AnalAK07_Recent Advances in Microencapsulation

Trends in Food Science & Technology 18 (2007) 240e251

Recent advances in

microencapsulation

of probiotics for

industrial

applications and

targeted delivery

Anil Kumar Anal* andHarjinder Singh

Riddet Centre, Massey University, AgHort Building

Block C, Riddet Road, Private Bag 11 222, Palmerston

North 4442, New Zealand (Tel.: D64 6 3505356;

fax: D64 6 3505655; e-mail: [email protected])

Because of their perceived health benefits, probiotics have

been incorporated into a range of dairy products, including

yoghurts, soft-, semi-hard and hard cheeses, ice cream, milk

powders and frozen dairy desserts. However, there are still

several problems with respect to the low viability of probiotic

bacteria in dairy foods. This review focuses mainly on current

knowledge and techniques used in the microencapsulation

of probiotic microorganisms to enhance their viability during

fermentation, processing and utilization in commercial

products. Microencapsulation of probiotic bacteria can be

used to enhance the viability during processing, and also for

the targeted delivery in gastrointestinal tract.

IntroductionProbiotics have been defined in several ways, depending

on our understanding of the mechanisms of action of theireffects on the health and well-being of humans. The mostcommonly used definition is that of Fuller (1989): probiot-ics are live microbial feed supplements that beneficiallyaffect the host by improving its intestinal microbial

* Corresponding author.

0924-2244/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.tifs.2007.01.004

Review

balance. Recently Food and Agriculture Organization(FAO) of the United Nations and the World Health Organi-zation (WHO) define probiotics as ‘‘Live microorganisms(bacteria or yeasts), which when ingested or locally appliedin sufficient numbers confer one or more specified demon-strated health benefits for the host’’ (FAO/WHO, 2001).The beneficial effects of probiotics on the human gut florainclude antagonistic effects and immune effects. The useof probiotic bacterial cultures stimulates the growth ofpreferred microorganisms, crowds out potentially harmfulbacteria and reinforces the body’s natural defense mechanisms(Dunne, 2001; Gismondo, Drago, & Lombardi, 1999). Themechanism of anti-pathogenic effect may be throughdecreasing the luminal pH by the production of short chainfatty acids such as acetic acid, lactic acid or propionic acid,rendering vital nutrients unavailable to pathogens, alteringthe redox potential of the environment, producing hydrogenperoxide or producing bacteriocins or other inhibitorysubstances (Kailasapathy & Chin, 2000).

Probiotics may cause cell-mediated immune responses,including activation of the reticulo-endothelial system,augmentation of cytokine pathways and stimulation ofpro-inflammatory pathways such as tumour necrosis factorsand interleukin regulation, without being a target of thehost immune system (Gill, Cross, Rutherfurd, & Gopal,2001; Isolauri, 2000; Isolauri, Arvola, Sutas, Moilanen, &Salminen, 2000). Probiotics may even activate macro-phages directly (Tejada-Simon, Ustunol, & Pestka, 1999).Recently, probiotics have been proposed for various treat-ments of human intestinal barrier dysfunctions such aslactose intolerance, acute gastroenteritis, food allergy, atopicdermatitis, Crohn’s disease, rheumatoid arthritis, and coloncancer (Kalliomaki, Salminen, Poussa, Arvilommi, &Isolauri, 2003; Lee, Puong, Ouwehand, & Salminen, 2003;Rinkinen, Jalava, Westermarck, Salminen, & Ouwehand,2003; Salminen et al., 1998).

Lactic acid bacteria (LAB) are the most important pro-biotic microorganisms typically associated with the humangastrointestinal tract. These bacteria are Gram-positive,rod-shaped, non-spore-forming, catalase-negative organ-isms that are devoid of cytochromes and are of non-aerobichabit but are aero-tolerant, fastidious, acid-tolerant andstrictly fermentative; lactic acid is the major end-productof sugar fermentation (Axelsson, 1993). A few of theknown LAB that are used as probiotics are Lactobacillusacidophilus, Lactobacillus amylovorous, Lactobacillus

mailto:[email protected]

241A.K. Anal, H. Singh / Trends in Food Science & Technology 18 (2007) 240e251

casei, Lactobacillus crispatus, Lactobacillus delbrueckii,Lactobacillus gasseri, Lactobacillus johnsonoo, Lacto-bacillus paracasei, Lactobacillus plantarum, Lactobacillusreuteri, Lactobacillus rhamnosus etc. (Makinen & Bigret,1993).

Other common probiotic microorganisms are the bifido-bacteria. Bifidobacteria are also Gram-positive and rod-shaped but are strictly anaerobic. These bacteria cangrow at pH in the range 4.5e8.5. Bifidobacteria activelyferment carbohydrates, producing mainly acetic acid andlactic acid in a molar ratio of 3:2 (v/v), but not carbon di-oxide, butyric acid or propionic acid. The most recognizedspecies of bifidobacteria that are used as probiotic organ-isms are Bifidobacterium adolescentis, Bifidobacteriumanimalis, Bifidobacterium bifidum, Bifidobacterium breve,Bifidobacterium infantis, Bifidobacterium lactis and Bifido-bacterium longum. Other than these bacteria, Bacilluscereus var. toyoi, Escherichia coli strain nissle, Propionio-bacterium freudenreichii, and some types of yeasts, e.g.Saccharomyces cerevisiae and Saccharomyces boulardiihave also been identified as having probiotic effects (Hol-zapfel, Haberer, Geisen, Bjorkroth, & Schillinger, 2001).

Because of their perceived health benefits, probiotic bac-teria have been increasingly included in fermented dairyproducts, including yoghurts, soft-, semi-hard and hardcheeses, ice cream and frozen fermented dairy desserts(Desmond et al., 2005; Dinakar & Mistry, 1994; Stanton,Desmond, Fitzgerald, & Ross, 2003; Stanton et al., 2001;Stanton, Ross, Fitzgerald, & Van Sinderen, 2005).

The ability of probiotic microorganisms to survive andmultiply in the host strongly influences their probiotic ben-efits. The bacteria should be metabolically stable and activein the product, survive passage through the upper digestivetract in large numbers and have beneficial effects when inthe intestine of the host (Gilliland, 1989). The standardfor any food sold with health claims from the additionof probiotics is that it must contain per gram at least106e107 cfu of viable probiotic bacteria (FAO/WHO,2001). However, there are still several problems with re-spect to the low viability of probiotic bacteria in dairyfoods. Several factors have been reported to affect the via-bility of probiotics in fermented dairy products, includingtitratable acidity, pH, hydrogen peroxide, dissolved oxygencontent, storage temperature, species and strains of associa-tive fermented dairy product organisms, concentration oflactic and acetic acids and even whey protein concentration(Dave & Shah, 1997; Kailasapathy & Supriadi, 1996; Lan-kaputhra, Shah, & Britz, 1996). Survival is, of course, es-sential for organisms targeted to populate the human gut,one of the most important issues in health benefit provisionby probiotic bacteria.

Different approaches that increase the resistance of thesesensitive microorganisms against adverse conditions havebeen proposed, including appropriate selection of acid- andbile-resistant strains, use of oxygen-impermeable containers,two-step fermentation, stress adaptation, incorporation of

micronutrients such as peptides and amino acids, and micro-encapsulation (Gismondo et al., 1999).

