Microencapsulated bile salt hydrolase producing Lactobacillus reuteri for oral targeted delivery in the gastrointestinal tract
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- Martoni, C., Bhathena, J., Urbanska, A.M. et al. Appl Microbiol Biotechnol (2008) 81: 225. doi:10.1007/s00253-008-1642-8
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This is the first study of its kind to screen probiotic lactic acid bacteria for the purpose of microencapsulating a highly bile salt hydrolase (BSH)-active strain. A Lactobacillus reuteri strain and a Bifidobacterium longum strain were isolated as the highest BSH producers among the candidates. Microcapsules were prepared with a diameter of 619 ± 31 μm and a cell load of 5 × 109 cfu/ml. Post de Man, Rogosa, and Sharpe broth-acid challenge, L. reuteri microcapsules metabolized glyco- and tauro-conjugated bile salts at rates of 10.16 ± 0.46 and 1.85 ± 0.33 μmol/g microcapsule per hour, respectively, over the first 2 h. Microencapsulated B. longum had minimal BSH activity and were significantly (P < 0.05) more susceptible to acid challenge. Further testing of L. reuteri microcapsules in a simulated human gastrointestinal (GI) model showed an improved rate, with 49.4 ± 6.21% of glyco-conjugates depleted after 60 min and complete deconjugation after 4 h. Microcapsules protected the encased cells in the simulated stomach maintaining L. reuteri viability above 109, 108, and 106 cfu/ml after 2 h at pH 3.0, 2.5, and 2.0, respectively. Results show excellent potential for this highly BSH-active microencapsulation system in vitro, highlighted by improved viability and substrate utilization in simulated GI transit.
KeywordsMicrocapsuleProbiotic bacteriaL. reuteriBile salt hydrolaseOral deliveryGastrointestinal tract
Bifidobacteria and lactobacilli are important residents of the gastrointestinal (GI) microflora and have been the subjects of intense and growing interest due to their possible role in the maintenance of health (Naidu et al. 1999; Young 1998). On this basis, there has been much interest in developing food products which include these bacteria as dietary supplements. Lactic acid bacteria (LAB) are frequently used in products for human consumption and can be found as probiotics in foods, cultured milks, and various pharmaceutical preparations (Sanders and in‘t Veld 1999). In recent years, various lactobacilli strains have been proven to lower total or low-density lipoprotein (LDL) cholesterol in humans and animals (Gilliland et al. 1985; Grunewald 1982). This effect can at least partially be attributed to the enzymatic deconjugation of bile salts. Most often, bile salt hydrolase activity has been detected in strains indigenous to the gastrointestinal tract (du Toit et al. 1998)—an environment rich in conjugated and unconjugated bile acids. A prerequisite for any effect of ingested bacteria on either the endogenous flora or the whole human organism is that these bacteria remain viable during gastric transit. However, insufficient survival when passing from the mouth to the intestine has limited the potential of many bacterial strains from clinical or commercial use (Holzapfel et al. 1998; Huang and Adams 2004). Artificial cell microencapsulation, a concept wherein biologically active materials are encapsulated in specialized ultrathin semipermeable polymer membranes, has been proposed as a means to improve cell viability in the GI and act as a vehicle for targeted delivery of highly bile salt hydrolase (BSH)-active microorganisms.
Until now, no study has performed a screening of naturally occurring lactic acid bacteria for the isolation and microencapsulation of highly BSH-active microorganisms. The enclosed material can be retained inside and separated from the harsh external environment while carrying out its function, making microencapsulation particularly useful for biochemical and clinical applications. In the current study, the bioactivity of BSH-active microorganisms will be studied in a simulated human GI model. The computer-controlled bioreactor in series simulates the complex GI environment in the stomach, small intestine, and colon. Specifically, the apparatus mimics the gradual transit of ingested food products and therapeutics through the human digestive tract in which in vivo conditions with regard to pH, temperature, bacteria, enzyme types and activities, volume, agitation, and food particles are closely simulated.
Materials and methods
Microorganisms and culture conditions
In the present study, microorganisms chosen for BSH screening all had established probiotic potential. Lactobacillus plantarum 80 wild type and Lactobacillus reuteri BSH+ were obtained from LabMet, Belgium. Lactobacillus acidophilus ATCC # 8287 was purchased from American Type Culture Collection (ATCC), Manassas, VA, USA. Bifidobacterium bifidum WD0035, Bifidobacterium breve WA0019, Bifidobacterium longum VL0030, and Bifidobacterium infantis WB0311 were kindly provided by Rosell-Lallemand, Montreal, Canada. Bacteria were serially propagated three times in the appropriate medium before experimental use. Lactobacilli were cultivated in de Man, Rogosa, and Sharpe (MRS) broth (Fisher Scientific, Fair Lawn, NJ, USA) followed by serial passages in MRS supplemented with 5 mM glycodeoxycholic acid (GDCA) and 5 mM taurodeoxycholic acid (TDCA; Sigma, St. Louis, MO, USA). A 1% inoculum was used, and incubations were performed at 37°C for 20 h in microaerophilic conditions. Bifidobacteria were passaged anaerobically at 37°C for 20 h in MRS broth supplemented with 0.05% cysteine.
