Transcript
Page 1: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

20Micro-Encapsulation ofProbioticsJean-Antoine Meiners

20.1 Introduction

Micro-encapsulation is defined as the technology for packaging with the help of

protective membranes particles of finely ground solids, droplets of liquids or

gaseous materials in small capsules that release their contents at controlled rates

over prolonged periods of time under the influences of specific conditions (Boh,

2007). The material encapsulating the core is referred to as coating or shell.

The majority of microcapsules have spherical shapes and their diameter

varies from a few microns to 1 mm. However, some authors consider particles

of even more than one millimeter as microcapsules. For the purpose of this

chapter the term microcapsule refers to capsules whose aim is the protection

and controlled release of the active substance, and the term microsphere, a

commonly used term in the scientific literature, refers to granules, that do not

have a core-shell morphology, and can be simply defined as the embedding of an

active substance in a matrix (Watheley, 1996).

In general, encapsulation can be used to improve the stability of the active

substance during processing and storage, mask unpleasant flavors and odors,

control possible oxidative reactions, and provide controlled release at the right

place and the right time. As such, it has numerous applications in the food,

pharmaceutical, cosmetic, agricultural, textile, paper and paint industries. One of

the earliest published inventions was the carbonless copy paper in which tiny

microcapsules were fixed on the backside of a sheet of paper; these were crushed

by the pressure of writing, thus releasing their dye (Green, 1957). In the areas of

pharmaceuticals and chemicals, many products, e.g., enzymes, can cause health

and safety hazards when manipulated in a very fine powder form, due to excessive

dust formation; granulation into larger size particles and coating can be used to

alleviate such handling problems (Meesters, 2006). In terms of food applications,

the aims of encapsulation techniques are to improve the stability of bioactive

# Springer ScienceþBusiness Media, LLC 2009

Page 2: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

ingredients during processing and storage and to prevent undesirable interactions

with the food matrix (Champagne and Fustier, 2008). An example is the case of

ascorbic acid, a highly reactive, water soluble and heat-labile compound, encap-

sulation of which can double its shelf life compared to the free form (Wilson and

Shah, 2007).

Various approaches can be used to open up the microcapsules to release the

active substance. These include (i) mechanical rupture of the membrane, e.g., in the

case of mastication of micro-encapsulated flavours in chewing gums, (ii) exposure

to high temperatures to make the coating material melt, a technology frequently

used for encapsulated chemical leavening agents in baked goods, (iii) dissolution of

the capsules when placed in solvents, (iv) exposure to specific pH, (v) biodegrada-

tion of the polymer coating by enzymes, (vi) diffusion of the active substance

through the polymer coating, (vii) high osmotic pressure inside the microcapsule

and (viii) combinations of the above (Pothakamury and Barnosa-Canovas, 1995).

In the area of microbial products, micro-encapsulation is used in order to

enhance the delivery of probiotic microorganisms into foods during processing

and storage, or to protect against the acidic conditions in the stomach and ensure

delivery into the intestine. In addition, microbial cells can also be immobilized

onto polymer matrices and used as biocatalysts in fermentation processes. The

main difference between encapsulation and immobilization is that in the latter

the polymer beads produced allow fast and easy diffusion of water and other

fluids, and thus the cells are biochemically active (Klein and Vorlop, 1985).

20.2 Micro-encapsulation Techniques and Processes

A variety of encapsulation techniques are available and include both chemical

processes, such as phase microseparation, coacervation, liposome encapsulation

molecular inclusion, as well as physical processes, such as spray drying, spray

chilling, prilling, spinning disk, fluidized bed coating, and extrusion. Certain

steps are common to many of these processes.

Basically the initial step is to introduce the active substance inside the shell;

this involves dispersion or atomization in order to position the membrane on the

outside of the microcapsule and the active substance in the core.

In the case of microcapsules containing a liquid core active substance an

emulsion is prepared, whose surface is polymerized in a subsequent step, the

interface condensed and/or the solvent evaporated. In the section below, the

different types of encapsulation methods are presented in more detail.

806 20 Micro-Encapsulation of Probiotics

Page 3: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

20.2.1 Spray Drying

Spray drying is one of the most commonly used encapsulation method, as it is a

well established and cost-efficient technology; it is basically designed to evaporate

water from the dry matter. The solution is injected in a hot air stream in a closed

vessel and the solvent, whichmost of the times is water, is evaporated. The energy is

absorbed to evaporate the water and consequently the powder temperature can be

controlled. The residence time in the tower is one of the limiting factors; the

majority of the moisture has to evaporate during the fall time in the tower of the

particles (Adamiec and Marciniak, 2004). In order to be efficient, the total surface

area should be as large as possible and consequently the droplet sizes small, and if

possible, of the same size.