Microencapsulation technologyMicroencapsulation is defined as a technology of packag-

ing solids, liquids or gaseous materials in miniature, sealedcapsules that can release their contents at controlled ratesunder the influences of specific conditions (Anal & Stevens,2005; Anal, Stevens, & Remunan-Lopez, 2006; Kailasapathy& Masondole, 2005). A microcapsule consists of a semi-permeable, spherical, thin, and strong membrane surroundinga solid/liquid core, with a diameter varying from a few mi-crons to 1 mm. A brief description of microencapsulationtechniques for encapsulation probiotic microorganisms isgiven in Table 1. In a broad sense, encapsulation can beused for many applications in the food industry, includingstabilizing the core material, controlling the oxidative reac-tion, providing sustained or controlled release (both temporaland time-controlled release), masking flavours, colours orodours, extending the shelf life and protecting componentsagainst nutritional loss. Food-grade polymers such as algi-nate, chitosan, carboxymethyl cellulose (CMC), carrageenan,gelatin and pectin are mainly applied, using various micro-encapsulation technologies (Table 2).

Microcapsules and microspheres can be engineered togradually release active ingredients. A microcapsule maybe opened by many different means, including fracture byheat, solvation, diffusion, and pressure (Brannon-Peppas,1997). A coating may also be designed to open in thespecific areas of the body. A microcapsule containingacid-labile core materials that will be consumed by gastro-intestinal fluids must not be fractured until after it passesthrough the stomach. A coating can therefore be used thatis able to withstand acidic conditions in the stomach acidsand allows those active ingredients to pass through thestomach (Anal, Bhopatkar, Tokura, Tamura, & Stevens,2003; Anal & Stevens, 2005). Fig. 1 illustrates the swelling,erosion and disintegration of milk proteinepolysaccharides’microcapsules containing probiotics (Unpublished). Thesemicrocapsules were first incubated in simulated gastricfluid (SGF, pH 1.2) for 2 h and then transferred into simu-lated intestinal fluid (SIF, pH 7.4).

This review focuses on the current knowledge and tech-niques used in the microencapsulation of probiotic micro-organisms to enhance the performance of these organismsduring fermentation, downstream processing and utilizationin commercial products.

Spray- and freeze-dried probiotic productsProbiotic cultures for food applications are frequently

supplied in frozen or dried form, as either freeze-dried orspray-dried powders (Holzapfel et al., 2001). The success-ful spray drying of Lactobacilli and Bifidobacteria has pre-viously been reported for a number of different strains,including L. paracasei (Desmond, Ross, O’Callaghan,

242 A.K. Anal, H. Singh / Trends in Food Science & Technology 18 (2007) 240e251

Table 1. Techniques and processes used for encapsulating probiotic microorganisms

Microencapsulationtechniques

Types of materials for coating Major steps in processes

Spray-drying Water-soluble polymers (i) Preparation of the solutions including microorganisms(ii) Atomization of the feed into spray(iii) Drying of spray (moisture evaporation)(iv) Separation of dried product form

Spray-congealing Waxes, fatty acids, water-solubleand water-insoluble polymers,monomers

(i) Preparation of the solutions containing core (e.g. probiotics)(ii) Solidification of coat by congealing the molten coating materials intonon-solvent(iii) Removal of non-solvent materials by sorption, extraction orevaporation techniques

Fluidized-bed coating/air-suspension

Water-insoluble and water-solublepolymers, lipids, waxes

(i) Preparation of coating solutions(ii) Fluidization of core particles(iii) Coating of core particles with coating solutions

Extrusion Water-soluble and water-insoluble polymers

(i) Preparation of coating solution materials(ii) Dispersion of core materials(iii) Cooling or passing of core-coat mixtures through dehydrating liquid

Coacervation/phaseseparation technique

Water-soluble polymers (i) Core material is dispersed in a solution of coating polymer, the solventfor the polymer being the liquid manufacturing vehicle phase(ii) Deposition of the coating, accomplished by controlled, physicalmixing of the coating and core materials in the vehicle phase(iii) Rigidifying the coating by thermal, cross-linking or desolvationtechniques, to form self-sustaining microcapsules

Electrostatic method Oppositely charged polymers/compounds

(i) Mixing of core and coating materials(ii) Extrusion of mixtures of core-coating materials in oppositely chargedsolutions(iii) Freeze-dry or oven-dry of microcapsules/microspheres/beads

Fitzgerald, & Stanton, 2002; Gardiner et al., 2000), Lacto-bacillus curvatus (Mauriello, Aponte, Andolfi, Moschetti,& Villani, 1999), L. acidophilus (Prajapati, Shah, &Dave, 1987), L. rhamnosus (Corcoran, Ross, Fitzgerald,& Stanton, 2004) and Bifidobacterium ruminantium(O’Riordan, Andrews, Buckle, & Conway, 2001). However,most probiotic bacteria do not survive well during the tem-perature and osmotic extremes to which they are exposedduring the spray drying process (Selmer-Olsen, Sorhaug,Birkeland, & Pehrson, 1999; Teixeira, 1979). When spraydrying is used for the preservation of potential probioticcultures, much of their activity is typically lost aftera few weeks of storage at room temperature. This is associ-ated with stress that is induced by temperature changes,phase changes and drying, a combination of which tendsto damage cell membranes and associated proteins.

One approach used by a number of researchers to im-prove probiotic survival is the addition of protectants tothe media prior to drying. For example, the incorporationof thermoprotectants, such as trehalose (Conrad, Miller,Cielenski, & de Pablo, 2000), non-fat milk solids and/or adnitol (Selmer-Olsen et al., 1999), growth promotingfactors including various probiotic/prebiotic combinations(Desmond et al., 2002) and granular starch (Crittendenet al., 2001) have been shown to improve culture viabil-ity during drying and storage. Recently, incorporation ofthe soluble fiber, gum acacia, into a milk-based mediumprior to spray drying the probiotic L. paracasei was

found to increase its viability during storage, comparedwith milk powder alone (Desmond et al., 2002). How-ever, other soluble fibers investigated, including inulinand polydextrose, did not enhance probiotic viabilityduring spray drying or powder storage (Corcoran, Stanton,Fitzgerald, & Ross, 2005).

Microencapsulation by spray drying is a well-establishedprocess that can produce large amounts of material. Never-theless, this economical and effective technology for protect-ing materials is rarely considered for cell immobilizationbecause of the high mortality resulting from simultaneousdehydration and thermal inactivation of microorganisms.In response to these limitations, a low cost microencapsula-tion method, which can be easily scaled up, to improve thestability of probiotic lactic cultures has been proposed (Picot& Lacroix, 2003a, 2003b). The technique consists of coatingmilk fat droplets containing powder particles of freeze-driedbacteria with whey protein polymers, using emulsificationand spray drying in a continuous two-step process. Success-ful production of the resulting multiphase low diameter mi-crocapsules requires rigorous control of the size distributionof the different elements constituting the capsules. In partic-ular, the material dispersed in the hydrophobic phase must belarger than the bacterial cells and smaller than the fat glob-ules. In order to decrease the powder particle size, in laterstudies, Picot and Lacroix (2003c) micronized the powderparticles, produced by spray drying and emulsificationmethods, using a spiral jet mill as a grinding system. The

Table

Bacter gy Functionality References

Lactob Biomass production Klein and Vorlop (1985)S. ther Biomass production Audet et al. (1988, 1990, 1991)L. bulg Biomass production Ouellette et al. (1994)B. infaLactob Biomass production

Doleyres, Fliss et al. (2002),Doleyres et al. (2004), Doleyres,Paquin et al. (2002)

Bifidob

Bifidob Biomass production Kebary (1996)Lactob Acid stable Chandramouli et al. (2004)BifidobBifidob Acid and bile salt stable Rao et al. (1989)L. delb Biomass production Sheu et al. (1993)L. case Acid stable Chan and Zhang (2002)L. lact Biomass production Hyndman et al. (1993)Lactob Acid stable Chandramouli et al. (2004)L. acid Acid stable Chan and Zhang (2002)B. bre Acid stable Hansen et al. (2002)B. LonPedico

Acid stable Picot and Lacroix (2003a, 2003b, 2004)

Bifidob Acid stable/storage Lee et al. (2004)Bifidob Acid stable/storage Guerin et al. (2003)Bifidob Acid stable/storage Crittenden et al. (2001)Lactob Acid stable/storageBifidob Acid stable/storage O’Riordan et al. (2001)Lactob Acid stable

and stable during storageSultana et al. (2000), Kailasapathyand Chin (2000)Bifidob

24

3A

.K.

