International Dairy Journal 13 (2003) 313

Review

Evaluation of encapsulation techniques of probiotics for yoghurt

Wunwisa Krasaekoopt, Bhesh Bhandari*, Hilton Deeth
School of Land and Food Sciences, The University of Queensland, St. Lucia, Qld. 4072, Australia Received 19 June 2001; accepted 10 October 2002

Abstract The health benets provided by probiotic bacteria have led to their increasing use in fermented and other dairy products. However, their viability in these products is low. Encapsulation has been investigated to protect the bacteria in the products environment and improve their survival. There are two common encapsulation techniques, namely extrusion and emulsion, to encapsulate the probiotics for their use in the fermented and other dairy products. This review evaluates the merits and limitations of these two techniques, and also discusses the supporting materials and special treatments used in encapsulation processes. r 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Probiotics; Microencapsulation; Survival; Extrusion; Emulsion

Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Techniques for microencapsulation 2.1. Extrusion technique . . . . 2.1.1. Supporting material 2.2. Emulsion technique . . . . . 2.2.1. Continuous phase . 2.2.2. Supporting material of bacterial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . in hydrocolloid beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 5 5 6 6

3.

Special treatments . . . . . . . . . . . . . 3.1. Cross-linking with cationic polymers . 3.2. Coating with other polymers . . . . . 3.3. Mixing with starch . . . . . . . . . . 3.4. Incorporation of additives . . . . . .

. . 9 . . 9 . 10 . 10 . 10 10 10 11 11

4. 5. 6.

Advantages and limitations of extrusion and emulsion encapsulation techniques . . . . . . . . . . . . . . Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Probiotics are live microbial supplements, which benecially affect the host by improving its intestinal
*Corresponding author. Tel.: +617-33469192; fax: +617-33651177. E-mail address: b.bhandari@mailbox.uq.edu.au (B. Bhandari).

microbial balance (Fuller, 1992). Claimed benets include controlling intestinal infection, controlling serum cholesterol levels, improving lactose utilization in persons who are lactose maldigestors, and possessing anticarcinogenic activity. Probiotic bacteria, which are commensals of the human gut, have been reported to inhibit the growth

0958-6946/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 2 ) 0 0 1 5 5 - 3

W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313

of undesirable microorganisms and food poisoning bacteria, such as Salmonella, that can be encountered in the gastrointestinal tract (Huges & Hoover, 1991; Lim, Huh, & Baek, 1993). The probiotic effect has been attributed to production of acid (Rasic & Kurmann, 1983), production of bacteriocins (Tagg, Dajani, & Wannamaker, 1976), competition with pathogens (Gurr, 1987), and enhancement of the immune system (Fuller, 1992). Probiotic bacteria produce b-galactosidase which is benecial for people with lactose intolerance. The normal yoghurt cultures, Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus, produce bgalactosidase in yoghurt, but these bacteria cannot survive and grow in the intestinal tract due to their low bile salt tolerance. In contrast, probiotic bacteria such as Lb. acidophilus and Bidobacterium bidum can survive and grow in the intestinal tract and produce bgalactosidase in the presence of bile (Noh & Gilliland, 1993). Kim and Gilliland (1983) concluded that improvement in lactose digestion resulted from this intestinal enzyme production and not from hydrolysis of lactose before consumption of milk containing Lb. acidophilus. Probiotics have also been claimed to have anticarcinogenic or antimutagenic activities. This may result from one or more factors, such as inhibition of the carcinogen and/or procarcinogen, inhibition of bacteria that convert procarcinogens to carcinogens, activation of the hosts immune system, and reduction of the intestinal pH to reduce microbial activity (Gilliland, 1989; Rasic & Kurmann, 1983). In addition, hypocholesterolemic effects (lowering of blood cholesterol) have been reported, especially for Lb. acidophilus strains (Buck & Gilliland, 1994; Gilliland, 1989). Many different microorganisms are added to dairy products for their probiotic potential (Fuller, 1997; Gibson & Fuller, 1998). These include Lactobacilli such as Lb. acidophilus, Lb. casei, Lb. delbrueckii ssp. bulgaricus, Lb. reuteri, Lb. brevis, Lb. cellobiosus, Lb. curvatus, Lb. fermentum, and Lb. plantarum; Grampositive cocci such as Lactococcus lactis ssp. cremoris, Str. thermophilus, Enterococcus faecium, Str. diacetylactis, and Str. intermedius; and Bidobacteria such as B. bidum, B. adolescentis, B. animalis, B. infantis, B. longum, and B. thermophilum. In many countries standards have been developed for the numbers of the probiotic bacteria in fermented products. For example, a minimal content (106 cfu g1) was established for bidobacteria added to fermented milks by regulation recently approved by the countries of MERCOSUR (Argentina, Paraguay, Brazil, and Uruguay) (Pagano, 1998). In Japan, a standard has been developed by the Fermented Milks and Lactic Acid Bacteria Beverages Association, which requires a minimum of 107 viable probiotic bacteria cells per millilitre to be present in fresh dairy products (Robinson, 1987). A suggested minimum level for probiotic bacteria in

