#METABOLOMICS WORKBENCH Synbio355_20250908_075807 DATATRACK_ID:6394 STUDY_ID:ST004319 ANALYSIS_ID:AN007197 PROJECT_ID:PR002733
VERSION             	1
CREATED_ON             	October 29, 2025, 9:36 pm
#PROJECT
PR:PROJECT_TITLE                 	Community structure and metabolite profiles of psychrophiles and thermophiles
PR:PROJECT_TITLE                 	isolated from raw cow’s milk in Xinjiang, China.
PR:PROJECT_SUMMARY               	This project characterizes the bacterial communities and metabolomic signatures
PR:PROJECT_SUMMARY               	associated with psychrophilic and thermophilic isolates from raw cow’s milk
PR:PROJECT_SUMMARY               	collected across seven dairy farms in Xinjiang, China. We performed 16S rRNA
PR:PROJECT_SUMMARY               	gene sequencing on milk and associated environmental samples and carried out
PR:PROJECT_SUMMARY               	untargeted LC–MS metabolomics on isolates re-inoculated into sterilized milk
PR:PROJECT_SUMMARY               	(positive and negative ion modes). The dataset supports microbial source
PR:PROJECT_SUMMARY               	tracking, strain-specific metabolite profiling, and assessment of potential
PR:PROJECT_SUMMARY               	risks to milk quality and safety.
PR:INSTITUTE                     	Xinjiang University
PR:LAST_NAME                     	Yue
PR:FIRST_NAME                    	Haitao
PR:ADDRESS                       	Yushugou Street, Shuimogou District, Urumqi City, Xinjiang Province
PR:EMAIL                         	yuehaitao@tsinghua.org.cn
PR:PHONE                         	13639932226
#STUDY
ST:STUDY_TITLE                   	Community structure and metabolite profiles of psychrophiles and thermophiles in
ST:STUDY_TITLE                   	bovine raw milk from arid inland areas
ST:STUDY_SUMMARY                 	This project characterizes the bacterial communities and metabolomic signatures
ST:STUDY_SUMMARY                 	associated with psychrophilic and thermophilic isolates from raw cow’s milk
ST:STUDY_SUMMARY                 	collected across seven dairy farms in Xinjiang, China. We performed 16S rRNA
ST:STUDY_SUMMARY                 	gene sequencing on milk and associated environmental samples and carried out
ST:STUDY_SUMMARY                 	untargeted LC–MS metabolomics on isolates re-inoculated into sterilized milk
ST:STUDY_SUMMARY                 	(positive and negative ion modes). The dataset supports microbial source
ST:STUDY_SUMMARY                 	tracking, strain-specific metabolite profiling, and assessment of potential
ST:STUDY_SUMMARY                 	risks to milk quality and safety. We generated 16S rRNA amplicon sequencing data
ST:STUDY_SUMMARY                 	from 21 raw milk samples and associated environmental samples to profile
ST:STUDY_SUMMARY                 	microbial community composition and potential contamination sources. From these
ST:STUDY_SUMMARY                 	samples we isolated representative psychrophilic and thermophilic strains (7 +
ST:STUDY_SUMMARY                 	7), re-inoculated them into sterilized milk, and performed untargeted LC–MS
ST:STUDY_SUMMARY                 	metabolomics in both positive and negative ion modes. The metabolomics data
ST:STUDY_SUMMARY                 	reveal strain-specific metabolic signatures and identify metabolites potentially
ST:STUDY_SUMMARY                 	relevant to milk spoilage and food safety. Data deposited include raw 16S reads,
ST:STUDY_SUMMARY                 	isolate metadata, and LC–MS raw data and processed metabolite tables.
