Bile salts and cholesterol induce changes in the lipid cell membrane of Lactobacillus reuteri.

AIMS: The objective of this study was to evaluate the effect of bile salts and cholesterol in the lipid profile of Lactobacillus reuteri CRL 1098 and to determine the relationship existing between these changes: the in vitro removal of cholesterol and the tolerance of the cells to acid and cold stress. METHODS AND RESULTS: Lactobacillus reuteri CRL 1098 was grown in the following media: MRS (deMan Rogosa Sharpe; MC, control medium), MB (MC with bile salts), MCH (MC with sterile cholesterol) and MBCH (MC with bile salts and cholesterol). Fatty acids were determined by analytical gas-liquid chromatography, and phospholipids and glycolipids by colorimetric techniques. The cells from different culture media were subjected to cold and acid stress. The MB cultures displayed a decrease in phospholipids and a low ratio of saturated : unsaturated fatty acids. The presence of the unusual C18 : 0,10-OH and C18 : 0,10-oxo fatty acids was the prominent characteristic of the bile salts growing cells. The relative increase in glycolipids and the changes in the fatty acids profiles of the MB cells would be responsible for the cholesterol remotion. The changes induced by bile salts in the lipid profile did not improve the tolerance of L. reuteri CRL 1098 to freezing and acid stress. CONCLUSIONS: The changes in lipid profiles reported in this study would play a key role in the response of Lactobacilli to environmental stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides useful information about the effect of bile salts on the cell membrane of L. reuteri, a probiotic enterolactobacillus. The steady-state response of the cells subjected to bile stress seems to be the appropriate model for evaluating the bacterial behaviour in detergent-containing gastrointestinal tracts, where the bile salts stress would presumably be continuous.

Effect of lipid composition on the stability of cellular membranes during freeze-thawing of Lactobacillus acidophilus grown at different temperatures.

Lactobacillus acidophilus CRL 640 grown at the optimal temperature of 37 degrees C (M37) appeared more sensitive to freeze-thawing than when it was grown at 25 degrees C (M25). In the first case, 87% of the cells died, in contrast to 33% for cells grown at 25 degrees C. All the surviving M37 cells showed sensitivity to NaCl. However, among the surviving M25 cells, only 85% were sensitive to NaCl. The rest of the cells were considered uninjured. Freeze-thawing in cells grown at 25 degrees C showed a liberation of nucleic acids and proteins. However, the leakage was higher in M37 cells after freeze-thawing. The greater fraction of damaged cells were observed in M25 culture after freeze-thawing. A relative increase of 81% in cardiolipid (CL), with respect to total phospholipids and 72% triglycosyldiglyceride (TGDG) with respect to the total glycolipids was observed in M37. In addition, a decrease of palmitoyl (C16:0), oleoyl (C18:0) fatty acids at CL, phosphatidylglycerol (PG), and diglicosyldiglyceride (DGDG) fractions and the increase of C19 cyc and C18:0, 10-OH fatty acids in neutral lipid, and CL fractions was also apparent. In M25 cells, the concentration of DGDG and PG was higher than in M37 cells. The difference in cryotolerance between the frozen cultures emphasizes the importance of selecting appropriate conditions of growth of microorganisms for use as dietary adjuncts.

Permeability and stability properties of membranes formed by lipids extracted from Lactobacillus acidophilus grown at different temperatures.

Lactobacillus acidophilus CRL 640 grown at 25 and 37 degrees C showed a high content of cardiolipin, phosphatidylglycerol, and glycolipids. Cultures grown at 25 degrees C showed a twofold increase in glycolipids in relation to phospholipids, a twofold increase in the C16:0 and a fourfold increase in the C18:2 fatty acids. In contrast, the C19-cyc and the 10-hydroxy acid (C18:0-10 OH) species showed a noticeable decrease. Extracts of total lipids of bacteria grown at 25 and 37 degrees C dispersed in water yielded particles having a high negative surface potential as measured by electrophoretic mobility. Vesicles prepared by extrusion of these dispersions through polycarbonate membranes of 100-nm pore diameter showed high trapping of carboxyfluorescein (CF), which remained unchanged for at least 20 h. The fluorescence anisotropy measured with diphenylhexatriene (DPH) and the generalized polarization of Laurdan were significantly lower in vesicles prepared with lipids containing the highest glycolipid ratio, in comparison to those of bacteria grown at 37 degrees C. No phase transition was detected between 5 and 50 degrees C as measured with both probes. In accordance with these results, no significant release of the trapped CF in this range of temperature was detected. Bile salts and NaCl promoted an increase in the fluorescence, which is interpreted as a change in the permeability properties of the membrane. This effect was lower with KCl, while CaCl2 did not cause any change. The greater permeability change was observed in vesicles with a low glycolipid/phospholipid ratio. NaCl did not affect the packing of the interface as measured with Laurdan, in contrast to CaCl2. The action of Ca+2 may be ascribed to the binding to the negatively charged lipids, such as phosphatidyl glycerol and cardiolipin. It is concluded that the higher glycolipid/phospholipid ratio and the fatty acids C18:2 and C16:0 enhance the lipid membrane stability and decrease the organization in the interfacial and hydrocarbon zones. These results are congruent with the behavior of entire bacteria subject to osmotic and freeze/thaw stresses.

Influence of the incubation temperature on the autolytic activity of Lactobacillus acidophilus.

The effect of temperature and growth phase on the autolysis of Lactobacillus acidophilus CRL 640 was studied. The maximal rate of autolytic activity (ca 48% cell lysis) was found at 45 degrees C. At this temperature, two peaks were detected: the first one at the early exponential phase of growth and the second lysis peak during the transition stage from the exponential to the stationary phase. The release of intracellular compounds absorbing at 260 and 280 nm was also detected at 45 degrees C. The microscopic observations revealed morphological changes and the presence of ghost cells. At 37 degrees C, the low autolytic activity obtained would be related to the normal cell cycle of growth.