Bile Salt Hydrolase Activity of Three Strains of Lactobacillus acidophilus1

Bile Salt Hydrolase Activity of Three Strains of Lactobacillus acidophilus1 G. CORZO2 and S. E. GILLILAND3 Department of Animal Science, Oklahoma State University, Stillwater 74078

ABSTRACT Three strains of Lactobacillus acidophilus, two from human intestinal origin (016 and L1) and one from porcine intestinal origin (ATCC 43121), were tested for their bile salt deconjugation activity. The L. acidophilus ATCC 43121 had more deconjugating activity of both sodium glycocholate and sodium taurocholate at pH 6.5 than did either L. acidophilus 016 or L1. The activity of intracellular bile salt hydrolase found in strain ATCC 43121 was 14-fold higher than that in either of the other two strains. The optimum pH for deconjugation of sodium glycocholate was between 4 and 5.5 for all three strains. For deconjugation of sodium taurocholate, the optimum pH was between 3.5 and 4.5 for strains L1 and ATCC 43121 and was between pH 5 and 6 for strain O16. The molecular mass of the enzyme in all three strains of L. acidophilus was estimated to be 126 kDa by Sephadex G-200 gel filtration. All three strains exhibited more bile salt hydrolase activity towards sodium glycocholate than towards sodium taurocholate. ( Key words: Lactobacillus acidophilus, bile salt hydrolase, deconjugation) Abbreviation key: BSH = bile salt hydrolase, GIT = gastrointestinal tract. INTRODUCTION The importance of bile salt deconjugation by intestinal microorganisms has potential importance in reducing serum cholesterol (10, 13, 15). The major route of cholesterol excretion from humans and other

Received June 19, 1998. Accepted November 16, 1998. 1Approved for publication by the director, Oklahoma Agricultural Experiment Station. This research was supported under Project H-2293. The senior author was supported on a CONACyTFulbright Fellowship. 2Present address: Suntory Institute of Biorganic Research (SUNBOR), Osaka, Japan. 3Corresponding author. 1999 J Dairy Sci 82:472–480

mammals is via feces. Cholesterol is the precursor of primary bile salts that are formed in the liver and are stored as conjugated bile salts in the gallbladder for subsequent secretion in the gastrointestinal tract ( GIT) . Between 750 and 1250 mg of cholesterol and between 5500 and 35,500 mg of conjugated bile salts are emptied into the small intestine of humans daily (20). Conjugated bile salts are secreted into the small intestine to help in the absorption of dietary fat (22), cholesterol (36), hydrophobic vitamins (24), and other fat-soluble compounds. Conjugated bile salts are absorbed from the small intestine (ca. 97%) and are returned to the liver by the hepatic portal circulation (18). A small fraction of the bile salts (250 to 400 mg), which is not absorbed in the process, is lost from the human body as free bile salts in feces ( 6 ) . Free bile salts are less soluble and are less likely to be absorbed by the intestinal lumen than are conjugated bile salts (4, 5, 35). Thus, in a steady-state situation, deconjugation of bile acids can lead to the reduction of serum cholesterol by increasing the formation of new bile acids that are needed to replace those that have escaped the enterohepatic circulation or by reducing the absorption of cholesterol throughout the intestinal lumen (31). Bile salt hydrolase ( BSH) , the enzyme responsible for bile salt deconjugation, is present in several bacterial species of the GIT such Lactobacillus sp. (27), Bifidobacterium longum (19), Clostridium perfringens (17), and Bacteroides fragilis ssp. fragilis (37). Bile salt hydrolases are active on both glycine and taurine conjugated bile salts. Lactobacillus acidophilus, a normal component of the microflora of the small intestine, deconjugates bile salts (8, 15). Lactobacillus acidophilus also has potential for exerting other health or nutritional benefits such as prevention of carcinogenesis (16), treatment of encephalopathy (30), and prevention of gastrointestinal infections (1). This study compared the bile salt deconjugation activity from three strains of L. acidophilus and determined the effect of pH on activity of their bile salt hydrolases.

