Proteomics as a Quality Control Tool of Pharmaceutical Probiotic Bacterial Lysate Products

Abstract

Probiotic bacteria have a wide range of applications in veterinary and human therapeutics. Inactivated probiotics are complex samples and quality control (QC) should measure as many molecular features as possible. Capillary electrophoresis coupled to mass spectrometry (CE/MS) has been used as a multidimensional and high throughput method for the identification and validation of biomarkers of disease in complex biological samples such as biofluids. In this study we evaluate the suitability of CE/MS to measure the consistency of different lots of the probiotic formulation Pro-Symbioflor which is a bacterial lysate of heat-inactivated Escherichia coli and Enterococcus faecalis. Over 5000 peptides were detected by CE/MS in 5 different lots of the bacterial lysate and in a sample of culture medium. 71 to 75% of the total peptide content was identical in all lots. This percentage increased to 87–89% when allowing the absence of a peptide in one of the 5 samples. These results, based on over 2000 peptides, suggest high similarity of the 5 different lots. Sequence analysis identified peptides of both E. coli and E. faecalis and peptides originating from the culture medium, thus confirming the presence of the strains in the formulation. Ontology analysis suggested that the majority of the peptides identified for E. coli originated from the cell membrane or the fimbrium, while peptides identified for E. faecalis were enriched for peptides originating from the cytoplasm. The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP). Sequence analysis also identified the presence of soybean, yeast and casein protein fragments that are part of the formulation of the culture medium. In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots.

Review of the CE‐MS platform as a powerful alternative to conventional couplings in bio‐omics and target‐based applications

“…The discussion now focuses on proteomics analysis for kidney diseases [108][109][110] and quality control (QC) of intact proteins [111][112][113][114]. There is an absence of standard studies on individual peptides and proteins.…”

“…A study developed by Klein et al [112] evaluated the suitability of CE-TOF-MS to measure the consistency of different lots of the probiotic formulation Pro-Symbioflor, which is a bacterial lysate of heat-inactivated Escherichia coli and Enterococcus faecalis. Probiotics are defined as micro-organisms that, when obtained in sufficient active quantities in the gut, lead to positive effects on health; most of the proven health effects probiotics elicit are provided in the gastrointestinal tract.…”

“…There is an absence of standard studies on individual peptides and proteins. The discussion now focuses on proteomics analysis for kidney diseases [108][109][110] and quality control (QC) of intact proteins [111][112][113][114].…”

Probiotical Cell Fragments (PCFs) as “Novel Nutraceutical Ingredients”

“…Moreover, paraprobiotics have been found to contain about 2939 to 3381 different host defense peptides (HDPs) [30]. In general, the HDPs are known to influence the host response to infections in several ways, including direct antimicrobial killing, promoting wounds healing, production of cytokines, modulation of chemokines, enhance angiogenesis [31], limiting inflammation, anti-sepsis, enhancing vaccine responses, and regulating metabolism [32].…”

“…This instance demonstrates that each peptide may have various biological activities. The mechanisms by which the PCFs exert their immunomodulatory effects, including individual thousands of HDPs [30], are stipulated in Figure 3. It can be clearly seen that the immunomodulatory effects are all related to the NLRs and TLRs.…”