Annals of Microbiology, 59 (3) 469-475 (2009)

A BSH volumetric activity dependent method for determination of coprecipitated cholesterol and the assimilation/coprecipitation proportion of cholesterol removal by Lactobacillus plantarum Guijie LI1, Xiaomin HANG2, Jing TAN1, Min ZHANG2, Xianglong LIU1, Daotang LI1, Hong YANG1* 1MOE Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, No. 800 Dongchuan Rd., Shanghai, 200240, P. R. China 2Institute of Bio-medicine, Shanghai Jiao Da Onlly Company Ltd., Shanghai, 200233, P. R. China

Received 20 February 2009 / Accepted 18 August 2009

Abstract - The in vitro removal of cholesterol by probiotics has been categorized into two phases: assimilation by bacterial cells, and coprecipitation with deconjugated bile salts. Bile salt hydrolase (BSH) produced by probiotics catalyzes the deconjugation reaction of bile salts. A novel method that depends on the BSH volumetric activity (μg min-1 ml-1 bacteria culture) was developed to evaluate the cholesterol coprecipitation in MRS culture supplemented with synthetic human bile and cholesterol. Six probiotic Lactobacillus plantarum strains from the healthy youth intestinal tract were screened. The amount of cholesterol coprecipitation that occurred in each L. plantarum culture was then determined according to the method developed here and verified by a modified redissolution method. Next, the assimilation/coprecipitation proportion of cholesterol removal for each strain was deduced. The results revealed that after 12 h of incubation, all six strains of L. plantarum removed a larger amount of cholesterol by assimilation (63.45-81.62%) than by coprecipitation (18.3836.55%). Finally, we investigated the effects of synthetic human bile on BSH specific activity (μg min-1 mg-1 protein) and found that the BSH specific activity of all strains showed a rapid, limited and reversible decrease in response to synthetic human bile stress. We also found that variations in the BSH specific activity were related to the growth phases and that the maximum emerged after approximately 8 h (middle exponential phase) of growth. Key words: BSH volumetric activity, Lactobacillus plantarum, cholesterol coprecipitation, BSH specific activity.

INTRODUCTION Probiotics are defined as living microorganisms that, when ingested in sufficient numbers, favourably influence the health of the host (Fuller, 1992). The reduction of serum cholesterol in humans is an important effect of probiotics (Mann, 1977; Ashar and Prajapati, 2000; Pawan and Bhatia, 2007) because the risk of cardiovascular disease is reduced as the hyper-cholesterol level decreases (Manson et al., 1992; Law et al., 1994). Several studies have shown that bacteria that lead to marked reductions in cholesterol in vitro, also have a significant effect on serum cholesterol reduction in vivo (Gilliland et al., 1985; Anderson and Gilliland, 1999). Different hypotheses have been advanced to explain the possible mechanisms of cholesterol removal by probiotics. These have generally suggested that the effects occur via assimilation by bacterial cells and deconjugation of bile salts (Suvarna and Boby, 2005). During assimilation, cholesterol that is incorporated * Corresponding Author. Phone: +86-21-34205343; E-mail: [email protected]

into or attached to probiotic cells becomes unavailable for intestinal absorption into the blood (Pereira and Gibson, 2002). During bile salt deconjugation, the bile salt hydrolase (BSH) activity of the probiotics results in the peptide bonds of glycine- or taurineconjugated bile salts being broken down, thereby releasing the amino acid residues and deconjugated bile salts (bile acids). In vivo, bile acids are more readily excreted in the feces than their conjugated counterparts (De Smet et al., 1995; Hofmann, 1976), which in turn increases the synthesis of bile salts from serum cholesterol to maintain the physiologic amount of bile (Reynier et al., 1981). In vitro, deconjugated bile salts separate cholesterol from the medium by coprecipitation below pH 5.4 (Tahri et al., 1996). The in vivo mechanism of cholesterol removal is essentially consistent with the in vitro coprecipitation, as they are both based on the BSH directed deconjugation of bile salts. Thus, the variations in BSH activity that occur during in vivo colonization and in vitro cultivation would necessarily affect the reduction of cholesterol. To more precisely evaluate the in vitro removal of cholesterol, attempts have been made to differentiate assimilation from coprecipitation. The amount of assimilated cholesterol was com-