472

473

LACTOBACILLUS BILE SALT HYDROLASE

MATERIALS AND METHODS Sources and Maintenance of Cultures The three strains of L. acidophilus, L1 and O16 from human origin and ATCC 43121 from porcine origin, that were used in this study were obtained from the stock culture collection of the Food Microbiology Laboratory in the Department of Animal Science at Oklahoma State University (Stillwater). All cultures were maintained by subculturing in lactobacilli MRS broth (Difco Laboratories, Detroit, MI) using 1% inocula and 18- to 24-h incubation at 37°C. Stock cultures were maintained in MRS agar (MRS broth plus 1.5% agar) stabs. All cultures were stored at 5 to 7°C between subcultures. Bile Salt Deconjugation During Growth Without pH Control The MRS broth (100 ml) supplemented with 1 mM sodium taurocholate and 1 mM sodium glycocholate (Sigma Chemical Co., St. Louis, MO) was prepared and was placed in 100-ml volumes into dilution bottles of about 180-ml capacity. The bottles containing the broth were autoclaved at 121°C for 15 min and were cooled. Freshly prepared MRS broth cultures of L. acidophilus (i.e., subcultured on each of 2 successive d just prior to the experiment) were inoculated ( 1 % ) into the medium and were mixed for 1 min. A series of 10-ml aliquots was withdrawn aseptically from the bottle for each culture, placed into sterile screw-cap test tubes (1.8 × 12 cm), and incubated at 37°C for 24 h. A tube was taken for analyses every 2 h for 14 h and at the end of the 24-h incubation. Growth was monitored by plate count on MRS agar. Samples also were analyzed for pH and bile salts. Preliminary experiments showed that deconjugation was not affected by heat sterilization of the medium and that low oxidation-reduction potential was not an important factor for deconjugation by any of the three strains of L. acidophilus in this study. Bile Salt Deconjugation During Growth with pH Control The MRS broth (300 ml), supplemented with 1 mM sodium taurocholate and 1 mM sodium glycocholate, was prepared and placed into a fermentor of about 1-L capacity. The fermentor was equipped with an autoclavable combination pH electrode (Ingold Electrodes, Inc., Wilmington, MA). It also was

equipped with a port for addition of neutralizer and a line to permit continuous sparging with nitrogen gas. The fermentor containing the broth was autoclaved at 121°C for 15 min, was cooled, and was placed in a 37°C water bath. The pH was controlled by using a mixed solution of 10% sodium carbonate in 10% ammonium hydroxide (14). The neutralizer was delivered as required from a flask to the fermentor by a peristaltic pump (Masterflex; Cole-Parmer Inst. Co., Chicago, IL) connected to an automatic pH controller (model 5997; Horizon Ecology Co., Chicago, IL) that was adjusted to maintain the desired pH in the broth. Nitrogen gas was continuously sparged through the broth from bottom to top at about 5 ml/min. After mixing the broth for 5 min, a freshly prepared MRS broth culture of L. acidophilus was inoculated ( 1 % ) into the fermentor and then was mixed for 1 min. Ten milliliters were withdrawn aseptically from the fermentor and were placed into a sterile test tube held in an ice-water bath to serve as the initial sample (i.e., 0 h). The fermentor containing the culture was incubated at 37°C for 24 h. Samples were taken aseptically every 3 h for 12 h and at the end of the 24 h. All samples were held in an ice-water bath until analyzed. Growth was monitored by plate count and absorbance at 620 nm. Samples also were analyzed for bile salts. Measuring Culture Growth Growth was measured by plate count using MRS agar. Appropriate dilutions, prepared using 0.1% sterile peptone (Difco Laboratories) dilution blanks containing 0.01% silicone antifoamer (Sigma Chemical Co.), were plated by the pour-plate method with MRS agar. The plates were overlayed with the same medium and were incubated for 48 h at 37°C, after which colonies were counted with the aid of a Quebec Colony Counter (American Optical Co., Buffalo, NY). Colony-forming units per milliliter, expressed as log10, were plotted against incubation time. BSH Activity To recover bile salts from MRS broth cultures, cells were removed from 5-ml samples of culture by centrifugation (10,000 × g for 10 min at 5°C). The method of Ruben and Berge-Henegouwen ( 3 2 ) was modified to recover the bile salts from the spent broth. By filtering it through a 0.45-mm polysulfone filter (Whatman Inc., Clifton, NJ), 8 ml of 0.2 M NaOH in 0.9% NaCl were added to 1 ml of the filtrate and were mixed using a vortex mixer. The solution Journal of Dairy Science Vol. 82, No. 3, 1999

