#METABOLOMICS WORKBENCH Jing0319_20260103_044240 DATATRACK_ID:6947 STUDY_ID:ST004508 ANALYSIS_ID:AN007558 PROJECT_ID:PR002837 VERSION 1 CREATED_ON January 3, 2026, 6:30 pm #PROJECT PR:PROJECT_TITLE Silage-derived flavonoids reprogram rumen microbiota to improve milk fat PR:PROJECT_TITLE synthesis via rumen-mammary gland axis PR:PROJECT_TYPE Research PR:PROJECT_SUMMARY Inoculation with homofermentative lactic acid bacteria (LAB) effectively PR:PROJECT_SUMMARY enhances the silage quality of forages. Moreover, feeding such LAB-inoculated PR:PROJECT_SUMMARY silage modulates rumen microbiota composition and metabolites, thereby improving PR:PROJECT_SUMMARY ruminant production performance. Nevertheless, the specific mechanism through PR:PROJECT_SUMMARY which LAB inoculants regulate the silage–rumen–mammary gland axis remains PR:PROJECT_SUMMARY unclear. PR:INSTITUTE School of Life Sciences, Lanzhou University PR:DEPARTMENT State Key Laboratory of Grassland and Agro-ecosystems PR:LAST_NAME Ma PR:FIRST_NAME Jing PR:ADDRESS No. 222, Tianshui South Road, Lanzhou, Gansu, 730000, China PR:EMAIL mjing2023@lzu.edu.cn PR:PHONE +86 19852806670 #STUDY ST:STUDY_TITLE Silage-derived flavonoids reprogram rumen microbiota to improve milk fat ST:STUDY_TITLE synthesis via rumen-mammary gland axis ST:STUDY_TYPE Research ST:STUDY_SUMMARY Inoculation with homofermentative Lactiplantibacillus plantarum BX62 improved ST:STUDY_SUMMARY the alfalfa silage quality. Dairy goats fed the BX62 group silage showed ST:STUDY_SUMMARY significantly higher milk fat content compared to the control group (no ST:STUDY_SUMMARY inoculation) (P < 0.05). Integrated analysis of silage microbial metabolomics ST:STUDY_SUMMARY and experimental validation revealed a significant increase in flavonoid content ST:STUDY_SUMMARY in the BX62 silage. This was attributed to microbial community restructuring and ST:STUDY_SUMMARY secretion of carbohydrate-active enzymes (CAZymes), which facilitated plant cell ST:STUDY_SUMMARY wall degradation and flavonoid release. ST:INSTITUTE School of Life Sciences, Lanzhou University ST:DEPARTMENT State Key Laboratory of Grassland and Agro-ecosystems ST:LAST_NAME Ma ST:FIRST_NAME Jing ST:ADDRESS No. 222, Tianshui South Road, Lanzhou, Gansu, 730000, China ST:EMAIL mjing2023@lzu.edu.cn ST:PHONE +86 19852806670 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Medicago sativa L. #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 ATCC1 POS_ATCC1 Sample source:BX62 Silage | factor:BX62 inoculation RAW_FILE_NAME(Raw file name)=POS_ATCC1.mzXML SUBJECT_SAMPLE_FACTORS ATCC2 POS_ATCC2 Sample source:BX62 Silage | factor:BX62 inoculation RAW_FILE_NAME(Raw file name)=POS_ATCC2.mzXML SUBJECT_SAMPLE_FACTORS ATCC3 POS_ATCC3 Sample source:BX62 Silage | factor:BX62 inoculation RAW_FILE_NAME(Raw file name)=POS_ATCC3.mzXML SUBJECT_SAMPLE_FACTORS ATCC4 POS_ATCC4 Sample source:BX62 Silage | factor:BX62 inoculation RAW_FILE_NAME(Raw file name)=POS_ATCC4.mzXML SUBJECT_SAMPLE_FACTORS CKM1 POS_CKM1 Sample source:CK Silage | factor:No inoculation RAW_FILE_NAME(Raw file name)=POS_CKM1.mzXML SUBJECT_SAMPLE_FACTORS CKM2 