International Dairy Journal 14 (2004) 737–743

The influence of coating materials on some properties of alginatebeads and survivability of microencapsulated probiotic bacteria

Wunwisa Krasaekoopt, Bhesh Bhandari*, Hilton Deeth

School of Land and Food Sciences, The University of Queensland, St. Lucia, Qld. 4072, Australia

Received 29 April 2003; accepted 16 January 2004

Abstract

The probiotics, Lactobacillus acidophilus 547, Bifidobacterium bifidum ATCC 1994, and Lactobacillus casei 01, were encapsulated

into uncoated calcium alginate beads and the same beads were coated with three types of material, chitosan, sodium alginate, and

poly-l-lysine in combination with alginate. The thickness of the alginate beads increased with the addition of coating materials. No

differences were detectable in the bead strength by texture analysis or in the thickness of the beads with different types of coating

materials by transmission electron microscopy. The survivability of three probiotics in uncoated beads, coated beads, and as free

cells (unencapsulated) was conducted in 0.6% bile salt solution and simulated gastric juice (pH 1.55) followed by incubation in

simulated intestinal juice with and without 0.6% bile salt. Chitosan-coated alginate beads provided the best protection for

L. acidophilus and L. casei in all treatments. However, B. bifidum did not survive the acidic conditions of gastric juice even when

encapsulated in coated beads.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Probiotics; Microencapsulation; Alginate bead; Coating; Gastric juice

1. Introduction

Microencapsulation is a process in which the cellsare retained within an encapsulating matrix or mem-brane. Microencapsulation of probiotics has beeninvestigated for improving their viability in foodproducts and the intestinal tract (Rao, Shiwnarain, &Maharaj, 1989). The most widely used encapsulatingmaterial is alginate, a linear heteropolysaccharide ofd-mannuronic and l-guluronic acid extracted fromvarious species of algae (Smidsrod, Haug, & Lian,1972). Alginate beads can be formed by both extrusionand emulsion methods (Krasaekoopt, Bhandari, &Deeth, 2003). The use of alginate is favoured becauseof its cheapness, simplicity, and biocompatibility (Klein,Stock, & Vorlop, 1983; Tanaka, Masatose, & Veleky,1984; Martinsen, Skjak-Braek, & Smidsrod, 1989).Bacteria (1–3 mm size) are well retained in the alginate

gel matrix which is estimated to have a pore size of lessthan 17 nm (Klein et al., 1983). However, the gel issusceptible to disintegration in the presence of excess

monovalent ions, Ca2+-chelating agents and harshchemical environments (Smidsrod & Skjak-Braek,1990). A crosslinked alginate matrix system at verylow pH is reported to undergo a reduction in alginatemolecular weight causing a faster degradation andrelease of active ingredients (Gombotz & Wee, 1998).Poly-cations, such as chitosan or poly-amino acids (forexample, poly-l-lysine (PLL), form strong complexeswith alginates which are stable in the presence of Ca2+

chelators and reduce the porosity of the gel (Smidsrod &Skjak-Break, 1990; Gombots & Wee, 1998). Thus,coating alginate beads with poly-cations can improvethe chemical and mechanical stability of the alginatebeads, consequently improving the effectiveness ofencapsulation.Dropping an alginate solution into a solution contain-

ing a mixture of calcium chloride and chitosan (Over-gaard, Scharer, Moo-Young, & Bols, 1991) or soakingalginate beads in 0.1% chitosan at pH 6.5 for 20min(McKnight, Ku, & Goosen, 1988) can form alginatebeads with a chitosan coating. The chitosan film, a semi-permeable membrane (Murata, Maeda, Miyamoto,& Kawashima, 1993), is a poly-electric complex. Low-molecular-weight chitosan provides a denser membrane

ARTICLE IN PRESS

*Corresponding author. Tel.: +61-7-33469192; fax: +61-7-33651177.

E-mail address: b.bhandari@uq.edu.au (B. Bhandari).