474

CORZO AND GILLILAND

was passed through a Sep-Pak® C18 Cartridge (Waters Associates, Milford, MA) that had been prepared according to the manufacturer’s instructions. Subsequently, the Sep-Pak® cartridge was washed once with 10 ml of water, once with 5 ml of 10% acetone, and again with 10 ml of water. The conjugated and free bile acids were eluted from the cartridge with 5 ml of methanol. The methanolic filtrate was evaporated to dryness at 60°C under a stream of nitrogen gas. The residue was dissolved in 1 ml of mobile phase and was filtered through a 0.45-mm polysulfone filter prior to HPLC analysis. The HPLC method described by Corzo and Gilliland ( 7 ) was used to quantitate the individual bile salts. The BSH activity was based on the disappearance of sodium glycocholate, or sodium taurocholate, or both from the medium. The BSH activity of cell-free extracts and partially purified enzyme preparations was measured as described by Corzo and Gilliland ( 7 ) . The activity in these assays also was based on the disappearance of sodium glycocholate, or sodium taurocholate, or both. Partial Purification of BSH After 24 h of growth in MRS broth without conjugated bile salts, the broth culture (200 ml) was centrifuged at 10,000 × g for 10 min at 5°C. The spent broth was transferred to a clean tube. The cell pellet was resuspended and was washed twice with acetate buffer (50 mM, pH 4.0, 20 ml) and then was centrifuged at 10,000 × g for 10 min at 5°C. The washed cells were resuspended in 50 mM acetate buffer (pH 5.4) to 0.1 of the volume of the original culture. The cells were disrupted by sonication ( 5 mm from peak to peak of amplitude, and 85 W delivered to the cell suspension) five times for 2 min each at 5°C with a Sonic Dismembrator (model 550; Fisher Scientific, Pittsburgh, PA). The lysed cell suspension was centrifuged at 10,000 × g for 10 min at 5°C, and the resulting supernatant fluid was filtered through a 0.45-mm polysulfone filter (Whatman Inc.) and was used for enzyme isolation (cell-free extract). Cell-free extracts were mixed with methanol in a ratio of 2 to 1 to obtain a final concentration of 33% methanol (vol/vol). After 1 h, the spent brothmethanol or cell-free extract-methanol mixture was centrifuged at 10,000 × g for 10 min at 5°C to pellet the precipitate. The precipitate was dissolved in 50 mM acetate and 1 mM EDTA buffer (pH 5.4). (Related experiments in our laboratory showed that methanol precipitation did not adversely influence the activity of BSH of L. acidophilus) . Journal of Dairy Science Vol. 82, No. 3, 1999

The dissolved methanol precipitate was fractionated by ammonium sulfate precipitation (40 to 80% saturation). The precipitated fraction was harvested by centrifugation at 5000 × g for 15 min at 5°C and was resuspended in 50 mM sodium acetate buffer containing 1 mM EDTA (pH 5.4), vortexed to complete dissolution, and dialyzed for 18 h against the acetate-EDTA buffer ( 2 L ) through dialysis membranes (Spectra/Por; Spectrum Medical Instruments, Inc., Los Angeles, CA) with a molecular mass cutoff from 12 to 14 kDa. The dialyzed ammonium sulfate fraction was stored at –20°C for no more than 4 wk. Gel filtration was performed using Sephadex G-200 columns (2.5 × 50 cm) that were prepared according to Stellwagen (38). A mobile phase comprised 50 mM sodium acetate (pH 5.4) with 1 mM EDTA and 0.02% sodium azide and was used to elute the proteins. The flow rate of the mobile phase was controlled at 0.16 ml/min by using a peristaltic pump (Cole-Parmer Inst.) attached to the column inlet. The sample with a protein concentration of no more than 2 mg/ml and a volume of no more than 2 ml was loaded onto the column, and samples of 0.8 ml each were collected by an automatic fraction collector (Retriever II; ISCO Co., Lincoln, NE) and were monitored manually for absorbance at 280 nm (Spectronic 21D; Milton Roy, Rochester, NY). The fractions also were analyzed for BSH activity. Fractions with BSH activity from spent broth were pooled and concentrated with a Centricell 60 membrane (Polysciences, Warrington, PA) with molecular cutoff of 30 kDa. The pooled fractions were centrifuged at 1500 × g for 20 to 60 min at 4°C or until the desired concentration was reached. Volumes of pooled fractions and filtrates were recorded. The molecular mass of the enzymes was determined by Sephadex G-200 using blue dextran, immunoglobulin G, bovine serum albumin, and lyzozyme as markers (Sigma Chemical Co.). Effect of pH on BSH Activity and Substrate Specificity The effect of pH on activity of the partially purified BSH was determined over a range of pH 3.5 to 7 in 0.1 M sodium acetate buffer (pH 3.5 to 5.5) and 0.1 M sodium phosphate buffer (pH 6 to 7). A control without BSH was included for each pH. Protein Determination The protein content of the fraction(s) at each step of purification as well as fractions from the gel chromatography was determined by the method of Bradford ( 2 ) . Bovine serum albumin (Sigma Chemical Co.) was used as the protein standard.

LACTOBACILLUS BILE SALT HYDROLASE

Statistical Analysis Bile salt deconjugation, growth, and pH were analyzed using the general linear model correlation procedure from SAS® ( 3 4 ) to determine whether significant relationships occurred among these variables. Bile salt deconjugation and specific growth rates of L. acidophilus were analyzed by the modified logistic model ( 4 1 ) using the nonlinear model procedure from SAS® (34). The least significant difference method was used to determine whether statistically significance differences occurred among means. RESULTS Bile Salt Deconjugation and Cell Growth in Static Cultures All three strains of L. acidophilus that were grown in MRS broth supplemented with 1 mM each of sodium glycocholate and sodium taurocholate without