yoghurt is 106 (Robinson, 1987; Kurman & Rasic, 1991) or daily intake should be about 108 (Anonymous, 1992). The Australian Food Standards Code does not specify any requirements regarding the number of probiotic bacteria in fermented products. The ability of microorganisms to survive and multiply in the host strongly inuences their probiotic benets. The bacteria should be metabolically stable and active in the product, survive passage through the upper digestive tract in large numbers, and have benecial effects when in the intestine of host (Gilliland, 1989). Many studies have shown low viability of probiotics in yoghurt and fermented milk (Gilliland & Speck, 1977; Anonymous, 1992; Iwana, Masuda, Fujisawa, Suzuki, & Mitsuoka, 1993; Shah, Lankaputhra, Britz, & Kyle, 1995; Dave & Shah, 1997; Shah & Lankaputhra, 1997; Gardini, Lanciotti, Guerzoni, & Torriani, 1999; Schillinger, 1999; Vinderola, Bailo, & Reinheimer, 2000). Ravula and Shah (1998) also reported that very high levels of probiotic bacteria do not survive in fermented frozen dairy desserts. They reported that the count declined by 56 log cycles within 812 weeks of storage at 181C. Other studies of survival of probiotics have produced similar results (Holcomb, Frank, & McGregor, 1991; Laroia & Martin, 1991; Hekmat & McMahon, 1992). In addition, Lankaputhra and Shah (1995) found that survival of Lb. acidophilus and Bidobacterium spp. is low in the presence of acid and bile salts. Protection of probiotics by microencapsulation in hydrocolloid beads has been investigated for improving their viability in food products and the intestinal tract (Rao, Shiwnarain, & Maharaj, 1989). This has been proposed for various dairy fermentations (Champagne, Gaudy, Poncelet, & Neufeld, 1992a; Champagne et al., 1992b; Champagne, Girard, & Rodrigue, 1993; Champagne, Lacroix, & Sodini-Gallot, 1994) such as fermentation of whey (Audet, Paquin, & Lacroix, 1989) and continuous inoculation of milk for yoghurt manufacture (Prevost & Divies, 1988). Additional benets of microencapsulation of cells include: protection of cells inside the beads from bacteriophages (Steenson, Klaenhammer, & Swaisgood, 1987); increased survival during freeze drying and freezing (Kearney, Upton, & Loughlin, 1990; Sheu & Marshall, 1993; Sung, 1997; Kim & Yoon, 1995); and greater stability during storage (Kim, Kamara, Good, & Enders, 1988; Kebary, Hussein, & Badawi, 1998; Reuter, 1990). Microencpasulation of various bacterial cultures including probiotics has been a common practice for extending their storage life and converting them into a powder form for ease of their use. There are several techniques such as spray drying, freeze drying, uidized bed drying for encapsulating the cultures and converting them into a concentrated powdered form. However, the bacteria encapsulated by these techniques are

W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313

completely released in the product. In this case, the cultures are not protected from the product environment or during the passage through the stomach or intestinal tract. Encapsulation in hydrocolloid beads entraps or immobilizes the cells within the bead matrix, which in turn provides protection in such an environment. This paper reviews the techniques for encapsulation of probiotic bacteria in hydrocolloid beads, particularly for the use in yoghurt. Application of immobilized cells in hydrocolloids for probiotic biomass production is been described.

2. Techniques for microencapsulation of bacterial cells in hydrocolloid beads Microencapsulation is a process in which the cells are retained within an encapsulating membrane to reduce cell injury or cell loss. The encapsulation techniques applied to probiotics for the use in fermented milk products or biomass production can be classied into 2 groups, depending on the method used to form the beads: extrusion (droplet method) and emulsion or twophase system. Both extrusion and emulsion techniques increase the survival of probiotic bacteria by up to 8095% (Audet et al., 1988; Rao et al., 1989; Sheu & Marshall, 1991; Sheu & Marshall, 1993; Sheu, Marshall, & Heymann, 1993; Jankowski, Zielinska, & Wysakowska, 1997; Kebary et al., 1998). 2.1. Extrusion technique Extrusion is the oldest and most common approach to making capsules with hydrocolloids (King, 1995). It simply involves preparing a hydrocolloid solution, adding microorganisms to it, and extruding the cell suspension through a syringe needle in the form of droplets to free-fall into a hardening solution or setting bath (Fig. 1). The size and shape of the beads depend on the diameter of the needle and the distance of free-fall, respectively (Table 1). This method is the most popular due to its ease, simplicity, low cost, and gentle formulation conditions ensuring high retention of cell viability. 2.1.1. Supporting material The supporting material used for extrusion is alginate, which is a linear heteropolysaccharide of d-mannuronic and l-guluronic acid extracted from various species of algae (Smidsrod, Haug, & Lian, 1972). Depending on the source, the composition and the sequence in lguluronic acid and d-mannuronic acid vary widely. The functional properties of alginate as supporting material correlate strongly with the composition and sequence of l-guluronic acid and d-mannuronic acid. Divalent cations such as Ca2+ bind preferentially to the polymer

of l-guluronic acid. The length of the polymer of dmannuronic acid is, therefore, the main structural feature contributing to gel formation (Smidsrod et al., 1972; Skjak-Braek, Larsen, & Smidsrod, 1986). To form beads, a cell suspension is mixed with a sodium alginate solution, and the mixture dripped into a solution containing a multivalent cation (usually Ca2+ in the form of CaCl2). The droplets form gel spheres instantaneously, entrapping the cells in a three-dimensional lattice of ionically cross-linked alginate. The success of the alginate gel encapsulation technique is due to the gentle environment it provides for the entrapped material, cheapness, simplicity, and its biocompatibility (Klein, Stock, & Vorlop, 1983; Tanaka, Masatose, & Veleky, 1984; Martinsen, Skjak-Braek, & Smidsrod, 1989). The concentrations of alginate used to form the gel vary. Jankowski et al. (1997) used a very low concentration of 0.6% to form a gel with 0.3 m CaCl2. Others have used 12% alginate and 0.051.5 m CaCl2 (Table 1). The size of the beads is approximately 23 mm in diameter. Moreover, the size and sphericity of the bead depend mainly on the viscosity of the sodium alginate solution and the distance between the syringe and the calcium chloride collecting solution (Smidsrod & Skjak-Braek, 1990). As the concentration, and hence viscosity, of sodium alginate increases, the size of the beads decreases. The extruder orice diameter is another important factor, which regulates droplet size. Using a 0.27-mm syringe, Smidsrod and Skjak-Braek (1990) obtained a bead size of 23 mm. The composition of the alginate also inuences bead size; small beads result from low guluronic alginates (Martinsen et al., 1989).

