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International Dairy Journal 16 (2006) 1190–1195

www.elsevier.com/locate/idairyj

Effect of alginate concentrations on survival of microencapsulatedLactobacillus casei NCDC-298

S. Mandal�, A.K. Puniya, K. Singh

Dairy Microbiology Division, National Dairy Research Institute, Karnal-132001, Haryana, India

Received 15 January 2005; accepted 13 October 2005

Abstract

This study reports the tolerance of Lactobacillus casei NCDC-298 encapsulated in different alginate concentrations (2%, 3% or 4%),

to low pH (1.5), high bile salt concentration (1% or 2%) and heat processing (55, 60 or 65 1C for 20min). The release of encapsulated

cells in simulated aqueous solution of colonic pH was also assessed. The survival of encapsulated L. casei was better at low pH, high bile

salt concentration and during heat treatment as compared to free cells. The survival increased proportionately with increasing alginate

concentrations without affecting the release of entrapped cells in solution of colonic pH.

r 2005 Published by Elsevier Ltd.

Keywords: Microencapsulation; Lactobacilli; Probiotics; Survival

1. Introduction

Probiotics upon ingestion exert health benefits beyondinherent nutrition and should be at the level of 106–107 livemicroorganisms per gram of product at the time ofconsumption (Guarner & Schaafsma, 1998; Ouwehand &Salminen, 1998; Shah, 2000). However, the bacteria do notsurvive during processing in high numbers in dairyproducts (Dave & Shah, 1996; Hamilton-Miller, Shah, &Winkler, 1999; Kailasapathy & Rybka, 1997) and furtherresearch is required to increase the survival.

Since the viability and activity of probiotics are neededat the site of action, these should withstand the host’snatural barriers the gastrointestinal tract (GIT) transit.Lactobacillus spp. lack the ability to survive the harshacidity and bile concentration commonly encountered inthe GIT and also the high temperature of dairy processing(Conway, Gorbach, & Goldin, 1987; Gardiner et al., 2000;Hood & Zottola, 1988; Lankaputhra & Shah, 1995; Shah &Jelen, 1990; Silva, Carvahlo, Teixeira, & Gibbs, 2002).However, the stabilization of probiotics using a carrier mayimprove survival of these microbes in products, both

e front matter r 2005 Published by Elsevier Ltd.

airyj.2005.10.005

ing author.

ess: msurajit77@rediffmail.com (S. Mandal).

during processing and GIT transition (Goderska, Zybals,& Czarnecki, 2003).Microencapsulation of probiotics in hydrocolloid beads

has been tested for improving their viability in foodproducts and during GIT transit (Kebary, Hussein, &Badawi, 1998; Khalil & Mansour, 1998; Krasaekoopt,Bhandari, & Deeth, 2003). Microencapsulation usinggelatin or vegetable gum provides protection to acid-sensitive bifidobacteria (Rao, Shiwnarain, & Mahraj,1989); however, the most widely used matrix for micro-encapsulation is alginate (Kailasapathy, 2002). Alginatebeads have been found to increase the survival ofprobiotics by up to 80–95% (Audet, Paquin, & Lacroix,1988; Jankowski, Zielinska, & Wysakowska, 1997;Krasaekoopt et al., 2003; Sheu & Marshall, 1991; Sheu,Marshall, & Heymann, 1993). Encapsulation of Lactoba-

cillus rhamnosus in alginate improved survival at pH 2.0 upto 48 h, while the free cells were destroyed completely(Goderska et al., 2003). Similarly, the death rate ofBifidobacterium longum, immobilized in alginate decreasedproportionately with increasing alginate concentrations(2–4%) and bead size (Lee & Heo, 2000). Microencapsula-tion of bifidobacteria also exhibited a lower populationreduction during exposure to simulated gastric environ-ment and bile solution (Picot & Lacroix, 2004).

