Lactobacillus delbrueckii subsp. lactis: interaction with enterocyte -like cells and inhibition of Escherichia coli

HUGO, A. A.; KOCIUBINSKI, G.; DE ANTONI. G.L; PÉREZ. P.F

París. Francia.

Simposio; Functional Foods; 2001

Microorganisms belonging to the genus Lactobacillus have shown to exert health benefits on human beings. This probiotic effect could be in part ascribed to both their ability to interact with intestinal epithelia and their inhibitory effect on intestinal pathogens. In a survey of the capability of different lactic acid bacteria to inhibits gram negative microorganisms, Lactobacillus delbrueckii subsp. lactis strain CIDCA 133 has been selected because of its high inhibitory power against different microorganisms The aim of the present work was to gain further insight on the kinetics of inhibition as well as on the interaction with enterocyte-like cells of strain CIDCA 133 (Lactobacillus delbrueckii subsp lactis). Bacterial cultures: Strain CIDCA 133 belongs to the culture collection of the CIDCA and was grown in MRS broth for 16 h at 37°C. The strain of Escherichia coli belongs to the culture collection of the Cátedra de Microbiología de la Facultad de Ciencias Exactas de la Universidad Nacional de La Plata. This strain was grown in nutrient broth. Adhesion assays Cultures were centrifuged and the pellets were suspended in phosphate buffered saline (PBS). Optical density (600 nm) was adjusted to 1 unit and 0.5 ml of the bacterial suspension was coincubated with differentiated human enterocytes (Caco-2 cells) at 37°C cultured in 24-wells. Cells were washed three times with PBS and the percentage of adherence was evaluated by means of two methods: a) viable counts (strain CIDCA 133) and b) determination of the nitrate reductase activity (E. coli). Inhibition assays. Spent culture supernatants were collected by centrifugation. After being filtered (0.45 µm pore size), supernatants were mixed in different proportions with nutrient broth. Afterwards, the strain of E. coli under study was inoculated. Bacterial growth was evaluated by viable counts and measure of the optical density. Different concentrations and pH of the spent culture supernatants were used. Concentration of fatty acids in the spent culture supernatants were evaluated by HPLC. After coincubation of strain CIDCA 133 with Caco-2 monolayers, a high amount of lactobacilli became adhered to the enterocytes. This interaction did not produce any damage to the epithelial cells. Growth of E. coli was strongly inhibited by culture filtrate supernatants of strain CIDCA 133. When 1/6 dilutions were assayed, a bactericidal effect was observed. Higher dilutions (1/12 and 1/20) produced a bacteriostatic effect. Lag phase, growth rate and maximal bacterial concentration were affected in a dose-dependent manner. The main metabolic product of strain CIDCA 133 was lactic acid. Further analysis revealed that the inhibitory effect could be due mainly to the action of lactic acid although the effect of other exocellular factors cannot be ruled out. The results of the present work shows that L. delbrueckii subsp. lactis CIDCA 133, a strain isolated from milk, is able to adhere to enterocyte-like cells and has a strong inhibitory power against E. coli. Although, the inhibitory power could be adscribed to the production of lactic acid the effect of other exocellular factors could be also involved. Interestigly enough, the inhibition of E.coli by spent culture supernatant of strain CIDCA 133, ocurred even at low concentrations.Taken together, our results suggest  that this microorganism fullfil the main requirements for a probiotic strain.