2.2. Emulsion technique In this technique, a small volume of the cell-polymer suspension (discontinuous phase) is added to a large volume of a vegetable oil (continuous phase) such as soybean oil, sunower oil, canola oil or corn oil. The mixture is homogenized to form a water-in-oil emulsion. Once the water-in-oil emulsion is formed, the watersoluble polymer must be insolubilized (cross-linked) to form tiny gel particles within the oil phase (Fig. 1). The smaller the internal phase particle size of the emulsion, the smaller the nal microparticles will be. The insolubilization method of choice depends on the type of supporting material used. The beads are harvested later by ltration. The size of the beads is controlled by the speed of agitation, and can vary between 25 mm and 2 mm. This technique has been used successfully to encapsulate lactic acid bacteria for batch (Lacroix, Paquin, & Arnaud, 1990) and continuous fermentation (Audet, Lacroix, & Paquin, 1992) (Table 2).

W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313

Sodium alginate

Microbial cell suspension

Mix Extrusion Technique Emulsion Technique

Emulsification in vegetable oil Cell suspension CaCl2

Drop in Calcium chloride solution

Addition of calcium chloride to Break emulsion and form gel Microbial cell Liquid core Alginate

Calcium alginate bead

Fig. 1. Flow diagram of encapsulation of bacteria by the extrusion and emulsion techniques.

2.2.1. Continuous phase For food applications, vegetable oils are used as the continuous phase. Some studies have used white light parafn oil (Rao et al., 1989) and mineral oil (Groboillot et al., 1993). In some cases emulsiers are added to form a better emulsion, because the emulsiers lower the surface tension, resulting in smaller spheres (Adamson, 1982). The most common emulsier used is Tween 80 at 0.2% (Sheu & Marshall, 1993; Sheu et al., 1993; Kebary et al., 1998). Sheu et al. (1993) used Tween 80 together with 0.5% sodium lauryl sulphate, which produced a bead size of 2535 mm.

2.2.2. Supporting material There are many supporting materials used with the emulsion technique. These include a mixture of kcarageenan and locust bean gum (Audet et al., 1988, 1989; Arnaud et al., 1992), cellulose acetate phthalate (Rao et al., 1989), alginate (Sheu et al., 1991, 1993; Sheu & Marshall, 1993; Larisch et al., 1994; Kebary et al., 1998), chitosan (Groboillot et al., 1993), and gelatin (Hyndman, Groboillot, & Poncelet, 1993). 2.2.2.1. Carrageenan. k-Carageenan is a natural polysaccharide extracted from marine macroalgae, commonly used as a food additive. Elevated temperatures

Table 1 Encapsulation of lactic acid and probiotic bacteria by the extrusion technique Conc. of alginate (%) 1.875 2.5 Yoghurt 1.5 Na Conc. of CaCl2 (m) Special treatment Diameter of bead (mm) Application Reference Prevost, Divies, and Rousseau (1985)

Bacteria

Supports used

Lactobacillus delbrueckii ssp. bulgaricus Streptococcus thermophilus 1.875 1.5 N 2.6 Cheese

Alginate

Str. lactis ssp. diacetylactis Str. cremoris 1 1.875 0.1 1 N N b 2.5

Alginate

Prevost and Divies (1987)

Str. cremoris Lb. delbrueckii ssp. bulgaricus Str. Thermophillus 2 2 0.05 Coated with low MWc chitosan

Alginate Alginate

Phage protection Yoghurt

Steenson et al. (1987) Prevost and Divies (1988)

Lactococcus lactis ssp. cremoris

Alginate

Biomass production

Zhou, Martins, Groboillot, Champagne, and Neufeld (1998) Biomass production Kearney et al. (1990)

Lb. plantarum 2 0.1 Added glycerol 2

Alginate

Lc. lactis ssp. lactis bv. diacetylactis Lc. lactis ssp. cremoris 1.5 2 0.1 0.2 Coated with alginate N

Alginate

Cream Biomass production Biomass production

Prevost and Divies (1992) Morin, Bernier-Cardou, and Champagne (1992) Champagne et al. (1993)

Alginate

W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313

Str. thermophilus 2 0.1 N

Alginate

Lb. delbrueckii ssp. bulgaricus Lc. lactis ssp. lactis bv. diacetylactis Lb. acidophillus 1.8 0.6 0.3 1.5

Alginate

N Mixed with starch

Biomass production

Cachon and Divies (1993) Jankowski et al. (1997)

Alginate

N, no treatment. , no record. c MW, molecular weight.

Table 2 Encapsulation of probiotic bacteria by the emulsion technique Continuous phase Soy oil 0.52 mm Yoghurt N
a

Bacteria Audet et al. (1988)

Support used

Special treatment

Diameter of bead

Application

Reference

Streptococcus thermophilus

3% k-carrageenan and locust bean gum (2:1)

Lactobacillus delbrueckii ssp. bulgaricus Soy oil N 0.51 mm b Audet et al. (1989)

Str. Thermophilus

3% k-carrageenan and locust bean gum (2:1)

Lactococcus lactis ssp. lactis Lb. delbrueckii ssp. bulgaricus White light parafn oil Vegetable oil with 2% emulsier Vegetable oil N 12 mm 2% emulsier added N Rao et al. (1989)

Bidobacterium pseudolongum

10% Cellulose acetate phthalate

Lb. delbrueckii ssp. bulgaricus

3% Alginate

Ice cream

Sheu and Marshall (1991) Biomass production Arnaud, Lacroix, and Choplin (1992)

Lb. casei ssp. casei

3% k-carrageenan and locust bean gum (11:1) Mineral oil Cross-linked with hexamethylene diisocyanate or glutaldehyde 6% glycerol or mannitol added 0.5% sodium lauryl sulphate added 150 mm

Lc. lactis ssp. cremoris

Chitosan (4%)

