Encapsulation in alginate-coated gelatin microspheres improves survival of the probiotic Bifidobacterium adolescentis 15703T during exposure to simulated gastro-intestinal conditions

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Food Research International 41 (2008) 184–193 www.elsevier.com/locate/foodres

Encapsulation in alginate-coated gelatin microspheres improves survival of the probiotic Bifidobacterium adolescentis 15703T during exposure to simulated gastro-intestinal conditions N.T. Annan, A.D. Borza, L. Truelstrup Hansen * Food Science Program, Department of Process Engineering and Applied Science, Dalhousie University, 1360 Barrington Street, Halifax, Nova Scotia, Canada B3K 4Y3 Received 30 July 2007; accepted 4 November 2007

Abstract Alginate-coated gelatin microspheres were produced to encapsulate the probiotic Bifidobacterium adolescentis 15703T with the objective of enhancing survival during exposure to the adverse conditions of the gastro-intestinal tract. Gelatin microspheres were cross-linked with the non-cytotoxic genipin and coated with alginate cross-linked by Ca2+ from external or internal sources. The alginate coat prevented pepsin-induced degradation of the gelatin microspheres in simulated gastric juice (pH 2.0, 2 h), resulting in significantly (P < 0.05) higher numbers of survivors due to the buffering effect of intact microspheres. After sequential incubation in simulated gastric (1 h) and intestinal juices (pH 7.4, 4 h), number of surviving cells were 7.6 and 7.4 log cfu ml1 for alginate coated microspheres by the internal and external Ca2+-source methods, respectively, while 6.7 and 6.4 log cfu ml1 were obtained for cells in uncoated gelatin microspheres and free cells, respectively. This study presents a novel microencapsulation method, which protects probiotic bifidobacteria during exposure to adverse environmental conditions. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Gelatin; Alginate; Microencapsulation; Probiotics; Bifidobacterium adolescentis; Simulated gastro-intestinal juices; Functional foods

1. Introduction Probiotic bacteria are defined as ‘live microorganisms which, when administered in adequate amounts, confer a health benefit on the host’ (Araya et al., 2002). Due to the fastidious nature of many probiotic bacteria, survival in sufficiently high numbers during passage through the human gastro-intestinal tract (GIT) remains a major challenge for effective delivery of these beneficial bacteria. Also, probiotic survival during the processing and storage of functional food products is of concern for the development of products with a guaranteed content of bioactive cells (Anal & Singh, 2007). Colonization of the intestine by an exogenous probiotic bacterium is influenced by many fac-

*

Corresponding author. Tel.: +1 902 494 3145; fax: +1 902 420 0219. E-mail address: [email protected] (L.T. Hansen).

0963-9969/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2007.11.001

tors including the size of the inoculum, physiological state of the bacteria, buffering capacity of the delivery food and the capacity of the microorganisms to resist acid and bile encountered in the upper segments of the GIT (Conway, Gorbach, & Goldin, 1987). Bifidobacteria, one group of commonly selected probiotics, are indigenous to the human intestine, where they preferentially colonise the colon (Fuller, 1991). The bacteria must therefore survive exposure to the acid in the human stomach and bile in the intestine in order to be effective (Shah, 2000). Bifidobacterium strains, however, vary greatly in their sensitivity to the harsh acidic environment of the stomach and many foods (Charteris, Kelly, Morelli, & Collins, 1998; Clark & Martin, 1994; Truelstrup Hansen, Allan-Wojtas, Jin, & Paulson, 2002). Lankaputhra and Shah (1997) found that there were more sensitive than resistant probiotic strains and this has led to several studies on microencapsulation using food grade

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biopolymers such as alginate and whey proteins to protect the acid sensitive bifidobacteria with varying successes (e.g., Gue´rin, Vuillemard, & Subirade, 2003; Lee, Cha, & Park, 2004; Rao, Shiwnarain, & Maharaj, 1989; Ravula & Shah, 1998; Reid, Champagne, Gardner, Fustier, & Vuillemard, 2007; Sheu & Marshall, 1993; Truelstrup Hansen et al., 2002). For example, entrapment in alginate microspheres with diameters of 40–80 lm resulted in insignificant protection of bifidobacteria during exposure to simulated gastric juice at pH 2.0 (Truelstrup Hansen et al., 2002) while larger alginate (1–3 mm) microspheres protected entrapped cells (Lee & Heo, 2000). However, the latter microsphere sizes are too big for incorporation into food products without affecting the texture (Champagne & Fustier, 2007) and diameters below 100 lm are preferred for most applications. Other factors that affect the success of probiotic encapsulation formulations in gastric environments include the method of microencapsulation, presence of single or multiple coatings, biopolymer concentration and increase in initial cell loads (Chandramouli, Kailasapathy, Peiris, & Jones, 2004; Krasaekoopt, Bhandari, & Deeth, 2003). In an earlier study, we optimized the processing parameters for entrapment of bifidobacteria in a novel gelatin microsphere matrix cross-linked with the non-cytotoxic genipin but found that micropheres cross-linked with 10 mmol genipin disintegrated after 10 min when exposed to simulated gastric juice with 3 g l1 pepsin (Annan, Borza, Moreau, Allan-Wojtas, & Truelstrup Hansen, 2007). As survival of the probiotic cell load is related to the structural integrity of the microspheres, this result clearly indicated the need to coat the pepsin-sensitive gelatin matrix with a biocompatible non-toxic and pepsin-resistant polymer, which is insoluble at acidic pH but dissolves in the alkaline pH of the intestine. Several pH-sensitive polymers, including alginates, have been investigated for use in drug delivery systems (Narayani & Rao, 1995a, 1995b), however, only few studies report on the use of these polymers for targeted delivery of encapsulated bacteria. Alginates are natural anionic polysaccharides made up of D-mannuronic and L-guluronic acid residues joined linearly by (1–4)-glycosidic linkages. The variable proportional and sequential arrangement of the D-mannuronic and Lguluronic units results in distribution of negative charges along the polymer backbone in aqueous solution (Thu et al., 1996), potentially allowing for polyion complexation with positively charged gelatin polymers. As alginate gels are stable in low pH solutions but swell in weakly basic solutions, alginate coating of gelatin capsules and microspheres can be used to protect drugs from the acidity of gastric juice while allowing subsequent release in the basic environment of intestinal fluids (Narayani & Rao, 1995a; Rao & Rao, 1997). Moreover, when pH is lowered below the pKa values of D-mannuronic and L-guluronic acid (3.6 and 3.7, respectively), alginate is converted to alginic acid with release of calcium ions and the formation of a more dense gel due to water loss, however, the entrapped

