Prebiotics and Probiotics Science and Technology || Micro-Encapsulation of Probiotics

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<ul><li><p>20Micro-Encapsulation ofProbioticsJean-Antoine Meiners</p><p>20.1 Introduction</p><p>Micro-encapsulation is defined as the technology for packaging with the help of</p><p>protective membranes particles of finely ground solids, droplets of liquids or</p><p>gaseous materials in small capsules that release their contents at controlled rates</p><p>over prolonged periods of time under the influences of specific conditions (Boh,</p><p>2007). The material encapsulating the core is referred to as coating or shell.</p><p>The majority of microcapsules have spherical shapes and their diameter</p><p>varies from a few microns to 1 mm. However, some authors consider particles</p><p>of even more than one millimeter as microcapsules. For the purpose of this</p><p>chapter the term microcapsule refers to capsules whose aim is the protection</p><p>and controlled release of the active substance, and the term microsphere, a</p><p>commonly used term in the scientific literature, refers to granules, that do not</p><p>have a core-shell morphology, and can be simply defined as the embedding of an</p><p>active substance in a matrix (Watheley, 1996).</p><p>In general, encapsulation can be used to improve the stability of the active</p><p>substance during processing and storage, mask unpleasant flavors and odors,</p><p>control possible oxidative reactions, and provide controlled release at the right</p><p>place and the right time. As such, it has numerous applications in the food,</p><p>pharmaceutical, cosmetic, agricultural, textile, paper and paint industries. One of</p><p>the earliest published inventions was the carbonless copy paper in which tiny</p><p>microcapsules were fixed on the backside of a sheet of paper; these were crushed</p><p>by the pressure of writing, thus releasing their dye (Green, 1957). In the areas of</p><p>pharmaceuticals and chemicals, many products, e.g., enzymes, can cause health</p><p>and safety hazards when manipulated in a very fine powder form, due to excessive</p><p>dust formation; granulation into larger size particles and coating can be used to</p><p>alleviate such handling problems (Meesters, 2006). In terms of food applications,</p><p>the aims of encapsulation techniques are to improve the stability of bioactive</p><p># Springer ScienceBusiness Media, LLC 2009</p></li><li><p>ingredients during processing and storage and to prevent undesirable interactions</p><p>with the food matrix (Champagne and Fustier, 2008). An example is the case of</p><p>ascorbic acid, a highly reactive, water soluble and heat-labile compound, encap-</p><p>sulation of which can double its shelf life compared to the free form (Wilson and</p><p>Shah, 2007).</p><p>Various approaches can be used to open up the microcapsules to release the</p><p>active substance. These include (i) mechanical rupture of the membrane, e.g., in the</p><p>case of mastication of micro-encapsulated flavours in chewing gums, (ii) exposure</p><p>to high temperatures to make the coating material melt, a technology frequently</p><p>used for encapsulated chemical leavening agents in baked goods, (iii) dissolution of</p><p>the capsules when placed in solvents, (iv) exposure to specific pH, (v) biodegrada-</p><p>tion of the polymer coating by enzymes, (vi) diffusion of the active substance</p><p>through the polymer coating, (vii) high osmotic pressure inside the microcapsule</p><p>and (viii) combinations of the above (Pothakamury and Barnosa-Canovas, 1995).</p><p>In the area of microbial products, micro-encapsulation is used in order to</p><p>enhance the delivery of probiotic microorganisms into foods during processing</p><p>and storage, or to protect against the acidic conditions in the stomach and ensure</p><p>delivery into the intestine. In addition, microbial cells can also be immobilized</p><p>onto polymer matrices and used as biocatalysts in fermentation processes. The</p><p>main difference between encapsulation and immobilization is that in the latter</p><p>the polymer beads produced allow fast and easy diffusion of water and other</p><p>fluids, and thus the cells are biochemically active (Klein and Vorlop, 1985).</p><p>20.2 Micro-encapsulation Techniques and Processes</p><p>A variety of encapsulation techniques are available and include both chemical</p><p>processes, such as phase microseparation, coacervation, liposome encapsulation</p><p>molecular inclusion, as well as physical processes, such as spray drying, spray</p><p>chilling, prilling, spinning disk, fluidized bed coating, and extrusion. Certain</p><p>steps are common to many of these processes.</p><p>Basically the initial step is to introduce the active substance inside the shell;</p><p>this involves dispersion or atomization in order to position the membrane on the</p><p>outside of the microcapsule and the active substance in the core.</p><p>In the case of microcapsules containing a liquid core active substance an</p><p>emulsion is prepared, whose surface is polymerized in a subsequent step, the</p><p>interface condensed and/or the solvent evaporated. In the section below, the</p><p>different types of encapsulation methods are presented in more detail.