Principles and Methods of Microencapsulation of Probiotic Microorganisms

Document Type: Review Paper

Authors

Department of Food Science and Engineering, Faculty of Biosystem Engineering, Campus of Agriculture, University of Tehran, P.O. Box 31587 -78659, Karaj, I.R. Iran

Abstract

Worldwide popularity of probiotic- microorganisms and their products on the one hand, and their general weak viability in food products (especially fermented types) as well as gastrointestinal conditions on the other hand, has encouraged researchers to innovate different methods of probiotics viability improvement.  Microencapsulation of the probiotic cells is one of the newest and highly efficient methods, which is now under the especial attention and is being developed by various researchers. This article reviews the principles and methods of probiotic cell microencapsulation.

Keywords


Table of contents:

1. Introduction
2. Structural details of microbeads
3. Main components used for microencapsulation of probiotics
4. Applications and Advantages of probiotics microencapsulation
5. Factors affect microencapsulation effectiveness of probiotics
6. Methods of probiotics microencapsulation
7. Methods for evaluating the microencapsulation efficiency
   of probiotics
8. Conclusions

 


INTRODUCTION

Probiotics are microorganisms which settle in the intestine medium and render healthful effects on the host (humans or animals), substantially via maintenance and improvement of the microbial balance (between the healthful and harmful microorganisms) of the intestine (Fuller 1989; 1991; Goldin 1998; Gismondo et al. 1999). Various health benefits have been attributed to probiotics such as antimutagenic and anticarcinogenic properties, antiinfection properties, immune system stimulation, serum cholesterol reduction, alleviation of lactose intolerance and nutritional enhancement (Gilliland and Speck, 1977; Kim and Gilliland, 1983; Rasic and Kurmann, 1983; Gurr, 1987; Gilliland, 1989; Surawicz et al., 1989; Fuller, 1992; Buck and Gilliland, 1994; Lancaputhra and Shah, 1995; Daly and Davis, 1998; Klein et al., 1998; Macfarlane and Cummings, 1999; Mombelli and Gismondo, 2000). Species of Lactobacillus acidophilus, L. casei, Bifidobacterium bifidum,                 B. longum, B. breve, B. infantice and B. lactis (B. animalis) are the most popular bacteria applied food probiotic products (Daly and Davis, 1998; Klein et al., 1998; Macfarlane and Cummings, 1999). Saccharomyces boulardii is the only probiotic fungus which has been successfully used for curing the intestinal infections, especially diarrhea (Surawicz et al., 1989; Mombelli and Gismondo, 2000). Extensive research carried out on the viability and survivability of probiotics in gastrointestinal tract and food products (especially dairy fermented products) have revealed that in general, their viability dramatically decreases due to exposure to detrimental environmental factors such as organic acids, hydrogen ions, molecular oxygen and antibacterial components (Gilliland and Speck, 1977; Hamilton-Miller, 1999; Iwana et al., 1993; Lancaputhra and Shah, 1995; Shah et al., 1995; Dave and Shah, 1996; Dave and Shah, 1997;  Kailasapathy and Rybka, 1997; Shah and Lankaputhra, 1997; Kebary et al., 1998; Beal et al., 1999; Gardini et al. 1999; Hamilton-Miller et al., 1999; Schillinger, 1999; Vinderola et al., 2000; Sultana et al., 2000; Mortazavian et al., 2006a,b,c,d). In addition, the beneficial effects of probiotic microorganisms appear when they arrive in the intestinal medium, viable and in high enough number, after surviving the above mentioned harsh conditions (Gilliland, 1989). The minimum number of probiotic cells (cfu/g) in the product at the moment of consumption that is necessary for the fruition of beneficial pharmaceutical (preventive or therapeutic) effects of probiotics has been suggested to be represented by the minimum of bio-value (MBV) index (Mortazavian et al., 2006c). According to the International Dairy Federation (IDF) recommendation, this index should be ³107 cfu/g up to the date of minimum durability (Ouwehand and Salminen, 1998). In some countries such as Argentina, Prague and Brazil, the standard of ³106 cfu/g has been accepted in the case of bifidobacteria. This standard has been prescribed >107 cfu/g in Japan (Robinson, 1987). Also, various recommendations have been presented by different researchers such as >106 cfu/g by all probiotics in yogurt (Robinson, 1987; Kurman and Rasic, 1991) and >107 cfu/g in the case of bifidobacteria (Holcomb et al., 1991). Apart from the MBV index, daily intake (DI) of each food product is also determinable for their probiotic effectiveness. The minimum amount of the latter index has been recommended as approximately 109 viable cells per day (Shah et al., 1995; Kurman and Rasic, 1991; Mortazavian, 2006c). The type of culture media used for the enumeration of probiotic bacteria is also an important factor for determination of their viability, as the cell recovery rate of various media are different (Mortazavian, 2006c,d).
   Viability loss of probiotics in food products (especially fermented types) and acidic-bile conditions of gastrointestinal tract has always encouraged researchers to find new efficient methods of viability improvement. Microencapsulation, as one of the newest and most efficient methods, has recently been under especial consideration and investigation. From a microbiological point of view, microencapsulation can be defined as the process of entrapment/enclosure of microorganisms cells by means of coating them with proper hydrocolloid(s) in order to segregate the cells from the surrounding environment; in a way that results in appropriate cell release in the intestinal medium (Sultana et al., 2000; Krasaekoopt et al., 2003; Picot and Lacroix, 2003a). Among the releasing agents (triggers), pH changes, mechanical tensions, heat, enzymatic activities, osmotic pressure, slow diffusion of the moisture through the capsule layers, presence of some chemical components and storage time can be mentioned (Gouin, 2004). Micropropagation of probiotic cells has been shown preserve them from detrimental environmental factors such as high acidity and low pH (Wenrong and Griffiths, 2000), bile salts (Lee and Heo, 2000), cold shocks induced by the process conditions such as deep freezing and freeze drying (Shah and Rarula, 2000), molecular oxygen in case of obligatory anaerobic microorganisms (Sunohara et al., 1995), heat shocks caused by process conditions such as spray drying, bacteriophages (Steenson et al., 1987) and chemical antimicrobial agents (Sultana, 2000). However, other advantages such as increase of sensory properties stability and/or its improvement (Gomes and Malcata, 1999) and immobilization of the cells for their homogeneous distribution throughout the product (Steenson et al., 1987; Krasaekoopt et al., 2003) can also be achieved.
    Importance of the microencapsulation method, as an efficient manner for increasing probiotics viability, justifies reviewing the newest achievements in this regard. The present article reviews principles and methods of probiotics microencapsulation including discussions of microbeads structure, components used for microencapsulation, its applications and advantages regarding probiotics, factors affecting microencapsulation effectiveness, microencapsulation methods and technology and methods of microencapsulation efficiency evaluation (by the assessment of its qualitative factors).