ARTICLE IN PRESSS. Mandal et al. / International Dairy Journal 16 (2006) 1190–1195 1191

Little research has been carried out with an aim toincorporate probiotics into heat-treated foods, due todestruction of live culture during heat treatment. Thoughencapsulating lactobacilli in calcium-alginate, beads havebeen found to improve their heat tolerance (Selmer-Olsen,Sorhaug, Birkeland, & Pehrson, 1999), there are nosystematic reports on survival of encapsulated probioticsin varying alginate concentrations and release of bacteriafrom alginate matrix. This paper reports the effect ofvarying concentrations of alginate on survival of micro-encapsulated probiotic bacteria after heat treatments (55,60 or 65 1C for 20min), low pH and high bile saltconcentration.

2. Materials and methods

2.1. Microorganisms

A freeze-dried ampoule of Lactobacillus casei NCDC-298 procured from National Collection of Dairy Cultures(NCDC), Karnal, India was activated in chalk litmus milk(37 1C, 24 h) and maintained in a refrigerator (771 1C)before being sub-cultured monthly. The culture wasreactivated by transferring 2–3 times in MRS broth andthe cells were harvested (80mL) by centrifugation at 2000g

for 10min at 4 1C (Kubota High Speed Centrifuge, Osaka,Japan). The cells were washed twice before resuspendingthem in 5mL normal saline. The final cell concentrationwas adjusted to 1.0� 1011 cfumL�1.

2.2. Microencapsulation

L. casei NCDC-298 cells were encapsulated in sodiumalginate matrix as described by Sheu et al. (1993). Sodiumalginate solutions (2%, 3% or 4%) were prepared,sterilized by autoclaving (1201C for 15min) and cooled to38–40 1C. Twenty millilitres of this solution and 4mL ofcell suspension were transferred into a centrifuge tube(40mL) and the content was vortexed to homogeneity.Soybean oil (100mL) containing 0.2% Tween 80 (emulsi-fier) was taken in a beaker (500mL) and to this thealginate–cell mixture was added dropwise while stirringmagnetically. After 5min, a uniformly turbid emulsion wasobtained to which 0.1M calcium chloride (100mL) wasquickly added for hardening of microcapsules and break-ing the emulsion. The capsules were harvested by centrifu-ging at 350g for 10min at 4 1C and washed twice withdistilled water. The beads were separated by filtration usingWhatman filter paper (No. 1), transferred to a sterile Petridish and stored in a refrigerator (771 1C).

2.3. Survival of microencapsulated and free cells at low pH

and high bile salt concentration

Simulated gastric solution (pH 1.5) containing 0.2%NaCl was prepared as per Rao et al. (1989). Freshlyprepared microcapsules (1 g) or 1mL of the free cell

suspension (1010 cells mL�1), placed separately in test tubescontaining 10mL simulated gastric pH solution, wereincubated at 37 1C (Lee & Heo, 2000). At the end of 1 and3 h (Charteris, Kelly, Morelli, & Collins, 1998), beads wereharvested, washed and immediately used for enumerationof viable cells after depolymerization of the capsules in10mL phosphate buffer (0.1 M, pH 7.170.2), followed byplating using MRS agar at 37 1C for 48 h (Sheu &Marshall, 1993).Tolerance of microencapsulated lactobacilli to simulated

bile salt concentration of small intestine of human wascarried out as per Lee and Heo (2000). Similar to low pHtolerance, 1 g of microcapsules or 1mL of the free cellsuspension (1010 cellsmL�1) were transferred in test tubescontaining 10mL of 1% or 2% bile salt and incubated at37 1C. At the end of 3 and 12 h (Clark & Martin, 1994),enumeration of viable cells was carried out.

2.4. Survival of free and encapsulated cells under heat

treatments

Tolerance of encapsulated L. casei NCDC-298 to heattreatment (55, 60 or 65 1C for 20min) was studied usingdistilled water (pH 6.470.2) as a suspending medium. Onegram of microcapsules or 1mL of the free cell suspension(1010 cellsmL�1) was transferred in test tubes containing10mL of distilled water. After the heat treatment, thecontent was cooled to room temperature (�30 1C) andviable cells were enumerated as described in Section 2.3.