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monly determined by eliminating the influence of coprecipitation by controlling the growth media at pH 6.0 (Klaver and Van der Meer, 1993; Noh et al., 1997). The amount of coprecipitated cholesterol was then routinely determined by redissolving the precipitate at pH 7.0 (Tahri et al., 1996). However, each of these approaches has some potential shortcomings. For example, when the pH of a culture is controlled at 6.0, the bacterial behaviour, including cholesterol assimilation, might deviate from the behaviour that occurs in free growing conditions. In the redissolution method, some of the assimilated cholesterol may be lost during the washing process. Another common problem in such tests has been that the bile used for deconjugation was either a single bile salt (taurocholate or glycocholate) or oxgall, whereas in vivo removal of cholesterol by probiotic organisms occurs in the environment of human bile. To avoid these limitations, a novel method to determine the amount of cholesterol that coprecipitates with deconjugated bile salts was developed in this study. To this regard, six strains of Lactobacillus plantarum were screened from the intestinal tract of healthy young men for their ability to remove cholesterol. Lactobacillus plantarum is a lactic acid bacterium that is widely used as probiotic; however, it has still not been studied as intensively as the more popular probiotic species Lactobacillus acidophilus or Lactobacillus casei, especially with respect to the mechanism of cholesterol removal. In this study, the variation in the BSH specific activity of each strain in the presence of synthetic human bile was also investigated.

MATERIALS AND METHODS Bacteria and culture media. Six strains of Lactobacillus plantarum with cholesterol-removal potential were used in this study. Lp501 (DQ235649), Lp529 (DQ235650), LS12 (DQ235651), LS31 (AY851751) and Re1 (DQ239698) were screened from the intestinal tract of healthy youth, and a commercial strain LpOnlly (AY590777) was supplied by Shanghai Jiao Da Onlly Co., Ltd. The stock bacterial culture was stored in 40% (v/v) glycerol at -20 °C. Prior to use, all strains were activated three times in MRS (De Mann et al., 1960) broth. Each incubation was performed using 1% (v/v) inoculum at 37 °C for 16 h. The synthetic human bile stock at a concentration of 120 μmol ml-1 was made of sodium glycocholic and taurocholic acid (Sigma Chemical Co., St. Louis, US) in the molar ratio of 3:1. Enriched media used in this study included: a. MRSt broth: MRS with 1% (w/v) sodium thioglycolate (Oxoid Ltd, Basingstoke, UK); b. MRSb broth: MRSt supplemented by 6 μmol ml-1 synthetic human bile, which is considered the average concentration of bile salt prevailing in the intestine (Brashears et al., 1998; De Boever et al., 2000); and c. MRSbc broth: MRSb added with 120 μg ml-1 polyoxyethanyl-cholesteryl sebacate (Sigma). BSH assay. As described by Tanaka et al. (1999), cell extracts were prepared and BSH was determined by a two-step procedure in which amino acid liberated from conjugated bile salt was measured. Protein content was assayed by Lowry method (Lowry et al., 1951). BSH volumetric activity was expressed as the liberated amount (μmol) of amino acid per min per ml of culture. BSH specific activity was defined as the liberated amount (μmol) of amino acid per min per mg protein. Cholesterol assay. The amount of dissolved cholesterol was determined by the o-phthaladehyde method (Rudel and Morris, 1973).