0958-6946/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.idairyj.2004.01.004

than high-molecular-weight chitosan (McKnight et al.,1988) and cell release is reduced by 40% (Zhou,Martins, Groboillot, Champagne, & Neufeld, 1998).Coating of beads with a poly-amino acid, such as

PLL, has potential application in foods (Champagne,Gaudy, Poncelet, & Neufeld, 1992; Larisch, Poncelet, &Champagne, 1994). The positively charged PLL forms acomplex with surface alginate to form a semi-permeablemembrane. Coating with a second layer of alginateneutralizes any excess PLL on the surface and increasesbead strength.This paper reports a study of the influence of coating

materials on the properties of alginate beads (such assize and internal appearance by transmission electronmicroscopy (TEM) and mechanical strength by textureanalysis) and the survivability in simulated digestivesystems of microencapsulated probiotic bacteria in thebeads coated with various types of coating materials.

2. Materials and methods

2.1. Preparation of microorganisms

A slant of Lactobacillus acidophilus 547 (LA 547)(Culture collection of the University of Queensland,Australia) and a slant of L. casei 01 (LC 01) (Chr.Hansen Pty Ltd., Australia) were inoculated into 10mLMRS (de Man, Rogosa, Sharpe) broth (Oxoid,Australia) and incubated at 37�C for 24 h under aerobicconditions. Bifidobacterium bifidum (BB) (ATTC 1994or CSCC 1909, CSIRO Starter Culture Collection,Australia) was grown in 18mL MRS broth, supplemen-ted with 0.05% l-cysteine hydrochloride (Sigma,Australia) MMRS (Ventling & Mistry, 1993) to providean anaerobic environment, at 37�C for 48 h underanaerobic conditions using the Gas Pak system (Mitsu-bishi Gas Chemical Company Inc., Australia). Thecultures were transferred into 95mL MRS broth forL. acidophilus and L. casei, and 180mL MMRS forB. bifidum and incubated under the same conditions.The cells were harvested by centrifuging at 1500g for15min at 25�C and washed twice with sterile 0.1%peptone solution.

2.2. Microencapsulation of microorganisms

The extrusion technique of microencapsulation wasused (Zhou et al., 1998; Krasaekoopt et al., 2003). Afterwashing, the cultures were suspended in 5mL of sterile0.1% peptone solution and mixed with 20mL of 2%(w/v) sodium alginate solution (D 3247 AJAX Chemi-cals Ltd., Australia), sterilized at 121�C for 15min. Thecell suspension was injected through a 0.11-mm needleinto sterile 0.05m CaCl2. The beads were allowed tostand for 30min for gelification, and then rinsed with,

and subsequently kept in, sterile 0.1% peptone solutionat 4�C.

2.3. Coating of alginate beads

Low-molecular-weight chitosan, low-molecular-weight PLL in combination with alginate, and alginatewere selected for this experiment. Coating proceduresfollowed the methods reported by Zhou et al. (1998) andChampagne et al. (1992).

2.3.1. Coating with chitosan

Low-molecular-weight chitosan (0.4 g; specification:low viscosity 14mPa in 1% w/v solution; Fluka,Australia) was dissolved in 90mL distilled water,acidified with 0.4mL of glacial acetic acid to achieve afinal concentration of 0.4% (w/v). The pH was thenadjusted to between 5.7 and 6 by adding 1m NaOH. Themixture was filtered through Whatman #4 filter paperand the volume adjusted to 100mL before autoclavingat 121�C for 15min. Then 15 g of washed beads wereimmersed in 100mL of chitosan solution and shaken at100 rpm for 40min on an orbital shaker for coating. Thechitosan-coated beads were washed with, and kept in0.1% peptone solution at 4�C.

2.3.2. Coating with poly-l-lysine and alginate

Uncoated beads (15 g) were suspended in 100mL of0.05% (w/v) low-molecular-weight (1000–4000) PLL(Sigma, Australia), sterilized by using a 0.22 mm Milli-pore filter. The suspension was agitated at 100 rpmfor 20min on an orbital shaker. The beads were rinsedwith 0.1% peptone solution, added into 100mL of0.17% alginate solution (sterilized at 121�C), andshaken at 100 rpm for 20min on an orbital shaker.The beads were washed with, and kept in 0.1% peptonesolution at 4�C.

2.3.3. Coating with alginate

Washed beads (15 g) were mixed with 100mL of0.17% sterile sodium alginate solution at 100 rpm for20min on an orbital shaker, then rinsed with, and keptin 0.1% peptone solution at 4�C.

