.56 107348866.5 99733776.79 112355307.7 118896791.9 114102731.8 104814812.7 pro-val 192408562.3 201166233.1 205431157.3 205120945 209949437.9 191916194.6 232395616 218368001.1 229459920.7 221824127.7 229913873 220661568.9 sebacic acid 15067764.34 16296211.43 20639320.89 18522872.63 17662495.38 19702771.19 28271762.82 25659066.87 28750369.89 24427879.62 24435709.7 23181648.78 5-(4-morpholino)-2-nitroanisole 30406091.86 28392579.05 28084596.86 28370398.1 28318394.37 28496779.72 33278993.78 31913539.49 31196333.22 31436796.27 30847871.52 33675115.98 N,N'-diisobutylurea 5670745.355 5892746.861 5840993.33 5840370.528 5625319.831 6212471.428 5122401.631 4745435.617 5150614.151 4465266.74 4959945.63 5139882.262 Monocillin Ii 33434014.24 32955184.72 40659246.69 38755756.17 36968589.73 35192202.82 44586879.93 45417828.76 47190230.67 53405757.23 43260661.34 46646537.41 Ile-His 11626259.35 12417396.82 11627414.04 12179422.79 12014141.44 12646138.05 15007927.31 13480274.27 14108355.13 13012768.84 14496987.57 14021822.69 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quantified m/z KEGG ID Xanthine 153.04068 C00385 Ser-Val 205.11818 D-Valyl-L-threonine 219.13374 - (1R,3S,4S,5R,7R)-4-(3-hydroxybutyl)-5-methyl-10-methylidene-8-oxatricyclo[5.3.0.0l,]decan-9-one 233.14961 - 5-hydroxy-2,2,6,6-tetramethyl-4-{2-methyl-1-[2,4,6-trihydroxy-3-(2-methylpropanoyl)phenyl]propyl}cyclohex-4-ene-1,3-dione 415.21152 - Ethyl 2-naphthoate 201.08694 - L-Isoleucyl-L-serine 203.13885 - Ala-Leu 203.13899 - L-alanyl-D-glutamic acid 219.0974 - Lauryldimethylamine oxide 230.24782 - Ile-Glu 261.14438 - Ile-Ile 245.18591 - Leu-Glu 261.14423 - L-Isoleucyl-L-alanine 203.13885 - pro-val 215.13885 - sebacic acid 203.13898 C08277 5-(4-morpholino)-2-nitroanisole 239.10248 - N,N'-diisobutylurea 173.16483 - Monocillin Ii 301.1506 - Ile-His 269.16095 - METABOLITES_END #END