The principle of the technique is based on a spray, which is created by forcing

the fluid through an orifice. The energy required to overcome the pressure drop

upon exiting the orifice is supplied by the spray dryer feed pump. Pressure nozzles

coupled to high pressure feed pumps, which produce pressures of as much as 3000

psig, have the advantage of producing a narrow particle size distribution, but can

have severe damaging effects on a microorganism’s cell structure. Despite the fact

that they are less energy efficient and produce narrower particle size distribution,

the two-fluid nozzle is very popular for laboratory equipment, probably since it

allows working with small flow rates. The most popular nozzle type for industrial

spray drying is the centrifugal atomizer, where a spray is created by passing the fluid

across or through a rotating wheel or disk. The benefit for micro-encapsulation

purposes is that rather large particles, up to 100 mm, can be produced this way.

20.2.2 Spray Chilling and Cooling

Spray chilling and spray cooling (or congealing), the second being operated at

lower temperatures, involve mixing thoroughly the core and amolten shell material

with a melting temperature well above the operating temperature (usually lipids),

and atomizing through a two media nozzle into a cooling chamber in order to

solidify the droplet instantaneously. One of the limitations is the speed in heat

transfer of the energy freed during the re-crystallization. The use of cooling media

has allowed high volume and high speed production. Another limiting factor is the

difference in surface tension between the matrix and the core material. Conse-

quently, the core is not always placed in the center of the microcapsule, which may

affect the protective properties of the microcapsule (Meiners, 2004).

Micro-Encapsulation of Probiotics 20 807

Page 4: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

20.2.3 Prilling

Hawley’s Condensed Chemical Dictionary says that prills are ‘small round or

acicular aggregates of a material’. Prilling can be a useful technology, when solidifi-

cation of the shell material is an instantaneous reaction. The core and the shell

material are mixed thoroughly and the dispersion is pumped to flow over a sonically

vibrating dispersion head. Gravity allows the droplets to fall into the collection

device for cooling or polymerization (Wu et al., 2007). More advanced and sophis-

ticated equipment have been developed that limit considerably the residence time

of the dispersion and the need for large cooling towers (Meiners, 2004).

20.2.4 Spinning Disk

A comparable technology to prilling is the spinning disk technology, which uses

centrifugal forces for droplet separation.

The droplet volumes and their mutual spacing are governed by the channel

geometry and the frequency of rotation. Devices exist that combine spinning disk

forces with high frequency droplet separation (Chesnokov, 2001).

20.2.5 Fluidized Bed

Fluidized bed technology is based on the separation of individual particles in a

gas stream and the fixation of the membrane substance by polymerization, drying

or crystallization around the core. The solvation and drying steps can be avoided,

which for thermo-sensitive materials may represent a major advantage. Agglom-

eration and retention in the filter system cause the use of the fluidized bed system

to be difficult with products having strong adhesive properties (Guignon, 2002).

20.2.6 Extrusion

Micro-encapsulation by extrusion involves projecting an emulsion core and

coating material through a nozzle at high pressure. It involves preparing a

hydrocolloidal solution, adding the active substance and extrusing the suspension

through a nozzle in the form of droplets into a hardening solution or setting bath

(Krasaekoopt et al., 2003). Carbohydrate matrices in the glassy state have very

808 20 Micro-Encapsulation of Probiotics

Page 5: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

good barrier properties and extrusion is a convenient process enabling the

encapsulation of active substances in such matrices (Gouin, 2004).

20.2.7 Coacervation

Coacervation consists of the separation of colloid particles from a solution, which

then agglomerate into a separate liquid phase called coarcevate. A number of hydro-

colloid systems have been evaluated for coacervation micro-encapsulation including

among others the gelatine/gum acacia, heparin/gelatine, carageenan, chitosan, soy

protein, lactogloboulin/gum acacia and the guar/dextran system (Gouin, 2004).

20.2.8 Liposomes

Liposomes are artificially made microscopic membrane vesicles consisting of one

or more concentric layers of lipids. They are formed by dispersing the lipid

formulation in a solvent system, decreasing the solvent volume and then re-

dispersing the film of lipid/solvent in an aqueous phase (Bangham, 1995).

20.2.9 Inclusion Complexation

Cyclodextrins are cyclic oligomers, who have the ability to form inclusion com-

plexes with the active substances. They are typically used for the protection of

unstable and high value chemicals, such as flavours. Oil-in-water emulsions using

cyclodextrins can also be subsequently spray dried (Astray et al., 2009).

20.3 Technologies used for the Immobilization andMicro-encapsulation of Microganisms

Immobilization of living cells was the first form of micro-encapsulation and

was pioneered about 50 years ago for medical purposes, and (Chang, 1964).

The first patent demonstrating the biocompatibility of polymers and the resis-

tance of the encapsulated material to sterilization conditions was granted in 1965

(Mauvernay, 1965).

Immobilization of microorganisms for bioprocessing purposes became im-

portant for the development of the continuous fermentation process. Much of the

Micro-Encapsulation of Probiotics 20 809

Page 6: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

research work was done by the brewing industry using immobilized yeast cells

and the dairy industry using immobilized lactic acid producing bacteria (Prevost

and Divies, 1988).

The pore size of the microcapsules produced has an important effect on the

cellular biochemical activities, as it controls the rate of retention/passage of

undesirable metabolites (Klein et al., 1983). Different methods have been used

to immobilize microorganisms, including, physical entrapment in a polymeric

network, attachment or adsorption to a carrier, and membrane entrapment.