G. LI et al.

BSH volumetric activity and cholesterol removal. Bacterial cultures were 1% (v/v) inoculated into MRSbc broth and then incubated anaerobically (GENbox Jar 2.5 L; GENbox anaer bioMérieux, France) at 37 °C for 24 h. From inoculation (0 h), viable cell counts by the agar-plate method and the BSH volumetric activity of each culture were determined at 2 h intervals. After 12 h of incubation, when the six-strain cultures had first entered the stationary growth phase, the samples were centrifuged (12000 x g, 10 min and 4 °C) and the supernatant and pellets were then collected for further analysis of cholesterol. A decreased cholesterol level in the supernatant fluid when compared to the uninoculated MRSbc broth was the amount that was removed by bacteria. The collected pellets were assayed in the next experiment for the redissolution of precipitated cholesterol. Modified redissolution of precipitated cholesterol. The coprecipitated cholesterol was redissolved and assayed using a modified version of the method described by Tahri et al. (1996). Briefly, the pellet collected in the previous procedure was immersed in 3 ml of 95% (v/v) ethanol and shaken gently for 20 s. This treatment dehydrated the peptidoglycan cell wall of L. plantarum, which caused the pores in the wall to shrink, thereby preventing the assimilated cholesterol that was embedded or bound to the membrane from escaping during the following washes. The resuspension was then immediately centrifuged (12,000 g, 30 s; 4 °C), after which the pellet was sequentially washed with 3 ml of 0.1 mol ml -1 phosphate buffer (pH 6.5) three times, with the suspension being centrifuged (12 000 x g, 10 min; 4 °C) after each wash. All supernatants from the four centrifugations were then combined and assayed for cholesterol. The washed pellet was finally dried (103 °C) to a constant weight to determine the dry mass. Cholesterol coprecipitation based on cell-free BSH activity. The stationary-phase cell extract of Lp529 was prepared by sonication, after which the BSH volumetric activity was determined. Different volumes of the cell extract of Lp529 (0-1.2 ml by 75 μl interval) were then added to 0.1 ml of synthetic human bile stock (or distilled water for the control) and 0.7 ml of coprecipitation reagent. Every 70 ml of coprecipitation reagent was composed of 30 ml of 0.5 mmol ml-1 citrate buffer (pH 4.7), 20 ml of 0.1 mmol ml-1 thioglycol (Oxoid), 18 ml of 55 μmol ml-1 EDTA and 2 ml of 12 mg ml-1 polyoxyethanyl-cholesteryl sebacate solution. Phosphate buffer was then used to adjust each mixture to 2.0 ml. The final concentrations of bile salt and cholesterol were 6 μmol ml-1 and 120 μg ml-1, respectively, and the final pH was 5.0, which simultaneously met the pH necessary for coprecipitation (< 5.4; Tahri et al., 1996), the optimal pH of BSH (5-6; Begley et al., 2006) and the protoplasmic pH of stationary-phase cells of L. plantarum (5.0-5.3; McDonald et al., 1990). Incubation of the mixtures was conducted anaerobically at 37 °C for 8 h. The cholesterol-bile acid coprecipitate was then removed by centrifugation (20000 x g, 10 min), after which the supernatant was subjected to a cholesterol assay. A decreased level of cholesterol in the supernatant as compared to the control was taken to indicate coprecipitation in response to the activity of the cell-free BSH enzyme. The coprecipitation curve was plotted as coprecipitated cholesterol vs. BSH volumetric activity. BSH specific activity in response to bile salt stress. This experiment investigated variations in the BSH specific activity in the presence of synthetic human bile (Step 1) and after the removal of bile salts (Step 2). In the preparation procedure, bacteria were cultivated in MRSt broth until reaching the sta-