475

pH control exhibited deconjugation activity (Figure 1). Figure 1A shows the growth, pH, and deconjugation of sodium taurocholate and sodium glycocholate by L. acidophilus O16 during incubation at 37°C for 24 h. As expected, the pH dropped as growth of the culture occurred. Plate counts increased during the first 8 to 12 h of incubation followed by a decrease after 12 h. The reduction in plate count beyond 12 h might have resulted from the effect the free bile salts resulting from deconjugation of the sodium glycocholate, or sodium taurocholate, or both. Sodium cholate, a product of the deconjugation, is more toxic for L. acidophilus and other intestinal microorganisms than are the conjugated bile salts (11, 12). The other two strains (L1 and ATCC 43121) yielded similar results except that L1 more rapidly deconjugated sodium glycocholate than it did sodium taurocholate (Figure 1B). Additionally, the viability (based on plate count) of ATCC 43121 declined at least one log cycle between 8 and 24 h, indicating a greater sensitivity to free bile salts than observed for the other two strains

Figure 1. Disappearance of conjugated bile salts, change in pH ( ⁄) , and growth (plate count) ( ÿ) of Lactobacillus acidophilus O16 ( A ) , L1 ( B ) , and ATCC 43121 ( C ) in MRS broth supplemented with 1 mM sodium glycocholate ( ◊) and 1 mM sodium taurocholate ( o) . Each point on each graph represents a mean of three independent trials. Journal of Dairy Science Vol. 82, No. 3, 1999

476

CORZO AND GILLILAND

TABLE 1. Deconjugation rate of Lactobacillus acidophilus 016, L1, and ATCC 43121 in static cultures.1 Strain

L1 016 ATCC 43121

Glycocholate X 0.26a,A 0.18a,A 0.20a,A

SE 0.02 0.04 0.03

Taurocholate ( m M/h) X 0.07b,B 0.17a,A 0.18a,A

SE 0.01 0.02 0.03

1Based on growth in MRS broth supplemented with 1 mM glycocholate and 1 mM taurocholate. Each value is the average of three independent trials. a,bMeans in the same column without common superscript letters differ ( P < 0.05). A,BMeans in the same row without common superscript letters differ ( P < 0.05).

(Figure 1C). Such declines in viability for this culture were not obsrved in related experiments in a medium having the same composition except for the absence of bile salts (data not shown). The bile salt deconjugation data for all three strains of L. acidophilus were analyzed by the modified logistic equation (41). The purpose of such analysis was to determine whether there were any significant differences in the deconjugation rate between sodium glycocholate and sodium taurocholate. The deconjugation rates of sodium glycocholate did not differ ( P > 0.05) among strains of L. acidophilus growing under static conditions (Table 1). The deconjugation rates of sodium taurocholate were not differ-

ent ( P > 0.05) for strains ATCC 43121 and O16. However, L. acidophilus L1 exhibited a lower ( P < 0.05) deconjugation rate for sodium taurocholate than did the other two strains. Sodium glycocholate was totally deconjugated by all strains during the 24 h of incubation (Figure 1). However, sodium taurocholate was totally deconjugated only by strains O16 and ATCC 43121 (Figure 1, A and C). Lactobacillus acidophilus L1 had deconjugated only 76% of sodium taurocholate after 24 h of incubation (Figure 1B). Bile salt hydrolase activity also was detected in the spent broths from each strain. Spent broth from strain ATCC 43121 exhibited more ( P < 0.05) BSH activity than did the other two strains (data not shown). Bile Salt Deconjugation During Growth at pH 6.5 Additional experiments were conducted for all three strains of L. acidophilus in which the growth medium was maintained at pH 6.5. This pH level is similar to that of the small intestine in healthy humans (29). For these experiments, the concentrations of sodium glycocholate and sodium taurocholate were modified to resemble more closely the ratio encountered in healthy humans, which involved a sodium glycocholate to sodium taurocholate molar ratio of 2.3 (21, 28, 33) so that the growth medium was supplemented with 2.8 mM sodium glycocholate and 1.2 mM sodium taurocholate. All three cultures exhibited

Figure 2. Disappearance of conjugated bile salts and growth (plate counts) ( ÿ) of Lactobacillus acidophilus O16 ( A ) , L1 ( B ) , and ATCC 43121 ( C ) in MRS broth supplemented with 2.8 mM sodium glycocholate ( ◊) and 1.2 mM sodium taurocholate ( o) at pH 6.5. Each point on each graph represents a mean of two independent trials. Journal of Dairy Science Vol. 82, No. 3, 1999

477

LACTOBACILLUS BILE SALT HYDROLASE TABLE 2. Deconjugation rates of Lactobacillus acidophilus 016, L1, and ATCC 43121 at pH 6.5.1 Strain

Glycocholate X 0.09b,A 0.10b,A 0.28a,A

L1 016 ATCC 43121

SE 0.01 0.02 0.02

Taurocholate ( m M/h) X 0.011c,B 0.058b,B 0.107a,B

SE 0.01 0.01 0.01

of sodium taurocholate ( P < 0.05). These results suggest that L. acidophilus ATCC 43121, O16, and L1 have a tendency for more efficient deconjugation of sodium glycocholate than of sodium taurocholate. Partial Purification of BSH

1Based on growth in MRS broth supplemented with 2.8 mM glycocholate and 1.2 mM taurocholate. Each value is the mean of two independent trials. a,b,cMeans in the same column without common superscript letters differ ( P < 0.05). A,BMeans in the same row without common superscript letters differ ( P < 0.05).