In order to entrap the biomass in microcapsules simultaneous with the

membrane formation, it is important for the that droplets to be generated simul-

taneously with the membrane. For this purpose, specific equipment has been

developed and polymers have been selected. The polymers generally form a gel

under the influence of ionization or thermosetting.

For the purpose of droplet separation with a narrow size distribution, a

pumping system under gravity provides a constant uninterrupted flow of the

liquid. In the early droplet generators, the liquid consisted of a solution of the

biomass and the dissolved polymer. Upon falling into the collection bath filled

with a solution containing the ionic solution, the polymer membrane was cross-

linked to form self sustaining microcapsules, encapsulating the biomass droplet

within the membrane (Sheu and Marshall, 1993). The more recent versions use a

nozzle designed for co-extrusion, creating a polymer network around the droplet

during its fall into the collection bath. The different dripping systems can be

identified according to the principles below. > Figures 20.1–20.4 depict the

various types of nozzles used.

� Dripping without assistance by gravity into the collection bath

� Dripping assisted by an air stream

� Dripping assisted by an electrostatic force

� Laminar jet break up assisted by vibration

� Jet break up assisted by rotation and vibration

� Co-extrusion assisted by laminar jet break up.

The use of static mixers has also appeared to be very promising, since it allows

large volume and cost efficient production. The device consists of mixer elements

contained in a cylindrical (tube) or squared housing. These can vary from 6 mm

to 6 meters diameter. Static mixer elements consist of a series of baffles. As the

streams move through the mixer, the non-moving elements continuously blend the

materials (Maa and Hsu, 1996).

810 20 Micro-Encapsulation of Probiotics

Page 7: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

20.4 Objectives for the Micro-encapsulation ofProbiotics

A major function of micro-encapsulation is to provide protection against the high

acidity of the gastric fluids. A microcapsule containing the probiotic must not be

fractured until it passes through the stomach. Since the biological release mecha-

nism is triggered by the higher pH in the upper intestine, a coating can be used that

. Figure 20.1Dripping assisted by an electrostatic force (courtesy of Nisco Engineering AG).

. Figure 20.2Dripping assisted by aerodynamically jetting (courtesy of Nisco Engineering AG).

Micro-Encapsulation of Probiotics 20 811

Page 8: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

withstands the low pH and can release its content at a pH similar to that of the large

intestine (e.g., pH 5.5 to 7). The survival of commercial probiotics in conditions

of very low pH was recently investigated; the researchers assessed in vitro the

survival of 32 probiotic strains, all isolated from commercially available products

. Figure 20.3Dripping assisted by co-axial air (courtesy of Nisco Engineering AG).

. Figure 20.4Jet cutter (courtesy of Genialab AG).

812 20 Micro-Encapsulation of Probiotics

Page 9: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

in simulated gastric contents. Approximately 50% of them were viable after

20 minutes exposure; eighteen strains were further tested in respect to their bile

tolerance; five of them showed poor tolerance, whereas seven of them showed

moderate tolerance and the rest high tolerance (Gibson et al., 2005). The above

highlight the importance of developing effective micro-encapsulation methods,

especially for strains that are less robust than others. Another function of micro-

encapsulation of probiotics is to enhance the viability of the cells during processing

and storage. To this end, it is important to highlight that the final application of

the product may dictate the use of totally different technologies, or different shell

or carrier materials. For example, in the case of dried products (e.g., foods or

neutraceuticals), the water content, and in particular the water activity, is one of

the most important factors influencing cell survival, as it has a direct effect on

the metabolic activity of microorganisms. Water activity (aw) describes the

(equilibrium) amount of water available for the hydration of materials. Water

activity is the effective mole fraction of water, defined as aw¼lwXw ¼ p/p0,

where lw is the activity coefficient of water, Xw is the mole fraction of water in

the aqueous fraction, p is the partial pressure of water above the material and p0 is

the partial pressure of pure water at the same temperature. Based on the above, a

non-water permeable capsule material would be the first type of material to

evaluate for using in a dried product. If the capsule material is chosen from

non-ionisable polymers, the protection against the gastric acidity would likely

improve too.

In addition to the above, it must be noted that the suitability of a particular

encapsulation technology or material depends on the properties of the specific

probiotic strain (e.g., acid tolerance, oxygen tolerance, bile tolerance). It is generally

difficult to simply transfer the results from one strain to another; significant amount

of experimental work is needed to identify and optimise suitable encapsulation

methods. Below is a list of applications of encapsulated probiotics in the food and

non-food sectors.

� Cell immobilisation (e.g., industrial fermentations)

� Micro-encapsulation of probiotics in dairy and beverage applications

� Micro-encapsulation of probiotics in dry food applications

� Micro-encapsulation of probiotics in nutritional or medical supplements

>Table 20.1 lists the encapsulation technologies and the types of shell

materials commonly used for the micro-encapsulation of probiotics.