Biomass production

Groboillot, Champagne, Darling, and Poncelet (1993)

Lb. delbrueckii ssp. bulgaricus

3.6% Alginate

30 mm

Frozen dessert

Sheu et al. (1993)

W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313

Lb. delbrueckii ssp. bulgaricus

3% Alginate

Vegetable oil containing 0.2% Tween 80 Vegetable oil containing 0.2% Tween 80 Sunower seed oil

2535 mm

Frozen ice milk

Sheu and Marshall (1993)

Lc. lactis ssp. cremoris

24% Gelatin

Cross-linked with toluene-2,4diisocyanate Coated with poly-llysine Added glycerol

271+168 mm

Biomass production

Hyndman, Groboillot, and Poncelet (1993) 50 mm1 mm Biomass production Ice milk Larisch, Poncelet, and Champagne (1994) Kebary et al. (1998)

Lc. lactis ssp. cremoris Canola oil

2% Alginate

B. bidum B. infantis

3% Alginate

orn oil containing 0.2% Tween 80

N, no treatment. , no record.

W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313

(60801C) are needed to dissolve the polymer at concentrations ranging from 2% to 5% (Klein & Vorlop, 1985). Gelation of carrageenan is induced by temperature changes. The cell slurry is added to the heat-sterilized polymer solution at 40451C and gelation occurs by cooling to room temperature. After the beads are formed, K ions (in the form of KCl) are used to stabilize the gel and to prevent swelling, or to induce gelation. Audet et al. (1988) reported that KCl has an inhibitory effect on some lactic acid bacteria such as Str. thermophilus and Lb. delbrueckii ssp. bulgaricus. Monovalent ions such as Rb+, Cs+ and NH+ result in 4 stronger gels (Tosa et al., 1979). Locust bean gum, at a ratio of carrageenan to locust bean gum of 2:1, increases the strength of gels through specic interaction of its galactomannan chains with carrageenan (Takata, Tosa, & Chibata, 1977; Miles, Morris, & Carroll, 1984). 2.2.2.2. Cellulose acetate phthalate (CAP). Due to the presence of ionisable phthalate groups, this polymer is insoluble in acid media at a pH of 5 or lower but is soluble when the pH is increased to 6 or higher (Malm, Emerson, & Hiatt, 1951). In addition, CAP is physiologically inert when administered in vivo and is, therefore, widely used as an enteric coating material for the release of drugs and other pharmaceutical substances in the intestine. Rao et al. (1989) studied a procedure for microencapsulation of B. pseudolongum with CAP, using the emulsion technique. Microencapsulated B. pseudolongum survived the simulated gastric environment in larger numbers (109 cfu mL1) than non-encapsulated organisms, which did not retain any viability when exposed to a simulated gastric environment for 1 h. 2.2.2.3. Chitosan. Chitosan is a positively charged linear polysaccharide formed by deacetylation of chitin extracted from crustacean shells. It is water soluble below pH 6, and, like alginate, forms a gel by ionotropic gelation. Chitosan, a polycation with amine groups, can be cross-linked by anions or polyanions, such as poly phosphates, [Fe(CN)6]4, [Fe(CN)6]3, polyaldehydrocarbonic acid (Klein & Vorlop, 1983). Chitosan exhibited inhibitory effects on different types of lactic acid bacteria (Groboillot et al., 1993). To overcome viability problems with chitosan, Zhou, Martins, Groboillot, Champagne, and Neufeld (1998) used this polymer for cell encapsulation to coat alginate beads by soaking the beads in chitosan solution (0.4%) with gentle shaking for 40 min. Cell loading in beads ranged from 108 to 1010 cfu g1. 2.2.2.4. Gelatin. Gelatin, a protein, is useful as a thermally reversible gelling agent for encapsulation. Because of its amphoteric nature, it also is an excellent candidate for cooperation with anionic polysaccharides like gellan gum. These hydrocolloids are miscible at pH

> 6, since they both carry net negative charges and repel one another. However, when the pH is adjusted below gelatins isoelectric point, the net charge on the gelatin becomes positive, causing an interaction with the negatively charged gellan gum (King, 1995). Hyndman et al. (1993) used high concentration gelatin (24%) to encapsulate Lc. lactis ssp. cremoris by cross-linking with toluene-2, 4-diisocyanate for biomass production. 2.2.2.5. Alginate. In the emulsion technique, alginate is added prior to emulsion formation. The addition of an oil-soluble acid, such as acetic acid, reduces the alginate pH from 7.5 to approximately 6.5 and initiates gel formation with Ca2+ (Poncelet, Poncelet, Beaulieu, & Neufeld, 1993). Following gelation, the beads are partitioned into water and washed to remove residual oil.

3. Special treatments Despite the suitability of alginate as the entrapment matrix material, gel entrapment in alginate has some limitation due to low stability in the presence of chelating agents such as phosphate, lactate, and citrate. The chelating agents share afnity for calcium and destabilize the gel (Smidsrod & Skjak-Braek, 1990). Thus, stability problems are encountered during lactic acid fermentation (Roy, Goulet, & Duy, 1987) and cause cell release from the beads. In the case of other matrix material, such as chitosan, the entrapped cells can be released from the beads during fermentation and cause low initial loading for the next fermentation. Therefore, special treatments, such as coating the beads, are applied in order to improve the properties of encapsulated beads. Coated beads not only prevent cell release but also increase mechanical and chemical stability. Cross-linking with cationic polymers, coating with other polymers, mixing with starch, and incorporating additives can improve stability of beads. 3.1. Cross-linking with cationic polymers Alginate beads have been stabilized by cross-linking with cationic polymers such as polyethyleneimine and polypropyleneimine, or polyethyleneimine and glutaraldehyde (Marx, 1989). Formation of a membrane around the beads and the spraying of the beads with glutaraldehyde have been proposed as stabilizing techniques to minimize cell release (Kolot, 1988). Groboillot et al. (1993) showed that the membrane formed with 4% chitosan cross-linked with hexamethylene diisocyanate or glutaraldehyde resulted in stronger microcapsules. The reaction of the bifunctional reagent with chitosan resulted in bridge formation linking the chitosan molecules. The length of the bridge depends on