BSH activity under these conditions (Figure 2). Strain ATCC 43121 appeared more active toward sodium glycocholate than did the other two strains. All three strains were more active on sodium glycocholate than on sodium taurocholate. Strain L1 showed very little if any activity toward sodium taurocholate (Figure 2B). The plate count for strain O16 (Figure 2A) declined after 6 h of incubation, whereas no decline was evident for the other two strains. Analysis of the data by the modified logistic equation ( 4 1 ) showed the deconjugation rates for both sodium glycocholate and sodium taurocholate by L. acidophilus ATCC 43121 growing at pH 6.5 were higher ( P < 0.05) than those for strains L1 and O16 (Table 2). The deconjugation rate of sodium glycocholate was higher for all three strains of L. acidophilus than that

Intracellular BSH activity (cell-free extracts) of strain ATCC 43121 was higher than that of strains O16 and L1 (Table 3), which was true for both total and specific activity. Cell disruption was 91% for strain ATCC 43121, 93% for strain L1, and 96% for strain O16 as determined by colony-forming units per milliliter before and after sonication. Methanolic and ammonium sulfate fractionation resulted in 4- to 8-fold purification of the enzyme for each strain. Specific activity increased with each step in purification. After Sephadex G-200 chromatography, specific activity was 13- to 14-fold higher for strain ATTC 43121 than for strains L1 and O16. Elution profiles from Sephadex G-200 chromatography of ammonium sulfate fractions for all three strains followed a similar pattern (data not shown). Their molecular masses were estimated to be 126 ± 11 kDa by gel filtration. Effect of pH on BSH Activity and Substrate Specificity The effect of pH was measured in a range from 3.5 to 7 for the partially purified enzymes of all three strains of L. acidophilus. The pH ranges supporting optimum BSH activity from all three strains of L.

TABLE 3. Purification of bile salt hydrolase1 from three strains of Lactobacillus acidophilus. Step Strain L 1 Cell-free extracts Methanol Ammonium sulfate Sephadex G-200 Strain O16 Cell-free extracts Methanol Ammonium sulfate Sephadex G-200 Strain ATCC 43121 Cell-free extracts Methanol Ammonium sulfate Sephadex G-200 1Based

Total protein

Total activity

Specific activity

Recovery

Purification

(mg)

(U)

(U/mg)

(%)

(fold)

44.8 9.1 6.0 0.4

1696 1571 1172 793

38 173 195 1983

100.0 92.6 69.1 46.7

1.0 4.6 5.2 52.5

53.6 4.8 3.1 0.4

2731 1427 977 628

51 293 315 1570

100 52.2 35.7 22.9

1.0 5.8 6.2 30.8

66.3 9.0 3.1 0.1

53726 35588 22324 2580

810 3954 7201 25800

100.0 66.2 41.5 5

1.0 4.9 8.9 31.8

on activity on sodium glycocholate. Journal of Dairy Science Vol. 82, No. 3, 1999

478

CORZO AND GILLILAND

acidophilus on both sodium glycocholate and sodium taurocholate varied (Figure 3). In general, the enzymes from all three strains exhibited optimum BSH activity at relatively low pH. The optimum pH was not the same for each conjugated bile salt for each strain. Bile salt hydrolase from L. acidophilus O16 was most active on sodium taurocholate at pH 5 to 6 and on sodium glycocholate at pH 3.5 to 5.5. However, strains L1 and ATCC 43121 were most active on

Figure 3. Optimum pH of bile salt hydrolase on sodium glycocholate ( A ) and on sodium taurocholate ( B ) from three strains of Lactobacillus acidophilus O16 ( ◊) , L1 ( ÿ) , and ATCC 43121 ( o) . Each value is the mean of two independent trials. Journal of Dairy Science Vol. 82, No. 3, 1999

