Monitoring of multiple bacteriocins through a developed dual extraction protocol and comparison of HPLC-DAD with turbidometry as their quantification system

• Published in: Journal of microbiological methods 2016-08, Vol.127, p.123-131
• Main Authors: Katharopoulos, Efstathios, Touloupi, Katerina, Touraki, Maria
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - isolation & purification; Anti-Bacterial Agents - pharmacology; Bacteria; Bacteriocins; Bacteriocins - chemistry; Bacteriocins - isolation & purification; Bacteriocins - pharmacology; Chromatography, High Pressure Liquid - methods; Gram-Negative Bacteria - drug effects; Gram-Positive Bacteria - drug effects; Hot Temperature; HPLC; Hydrogen-Ion Concentration; Lactococcus lactis; Lactococcus lactis - chemistry; Nephelometry and Turbidimetry - methods; Profiling; Quantification; Turbidometry
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2016.06.003

The present study describes the development of a simple and efficient screening system that allows identification and quantification of nine bacteriocins produced by Lactococcus lactis. Cell-free L. lactis extracts presented a broad spectrum of antibacterial activity, including Gram-negative bacteria, Gram-positive bacteria, and fungi. The characterization of their sensitivity to pH, and heat, showed that the extracts retained their antibacterial activity at extreme pH values and in a wide temperature range. The loss of antibacterial activity following treatment of the extracts with lipase or protease suggests a lipoproteinaceous nature of the produced antimicrobials. The extracts were subjected to a purification protocol that employs a two phase extraction using ammonium sulfate precipitation and organic solvent precipitation, followed by ion exchange chromatography, solid phase extraction and HPLC. In the nine fractions that presented antimicrobial activity, bacteriocins were quantified by the turbidometric method using a standard curve of nisin and by the HPLC method with nisin as the external standard, with both methods producing comparable results. Turbidometry appears to be unique in the qualitative determination of bacteriocins but the only method suitable to both separate and quantify the bacteriocins providing increased sensitivity, accuracy, and precision is HPLC. [Display omitted] •L. lactis extracts present antibacterial activity against bacteria and fungi.•The produced lipoproteinic antimicrobials are pH and temperature tolerant.•Purification: extractions (ammonium sulfate and organic solvent), IEC, SPE, HPLC.•Antimicrobial activity in nine HPLC fractions quantified by turbidometry and HPLC.•Turbidometry: qualitative determination, HPLC: profiling and quantitation.