Ann. Microbiol., 59 (3), 469-475 (2009)

tionary growth phase, after which the culture was divided into three aliquots, which were denoted as Fractions 1, 2 and 3. The spent broth of Fraction 1 was saved. In Step 1, Fraction 2 was added to synthetic human bile stock to produce a MRSb culture. After anaerobic incubation for 0, 0.5, 1, 3, 5, 10, 15 and 20 min at 37 °C, the samples were immediately quenched at -20 °C and then assayed for BSH specific activity. In Step 2, the residual portion of Fraction 2 was equally divided into two portions, denoted Portion 1 and 2. Cells from Portion 1 were then transferred into the spent broth of Fraction 1 that was collected during the preparation procedure so that the stationary MRSt culture was restored. Portion 2 was added to cholesterol to produce an MRSbc culture. Incubation and the BSH assay of the two cultures were then conducted under the same conditions as those described in Step 1. Fraction 3 served as an MRSt control whose BSH during the entire process was defined as 100% of the specific activity. The relative ratio of BSH specific activity (percentage of samples/control) was plotted against the incubation time. BSH specific activity during growth in MRSb media. Cultures of L. plantarum were 1% (v/v) inoculated into MRSb broth and incubated anaerobically at 37 °C for 24 h. The BSH specific activity and viable cell count were determined at 2 h intervals. Statistical analysis. Experiments were performed in triplicate and carried on three occasions. Data analyses were carried out with SPSS inc. software (ver.10.0). One-way ANOVA was used to determine whether significant (P < 0.05) variation occurred among means.

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RESULTS AND DISCUSSION BSH volumetric activity dependent determination of coprecipitated cholesterol Generally, in MRSbc cultures that contain an excess of bile and cholesterol, the amount of cholesterol that coprecipitates at any moment depends on the output of deconjugated bile salt, which is ultimately determined by the activity of bacterial BSH. In this study, the BSH volumetric activity and growth conditions of the L. plantarum MRSbc culture were investigated. As shown in Fig. 1, the log BSH volumetric activity produced a sigmoid curve as a function of incubation time. After 12 h of incubation, all of the strains entered the stationary-phase of growth and the rapid increase in BSH volumetric activity stopped. In addition, The orocecal transit time in humans is not likely to be longer than 12 h; therefore, the first 12 h of incubation were chosen for the mathematical evaluation of the BSH volumetric activity and cholesterol removal. The BSH volumetric activity was defined as BVA (t) (t for time, hour). Log BVA (t) during the first 12 h of incubation formed a curve with two segments: 0-4 h, which was the latency period in which BVA (t) retained less than 1% of the plateau value, and 4-12 h, which was the exponential phase in which BVA (t) proliferated by approximately 80 times until eventually reaching a plateau. Linear regression of the exponential phase (forced through point BVA (12) with the regression slope, k) gave the following regression function: ,: log BVA (t) = k t + [log BVA (12) - 12k]

(t = 4 -12 h)

Therefore, at any time, t, within 0-12 h: ϩ: BVA (t)

FIG. 1 - BSH volumetric activity during 24 h of incubation in MRSbc culture. The log CFU ml-1 (ƻ/●), log BSH volumetric activity (r/p) and linear regression for the exponential phase (4-12 h) of the log BSH volumetric activity (---/—) for Lp529/LpOnlly, respectively.

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According to the definition of BSH volumetric activity, the amount (μmol ml-1) of bile salt deconjugation was expressed as ∫BVA (t) dt. Because BVA (t) during the first 4 h was close to zero, deconjugation at that period can be omitted. Therefore, BSH only had a significant effect in the following eight hours, which was described by the following equation: 12

Ϫ:

°

0

12

BVA (t) d t ≈

°

4

12

BVA (t) d t =

°

4

BVA (12) × 10 – k (12 – t) d t

Assuming that the cell-free coprecipitation system, which has a constant equivalent BSH volumetric activity (noted eq.-BVA), exerted the same deconjugation effect as the growing culture did within the eight hours: 12

ϫ: eq.-BVA × t =

°

4

BVA (12) × 10 – k (12 – t) d t

(t = 8 h)

then Eq.-BVA can be calculated by BVA (12) and k Ϭ:

eq.-BVA 

BVA (12) s (1 10 8 s ln10 s k

8k

)