ST:INSTITUTE                     	Xinjiang University
ST:LAST_NAME                     	Yue
ST:FIRST_NAME                    	Haitao
ST:ADDRESS                       	Yushugou Street, Shuimogou District, Urumqi City, Xinjiang Province
ST:EMAIL                         	yuehaitao@tsinghua.org.cn
ST:PHONE                         	13639932226
#SUBJECT
SU:SUBJECT_TYPE                  	Food item
#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           	A40_pos	A40_pos	Sample source:Raw milk isolate | Bacteria type:Psychrophile	RAW_FILE_NAME(Raw_file_name)=A40_pos.raw
SUBJECT_SAMPLE_FACTORS           	A40_neg	A40_neg	Sample source:Raw milk isolate | Bacteria type:Psychrophile	RAW_FILE_NAME(Raw_file_name)=A40_neg.raw
SUBJECT_SAMPLE_FACTORS           	B61_pos	B61_pos	Sample source:Raw milk isolate | Bacteria type:Psychrophile	RAW_FILE_NAME(Raw_file_name)=B61_pos.raw
SUBJECT_SAMPLE_FACTORS           	B61_neg	B61_neg	Sample source:Raw milk isolate | Bacteria type:Psychrophile	RAW_FILE_NAME(Raw_file_name)=B61_neg.raw
SUBJECT_SAMPLE_FACTORS           	C5_pos	C5_pos	Sample source:Raw milk isolate | Bacteria type:Psychrophile	RAW_FILE_NAME(Raw_file_name)=C5_pos.raw
SUBJECT_SAMPLE_FACTORS           	C5_neg	C5_neg	Sample source:Raw milk isolate | Bacteria type:Psychrophile	RAW_FILE_NAME(Raw_file_name)=C5_neg.raw
SUBJECT_SAMPLE_FACTORS           	D73_pos	D73_pos	Sample source:Raw milk isolate | Bacteria type:Psychrophile	RAW_FILE_NAME(Raw_file_name)=D73_pos.raw
SUBJECT_SAMPLE_FACTORS           	D73_neg	D73_neg	Sample source:Raw milk isolate | Bacteria type:Psychrophile	RAW_FILE_NAME(Raw_file_name)=D73_neg.raw
SUBJECT_SAMPLE_FACTORS           	E63_pos	E63_pos	Sample source:Raw milk isolate | Bacteria type:Psychrophile	RAW_FILE_NAME(Raw_file_name)=E63_pos.raw
SUBJECT_SAMPLE_FACTORS           	E63_neg	E63_neg	Sample source:Raw milk isolate | Bacteria type:Psychrophile	RAW_FILE_NAME(Raw_file_name)=E63_neg.raw
SUBJECT_SAMPLE_FACTORS           	F22_pos	F22_pos	Sample source:Raw milk isolate | Bacteria type:Psychrophile	RAW_FILE_NAME(Raw_file_name)=F22_pos.raw
SUBJECT_SAMPLE_FACTORS           	F22_neg	F22_neg	Sample source:Raw milk isolate | Bacteria type:Psychrophile	RAW_FILE_NAME(Raw_file_name)=F22_neg.raw
SUBJECT_SAMPLE_FACTORS           	G5_pos	G5_pos	Sample source:Raw milk isolate | Bacteria type:Psychrophile	RAW_FILE_NAME(Raw_file_name)=G5_pos.raw
SUBJECT_SAMPLE_FACTORS           	G5_neg	G5_neg	Sample source:Raw milk isolate | Bacteria type:Psychrophile	RAW_FILE_NAME(Raw_file_name)=G5_neg.raw
SUBJECT_SAMPLE_FACTORS           	AR1_pos	AR1_pos	Sample source:Raw milk isolate | Bacteria type:Thermophile	RAW_FILE_NAME(Raw_file_name)=AR1_pos.raw
SUBJECT_SAMPLE_FACTORS           	AR1_neg	AR1_neg	Sample source:Raw milk isolate | Bacteria type:Thermophile	RAW_FILE_NAME(Raw_file_name)=AR1_neg.raw
SUBJECT_SAMPLE_FACTORS           	BR2_pos	BR2_pos	Sample source:Raw milk isolate | Bacteria type:Thermophile	RAW_FILE_NAME(Raw_file_name)=BR2_pos.raw