Micro-Encapsulation of Probiotics 20 813

Page 10: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

. Table 20.1

Technologies used for the micro-encapsulation of probiotics (Adapted from Anal and Singh,

2007)

Technology Shell material Micro-encapsulation steps

Spray drying Water soluble polymers (a) Incorporate the microorganisms intothe solution

(b) Atomization into spray

(c) Evaporation of solvent

(d) Separation of powder

Spray chilling Lipids, waxes (a) Incorporate the micro organisms intothe melt

(b) Atomizing into spray

(c) Solidification of the coating by chillingbelow the melting temperature

Air suspensioncoating

Water insoluble and watersoluble polymers, lipids, waxes

(a) Preparation of the coating melt orsolution

(b) Fluidizing the core particles

(c) Atomizing small droplets of thecoating material around the core particles

(d) Drying, solidifying, crystallizing thecoating with core in the center

Extrusion; Jetcutter; Staticmixer

Water soluble polymers (a) Active substance is dissolved into thepolymer solution

(b) Dripping the solution into thecollection bath

(C1) Cross linking the polymer withdivalent ions

(C2) Gelling the polymer bythermosetting

(C3) Complexing with a polyelectrolyte

Co-extrusion Water soluble polymers (A1) Active substance is pumped to theinner nozzle port

(A2) Polymer is pumped to the outernozzle port

(b) Dripping the active substance andpolymer into the collection bath

(C1) Cross linking the polymer withdivalent ions

(C2) Gelling the polymer bythermosetting

814 20 Micro-Encapsulation of Probiotics

Page 11: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

20.5 Biopolymers

It is obvious that the biopolymers applied must be biocompatible and non toxic

to the cells. The most popular biopolymers for micro-encapsulation of micro-

organisms are discussed below:

Alginate Alginate is one of the most used polymers for the encapsulation of

microorganisms. It can be found in the cell walls and intercellular spaces of brown

algae. It is a linear co-polymer with homo-polymeric blocs, covalently linked in

different sequences, depending to the source of algae. Alginic acid, the free acid from

alginate is the intermediate product in the commercial alginates and has limited

stability. In order to make stable water soluble products alginic acid is transformed

into a range of salts i.e., Ca-alginate; Na-alginate; K-alginate; Mg-alginate; NH4-

alginate.The ratio of mannuronic acid to gluconic acid and the structure of the

polymer determine the properties of alginate in solution. Alginates may be

prepared with a wide range of molecular weights. Alginate capsules are formed

by dripping an aqueous alginate solution into a solution containing a multi-

valemt cation, usually a calcium salt. The calcium ion attaches to two polymer

strands by replacing the salt bond, and can thus form a very fine network.

Carrageenan Carrageenan is family of a naturally occurring sulphated poly-

saccharide which fill the voids of the cellulosic structure of red seaweed. Carragee-

nans are made up of repeating galactose units and 3,6 anhydrogalactose, joined by

alternating glycosidic linkages. They are divided into three classes, k-, i- and

l- carrageenan, which have different properties. The plant source and the extraction

method determine to a large extent the molecular structures and properties; e.g.,

carrageenans with different amounts of sulphate ester groups will associate differ-

ently with metal ions. All types are all soluble in hot water, but only the k- type is

soluble in cold water. Gelation of carrageenan involves helix formation, and is

induced by cooling down to room temperature from a hot solution. Potassium and

calcium ions are also essential for gelling, as they stabilize the gel, prevent swelling,

or induce gelation (Krasaekoopt et al., 2003).

The strength of the carrageenan capsules can be improved using locust bean

gum (LBG) at a ratio of carrageenan to LBG of 2:1. LBG is part of the galacto-

mannan family. It is extracted from the kernels of the carob tree; it forms a food

reserve for the seeds and helps to retainwater under arid conditions. LBG consists of

a backbone of b-(1,4)-D-mannose units; approximately every fourthmannose unit

there is a substitution by a a-D-galactose side chain. The level of substitution is

important for the properties of the gum, as the galactomannan can associate and

self cross-link. LBG requires heating to dissolve in water and is rarely used as a

Micro-Encapsulation of Probiotics 20 815

Page 12: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

single shell material (Rees, 1972). The increased strength of carrageenan/LBG

capsules is due to synergistic effects, which are attributed to the interaction

between the double stranded helix of k- carrageenan and the un-branched

segments of the D- mannose backbone of the LBG molecule.

Chitosan Chitosan is a weak anionic polysaccharide consisting mainly of

b(1,4) linked glucosamine units together with some N-acetylglucosamine units.

It is produced by de-acetylation of chitin extracted from crustacean shells. It is

positively charged and water soluble at pH below 6.5, and this enables it to form

polyelectrolyte complexes with negatively charged materials, such as polypho-

sphates, [Fe(CN)6]4- and [Fe(CN)6]3-, and citrate (Peniche et al., 2003).