2.4. Analysis of number of entrapped cells, size, and

mechanical strength of alginate beads

Freshly prepared beads (1 g), except for chitosan-coated beads, were liquefied in 99mL of 1% (w/v) sterilesodium citrate solution at pH 6.0 by gently shaking atroom temperature for 10min. Since chitosan-coatedbeads did not dissolve in sodium citrate solution, theywere blended in a stomacher for 1min and then allowedto stand for 10min to dissolve. L. acidophilus andL. casei were enumerated on MRS agar at 37�C underaerobic conditions for 72 h, while B. bifidum was

ARTICLE IN PRESSW. Krasaekoopt et al. / International Dairy Journal 14 (2004) 737–743738

cultured on reinforced clostridia medium agar (RCM)(Oxoid, Australia) at 37�C under anaerobic conditionsfor 72 h.The diameters of 120 randomly selected beads of

each treatment were measured with an eyepiece micro-meter on an optical microscope at a magnification of100� . A texture analyser (Model TA-XT2, MicrostableSystem, UK) was used to measure the gel strength ofthe beads. The 35mm diameter cylindrical aluminiumprobe at a speed of 0.1mm s�1 in a compression mode,and the rupture distance of 1.0mm was used. The peakforce was measured in grams. Ten beads were testedeach time and 10 replications were applied for eachtreatment.

2.5. Survival of microencapsulated cells in bile salt

solution

Freshly prepared beads (1 g) were placed into a tubecontaining 10mL of 0.6% bile salt solution (Oxoid,Australia) at pH 8.25, sterilized by autoclaving at 121�Cfor 15min, and incubated at 37� for up to 2 h. The beadswere removed and rinsed with 0.1% peptone solution atvarious time intervals. The viable cells of each bacteriumwere then enumerated using methods described inSection 2.4.

2.6. Survival of microencapsulated cells after sequential

incubation in simulated gastric juice and simulated

intestinal juice with and without bile salt

This analysis was based on the method describedby Rao et al. (1989). Freshly prepared beads (1 g)were placed in a tube containing 10mL of sterilesimulated gastric juice (0.08m HCl containing 0.2%NaCl, pH 1.55) without pepsin and incubated at 37�Cfor 30, 60, 90, and 120min. After incubation, thebeads were removed and placed in 9mL of sterilesimulated intestinal juice (0.05m KH2PO4, pH 7.43)with or without 0.6% bile salt. The tubes were thenincubated at 37�C for 150min. After incubation, a1.0-mL aliquot of dissolved beads of each bacteriumwas removed and assayed using method described inSection 2.4.

2.7. Statistical analysis

A randomized block design with three replicationsand analysis of variance (ANOVA) were used except forthe study of survival of microencapsulated cells aftersequential incubation in simulated gastric juice andsimulated intestinal juice with and without bile saltwhere a 2� 5 factorial design with three replications wasused. Comparisons of means were carried out usingDuncan’s multiple range tests.

3. Results and discussion

3.1. Number of cells entrapped, size, internal appearance

and mechanical strength of alginate beads

The initial cell count before encapsulation was in therange of 9.01–10.01 log cfumL�1. High cell loading inthe range of 9.0–9.2 (log cfu g�1 beads) was achieved inboth uncoated and coated beads. There were nosignificant differences (p > 0:05) between coated anduncoated beads. The results represented a yield afterencapsulation and coating of 99.9%. The loss duringencapsulation and coating was very low due to thegentle methods used. This result implies that theencapsulation and coating methods had no effect oncell viability.The beads were globular in shape. The type of coating

materials had no influence on the size of the beads in thisexperiment. The diameter of uncoated beads was1.62mm, which was significantly lower than that ofcoated beads (1.89mm) for all encapsulated probiotics.Using the same extrusion technique as used in thisexperiment, Prevost, Divies, and Rousseau (1985) andPrevost and Divies (1987, 1988) obtained a larger beadsize of 2.5mm. Moreover, Koo, Cho, Huh, Baek, andPark (2001) reported that the shape and size of the beadswere not changed when chitosan was added to alginatebeads.The internal appearances of coated and uncoated

alginate beads are shown in Fig. 1. Formation of a skinaround the bead periphery was observed. The thicknessof the calcium alginate membrane increased as a resultof the addition of coating materials. However, differ-ences among beads with different coating materials wereundetectable by a TEM.The mechanical strength of uncoated and coated

alginate beads was 13.6671.13 g. There were no detect-able differences among the treatments according tomeasurements by the texture analysis. Therefore coatingdid not appear to increase the strength of the beads.

