Single‐cell rapid identification, in situ viability and vitality profiling, and genome‐based source‐tracking for probiotics products

Rapid expansion of the probiotics industry demands fast, sensitive, comprehensive, and low‐cost strategies for quality assessment. Here, we introduce a culture‐free, one‐cell‐resolution, phenome‐genome‐combined strategy called Single‐Cell Identification, Viability and Vitality tests, and Source‐tracking (SCIVVS). For each cell directly extracted from the product, the fingerprint region of D2O‐probed single‐cell Raman spectrum (SCRS) enables species‐level identification with 93% accuracy, based on a reference SCRS database from 21 statutory probiotic species, whereas the C–D band accurately quantifies viability, metabolic vitality plus their intercellular heterogeneity. For source‐tracking, single‐cell Raman‐activated Cell Sorting and Sequencing can proceed, producing indexed, precisely one‐cell‐based genome assemblies that can reach ~99.40% genome‐wide coverage. Finally, we validated an integrated SCIVVS workflow with automated SCRS acquisition where the whole process except sequencing takes just 5 h. As it is >20‐fold faster, >10‐time cheaper, vitality‐revealing, heterogeneity‐resolving, and automation‐prone, SCIVVS is a new technological and data framework for quality assessment of live‐cell products. Highlights We introduce a culture‐free, one‐cell‐resolution, phenome‐genome‐combined strategy called Single‐Cell Identification, Viability and Vitality tests, and Source‐tracking (SCIVVS) for rapid quality assessment of probiotics products. SCIVVS enables species‐level identification with 93% accuracy and quantifies viability, metabolic vitality plus their intercellular heterogeneity. For source‐tracking a cell, SCIVVS can reach ~99.40% genome‐wide coverage for various Lactobacillus, Bifidobacterium, and Streptococcus spp. SCIVVS is >20‐fold faster, >10‐time cheaper, vitality‐revealing, heterogeneity‐resolving, and automation‐prone. SCIVVS is a new technological and data framework that can transform quality evaluation, biomanufacturing‐process monitoring, and intellectual‐property protection of probiotics and other live‐cell products. The rapid expansion of the probiotics industry demands fast, sensitive, comprehensive, and low‐cost methods for quality assessment of such live‐cell products. However current methods are slow, tedious, and incapable of quantifying in situ vitality or its heterogeneity. Here, we introduce a culture‐free, one‐cell‐resolution, phenome‐genome‐combined strategy called Single‐Cell Identification, Viability and Vitality tests, and So