Starch Starch is a polysaccharide consisting of a large number of glucose

monosaccharide units joined together by glycoside bonds. Starch consists of two

types of molecules, amylose (normally 20 – 30%) and amylopectin (normally

70 – 80%). Both consist of polymers of a-D-glucose units. Of the two components

of starch, amylose has the most useful functions as a hydrocolloid. Chemical

modification of starches, such as cross-linking, oxidization, acetylation, and hydro-

xypropylation may confer interesting changes in functionality. For example, octe-

nylsuccinic acid anhydride (OSAN)-modified starches are popular for their

emulsifying properties, as they contain both hydrophobic and hydrophillic groups.

In addition, resistant starch, i.e., the indigestible form of starch, offers an ideal

surface for adherence for the probiotic microorganisms during processing, storage

and their passage through the gastrointestinal tract (GIT) (Anal and Singh, 2007).

Mixing starch with k-carrageenan, alginate, xanthan gum and low molecular

weight sugars is a popular practice in micro-encapsulation as they reduce starch

retrogradation. Starch derivatives mostly hydrolyzed forms such as dextrins and

maltodextrins, are also frequently used as carrier material for spray and freeze

drying (Anal and Singh, 2007).

Gum arabic Gumarabic is a hydrocolloid produced by the natural exudation

of acacia trees. Because it is a complex mixture of molecules and the fact that the

material varies significantly with the source, the investigation of the exact molecu-

lar structures still attracts considerable research interest. It is generally composed

of a highly branched polysaccharide fraction consisting of galactose, arabinose,

rhamnose and glucuronic acids. It also contains an arabinogalactan-protein

complex in which arabinogalactan chains are covalently linked to a protein chain

through serine and hydroxyproline groups (Dror et al., 2006). The protein plays an

important role in the functionality of the gum. The simultaneous presence of

hydrophilic carbohydrates and hydrophobic protein gives the molecule its emul-

sification and stabilization properties (Randall et al., 1988).

816 20 Micro-Encapsulation of Probiotics

Page 13: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

Gum arabic is used to produce gum arabic / gelatine coacervates, which have

been used as encapsulation materials. The formation of coacervates is possible

only at pH values below the isoelectric point of gelatine, which is approximately

at pH 8. It is at these values that gelatine becomes positively charged, whereas

gum arabic continues to be negatively charged. The formation of stable micro-

capsules using mixtures of gum arabic and whey protein has also been reported

(Weinbreck et al., 2004).

Pectin Pectin is a major cell wall component in plants, playing a role in the

control of cell growth and the defence against the invasions of microorganisms.

Pectins are composed of a a-D-galacturonic acid, which are interrupted by single

a-L-rhamnose residues. A major difference between pectins is their content in

methyl esters. The degree of esterification (DE) is defined by the numberof esterified

D-galacturonic acid residues. High methoxyl pectin forms gels due to hydropho-

bic interactions and hydrogen bonding between pectin molecules. Low methoxyl

pectin forms gels in the presence of di- and polyvalent cations, which cross-link

and neutralise the negative charges of the pectin molecule (Wher et al., 2004).

Gelatin Gelatin is a high molecular mass polypeptide derived from con-

nective animal tissue, such as bone and skin. The protein chain unfolds upon

melting and upon cooling it forms a coil-helix structure and entraps water,

forming a reversible gel. Gelatin is extracted after acid pre-treatment and has

an isoelectric point (IP) of 7–9.4; gelatin after lime pre-treatment has an IP of

4.5–5.3. The possibility to vary the IP, and thus the charge by adjusting the

pH makes gelatin a favourite candidate for micro-encapsulation. However, in

order for the capsules to be self sustainable cross-linking is required; and the

cytotoxity of the traditionally applied organic solvents makes the process less

suitable for microorganisms (Hyndman et al., 1993). However, as mentioned

previously, gelatin is used due to its amphoteric nature in co-operation with

anionic carbohydrates to form gum/gelatine coacervates.

Whey protein Whey protein is the protein obtained from whey during

cheese making. Whey proteins are obtained by ultrafiltration, during which,

the low molecular weight compounds such as lactose, minerals, vitamins and

non-protein nitrogen are removed in the permeate while the proteins become

concentrated in the retentate. After ultrafiltration, the retentate is pasteurized,

sometimes evaporated, and then dried usually by spray drying at low tempera-

tures in order to avoid significant protein denaturation.

Whey protein is very popular for its film forming characteristics and is used

as a protective material in spray drying, resulting in a water soluble microcapsule

system (Picot and Lacroix, 2004).

Micro-Encapsulation of Probiotics 20 817

Page 14: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

Other polymers There is a large choice of acid resistant polymers with

designed release at intestinal pH values. Most of them have been approved for

pharmaceutical purposes, while only few of those have food approval: hydro-

xypropyl methylcellulose; methylcellulose; ethylcellulose; hydroxypropyl methyl-

cellulose phthalate; hydroxypropyl methylcellulose acetate succinate; poly(methyl

methacrylate); carboxymethyl cellulose; polyvinyl acetate phthalate; methylcellu-

lose phthalate; cellulose acetate phthalate; polyvinyl acetate phthalate; polyvinyl

pyrrolidone; carboxypolymethylene. Most of these products are only soluble

in alcohol and cannot be brought into direct contact with the culture; therefore

they can be applied as an outer top coating only. Finished products are in general

found in the pharmaceutical or the nutritional supplement markets.