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pay loads, cells and/or drugs, were found to still remain in the intact alginate only beads (Doume`che et al., 2004). Genipin is a new cross-linker derived from Gardenia jasminidides or Genipa americana. The fruits from these plants are eaten raw and extracts used for medicinal purposes and as food colourants (Butler, Ng, & Pudney, 2003). Genipin has been approved for use as an additive in pharmaceuticals and foods in countries such as Japan, Korea and Taiwan (Nickerson et al., 2006) but has yet to be approved in Canada and USA. The aim of the study was to develop a method for producing alginate-coated genipin cross-linked gelatin microspheres using food grade polymers and to investigate the effect of this alginate-coating on the survival of probiotic bifidobacteria during exposure to the adverse environment found in the upper segments of the gastro-intestinal tract. We report here for the first time that encapsulation in alginate-coated gelatin microspheres significantly (P < 0.05) improved the survival of Bifidobacterium adolescentis 15703T in simulated gastric and intestinal juices in comparison to the survival obtained for free cells or cells entrapped in uncoated gelatin microspheres. 2. Materials and methods 2.1. Materials Gelatin (type A, from porcine skin, 300 Bloom), alginic acid sodium salt (medium viscosity), Tween 80, Span 85, glycerol, L-cysteine hydrochloride monohydrate, pancreatin (P-1750) and pepsin (P-7000), 5-aminofluorescein (HPLC grade), diphosphorpentoxide and EDC(1-[3dimethylaminopropyl]-3-ethyl-carbodiimide hydrochloride) were all supplied by Sigma Aldrich Co. (Oakville, ON, Canada). Genipin was obtained from Challenge Bioproducts Co. Ltd. (CBC, Taiwan, R.O.C.), while the deMan, Rogosa and Sharpe (MRS) broth, MRS agar, bile salts (Oxgall) and bacteriological peptone were supplied by Oxoid Inc. (Nepean, ON, Canada). Gas Pak Plus system (gas generator envelopes and anaerobic jars from BBL), 1,4-dioxane and sodium chloride were supplied by Fisher Scientific (Nepean, ON, Canada) and Bifidobacterium adolescentis 15703T was supplied by American Type Culture Collection (Manassas, VA, USA). Canola oil was obtained from LOBLAWS Inc. (Montre´al, QC, Canada). 2.2. Preparation of cells for microencapsulation Frozen stock cultures (2 ml, MRS with 20% v/v glycerol) of B. adolescentis 15703T were inoculated in 500 ml MRS broth containing filter sterilized L-cysteine (0.5 g l1) and incubated at 37 °C for 24 h to obtain a cell density of about 109–1010 colony forming units per ml (cfu ml1). Harvesting of cells was done by centrifugation at 8000 rpm (3578g) for 10 min at 4 °C and after discarding the supernatant of spent culture broth, the cell pellet was resuspended in peptone saline (PS, 1 g l1 peptone, 8.5 g l1 NaCl) and centrifuged

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again under the same conditions. Washed cells were then suspended in a total of 10 ml PS and stored at 4 °C until usage. Fresh cell suspensions were prepared for each experiment and enumerated by pour plating in MRS agar containing filter sterilized L-cysteine (0.5 g l1, MRS-cys agar). Plates were incubated anaerobically at 37 °C for three days using the GasPak Plus system. 2.3. Preparation of microspheres with encapsulated B. adolescentis 15703T Uncoated genipin cross-linked gelatin microspheres were prepared by dissolving gelatin powder in NaCl solution (0.5% w/v) at 50 °C to obtain a final gelatin concentration of 13% (w/v) in the microspheres. One millilitre of washed cell suspension was added to aqueous gelatin to obtain a cell to polymer solution ratio of 1:10 and the cell-gelatin aqueous mixture was then emulsified into tempered oil (45 °C, aqueous to oil phase volume ratio of 1:5) containing 0.5% Span 85 (w/w). The emulsion was produced by stirring for 30 min using a Caframo Real Torque Digital Stirrer (Caframo Ltd., Wiarton, ON, Canada) at a constant speed (900 rpm). Genipin solution giving a final concentration of 1.25 mmol l1 in the gelatin microspheres was subsequently added and the emulsion was left stirring overnight at room temperature (22 °C) to allow for the completion of the genipin-gelatin cross-linking reaction. A washing mixture (8.5 g l1 NaCl, 5 g l1 Tween 80 in distilled water) was added at a ratio of 1:1 to the aqueous gelatin phase and the microspheres were harvested with two subsequent washings in a separatory funnel to remove the oil phase, filtered (Whatman No. 4, filter paper, Fisher Scientific) and stored in PS at 4 °C until ready for use. Procedures for alginate-coating of the gelatin microspheres were developed using two techniques, namely the (a) external and (b) internal Ca2+-source gelation methods. (a) For the external Ca2+-source method, gelatin microspheres with encapsulated B. adolescentis were prepared as described above. Ten gram of the filtered microspheres were added to 40 g of 1% (w/v) medium viscosity alginate solution and stirred at an initial speed of 900 rpm to disperse the beads. The microspheres were left to stir in alginate for 20 min at 600 rpm before being filtered, collected and resuspended in 60 g of oil containing 5 g l1 Tween 80 and 62.5 mmol l1 CaCl2 for 20 min to initiate the external Ca2+ cross-linking of the peripheral alginate layer. To break the emulsion, 40 ml of 0.05 mol l1 CaCl2 solution prepared in PS was added followed by transfer to a separatory funnel. The Ca2+ cross-linked alginate coated microspheres were collected and stored in PS with 0.05 mol l1 CaCl2 until further analysis. (b) An internal Ca2+-source method was developed based on a modification of Rosenberg and Lee’s (2004) method for alginate coated whey protein based microspheres. Here the formation of the Ca2+-alginate coating depends on the diffusion of Ca2+ ions dissolved in the aqueous gelatin to the surface of the gelatin matrix to cross-link