Anal,

H.

Singh

/Tren

ds

inFo

od

Science

&Tech

nolo

gy18

(2007)

240

e251

2. Encapsulation of probiotic bacteria in various polymer systems

ia systems Polymers Microencapsulation technolo

acillus Carrageenan Gel beadsmophilus Carrageenan Gel beadsaricus Carrageenan/locust bean gum Gel beadsntisacillus

Carrageenan/locust bean gumGel beads

acterium

acterium Alginate/glycerol Gel beadsacillus Alginate Gel beadsacteriumacterium pseudolongum Cellulose acetate phthalate Gel beadsrueckii Alginate/sodium lauryl sulphate Gel beadsi Carrageenan/locust bean gum Emulsification

is Gelatin/toluene-2-4-diisocyanate Gel beadsacillus Alginate Gel beadsophillus Alginate Direct compression

ve Alginate microspheres Emulsificationgumccus acidilactei

Whey protein Micronization

acterium Alginate/chitosan Gel beadsacterium Alginate/pectin/whey protein Gel beadsacterium Resistant starch Gel beadsacillus Starch Gel beadsacterium Waxy maize starch Gel beads/emulsificationacillus

Alginate/starchGel beads

acterium


Fig. 1. Photographs showing the disintegration mechanism (swelling and burst effect) of whey protein isolateealginateechitosanecalcium chloridemicrocapsules incubated in simulated gastrointestinal fluids; simulated gastric fluid (SGF, pH 1.2), and simulated intestinal fluid (SIF, pH 7.4) at37 �C: (a) dried microcapsules; (b) microcapsules incubated in SGF for 2 h; (c) microcapsules incubated in SIF for 2 h; (d) microcapsules incubated

in SIF for 8 h (Unpublished).

effect of micronization on cell viability was governed mainlyby the final particle size of the processed culture. They re-vealed that micronization is an effective means of reducingthe powder particle size of freeze-dried cultures with an ac-ceptable mortality rate before producing microcapsules withlow diameters for the protection of sensitive probiotic bacte-ria. However, the use of micronized cultures in cell encapsu-lation technologies that require heat treatment, such as spraydrying, may reduce cell viability.

Direct dispersion of fresh cells in a heat-treated wheyprotein suspension followed by spray drying was found tobe an alternative and less destructive microencapsulationmethod, with survival rates after spray drying of 26% forB. breve and 1.4% for the more heat-sensitive B. longum

(Picot & Lacroix, 2004). Even though the viability of thebacteria after spray drying remained low, these microparti-cles showed cell protection in gastric juice and controlledrelease of probiotic bacteria under simulated intestinalconditions.

Furthermore, the addition of cryoprotectants during thefreeze drying of lactobacilli has been used to help overcomeinactivation during drying and stabilization during storage.In a recent study, freeze-dried Lactobacillus bulgaricus sur-vived better during storage at �20 �C over 10 months whencells had been grown in the presence of fructose, lactose ormannose or when glucose, fructose, monosodium glutamateor sorbitol was added to the drying medium (Carvalho et al.,2002, 2003, 2004a, 2004b). In particular, trehalose,


a disaccharide of glucose, has been found to be effective atprotecting the bacterial cells during freezing and drying(Garcia De Castro, Bredholt, Strom, & Tunnaclife, 2000).

Encapsulation of probiotics in polymer systemsEncapsulation of probiotics in a biodegradable polymer

matrix has a number of advantages. Once entrapped/encapsulated in matrix beads or in microcapsules, the cellsare easier to handle than in a suspension or in slurry. Thenumber of cells in beads or microparticles can be quanti-fied, allowing the dosage to be readily controlled. Cryo-and osmo-protective components can be incorporated intothe matrix, enhancing the survival of cells during process-ing and storage. Finally, once the matrix beads/microcap-sules have been dried, a further surface coating can beapplied. This outer layer can be used to alter the aestheticand sensory properties of the product and may also be func-tional, providing an extra level of protection to the cells. Inaddition, the coating layer can have desirable dissolutionproperties, which permit delayed release of the cells orrelease upon, for example, a change in pH.

Various polymer systems have been used to encapsulateprobiotic microorganisms to protect against low pH andhigh bile concentrations and to enhance physical stabilityduring downstream processing. Microcapsule or bead sys-tems using various biopolymers are very easy to prepareon a lab-scale, and any ingredients can be encapsulated,whether it is hydrophilic, hydrophobic, liquid, or a viscousoil, a solid etc. However, the scaling up of the process isvery difficult and processing costs are very high. Moreover,most of the conventionally produced microcapsules (e.g.calcium alginate beads/microcapsules), tend to be very po-rous which allows fast and easy diffusion of water and otherfluids in and out of the matrix.

Spherical polymer beads with diameters ranging from0.3 to 3.0 mm and immobilizing active biomass are pro-duced using extrusion or emulsification techniques, by ther-mal (k-carrageenan, gellan, agarose, gelatin) or ionotropic(alginate, chitosan) gelation of the droplets. Some of thesesystems are discussed in more detail in the followingsections.

Encapsulation of probiotics in k-carrageenanCarrageenan is a natural polysaccharide that is extracted

from marine macroalgae and is commonly used as a foodadditive. Elevated temperatures (60e80 �C) are needed todissolve the polymer at concentrations ranging from 2 to5% (Klein & Vorlop, 1985). Gelation of k-carrageenan isgenerally dependent on a change in temperature. The cellslurry is added to the heat-sterilized carrageenan solutionat 40e45 �C and gelation occurs by cooling to room temper-ature. The beads are formed after dropping the mixture ofpolymer and cells into a potassium chloride (KCl) solution.Audet, Paquin, and Lacroix (1988) reported the inhibitoryeffect of KCl on some bacteria such as Streptococcus ther-mophilus and L. bulgaricus. Later they used a combination

of k-carrageenan and locust bean gum to encapsulate LABto enhance their stability during biomass production in dairyproducts (Audet, Paquin, & Lacroix, 1990, 1991). The gelbead strength can be enhanced using another polymer,such as locust bean gum. A ratio of carrageenan to locustbean gum of 2:1, through specific interaction of the galacto-mannan chains of locust bean gum with carrageenan, hasbeen found to give the synergistic effects and to form thestrong gel beads. Ouellette, Chevalier, and Lacroix (1994)immobilized a pure culture of B. infantis in k-carrageenan/locust bean gum beads and also used this system to contin-uously ferment skimmed milk supplemented with 1% yeastextract. Immobilized cell growth and cell release from thegel beads into the circulating milk allowed for a steady in-oculation of the feed for a maximal cell volumetric produc-tivity of approximately 1� 1012 cfu/mL.