20.6 Applications of Micro-encapsulation ofProbiotics

In this section, some examples from the various studies on micro-encapsulation

of probiotics are presented. The area of cell immobilization for use in fermenta-

tion systems has attracted considerable interest recently for both probiotic and

non probiotic lactic acid bacteria (LAB). The advantages of immobilized cells

over free cells include enhanced biological stability, high biomass concentration,

increased product yields, increased product stability, and the ability to separate

and re-use cells (Dervakos and Webb, 1991; Lacroix and Yidirim, 2007). As such,

immobilisation techniques have the potential for enhancing the performance of

probiotics and produce strains with specific physiological and functional char-

acteristics, as well as improved technological properties.

The alginate system has been studied extensively for the encapsulation of

probiotics. The research suggests that the size of the beads affects considerably cell

survival under simulated gastrointestinal conditions (Lee et al., 2000; Chandramouli

et al., 2004). Beads of less than 100 mm in size did not significantly protect

Bifidobacterium cells; the protection was increased as the beads became bigger,

especially for very large beads with sizes higher than 1 mm. However, such large

beads cause a coarseness of texture in food systems (Hansen et al., 2002).

Starch has also been investigated as a potential encapsulation material for

probiotics. Researchers have developed a micro-encapsulation technology that

involves encasing bifidobacteria in the hollow core of partially hydrolyzed

granular starch, which is then encapsulated with an outer coating of amylose.

818 20 Micro-Encapsulation of Probiotics

Page 15: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

This technology is designed to protect the probiotic bacteria from adverse

environmental conditions during processing, in products during storage, and

during passage through the upper GIT for delivery in the large intestine (Mattila-

Sandholm et al., 2002).

Mixtures of various polysaccharides and in some cases proteins have also been

investigated as a potential encapsulation materials for probiotics. For example,

alginate was combined with pectin and whey proteins in order to protect Bifido-

bacterium bifidum during transit through the GIT. A clear improvement in cell

survival was observed compared to free cells, although still a significant percentage

of the cells were dying (Guerin et al., 2003). In another study alginate-starch gel

beads were produced to protect Lactobacillus acidophilus and Bifidobacterium

lactis; it was found that encapsulation prevented cell death from oxygen toxicity

(Talwalkar and Kailasapathy, 2003). It was also found that chitosan-coated alginate

beads were offering protection to various LAB strains during storage in milk

(Krasaekoopt et al., 2004), as well as in simulated gastrointestinal fluids (Lee

et al., 2004). In another study, a polysaccharide from kiwifruit was combined with

alginate and chitosan and shown to improve the survival of Lactobacillus rham-

nosus at low pH (Ying et al., 2007). A low-cost micro-encapsulation technique has

been recently proposed, which consists of coating milk fat droplets containing

powder particles of freeze dried cultures with whey protein and polymers, using

emulsification and spray drying in a continuous two step process. Rigorous control

of the size distribution of the different elements constituting the microcapsule is

required. In particular, the size of the material dispersed in the hydrophobic phase

must be larger than that of the globule (Picot and Lacroix, 2004).

The use of polymers as immobilisation matrices for probiotics during freeze

and spray drying has also been investigated. It was shown that immobilization of

Lactobacillus acidophilus with k-carrageenan could enhance the temperature

tolerance of the freeze dried cells and improve their storage stability (Tsen et al.,

2002). The results from other studies regarding the role of specific polymers on

cell survival during storage were less conclusive, and were highly dependent on

the species (Champagne et al., 1996a, 1996b).

In the case of spray drying, most of the probiotic strains do not survive

well the high temperatures and the osmotic stress to which they are exposed to

in the process. The temperature and phase changes, and drying, stress the cells

and damage their membranes. As a result their activity is typically lost after a

few weeks of storage at room temperature (Anal and Singh, 2007). One of the

methods that has been proposed to circumvent this is the incorporation of

Micro-Encapsulation of Probiotics 20 819

Page 16: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

acacia gum into the drying medium; it was shown that this increased significanlty

the survival rate of Lactobacillus paracasei during storage at a variety of tempera-

tures (Desmond et al., 2002).

The size of the market for probiotics in animal feed applications has increased

significantly after the ban on antibiotics as growth promoters. Since feed additives

are generally stored under ‘barn conditions’, i.e., ambient temperature, oxygen and

moisture, micro-encapsulation can help to improve the survival of probiotics.

In this regard, and taking into account the relationship between high moisture

content and death, a watertight shell is required to achieve high protection. Fat

encapsulated probiotics have shown to be ‘barn stable’ for a period of over 2 years

without refrigeration (Meiners, 2004). In this process, also called ‘hot melt’

process, very thin layers of molten lipids are applied to the surface of freeze

dried cultures in fluidized bed equipment. After re-crystallization, very tight

microcapsules are formed. The properties of the microcapsules depend on the

choice of lipids, the moisture residue in the fluidizing air stream and the process

parameters, such as the droplet size of the molten lipid and the temperature of the

atomizing air.