taurocholate at pH 3.5 to 4.5 and on glycocholate at pH 3.5 to 5.5. DISCUSSION Bile salt deconjugation, as a result of BSH activity, increased only when the cultures reached their maximum cell growth in static culture conditions. High BSH activity was observed at the stationary phase of growth of B. fragilis ssp. fragilis (37), of Lactobacillus sp. strain 100-100 (27), and of Lactobacillus acidophilus RP32 (26). Lundeen and Savage ( 2 7 ) suggested that the BSH activity was regulated by the growth phase in Lactobacillus sp. strain 100-100. Because the lowest pH of all strains of L. acidophilus in static cultures was always found in the stationary phase and the BSH activity occurred preferentially at low pH, the higher BSH deconjugation activity associated with the stationary phase might not have been entirely the result of the growth phase but the result of the pH. The pH and the cell growth were positively and negatively correlated, respectively, with both sodium taurocholate and sodium glycocholate bile salt deconjugation ( P < 0.0001). The disappearance of sodium glycocholate from the growth media could have been due to precipitation caused by the acidic condition (40). However, based on a related study in our laboratory, the pH of the culture media in the present study would not affect the solubility of sodium glycocholate and sodium taurocholate ( 7 ) . When sodium glycocholate was added to buffers ranging in pH from 1 to 7, 80 and 75% of sodium glycocholate was precipitated at pH 1 and 2, respectively, but no precipitation occurred at pH ≥3 ( 7 ) . Conjugated bile salts are found in a 2.2 to 3 M ratio of glycocholate per taurocholate in the gallbladder of the human adult, and the total amount of conjugated bile salts secreted into the GIT is about 10 to 15 mmol for each of six daily cycles (23). Because the volume of the gallbladder is smaller than that of the GIT, the conjugated bile salts become diluted when entering into the upper part of the small intestine. Therefore, the highest bile salt molarity that might occur throughout the small intestine is between 2.8 and 4 mM as a consequence of such a dilution (23). However, a higher concentration might be expected in the duodenum where the bile is secreted, and a lower concentration might be found at the end of the ileum because of diffusion mechanisms, microbial transformations, and absorption of bile salts to the portal vein throughout the intestinal wall. Because the stomach has a pH between 1 and 3 and its content is mixed with an alkaline pH between

479

LACTOBACILLUS BILE SALT HYDROLASE

7 and 9 of pancreatic and bile secretions, a variable pH occurs in the upper part of the GIT. However, a pH of 6.5 to 7.1 is expected because of the digestive buffer system in the intestinal lumen (29). All three strains of L. acidophilus deconjugated both sodium taurocholate and sodium glycocholate at pH 6.5 even though optimum activity was observed at lower levels of pH. The two strains of human origin (L1 and O16) had lower deconjugation rates than strain ATCC 43121 of porcine origin. Studies by De Rodas et al. ( 9 ) and Gilliland et al. ( 1 3 ) have shown that strain ATCC 43121 of porcine origin reduces the serum cholesterol in pigs. De Rodas et al. ( 9 ) observed a significant relationship between levels of bile salts and cholesterol in the blood serum of pigs that were fed L. acidophilus ATCC 43121. Those researchers concluded that the hypocholesterolemic effect of consuming L. acidophilus was at least in part due to deconjugation of bile salts in the small intestine that resulted in less reabsorption of bile salts via the enterohepatic circulation. This strain also assimilated cholesterol and was bile tolerant ( 1 3 ) as were the two strains of human origin ( 3 ) . Cholesterol assimilation is not correlated with bile salt deconjugation (39); however, high bile salt deconjugation at neutral pH is another important characteristic to consider when selecting strains for their potential use to help reduce serum cholesterol. Strains L1 and O16 had three times less deconjugation activity than did strain ATCC 43121 when grown at pH 6.5, and their intracellular BSH activity was 14 times less than that of strain ATCC 43121. The molecular mass of the BSH from all three strains of L. acidophilus was approximately 126 kDa, which is smaller than the molecular mass of 250 kDa that has been reported for BSH from other organisms such as B. fragilis ssp. fragilis (37), B. longum (19), and C. perfringens (17), but it is in the range of that reported for Lactobacillus sp. strain 100-100 ( 2 7 ) and Bacteroides vulgatus (25). We found no reports in the literature on differences in the optimum pH for deconjugation of sodium taurocholate or sodium glycocholate by BSH from different strains of the same bacterial species. In this study, the optimum pH for BSH of both L. acidophilus strains ATCC 43121 and L1 was in the range of 4 to 5. The optimum pH for BSH of L. acidophilus strain O16 was between 5.5 and 6.5. Although there were apparent differences among the three strains, the values were similar to the optimal values of BSH reported by others. Lundeen and Savage (27), who studied BSH from Lactobacillus spp., found an optimum pH between 3.8 and 4.5 when using taurocho-