Screening of cultures for BSH activity
BSH activity of the growing cultures was evaluated using the direct plate assay method previously described (du Toit et al. 1998; Pereira and Gibson 2002) with modifications. Briefly, after three passages, a 1% inoculum of bacteria was grown for 20 h in MRS-bile broth containing 5 mM GDCA and 5 mM TDCA. Three autoclaved sterile filter disks (Whatman #3) were incubated along with the growing culture. The isolates were screened for BSH activity by placing the impregnated disks on MRS agar plates supplemented with 5 mM TDCA, 5 mM GDCA, and 0.37 g/l CaCl2. Cysteine (0.05%) was added for screening of bifidobacteria. The plates were incubated anaerobically at 37°C for 72 h. Precipitation zone formation around the disks was measured as an indication of BSH activity. MRS agar plates with incubated sterile filter paper disks were used as controls.
Preparation of microcapsules containing highly BSH-active microorganisms
The bacterial strains exhibiting the highest BSH activity in screening were isolated for microencapsulation and further testing. To prepare microcapsules, grown cultures were centrifuged at 8,000 rpm for 20 min at 15°C. After removal of supernatant, the pelleted cell isolate was washed several times and re-suspended in sterile saline and gently added to a lightly stirred sterile low-viscosity sodium alginate (Sigma) solution with a final concentration of 1.5% (w/v). In a sterile environment, the bacterial alginate suspension was passed through an Inotech Encapsulator IER-20 (Inotech Biosystems International, Rockville, MD, USA). Encapsulation parameters were as follows: nozzle diameter 300 μm, cell loading 4.17 g cell wet weight per 100 ml, vibrational frequency 918 Hz, flow rate 11.3 ml/min, voltage 1.50 kV, and drop height 16.0 cm. After extrusion, the droplets were allowed to gel for 5 min in a gently stirred sterile 0.1 M CaCl2 solution. The Ca-alginate beads were coated with 0.1% (w/v) poly-l-lysine (hydrobromide, MW 21,320, Sigma) and 0.1% (w/v) Na-alginate for 10 and 5 min, respectively. Microcapsules were washed in sterile saline between each coat and after the encapsulation procedure. Empty microcapsules were prepared as above without bacterial component. All batches of microcapsules were stored in minimal solution (90% saline, 10% MRS broth) at 4°C.
Real-time BSH assay in flask media
Microcapsules were incubated in MRS media adjusted to stomach and intestinal conditions to determine the effect of microencapsulation on bile-salt-hydrolyzing activity of encased cells. Microcapsules (2.5 g) containing L. reuteri, B. longum, or nothing were incubated in 15 ml of MRS medium pH adjusted to 2.0 by 1.0 M HCl for 60 min at 37°C and 100 rpm agitation. Viability of encased cells was determined before and after MRS-acid challenge. After 60 min, microcapsules were removed from acid MRS and transferred to 15 ml of MRS supplemented with 5 mM GDCA and 5 mM TDCA for 10 h at 37°C and 100 rpm agitation. Supernatant was sampled at intervals of 1 h and processed to determine bile salt deconjugation of GDCA and TDCA over time. Viable cell count and pH conditions were monitored before and after bile MRS exposure. Empty microcapsules were used as controls. The unpaired Student’s t test was used to determine which means differed significantly (P < 0.05).