2.5. Release of encapsulated cells

In vitro release of encapsulated L. casei NCDC-298 atsimulated colonic pH was studied out as described by Raoet al. (1989). Microcapsules (1 g) were transferred into10mL simulated colonic pH solution (0.1 M KH2PO4, pH7.470.2), mixed gently and incubated at 37 1C. At 0.5, 1, 2and 3 h time intervals, aliquots (1mL) were taken andviable counts were enumerated as described in Section 2.3.

2.6. Analysis of data

Data were recorded as mean7S.E. of four independentreplicates carried out on different days with freshlyprepared cultures and media. Analysis of variance wascarried out using ‘General Linear Models’ of SYSTATVersion 6.0.1 (SPSS Inc., 1996) and treatment means werecompared by ‘Fisher’s Least Significant Difference’ at 5%level.

3. Results and discussions

3.1. Survival of encapsulated cells in simulated gastric pH

and intestinal bile salt solutions

No significant reduction in viable count was observed infree as well encapsulated cells in distilled water (pH 6.5) on

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0 3 120

2

4

6

8

10

0% bile 1% bile 2% bile

ap ap ap ap ap

bqcr

bq

cr

Incubation period (h)

Log

cou

nt (c

fu m

L-1

)

Fig. 1. Effect of bile salt on viable counts of free L. casei NCDC-298

(log cfumL�1). Error bars indicate the standard error of the mean (n ¼ 4).

Mean bars with different letters (a–c) in same bile salt concentration at

different incubation periods differ significantly ðPo0:05Þ. Mean bars with

different letters (p–r) at same incubation period in different bile salt

concentrations differ significantly ðPo0:05Þ.

S. Mandal et al. / International Dairy Journal 16 (2006) 1190–11951192

incubation for up to 3 h. However, there were significantreductions ðPo0:05Þ of free as well 2% alginate encapsu-lated cells on immediate exposure to pH 1.5. Free cells weredrastically reduced to 4.24 and 3.38 log cfumL�1 at the endof 1 and 3 h at pH 1.5. After 1 h of incubation, viability wassignificantly higher in 2% alginate beads, than free cells,but was lower as compared with 3% and 4% alginate beadsðPo0:05Þ. Further, at 3 h exposure, the highest survival ofcell was recorded in 4% alginate beads, followed by 3%and 2%. Thus, viability of encapsulated L. casei NCDC-298 cells improved with increasing alginate concentration(Table 1). Chandramouli, Kailasapathy, Peiris, and Jones(2004) reported a significant increase in viable L. acid-

ophilus at pH 2.0 when encapsulated in alginate. Highersurvival of immobilized bifidobacteria at acidic pH of GITwas observed (Guerin, Vuillemard, & Subirade, 2003; Picot& Lacroix, 2004; Wenrong & Griffiths, 2000). Micro-encapsulated cells of L. acidophilus in alginate beadssurvived better after sequential incubation in simulatedgastric and intestinal juices (Krasaekoopt, Bhandari, &Deeth, 2004). Higher survival was also reported whenlactobacilli immobilized in alginate beads were incubatedin simulated gastric fluid (Lee, Cha, & Park, 2004; Le-Tien,Millette, Mateescu, & Lacroix, 2004). The death rate ofB. longum entrapped in alginate beads decreased propor-tionately with increased capsules size and alginate con-centration (Lee & Heo, 2000). However, our results are incontrast to other workers (Hansen, Allan-Wojtas, Jin, &Paulson, 2002; Sultana et al., 2000; Trindade & Grosso,2000) who reported that microencapsulation in alginatebead did not effectively protect the microorganisms fromlow pH.