3.2. Survival of microencapsulated cells in bile salt

solution

Although, bile tolerance is often used as a criterionfor probiotic strain selection, bile salt solution was usedhere to determine whether coating of the alginate beadswould increase survival of cells in this environment,which is similar to that of the digestive system. Thesurvivability of the probiotics, L. acidophilus, B. bifidum,and L. casei, was expressed as the destructive value(D-value), which is the time required to destroy 90% orone log cycle of the organism. The D-values of free cellsand microencapsulated probiotics in both uncoated andcoated beads in 0.6% bile salt solution (pH 8.25) at37�C for 2 h are shown in Table 1.

ARTICLE IN PRESSW. Krasaekoopt et al. / International Dairy Journal 14 (2004) 737–743 739

For L. acidophilus, with initial cell counts in the rangeof 1.171.2� 109 to 1.970.1� 109, the survival of cellsin both coated and uncoated beads was significantly

(po0:05) better than that of free cells. Chitosan andalginate coating provided the best protection, followedby PLL-alginate coating, uncoated, and free cells. ForB. bifidum, with initial cell counts in the range of8.270.4� 108 to 4.170.3� 109, only chitosan-coatedbeads were significantly better than the others, whichwere not significantly different from each other. ForL. casei, with initial cell counts in the range of4.270.6� 109 to 9.470.3� 109, chitosan coating pro-vided the best protection followed by alginate and PLL-alginate coatings, which provided similar protection. Inall cases the uncoated alginate beads provided lessprotection then the coated beads (Table 1).The chitosan coating provides the best protection in

bile salt solution because an ion-exchange reaction takesplace when the beads absorb bile salt (Murata, Toniwa,Miyamoto, & Kawashima, 1999). An insoluble complexbetween chitosan and bile salt forms in the chitosan–alginate membrane. Therefore, the diffusion of bile saltinto the beads may be limited. This will protectencapsulated cells from interacting with the bile salt.Yu, Yim, Lee, and Heo (2001) and Koo et al. (2001) alsoreported that bifidobacteria and L. casei entrapped inalginate beads containing chitosan had higher viabilitythan in alginate without chitosan.In the case of chitosan-coated beads, L. acidophilus

(D-value=31.7min) showed less resistance to bile saltcompared to B. bifidum (86.0min) and L. casei

(81.5min). Sultana et al. (2000) reported that alginateencapsulated L. acidophilus and L. casei decreased by

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Fig. 1. TEM images of uncoated and coated alginate beads at � 6000

magnification: (a) uncoated bead, (b) chitosan-coated bead, (c)

alginate-coated bead, and (d) PLL+alginate-coated bead.

Table 1

Number of survival cells and D-values of free and microencapsulated cells of Lactobacillus acidophilus, Bifidobacterium bifidum, and Lactobacillus

casei after incubation in 0.6% bile salt solutions (pH 8.25) at 37�C for 2 h, with three replications

Microorganism Coating

material

No. of survival cells after expose in 0.6% bile salt solution at different times

(cfu g�1 bead)

D-value (min) Regression

coefficient (r2)

0min 30min 60min 90min 120min

Lactobacillus

acidophilus

Uncoated 1.970.1� 109 1.970.5� 107 6.371.8� 105 4.470.7� 104 3.971.1� 103 22.270.5c� 0.98

Chitosan 1.270.4� 109 1.470.1� 108 1.070.1� 107 4.170.7� 106 1.170.4� 105 31.771.5a 0.97

Alginate 1.171.2� 109 1.570.1� 108 1.270.1� 107 3.770.2� 106 1.670.9� 105 32.072.0a 0.98

PLL-alginate 1.370.2� 109 5.471.9� 107 1.770.9� 106 1.171.0� 106 5.771.7� 104 27.773.2b 0.96

Free cell 1.470.5� 109 8.970.3� 106 6.071.8� 104 4.670.9� 103 1.070.2� 102 16.970.5d 0.98