SUBJECT_SAMPLE_FACTORS           	BR2_neg	BR2_neg	Sample source:Raw milk isolate | Bacteria type:Thermophile	RAW_FILE_NAME(Raw_file_name)=BR2_neg.raw
SUBJECT_SAMPLE_FACTORS           	CR2_pos	CR2_pos	Sample source:Raw milk isolate | Bacteria type:Thermophile	RAW_FILE_NAME(Raw_file_name)=CR2_pos.raw
SUBJECT_SAMPLE_FACTORS           	CR2_neg	CR2_neg	Sample source:Raw milk isolate | Bacteria type:Thermophile	RAW_FILE_NAME(Raw_file_name)=CR2_neg.raw
SUBJECT_SAMPLE_FACTORS           	DR1_pos	DR1_pos	Sample source:Raw milk isolate | Bacteria type:Thermophile	RAW_FILE_NAME(Raw_file_name)=DR1_pos.raw
SUBJECT_SAMPLE_FACTORS           	DR1_neg	DR1_neg	Sample source:Raw milk isolate | Bacteria type:Thermophile	RAW_FILE_NAME(Raw_file_name)=DR1_neg.raw
SUBJECT_SAMPLE_FACTORS           	ER1_pos	ER1_pos	Sample source:Raw milk isolate | Bacteria type:Thermophile	RAW_FILE_NAME(Raw_file_name)=ER1_pos.raw
SUBJECT_SAMPLE_FACTORS           	ER1_neg	ER1_neg	Sample source:Raw milk isolate | Bacteria type:Thermophile	RAW_FILE_NAME(Raw_file_name)=ER1_neg.raw
SUBJECT_SAMPLE_FACTORS           	FR1_pos	FR1_pos	Sample source:Raw milk isolate | Bacteria type:Thermophile	RAW_FILE_NAME(Raw_file_name)=FR1_pos.raw
SUBJECT_SAMPLE_FACTORS           	FR1_neg	FR1_neg	Sample source:Raw milk isolate | Bacteria type:Thermophile	RAW_FILE_NAME(Raw_file_name)=FR1_neg.raw
SUBJECT_SAMPLE_FACTORS           	GR1_pos	GR1_pos	Sample source:Raw milk isolate | Bacteria type:Thermophile	RAW_FILE_NAME(Raw_file_name)=GR1_pos.raw
SUBJECT_SAMPLE_FACTORS           	GR1_neg	GR1_neg	Sample source:Raw milk isolate | Bacteria type:Thermophile	RAW_FILE_NAME(Raw_file_name)=GR1_neg.raw
#COLLECTION
CO:COLLECTION_SUMMARY            	Bacterial isolation and culture methods: Raw milk samples (100 mL each) were
CO:COLLECTION_SUMMARY            	collected from seven livestock farms (A–G) in July 2021. For isolation, 1 mL
CO:COLLECTION_SUMMARY            	of each milk sample was serially diluted in sterile normal saline to 10^−7.
CO:COLLECTION_SUMMARY            	Each dilution (100 µL) was spread onto MPC agar plates (n = 3 per dilution).
CO:COLLECTION_SUMMARY            	Psychrophilic isolates were selected by incubation at 6.5 ± 0.5 °C for 10
CO:COLLECTION_SUMMARY            	days, while thermophilic isolates were selected by incubation at 55 ± 1 °C for
CO:COLLECTION_SUMMARY            	48 ± 2 h. Distinct colonies were picked randomly, purified twice on MPC medium,
CO:COLLECTION_SUMMARY            	and propagated in TSB medium at 150 rpm for characterization. Purified strains
CO:COLLECTION_SUMMARY            	used for downstream analyses were maintained in culture in the laboratory (see
CO:COLLECTION_SUMMARY            	Methods for details). For taxonomic identification, genomic DNA from purified
CO:COLLECTION_SUMMARY            	isolates was extracted using a bacterial DNA extraction kit and the 16S rRNA
CO:COLLECTION_SUMMARY            	gene was PCR-amplified (27F / 1492R) and Sanger-sequenced for BLAST-based
CO:COLLECTION_SUMMARY            	identification.
