Survival of Microencapsulated Bifidobacterium pseudolongum in Simulated Gastric and Intestinal Juices

Can. Inst. FoodSci. Technol. J. Vo!. 22, No. 4, pp. 345-349,1989

RESEARCH

Survival of Microencapsulated Bifidobacterium pseudo[ongum in Simulated Gastric and Intestinal Juices A.V. Rao, N. Shiwnarain and 1. Maharaj Department of Nutritional Sciences University of Toronto Toronto. Ontario M5S IA8

Abstract A preliminary procedure for the microencapsulation of bifidobaeteria with cellulose acetate phthalate (CAP), using phase separation-coacervation was developed. In vitro studies were conducted on microencapsulated Bifidobacterium pseudolongum, to determine the effect of gastric and intestinal pH on the release of the bacteria by sequential incubation in simulated gastric and intestinal juices, without enzymes. Microbiological analyses indicated that microencapsulated B. pseudolongum survived the simulated gastric environment in larger numbers than non-encapsulated B. pseudolongum.

Resume Une procedure preliminaire a ete developpee pour la microencapsulation des bacteries bifido avec du phthalate acetate de cellulose (CAP). a I'aide de la technique de coacervation et separation de phases. Des etudes furent conduites in vitro sur des Bifidobacterium pseudolongum microencapsules pour etablir I'effet des pHs gastrique et intestinal sur la liberation des baeteries par incubation sequentielle dans des jus gastriques et intestinaux, sans enzymes. Les analyses mierobiologiques ont indique que B pseudolongum microeneapsule a survecu en plus grand nombre dans le milieu gastrique simule que les bacteries non encapsulees.

Introduction Bifidobacteria have been shown to play an important beneficial role in human and animal health (Rasic and Kurmann, 1983; Mutai and Tanaka, 1987). Distribution of bifidobacteria in humans changes dramatically with age from being the predominant intestinal microorganism in infants (> 99070) to being the fourth largest bacterial group « 15%) in adults (Mitsuoka, 1982). Although the composition of the intestinal microflora of healthy adults is quite stable, factors such as stomach and intestinal disorders, antibiotic therapy and other stress conditions can result in significant changes in the intestinal micro flora (Drasar and HilI, 1974; Bornside, 1978), including a reduction or disappearance of bifidobacteria (Mata and Urrutia, 1971; Mataetal., 1969; Mitsuoka, 1982). A reduction in the number of bifidobacteria, accompanied by a significant increase in the number of

pathogenic bacteria, has been shown during senescence (Mitsuoka, 1982). It is possible, however, to maintain a favourable distribution of the intestinal microflora by the dietary addition of bifidobacteria and bifidogenic products (Muting, 1977; Kawase et al., 1983; Tanaka et al., 1983; Kageyama et al., 1984; Shimoyama et al., 1984; Hidaka et al., 1986). The usefulness of bifidobacteria has been suggested in replacement therapy for several bacterial-induced gastrointestinal disorders. It is thought to compete with potential intestinal pathogens for mucosal cell attachment. However, one of the major barriers to the survival of ingested microorganisms is the low pH of the stomach. In a recent report Kim et al. (1988) described a method for the preparation of stable microencapsulated lactic acid bacteria using polyvinyl acetate phthalate. In this study a method of microencapsulation was investigated using cellulose acetate phthalate (CAP), as an enteric coating, on the stability of bifidobacteria. Due to the presence of ionizable phthalate groups, this polymer is insoluble in acid media at a pH of 5 or lower but is soluble when the pH is increased to 6 or higher (Maim et al., 1951). In addition, CAP is physiologically inert when administered in vivo and is, therefore, widely used as an enteric coating material for the release of drugs and other pharmaceutical substances in the intestine. CAP was successfully used to prepare microcapsules of active viral antigens and other proteins for oral consumption (Maharaj et al., 1984). In this paper, a preliminary procedure for the microencapsulation of freeze-dried Bifidobacterium pseudolongum using CAP is described. The release and viability of microencapsulated B. pseudolongum were investigated in vitro.

