The Impact of Storage Conditions on the Microbiome Composition and Metabolites of Fecal Samples

To investigate the effects of storage time and temperature on the quality of fecal samples and to provide a reference for clinical laboratories and biobanks in formulating sample storage operation guidelines.

Fresh fecal samples were collected from healthy volunteers and immediately aliquoted into aliquots. Different temperature and time gradients were established to simulate common pre-analytical storage processes in clinical practice, with samples snap-frozen in liquid nitrogen immediately after collection as the control group. 16S rRNA gene sequencing and untargeted lipid metabolomics were employed to determine changes in microbial diversity, species abundance, and metabolite concentrations under different storage conditions, and sample quality was evaluated based on these indicators.

Storage at 4°C significantly minimized fluctuations in α-diversity indices, with the most pronounced protective effect observed within 2-4 hours; beyond 4 hours, changes in microbial community structure intensified. β-diversity analysis revealed that 4°C storage delayed the increase in microbial dissimilarity between samples and the liquid nitrogen-frozen control group, among which samples stored for 2-4 hours exhibited the highest similarity to the control. At the phylum level, the abundance of Firmicutes increased significantly after 6 hours of storage at room temperature, while 4°C storage effectively delayed this change. Metabolomic analysis identified more metabolomic differences (including bile acids and amino acids) in samples stored at room temperature, whereas only minor changes in fatty acid metabolites were observed at 4°C. 3β-hydroxy-5-cholenic acid exhibited a continuous upward trend with prolonged storage at both temperatures, suggesting its potential as a biomarker for evaluating sample storage quality.

Short-term storage at 4°C (≤4 hours) can effectively delay the quality degradation of fecal microbial communities and metabolites, making it the optimal transitional storage strategy when immediate liquid nitrogen freezing is not feasible in clinical practice. These findings provide critical experimental data for the establishment of standardized fecal sample storage protocols.