Community structure and metabolite profiles of psychrophiles and thermophiles in bovine raw milk from arid inland areas.

This study provides a regional assessment of bacterial communities and associated metabolite profiles in raw cow's milk collected from seven commercial farms across southern and northern Xinjiang, an arid inland milk-producing region of China. By comparatively analyzing psychrophilic and thermophilic bacterial assemblages, this work aimed to clarify their community composition, explore their potential environmental origins, and identify metabolites that differ between these two microbial groups with possible implications for milk quality and safety. To achieve this, we combined 16S rRNA gene sequencing, culture-based isolation of representative strains, and untargeted liquid chromatography-mass spectrometry metabolomics. At the phylum level, three dominant bacterial phyla were identified in samples from southern farms, while four dominant phyla were observed in northern farms; at the genus level, Pseudomonas and Acinetobacter were among the most abundant taxa associated with psychrophilic or thermophilic conditions. Differential metabolomic analysis revealed 59 metabolites that were consistently upregulated in samples associated with thermophilic communities and 2 that were downregulated, whereas samples associated with psychrophilic communities showed 1consistently downregulated metabolite. One metabolite feature was annotated as 3,4-methylenedioxyamphetamine based on database matching; given the inherent uncertainty of untargeted annotations, this identification requires further targeted validation before definitive safety conclusions can be drawn. Pathway enrichment analysis implicated arginine biosynthesis and galactose metabolism, which may be related to enzymatic activities involved in milk spoilage. Overall, this study offers a regionally focused characterization of psychrophilic and thermophilic microbial and metabolite patterns in raw milk from arid inland Xinjiang and identifies microbial and metabolic features that warrant further targeted investigation for dairy quality control.IMPORTANCERaw milk from arid inland regions represents a unique ecological niche in which microbial survival, metabolic activity, and contamination dynamics differ from those in humid or temperate dairy systems. By jointly characterizing psychrophilic and thermophilic microbial communities and their associated metabolite profiles, this study provides an integrated microbe-metabolite perspective on raw milk quality under arid production conditions. The coupling of microbial source tracking with metabolomic analysis offers a framework for linking contamination sources to functional and chemical consequences, extending beyond traditional taxonomic surveys. The detection of metabolites with potential toxicological relevance highlights the importance of integrating chemical risk screening into routine microbiological assessments, while also underscoring the need for targeted confirmation in food safety surveillance. More broadly, this work demonstrates how combined microbial-metabolite approaches can support evidence-based hygiene management and risk prevention strategies in dairy production systems, with particular relevance for arid and semi-arid agricultural regions.