10

W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313 Table 3 Positive and negative features of extrusion and emulsion techniques Extrusion Technological feasibility Cost Simplicity Survival of microorganism Size of bead Difcult to scale up Low High 8095% 25 mm Emulsion Easy to scale up High Low 8095% 25 mm2 mm

the type of cross-linking agent. Hyndman et al. (1993) obtained a similar result when toluene diisocyanate was used to cross-link the gelatin. The microcapsules were more resistant to breakage at the higher concentration. 3.2. Coating with other polymers Overgaard, Scharer, Moo-Young, and Bols (1991) produced alginate beads coated with a chitosan lm. The beads were obtained by dropping an alginate solution into a mixture of calcium chloride and chitosan solution. McKnight, Ku, and Goosen (1988) reported the coating of alginate beads with chitosan while Zhou et al. (1998) found that suspending alginate beads in a low molecular weight chitosan solution formed a membrane, which reduced cell release by 40%. Chitosan, a positively charged polyamine, forms a semipermeable membrane around a negatively charged polymer such as alginate. This membrane does not dissolve in the presence of Ca2+ chelators or antigelling agents and thus enhances stability of the gel (Smidsrod & Skjak-Braek, 1990), and provides a barrier to cell release. It was observed that low-molecular-weight chitosan diffuses more readily into the alginate gel matrix, resulting in a denser membrane than with highmolecular-weight chitosan (McKnight et al., 1988). Tanaka, Irie, and Ochi (1989) reported the coating of gel beads by a cell-free alginate gel layer. Furthermore, coating of beads with a poly amino acid such as poly llysine is of interest due to its better biocompatibility (Larisch et al., 1994), and potential for application in the food industry, such as yoghurt production (Champagne et al., 1992a). Champagne et al. (1992a) showed that coating alginate beads with a single layer of poly llysine did not signicantly reduce the release of cells but double coating with poly l-lysine and alginate reduced cell release by a factor of approximately 50. 3.3. Mixing with starch Alginate/starch liquid core capsules offer the ability to encapsulate Lb. acidophilus without loss of viability and fermentation ability (Jankowski et al., 1997). Capsule membranes allow sufcient diffusion of nutrients and metabolites to maintain growth of encapsulated cells. 3.4. Incorporation of additives With incorporation of cryoprotectants such as glycerol, survival of encapsulated cells after lyophilisation and rehydration is enhanced because these agents confer additional protection (Kearney et al., 1990). The survival of bidobacteria increased signicantly to 88.5% (Kebary et al., 1998), and Lb. delbrueckii ssp. bulgaricus to 90% (Sheu et al., 1993) because these agents reduced ice crystal formation by binding with

water. The beads with glycerol also exhibited a 43% decrease in size due to the higher alginate concentration per unit volume in the beads with glycerol binding with water.

4. Advantages and limitations of extrusion and emulsion encapsulation techniques For encapsulation of probiotics, both extrusion and emulsion techniques can be applied. Advantages and disadvantages of these techniques are shown in Table 3. Extrusion is a relatively simple technique. It usually produces entrapped, rather than encapsulated core material, although encapsulation can be achieved through co-extrusion devices or dropping into a bath of coating material which react at the droplet surface. This method can be difcult for large-scale production because of slow formation of beads compared with the emulsion technique. On the other hand, the emulsion technique is relatively new to the food industry and easy to scale up for large-scale production. It provides both encapsulated and entrapped core materials. The size of the beads formed by this method is smaller (25 mm to 2 mm) than that of beads produced by the extrusion method (25 mm). The size of beads from the extrusion method depends mainly on the size of the needle used, while the size of beads from the emulsion method depends on the speed of agitation and the type of emulsier used. Due to the need for a vegetable oil, the operating cost of the emulsion technique may be higher than that of the extrusion technique.

5. Applications Because of the many benets offered by encapsulation, entrapped microorganisms can be used to advantage for producing dairy products such as yoghurt, cheese and frozen milk products, as well as for biomass production. Prevost, Divies and Rousseau (1985) reported that the continuous manufacture of yoghurt with entrapped microorganisms (Lb. delbrueckii ssp. bulgaricus and Str. thermophilus) is more complicated than the traditional batch method, but presents many advantages. It is possible to obtain a product with

W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313

11

constant characteristics because the residence time, acidity and continuous inoculation of milk with a constant bacilli/cocci ratio can be controlled at a desired pH. Prevost and Divies (1988) also obtained a similar result. Audet et al. (1988) used encapsulated Str. thermophilus and Lb. delbrueckii ssp. bulgaricus to produce yoghurt. The viability of these bacteria remained very high throughout the entrapment steps and subsequent storage. The bead diameter inuenced the fermentation rate; smaller beads (0.51.0 mm) had higher cell release rates, lactose utilization and acid production by the entrapped cells, reaching values found for free cells. The use of encapsulated microorganisms reduced the incubation time by 50% and 60% for fresh fermented cheese production (Prevost & Divies, 1987) and cream fermentation (Prevost & Divies, 1992), respectively. Sheu and Marshall (1993) reported that about 40% more lactobacilli survived freezing of ice cream when they were entrapped in calcium alginate than when they were not entrapped. Moreover, encapsulation protected the microorganisms in batch-frozen and continuously frozen ice milk mixes (Sheu et al., 1993). Kebary et al. (1998) reported that encapsulation of bidobacteria signicantly improved their survival throughout storage from 4344% to about 5060% while Rao et al. (1989) showed that encapsulated B. pseudolongum could survive in a simulated gastric environment in larger numbers than non-encapsulated microorganisms. Encapsulation can also be applied for biomass production since the culture is protected from attack by bacteriophage due to the exclusion of phage particles from the gel matrix (Steenson et al., 1987). Moreover, encapsulated culture provides high stability of cells and high productivity for metabolite production with high agitation rates (Arnaud et al., 1992). Champagne et al. (1993) reported that it was possible to use encapsulated microorganisms to produce bacterial densities (123.1 108 mL1) 6 times higher than with classical cell free suspensions (18.6 108 mL1). Morin, BernierCardou and Champagne (1992) suggested that addition of CaCO3 to the fermentation medium for biomass production improved bead stability. On the other hand, the encapsulated culture may increase the processing time. For example, encapsulated Lc. lactis ssp. cremoris took 17% longer than free cells to reduce the pH of milk to 4.5 (Larisch et al., 1994). Groboillot et al. (1993) reported that loss of acidication activity during encapsulation was recovered in subsequent fermentation to levels similar to that of free-cell fermentation.