Bifidobacterium

bifidum

Uncoated 4.170.3� 109 6.770.4� 107 7.670.4� 105 6.670.3� 104 1.170.1� 103 18.470.5b 0.99

Chitosan 1.771.1� 109 7.770.7� 108 5.6 71.0� 108 1.5 70.8� 108 6.97 0.4� 107 86.070.9a 0.97

Alginate 8.270.4� 108 1.170.6� 108 7.470.3� 106 1.171.5� 105 2.671.1� 104 24.070.7b 0.98

PLL-alginate 1.071.5� 109 8.670.4� 107 1.270.8� 107 9.370.9� 104 3.371.1� 103 22.071.2b 0.98

Free cell 2.770.2� 109 7.571.0� 106 1.370.6� 106 3.471.2� 105 4.271.4� 102 19.770.8b 0.93

Lactobacillus

casei

Uncoated 9.470.3� 109 2.170.7� 109 3.671.0� 108 3.071.4� 107 3.271.2� 103 19.571.1c 0.84

Chitosan 5.870.4� 109 4.671.1� 109 4.170.5� 109 1.370.2� 109 1.770.3� 108 81.571.7a 0.81

Alginate 4.270.6� 109 2.370.5� 109 6.471.4� 108 4.571.5� 107 1.771.0� 106 33.671.2b 0.92

PLL-alginate 5.270.7� 109 3.470.4� 109 1.170.4� 109 1.070.5� 108 1.970.9� 106 34.371.7b 0.88

Free cell 6.170.1� 109 6.571.2� 106 1.170.2� 106 8.170.4� 105 1.570.1� 102 18.570.6c 0.88

�Values followed by the same letters are not significantly different (p > 0:05). Statistical analysis of each bacterium was done separately.

W. Krasaekoopt et al. / International Dairy Journal 14 (2004) 737–743740

two log cycles compared to the initial cell count in 1%and 2% bile salt solutions. Moreover, in this experimentthe survival decreased proportionately with the time thecells were exposed to bile salt solutions, which wassimilar to that reported by Lee and Heo (2000).

3.3. Survival of probiotics after sequential incubation in

simulated gastric juice and simulated intestinal juice with

and without bile salt

To determine the effect of the acidic pH of thestomach on the survival of encapsulated probiotics, anin vitro system was utilized. The cultures were put intosimulated gastric juice for 2 h, followed by in intestinaljuice with or without 0.6% bile salt for 2.5 h. The resultsare shown in Table 2. There was no survival ofB. bifidum, either encapsulated or as free cells, in thepresence of gastric juice due to its low acid resistance.The cell numbers decreased to an undetectable levelwithin 15min. This concurs with the studies of Sun andGriffiths (2000) and Sultana et al. (2000) on B. infantis

(the most acid-resistant strain). On the other hand, Raoet al. (1989) reported that the microencapsulatedB. pseudolongum resisted the simulated gastric andintestinal juices up to 60min but after that time, noneof the organisms survived. Many scientists have also

reported that survival of microencapsulated B. longum

(Woo, Lee, & Heo, 1999; Lee & Heo, 2000; Lee et al.,2001a), and B. breve (Lee et al., 2001b) was higher thanthat of the free cells. Moreover, Yu et al. (2001) foundthat the survival rate of bifidobacteria in alginate beadscontaining chitosan was higher than that of alginatebeads.The initial cell count of L. acidophilus was in the range

of 5.970.2� 108 to 2.570.5� 109. Microencapsulatedcells survived better than free cells after sequentialincubation in simulated gastric and intestinal juices,with and without bile salt, and chitosan coatingenhanced the survivability of cells more than othercoating materials (Table 2).The D-values of the cells in the alginate, PLL-alginate

coated, and uncoated beads were not significantlydifferent (p > 0:05) for incubation without bile salt. Thisimplies that alginate and PLL-alginate coating could notimprove the survival of L. acidophilus under theseconditions. This result of uncoated beads is in contrastto that of Sultana et al. (2000), who found thatencapsulation of bacteria in alginate beads did noteffectively protect the organisms from high acidity. Onthe other hand, for the incubation with bile salt, the cellsin uncoated beads (D-value=33.8min) survived betterthan those in alginate (26.3min) and PLL-alginate

