Physiological, biochemical and structural damages in cells of Propionibacterium freudenreichii subjected to in vitro gastrointestinal digestion

VERÓNICA HAEL CONRAD, GABRIELA ZÁRATE, ADRIANA PÉREZ CHAIA

San Miguel de Tucumán

Congreso; VII Congreso Argentino de Microbiología General Samige del bicentenario; 2011

Sociedad Argentina de Microbiología General

Dairy propionibacteria have a long history as starter cultures in the food industry, and today, they are also studied as probiotic microorganisms. The successful performance as probiotics depends on achieving the intestine metabolically active and in high number. They must recover in short time their activity after exposure to the aggressive conditions of the gastrointestinal tract (GIT). For their protection various strategies have been developed, however, to design appropriate methods is necessary to identify  the stages of the digestive process responsible for major damage to these bacteria. The aims of the study were to evaluate how aggressive digestion conditions affect physiological, biochemical and structurally a strain of Propionibacterium freudenreichii, often used in the manufacture of Emmental cheese. Propionibacterium freudenreichii CRL 757 was cultivated in LAPT broth with 1% lactose (LAPTl) for 24 hours at 37C. A suspension of 108 bacteria in phosphate buffer saline (PBS) was subjected to artificial gastric (DG) and intestinal (DI) digestion protocols and to the sequence of both digestions (DGI). The cells subjected to treatments were washed and the percentage of cells injurred or death was determined by staining with iodide propidium (PI) and counted by fluorescence microscopy. An aliquot was used as untreated control. The count was repeated after subjecting the samples to an additional stress of heat (100C, 0 to 120 min). The cell injury was represented as IP (+) cells vs. heating time and the decimal reduction time (D) in each case was calculated. In other aliquots of cells subjected to digestion, we determined the changes in physical-chemical properties of the cell wall through their affinity to  organic solvents. We also assessed the biochemical activity of B-galactosidase and physiological changes by studying the recovery of cells subjected to digestion and cultured in LAPTl. The in vitro digestion increased the number of injured or death cells in a log unit, but the differences between treatments were evident by detection of D (without digestion >DG>DGI). DG increased the affinity to chloroform and DI to hexane and chloroform. The B-galactosidase activity was significantly decreased after DI, but not DG. Cultures inoculated with treated cells showed increased lag phase and arrested their growth in less time and with lower biomass production. The effects were similar in the DG and DI treatment, but greater delay and lower biomass was obtained with DGI. Therefore, both stages of digestion caused reduction on cells number or changes in the permeability of propionibacteria and modified properties related to adherence to epithelial cells. The DI, but not the DG affected the biochemical activity. The sequence of the DGI caused major growth delay than DG or DI.