late as a substrate. Kawamoto et al. ( 2 5 ) and Gilliland and Speck ( 1 5 ) showed that the optimum pH for taurocholate deconjugation by B. vulgatus was in the range of 5.6 to 6.4, and the optimum pH for deconjugation by L. acidophilus NCFM was 6. The pH optimum in B. longum ( 1 9 ) and C. perfringens ( 1 7 ) was in the range of 5.5 to 6.5. Stellwag and Hylemon ( 3 7 ) reported an optimum pH of 4.2 for BSH of B. fragilis ssp. fragilis. During the purification of BSH, sodium glycocholate was used as the principal substrate for measuring enzyme activity because of the high affinity of enzyme from each strain toward this substrate. Lundeen and Savage ( 2 7 ) and Grill et al. ( 1 9 ) did not find any significant differences in BSH deconjugating sodium taurocholate or sodium glycocholate by Lactobacillus sp. 100-100 or B. longum BB536, respectively. However, the BSH from C. perfringens showed higher substrate specificity toward glycine conjugates than toward taurine conjugates (17). In this study, bile salt deconjugation by all three strains of L. acidophilus was pH dependent. However, the high content of bile salt hydrolase in strain ATCC 43121 (of porcine origin) was enough to hydrolyze almost all conjugated bile salts in medium during growth at pH 6.5. Lactobacillus acidophilus L1 had a higher deconjugation rate on sodium glycocholate than on sodium taurocholate as did strain O16. Therefore, these two strains might be important for potential hypocholesterolemic activity based on their capacity to hydrolyze sodium glycocholate because it is the major conjugated bile salt in humans. ACKNOWLEDGMENTS This research was partially supported by the Oklahoma Agricultural Experiment Station (H-2293). The senior author was supported on a CONACyTFulbright Fellowship. We thank E. C. Nelson and F. Leach of the Department of Biochemistry and Molecular Biology (Oklahoma State University, Stillwater) for their critical review of the manuscript. We also thank C. L. Goad of the Department of Statistics for assistance with the statistical analyses. REFERENCES 1 Alm, L. 1983. The effect of Lactobacillus acidophilus administration upon the survival of Salmonella in randomly selected humans carriers. Prog. Food Nutr. Sci. 7:13–17. 2 Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72:248–254. 3 Buck, L. M., and S. E. Gilliland. 1994. Comparisons of freshly isolated strains of Lactobacillus acidophilus of human intestinal origin for ability to assimilate cholesterol during growth. J. Dairy Sci. 77:2925–2933. Journal of Dairy Science Vol. 82, No. 3, 1999

480

CORZO AND GILLILAND

4 Center, S. A. 1993. Serum bile acid in companion animal medicine. Pages 625–657 in: Gastroenterology: The 1990s. S. L. Michael ed., Saunders Co., Ltd., Philadelphia, PA. 5 Chickai, T., H. Nakao, and K. Uchida. 1987. Deconjugation of bile acids by human intestinal bacteria implanted in germ-free rats. Lipids 22:669–671. 6 Connor, W. E., D. T. Witiak, D. B. Stone, and M. L. Armstrong. 1969. Cholesterol balance and fecal neutral steroid and bile acid excretion in normal man fed dietary fats of different fatty acid composition. J. Clin. Invest. 48:1363–1375. 7 Corzo, G. A. and S. E. Gilliland. 1999. Measurement of bile salt hydrolase activity from Lactobacillus acidophilus based on disappearance of conjugated bile salts. J. Dairy Sci. 82:466–471. 8 Dashkevics, M. P., and S. D. Feighner. 1989. Development of a differential medium for bile salt hydrolase-active Lactobacillus spp. Appl. Environ. Microbiol. 55:11–16. 9 De Rodas, B. Z., S. E. Gilliland, and C. V. Maxwell. 1996. Hypocholesterolemic action of Lactobacillus acidophilus ATCC 43121 and calcium in swine with hypercholesterolemia induced by diet. J. Dairy Sci. 79:2121–2128. 10 De Smet, I., L. Van Hoorde, M. De Saeyer, M. Vande Woestyne, and W. Verstraete. 1994. In vitro study of bile salt hydrolase (BSH) activity of BSH isogenic Lactobacillus plantarum 80 strains and estimation of cholesterol lowering through enhanced BSH activity. Microb. Ecol. Health Dis. 7:315–329. 11 Floch, M. H., H. J. Binder, B. Filburn, and W. Gershengoren. 1972. The effect of bile acid on intestinal microflora. Am. J. Clin. Nutr. 251:1418–1426. 12 Floch, M. H., W. Gershengoren, S. Diamond, and T. Hersh. 1970. Cholic acid inhibition of intestinal bacteria. Am. J. Clin. Nutr. 23:8–10. 13 Gilliland, S. E., C. R. Nelson, and C. Maxwell. 1985. Assimilation of cholesterol by Lactobacillus acidophilus. Appl. Environ. Microbiol. 49:377–381. 14 Gilliland, S. E., and C. N. Rich. 1990. Stability during frozen and subsequent refrigerated storage of Lactobacillus acidophilus grown at different pH. J. Dairy Sci. 73:1187–1192. 15 Gilliland, S. E., and M. L. Speck. 1977. Deconjugation of bile acids by intestinal lactobacilli. Appl. Environ. Microbiol. 33: 15–18. 16 Goldin, B. R., and S. L. Gorbach. 1977. Alterations in fecal microflora enzymes related to diet, age, lactobacillus supplements, and dimethylhydrazine. Cancer 40:2421–2426. 17 Gopal-Srivastava, R., and P. B. Hylemon. 1988. Purification and characterization of bile salt hydrolase from Clostridium perfringens. J. Lipid Res. 29:1079–1085. 18 Gorbach, S. L. 1969. Bacteria, bile and the small bowel. Gut 10: 963–969. 19 Grill, J. P., F. Schneider, J. Crociani, and J. Ballongue. 1995. Purification and characterization of conjugated bile salt hydrolase from Bifidobacterium longum BB536. Appl. Environ. Microbiol. 61:2577–2582. 20 Grundy, S. M., and A. L. Metzeger. 1972. Physiological method for estimation of hepatic secretion of biliary lipid in man. Gastroenterology 62:1200–1217. 21 Haslewood, G.A.D. 1978. The biological importance of bile salts. Elsevier/North-Holland Biomedical Press, Amsterdam, The Netherlands. 22 Hofmann, A. F., and B. Borgstrom. 1964. The intraluminal phase of fat digestion in man. The lipid content of the micellar and oil phases of intestinal content obtained during fat digestion and absorption. J. Clin. Invest. 43:247–257.