cultured cream and frozen dairy desserts, and for biomass production. In the encapsulated form, the probiotics are protected from bacteriophage and harsh environments, such as freezing and gastric solutions. Thus, encapsulation facilitates the manufacture of fermented dairy products in which the bacteria have constant characteristics, higher stability during storage, and higher productivity than non-encapsulated bacteria.

References
Adamson, A. W. (1982). Physical chemistry of surfaces. New York: Wiley Inc. Anonymous (1992). Yoghurt and probiotics. Choice, 32(11), 3235. Arnaud, J. P., Lacroix, C., & Choplin, L. (1992). Effect of agitation rate on cell release rate and metabolism during continuous fermentation with entrapped growing. Biotechnology Techniques, 6(3), 265270. Audet, P., Lacroix, C., & Paquin, C. (1992). Continuous fermentation of a supplemented whey permeate medium with immobilized Steptococcus salivarius ssp. thermophilus. International Dairy Journal, 2(1), 115. Audet, P., Paquin, C., & Lacroix, C. (1988). Immobilized growing lactic acid bacteria with k-carrageenan-locust bean gum gel. Applied Microbiology and Biotechnology, 29(1), 1118. Audet, P., Paquin, C., & Lacroix, C. (1989). Sugar utilization and acid production by free and entrapped cells of Streptococcus salivarius ssp. thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, and Lactococcus lactis ssp. lactis in a whey permeate medium. Applied and Environmental Microbiology, 55(1), 185189. Buck, L. M., & Gilliland, S. E. (1994). Comparisons of freshly isolated strains of Lactobacillus acidophilus of human intestinal for ability to assimilate cholesterol during growth. Journal of Dairy Science, 77(10), 29252928. Cachon, R., & Divies, C. (1993). Localization of Lactococcus lactis ssp. lactis bv. diacetylactis in alginate gel beads affects biomass density and synthesis of several enzymes involved in lactose and citrate metabolism. Biotechnology Techniques, 7(6), 453456. Champagne, C. P., Gaudy, C., Poncelet, D., & Neufeld, R. J. (1992a). Lactococcus lactis release from calcium alginate beads. Applied and Environmental Microbiology, 58(5), 14291434. Champagne, C. P., Girard, F., & Rodrigue, N. (1993). Production of concentrated suspensions of thermophilic lactic acid bacteria in calcium-alginate beads. International Dairy Journal, 3(3), 257275. Champagne, C. P., Lacroix, C., & Sodini-Gallot, I. (1994). Immobilized cell technologies for the dairy industry. Critical Reviews in Biotechnology, 14(2), 109134. Champagne, C. P., Morin, N., Couture, R., Gagnon, C., Jelen, P., & Lacroix, C. (1992b). The potential of immobilized cell technology to produce freeze-dried, phage-protected cultures of Lactococcus lactis. Food Research International, 25(6), 419427. Dave, R. I., & Shah, N. P. (1997). Viability of yoghurt and probiotic bacteria in yoghurts made from commercial starter cultures. International Dairy Journal, 7(1), 3141. Fuller, R. (1992). Probiotics: the scientic basis. London: Chapman & Hall. Fuller, R. (1997). Probiotics 2applications and practical aspects. London: Chapman & Hall. Gardini, F., Lanciotti, R., Guerzoni, M. E., & Torriani, S. (1999). Evaluation of aroma production and survival of Streptococcus thermophilus, Lactobacillus delbrueckki ssp. bulgaricus and Lactobacillus acidophilus in fermented milks. International Dairy Journal, 9(2), 125134.