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Table 2

Number of survival cells and D-values of free and microencapsulated cells of Lactobacillus acidophilus and L. casei after sequential incubation in

simulated gastric juice (pH 1.55) for 2 h and simulated intestinal juice (pH 7.43), with and without 0.6% bile salt, at 37�C for 150min, with three

replications

Microorganism Condition Coating

material

No. of survival cells (cfu g�1 bead) D-value

(min)

Regression

coefficient (r2)

0min 30min 60min 90min 120min

Lactobacillus

acidophilus

Bile Uncoated 8.670.2� 108 9.770.2� 106 4.671.8� 106 1.371.5� 106 3.071.7� 105 33.871.3b� 0.88

Chitosan 1.170.4� 109 1.870.9� 107 8.170.2� 106 1.870.8� 106 1.570.9� 106 46.172.0a 0.84

Alginate 1.470.2� 109 1.570.2� 108 1.570.7� 107 5.371.3� 106 1.370.4� 104 26.371.5cd 0.92

PLL-alginate 1.271.1� 109 8.370.1� 107 1.270.2� 106 1.571.2� 105 1.071.3� 104 22.072.6de 0.98

Free cell 2.070.7� 109 1.671.1� 106 1.270.8� 105 3.471.7� 103 3.270.5� 102 18.471.0e 0.95

No bile Uncoated 1.670.1� 109 7.971.2� 108 2.070.2� 108 2.370.4� 107 8.571.0� 105 35.374.2b 0.93

Chitosan 1.070.5� 109 6.670.6� 108 3.870.6� 108 3.471.8� 107 5.870.3� 106 50.074.1a 0.91

Alginate 5.970.3� 108 2.871.3� 107 1.270.2� 107 1.671.1� 106 2.071.7� 105 32.671.1b 0.97

PLL-alginate 2.570.5� 109 2.570.2� 107 1.070.2� 106 3.871.4� 105 2.171.0� 105 31.070.9bc 0.87

Free cell 2.070.2� 109 4.270.9� 106 2.870.7� 105 4.071.5� 104 2.370.9� 103 18.871.4e 0.94

Lactobacillus

casei

Bile Uncoated 9.070.6� 109 1.770.8� 109 1.871.9� 108 1.270.6� 106 9.270.7� 103 26.071.4c 0.94

Chitosan 9.870.3� 109 3.470.9� 109 2.270.6� 108 4.471.5� 107 1.671.8� 106 30.471.1b 0.97

Alginate 8.470.4� 109 4.870.4� 108 9.771.0� 105 2.272.1� 104 6.770.7� 103 18.670.5d 0.95

PLL-alginate 7.870.2� 109 4.470.6� 107 6.270.9� 106 6.670.5� 105 7.070.6� 103 20.372.5d 0.97

Free cell 1.770.1� 1010 1.07� 0.4108 4.87� 0.7106 2.270.8� 104 2.371.3� 102 15.270.4e 0.99

No bile Uncoated 6.671.2� 109 1.570.4� 106 6.270.6� 105 1.670.6� 105 4.371.6� 104 24.773.5c 0.77

Chitosan 7.970.5� 109 7.070.7� 109 9.870.2� 107 7.071.5� 107 4.270.9� 106 34.872.1a 0.91

Alginate 1.070.4� 1010 7.970.2� 109 2.171.3� 108 3.171.4� 105 2.271.4� 104 18.570.8d 0.92

PLL-alginate 8.070.7� 109 6.170.3� 109 1.871.6� 107 1.771.0� 105 1.671.6� 104 18.371.1d 0.94

Free cell 1.170.2� 1010 7.371.8� 109 9.870.6� 108 2.370.4� 105 1.170.1� 104 18.170.4e 0.88

�Values followed by the same letters are not significantly different (p > 0:05). Statistical analysis of each bacterium was done separately.