Recently, Doleyres, Fliss, and Lacroix (2002, 2004) andDoleyres, Paquin, LeRoy, and Lacroix (2002) immobilizedprobiotic cells in carrageenan and locust bean gum gelbeads by ionotropic gelation method to produce a mixedlactic culture containing a non-competitive strain of bifido-bacteria and a competitive LAB strain, during repeatedbatches and continuous cultures. The two-stage continuousfermentation system, composed of a first reactor containingcells of the two strains separately immobilized in carra-geenan/locust bean gum gel beads and a second reactor op-erated with free cells released from the first reactor, allowedthe continuous production of a concentrated mixed culturewith a strain ratio of a composition that depended ontemperature. Cells produced by this technology exhibitedimportant physiological changes and increased stress toler-ance. The tolerance of these cells to stresses, such as freezedrying, hydrogen peroxide and simulated gastrointestinalconditions, increased markedly with culture time and after15 days was higher than the tolerance of cells produced bya conventional method.

Encapsulation of probiotics in alginate systemsAlginic acid, a natural polymer, is a polyuronic acid that

is extracted from seaweeds and is composed of variousproportions of 1e4 linked b-D-mannuronic (M) and a-L-guluronic (G) acids. These residues are present in variousproportions depending on the source of the alginic acid. Al-ginic acid and its salts are block copolymers, containingboth MM and GG homopolymer blocks and mixed blockscontaining irregular sequences of M and G units. The bind-ing of divalent cations and the subsequent gel formation aredependent on the composition and arrangement of theblocks of residues (Gemeiner, Rexova-Benkova, Svec, &Norrlow, 1994). The GG blocks have preferential bindingsites for divalent counter-ions, such as Ca2þ, and the boundions interact with other GG blocks to form linkages thatlead to gel formation. On addition of sodium alginate solu-tion to a calcium solution, interfacial polymerization is in-stantaneous, with precipitation of calcium alginate followedby a more gradual gelation of the interior as calcium ions


permeate through the alginate systems. The size of thebeads is generally dependent on the viscosity of the poly-mer solution, the diameter of the orifice and the distancebetween the outlet and the coagulation solution (Analet al., 2003; Anal & Stevens, 2005).

Various researchers (Chandramouli, Kailasapathy,Peiris, & Jones, 2004; Lee, Cha, & Park, 2004; Sheu,Marshall, & Heymann, 1993) have studied factors affectingbead preparation, such as concentrations of alginate andCaCl2, timing of hardening of the beads and cell concentra-tions on encapsulation of probiotics. The conventionalencapsulation method, with sodium alginate in calciumchloride (CaCl2), has been used to encapsulate L. acidophi-lus to protect this organism from the harsh acidic conditionsin gastric fluid. Studies have shown that calcium-alginateeimmobilized cell cultures are better protected, shown by anincrease in the survival of bacteria under different condi-tions, than the non-encapsulated state. The results fromthese studies indicate that the viability of encapsulatedbacteria in simulated gastric fluid increases with an increasein capsule size.

However, Hansen, Allan-Wojtas, Jin, and Paulson (2002)reported that very large calcium alginate beads (>1 mm)cause a coarseness of texture in live microbial feed supple-ments and that small beads of size less than 100 mm do notsignificantly protect the bacteria in simulated gastric fluid,compared with free cells. These studies indicate that thesebacteria should be encapsulated within a particular sizerange. They tested nine different strains of Bifidobacteriumspp. for their tolerance to simulated gastrointestinal condi-tions, and observed some variations among the strains forresistance to gastric fluid (pH 2e3) and bile salts (5 and10 g/L). Among these strains, only a strain B. lactisBb-12 was found to be resistant to low pH and bile salts.They also encapsulated some of the strains in alginatemicrospheres to evaluate their resistance properties in gas-tric fluid and to bile salts. They obtained alginate micro-spheres (20e70 mm) by emulsifying the mixture of cellsand sodium alginate in vegetable oil and subsequentlycross-linking with CaCl2. Cryo-scanning electron micro-scopy revealed that these microparticles were denselyloaded with probiotic bacteria and were porous. The loadedalginate microparticles remained stable during storage at4 �C in 0.05 M CaCl2 and in milk (2% fat), sour creamand yoghurt for up to 16 days and in simulated gastric fluid(pH 2.0) for 1 h at 37 �C. However, the microparticles ex-posed to low pH did not improve the survival of acid-sensitive bifidobacteria. Kebary (1996) also showed that B.bifidum survived in higher numbers in frozen milk in beadsmade from alginate than in beads made from k-carrageenan.

Recently, Chen, Chen, Liu, Lin, and Chiu (2005) usedprebiotics (fructooligosaccharides or isomaltooligosacchar-ides), a growth promoter (peptide) and sodium alginate ascoating materials to microencapsulate different probioticssuch as L. acidophilus, L. casei, B. bifidum and B. longum.A mixture containing sodium alginate (1% w/v) mixed with

peptide (1% w/w) and fructooligosaccharides (3% w/w) ascoating materials produced the highest survival in terms ofprobiotic count.

Chan and Zhang (2002) developed an encapsulationtechnique of compression coating, which permits the stabi-lization of lyophilized cells during storage. This techniqueinvolves compressing the lyophilized cell powder intoa core tablet and then compressing coating materials aroundthis core to form the final compact. The objective of thiswork was to investigate the use of methacrylic acid copoly-mer as an enteric coating material for the compressioncoating of an industrially sourced strain of L. acidophilus.It was hoped that this enteric coating material would pro-tect the cells as they passed through the stomach, and,when used together with pectin, could be used to targetthe release of the probiotics to the terminal ileum and thebeginning of the colon in the human gastrointestinal tract.The release profile and the viability of the probiotic cellsduring passage through the simulated gastrointestinal tractwere investigated. The coating material used was a mixtureof sodium alginate and hydroxypropyl cellulose in theweight ratio 9:1. The encapsulated cells showed a 104e105-fold increase in cell survival compared with free cells underacidic conditions. The formation of a hydrogel barrier bythe compacted sodium alginate layer was shown to retardthe permeation of the acidic fluid into the cells. In vitrotests further revealed that the release of encapsulated cellsin the human digestive tract could occur near the end ofthe ileum and the beginning of the colon. The mechanismof cell release was considered to be primarily due to erosionof the alginate gel layer.

Cui, Goh, Kim, Choi, and Lee (2001) prepared poly-L-lysineecross-linked alginate microparticles loaded withbifidobacteria. They used an air atomization method tospray the alginateebacteria culture in a coagulation bathcontaining CaCl2. The microparticles were further cross-linked with poly-L-lysine. The survival of bifidobacteriafrom the alginateepoly-L-lysine microparticles was muchhigher, even in the lower pH media. Due to stability ofpoly-L-lysineecross-linked alginate microparticles in gas-tric fluid, bifidobacteria can be protected without losingtheir survivability. The survival of bifidobacteria loaded inthe particles remained highest (2.67� 109 cfu/g) at pH 6.8while the number is reduced at lower pH (1.5, exposuretime, 2 h) to 5.0� 107 cfu/g. However, only 1e3% of theunencapsulated bifidobacteria can survive in lower pH.The stability of the free-flowing bifidobacteriaealginateepoly-L-lysine microparticles was also improved duringstorage at 4 �C in a refrigerator, compared with freecultures.

