Development of a quality control method for postbiotics based on stable isotope dimethyl labeling and liquid chromatography-tandem mass spectrometry of heat-killed Lacticaseibacillus paracasei subsp. paracasei NTU 101

Postbiotics, including inactivated probiotics, retain immune modulatory and gut health benefits while offering advantages in storage and transport. However, quality control (QC) of heat-killed probiotics in complex matrices remains challenging. In this study, we used proteomics based on mass spectrometry (MS) for protein identification and batch-to-batch consistency assessment of heat-killed Lacticaseibacillus paracasei subsp. paracasei NTU 101. Subsequently, the signature peptides of the two target proteins, 50S ribosomal protein L7/L12 and cell wall hydrolysis protein p40, were selected based on taxonomic discrimination and functional relevance for absolute quantification using stable isotope dimethyl labeling with liquid chromatography-MS and multiple reaction monitoring (LC-MS-MRM). This method was validated for both peptides, with quantification ranges of 0.5–100 ng/mL. L7/L12 showed 94–104% accuracy and 1–3% coefficient of variation (CV), with a limit of quantification (LOQ) of 0.3 ng/mL and limit of detection (LOD) of 0.1 ng/mL. p40 demonstrated 100–111% accuracy and 1–9% CV, with an LOQ of 0.6 ng/mL and LOD of 0.2 ng/mL. Both proteins demonstrated good linearity and reproducibility, supporting their suitability for postbiotic QC. Recovery experiments conducted by spiking milk powder matrix containing 5% heat-killed NTU101 probiotics showed recoveries of 87.99–89.49% for L7/L12 and 102.68–108.72% for p40, indicating reliable detection in high-protein formulations. Overall, this method provides a robust analytical tool for postbiotic QC in complex product matrices. The platform may serve as a reference framework for developing regulatory or industrial quality standards for postbiotic ingredients in functional food applications. • A proteomics based QC workflow was developed for heat-killed L. paracasei NTU 101. • Label-free and isotopic labeling respectively assessed protein consistency and content. • Two peptides from L7/L12 and p40 exhibited high sensitivity and reproducibility. • Peptide quantification enables accurate dosing and improves postbiotic QC.