W. Krasaekoopt et al. / International Dairy Journal 14 (2004) 737–743 741

(22.0min) coated beads; these two D-values werenot significantly different (p > 0:05). These results alsoindicate that alginate and PLL-alginate coating couldnot increase the survivability of encapsulated cells. TheD-value of L. acidophilus in chitosan-coated beads in0.6% bile salt solution (31.7min) was lower than theD-value in simulated digestive juice containing bile salt(46.1min). This may be due to the fact that the pH ofthe bile salt testing solution was 8.25, which may not besuitable for this organism as it grows and survives betterin an acidic environment. After exposure to simulatedgastric juice, the organisms were put into simulatedintestinal juice containing bile salt at pH 7.43 (similar tothe pH in the small intestine). Moreover, the use ofdifferent batches of encapsulated organisms for thosetwo experiments may have contributed to the differenttolerances observed.In the case of L. casei, the initial cell count was in the

range of 6.670.2� 109 to 1.770.1� 1010. Chitosanpresented the best protection of cells both with andwithout bile salt (Table 2). For incubation without bilesalt, there were no significant differences (p > 0:05)among alginate-coated beads, PLL-alginate-coatedbeads, and free cells. In contrast, for the incubationwith bile salt, the survival of cells in the alginate-coatedbeads did not significantly (p > 0:05) differ from that ofcells in PLL-alginate-coated beads. Thus, the presenceof bile salt in the simulated intestinal juice had no effecton the survival of this organism in any of the treatmentsexcept in chitosan-coated beads.

4. Conclusion

Coating of alginate beads with various materials(chitosan, poly-l-lysine/alginate, and alginate) increasedthe bead size by approximately 17%. Negligiblestructural differences were discernable by TEM amongthe beads with different coating materials, and themechanical strength of the beads was not affected bythe coatings. There was no survival of B. bifidum in thepresence of gastric juice due to its low acid resistance.Among three coating methods applied in this research,chitosan coating provided the best protection ofcells. For example, in bile salt solution, the chitosancoating increased the D-values of L. acidophilus,B. bifidum, and L. casei by approximately 1.5, 4.7, and4.2 times, respectively, as compared to the D-values inuncoated beads. Investigation on the stability of theseprobiotics in coated beads during a longer storageperiod is being pursued. Development of an improvedtechnique to produce micron size beads (rather thanmillimetre size produced in this research) to give asmooth texture when the beads are incorporated intoproducts is another area which needs further attentionfrom researchers.

References

Champagne, C. P., Gaudy, C., Poncelet, D., & Neufeld, R. J. (1992).

Lactococcus lactis release from calcium alginate beads. Applied and

Environmental Microbiology, 58(5), 1429–1434.

Gombotz, W. R., & Wee, S. F. (1998). Protein release from alginate

matrices. Advanced Drug Delivery Reviews, 31, 276–285.

Klein, J., Stock, J., & Vorlop, K. D. (1983). Pore size and properties of

spherical Ca-alginate biocatalysts. European Journal of Applied

Microbiology and Biotechnology, 18(1), 86–91.

Koo, S., Cho, Y., Huh, C., Baek, Y., & Park, J. (2001). Improvement

of the stability of Lactobacillus casei YIT 9018 by microencapsula-

tion using alginate and chitosan. Journal of Microbiology and

Biotechnology, 11(3), 376–383.

Krasaekoopt, W., Bhandari, B., & Deeth, H. (2003). Evaluation of

encapsulation techniques of probiotics for yoghurt: a review.

International Dairy Journal, 13(1), 3–13.

Larisch, B. C., Poncelet, D., & Champagne, C. P. (1994). Micro-

encapsulation of Lactococcus lactis subsp. cremoris. Journal of

Microencapsulation, 11(2), 189–195.

Lee, K., & Heo, T. (2000). Survival of Bifidobacterium longum

immobilized in calcium alginate beads in simulated gastric juices

and bile salt solution. Applied and Environmental Microbiology,

66(2), 869–873.

Lee, K., Kim, J., Lee, Y., Choi, E., Shin, D., & Heo, T. (2001a).

Estimating the viability of Bifidobacterium longum in Ca-alginate

beads against simulated gastroenteric juices. Journal of Microbiol-

ogy and Biotechnology, 11(1), 97–105.

Lee, K., Kim, J., Yu, W., Lee, Y., Yoon, S., & Heo, T. (2001b).

Survival of Bifidobacterium breve in acidic solutions and yogurt,

following immobilization in calcium alginate beads. Journal of

Microbiology and Biotechnology, 11(3), 412–417.