Encapsulation of probiotics in cellulose acetatephthalate (CAP)

Because of its ionizable phthalate groups, this cellulosederivative polymer is insoluble in acid media at pH 5 andlower but is soluble at pH higher than 6. In addition,


CAP is physiologically inert when administered in vivo,and is, therefore, widely used as an enteric coating materialfor the release of core substances for intestinal targeteddelivery systems. Rao, Shiwnarain, and Maharaj (1989) re-ported the encapsulation of B. pseudolongum in CAP usingan emulsion technique. Microencapsulated bacteria sur-vived in larger numbers (109 cfu/mL) in an acidic environ-ment than non-encapsulated organisms, which did notretain any viability when exposed to a simulated gastricenvironment for 1 h. Favaro-Trindale and Grosso (2002)encapsulated B. lactis and L. acidophilus in CAP polymerusing a spray drying method. This study evaluated the resis-tance of microencapsulated microorganisms in acid andhigh bile salt concentrations. Spray-dried microcapsulesof CAP containing B. lactis and L. acidophilus were effec-tive in protecting both these microorganisms when inocu-lated into media with pH values similar to those in thehuman stomach. Microencapsulated L. acidophilus suffereda reduction of only 1 log at pH 1 after 2 h of incubation,and the population of B. lactis was reduced by only 1 logimmediately after inoculation into a pH 1 medium and be-tween 1 and 2 h after inoculation into a pH 2 medium. Afterinoculation of the CAP microcapsules loaded with bacteriainto bile solution (pH 7), complete dissolution of the pow-der indicated that both the wall material and the processused in the preparation of the microcapsules were adequatein protecting the bacteria, to pass undamaged through theacidic conditions of the stomach, followed by their rapidliberation in the pH of the intestine.

Encapsulation of probiotics in proteins andpolysaccharide mixtures

Gelatin is useful as a thermally reversible gelling agentfor encapsulation. Because of its amphoteric nature, it is alsoan excellent candidate for incorporating with anionic-gel-forming polysaccharides, such as gellan gum. Thesehydrocolloids are miscible at pH >6, because they bothcarry net negative charges and repel one another. However,the net charge of gelatin becomes positive when the pH isadjusted below its isoelectric point and causes a stronginteraction with the negatively charged gellan gum (King,1995). Hyndman, Groboillot, Poncelet, Champagne, andNeufeld (1993) used high concentrations of gelatin (24%w/v) to encapsulate Lactobacillus lactis by cross-linkingwith toluene-2,4-diisocyanate for biomass production.

In a recent study, Guerin, Vuillemard, and Subirade(2003) encapsulated Bifidobacterium cells in a mixed gelcomposed of alginate, pectin and whey proteins. They in-vestigated the protective effects of gel beads without extramembrane and gel beads coated with extra membranes,formed by the conjugation of whey protein and pectin, insimulated gastric pH and bile salt solutions on the survivalof free and encapsulated B. bifidum. After 1 h of incubationin acidic solution (pH 2.5), the free cell counts decreasedby 4.75 log, compared with a decrease of <1 log for entrap-ped cells. The free cells did not survive after 2 h of

incubation at pH 2.5, whereas the immobilized cells de-creased by about only 2 log. After incubation (1 or 3 h)in 2 and 4% bile salt solutions, the mortality for B. bifidumcells in membrane-free gel beads (4e7 log) was greaterthan that for free cells (2e3 log). However, the counts ofcells immobilized in membrane-coated gel beads decreasedby <2 log. The double membrane coating enhanced the re-sistance of the cells to acidic conditions and higher bile saltconcentrations.

Encapsulation of probiotics in chitosanThe biopolymer chitosan, the N-deacetylated product of

the polysaccharide chitin, is gaining importance in the foodand pharmaceutical field because of its unique polymericcationic character, good biocompatibility, non-toxicity andbiodegradability. Chitosan can be isolated from crustaceanshells, insect cuticles and the membranes of fungi. Theproperties of chitosan vary with its source. The terms chitinand chitosan refer not to specific compounds but to twotypes of copolymers, containing the two monomer residuesanhydro-N-acetyl-D-glucosamine and anhydro-D-glucosamine,respectively. Chitin is a polymer of b-(1-4)-2-acetamido-2-deoxy-D-glucopyranose and is one of the most abundant or-ganic materials on earth and second to cellulose and murein,which is the main structural polymer of the bacterial cellwall. In order to achieve sufficient stability, chitosan gelbeads and microspheres can be ionically cross-linked withpolyphosphates (Anal & Stevens, 2005) and sodium algi-nate (Anal et al., 2003).

Krasaekoopt, Bhandari, and Deeth (2003, 2004) evaluatedthe survival of probiotics encapsulated in chitosan-coatedalginate beads in yoghurt and in UHT- and conventionallytreated milk during storage. They used L. acidophilus 547,L. casei 01 and B. bifidum 1994 as model organisms for theirstudy. The survival of the encapsulated bacteria was higherthan that of the free cells by approximately 1 log. The numberof probiotic bacteria was maintained above the recommen-ded therapeutic minimum (107 cfu/g) throughout storagefor the lactobacilli but not for the bifidobacteria. Lee et al.(2004) carried out a similar study and compared various chi-tosans (different molecular weights) for coating conventionalalginate beads. They investigated the effects of chitosanealginate microparticles on the survival of L. bulgaricusKFRI763 in simulated gastric juices and simulated intestinalfluid and on their stability during storage at 4 and 22 �C. Theprobiotic loaded in alginate microparticles was prepared byspraying a mixture of sodium alginate and cell culture intoa CaCl2echitosan solution using an air-atomizing device.When the microorganism was exposed to gastric fluid (pH2.0) for 1 h, none survived. In contrast, an impressive andhigh survival rate was obtained when the sprayed particleswere coated with chitosan. They concluded that the microen-capsulation of LAB with alginate and a chitosan coating of-fers an effective means of delivering viable bacterial cells tothe colon and maintaining their survival during refrigeratedstorage.


Encapsulation of probiotics in starchStarch is a dietary component that has an important role

in colonic physiology and functions and a potential protec-tive role against colorectal cancer (Cassidy, Bingham, &Cummings, 1994). Resistant starch is the starch that isnot digested by pancreatic amylases in the small intestineand reaches the colon, where it can be fermented by humanand animal gut microflora. The fermentation of carbohy-drates by anaerobic bacteria produces short chain fattyacids and lowers the pH in the lumen (Kleessen et al.,1997; Le Blay, Michel, Blottiere, & Cherbut, 1999). Resis-tant starch can be used to ensure the viability of probioticpopulations from the food to the large intestine. Resistantstarch also offers an ideal surface for adherence of the pro-biotics to the starch granule during processing, storage andtransit through the upper gastrointestinal tract, providingrobustness and resilience to environmental stresses. Bacte-rial adhesion to starch may also provide advantages in newprobiotic technologies to enhance delivery of viable andmetabolically active probiotics to the intestinal tract(Crittenden et al., 2001).

A group of researchers (Mattila-Sandholm et al., 2002)worked on stabilization of LAB and to formulate new typesof foods fortified with encapsulated health-promoting bac-teria in the starch system. In their study, large potato starchgranules (50e100 mm), which were enzymatically treatedto obtain a porous structure, were used as a carrier. Sub-sequently, amylose, the linear polymer of starch, was solu-bilized, cooled and precipitated over the bacteria-filledstarch granules. Finally, the whole product, together withthe growth medium, was freeze dried to a powder form.They found the encapsulated LAB can survive at least6 months at room temperature under normal atmospherichumidity, and at least 18 months when frozen.