6. Conclusion Encapsulated probiotic bacteria can be used in many fermented dairy products, such as yoghurt, cheese,

12

W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313 Klein, J., & Vorlop, D. K. (1985). Immobilization techniques cells. In M. Moo-Young, C. L. Cooney, & A. E. Humphrey (Eds.), Comprehensive biotechnology (pp. 542550). Oxford: Pergamon Press. Kolot, F. B. (1988). Immobilized microbial system: principles, techniques, and industrial applications. Cambridge: Cambridge University Press. Kurman, J. A., & Rasic, J. L. (1991). The health potential of products containing bidobacteria. In R. K. Robinson (Ed.), Therapeutic properties of fermented milks (pp. 117158). London: Elsevier Application Food Science Series. Lacroix, C., Paquin, C., & Arnaud, J. P. (1990). Batch fermentation with entrapped growing cells of Lactobacillus casei. I. Optimisation of the rheological properties of the entrapment. Applied Microbiology and Biotechnology, 32(4), 403408. Lankaputhra, W. E. V., & Shah, N. P. (1995). Survival of Lactobacillus acidophilus and Bidobacterium spp. in the presence of acid and bile salts. Cultured Dairy Products Journal, 30(3), 27. Larisch, B. C., Poncelet, D., & Champagne, C. P. (1994). Microencapsulation of Lactococcus lactis ssp. cremoris. Journal of Microencapsulation, 11(2), 189195. Laroia, S., & Martin, J. H. (1991). Methods for enumerating and propagating bidobacteria. Cultured Dairy Products Journal, 26(2), 3233. Lim, K., Huh, C. S., & Baek, Y. J. (1993). Antimicrobial susceptibility of bifodobacteria. Journal of Dairy Science, 76(8), 21682174. Malm, C. J., Emerson, J., & Hiatt, G. D. (1951). Cellulose acetate phthalate as an enteric-coating material. Journal American Pharmaceutical Association Science, 10(3), 520525. 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), 7989. Marx, J. L. (1989). A revolution in biotechnology. Cambridge: Cambridge University Press. McKnight, C. A., Ku, A., & Goosen, M. F. A. (1988). Synthesis of chitosan-alginate microencapsule membranes. Journal of Bioactive and Compatible Polymers, 3(8), 334354. Miles, M. J., Morris, V. J., & Carroll, V. (1984). Carob gum kcarrageenan mixed gels: Mechanical properties and X-ray bre diffraction studies. Macromolecules, 17(19), 24432447. Morin, N., Bernier-Cardou, M., & Champagne, C. P. (1992). Production of Lactococcus lactis biomass by immobilized cell technology. Journal of Industrial Microbiology, 9(2), 131135. Noh, D. O., & Gilliland, S. E. (1993). Inuence of bile on cellular integrity and beta-galactosidase of Lactobacillus acidophilus. Journal Dairy Science, 76(5), 12531259. 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), 439443. Pagano, J. C. (1998). Nueva Legislacion del MERCOSUR para leches fermentadas. Industria Lechera, 7(13), 813. Poncelet, D., Poncelet, S. B., Beaulieu, C., & Neufeld, R. J. (1993). Scale-up of gel bead and microcapsule production in cell immobilization. In M. F. A. Goosen (Ed.), Fundamentals of animal cell encapsulation and immobilization (pp. 532547). Boca Raton: CRC Press Inc. Prevost, H., & Divies, C. (1987). Fresh fermented cheese production with continuous pre-fermented milk by a mixed culture of mesophilic lactic streptococci entrapped in Ca-alginate. Biotechnology Letters, 9(11), 789794. Prevost, H., & Divies, C. (1988). Continuous pre-fermentation of milk by entrapped yoghurt bacteria. I. Development of the process. Milchwissenschaft, 43(10), 621625. Prevost, H., & Divies, C. (1992). Cream fermentation by a mixed culture of lactococci entrapped in two-layer calcium alginate gel beads. Biotechnology Letters, 14(7), 583588.

Gibson, G. R., & Fuller, R. (1998). The role of probiotics and prebiotics in the functional food concept. In M. J. Sadler, & M. Saltmarsh (Eds.), Functional foods: The consumer, the products and the evidence (pp. 314). Cambridge: Royal Society of Chemistry. Gilliland, S. E. (1989). Acidophilus milk products: A review of potential benets to consumers. Journal of Dairy Science, 72(10), 24832494. Gilliland, S. E., & Speck, M. L. (1977). Instability of Lactobacillus acidophilus in yoghurt. Journal of Dairy Science, 60(9), 13941398. Groboillot, A. F., Champagne, C. P., Darling, G. D., & Poncelet, D. (1993). Membrane formation by interfacial cross-linking of chitosan for microencapsulation of Lactococcus lactis. Biotechnology and Bioengineering, 42(10), 11571163. Gurr, M. I. (1987). Nutritional aspects of fermented milk products. Milk: The vital force Proceedings of the XXII international dairy congress, Dordrecht, Netherlands, September 29October 3, 1986. Hekmat, S., & McMahon, D. J. (1992). Survival of Lactobacillus acidophilus and Bidobacterium bidum in ice cream for use as probiotic food. Journal of Dairy Science, 75(6), 14151422. Holcomb, J. E., Frank, J. F., & McGregor, J. U. (1991). Viability of L. acidophilus and Bifodobacterium bidum in soft serve frozen yoghurt. Cultured Dairy Product Journal, 26(3), 45. Huges, D. B., & Hoover, D. G. (1991). Bidobacteria: Their potential for use in American dairy products. Food Technology, 45(4), 7483. Hyndman, C. L., Groboillot, A. F., & Poncelet, D. (1993). Microencapsulation of Lactococcus lactis within cross-linked gelatin membranes. Journal of Chemical Technology and Biotechnology, 56(3), 259263. Iwana, H., Masuda, H., Fujisawa, T., Suzuki, H., & Mitsuoka, T. (1993). Isolation and identication of Bidobacterium ssp. in commercial yoghurt sold in Europe. Bidobacteria Microora, 12(1), 3945. Jankowski, T., Zielinska, M., & Wysakowska, A. (1997). Encapsulation of lactic acid bacteria with alginate/starch capsules. Biotechnology Techniques, 11(1), 3134. Kearney, L., Upton, M., & Loughlin, A. (1990). Enhancing the viability of Lactobacillus plantarum inoculum by immobilizing the cells in calcium-alginate beads. Applied and Environmental Microbiology, 56(10), 31123116. Kebary, K. M. K., Hussein, S. A., & Badawi, R. M. (1998). Improving viability of Bidobacteria and their effect on frozen ice milk. Egyptian Journal of Dairy Science, 26(2), 319337. Kim, H. S., & Gilliland, S. (1983). Lactobacillus acidophilus as a dietary adjunct for milk to aid lactose digestion in humans. Journal of Dairy Science, 66(5), 959966. Kim, H. S., Kamara, B. J., Good, I. C., & Enders, G. L. (1988). Method for the preparation of stabile microencapsulated lactic acid bacteria. Journal of Industrial Microbiology, 3(4), 253257. Kim, K. I., & Yoon, Y. H. (1995). A study on the preparation of direct vat lactic acid bacterial starter. Korean Journal of Dairy Science, 17(2), 129134. King, A. H. (1995). Encapsulation of food ingredients: a review of available technology, focusing on hydrocolloids. In S. J. Risch, & G. A. Reineccius (Eds.), Encapsulation and controlled release of food ingredients (pp. 213220). Washington DC: American Chemical Society. Klein, J., Stock, J., & Vorlop, K. D. (1983). Pore size and properties of spherical Ca-alginate biocatalysts. European Journal Applied Microbiology Biotechnology, 18(1), 8691. Klein, J., & Vorlop, K. D. (1983). Immobilized cells, catalyst preparation and reaction performance. In H. Blanch, E. T. Papoutsakis & G. Stephanopoulos (Eds.), Foundations of biochemicals engineering kinetics and thermodynamics in biological systems, ACS Symposium, Series No. 207 (pp. 475486). Washington DC: American Chemical Society.