Materials and Methods Cellulose-acetate-phthalate (CAP), was obtained from Eastman Kodak Co., Rochester, NY. Freeze-

Copyright iD 1989 Canadian Institute of Food Science and Technology

345

dried B. pseudolongum (108 -109 CFU/g) in skim milk powder was a donation from Gelda Scientific and Industrial Development, Mississauga, Ont., Canada. The procedures outlined were performed using aseptic techniques.

Survival of Freeze-dried B. pseudolongum at Different pH Levels The stability of freeze-dried B. pseudolongum was studied under pH conditions simulating those of the gastric environment. B. pseudolongum was assayed by serially diluting 1.0 mL aliquots of the test material in peptone water (15g/L) (London Analytical and Bacteriological Media Ltd., Salford, England), and spread-plating 0.1 mL on MRS agar (Gibco, Madison, WI, USA) containing 0.03070 (w/v) cysteine-hydrochloride. The plates were incubated anaerobically (Anaerocult C, E. Merck, Darmstadt, Germany) at 37°C for 48h. To determine the survival of freeze-dried B. pseudolongum at different pH levels, flasks containing 20 mL aliquots of (i) simulated gastric juice (USP) without pepsin, pH 1.33 (0.08 M HCI containing 0.2% (w/v) NaCl); (ii) MRS broth with 0.03% (w/v) cysteine-hydrochloride, pH 6.06; (iii) peptone water, pH 7.13, were pre-incubated at 37°C for lOmin and inoculated with 1.0 mL aliquots of the bacterial suspension. The suspension was prepared by dissolving 2g freeze-dried B. pseudolongum in 18 mL physiological saline. The flasks were then incubated anaerobically at 37°C and were shaken manually every 15 min. At time intervals of 0,20,40,60,90 and 120 min, 1.0 mL aliquots were removed from the flasks and assayed for B. pseudolongum.

Release of Microencapsulated B. pseudolongum in Simulated Intestinal Juice Two sets of flasks containing simulated (20 mL) intestinal juice USP without pancreatin (0.05M KH 2P04 adjusted to pH 7.43 with 0.1 N NaOH) were inoculated with 1 g samples of microspheres and core material, respectively, and incubated anaerobically at 37°C. At time intervals of 0, 20, 40, 60, 90, 120 min, 1.0 mL aliquots were removed and assayed for B. pseudolongum. At the same time, the pH of the intestinal juice was readjusted to pH 7.43 with 0.1 N NaOH.

Survival of Microencapsulated B. pseudolongum After Sequential Incubation in Simulated Gastric and Intestinal Juices One gram samples of microspheres were added to flasks containing 20 mL simulated gastric juice and incubated anaerobically at 37°C for 0,30,90, and 180 min. After incubation, the microspheres were removed by filtration and were subsequently placed in 20 mL of simulated intestinal juice and the pH adjusted to 7.43 with O.lN NaOH. Flasks were then incubated anaerobically at 37°C. During incubation, the flasks were manually shaken and the pH was periodically readjusted to 7.43. After incubation in the simulated intestinal juice, 1.0 mL aliquots were removed and assayed for B. pseudolongum.

Photography of Microencapsulated B. pseudolongum Samples of microencapsulated B. pseudolongum were photographed at 34x magnification with a Nikon Multiphot microscope (Nippon, Kogaku, Japan) for visual representation.

Microencapsulation of B. pseudolongum

Results and Discussion

The microencapsulating procedure as outlined by Maharaj et al. (1984) was modified in order to make it more suitable for a bacterial preparation. Corematerials, i.e., materials to be encapsulated (70% freeze-dried B. pseudolongum: 30070 starch), were mixed and finely ground using a sterile mortar and pestle. The microspheres were prepared in 3g batches; each batch of core material was then suspended in 300 mL white light paraffin oil in a 600 mL beaker. The mixture was dispersed by stirring at 260 rpm with a 44-mm polyethylene 3-blade paddle fitted to a hightorque stirrer for 2-3 min. Thirty mL of 10% (w/v) CAP in 100% acetone-95% ethanol (9:1) was added to the suspension and stirred for I h to allow the microspheres to form. The suspending medium was decanted and the microspheres were filtered through a Whatman No. 3 filter paper and collected. The microspheres were then resuspended in 1% (w Iv) beeswax at 37°C for 10-15 min, and refiltered. The microspheres were stored in tightly closed sterile glass vials at ambient temperature.