Talwalkar and Kailasapathy (2003) produced alginateestarch gel beads by dropping a mixture of alginateestarchebacteria into a CaCl2 coagulation bath. The probioticbacteria used for this study were L. acidophilus and B. lactis.They found that encapsulation prevented cell death fromoxygen toxicity. It is known that alginate gel beads restrictthe diffusion of oxygen through the gel, creating anoxic re-gions in the centre of the beads. In another report, they useda modified method to encapsulate probiotic bacteria in analginateestarch system. The incorporation of Hi-Maizestarch improved the encapsulation of viable bacteriacompared with the bacteria encapsulated without starch(Iyer & Kailasapathy, 2005; Sultana et al., 2000).

Conclusions and future directionsSophisticated shell materials and technologies have been

developed and an extremely wide variety of functionalitiescan now be achieved through microencapsulation. Any typeof triggers can be used to prompt the release of the encap-sulated ingredients, such as pH changes, mechanical stress,temperature, enzymatic activity, time, osmotic force, etc.Encapsulated probiotic bacteria can be used in many

fermented dairy products, such as yoghurt, cheese, culturedcream and frozen dairy desserts, and for biomass pro-duction. In the encapsulated form, the probiotics are pro-tected from bacteriophage and harsh environments, suchas freezing and gastric solutions. Thus, encapsulation facil-itates the manufacture of fermented dairy products in whichthe bacteria have consistent characteristics and higher sta-bility during storage and higher productivity than non-encapsulated bacteria. With the encapsulated products, theresidence time, acidity and continuous inoculation of milkwith a constant bacilli/cocci ratio can be controlled at adesired pH.

The use of microencapsulated probiotics for controlled-release applications is a promising alternative to solving themajor problems of these organisms that are faced by foodindustries. Even so, the challenges are to select the appro-priate microencapsulation technique and encapsulatingmaterials. To date, the research on the encapsulation of pro-biotics has focused mainly on maintaining the viability ofthe probiotic bacterial cells at low pH and high bileconcentrations.

One important challenge for cell encapsulation is thelarge size of microbial cells (typically 1e4 mm) or particlesof freeze-dried culture (more than 100 mm). This character-istic limits cell loading for small capsules or, when largesize capsules are produced, can negatively affect the tex-tural and sensorial properties of food products in whichthey are added. In almost all cases, gel entrapment usingnatural biopolymers, such as calcium alginate, carrageenan,gellan gum, and chitosan are favored by researchers. How-ever, although promising on a laboratory scale, the devel-oped technologies for producing gel beads still presentserious difficulties for large-scale production of food-grademicroencapsulated microorganisms.

Another major challenge is to improve the viability ofprobiotics during the manufacturing processes, particularlyheat processing. Consequently, there appears to be no com-mercial probiotic products available that are stable at hightemperatures. Keeping in view the importance of producingthermoresistant probiotic microorganisms, as well as theinterests of food and pharmaceutical companies, newapproaches are needed in further research. There are at leasttwo options: (1) discovering new strains of probiotic bacte-ria that are naturally heat stable or that have been geneti-cally modified and (2) developing an encapsulationsystem that effectively acts like an ‘‘insulation material’’.Our group is currently exploring approaches to tackle thischallenging area and is focusing on developing novel en-capsulation systems. This is based on an understanding ofthe thermal conductivity properties of several food-gradebiopolymers and lipids that are used as encapsulating shellmaterials, individually and in combination. These microen-capsulation systems may also control the diffusion of oxy-gen across the wall and may ensure a smaller log reductionin the viability of cells in foods. Coating of capsules withsome lipids, with high melting points, may also provide


low moisture conditions and an anaerobic environmentfor probiotic bacteria and may possibly improve thermalstability.

Application of this research could be particularlyimportant for the production of functional dairy productscontaining high concentrations of viable bacteria andbioingredients from LAB. Immobilization can efficientlyprotect cells, making this approach potentially useful fordelivery of viable bacteria to the lower gastrointestinal tractof humans via fermented, beverages and other functionalfood products.

AcknowledgementThe authors would like to thank Fonterra Co-operative

Group Limited, New Zealand for financial support of thiswork.

References

Anal, A. K., Bhopatkar, D., Tokura, S., Tamura, H., & Stevens, W. F.(2003). Chitosan-alginate multilayer beads for gastric passage andcontrolled intestinal release of protein. Drug Development andIndustrial Pharmacy, 29, 713e724.

Anal, A. K., & Stevens, W. F. (2005). Chitosan-alginate multilayerbeads for controlled release of ampicillin. International Journal ofPharmaceutics, 290, 45e54.

Anal, A. K., Stevens, W. F., & Remunan-Lopez, C. (2006). Ionotropiccross-linked chitosan microspheres for controlled release ofampicillin. International Journal of Pharmaceutics, 312, 166e173.

Audet, P., Paquin, C., & Lacroix, C. (1988). Immobilized growing lacticacid bacteria with k-carrageenan-locust bean gum gel. AppliedMicrobiology and Biotechnology, 29, 11e18.

Audet, P., Paquin, C., & Lacroix, C. (1990). Batch fermentations witha mixed culture of lactic acid bacteria immobilized separately ink-carrageenan locust bean gum gel beads. Applied Microbiologyand Biotechnology, 32, 662e668.

Audet, P., Paquin, C., & Lacroix, C. (1991). Effect of medium andtemperature of storage on viability of LAB immobilized ink-carrageenan-locust bean gum gel beads. BiotechnologyTechniques, 5, 307e312.

Axelsson, L. T. (1993). Lactic acid bacteria: classification andphysiology. In S. Salminen, & A. von Wright (Eds.), Lactic acidbacteria (pp. 1e64). New York, USA: Marcel Dekker.

Brannon-Peppas, L. (1997). Polymers in controlled drug delivery.Biomaterials, 11, 1e14.

Carvalho, A. S., Silva, J., Ho, P., Teixeira, P., Malcata, F. X., & Gibbs, P.(2002). Survival of freeze-dried Lactobacillus plantarum andLactobacillus rhamnosus during storage in the presence ofprotectants. Biotechnology Letters, 24, 1587e1591.

Carvalho, A. S., Silva, J., Ho, P., Teixeira, P., Malcata, F. X., & Gibbs, P.(2003). Protective effect of sorbitol and monosodium glutamateduring storage of freeze-dried LAB. Lait, 83, 203e210.

Carvalho, A. S., Silva, J., Ho, P., Teixeira, P., Malcata, F. X., & Gibbs, P.(2004a). Effects of various sugars added to growth and dryingmedia upon thermotolerance and survival throughout storage offreeze-dried Lactobacillus delbrueckii spp bulgaricus.Biotechnology Progress, 20, 248e254.

Carvalho, A. S., Silva, J., Ho, P., Teixeira, P., Malcata, F. X., & Gibbs, P.(2004b). Relevant factors for the preparation of freeze-dried LAB.International Dairy Journal, 14, 835e847.

Cassidy, A., Bingham, S. A., & Cummings, J. H. (1994). Starch intakeand colorrectal cancer risk: an international comparison. BritishJournal of Cancer, 69, 119e125.

Chan, E. S., & Zhang, Z. (2002). Encapsulation of probiotic bacteriaLactobacillus acidophilus by direct compression. Food andBioproducts Processing, 80, 78e82.

Chandramouli, V., Kailasapathy, K., Peiris, P., & Jones, M. (2004). Animproved method of microencapsulation and its evaluation toprotect Lactobacillus spp. in simulated gastric conditions. Journalof Microbiological Methods, 56, 27e35.

Chen, K. N., Chen, M. J., Liu, J. R., Lin, C. W., & Chiu, H. Y. (2005).Optimization of incorporated prebiotics as coating materialsfor probiotic microencapsulation. Journal of Food Science, 70,260e266.