</p><p>806 20 Micro-Encapsulation of Probiotics</p></li><li><p>20.2.1 Spray Drying</p><p>Spray drying is one of the most commonly used encapsulation method, as it is a</p><p>well established and cost-efficient technology; it is basically designed to evaporate</p><p>water from the dry matter. The solution is injected in a hot air stream in a closed</p><p>vessel and the solvent, whichmost of the times is water, is evaporated. The energy is</p><p>absorbed to evaporate the water and consequently the powder temperature can be</p><p>controlled. The residence time in the tower is one of the limiting factors; the</p><p>majority of the moisture has to evaporate during the fall time in the tower of the</p><p>particles (Adamiec and Marciniak, 2004). In order to be efficient, the total surface</p><p>area should be as large as possible and consequently the droplet sizes small, and if</p><p>possible, of the same size.</p><p>The principle of the technique is based on a spray, which is created by forcing</p><p>the fluid through an orifice. The energy required to overcome the pressure drop</p><p>upon exiting the orifice is supplied by the spray dryer feed pump. Pressure nozzles</p><p>coupled to high pressure feed pumps, which produce pressures of as much as 3000</p><p>psig, have the advantage of producing a narrow particle size distribution, but can</p><p>have severe damaging effects on a microorganisms cell structure. Despite the fact</p><p>that they are less energy efficient and produce narrower particle size distribution,</p><p>the two-fluid nozzle is very popular for laboratory equipment, probably since it</p><p>allows working with small flow rates. The most popular nozzle type for industrial</p><p>spray drying is the centrifugal atomizer, where a spray is created by passing the fluid</p><p>across or through a rotating wheel or disk. The benefit for micro-encapsulation</p><p>purposes is that rather large particles, up to 100 mm, can be produced this way.</p><p>20.2.2 Spray Chilling and Cooling</p><p>Spray chilling and spray cooling (or congealing), the second being operated at</p><p>lower temperatures, involve mixing thoroughly the core and amolten shell material</p><p>with a melting temperature well above the operating temperature (usually lipids),</p><p>and atomizing through a two media nozzle into a cooling chamber in order to</p><p>solidify the droplet instantaneously. One of the limitations is the speed in heat</p><p>transfer of the energy freed during the re-crystallization. The use of cooling media</p><p>has allowed high volume and high speed production. Another limiting factor is the</p><p>difference in surface tension between the matrix and the core material. Conse-</p><p>quently, the core is not always placed in the center of the microcapsule, which may</p><p>affect the protective properties of the microcapsule (Meiners, 2004).</p><p>Micro-Encapsulation of Probiotics 20 807</p></li><li><p>20.2.3 Prilling</p><p>Hawleys Condensed Chemical Dictionary says that prills are small round or</p><p>acicular aggregates of a material. Prilling can be a useful technology, when solidifi-</p><p>cation of the shell material is an instantaneous reaction. The core and the shell</p><p>material are mixed thoroughly and the dispersion is pumped to flow over a sonically</p><p>vibrating dispersion head. Gravity allows the droplets to fall into the collection</p><p>device for cooling or polymerization (Wu et al., 2007). More advanced and sophis-</p><p>ticated equipment have been developed that limit considerably the residence time</p><p>of the dispersion and the need for large cooling towers (Meiners, 2004).</p><p>20.2.4 Spinning Disk</p><p>A comparable technology to prilling is the spinning disk technology, which uses</p><p>centrifugal forces for droplet separation.</p><p>The droplet volumes and their mutual spacing are governed by the channel</p><p>geometry and the frequency of rotation. Devices exist that combine spinning disk</p><p>forces with high frequency droplet separation (Chesnokov, 2001).</p><p>20.2.5 Fluidized Bed</p><p>Fluidized bed technology is based on the separation of individual particles in a</p><p>gas stream and the fixation of the membrane substance by polymerization, drying</p><p>or crystallization around the core. The solvation and drying steps can be avoided,</p><p>which for thermo-sensitive materials may represent a major advantage. Agglom-</p><p>eration and retention in the filter system cause the use of the fluidized bed system</p><p>to be difficult with products having strong adhesive properties (Guignon, 2002).</p><p>20.2.6 Extrusion</p><p>Micro-encapsulation by extrusion involves projecting an emulsion core and</p><p>coating material through a nozzle at high pressure. It involves preparing a</p><p>hydrocolloidal solution, adding the active substance and extrusing the suspension</p><p>through a nozzle in the form of droplets into a hardening solution or setting bath</p><p>(Krasaekoopt et al., 2003). Carbohydrate matrices in the glassy state have very</p><p>808 20 Micro-Encapsulation of Probiotics</p></li><li><p>good barrier properties and extrusion is a convenient process enabling the</p><p>encapsulation of active substances in such matrices (Gouin, 2004).</p><p>20.2.7 Coacervation</p><p>Coacervation consists of the separation of colloid particles from a solution, which</p><p>then agglomerate into a separate liquid phase called coarcevate. A number of hydro-</p><p>colloid systems have been evaluated for coacervation micro-encapsulation including</p><p>among others the gelatine/gum acacia, heparin/gelatine, carageenan, chitosan, soy</p><p>protein, lactogloboulin/gum acacia and the guar/dextran system (Gouin, 2004).