the peripheral alginate layer. Briefly, genipin cross-linked gelatin microspheres were made as described above, except that a final concentration of 0.1 mol l1 CaCl2 was added to the aqueous gelatin phase. The resulting Ca2+-containing gelatin microspheres were harvested, filtered and washed twice with deionized water. Ten grams of the microspheres were weighed into 40 g of 1% (w/v) medium viscosity alginate solution and dispersed at an initial speed of 900 rpm followed by stirring for 20 min at 600 rpm. The by filtration recovered microspheres were again washed three times with deionized water and subsequently dispersed (900 rpm) in 60 ml PS with 0.05 mol l1 CaCl2 and left to stir gently (600 rpm) for 20 min. The microspheres were filtered, rinsed and stored in PS containing 0.05 mol l1 CaCl2 until further analysis. 2.4. Enumeration of microencapsulated bacteria Entrapped bacteria in uncoated gelatin microspheres were released by homogenizing 1.0 g of a filtered microsphere slurry in 9.0 ml of tempered PS (45 °C) for 30 s using a Polytron homogeniser (Brinkman Instruments, Rexdale, ON, Canada). For alginate-coated gelatin microspheres, tempered (45 °C) phosphate buffer (PB, 0.05 mol l1 NaH2PO4, pH 8.0) was used because phosphate ions chelate calcium thereby weakening the alginate coating for effective release of cells. The homogenized samples were diluted to appropriate concentrations and pour plated in MRS-cys agar. The plates were incubated anaerobically for three days at 37 °C and the encapsulated bacteria enumerated as cfu ml1. The encapsulation yield (EY), which is a combined measurement of the efficacy of entrapment and survival of viable cells during the microencapsulation procedure, was calculated as EY ¼

N  100 N0

where N is the number of viable entrapped cells released from the microspheres, and N0 is the number of free cells added to the biopolymer mix during the production of the microspheres. 2.5. Particle size measurement The particle sizes of microspheres were determined by measuring diameters of 300 microspheres at 100 magnification using an optical microscope (Optihot, Nikon Canada Inc., Mississauga, ON, Canada) fitted with a calibrated micrometer scale. The mean diameters of microspheres were calculated and presented with standard deviations (n  1). 2.6. Microscopy of microspheres to determine the structure of the alginate coat To determine the structure and thickness of alginate coating on gelatin microspheres by fluorescence and

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confocal laser scanning microscopy (CLSM), sodium alginate was labelled with fluorescein according to the procedure described by Blonk, van Eendenburg, Koning, Weisenborn, and Winkel (1995) while gelatin was not labelled. Briefly, 5 g of sodium alginate (medium viscosity) were dissolved in 400 ml of distilled water and drops of concentrated sulphuric acid (96%) added to precipitate the alginic acid. The mixture was centrifuged at 8000 rpm (3578g) for 10 min and the alginic acid pellets collected and washed twice with water and acetone. Washed pellets were dried overnight in vacuo in the presence of diphosphorpentoxide followed by mixing of the dried alginic acid with 80 ml distilled water, 30 ml 1,4-dioxane, 50 mg 5-aminofluorescein and 1 g EDC (1-[3-dimethylaminopropyl]-3-ethyl-carbodiimide hydrochloride). The mixture was stirred for three hours and left overnight to slowly turn orange before filtration through Whatman no. 1 filter paper followed by three washes with acetone to remove the unreacted alginic acid. The washed pellets were dried in vacuo and the resulting labelled alginate powder applied at a concentration of 0.3% (w/v) to the sodium alginate coating-solution with a final concentration of 1% (w/v). Gelatin microspheres were then coated with the fluorescein-labelled alginate as described above. Gelatin microspheres coated with fluorescein-labelled alginate were examined by an Axioplan 2 (Carl Zeiss Canada Ltd., Toronto, ON, Canada) fluorescence microscope (excitation: 450–490 nm, emission: 515–565 nm) and a Zeiss LSM 510 confocal laser scanning microscope with a Plan-Apochromat (100/1.4 oil DIC) objective. Confocal microscopy images were acquired using an argon laser emitting at 488 and 543 nm, a bypass (BP) filter detecting at 560–615 nm, a long pass (LP) filter detecting at 505 nm and were analyzed using the LSM 510 software (Carl Zeiss, Jena, Germany). For all microscopy work, the gelatin microspheres were prepared without genipin in order to eliminate background fluorescence emitted by genipin.