Conrad, P. B., Miller, D. P., Cielenski, P. R., & de Pablo, J. J. (2000).Stabilization and preservation of Lactobacillus acidophilus insaccharide matrices. Cryobiology, 41, 17e24.

Corcoran, B. M., Ross, R. P., Fitzgerald, G. F., & Stanton, C. (2004).Comparative survival of probiotic lactobacilli spray-dried in thepresence of prebiotic substances. Journal of Applied Microbiology,96, 1024e1039.

Corcoran, B. M., Stanton, C., Fitzgerald, G. F., & Ross, R. P. (2005).Survival of probiotic lactobacilli in acidic environments asenhanced in the presence of metabolizable sugars. Appliedand Environmental Microbiology, 71, 3060e3067.

Crittenden, R., Laitila, A., Forssell, P., Matto, J., Saarela, M., Mattila-Sandholm, T., et al. (2001). Adhesion of Bifidobacteria to granularstarch and its implications in probiotic technologies. Applied andEnvironmental Microbiology, 67, 3469e3475.

Cui, J. H., Goh, J. S., Kim, P. H., Choi, S. H., & Lee, B. J. (2001).Survival and stability of bifidobacteria loaded in alginate poly-L-lysine microparticles. International Journal of Pharmaceutics, 210,51e59.

Dave, R. I., & Shah, N. P. (1997). Viability of yoghurt and probioticbacteria in yoghurts made from commercial starter cultures.International Dairy Journal, 7, 31e41.

Desmond, C. B., Corcoran, M., Coakley, M., Fitzgerald, G. F.,Ross, R. P., & Stanton, C. (2005). Development of dairy-basedfunctional foods containing probiotics, and prebiotics. AustralianJournal of Dairy Technology, 60, 121e126.

Desmond, C., Ross, R. P., O’Callaghan, E., Fitzgerald, G., &Stanton, C. (2002). Improved survival of Lactobacillus paracaseiNFBC 338 in spray-dried powders containing gum acacia. Journalof Applied Microbiology, 93, 1003e1011.

Dinakar, P., & Mistry, V. V. (1994). Growth and viability of Bifido-bacterium bifidum in cheddar cheese. Journal of Dairy Science, 77,2854e2864.

Doleyres, Y., Fliss, I., & Lacroix, C. (2002). Quantitative determinationof the spatial distribution of pure- and mixed-strain immobilizedcells in gel beads by immunofluorescence. Applied Microbiologyand Biotechnology, 59, 297e302.

Doleyres, Y., Fliss, I., & Lacroix, C. (2004). Continuous production ofmixed lactic starters containing probiotics using immobilized celltechnology. Biotechnology Progress, 20, 145e150.

Doleyres, Y., Paquin, C., LeRoy, M., & Lacroix, C. (2002). Bifido-bacterium longum ATCC 15707 cell production during free- andimmobilized-cell cultures in MRS-whey permeate medium.Applied Microbiology and Biotechnology, 60, 168e173.

Dunne, C. (2001). Adaptation of bacteria to the intestinal niche:probiotics and gut disorder. Inflammatory Bowel Diseases, 7,136e145.

FAO/WHO Experts’ Report (2001). Health and nutritional properties ofprobiotics in food including powder milk with live lactic acidbacteria.

Favaro-Trindale, C. S., & Grosso, C. R. F. (2002). Microencapsulationof L acidophilus (La-05) and B lactis (Bb-12) and evaluation of their


survival at the pH values of the stomach and in bile. Journal ofMicroencapsulation, 19, 485e494.

Fuller, R. (1989). Probiotics in man and animals. Journal of AppliedBacteriology, 66, 365e378.

Garcia De Castro, A., Bredholt, H., Strom, A. R., & Tunnaclife, A.(2000). Anhydrobiotic engineering of gram-negative bacteria.Applied and Environmental Microbiology, 66, 4142e4144.

Gardiner, G. E., O’Sullivan, E., Kelly, J., Auty, M. A. E.,Fitzgerald, G. F., Collins, J. K., et al. (2000). Comparative survivalrates of human-derived probiotic Lactobacillus paracasei and L.salivarius strains during heat treatment and spray drying. Appliedand Environmental Microbiology, 66, 2605e2612.

Gemeiner, P., Rexova-Benkova, L., Svec, F., & Norrlow, O. (1994).Natural and synthetic carriers suitable for immobilization of viablecells, active organelles and molecules. In I. A. Veliky, &R. J. McLean (Eds.), Immobilized biosystems: Theory and practicalapplications (pp. 67e84). London, UK: Chapman and HallPublications.

Gill, H. S., Cross, M. L., Rutherfurd, K. J., & Gopal, P. K. (2001).Dietary probiotic supplementation to enhance cellular immunityin the elderly. British Journal of Biomedical Science, 58, 94e96.

Gilliland, S. E. (1989). Acidophilus milk-productsda review ofpotential benefits to consumers. Journal of Dairy Science, 72,2483e2495.

Gismondo, M. R., Drago, L., & Lombardi, A. (1999). Review ofprobiotics available to modify gastrointestinal flora. InternationalJournal of Antimicrobial Agents, 12, 287e292.

Guerin, D., Vuillemard, J. C., & Subirade, M. (2003). Protection ofBifidobacteria encapsulated in polysaccharide-protein gel beadsagainst gastric juice and bile. Journal of Food Protection, 66,2076e2084.

Hansen, L. T., Allan-Wojtas, P. M., Jin, Y. L., & Paulson, A. T. (2002).Survival of Ca-alginate microencapsulated Bifidobacterium spp. inmilk and simulated gastrointestinal conditions. Food Microbiology,19, 35e45.

Holzapfel, W. H., Haberer, P., Geisen, R., Bjorkroth, J., &Schillinger, U. (2001). Taxonomy and important features ofprobiotic microorganisms in food and nutrition. AmericanJournal of Clinical Nutrition, 73, 365e373.

Hyndman, C. L., Groboillot, A. F., Poncelet, D., Champagne, C. P., &Neufeld, R. J. (1993). Microencapsulation of Lactococcus lactiswithin cross-linked gelatin membranes. Journal of ChemicalTechnology and Biotechnology, 56, 259e263.

Isolauri, E. (2000). The use of probiotics in paediatrics. HospitalMedicine, 61, 6e7.

Isolauri, E., Arvola, T., Sutas, Y., Moilanen, E., & Salminen, S. (2000).Probiotics in the management of atopic eczema. Clinical andExperimental Allergy, 30, 1604e1610.

Iyer, C., & Kailasapathy, K. (2005). Effect of co-encapsulation ofprobiotics with prebiotics on increasing the viability ofencapsulated bacteria under in vitro acidic and bile salt conditionsand in yogurt. Journal of Food Science, 70, 18e23.

Kailasapathy, K., & Chin, J. (2000). Survival and therapeutic potential ofprobiotic organisms with reference to Lactobacillus acidophilus andBifidobacterium spp. Immunology and Cell Biology, 78, 80e88.

Kailasapathy, K., & Masondole, L. (2005). Survival of free and micro-encapsulated Lactobacillus acidophilus and Bifidobacterium lactisand their effect on texture of feta cheese. Australian Journal ofDairy Technology, 60, 252e258.

Kailasapathy, K., & Supriadi, D. (1996). Effect of whey proteinconcentrate on the survival of Lactobacillus acidophilus in lactosehydrolysed yoghurt during refrigerated storage. Milchwissenschaft,51, 565e569.

