Evolution of bacteria Lactobacillus plantarum strains from Croatian autochthonous fermented products during prolonged stationary phase

Abstract

The changes in bacterial metabolism and proteome were observed after prolonged stationary phase. Bacterial cells adapt to stress condition by stress response systems and gene mutation, which have a bacterial evolution as result. Objective: Studies in the lactic acid bacteria growth performance as well as adaptation to stress are of great interest because of the large use of these microorganisms in the food industry. The aim of this study was to investigate a long term growth and evolution of Croatian autochthonous Lactobacillus plantarum strains. The bacterial evolution was observed based on the changes on metabolism, proteome and bacterial competition during the prolonged stationary phase. Methods: The number of bacterial cells and bacterial competition were monitored during the long term growth by measuring colony-forming units per milliliter (CFU). High Performance Liquid Chromatography (HPLC) was used to observe differences in metabolism between 1-day-old strains and 10-day-old strains based on the production of organic acids. HPLC was also used in order to determine stability of a 10-day-old strain metabolism. To identify the proteins that were responsible for the metabolic differences between 1-day-old strain and 10-day-old strain and proteins involved in the adaptation of 10-day-old strain to long term growth, these proteins were separated by 2-DE. The differential display analysis of expressed proteins was performed by using MALDI-TOF/TOF. Tandem mass spectrometry MS/MS ion search data was used to search the Fimicutes (Gram-positive bacteria) by accessing the NCBInr database through MASCOT search engine. Protein-protein integrated map of differentially expressed proteins between 1-day-old cells and 10-day-old cells was obtained by using http://string-db.org/ analyzer. Results: After long term growth different percentages of long term survivors were obtained. Based on results we concluded that mutation and evolution are random processes. Differences between 1-day-old and 10-day-old strain’s metabolism and proteome were also obtained. 10-day old strains had lower organic acid production and different expressed proteins involved in bacterial metabolism and stress response. However bacterial competition against pathogen strain remained stable