20.7 Conclusion

The micro-encapsulation of probiotics has received a lot of attention, as it can help

to improve probiotic survival during processing and storage, as well as during

passage through the GI tract. A number of encapsulation techniques, such as spray

drying, spray coating, phase separation and extrusion, a well as natural biopoly-

mers, such as alginates, carrageenan, chitosan and pectin have been studied.

Although promising at a laboratory scale many of these technologies are difficult

to scale up. Further research is needed for the development of scalable and effective

technologies as well as for the design of controlled release delivery systems.

List of Abbreviations

DE Degree of Esterification

GIT Gastrointestinal Tract

IP Isoelectric Point

LAB Lactic Acid Bacteria

LBG Locust Bean Gum

820 20 Micro-Encapsulation of Probiotics

Page 17: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

References

Adamiec J, Marciniak E (2004) Microencapsu-

lation of Oil/Matrix/Water system during

spray drying process. Proceedings of

the 14th International Drying Sympo-

sium, Sao Paulo, Brazil, 22–25 Vol. C,

2043–2050

Anal AK, Singh H (2007) Recent advances

in microencapsulation of probiotics for

industrial applications and targeted deliv-

ery. Trends in Food Science & Technology

18: 240–251

Astray G, Gonzalez-Barreiro C, Mejuto JC,

Rial-Otero R, Simal-Gandara J (2009)

A review on the use of cyclodextrins in

food. Food Hydrocolloids (In presss)

Bangham AD (1995) Surrogate cells or trojan

horses - the discovery of liposomes. Bio-

essays 17: 1081–1088

Boh B (2007) Developements et applications

industrielles des microcapsules. In:

Vandamme, Thierry F. (ed.). Microencap-

sulation: des sciences aux technologies.

Paris: Lavoisier, pp. 9–22

Champagne CP, Fustier P (2007) Microencap-

sulation for the improved delivery of bio-

active compounds into foods. Current

Opinion in Biotechnol 18: 184–190

Champagne CP, Mondou F, Raymond Y,

Brochu E (1996a) Effect of immobi-

lization in alginate on the stability of

freeze-dried Bifidobacterium longum,

Bioscience Microflora 15: 9–15

Champagne CP, Mondou F, Raymond Y, Roy D

(1996b) Effect of polymers and storage

temperature on the stability of freeze-

dried lactic acid bacteria. Food Research

International 29: 555–562

Chandramouli V, Kailasapathy K, Peiris P,

Jones M (2004) An improved method of

microencapsulation and its evaluation to

protect Lactobacillus spp. in simulated

gastric conditions. J Microbiol Methods

56: 27–35

Chang TMS (1964) Semipermeable microcap-

sules. Science 146: 524–525

Chesnokov YJ (2001) Short capillary waves on

the surface of a stretching cylindrical jet of

a viscous liquid. J Applied Mechanics and

Technical Physics 42: 431–436

Dervakos GA, Webb C (1991) On the merits of

viable-cell immobilization. Biotechnol

Advances 9: 559–612

Desmond C, Ross RP, O’Callaghan E, Fitzgerald

G, Stanton C (2002) Improved survival

of Lactobacillus paracasei NFBC 338 in

spray-dried powders containing gum aca-

cia. J Appl Microbiol 93: 1003–1011

Dror Y, Cohen Y, Yerushalmi-Rozen R (2006)

Structure of gum arabic in aqueous solu-

tion. J Polymer Science Part B-Polymer

Physics 44: 3265–3271

Gibson GR, Rouzaud, G, Brostoff J, Rayment N

(2005) An evaluation of probiotic effects

in the human gut: microbial aspects,

Final Technical report for FSA project ref

G01022

Gouin S (2004) Microencapsulation: industrial

appraisal of existing technologies and

trends. Trends in Food Science & Techno-

logy 15: 330–347

Green BK (1957) U.S. Patent 2800457

Guerin D, Vuillemard JC, Subirade M (2003)

Protection of bifidobacteria encapsulated

in polysaccharide-protein gel beads against

gastric juice and bile. J Food Protection 66:

2076–2084

Guignon B, Duquenoy A, Dumoulin, ED

(2002) Fluid bed encapsulation of parti-

cles: Principles and practice. Drying Tech-

nology 20: 419–447

Hansen LT, Allan-Wojtas PM, Jin YL, Paulson

AT (2002) Survival of Ca-alginate micro-

encapsulated Bifidobacterium spp. in milk

and simulated gastrointestinal conditions.

Food Microbiol 19: 35–45

Hyndman CL, Groboillot AF, Poncelet D,

Champagne CP, Neufeld RJ (1993)

Microencapsulation of lactococcus-lactis

within cross-linked gelatin membranes.