POS_CKM2 Sample source:CK Silage | factor:No inoculation RAW_FILE_NAME(Raw file name)=POS_CKM2.mzXML SUBJECT_SAMPLE_FACTORS CKM3 POS_CKM3 Sample source:CK Silage | factor:No inoculation RAW_FILE_NAME(Raw file name)=POS_CKM3.mzXML SUBJECT_SAMPLE_FACTORS CKM4 POS_CKM4 Sample source:CK Silage | factor:No inoculation RAW_FILE_NAME(Raw file name)=POS_CKM4.mzXML SUBJECT_SAMPLE_FACTORS ATCC1 NEG_ATCC1 Sample source:BX62 Silage | factor:BX62 inoculation RAW_FILE_NAME(Raw file name)=NEG_ATCC1.mzXML SUBJECT_SAMPLE_FACTORS ATCC2 NEG_ATCC2 Sample source:BX62 Silage | factor:BX62 inoculation RAW_FILE_NAME(Raw file name)=NEG_ATCC2.mzXML SUBJECT_SAMPLE_FACTORS ATCC3 NEG_ATCC3 Sample source:BX62 Silage | factor:BX62 inoculation RAW_FILE_NAME(Raw file name)=NEG_ATCC3.mzXML SUBJECT_SAMPLE_FACTORS ATCC4 NEG_ATCC4 Sample source:BX62 Silage | factor:BX62 inoculation RAW_FILE_NAME(Raw file name)=NEG_ATCC4.mzXML SUBJECT_SAMPLE_FACTORS CKM1 NEG_CKM1 Sample source:CK Silage | factor:No inoculation RAW_FILE_NAME(Raw file name)=NEG_CKM1.mzXML SUBJECT_SAMPLE_FACTORS CKM2 NEG_CKM2 Sample source:CK Silage | factor:No inoculation RAW_FILE_NAME(Raw file name)=NEG_CKM2.mzXML SUBJECT_SAMPLE_FACTORS CKM3 NEG_CKM3 Sample source:CK Silage | factor:No inoculation RAW_FILE_NAME(Raw file name)=NEG_CKM3.mzXML SUBJECT_SAMPLE_FACTORS CKM4 NEG_CKM4 Sample source:CK Silage | factor:No inoculation RAW_FILE_NAME(Raw file name)=NEG_CKM4.mzXML #COLLECTION CO:COLLECTION_SUMMARY Alfalfa (Medicago sativa L.) was mowed at full-flowering stage from an alfalfa CO:COLLECTION_SUMMARY feld located in Dingxi, Gansu Province, China, and was chopped to 2-4 cm lengths CO:COLLECTION_SUMMARY using a forage harvester. The forage was divided into two groups for different CO:COLLECTION_SUMMARY silage group. L. plantarum BX62 (CGMCC No. 15779) was used to prepare the CO:COLLECTION_SUMMARY experimental silage. The inoculant was diluted in non-chloride water and sprayed CO:COLLECTION_SUMMARY evenly with an application rate of 1 × 105 colony forming unit (CFU)/g CO:COLLECTION_SUMMARY fresh matter (FM). The control silage was sprayed with the same water source CO:COLLECTION_SUMMARY with no inoculant. Evenly-sprayed alfalfa was tightly wrapped with 4–6 layers CO:COLLECTION_SUMMARY of 25 µm thick polypropylene films by a bale wrapper. The wrapped round bales CO:COLLECTION_SUMMARY were transported to a dry, ventilated storage place, and feeding trials were CO:COLLECTION_SUMMARY started after 30 days of ensiling. Silage samples were collected after opening CO:COLLECTION_SUMMARY and were immediately frozen at -20°C for metabolite analysis. CO:SAMPLE_TYPE Plant CO:COLLECTION_LOCATION Dingxi, Gansu Province, China CO:STORAGE_CONDITIONS -20℃ #TREATMENT TR:TREATMENT_SUMMARY No treatment applied. TR:PLANT_HARVEST_DATE 2024-07-01 TR:PLANT_GROWTH_STAGE full-flowering stage #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 25 mg of freeze-dried sample was weighed into an EP tube, homogenising beads and SP:SAMPLEPREP_SUMMARY 1000 μL of extraction solution (methanol: acetonitrile: water = 2:2:1 (v/v/v)) SP:SAMPLEPREP_SUMMARY containing isotope-labelled internal