W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313 Prevost, H., Divies, C., & Rousseau, E. (1985). Continuous yoghurt production with Lactobacillus bulgaricus and Streptococcus thermophilus entrapped in Ca-alginate. Biotechnology Letters, 7(4), 247252. Rao, A. V., Shiwnarain, N., & Maharaj, I. (1989). Survival of microencapsulated Bidobacterium pseudolongum in simulated gastric and intestinal juices. Canadian Institute of Food Science and Technology Journal, 22(4), 345349. Rasic, J. L., & Kurmann, J. A. (1983). Bidobacteria and their role. Basel: Birkhauser. Ravula, R. R., & Shah, N. P. (1998). Viability of probiotic bacteria in fermented frozen dairy desserts. Food Australia, 50(3), 136139. Reuter, G. (1990). Bidobacteria cultures as components of yoghurt like products. Bidobacteria Microora, 9(1), 107. Robinson, R. K. (1987). Survival of Lactobacillus acidophilus in fermented products. Suid Afrikaanse Tydskrif Vir Suiwelkunde, 19(1), 2510727. Roy, D., Goulet, J., & Duy, A. (1987). Continuous production of lactic acid from whey permeates by free and calcium alginate entrapped Lactobacillus helveticus. Journal of Dairy Science, 70(3), 506513. Schillinger, U. (1999). Isolation and identication of lactobacilli from novel-type probiotic and mild yoghurts and their stability during refrigerated storage. International Journal of Food Microbiology, 47(1/2), 7987. Shah, N. P., & Lankaputhra, W. E. V. (1997). Improving viability of Lactobacillus acidophilus and Bidobacterium ssp. in yoghurt. International Dairy Journal, 7(5), 349356. Shah, N. P., Lankaputhra, W. E. V., Britz, M., & Kyle, W. S. A. (1995). Survival of Lactobacillus acidophilus and Bidobacterium bidum in commercial yoghurt during refrigerated storage. International Dairy Journal, 5(5), 515521. Sheu, T. Y., & Marshall, R. T. (1991). Improving culture viability in frozen dairy desserts by microencapsulation. Journal of Dairy Science, 74(supplement 1), 107. Sheu, T. Y., & Marshall, R. T. (1993). Microencapsulation of lactobacilli in calcium alginate gels. Journal of Food Science, 54(3), 557107561. Sheu, T. Y., Marshall, R. T., & Heymann, H. (1993). Improving survival of culture bacteria in frozen desserts by microentrapment. Journal of Dairy Science, 76(7), 19021907.

13

Skjak-Braek, G., Larsen, B., & Smidsrod, O. (1986). Tailoring of alginates by enzymatic modication in vitro. International Journal of Biological Macromolecules, 8(6), 330336. Smidsrod, O., Haug, A., & Lian, B. (1972). Properties of poly (1,4heuronates) in the gel state. I. Evaluation of a method for the determination of stiffness. Acta Chemica Scandinavica, 26(1), 7178. Smidsrod, O., & Skjak-Braek, G. (1990). Alginate as immobilization matrix for cells. Trends in Biotechnology, 8(3), 7178. Steenson, L. R., Klaenhammer, T. R., & Swaisgood, H. E. (1987). Calcium alginate-immobilized cultures of lactic streptococci are protected from bacteriophages. Journal of Dairy Science, 70(6), 11211127. Sung, H. H. (1997). Enhancing survival of lactic acid bacteria in ice cream by natural encapsulation. Dissertation Abstracts International, 13(9), 5407. Tagg, J. R., Dajani, A. S., & Wannamaker, L. W. (1976). Bacteriocins of gram-negative bacteria. Bacteriogical Review, 40(3), 722 5407756. Takata, I., Tosa, T., & Chibata, I. (1977). Screening of matrix suitable for immobilization of microbial cells. Journal Solid-Phase Biochemistry, 2(2), 225236. Tanaka, H., Irie, S., & Ochi, H. (1989). A novel immobilization method for prevention of cell leakage from the gel matrix. Journal of Fermentation and Bioengineering, 68(3), 216219. Tanaka, H., Masatose, M., & Veleky, I. A. (1984). Diffusion characteristics of substrates in Ca-alginate beads. Biotechnology and Bioengineering, 26(1), 5358. Tosa, T., Sato, T., Mori, T., Yamamoto, K., Takata, I., Nishida, Y., & Chibata, I. (1979). Immobilization of enzymes and microbial cells using carrageenan as matrix. Biotechnology and Bioengineering, 21(2), 16971700. Vinderola, C. G., Bailo, N., & Reinheimer, J. A. (2000). Survival of probiotic microora in Argentinian yoghurts during refrigerated storage. Food Research International, 33(2), 97102. Zhou, Y., Martins, E., Groboillot, A., Champagne, C. P., & Neufeld, R. J. (1998). Spectrophotometric quantication of lactic bacteria in alginate and control of cell release with chitosan coating. Journal of Applied Microbiology, 84(3), 342348.