Proteomics as a quality control tool of pharmaceutical probiotic bacterial lysate products

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

Volatolomics approach by HS‐SPME‐GC‐MS and multivariate analysis to discriminate olive tree varieties infected by Xylella fastidiosa

Introduction: Xylella fastidiosa (Xf) is a pathogenic bacterium that causes diseases in olive trees. Therefore, analytical methods for both the characterisation of the host/pathogen interaction and infection monitoring are needed. Volatile organic compounds (VOCs) are emitted by plants relate to their physiological state, therefore VOCs monitoring can assist in detecting stress or infection states before visible signs are present. Objective: In this work, the headspace‐solid phase microextractiongaschromatography‐ mass spectrometry (HS‐SPME‐GC‐MS) technique was used for the first time to highlight VOCs differences between healthy and Xf‐infected olive trees. Methodology: VOCs from olive tree twig samples were extracted and analysed by HS‐SPME‐GC‐MS, and hence identified by comparing the experimental linear retention indexes with the reference values and by MS data obtained from NIST library. Data were processed by principal component analysis (PCA) and analysis of variance (ANOVA). Results: The HS‐SPME step was optimised in terms of adsorbent phase and extraction time. HS‐SPME‐GC‐MS technique was applied to the extraction and analysis of VOCs of healthy and Xf‐infected olive trees. More than 100 compounds were identified and the differences between samples were evidenced by the multivariate analysis approach. The results showed the marked presence of methyl esters in Xfinfected samples, suggesting their probable involvement in the mechanism of diffusible signal factor. Conclusion: The proposed approach represents an easy and solvent‐free method to evaluate the presence of Xf in olive trees, and to evidence volatiles produced by host/pathogen interactions that could be involved in the defensive mechanism of the olive tree and/or in the infective action of Xf