CO:SAMPLE_TYPE                   	Milk
CO:COLLECTION_METHOD             	Milk collection and storage prior to inoculation/treatment: Raw milk (100 mL per
CO:COLLECTION_METHOD             	sample) was aseptically collected from individual animals at seven farms (A–G)
CO:COLLECTION_METHOD             	in July 2021. Collected samples were frozen in the field, transported to the
CO:COLLECTION_METHOD             	laboratory on dry ice, and stored at −80 °C until downstream processing (DNA
CO:COLLECTION_METHOD             	extraction for 16S sequencing and metabolite extraction for LC–MS). Aliquots
CO:COLLECTION_METHOD             	of stored raw milk were later thawed as needed for culture isolation and
CO:COLLECTION_METHOD             	follow-up experiments.
#TREATMENT
TR:TREATMENT_SUMMARY             	Fourteen bacterial isolates (7 psychrophiles and 7 thermophiles) were obtained
TR:TREATMENT_SUMMARY             	from raw cow’s milk collected in Xinjiang, China. Each isolate was
TR:TREATMENT_SUMMARY             	re-inoculated into sterilized milk at 2% (v/v) and cultured under appropriate
TR:TREATMENT_SUMMARY             	conditions (psychrophiles at 6.5 ± 0.5 °C for 10 days; thermophiles at 55 ± 1
TR:TREATMENT_SUMMARY             	°C for 48 ± 2 h). After incubation, metabolites were extracted from the
TR:TREATMENT_SUMMARY             	culture supernatants and analyzed by untargeted LC–MS in both positive and
TR:TREATMENT_SUMMARY             	negative ion modes.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Samples were thawed at 4 °C and vortexed for 1 min to ensure homogeneity. An
SP:SAMPLEPREP_SUMMARY            	aliquot of each sample was mixed with 200 µL methanol and 200 µL methyl
SP:SAMPLEPREP_SUMMARY            	tert-butyl ether, vortexed, and centrifuged at 12,000 rpm for 10 min at 4 °C.
SP:SAMPLEPREP_SUMMARY            	The supernatant was filtered through a 0.22 µm membrane and used for metabolite
SP:SAMPLEPREP_SUMMARY            	detection. A pooled quality control (QC) sample was prepared by combining 10 µL
SP:SAMPLEPREP_SUMMARY            	from each extract.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um)
CH:SOLVENT_A                     	100% water; 5 mM ammonium formate
CH:SOLVENT_B                     	100% acetonitrile
CH:FLOW_GRADIENT                 	0~1min, 2% B;1~9min, 2%~50% B; 9~12min, 50%~98% B;12~13.5min, 98% B;13.5~14min,
CH:FLOW_GRADIENT                 	98%~2% B;14~20min, 2% B
CH:FLOW_RATE                     	0.3 mL/min
CH:COLUMN_TEMPERATURE            	40 ℃
CH:METHODS_FILENAME              	report_(2).pdf
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:ANALYSIS_PROTOCOL_FILE        	report_(2).pdf
#MS
MS:INSTRUMENT_NAME               	Thermo Orbitrap Exploris 120
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Data were acquired in full scan mode (m/z 70–1050) with data-dependent MS/MS.
MS:MS_COMMENTS                   	Capillary temperature: 320 °C; Spray voltage: 3.5 kV; Sheath gas: 35 arb;
MS:MS_COMMENTS                   	Auxiliary gas: 10 arb. Both positive and negative ESI modes were used. Raw
MS:MS_COMMENTS                   	LC–MS data were processed using Thermo Compound Discoverer for peak detection,
MS:MS_COMMENTS                   	alignment, and normalization. Metabolite annotation was based on accurate mass,
MS:MS_COMMENTS                   	MS/MS fragmentation patterns, and database comparison (HMDB and KEGG).
MS:MS_RESULTS_FILE               	ST004319_AN007197_Results.txt	UNITS:peak intensity	Has m/z:Yes	Has RT:Yes	RT units:Seconds
#END