In addition, investigation of the cell-free coprecipitation system revealed that the coprecipitated cholesterol and eq.-BVA could be fit to a Hill model (Fig. 2): ϭ:

´ ¥ 14.940 Coprecipitated cholesterol  12.983 ¦¦

1µµ § eq. - BVA ¶

0.348

Consequently, simultaneous solution of equation Ϭ and ϭ enables the amount of cholesterol that precipitated from the MRSbc culture of L. plantarum after 12 h of incubation to be determined according to BVA (12) and the slope, k, for each strain (Table 1). Modified redissolution for verification of coprecipitated cholesterol The value of coprecipitated cholesterol that was measured using the modified redissolution method agreed well with the value determined using the BSH volumetric activity dependent method. Specifically, the percentage difference between the two groups of data was within ± 7.21% (Table 1), and no significant discrepancy (P > 0.5) was observed. Furthermore, the modified redissolution method showed better repeatability than

FIG. 2 - The amount of cholesterol that coprecipitated with deconjugated bile acid in the cell-free system. The system initially contained 6 μmol ml-1 synthetic human bile, 120 μg ml-1 cholesterol, and a series of levels of equivalent BSH volumetric activity (eq.-BVA). Incubation was conducted at pH 5.0 for 8 h. The data were sigmoid-fitted by the Hill model.

the original method developed by Tahri et al. (1996), as indicated by coefficients of variation of 0.27-1.70% being obtained for the modified method and 4.17-23.1% being obtained for the original method.

TABLE 1 - Calculation of the proportion of cholesterol removed by assimilation and that of the cholesterol removed by coprecipitation for six Lactobacillus plantarum strains that were cultivated in MRSbc broth for 12 h Strains

Lp501

Lp529

LpOnlly

LS12

LS31

Re1

Slope ka

0.292

0.237

0.242

0.219

0.239

0.246

BVA (12)b

63.88

45.65

43.54

46.21

42.08

12.91

eq.-BVA

11.82

10. 32

9.65

11.25

9.44

2.82

Chol. precipitatedc

20.64

17.18

16.01

19.14

15.67

7.82

Proportion (%)

33.51

36.55

19.22

34.43

29.20

18.38

Chol. redissolved

20.90 1.26 61.60

16.51 -3.90 47.00

16.87 5.37 83.28

20.21 5.59

14.54 -7.21 53.66

40.96 66.49

29.82 63.45

67.27 80.78

7.49 -4.22 42.54 34.72 81.62

Differenced (%) Chol. total removal Chol. assimilated Proportion (%) a b c d

55.59 36.45 65.57

37.99 70.80

Linear regression slope of the log BSH volumetric activity during the exponential growth phase of each strain; BSH volumetric activity in μmol min-1 ml-1; The amount of cholesterol (chol. for abbreviation) in μg cholesterol per milliliter of culture; Difference = (Chol. Redissolved – Chol. Precipitated) / Chol. Precipitated × 100%.

Ann. Microbiol., 59 (3), 469-475 (2009)

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FIG. 3 - The BSH specific activity during 24 h of cultivation in MRSb. The BSH specific activity (v/t) and log CFU ml-1 (ƻ/●) of Lp529/LpOnlly, respectively.