Martinsen, A., Skjak-Braek, C., & Smidsrod, O. (1989). Alginate as

immobilization material. I. Correlation between chemical and

physical properties of alginate gel beads. Biotechnology and

Bioengineering, 33(1), 79–89.

McKnight, C. A., Ku, A., & Goosen, M. F. A. (1988). Synthesis of

chitosan–alginate microencapsule membranes. Journal of Bioactive

and Compatible Polymers, 3(8), 334–354.

Murata, Y., Meada, T., Miyamoto, E., & Kawashima, S. (1993).

Preparation of chitosan-reinforced alginate gel beads-effect of

chitosan on gel matrix erosion. International Journal of Pharma-

ceutics, 96(1–3), 139–145.

Murata, Y., Toniwa, S., Miyamoto, E., & Kawashima, S. (1999).

Preparation of alginate gel beads containing chitosan salt and

their function. International Journal of Pharmaceutics, 176(2),

265–268.

Overgaard, S., Scharer, J. M., Moo-Young, M., & Bols, N. C.

(1991). Immobilization of hybridoma cells in chitosan alginate

beads. The Canadian Journal of Chemical Engineering, 69(4),

439–443.

Prevost, H., & Divies, C. (1988). Continuous pre-fermentation of milk

by entrapped yoghurt bacteria. I. Development of the process.

Milchwissenschaft, 43(10), 621–625.

Prevost, H., Divies, C., & Rousseau, E. (1985). Continuous yoghurt

production with Lactobacillus bulgaricus and Streptococcus

thermophilus entrapped in Ca-alginate. Biotechnology Letters,

7(4), 247–252.

Rao, A. V., Shiwnarain, N., & Maharaj, I. (1989). Survival of

microencapsulated Bifidobacterium pseudolongum in simulated

gastric and intestinal juices. Canadian Institute of Food Science

and Technology Journal, 22(4), 345–349.

Smidsrod, O., Haug, A., & Lian, B. (1972). Properties of poly(1,4-

heuronates) in the gel state. I. Evaluation of a method for the

determination of stiffness. Acta Chemica Scandinavica, 26(1),

71–78.

ARTICLE IN PRESSW. Krasaekoopt et al. / International Dairy Journal 14 (2004) 737–743742

Smidsrod, O., & Skjak-Braek, G. (1990). Alginate as immobilization

matrix for cells. Trends in Biotechnology, 8(3), 71–78.

Sultana, K., Godward, G., Reynolds, N., Arumugaswamy, R., Peiris,

P., & Kailasapathy, K. (2000). Encapsulation of probiotics bacteria

with alginate-starch and evaluation of survival in simulated

gastrointestinal conditions and in yoghurt. International Journal

of Food Microbiology, 62(1–2), 47–55.

Sun, W., & Griffiths, M. (2000). Survival of bifidobacteria in yoghurt and

simulated gastric juice following immobilization in gellan–xanthan

beads. International Journal of Food Microbiology, 61(1), 17–25.

Tanaka, H., Masatose, M., & Veleky, I. A. (1984). Diffusion

characteristics of substrates in Ca-alginate beads. Biotechnology

and Bioengineering, 26(1), 53–58.

Ventling, B. L., & Mistry, V. V. (1993). Growth characteristics of

bifidobacteria in ultrafiltered milk. Journal of Dairy Science, 76(4),

962–971.

Woo, C., Lee, K., & Heo, T. (1999). Improvement of Bifidobacterium

longum stability using cell-entrapment technique. Journal of

Microbiology and Biotechnology, 9(2), 132–139.

Yu, W., Yim, T., Lee, K., & Heo, T. (2001). Effect of skim milk-

alginate beads on survival rate of bifidobacteria. Biotechnology and

Bioprocess Engineering, 6(2), 133–138.

Zhou, Y., Martins, E., Groboillot, A., Champagne, C. P., & Neufeld,

R. J. (1998). Spectrophotometric quantification of lactic bacteria in

alginate and control of cell release with chitosan coating. Journal of

Applied Microbiology, 84(3), 342–348.

ARTICLE IN PRESSW. Krasaekoopt et al. / International Dairy Journal 14 (2004) 737–743 743