Real-time BSH assay in simulated human GI model
To obtain more realistic information on the utility of microcapsules in the GI tract, microcapsules containing L. reuteri were examined for bile-salt-hydrolyzing activity in a simulated human GI model. The computer-controlled apparatus, consisting of five bioreactor vessels arranged in series, mimics the gradual transit of ingested food products and therapeutics through the human digestive tract. Microcapsules were first incubated in a stomach environment containing food component of a human western diet acidified to pH 2.0 with 1.0 M HCl. The food component contained starch 3.0 g/l; pectin 2.0 g/l; mucin 4.0 g/l; arabinogalactan 1.0 g/l; xylan 1.0 g/l; yeast extract 3.0 g/l; peptone 1.0 g/l; glucose 0.4 g/l; and cysteine 0.5 g/l. Agitation was provided by stirring at 150 rpm. After 60 min, microcapsules were transferred to the small intestine compartment consisting of the acidified food suspension re-adjusted to pH 6.5 with 0.5 M NaOH. After the pH level of 6.5 was reached, the simulated intestine was supplemented with pancreatic juice containing GDCA and TDCA, pancreatin, and sodium bicarbonate. Final concentrations in the intestine were 5 mM for GDCA and TDCA, 0.18 g/l for pancreatin, and 2.4 g/l for sodium bicarbonate. The total bile salt concentration was approximately 0.5% (w/v). Microcapsules were incubated in the small intestine contents for 10 h at 37°C and 100 rpm agitation. Supernatant was sampled at intervals of 1 h and processed to determine bile salt concentrations. Viable cell count and pH conditions were monitored pre-stomach exposure, pre-small intestine exposure, and post-small intestine exposure. Empty microcapsules were used as controls. Microcapsules were visualized microscopically to determine the effect of simulated GI transit on microcapsule integrity. The unpaired Student’s t test was used to determine which means differed significantly (P < 0.05).
Analysis of bile salt concentrations using high-performance liquid chromatography
Supernatant samples from the in-flask assay and the simulated human GI assay were prepared for high-performance liquid chromatography (HPLC) analysis using a modification of the procedure described by Jones et al. (2004). Briefly, 250 μl supernatant samples were acidified with 2.5 μl of 6 N HCl to arrest enzymatic activity and supplemented with 250 μl of methanol containing 4 mM glycocholic acid (Sigma) as internal standard. The samples were vortexed, shaken at 225 rpm for 10 min. and centrifuged at 1,000×g for 20 min at 10°C. The supernatant was filtered through a 0.22 μm polyvinylidene fluoride 4 mm filter (Millipore) and analyzed directly. Standards for calibration containing 0, 1, 2, 3, 4, 5, and 6 mM GDCA and TDCA were treated as above. A modification of the HPLC procedures described by Scalia (1988) was used to determine bile salt concentrations. Analyses were performed on a reverse-phase C-18 column: LiChrosorb RP-18 (250 × 4.6 mm, 5 μm) from HiChrom (Novato, CA, USA). The solvents used were 0.05 M sodium acetate buffer adjusted to pH 4.3 with o-phosphoric acid and filtered through a 0.22-μm filter (Nalgene; solvent A) and HPLC-grade methanol (solvent B). An isocratic elution was applied consisting of 30% solvent A and 70% solvent B at a flow rate of 1.0 ml/min. An injection loop of 20 μl was used, and sample detection occurred at 210 nm.
Acid tolerance study
Specific tolerance of the microcapsule formulation to gastric conditions was measured using the simulated human GI model. Microcapsules were exposed to gastric juices from the stomach compartment, consisting of the food suspension described above adjusted to pH 2.0, 2.5, and 3.0 at 37°C and 150 rpm. Aliquots were removed from the system after 0, 20, 40, 60, 90, 120, and 240 min. At each time point, the microcapsules were promptly washed and crushed, and the released bacteria were resuspended in physiological saline. Samples were serially diluted and plated on MRS agar. The plates were incubated at 37°C for 72 h.
Selection of bacterial cells for BSH activity
Microencapsulation of BSH candidates
Alginate-poly-l-lysine-alginate (APA) microcapsules containing either L. reuteri or B. longum were prepared with 2.5 g of bacterial cell isolate per 60 g of microcapsules, resulting in a bacterial concentration of 4.17% (w/v) and a cell count of 5 × 109 cfu/ml microcapsule. Microcapsules were spherical in shape with a milky white texture due to the encased bacteria. The average microcapsule diameter for all batches was 619 ± 31 μm.