2.7. Survival assay of free and microencapsulated B. adolescentis in simulated gastric and intestinal juice Simulated gastric juice (SGJ) was prepared by dissolving pepsin in saline (0.2% NaCl, w/v) to a final concentration of 0.3 g l1 and adjusting the pH to 2.0 with concentrated HCl. The mixture was sterile filtered through a membrane (0.45 lm, NalgeneÒ Brand Products, Nalge Co., Rochester, NY, USA). The authors note, however, that the simulation of gastric juices for in vitro studies tends to overestimate viability losses that would occur in vivo, as it is known that the presence of food components may temporarily elevate the gastric pH (Mainville, Arcand, & Farnworth, 2005). Also, the intake of foods with a content of carbohydrates may provide Bifidobacterium strains with energy to overcome the acid stress by

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usage of the F0F1 ATPase pump (Corcoran, Stanton, Fitzgerald, & Ross, 2005; Sanz, 2007). Simulated intestinal juice (SIJ) was prepared based on the method of Huang and Adams (2004). Pancreatin and bile salts were suspended in PB to final concentrations of 1 g l1 and 4.5 g l1, respectively. The mixture was adjusted to a pH of 7.4 with 0.1 mol l1 NaOH and sterile filtered. Both gastric and intestinal juices were prepared fresh for use on the same day. Washed cell suspensions of B. adolescentis (0.5 ml) or 0.5 g of microspheres with entrapped bacteria were added to 4.5 ml of tempered (37 °C) SGJ, mixed well by vortexing for 10 s and incubated for 5, 30, 60 and 120 min at 37 °C with periodical shaking and monitoring of pH in the SGJ. Entrapped cells were released from microspheres by homogenizing using a Polytron homogeniser and surviving bacteria after each set time interval enumerated by pour plate counts in MRS-cys agar at 37 °C for 72 h as described above. 2.8. Survival and stability assay of free and microencapsulated B. adolescentis after sequential incubation in simulated gastric and intestinal juices Washed cell suspensions of B. adolescentis (0.5 ml) or 0.5 g of microspheres with entrapped bacteria were added to 4.5 ml of tempered (37 °C) SGJ, mixed well by vortexing for 10 s and incubated for 60 min at 37 °C. Simulated intestinal juice tempered at 37 °C was then added and pH adjusted to 7.4 with 0.1 mol l1 NaOH. The final volume of the mixture was made up to 10 ml with phosphate buffer and incubated for 60, 120 and 240 min at 37 °C with periodical shaking. Surviving bacteria after set times of sequential incubation were enumerated by pour plate counts in MRS-cys agar at 37 °C for 72 h as described above. 2.9. Stability of alginate-coated and un-coated gelatin microspheres during exposure to simulated gastric and intestinal juices The stability of microspheres with entrapped bacteria during sequential incubation was assessed by observing the disintegration under an optical microscope (magnification 100) every 5 min. The time for complete disintegration of microspheres was recorded. Fluorescence and CLSM microscopy was also performed on alginate coated microspheres, prepared with fluorescein-labelled alginate as described above, which had been exposed to SGJ for 1 h and to SIJ for 1 h following exposure to SGJ for 1 h. 2.10. Statistical analysis Four independent experiments with duplicate samples were conducted for each treatment. Results were statistically analyzed where appropriate by Analysis of Variance (ANOVA) using the Systat software package (Systat Inc., Evanston, IL, USA).

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3. Results 3.1. Size and encapsulation yields of uncoated and alginate-coated gelatin microspheres The mean diameters of uncoated and alginate-coated gelatin microspheres containing B. adolescentis were not significantly (P > 0.05) different as the average diameters ranged from 49.0 lm to 53.1 lm (Table 1). Alginate coating of the microspheres resulted in a uniform thin exterior layer as shown in the confocal laser scanning micrographs in Fig. 1b. The thinness (1–2 lm) of the alginate coat explained the minimal change in size between coated and uncoated microspheres. Encapsulation yields (EY) for viable cells of B. adolescentis were similar (41–43%) for uncoated and alginatecoated gelatin microspheres produced by the internal Ca2+-source method, however, EY were significantly (P < 0.05) lower (30%) for alginate-coated gelatin microspheres made by the external Ca2+-source method (Table 1).

3.2. Fluorescence and confocal laser scanning microscopy (CLSM) of uncoated and alginate-coated gelatin microspheres Optical CLSM images taken through the equator of the microspheres showed bacteria immobilized in the gelatin matrix of the microspheres with the fluorescein-labelled alginate bound as a very thin uniform membrane (1– 2 lm) to the periphery of the gelatin matrix (Fig. 1b). Uncoated gelatin microspheres similarly contained entrapped bifidobacteria throughout the matrix but showed no evidence of an outer membrane (Fig. 1a). The alginate membrane could be distinguished from the gelatin core as the fluorescein-labelled alginate emitted an intense green fluorescence at the surface of the microsphere while the gelatin core in both the coated and uncoated microspheres had a dull green colour. The alginate coated microsphere shown in Fig. 1b was coated using the external Ca2+-source gelation method, but CLSM on microspheres coated using the internal Ca2+-source gelation method looked identical (results not shown). 3.3. Survival of free and encapsulated B. adolescentis in simulated gastric juice

Table 1 Size and encapsulation yields of B. adolescentis in uncoated and alginate coated gelatin microspheres Microsphere type

Microsphere size (lm)

Encapsulation yield (%)

Uncoated Internal Ca2+ External Ca2+

49.0 ± 12.7xa 52.8 ± 10.8x 53.1 ± 10.3x

41.1 ± 3.3x 43.5 ± 4.4x 29.9 ± 6.2y

a Means (n = 900 ± standard deviation (n  1)) with different letter in a column are significantly different (P < 0.05).

Encapsulation in alginate-coated gelatin microspheres significantly (P < 0.05) improved survival of bifidobacteria (Fig. 2). Cell survival after exposure to SGJ for 5 min was 54%, 20%, 2% and 1% of the initial populations found in alginate-coated microspheres produced by external and internal Ca2+-sources, uncoated gelatin microspheres and free cells, respectively. After the initial losses, the populations of bifidobacteria declined at the same rate for all treatments over the 2 h incubation period (Fig. 2) with final

Fig. 1. Confocal laser scanning microscopy (CLSM) photographs showing the structure of (a) uncoated and (b) external Ca2+-source alginate-coated gelatin microspheres containing B. adolescentis (bold arrows). Scale bars represent 5 lm.

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entrapped in uncoated 6.71 log cfu ml1).