standard were added. The vortex-mixed SP:SAMPLEPREP_SUMMARY samples were homogenised and then transferred to an ice-water bath for SP:SAMPLEPREP_SUMMARY sonication for 5 min, and this step was repeated three times. After standing at SP:SAMPLEPREP_SUMMARY -40℃ for 1 h, the samples were centrifuged at 4℃, 12000 rpm for 15 min. SP:SAMPLEPREP_SUMMARY The supernatant was taken into the injection bottle for assaying on the machine. SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACT_STORAGE On ice #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The compounds in silage sample were separated on a Phenomenex Kinetex C18 (2.1 CH:CHROMATOGRAPHY_SUMMARY mm × 50 mm, 2.6 μm) column using a Vanquish (Thermo Fisher Scientific) CH:CHROMATOGRAPHY_SUMMARY ultra-performance-liquid chromatograph (UPLC) for non-polar metabolites. The CH:CHROMATOGRAPHY_SUMMARY liquid chromatographic phase A was aqueous phase containing 0.01% acetic acid, CH:CHROMATOGRAPHY_SUMMARY and the phase B was isopropanol: acetonitrile (1:1, v/v). The temperature of the CH:CHROMATOGRAPHY_SUMMARY sample tray was 4 ℃, and the injection volume was 2 μL. Chromatographic CH:CHROMATOGRAPHY_SUMMARY separation was achieved with the following gradient elution program: initial CH:CHROMATOGRAPHY_SUMMARY hold at 1% B for 0.5 min, linear increase to 99% B over 3.5 min (0.5-4 min), CH:CHROMATOGRAPHY_SUMMARY maintained at 99% B for 0.5 min (4-4.5 min), rapidly returned to 1% B in 0.05 CH:CHROMATOGRAPHY_SUMMARY min (4.5-4.55 min), and final equilibration at 1% B for 1.45 min (4.55-6 min). CH:CHROMATOGRAPHY_SUMMARY The separation was performed at a controlled column temperature of 25°C with a CH:CHROMATOGRAPHY_SUMMARY flow rate of 0.3 mL/min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex HILIC (50 x 2.1 mm, 2.6 μm) CH:SOLVENT_A 100% Water; 0.01% acetic acid CH:SOLVENT_B 50% Isopropanol/50% Acetonitrile CH:FLOW_GRADIENT initial hold at 1% B for 0.5 min, linear increase to 99% B over 3.5 min (0.5-4 CH:FLOW_GRADIENT min), maintained at 99% B for 0.5 min (4-4.5 min), rapidly returned to 1% B in CH:FLOW_GRADIENT 0.05 min (4.5-4.55 min), and final equilibration at 1% B for 1.45 min (4.55-6 CH:FLOW_GRADIENT min) CH:FLOW_RATE 0.3 mL/min CH:COLUMN_TEMPERATURE 25℃ CH:SAMPLE_INJECTION 2 #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 The Orbitrap Exploris 120 mass spectrometer was used for its ability to acquire MS:MS_COMMENTS MS/MS spectra on information-dependent acquisition (IDA) mode in the control of MS:MS_COMMENTS the acquisition software (Xcalibur, Thermo). In this mode, the acquisition MS:MS_COMMENTS software continuously evaluates the full scan MS spectrum. The ESI source MS:MS_COMMENTS conditions were set as following: sheath gas flow rate as 50 Arb, Aux gas flow MS:MS_COMMENTS rate as 15 Arb, capillary temperature 320℃, Sweep Gas: 1 Arb, Vaporizer Temp: MS:MS_COMMENTS 350℃, full MS resolution as 60000, MS/MS resolution as15000, collision energy: MS:MS_COMMENTS SNCE 20/30/40, spray voltage as 3.8 kV (positive). MS:MS_RESULTS_FILE ST004508_AN007558_Results.txt UNITS:mz_rt Has m/z:Yes Has RT:Yes RT units:Seconds #END