J Chemical Technol Biotechnol 56: 259–263

Micro-Encapsulation of Probiotics 20 821

Page 18: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

Klein J, Stock J, Vorlop KD (1983) Pore-size

and properties of spherical ca-alginate

biocatalysts. European J Appl Microbiol

Biotechnol 18: 86–91

Klein J, Vorlop KD (1985) Immobilization

Techniques – Cells. In: Moo-Young, M

(ed.). Comprehensive Biotechnology,

vol. 2, Pergamon Press, Oxford, UK,

203–224

Krasaekoopt W, Bhandari B, Deeth H (2003)

Evaluation of encapsulation techniques of

probiotics for yoghurt. International

Dairy J 13: PII S0958–6946(0902)00155-

00153

Lacroix C, Yidirim S (2007) Fermentation

technologies for the production of pro-

biotics with high viability and function-

ality. Current Opinion in Biotechnol 18:

176–183

Lee JS, Cha DS, Park HJ (2004) Survival of

freeze-dried Lactobacillus bulgaricus KFRI

673 in chitosan-coated calcium alginate

microparticles. J Agricultural and Food

Chemistry 52: 7300–7305

Lee KY, Heo TR (2000) Survival of

Bifidobacterium longum immobilized in

calcium alginate beads in simulated gas-

tric juices and bile salt solution. Appl En-

viron Microbiol 66: 869–873

Maa YF, Hsu C (1996) Liquid-liquid emulsifi-

cation by static mixers for use in micro-

encapsulation. J Microencapsulation 13:

419–433

Mattila-Sandholm T, Myllarinen P, Crittenden

R, Mogensen G, Fonden R, Saarela M

(2002) Technological challenges for

future probiotic foods. International

Dairy Journal 12: PII S0958–6946(0901)

00099-00091

Mauvernay RY (1965) Brevet d’Invention, BE

66701

Meesters GMH (2006) Agglomeration of

enzymes, microorganisms and flavours.

In: Salman, Agba; Hounslow, Michael;

Seville, Jonathan P.K. (ed.). Granulation,

Volume 11 (Handbook of Powder Tech-

nology), 555-591

Meiners JA (2004) Some like it hot. Glatt Inter-

national Times, no. 18

Peniche C, Arguelles-Monal W, Peniche H,

Acosta N (2003) Chitosan: An attractive

biocompatible polymer for microencap-

sulation. Macromolecular Bioscience 3:

511–520

Picot A, Lacroix C (2004) Encapsulation of

bifidobacteria in whey protein-based

microcapsules and survival in simulated

gastrointestinal conditions and in yoghurt.

International Dairy Journal 14: 505–515

Pothakamury UR, BarbosaCanovas GV (1995)

Fundamental aspects of controlled release

in foods. Trends in Food Science & Tech-

nology 6: 397–406

Prevost H, Divies C (1988) Continuous pre-

fermentation of milk by entrapped yogurt

bacteria .1. development of the process.

Milchwissenschaft-Milk Science Interna-

tional 43: 621–625

Randall RC, Phillips GO, Williams PA (1988)

The role of the proteinaceous component

on the emulsifying properties of gum ara-

bic. Food Hydrocolloids 2: 131–140

Rees DA (1972) Shapely polysaccharides -

eighth colworth medal lecture. Biochemi-

cal Journal 126: 257–&

Sheu TY, Marshall RT (1993) Microentrapment

of lactobacilli in calcium alginate gels.

J Food Science 58: 557–561

Talwalkar A, Kailasapathy K (2004) Compari-

son of selective and differential media for

the accurate enumeration of strains of

Lactobacillus acidophilus, Bifidobacter-

ium spp. and Lactobacillus casei complex

from commercial yoghurts. International

Dairy Journal 14: 143–149

Tsen JH, Chen HH, King VA (2002) Survival of

freeze-dried Lactobacillus acidophilus

immobilized in kappa-carrageenan gel.

J General and Appl Microbiol 48: 237–241

Watheley TL (1996) Microcapsules: Preparation

by interfacial polymerization and interfacial

complexation and their applications. In:

Benita, Simon (ed.). Micro-capsules prepa-

ration, Micro-encapsulation methods and

822 20 Micro-Encapsulation of Probiotics

Page 19: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

industrial applications. Informa Health

Care: 349–366

Wehr JB, Menzies NW, Blamey FPC (2004)

Alkali hydroxide-induced gelation of

pectin. Food Hydrocolloids 18: 375–378

Weinbreck F, Minor M, De Kruif CG (2004)

Microencapsulation of oils using whey

protein/gum arabic coacervates. J Micro-

encapsulation 21: 667–679

Wilson N, Shah NP (2007) Microencapsulation

of vitamins. Asean Food Journal 14: 1–14

Wu Y, Bao C, Zhou Y (2007) An innovated

tower-fluidized bed prilling process. Chi-

nese Journal of Chemical Engineering 15:

424–428

Ying DY, Parkar S, Luo XX, Seelye R, Sharpe JC,

Barker D, Saunders J, Pereira R, Schroder

R (2007) Microencapsulation of probio-

tics using kiwifruit polysaccharide and al-

ginate chitosan. Proceedings of the 6th

International Symposium on Kiwifruit,

Vols 1 and 2: 801–808

Micro-Encapsulation of Probiotics 20 823

Page 20: Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

Top Related