Survival (%)

100

189

gelatin

microspheres

(6.41–

3.5. Stability of alginate-coated and un-coated gelatin microspheres during exposure to simulated gastric and intestinal juices

10

1

0.1

0.01

0

20

40

60

80

100

120

Time (minutes) Fig. 2. Survival of free and gelatin encapsulated B. adolescentis during exposure to simulated gastric juice (pH 2.0) at 37 °C. Symbols: (d) free cells, (s) uncoated, (.) alginate-coated with internal Ca2+ and (O) alginate-coated with external Ca2+. Survival (%) represents the percentage of cells surviving relative to the initial population. Means (n = 8) ± standard deviation (n  1).

decreases of 3.45 log (cfu ml1) for free cells, 2.55 log (cfu ml1) for uncoated microspheres and 1.21 log (cfu ml1) and 1.75 log (cfu ml1) for coated microspheres with alginate cross-linked by the external and internal Ca2+-source methods, respectively. The average pH of SGJ containing free cells after 120 min was 2.1 ± 0.1 while pH of SGJ containing encapsulated beads was 2.2 ± 0.2.

3.4. Viability of free and encapsulated cells of B. adolescentis during sequential incubation in simulated gastric and intestinal juices The sequential transfer of free cells and encapsulated bifidobacteria after 60 min incubation in SGJ resulted in an initial reduction of viable cells during the first hour of exposure to SIJ, however, cell numbers subsequently stabilised and no further reductions were observed after 120 and 240 min (Table 2). Overall, the sequential exposure to SGJ (60 min) followed by SIJ (240 min) resulted in significantly (P < 0.05) higher numbers of B. adolescentis surviving in the alginate-coated gelatin microspheres (7.35– 7.57 log cfu ml1) than were obtained for free cells and cells

Disintegration times for alginate-coated gelatin microspheres in SIJ were longer for microspheres produced by the external Ca2+-source method (240 min) than for those produced by the internal Ca2+-source method (120 min), indicating a difference in the alginate surface layer obtained by the two methods (Table 2). Uncoated gelatin microspheres were already disintegrated after 45 min in SGJ before reaching the SIJ. Fig. 3a shows a fluorescence microscope image of externally coated alginate–gelatin beads after 1 h in SGJ and it can be seen that this coating was effective in making the alginate–gelatin microspheres resistant to degradation by pepsin in the acidic gastric juice thereby maintaining the probiotic bacteria entrapped in the gelatin matrix. Fig. 3b shows a CLSM image of the beads after sequential incubation in SIJ for 1 h where the alginate coating, which maintained the integrity of the microspheres in SGJ, is beginning to erode under the neutral conditions found in the small intestine with a subsequent disintegration of the underlying gelatin matrix and release of the probiotic bacteria (Fig. 3b).

4. Discussion 4.1. Size, encapsulation yields and structure of uncoated and alginate-coated microspheres Using the same concentration of gelatin, Annan et al. (2007) found the size of uncoated gelatin microspheres cross-linked with 2.5 mmol l1 genipin to be 41.2 ± 13.2 lm compared to 49.0 ± 12.7 lm obtained in the present study (Table 1). A lower concentration of genipin (1.25 mmol l1) was used in the present study which may have increased swelling of microspheres due to the uptake of water. Cross-linking of gelatin microspheres controls water uptake and swelling (Ugwoke & Kinget, 1998), hence

Table 2 Microsphere disintegration time (min) and number of surviving cells (log cfu ml1) for free and encapsulated B. adolescentis during sequential incubation (37 °C) in simulated gastric (SGJ) and intestinal (SIJ) juices Treatment

Disintegration time

SGJ 0 min

60 min

60 min

120 min

240 min

Free cells Uncoated Internal Ca2+ External Ca2+

– 15 ± 5b 120 ± 15 240 ± 30

10.41 ± 0.17xa 9.67 ± 0.35y 9.60 ± 0.54y 9.76 ± 0.09y

7.12 ± 0.09x 7.13 ± 0.27x 8.00 ± 0.17y 8.92 ± 0.78y

5.48 ± 0.15x 6.59 ± 0.04y 7.11 ± 0.45z 7.38 ± 0.15z

6.40 ± 0.31x 6.52 ± 0.04x 6.82 ± 0.34xy 7.31 ± 0.40y

6.41 ± 0.20x 6.71 ± 0.08y 7.57 ± 0.28z 7.35 ± 0.41z

a b

SIJ

Means (n = 8 ± standard deviation (n  1)) with different letter in a column are significantly different (P < 0.05). Disintegrated after 45 ± 5 min in SGJ.

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Fig. 3. Stability of alginate-coated gelatin microspheres during exposure to simulated gastro-intestinal conditions. Fluorescence microscopy photographs of (a) whole alginate coated microspheres in SGJ (1 h, 37 °C) and (b) CLSM image of alginate coated microspheres disintegrating in SIJ (1 h, 37 °C) following initial incubation in SGJ (1 h, 37 °C). Scale bars represent 20 lm.

a higher cross-linker concentration will minimize changes in shape and size of microspheres. Encapsulation yields for uncoated and alginate-coated (internal Ca2+-source) microspheres were similar to yields obtained by Annan et al. (2007) for uncoated gelatin microspheres. However, EY for the alginate-coated microspheres prepared by the external Ca2+-source method was significantly lower (Table 1). These microspheres were physically stronger as indicated by their resistance to degradation in simulated gastro-intestinal juices (Table 2 and Fig. 3) and required longer homogenization times for complete disintegration. It is possible that entrapped cells were not released or were injured during homogenization thus leading to the decrease in the apparent EY obtained for these microspheres. Use of fluorescein labelled alginate and CLSM enabled us to locate and estimate the thickness of the alginate layer on the gelatin microspheres, similar to observations by Strand, Mørch, Espevik, and Skja˚k-Brk (2003) who used the method to determine the location of alginate in alginate-L-lysine-alginate microcapsules. Leung et al. (2005) produced alginate coats on collagen beads using an adaptation of the aqueous emulsion method developed by Calafiore and Basta (1999). Also using CLSM, they observed the fluorescein-labelled alginate coat (1% w/v, medium viscosity) as a uniform coherent membrane with a thickness of about 10–20 lm, which was considerably thicker than that obtained in the present study (1–2 lm) using a similar concentration of alginate. In their coating method, Leung et al. (2005) added collagen beads to an emulsion of alginate, polyethylene glycol and Ficoll, an uncharged and highly branched polymer of sucrose, to achieve a uniform distribution of emulsion droplets before