Bile-salt-hydrolyzing activity of microencapsulated strains in MRS media
Oral delivery performance and bile-salt-hydrolyzing activity of L. reuteri microcapsules in simulated human GI model
Conjugated bile salt depletion over time by L. reuteri microcapsules in MRS-bile media vs. simulated GI model intestinal compartment
Conjugated bile salt depletion over time by L. reuteri microcapsules (%)
Simulated GI model intestine
TDCA depletion rate
GDCA depletion rate
TDCA depletion rate
GDCA depletion rate
2.70 ± 5.26
21.4 ± 2.47
2.57 ± 4.96
49.4 ± 6.21
8.11 ± 4.84
52.3 ± 4.63
13.0 ± 6.57
71.4 ± 5.14
15.2 ± 4.01
86.3 ± 3.79
23.3 ± 3.64
22.0 ± 2.10
32.9 ± 4.49
35.1 ± 5.05
38.4 ± 6.19
Viability of microencapsulated L. reuteri in simulated stomach
One beneficial effect resulting from human consumption of lactic acid bacteria with active bile salt hydrolase, or products containing them, is a reduction in serum cholesterol levels (Pereira and Gibson 2002). LAB are projected to lower cholesterol levels through an interaction with host bile salt metabolism via the enzymatic deconjugation of bile salts (De Smet et al. 1994; De Smet et al. 1998; Klaver and van der 1993; Tahri et al. 1996; Tahri et al. 1997; Usman and Hosono 1999). In so doing, there is an increased demand for cholesterol as a precursor for de novo bile salt synthesis. Earlier studies in our lab have shown that microencapsulated genetically engineered L. plantarum 80 BSH-overproducing cells can be used to effectively deconjugate bile salts in media (Jones et al. 2004) and in simulated human GI transit (Martoni et al. 2007). However, there remain concerns, both publicly and scientifically, about the safety of gene manipulation technology. Despite being nonpathogenic, there is a possibility of repeated large doses of novel microorganisms resulting in the transfer of genes to organisms in the environment (Gruzza et al. 1994). In contrast, this in vitro study screened naturally occurring nongenetically engineered LAB with established probiotic properties for BSH activity. In comparison to earlier studies, GDCA and TDCA were incorporated into the medium to allow for a more accurate representation of the in vivo physiological situation. L. reuteri was found to exhibit the greatest bile-salt-deconjugating activity and grew unhindered in bile salt media as compared to other strains without the protection offered by microencapsulation. This was in agreement with previous studies showing that L. reuteri is acid- and bile-salt-tolerant and has significant cholesterol-lowering properties in vivo (Taranto et al. 2000). Among bifidobacteria, only B. breve failed to exhibit significant BSH activity. Bifidobacteria has been shown to possess higher BSH activity than other probiotics existing in the human intestine (Benno et al. 1989). A comprehensive screening of over 300 strains by Tanaka et al. showed that nearly all bifidobacteria species and strains had bile salt hydrolase activity, in comparison to only selected species of lactobacilli (Tanaka et al. 1999). Based on the zones of precipitation formed, B. longum and L. reuteri were selected for microencapsulation and further study.
The accurate identification and quantitative measurement of various bile acids by high- performance liquid chromatography has been described for determining and measuring the major conjugated bile acids in human bile (Jones et al. 2004). The influences of the entrapment membrane on survival and activity of the BSH candidates exposed to bile salt solutions was tested by incubating the LAB-containing microcapsules in MRS acid and bile solutions. In this study, microencapsulated L. reuteri and B. longum were effective in deconjugating physiologically relevant concentrations of bile acids in vitro. However, there was significantly increased substrate specificity towards glyco-conjugates. The BSH enzyme has a broad substrate range and is able to hydrolyze a variety of human and nonhuman bile salts. Previous studies have shown that glycine-conjugatated bile salts were more effectively deconjugated than taurine-conjugated bile salts by BSHs from L. acidophilus, Clostridium perfringens, Lactobacillus buchneri JCM 1069, and Lactobacillus kefir BCCM 9480 (De Smet et al. 1995; Tanaka et al. 2000). Glyco-conjugates are more toxic than tauro-conjugates, and it has been hypothesized that BSH is expressed as a protective mechanism against toxicity (De Smet et al. 1995; Grill et al. 2000). Recent studies have demonstrated that the BSH enzyme of lactic acid bacteria is located intracellularly (Tanaka et al. 2000). In this experiment, over 90% of APA microcapsules containing either L. reuteri or B. longum maintained structural integrity through both stages of MRS challenge. Therefore, the BSH activity was the direct result of retained cells acting on conjugated bile salts diffusing through the microcapsule membrane pores. Results indicated that microencapsulated L. reuteri had significantly higher bile-salt-deconjugation ability and BSH activity than B. longum. Furthermore, there were significant differences in encapsulated strain survival due to acid and bile stresses. Bifidobacteria, despite their excellent survival mechanisms in bile-associated media, are fastidious, noncompetitive, and sensitive to environmental parameters such as oxygen and low pH (Shah 2000). After 60 min of MRS acid challenge, encased B. longum failed to meet the criterion of a minimum of 106 viable probiotic cells per milliliter needed to gain a therapeutic benefit (Robinson 1989). In contrast, the log of surviving L. reuteri in microcapsules was above 108 cfu/ml microcapsules after MRS acid treatment for 60 min at pH 2.0. The protection offered by microencapsulation was found to be strain specific. When the treated microcapsules were re-incubated in bile-supplemented media, it was seen that recovery of LAB was positive, and in case of L. reuteri, the cells vigorously propagated in the MRS bile media. After the 10-h incubation, microencapsulated L. reuteri showed a significantly greater decrease in pH in comparison to microencapsulated B. longum, indicating improved substrate utilization as well as organic acid production on account of cell metabolism and growth. Based on the performance in the MRS-bile assay, microencapsulated L. reuteri was selected for testing in the simulated human GI model as a precursor to in vivo analysis.