For bifidobacteria to be beneficial to the host they must colonize the colon. Following ingestion, the microorganisms must survive transit through the gastric environment and reach the colon in quantities large enough to facilitate colonization. The germicidal effect of gastric juice is mainly attributable to its low pH (Gianella et al., 1972). To determine the effect of the acidic pH of the stomach on the survival of bifidobacteria, an in vitro system was utilized. When B. pseudolongum was exposed to a simulated gastric environment for 1 h none of the organisms survived. However, the bacterial population was fully maintained at pH 6.06 and 7.13 (Figure 1). The latter pH values were chosen to evaluate the pH effect of the growth medium, MRS + 0.03% (w/v) cysteinehydrochloride broth, and the diluent, peptone water, respectively. These results suggest that ingestion of unprotected bifidobacteria would reduce viability. To ensure greater survival under gastric conditions, a method for the microencapsulation of bifidobacteria was developed.

346 / Rao et al.

J. lnst. Can. Sei. Technal. Aliment. Val. 22, No. 4, 1989

.... pH 1.33 ::l

~

. - PH 6.06

5·00

...

o

.... pH 7.13

Cl

g

3·00

1·00

20

40 TIME (minI

Fig. I. Survival of freeze-dried B. pseudolongum in simulated gastric juice.

Microencapsulation is a process in which small discrete solid materials, liquid droplets or gases are completely enveloped by an intact membrane. Several methods of microencapsulation are currently used including air suspension, electrostatic deposition, pan coating and spray-drying (Bakan and Anderson, 1976; D'Onofrio et al., 1979; Dziezak, 1988). The method chosen to microencapsulate B. pseudolongum was the "phase separation - coacervation method" (Madan, 1978; Maharaj et al., 1984; Beyger and Nairn, 1986) because of its simplicity and gentle conditions. Attempts to encapsulate freeze-dried B. pseudolongum as is, resulted in clumping due to the presence of excess moisture in the freeze-dried preparation. This excess moisture was eliminated by the incorporation of starch, a drying agent, to the freeze-dried B. pseudolongum. Trials indicated that a core-material consisting of 70:30 (w/w) mixture of freeze-dried B. pseudolongum to starch was adequate to prevent clumping during the microencapsulation procedure and produced quasi-round microspheres approximately 1 mm in diameter (Figure 2). Survival of B. pseudolongum during various stages of the microencapsulating procedure as outlined by Maharaj et al. (1984) was evaluated. The results (Table 1) showed that chloroform, a solvent used to terminate the process, was bactericidal. Although chloroform

Table I. Survival of freeze-dried B. pseudolongum during the microencapsulation procedure. Sample Starting material (core) Addition of paraffin After 3 min in paraffin Addition of solvent (1000/0 acetone-95% ethanol, 9: I) 60 min after addition of solvent Addition of chloroform 15 min after addition of chloroform IMean ± standard deviation Can. Inst. Food Sei. Teehnol. J. Vo!. 22, No. 4, 1989

IOglO CFU/g spheres 8.70 ± 0.64 1 8.46 ± 0.36 8.51 ± 0.35 8.32 ± 0.59 7.91 7.06 6.63

0.46 0.28 0.20

Fig. 2. Photograph of microspheres containing B. pseudolongum. (Magnification 34X)

yielded a clean, oil-free, free-flowing product due to the solubilization of the paraffin layer surrounding the microspheres, it proved to be detrimental to the survival of the bacterial cells. In our modified procedur~, chloroform was eliminated to enhance viability of the encapsulated bacterial preparation (Figure 3). Other studies have been reported on the microencapsulation of microbial cells (Mohan and Li, 1978). However, these cells were used primarily as sources of enzyme activities and viability was not an important factor. The microencapsulation of other viable cells such as red blood cells, hepatoma cells and pancreatic endocrine cells has also been investigated (Urn and Moss, 1981). When freeze-dried and microencapsulated B. pseudolongum were placed directly into simulated intestinal juice, pH 7.43, the release of viable cells was similar (Figure 4). There was a complete dissolution of the microspheres within 20-40 min which allowed the release of encapsulated B. pseudolongum. However, when the microspheres were placed in simulated gastric juice, pH 1.33, they became rigid and required up to 160 min of incubation in simulated intestinal juice for complete dissolution (Table 2). Because the bacterial count had also decreased, it seems likely that gastric juice entered the microspheres through surface pinholes resulting in bacterial loss. To reduce this loss, an Rao et al. / 347