cross-linking the alginate with calcium chloride. This conceptually different method produced a thicker coating than was obtained with our method, where the electrostatic interaction between the oppositely charged biopolymers determined the thickness of the alginate layer. The latter method was developed in order to obtain pepsin and acid resistant alginate–gelatin microspheres of a smaller size and with maximum holding capacity for the cell load (i.e., the probiotic bacteria). The ability of the alginate coating to provide a barrier against pepsin and acid degradation was evidenced in the fluorescence image shown in Fig. 3a. The CLSM image in Fig. 3b showed entrapped cells being released from the microsphere due to the erosion of the alginate coating under the neutral pH conditions of the SIJ with the subsequent degradation of the underlying gelatin matrix by pancreatic enzymes. This testifies to the success of this targeted delivery system. 4.2. Comparison of survival of free and encapsulated cells in simulated gastric juice Smith (1995) reported that food remains in the stomach for between 2 and 4 h while liquids empty from the stomach in only about 20 min. With the objective of improving survival of probiotic bifidobacteria during the exposure to the low pH of the stomach, we tested the hypothesis that the buffering capacity of gelatin would lead to increased survival of the encapsulated cells in comparison to free cells. Encapsulation of bifidobacteria in uncoated gelatin microspheres only marginally improved the survival over that of free cells (Fig. 2), however, the gelatin only microspheres were structurally unstable in SGJ due to

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degradation by pepsin and completely disintegrated after 45 min (Table 2). The 1–2 lm thick alginate coating prevented pepsin from degrading the gelatin microspheres and the entrapment in intact gelatin microspheres significantly (P < 0.05) improved survival of the entrapped bifidobacteria throughout the two hour trial period (Fig. 2), indicating the protective buffering effect of intact gelatin microspheres. Gue´rin et al. (2003) also reported that the buffering effect of whey proteins contributed to higher survival rates for bifidobacteria encapsulated in a mixed gel of alginate, pectin and whey proteins in comparison to free cells when exposed to SGJ at pH 2.5. Narayani and Rao (1995a) found that gelatin capsules loaded with a cancer drug and coated with 20% alginate remained intact for up to 8 h in SGJ while uncoated microspheres disintegrated after 15 min, their microspheres were, however, crosslinked with the cytotoxic gluteraldehyde. The decrease in the viable population by 3.45 log units for free B. adolescentis cells was similar to findings by Charteris et al. (1998) and Truelstrup Hansen et al. (2002) who observed reductions of about 3 log cfu ml1 for B. adolescentis exposed to SGJ (pH 2.0) for 2 to 3 h. The strength and structure of alginate hydrogels have been shown to be influenced by increasing gelation time/ rate and/or Ca2+ concentration due to the formation of a greater number of cross-links (Bodmeier & Wang, 1993; Liu & Krishnan, 1999). In the present study, both the rate of diffusion and the Ca2+ concentration are likely to have differed between the two coating methods, resulting in the external Ca2+-source alginate-coated microspheres appearing stronger (more difficult to homogenize and longer time to disintegration in SGJ/SIJ (Table 2)) and more protective to entrapped cells (Fig. 2).

concentrations of 2%, Clark and Martin (1994) observed a five log decrease in viable cell counts of B. adolescentis after 12 h of incubation at 37 °C. Truelstrup Hansen et al. (2002) found that B. adolescentis decreased by about 2 log cfu ml1 after 2 h of incubation in 0.5% (w/v) bile at 37 °C. Amor et al. (2002) studied the viability of B. lactis and B. adolescentis during exposure to bile salt stress and found B. adolescentis to be more susceptible to the damaging effects of bile salts. They also observed that sub-lethal injuries caused by bile salts may result in a viable but non-culturable fraction of the population. Interestingly, the number of free cells in SIJ increased from 5.48 log cfu ml1 after 60 min to 6.40 log cfu ml1 after 120 min and remained stable up to 240 min, however, this increase was most likely due to cell recovery and not growth as there were no nutrients available in the simulated intestinal juice. This observation agrees with previous reports of the subsequent recovery of temporarily damaged bifidobacteria cells after exposure to low pH and bile salt stresses (Picot & Lacroix, 2004). Higher surviving numbers of cells in alginate-coated gelatin microspheres after incubation in SGJ contributed to more cells surviving the sequential incubation into SIJ and showed that the microencapsulation matrix was effective in protecting the entrapped cells with levels of survivors of 7.6 and 7.4 log cfu ml1 as opposed to levels of 6.4–6.7 log cfu ml1 found for free cells and cells entrapped in gelatin-only microspheres after four h in SIJ. Gue´rin et al. (2003) also found that an initial immobilized B. bifidum population of 1010 cells g1 in a mixed alginate, pectin and whey protein matrix could reach the small intestine in numbers of 7.5 log cells g1 and hence provide the host with a beneficial health effect.