There was no significant difference in the viability of freecells in distilled water, indicating that water had no effecton the survival. Free cells decreased from 9.45 to7.29 log cfumL�1 and 9.34 to 5.60 log cfumL�1 on expo-sure to 1% and 2% bile salt, respectively after 12 h (Fig. 1).Encapsulation with alginate concetration increasing from2 to 4% improved the viability of cells at similar bile saltconcentrations (Fig. 2). The viability of L. casei NCDC-298 decreased proportionately with time of exposure to bilesalt and higher survival of cells was obtained on

Table 1

Effect of pH on viable counts of free and microencapsulated L. casei NCDC-

Alginate concentration pH 6.5

0 h 1 h 3 h

0% (free cells) 9.3370.12ap 9.4570.12ap 9.2

2% 9.5770.14ap 9.3570.13ap 9.3

3% 9.3970.08ap 9.3970.03ap 9.2

4% 9.3770.13ap 9.4670.253ap 9.6

aData are mean7S.E., n ¼ 4. Means in same row with different superscript

superscripts (p–s) differ significantly ðPo0:05Þ.

encapsulation in alginate. The results of viability ofencapsulated cells are in accordance with other researchers(Krasaekoopt et al., 2004; Lee & Heo, 2000). Chandra-mouli et al. (2004) found that encapsulation ofL. acidophilus in alginate significantly increased theviability in 1% bile salt. In contrast, Guerin et al. (2003)reported higher mortality of bifidobacteria, immobilized inpolysaccharide–protein gel beads, when exposed to 2% and4% bile salt. Trindade and Grosso (2000) also reportedthat immobilization of B. bifidum and L. acidophilus in Ca-alginate beads was not effective in protecting the cells from2% and 4% bile salt.

3.2. Survival of encapsulated cells after heat treatments

Free cells in distilled water (9.20 log cfumL�1) weredrastically reduced to 5.55, 4.93 and 3.98 log cfumL�1 onheat treatments at 55, 60 or 65 1C for 20min, respectively

298 (log cfumL�1 for free cells and log cfu g�1 for encapsulated cells)a

pH 1.5

0 h 1 h 3 h

970.08ap 7.5670.17bq 4.2470.52cr 3.3870.21cs

470.10ap 8.4270.12bq 5.9670.27cq 5.3770.12cr

370.07ap 9.2670.47ap 7.0470.18bp 6.2770.08cq

770.12ap 9.4170.14ap 8.0770.32bp 7.5470.43bp

s (a–d) differ significantly ðPo0:05Þ. Means in same column with different

ARTICLE IN PRESS

Initial 0 200

2

4

6

8

1055°C 60°C 65°C

ap ap ap ap

bq

br

bp

cq

cr

Heating period (min)

Log

cou

nt (c

fu m

L-1

)

Fig. 3. Effect of heat treatments on viable counts of free L. casei NCDC-

298 (log cfumL�1). Error bars indicate the standard error of the mean

ðn ¼ 4Þ. Mean bars with different letters (a–c) at same temperature for

different heating period differ significantly ðPo0:05Þ. Mean bars with

different letters (p–r) for same heating period in different temperature

differ significantly ðPo0:05Þ.

0 3

3

12 0

2

4

6

8

10

2% Alginate

2% Alginate

2% Alginate

3 % Alginate

3 % Alginate

4% Alginate

4% Alginate

3 % Alginate 4% Alginate

apap ap ap ap ap ap ap ap

Incubation period (h)

Incubation period (h)

Incubation period (h)

Log

cou

nts

(cfu

g-1

)L

og c

ount

s (c

fu g

-1)

Log

cou

nts

(cfu

g-1

)

0 120

2

4

6

8

10 apap ap

ab p ap bpbq bpq bp

0 3 120

2

4

6

8

10ap ap ap

bqbpq

bp

bqbp bp

(A)

(B)

(C)

Fig. 2. Effect of bile salt on viable counts of encapsulated L. casei NCDC-

298 (log cfu g�1): (A) control (0% bile); (B) 1% bile and (C) 2% bile. Error

bars indicate the standard error of the mean ðn ¼ 4Þ. Mean bars with

different letters (a,b) in same bile salt and alginate concentration at

different incubation periods differ significantly ðPo0:05Þ. Mean bars with

different letters (p,q) in same bile salt concentration and at incubation

period with different alginate concentrations differ significantly ðPo0:05Þ.