Kalliomaki, M., Salminen, S., Poussa, T., Arvilommi, H., & Isolauri, E.(2003). Probiotics and prevention of atopic disease: 4-yearfollow-up of a randomised placebo-controlled trial. Lancet, 361,1869e1871.

Kebary, K. M. K. (1996). Viability of Bifidobacterium bifidum and itseffect on quality of frozen Zabady. Food Research International,29, 431e437.

King, A. H. (1995). Encapsulation of food ingredients: a review ofavailable technology, focusing on hydrocolloids. In S. J. Risch, &G. A. Reineccius (Eds.), Encapsulation and controlled release offood ingredients (pp. 213e220). Washington, DC, USA: AmericanChemical Society.

Kleessen, B., Stoof, G., Proll, J., Schmiedl, D., Noack, J., & Blaut, M.(1997). Feeding resistant starch affects fecal and cecal microfloraand short chain fatty acids in rats. Journal of Animal Science, 75,2453e2462.

Klein, J., & Vorlop, D. K. (1985). Immobilization techniques: cells.In C. L. Cooney, & A. E. Humphrey (Eds.), Comprehensivebiotechnology (pp. 542e550). Oxford, UK: Pergamon Press.

Krasaekoopt, W., Bhandari, B., & Deeth, H. (2003). Evaluation ofencapsulation techniques of probiotics for yoghurt. InternationalDairy Journal, 13, 3e13.

Krasaekoopt, W., Bhandari, B., & Deeth, H. (2004). The influence ofcoating materials on some properties of alginate beads andsurvivability of microencapsulated probiotic bacteria. InternationalDairy Journal, 14, 737e743.

Lankaputhra, W. E. V., Shah, N. P., & Britz, M. L. (1996). Survival ofBifidobacteria during refrigerated storage in the presence of acidand hydrogen peroxide. Milchwissenschaft, 51, 65e70.

Le Blay, G., Michel, C., Blottiere, H. M., & Cherbut, C. (1999).Enhancement of butyrate production in the rat caecocolonic tractby long-term ingestion of resistant potato starch. British Journal ofNutrition, 82, 419e426.

Lee, J. S., Cha, D. S., & Park, H. J. (2004). Survival of freeze-driedLactobacillus bulgaricus KFRI 673 in chitosan-coated calciumalginate microparticles. Journal of Agricultural and FoodChemistry, 52, 7300e7305.

Lee, Y. K., Puong, K. Y., Ouwehand, A. C., & Salminen, S. (2003).Displacement of bacterial pathogens from mucus and Caco-2cell surface by Lactobacilli. Journal of Medical Microbiology, 52,925e930.

Makinen, A. M., & Bigret, M. (1993). Industrial use and production ofLAB. In S. Salminen, & A. von Wright (Eds.), Lactic acid bacteria(pp. 65e96). New York, USA: Marcel Dekker.

Mattila-Sandholm, T., Myllarinen, P., Crittenden, R., Mogenden, G.,Fonden, R., & Saarela, M. (2002). Technological challenges forfuture probiotic foods. International Dairy Journal, 12, 173e182.

Mauriello, G., Aponte, M., Andolfi, R., Moschetti, G., & Villani, F.(1999). Spray-drying of bacteriocins-producing LAB. Journal ofFood Protection, 62, 773e777.

O’Riordan, K., Andrews, D., Buckle, K., & Conway, P. (2001).Evaluation of microencapsulation of a Bifidobacterium strain withstarch as an approach to prolonging viability during storage.Journal of Applied Microbiology, 91, 1059e1066.

Ouellette, V., Chevalier, P., & Lacroix, C. (1994). Continuousfermentation of a supplemented milk with immobilizedBifidobacterium infantis. Biotechnology Techniques, 8, 45e50.

Picot, A., & Lacroix, C. (2003a). Effects of micronization on viabilityand thermotolerance of probiotic freeze-dried cultures.International Dairy Journal, 13, 455e462.

Picot, A., & Lacroix, C. (2003b). Optimization of dynamic loop mixeroperating conditions for production of o/w emulsion for cellmicroencapsulation. Lait, 83, 237e250.

Picot, A., & Lacroix, C. (2003c). Production of multiphase water-insoluble microcapsules for cell microencapsulation using anemulsification/spray-drying technology. Journal of Food Science,68, 2693e2700.

Picot, A., & Lacroix, C. (2004). Encapsulation of bifidobacteria inwhey protein-based microcapsules and survival in simulatedgastrointestinal conditions and in yoghurt. International DairyJournal, 14, 505e515.


Prajapati, J. B., Shah, R. K., & Dave, J. M. (1987). Survival ofLactobacillus acidophilus in blended spray-dried acidophiluspreparations. Australian Journal of Dairy Technology, 42, 17e21.

Rao, A. V., Shiwnarain, N., & Maharaj, I. (1989). Survival ofmicroencapsulated Bifidobacterium pseudolongum in simulatedgastric and intestinal juices. Canadian Institute of Food Scienceand Technology Journal, 22, 345e349.

Rinkinen, M., Jalava, K., Westermarck, E., Salminen, S., &Ouwehand, A. C. (2003). Interaction between probiotic LABand canine enteric pathogens: a risk factor for intestinalEnterococcus faecium colonization. Veterinary Microbiology,92, 111e119.

Salminen, S., von Wright, A., Morelli, L., Marteu, P., Brassard, D.,de Vos, W., et al. (1998). Demonstration of safety of probioticsd

a review. International Journal of Food Microbiology, 44, 93e106.Selmer-Olsen, E., Sorhaug, T., Birkeland, S. E., & Pehrson, R. (1999).

Survival of Lactobacillus helveticus entrapped in Ca-alginate inrelation to water content, storage and rehydration. Journal ofIndustrial Microbiology & Biotechnology, 23, 79e85.

Sheu, T. Y., Marshall, R. T., & Heymann, H. (1993). Improving survivalof culture bacteria in frozen desserts by microentrapment. Journalof Dairy Science, 76, 1902e1907.

Stanton, C., Desmond, C., Fitzgerald, G., & Ross, R. P. (2003).Probiotic health benefitsdreality or myth? Australian Journal ofDairy Technology, 58, 107e113.

Stanton, C., Gardiner, G., Meehan, H., Collins, K., Fitzgerald, G.,Lynch, P. B., et al. (2001). Market potential for probiotics. AmericanJournal of Clinical Nutrition, 73, 476Se483S.

Stanton, C., Ross, R. P., Fitzgerald, G. F., & Van Sinderen, D. (2005).Fermented functional foods based on probiotics and their biogenicmetabolites. Current Opinion in Biotechnology, 16, 198e203.

Sultana, K., Godward, G., Reynolds, N., Arumugaswamy, R., Peiris, P.,& Kailasapathy, K. (2000). Encapsulation of probiotic bacteriawith alginate-starch and evaluation of survival in simulatedgastrointestinal conditions and in yoghurt. International Journal ofFood Microbiology, 62, 47e55.

Talwalkar, A., & Kailasapathy, K. (2003). Effect of microencapsulationon oxygen toxicity in probiotic bacteria. Australian Journal of DairyTechnology, 58, 36e39.

Teixeira, A. A. (1979). Conduction-heating considerations in thermal-processing of canned foods. Mechanical Engineering, 101, 96.

Tejada-Simon, M. V., Ustunol, Z., & Pestka, J. J. (1999). Effects of lacticacid bacteria ingestion on basal cytokine mRNA and immunoglob-ulin levels in the mouse. Journal of Food Protection, 62, 287e291.

AnalAK07_Recent Advances in Microencapsulation

Documents

Transcript of AnalAK07_Recent Advances in Microencapsulation