4.3. Survival of free and encapsulated cells of B. adolescentis during sequential incubation in simulated gastro-intestinal conditions

5. Conclusions

While the ionotropic alginate gel formed by Ca2+ crosslinking of carboxylate groups is insoluble at low pH, exposure to neutral pH or higher solubilises the alginate (LeTien, Millette, Mateescu, & Lacroix, 2004; Tønnesen & Karlsen, 2002). In our study, this pH-dependent behaviour of the biopolymer was used to control the degradation of alginate-coated gelatin microspheres and release of the microencapsulated cell load under the neutral conditions found in the small intestine (Table 2 and Fig. 3), where the transit time for food ranges between 1 and 4 h (Smith, 1995). The continued reduction of cell numbers in SIJ especially during the first h of incubation in this environment may be attributed to the sensitivity of B. adolescentis to bile and for the entrapped cells, also to physical integrity of the microsphere matrix. The sensitivity of many strains of Bifidobacterium spp. to bile concentrations encountered in the human gastro-intestinal tract has been reported by several authors (Amor et al., 2002; Chung, Kim, Chun, & Ji, 1999; Lankaputhra & Shah, 1997; Picot & Lacroix, 2004). At bile

Coating of gelatin microspheres with alginate provided significant protection forB. adolescentis from the harsh acidic conditions of simulated gastric juice. As a result, significantly higher numbers of bacteria survived sequential incubation from the simulated gastric juice into the simulated intestinal fluid where disintegration of the alginatecoated gelatin microspheres and release of entrapped cells occurred. The novel encapsulation matrix developed in this study was designed to increase numbers of probiotic bacteria surviving the upper segment of the GIT allowing for introduction into the colon where they may offer health benefits by effecting positive changes to the host’s immune defence and intestinal microflora. Future work will look at the development of specific food applications and methods for longterm preservation of the alginate-coated gelatin microspheres. Acknowledgements This work was supported by a research grant from the Advanced Foods and Materials Network (AFMNet), a

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Network Centre of Excellence, Canada, awarded to author Truelstrup Hansen. Author Annan is a recipient of a Natural Sciences and Engineering Council (NSERC, Canada) Post Doctoral Fellowship. The excellent technical assistance of Shahnaz Bano is acknowledged. References Amor, K. B., Breeuwer, P., Rombouts, P., Verbaarschot, F. M., Akkermans, A. D. L., De Vos, W. M., et al. (2002). Multiparametric flow cytometry and cell sorting for the assessment of viable, injured and dead bifidobacterium cells during bile salt stress. Applied and Environmental Microbiology, 68, 5209–5216. Anal, A. K., & Singh, H. (2007). Recent advances in microencapsulation of probiotics for industrial applications and targeted delivery. Trends in Food Science & Technology, 18, 240–251. Annan, N. T., Borza, A., Moreau, D. L., Allan-Wojtas, P. M., & Truelstrup Hansen, L. (2007). Effect of process variables on particle size and viability of Bifidobacterium lactis Bb-12 in genipin–gelatin microspheres. Journal of Microencapsulation, 24, 152–162. Araya, M., Morelli, L., Reid, G., Sanders, M.E., Stanton, C., Pineiro, M., et al. (2002). Guidelines for the evaluation of probiotics in food. Joint FAO/WHO working group report on drafting guidelines for the evaluation of probiotics in food, London, ON, Canada. Blonk, J. C. G., van Eendenburg, J., Koning, M. M. G., Weisenborn, P. C. M., & Winkel, C. (1995). A new CSLM-based method for determination of the phase behaviour of aqueous mixtures of biopolymers. Carbohydrate Polymers, 28, 287–295. Bodmeier, R., & Wang, J. (1993). Microencapsulation of drugs with aqueous colloidal polymer dispersions. Journal of Pharmaceutical Sciences, 82, 191–194. Butler, M. F., Ng, Y-F., & Pudney, P. A. (2003). Mechanisms and kinetics of the cross-linking reaction between biopolymers containing primary amine groups and genipin. Journal of Polymer Science, Part A. Polymer Chemistry, 441, 3941–3953. Calafiore, R., & Basta, G. (1999). Alginate/poly-L-ornithine microcapsules for pancreatic islet cell immunoprotection. In W. M. Kuhtreiber, R. P. Lanza, & W. L. Chick, Cell encapsulation technology and therapeutics, (pp. 138–150). Boston, USA, Birkhauser. Champagne, C. P., & Fustier, P. (2007). Microencapsulation for the improved delivery of bioactive compounds into foods. Current Opinion in Biotechnology., 18, 184–190. Chandramouli, V., Kailasapathy, K., Peiris, P., & Jones, M. (2004). An improved method of microencapsulation and its evaluation to protect Lactobacillus sp. in simulated gastric environments. Journal of Microbiological Methods, 56, 27–35. Charteris, W. P., Kelly, P. M., Morelli, L., & Collins, J. K. (1998). Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. Journal of Applied Microbiology, 84, 759–768. Chung, H. S., Kim, Y. B., Chun, S. L., & Ji, G. E. (1999). Screening and selection of acid and bile resistant bifidobacteria. International Journal of Food Microbiology, 47, 25–32. Clark, P. A., & Martin, J. H. (1994). Selection of bifidobacteria for use as dietary adjuncts in cultured dairy foods: III – Tolerance to simulated bile concentrations of human small intestines. Cultured Dairy Products Journal, 29, 18–21. Corcoran, B. M., Stanton, C., Fitzgerald, G. F., & Ross, R. P. (2005). Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars. Applied and Environmental Microbiology, 71, 3060–3067. Conway, P. L., Gorbach, S. L., & Goldin, B. R. (1987). Survival of lactic bacteria in the human stomach and adhesion to intestinal cells. Journal of Dairy Science, 70, 1–12.