S. Mandal et al. / International Dairy Journal 16 (2006) 1190–1195 1193

(Fig. 3). Survival of the lactobacilli was found to increaseon alginate encapsulation and also with increasingconcentrations of alginate in capsules. The slower diffusionof water in 4% alginate matrix during heat treatment mighthave led to the higher survival of L. casei NCDC-298 (Fig.4). Lower diffusion of glucose and ethanol was reported inconcentrated alginate gels due to decreased number and

length of pores rather than decrease in pore diameter(Hannoun & Stephanopoulos, 1986).

3.3. Release of encapsulated cells in simulated colonic pH

solution

The release of cells from microcapsules in colon isessential for growth and colonization of probiotics;otherwise the microorganisms in the beads will be washedout from the body without exerting any beneficial effect.The released cell counts were between 3.40 and3.70 log cfu g�1 on immediate exposure to the solution ofsimulated colonic pH from an initial count�9.40 log cfu g�1. With increased incubation, the releaseof cells was increased and beyond 60min there was nosignificant change ðP40:05Þ indicating no effect of alginateconcentrations on the release of cells from microcapsules(Fig. 5). An efficient release of viable and metabolicallyactive cells in the intestine is one of the aims ofmicroencapsulation (Suita-Cruz & Goulet, 2001). Similarto our observations, a progressive release of viable cellsfrom whey protein-based microcapsules in simulatedintestinal conditions was reported by Picot and Lacroix(2004).

4. Conclusions

Microencapsulation of L. casei NCDC-298 in alginatebeads resulted in better survival than for free cells at lowpH, high bile salt concentration and heat treatment.Increasing alginate concentrations also had a positiveeffect on the survival of L. casei in simulated harsh condi-tions of GIT and heat processing, without significantly

ARTICLE IN PRESS

(A)Initial 0 20

0

2

4

6

8

10

2% Alginate 3% Alginate 4% Alginate

2% Alginate 3% Alginate 4% Alginate

2% Alginate 3% Alginate 4% Alginate

ap ap apbp bp bp

Heating period (min)

Heating period (min)

Heating period (min)

Log

cou

nts

(cfu

g-1

)L

og c

ount

s (c

fu g

-1)

Log

cou

nts

(cfu

g-1

)

crcq

cp

(B)Initial 0 20

0

2

4

6

8

10 ap ap ap

bp bp bp

cq cqcp

(C)Initial 0 20

0

2

4

6

8

10 ap ap ap

brbq

bp

cqcp cp

Fig. 4. Effect of heat treatments on viable counts of encapsulated L. casei

NCDC-298 (log cfu g�1): (A) 55 1C; (B) 60 1C and (C) 65 1C. Error bars

indicate the standard error of the mean ðn ¼ 4Þ. Mean bars with different

letters (a–c) at same temperature and alginate concentration for different

heating period differ significantly ðPo0:05Þ. Mean bars with different

letters (p–r) at same temperature and heating period with different alginate

concentrations differ significantly ðPo0:05Þ.

0.0 0.5 1.0 2.0 3.00

2

4

6

8

10

2% Alginate 3% Alginate 4% Alginate

ap apap

bp

cpcp cp cpcp

cpcp

cp

Incubation period (h)

Rel

ease

d ce

ll co

unts

(log

cfu

g-1

)

bpbp

cp

Fig. 5. Release of alginate encapsulated L. casei NCDC-298 (log cfu g�1)

at different time intervals (0–3 h) in simulated colonic pH solution. Error

bars indicate the standard error of the mean ðn ¼ 4Þ. Mean bars with

different letters (a–c) for same alginate concentration at different

incubation period differ significantly ðPo0:05Þ.

S. Mandal et al. / International Dairy Journal 16 (2006) 1190–11951194

affecting the release of viable cells from microcapsules insimulated colonic pH solution. Further studies on survivalof encapsulated lactobacilli in dairy processing and efficacyin delivering the viable cells in vivo are needed for betterapplication of probiotics in the development of functionalfoods.

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Further reading

Teixeira, P., Castro, H., & Kirby, R. (1994). Inducible thermo-tolerance

in Lactobacillus bulgaricus. Letters in Applied Microbiology, 18,

218–221.