Table 2. Viability of microencapsulated B. pseudolongum in vitro. Incubation Time (min) Simulated gastric juice, pH 1.33

Simulated intestinal juice, pH 7.43

o 30 90 180 I Mean ± standard Eieviation

160 160 160 160

7.69 ± 0.59 4.89 ± 0.35 4.50 ± 0.70 4.00 _CORE

outer coating on the microspheres, using one or two percent stearic acid and one percent beeswax (Motycka and Nairn, 1978), was investigated. Microspheres which were coated with one percent beeswax exhibited the highest survival of B. pseudolongum after sequential incubation in simulated gastric juice for 30 min followed by intestinal juice (Figure 5). Since this improved outer coating should also render the microspheres less permeable to oxygen this additional step was then included in our microencapsulation procedure (Figure 3). Based on these preliminary results, future studies with microencapsulated B. pseudolongum will be used in rat feeding studies to investigate their survival in vivo, and their effect on fecal pH and fecal betaglucuronidase, mucinase and azoreductase activites. Preliminary trials indicated that rats fed lab-chow plus microencapsulated B. pseudolongum exhibited a slightly higher feed efficiency compared to rats fed lab-chow alone. Microencapsulation of bacteria with CAP would seem to offer an effective way of delivering large numbers of viable bacterial cells to the colon. Microencapsulation technology may, therefore, play an important role in food processing and pharmaceutical preparations. Core Material (3g) B. pseudolongum: starch 70:30 (wlW)

I Blending Mix and finely grind whh Slerlle morlar and peSlle

-

CORE+CAP

1__---'1.-_---::01:-'--~'~--.l.-_ 30

60

90

120

TIME (min)

Fig. 4. Effect of simulated intestinal juice on the survival of freezedried and encapsulated core material. The degree of survival has been corrected to represent 100010 bacterial content.

Acknowledgements The authors are grateful to D. Milovanovic, Faculty of Pharmacy, University of Toronto, for his technical assistance and helpful discussion.

References Bakan, LA. and Anderson, J.L. 1976. Microencapsulation. In: Theory and Practice of Industrial Pharmacy. L. Lachman, H.A. Lieberman, and J.L. Kanig(Eds.). p. 420. Lea and Febiger, Philadelphia, PA. Beyger, J.W., and Nairn, G.J. 1986. Some factors affecting the microencapsulation of pharmaceuticals with cellulose acetate phthalate. J. Pharm. Sci. 75:573. Bornside, G.H. 1978. Stability of human fecal flora. Am. J. Clin. Nutr. 31:5141. O'Onofrio, G.P., Oppenheim, R.e. and Bateman, N.E. 1979. Encapsulated microcapsules.lnt. J. Pharm. 2:91. Orasar, B.S., and Hill, M.J. 1974. In: Human Intestinal Flora. 1st ed. p. I. Academic Press Inc., London, England. Oziezak, J.O. 1988. Microencapsulation and encapsulated ingredients. Food Technol. 42: 136.

I Suspension

Add to 300 mL paraffin, sUr 2·3 mln, 25°C 30mLC jP(10%)

8·00

I Mixing SUr lh, 25°C

I

Decant

I

Supematant

. - . 1%

_l%STEARIC ACID

Immersion

-.2010 STEARIC ACID

Mlcrospheres Immersed In 1% beeSwax, 10.15 mln, 37°C

I

Decant

I

80

100

120

140

Supemalant

Mlcroencapsulaled B. pseudotongum

Fig. 3. Flow diagram outlining the procedure for microencapsulating B. pseudolongum. 348 / Rao et al.