Doume`che, B., Ku¨ppers, M., Stapf, S., Blu¨mich, B., Hartmeier, W., & Ansorge-Schumacher, M. B. (2004). New approaches to the visualization, quantification and explanation of acid-induced water loss from Ca-alginate hydrogel beads. Journal of Microencapsulation, 21, 565–573. Fuller, R. (1991). Probiotics in human medicine. Gut, 32, 439–442. Gue´rin, D., Vuillemard, J.-C., & Subirade, M. (2003). Protection of bifidobacteria encapsulated in polysaccharide–protein gel beads against gastric juice and bile. Journal of Food Protection, 66, 2076–2084. Huang, Y., & Adams, M. C. (2004). In vitro assessment of the upper gastrointestinal tolerance of potential probiotic dairy propionibacteria. International Journal of Food Microbiology, 91, 253–260. Krasaekoopt, W., Bhandari, B., & Deeth, H. (2003). Evaluation of encapsulation techniques of probiotics for yoghurt. International Dairy Journal, 13, 3–13. Lankaputhra, W. E. V., & Shah, N. P. (1997). Improving viability of Lactobacillus acidophilus and bifidobacteria in yogurt using two step fermentation and neutralized mix. Food Australia, 49, 363–366. Leung, A., Ramaswamy, Y., Munro, P., Lawrie, G., Nielsen, L., & Trau, M. (2005). Emulsion strategies in the microencapsulation of cells: Pathways to thin coherent membranes. Biotechnology and Bioengineering, 92, 45–53. Lee, K.-Y., & Heo, T.-R. (2000). Survival of Bifidobacterium longum immobilized in calcium-alginate beads in simulated gastric juices and bile salt solution. Applied and Environmental Microbiology, 66, 869–873. Lee, J. S., Cha, D. S., & Park, H. J. (2004). Survival of freeze-dried Lactobacillus bulgaricus KFRI 673 in chitosan-coated calcium alginate microparticles. Journal of Agricultural and Food Chemistry, 52, 7300–7305. Le-Tien, C., Millette, M., Mateescu, M.-A., & Lacroix, M. (2004). Modified alginate and chitosan for lactic acid bacteria immobilization. Biotechnology and Applied Biochemistry, 39, 347–354. Liu, P., & Krishnan, T. R. (1999). Alginate-pectin-poly-L-lysine particulate as a potential controlled release formulation. Journal of Pharmacy and Pharmacology, 51, 141–149. Mainville, I., Arcand, Y., & Farnworth, E. (2005). A dynamic model that stimulates the upper gastro-intestinal tract for the study of probiotics. International Journal of Food Microbiology, 99, 287–296. Narayani, R., & Rao, K. P. (1995a). Polymer-coated gelatin capsules as oral delivery devices and their gastrointestinal tract behaviour in humans. Journal of Biomaterials Science-Polymer Edition, 7, 39–48. Narayani, R., & Rao, K. P. (1995b). pH-responsive gelatin microspheres for oral delivery of anticancer drug methotrexate. Journal of Applied Polymer Science, 58, 1761–1769. Nickerson, M. T., Wagar, A. T., Paulson, E., Farnworth, R., Hodge, S. M., & Rosseau, D. (2006). Some physical properties of crosslinked gelatin-maltodextrin hydrogels. Food Hydrocolloids, 20, 1072–1079. Picot, A., & Lacroix, C. (2004). Encapsulation of bifidobacteria in whey protein-based microcapsules and survival in simulated gastrointestinal conditions and in yoghurt. International Dairy Journal, 14, 505–515. Rao, J. K., & Rao, K. P. (1997). Controlled release of FITC-BSA from polymer coated gelatin microspheres. Journal of Bioactive and Compatible Polymers, 12, 127–138. Rao, A. V., Shiwnarain, H., & Maharaj, I. (1989). Survival of microencapsulated Bifidobacterium pseudolongum in simulated gastric and intestinal juices. Canadian Institute of Food Science and Technology Journal, 22, 345–349. Ravula, R. R., & Shah, N. P. (1998). Viability of probiotic bacteria in fermented frozen dairy desserts. Food Australia, 50, 136–139. Reid, A. A., Champagne, C. P., Gardner, N., Fustier, P., & Vuillemard, J. C. (2007). Survival in food systems of Lactobacillus rhamnosus R011 microentrapped in whey protein gel particles. Journal of Food Science, 72, M31–M37. Rosenberg, M., & Lee, S.-J. (2004). Water-insoluble, whey protein-based microspheres prepared by an all-aqueous process. Journal of Food Science, 69, E50–E58.

N.T. Annan et al. / Food Research International 41 (2008) 184–193 Shah, N. P. (2000). Probiotic bacteria: selective enumeration and survival in dairy foods. Journal of Dairy Science, 83, 894–907. Sanz, Y. (2007). Ecological and functional applications of the acidadaptation ability of Bifidobacterium: A way of selecting improved probiotic strains. International Dairy Journal, 17, 1284–1289. Sheu, T. Y., & Marshall, R. T. (1993). Microentrapment of lactobacilli in calcium alginate gels. Journal of Food Science, 54, 557–561. Smith, T., (1995). The digestive system. In The human body (pp. 150–173). Collingwood, UK: Ken Fin Books. Strand, B. L., Mørch, Y. A., Espevik, T., & Skja˚k-Brk, G. (2003). Visualization of alginate-poly-L-lysine-alginate microcapsules by confocal laser scanning microscopy. Biotechnology and Bioengineering, 82, 386–394.

193

Thu, B., Bruheim, P., Espevik, T., Smidsrød, O., Soon-Shiong, P., & Skja˚k-Brk, G. (1996). Alginate polycation microcapsules. I. Interaction between alginate and polycation. Biomaterials, 17, 1031–1040. Tønnesen, H. H., & Karlsen, J. (2002). Alginate in drug delivery systems. Drug Development and Industrial Pharmacy, 28, 621–630. Truelstrup Hansen, L., Allan-Wojtas, P. M., Jin, Y. L., & Paulson, A. T. (2002). Survival of Ca-alginate microencapsulated Bifidobacterium spp. in milk and simulated gastrointestinal conditions. Food Microbiology, 19, 35–45. Ugwoke, M. I., & Kinget, R. (1998). Influence of processing variables on the properties of gelatin microspheres prepared by the emulsification solvent extraction technique. Journal of Microencapsulation, 15, 273–281.