Bile salt hydrolase (BSH) activity was shown to be constitutive and substrate-specific: the BSH isogenic Lactobacillus plantarum wild type (LP80 WT) and BSH overproducing LP80 (pCBH1) strains preferentially hydrolysed glycodeoxycholic acid (GDCA), whereas the hamster Lact. animalis isolates H362 and H364 showed a higher affinity for taurodeoxycholic acid (TDCA). In viability studies in the presence of nutrients, it was demonstrated that GDCA exerted a higher toxicity than TDCA in a pH-dependent manner. This toxicity was inversely proportionate to the BSH activity level of the strains tested, indicating that BSH activity contributed towards bile salt resistance when appropriate nutrients were available. The high toxicity of GDCA relative to TDCA was suggested to be caused by their weak and strong acid properties respectively. It was therefore hypothesized that the protonated form of bile salts exhibited toxicity as it imported protons in the cell. This puts an energy-burden on BSH- lactobacilli which undergo intracellular acidification. BSH+ cells primarily protect themselves through the formation of the weaker DCA compound, which can help negate the pH-drop by recapturing and exporting the co-transported proton. However, since DCA is more toxic than its conjugated counterparts, an additional energy-dependent detoxification of DCA is suggested.

Growth and bile salt hydrolase (BSH) activity of the isogenic LactobacilZus plantarum 80 (LP80) strains were studied in vitro. In pure culture experiments viability and growth performance of the BSH-LP80 strain was negatively affected by the presence of conjugated bile salts. The LP80 wild type (WT) and BSH overproducing LP80 (pCBH1) strains did not show a die-off upon supplementation of bile salts. The latter strains hydrolysed glyco-conjugated deoxycholate (GDCA) more readily than tauro-conjugated deoxycholate (TDCA), indicating substrate specificity of the enzyme. BSH activities towards TDCA of LP80 WT and LP80 @CBHl) stationary phase cells were 0.17 and 1.02 FmoUmg CDW.h respectively; activities towards GDCA of the respective strains were 3.52 and 54%0 FmoUmg CDW.h respectively. The study of BSH activity as a function of growth revealed a marked difference in behaviour between LP80 WT and LP80 (pCBH1) with LP80 WT hydrolysing GDCA when reaching the exponential phase, whereas LP80 (pCBH 1) immediately started to hydrolyse GDCA. TDCA hydrolysis increased after GDCA hydrolysis was completed. BSH activity of LP80 (pCBH1) in a mixed microbial association, resembling that of the small intestine, was comparable to that determined under pure culture conditions, indicating that BSH activity will probably not be influenced by the presence of the normal intestinal microbiota. Based on the BSH activity of LP80 (pCBH 1) and on physiological data on the bile salt-cholesterol metabolism interrelationship, it was calculated that a daily intake of a realistic amount of highly BSH active Lactobacillus cells, e.g. in the form of yoghurt, might lead to a significant reduction of cholesterol. Hence, this in vitro study indicates that altering BSH activity can be a valid (micro) biological alternative treatment for patients with severe hypercholesterolaemia.

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