BEESWAX

TIME (minI

Fig. 5. Release of B. pseudolongum from microspheres coated with stearic acid and beeswax.

J. Ins/. Can. Sci. Technol. Aliment. Vo!' 22, No. 4, 1989

G' ella R.A., Broitman, S.A., and Zamcheck, N. 1972. Gastric lan 'acid barrier to ingested microorganisms in man: studies in vivo and in vitro. Gut 13:251. Hidaka, H., Eida, T., a.nd Tak.izawa, T. 1986. Effects of fructooligosacchandes on intestinal flora and human health. Bifido. Microflora 5:37. Kageyama, T., Tomada, T., and. ~akano.' Y. 1984. T~e effectof Bifidobactenum admInIstration In patients with leukemia. Bifido. Microflora 3:29. Kawase, K., Suzuki, T., Kiyosawa, I., Okonogi, S., Kawashima, T., and Kuboyama, M. 1989. Effect of composition of infant formulas on the intestinal microflora of infants. Bifido. Microflora 2:25. Kim, H.S., Kamara, B.1., Good, I.C. and Enders, G.L., Jr. 1988. Method for the preparation of stabile microencapsulated lactic acid bacteria. J. Ind. Microbiol. 3:253. Urn, F., and Moss, R. 1981. Microencapsulation of living cells and tissues. J. Pharm. Sci. 70:351. Madan, P.L. 1978. Microencapsulation I. Phase separation or coacervation. Drug Dev. Ind. Pharm.4:95. Maharaj, I., Nairn, G.J., and Campbell, J.B. 1984. Simple rapid method for the preparation of enteric-coated microspheres. J. Pharm. Sci. 73:39. Maim, C.J., Emerson, J., and Hiatt, G.D. 1951. Cellulose acetate phthalate as an enteric-coating material. J. Am. Pharm. Assoc. Sci. 10:520. Mata, L.J., and Urrutia, J.J. 1971. Intestinal colonization of breastfed children in a rural area of the low socio-economic level. In: Neonatal Enteric infection caused by E. Coli. B. Tennat (Ed.). Ann. N. Y. Acad. Sci. 176:93. Mata, L.J., Urrutia, 1.J., Garcia, B., Fernandez, R. and Behar, M. 1969. Shigella infection in breast-fed Guatemalan Indian neonates. Am. 1. Dis. Child. 117: 142.

Can. Inst. Food Sei. Teehnol. J. Vo!. 22, No. 4, 1989

Mitsuoka, T. 1982. Recent trends in research on intestinal flora. Bifido. Microflora 1:3. Mohan, R.R., and Li, N.N. 1978. Nitrate and nitrite reduction by liquid membrane encapsulated whole cells. Biotechnol. Bioeng.17:1137. Motycka, S. , and Nairn, 1.G. 1978. Influence of wax coatings on release rate of anions from ion-exchange resin beads. J. Pharm. Sci. 67:500. Mutai, M. , and Tanaka, R. 1987. Ecology of Bifidobacterium in the human intestinal flora. Bifido. Microflora. 6:33. Muting, D. 1977. Clinical picture and therapy of portalsystemic encephalopathy. Leber, Magen and Darm (4):256. In: Bifidobacteria and Their Role. J.L. Rasic and J .A. Kurmann (Eds.). 1983, p. 45. Birkhauser Verlag, Basel, Switzerland. Rasic, J.L. and Kurmann, J .A. 1983. Bifidobacteria and Their Role. Microbiological, Nutritional, Physiological, Medical and Techological Aspects and Bibilography. Birkhauser Verlag, Basel, Switzerland. Shimoyama, T., Hori, S., Tamura, K., Yamamura, M., Tanaka, M., and Yamazaki, K. 1984. Microflora of patients with stool abnormality. Bifido. Microflora. 3:35. Tanaka, R., Takayama, H., Morotomi, M., Kuroshima, T., Ueyama, S., Matsumoto, K., Kurodo, A. and Mutai, M. 1983. Effects of administration of TOS and Bifidobacterium breve 4006 on human fecal flora. Bifido. Microflora. 2: 17.

Submitted August 9, 1988 Revised June 2, 1989 Accepted June 12, 1989

Rao et al. / 349