Journal of Dairy Science Vol. 82, No. 3, 1999

23 Hofmann, A. F. 1977. The enterohepatic circulation of bile acids in man. Pages 3–24 in Clinics in Gastroenterology. G. Paumgartner, ed. Saunders Co. Ltd., Philadelphia, PA. 24 Holt, R. 1972. The roles of bile acids during the process of normal fat and cholesterol absorption. Arch. Itern. Med. 130: 574–583. 25 Kawamoto, K., I. Horibe, and K. Uchida. 1989. Purification and characterization of a new hydrolase for conjugated bile acids, chenodeoxycholyltaurine hydrolase, from Bacteroides vulgatus. J. Biochem. 106:1049–1053. 26 Klaver, F.A.M., and R. Van der Meer. 1993. The assumed assimilation of cholesterol by Lactobacilli and Bifidobacterium bifidum is due to their bile salt-deconjugating activity. Appl. Environ. Microbiol. 59:1120–1124. 27 Lundeen, S., and D. C. Savage. 1990. Characterization and purification of bile salt hydrolase from Lactobacillus sp. strain 100-100. J. Bacteriol. 172:4171–4177. 28 Mallory, A., F. Kern, J. Smith, and D. Savage. 1973. Patterns of bile acids and microflora in the human small intestine. Gastroenterology 64:26–33. 29 Mitsuoka, T. 1978. Intestinal bacteria and health. Pages 81–82 in Harcourt Brace Jovanovich Japan, Inc., Tokyo, Japan. 30 Read, A. E., C. F. McCarthy, K. W. Heaton, and J. Laidlow. 1966. Lactobacillus acidophilus (ENPAC) in treatment of hepatic encephalopathy. Brit. Med J. 1:1267–1269. 31 Reynier, M. O., J. C. Montet, A. Gerolami, C. Marteau, C. Crotte, A. M. Montet, and S. Mathieu. 1981. Comparative effects of cholic, chenodeoxycholic, and ursodeoxycholic acids on micellar solubilization and intestinal absorption of cholesterol. J Lipid Res. 22:467–473. 32 Ruben, A. T., and G. P. Van Berge-Henegouwen. 1982. A simple reverse-phase high pressure liquid chromatographic determination of conjugated bile acids in serum and bile using a novel radial compression separation system. Clin. Chim. Acta, 119: 41–50. 33 Sandine, W. E. 1979. Roles of Lactobacillus in the intestinal tract. J. Food Prot. 42:259–262. 34 SAS® Procedures Guide for Personal Computers version 6 ed. 1985. SAS Inst. Inc., Cary, N.C. 35 Schiff, E. R., N. C. Small, and J. M. Dietschy. 1972. Characterization of the kinetics of the passive and active transport mechanisms for bile acid absorption in the small intestine and colon of the rat. J. Clin. Invest. 51:1351–1362. 36 Simmonds, W. J., A. F. Hofmann, and E. Theodor. 1967. Absorption of cholesterol from a micellar solution: Intestinal perfusion studies in man. J. Clin. Invest. 46:874–890. 37 Stellwag, E. J., and P. B. Hylemon. 1976. Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis. Biochim. Biophys. Acta. 452:165–176. 38 Stellwagen, E. 1990. Gel filtration. Methods Enzymol. 182: 317–328. 39 Walker, D. K., and S. E. Gilliland. 1993. Relationships among bile tolerance, bile salt deconjugation and assimilation of cholesterol by Lactobacillus acidophilus. J. Dairy Sci. 76: 956–961. 40 Zhu, X. X., and G. R. Brown. 1990. A simple method for the analysis of bile acids. Anal. Lett. 23:2011–2018. 41 Zwietering, M. H., F. M. Jongenburger, F. M. Rombouts, and K. Van’T Riet. 1990. Modeling of the bacterial growth curve. Appl. Environ. Microbiol. 56:1875–1881.