</p><p>20.2.8 Liposomes</p><p>Liposomes are artificially made microscopic membrane vesicles consisting of one</p><p>or more concentric layers of lipids. They are formed by dispersing the lipid</p><p>formulation in a solvent system, decreasing the solvent volume and then re-</p><p>dispersing the film of lipid/solvent in an aqueous phase (Bangham, 1995).</p><p>20.2.9 Inclusion Complexation</p><p>Cyclodextrins are cyclic oligomers, who have the ability to form inclusion com-</p><p>plexes with the active substances. They are typically used for the protection of</p><p>unstable and high value chemicals, such as flavours. Oil-in-water emulsions using</p><p>cyclodextrins can also be subsequently spray dried (Astray et al., 2009).</p><p>20.3 Technologies used for the Immobilization andMicro-encapsulation of Microganisms</p><p>Immobilization of living cells was the first form of micro-encapsulation and</p><p>was pioneered about 50 years ago for medical purposes, and (Chang, 1964).</p><p>The first patent demonstrating the biocompatibility of polymers and the resis-</p><p>tance of the encapsulated material to sterilization conditions was granted in 1965</p><p>(Mauvernay, 1965).</p><p>Immobilization of microorganisms for bioprocessing purposes became im-</p><p>portant for the development of the continuous fermentation process. Much of the</p><p>Micro-Encapsulation of Probiotics 20 809</p></li><li><p>research work was done by the brewing industry using immobilized yeast cells</p><p>and the dairy industry using immobilized lactic acid producing bacteria (Prevost</p><p>and Divies, 1988).</p><p>The pore size of the microcapsules produced has an important effect on the</p><p>cellular biochemical activities, as it controls the rate of retention/passage of</p><p>undesirable metabolites (Klein et al., 1983). Different methods have been used</p><p>to immobilize microorganisms, including, physical entrapment in a polymeric</p><p>network, attachment or adsorption to a carrier, and membrane entrapment.</p><p>In order to entrap the biomass in microcapsules simultaneous with the</p><p>membrane formation, it is important for the that droplets to be generated simul-</p><p>taneously with the membrane. For this purpose, specific equipment has been</p><p>developed and polymers have been selected. The polymers generally form a gel</p><p>under the influence of ionization or thermosetting.</p><p>For the purpose of droplet separation with a narrow size distribution, a</p><p>pumping system under gravity provides a constant uninterrupted flow of the</p><p>liquid. In the early droplet generators, the liquid consisted of a solution of the</p><p>biomass and the dissolved polymer. Upon falling into the collection bath filled</p><p>with a solution containing the ionic solution, the polymer membrane was cross-</p><p>linked to form self sustaining microcapsules, encapsulating the biomass droplet</p><p>within the membrane (Sheu and Marshall, 1993). The more recent versions use a</p><p>nozzle designed for co-extrusion, creating a polymer network around the droplet</p><p>during its fall into the collection bath. The different dripping systems can be</p><p>identified according to the principles below. &gt; Figures 20.120.4 depict the</p><p>various types of nozzles used.</p><p> 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.</p><p>The use of static mixers has also appeared to be very promising, since it allows</p><p>large volume and cost efficient production. The device consists of mixer elements</p><p>contained in a cylindrical (tube) or squared housing. These can vary from 6 mm</p><p>to 6 meters diameter. Static mixer elements consist of a series of baffles. As the</p><p>streams move through the mixer, the non-moving elements continuously blend the</p><p>materials (Maa and Hsu, 1996).</p><p>810 20 Micro-Encapsulation of Probiotics</p></li><li><p>20.4 Objectives for the Micro-encapsulation ofProbiotics</p><p>A major function of micro-encapsulation is to provide protection against the high</p><p>acidity of the gastric fluids. A microcapsule containing the probiotic must not be</p><p>fractured until it passes through the stomach. Since the biological release mecha-</p><p>nism is triggered by the higher pH in the upper intestine, a coating can be used that</p><p>. Figure 20.1Dripping assisted by an electrostatic force (courtesy of Nisco Engineering AG).</p><p>. Figure 20.2Dripping assisted by aerodynamically jetting (courtesy of Nisco Engineering AG).</p><p>Micro-Encapsulation of Probiotics 20 811</p></li><li><p>withstands the low pH and can release its content at a pH similar to that of the large</p><p>intestine (e.g., pH 5.5 to 7). The survival of commercial probiotics in conditions</p><p>of very low pH was recently investigated; the researchers assessed in vitro the</p><p>survival of 32 probiotic strains, all isolated from commercially available products</p><p>. Figure 20.3Dripping assisted by co-axial air (courtesy of Nisco Engineering AG).</p><p>. Figure 20.4Jet cutter (courtesy of Genialab AG).</p><p>812 20 Micro-Encapsulation of Probiotics</p></li><li><p>in simulated gastric contents. Approximately 50% of them were viable...</p></li></ul>

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