In the normal enterohepatic circulation, a bile salt pool of approximately 3.6 g is secreted into the duodenum six to eight times daily, primarily during meals (Bullock et al. 2001). As a result, daily bile acid secretions average 30 g, sustaining an intestinal bile salt concentration of 5–10 mM (Hofmann 1989). During intestinal transit, approximately 95% of secreted bile salts are actively reabsorbed in the terminal ileum and returned to the liver via portal circulation, resulting in a 5% fecal bile salt loss per cycle (Bullock et al. 2001). Bile salts undergoing intestinal bacteria transformations are less soluble and less efficiently reabsorbed from the intestinal lumen, resulting in an increase in fecal bile salt excretion (De Rodas et al. 1996). In the simulated intestine, stomach-treated microencapsulated L. reuteri deconjugated 49.4 ± 6.21% of glyco-conjugates over the first hour with complete deconjugation seen after 4 h. It could be extrapolated that twice daily therapy would effectively breakdown two complete cycles of glyco-conjugates in the average human. This would result in a significant increase in fecal bile salt loss/cycle, thus stimulating the synthesis of replacement bile acids from cholesterol. Comparatively, microencapsulated L. reuteri showed significantly higher BSH activity than microencapsulated genetically engineered L. plantarum 80 cells towards both glyco-conjugates and tauro-conjugates despite no significant differences in cell viability at each stage of transit. Furthermore, BSH activity of microencapsulated L. reuteri cells was found to have a sixfold preference of glyco-deconjugation over tauro-deconjugation in the simulated intestine, bearing potential significance in cholesterol lowering, since glyco-conjugates greatly outnumber tauro-conjugates in human bile (Hardison 1978).
Since large amounts of deconjugated bile salts may have undesirable effects for the human host, concerns may arise over the safety of administering such a therapy (Nagengast et al. 1995). Also, elevated intraluminal concentrations of deconjugated bile acids in the colon normally result in an increased secretion of electrolytes and water, causing diarrhea (Hofmann 1999). However, in the current study, no environmental concentration of DCA was observed in the simulated intestine after 10 h. It is believed that the deconjugated products are precipitated at the low pHs in the intestine caused by the fermentation products of lactic acid bacteria (Fig. 4). Furthermore, this localized phenomenon is potentially increased within the microcapsule membrane, allowing for a greater precipitation of deconjugation products which could then be bound and retained within the microcapsule membrane and excreted with the feces.
Further research is required to substantiate these results, in particular, in vivo affirmation of the cholesterol-lowering capacity of microencapsulated L. reuteri. Experiments in pigs have shown that nonencapsulated (free) L. reuteri can reduce total and LDL cholesterol by 15% and 24%, respectively, in comparison to control animals (De Smet et al. 1998). De Smet et al. concluded that the net positive effect of administering free L. reuteri to pigs was comparable with cholesterol reductions on account of therapeutic or surgical interruption of the enterohepatic circulation (De Smet et al. 1998). Microencapsulation ensures greater survival of L. reuteri in gastric and intestinal environments and renders the potentially harmful products of BSH deconjugation less bioavailable, while at the same time avoiding the problems associated with oral administration of free bacterial cells. Therefore, microencapsulated L. reuteri may well prove to be an exceptional choice as a cholesterol-lowering agent for use either alone or in a combination therapy with other lipid-lowering pharmacologic therapeutics. Several parameters must be considered when considering in vivo oral effectiveness, including dosage, frequency and timing of therapeutic administration, composition of microcapsule membrane, and potential side effects of byproducts. While these factors present challenges, we believe in the potential of this approach to be superior to free cell administration both in terms of safety and efficacy of naturally occurring nongenetically engineered bile salt hydrolase-active probiotics.
This work was supported by research grants from Micropharma Limited (Montreal, Quebec) and the Canadian Institutes of Health Research (CIHR). C.M. gratefully acknowledges a Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada (NSERC). J.B. gratefully acknowledges Postgraduate Scholarships from CIHR and the Canadian Liver Foundation (CLF). A.M.U. gratefully acknowledges a Postgraduate Scholarship from NSERC.