The assimilation/coprecipitation proportion of cholesterol removal The assimilated cholesterol was calculated as the total cholesterol removal minus the amount of coprecipitated cholesterol determined based on the BSH volumetric activity dependent method. As shown in Table 1, all six L. plantarum strains had a larger proportion of assimilated cholesterol (63.45-81.62%) than coprecipitated cholesterol (18.38-36.55%). As a result, it can be expected that when these six strains are ingested, the cholesterol reduction by bacterial assimilation may be far greater than the reduction that occurs as a result of bile salt deconjugation. This is because the capacity of in vitro coprecipitation represents the potential for in vivo cholesterol reduction, which are both driven by BSH dependent bile salt deconjugation. The ratio of the cholesterol that is assimilated to the cholesterol that is co-precipitated is a reflection of inherent bacterial behaviors. Nevertheless, this ratio is also influenced by the culture conditions. For example, Tahri et al. (1996) and Grill et al. (2000) reported that strains of Bifidobacteria spp. and Lactobacillus amylovorus cultured in the presence of different bile salts, including taurocholate, glycocholate and oxgall, had different proportions of assimilated and coprecipitated cholesterol. These differences may be explained by the fact that BSHs have different substrate preferences, and tend to prefer glycine-conjugated substrates to taurine-conjugated substrates (Begley et al., 2006). In another example, Parvez et al. (2006) investigated the influence of incubation time on the assimilation and coprecipitation proportions of Bifidobacterium bifidum and found that the assimilated cholesterol and coprecipitated cholesterol were partially interconvertible when bacteria were cultivated for different lengths of time. Thus, bile salts and incubation time are two critical conditions for that must be considered when analyzing the proportion of cholesterol removed by assimilation and coprecipi-

tation. In this study, incubation in the presence of 6 μmol ml-1 of synthetic human bile for 12 h was conducted because it partially simulated the intestinal conditions of humans after the ingestion of food. Variation in BSH specific activity in the presence of synthetic human bile We evaluated the effects of synthetic human bile on bacterial BSH specific activity under two conditions, short-term stress and long-term adaptation. In the short-term stress experiment, 6 μmol ml -1 of synthetic human bile immediately repressed the BSH specific activity of each of the six strains of L. plantarum evaluated in this study when the cell pellets were resuspended in MRSb broth (Fig. 3). This repression of BSH specific activity may be explained by the cytotoxicity of bile salts. For example, cholic acid, the product of BSH deconjugation, is more deleterious to microbes than the conjugated substrates (Taranto et al., 1999). The repression was limited and rapid, as indicated by the BSH specific activity decreasing by 14-21% within 5 min and then being maintained at an approximately stable level. Furthermore, the repression was shown to be reversible after the bile was removed either by replacing the broth or by the addition of cholesterol (co-precipitating), as indicated by 89-95% of the BSH specific activity being recovered after the removal of bile (Fig. 3). Evaluation of the long-term effects of synthetic human bile on the culture revealed a common tendency in which the BSH specific activity was represented as a peak flanked plateau, followed by a zone of declivity (Fig. 4). Specifically, the BSH specific activity increased in the middle of the exponential growth phase, reached its peak value after approximately 8 h of incubation, and then decreased by 5-9% and maintained a plateau from 12-20 h. When probiotics are ingested and enter the intestinal tract, evaluation of changes in their physiological characteristics to

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FIG. 4 - The stress effect of synthetic human bile on the BSH specific activity of stationary cells. Variations in the relative ratio of the BSH specific activity of Lp529/LpOnlly respectively, when cultured in: MRSb broth (□/Ƶ) during 0-20 min, followed by replacement of the MRSt broth (r/p) or in MRSbc broth (after the addition of cholesterol) (s/q) during 20-40 min.

adapt the biliary environment will be very informative. However, direct in vivo investigation of characteristics such as BSH specific activity or serum cholesterol-reducing properties is not widely applicable. Because in vitro coprecipitation is essentially consistent with the in vivo BSH directed cholesterol-reducing mechanism, our in vitro results may reflect the actual in vivo variation in BSH specific activity when probiotics are ingested as non-protected naked cells. Acknowledgements This work was supported by the National Hi-Tech Research and Development Program of China (“863” Program, grant No. 2007AA10Z355), National Basic Research Program of China (“973” Program, grant No.2007CB714301), Science and Technology Commission of Shanghai Municipality (grant No. 07QB14021), and Medical-Engineering Joint Research Foundation of Shanghai Jiao Tong University (grant No. YG2009MS11).

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