2. Structural details of microbeads
Figure 1 represents structural characteristics of microbeads. Each microbead consists of hydrocolloids (also called capsule) coated around the bacterial cell(s). If the capsule has a gel-like structure, the microbead is named gel-bead. Because the geometrical shape of a microbead is usually spherical to elliptical, it is also called a “microsphere”. Beads might have even/smooth or rough surfaces (Figure 1, part 1.1). Each bead might consist of one or several cells. When several cells are enclosed by the capsule, the interstitial liquid from solution fills the free spaces of the microbead. Superficial and/or deep cracks might appear in the beads (Figure 1, part 1.1). Extension of these cracks leads to pore formation, which considerably reduces the encapsulation efficiency. Microbeads can be coated with a second layer of chemical compounds in order to increase microencapsulation efficiency. The second layer is a so-called coat or support or shell. Microbeads with (Figure 1, part 1.3) or without the coat are named coated- and uncoated beads, respectively. The constituents entrapped within the coat are known as the “core” (Sultana et al., 2000; Truelstrup-Hansen et al., 2002; Dimantov et al., 2003; Krasaekoopt et al., 2003; Chandramouli et al., 2004).

3. Main components used for microencapsulation of probiotics
3.1. Alginate and its combinations: Alginate is a linear heteropolysaccharide extracted from different types of algae, with two structural units consisting of D-mannuronic and L-guluronic acids. Calcium alginate has been widely used for the encapsulation of lactic acid- and probiotic bacteria, mainly in the concentration range of 0.5-4% (Sheu and Marshall, 1991; Sheu and Marshall, 1993; Truelstrup-Hansen et al., 2002; Kim et al., 1996; Jankowski et al., 1997; Khalil and Mansour, 1998; Kebary et al., 1998; Lee and Heo, 2000; Shah and Rarula, 2000; Sultana et al., 2000; Truelstrup-Hansen, 2002; Krasaekoopt et al., 2004). Alginate capsules have some advantages as follows (Klien et al., 1983; Tanaka et al., 1984; Martinsen et al., 1989; Prevost and Divies, 1992; Dimantov et al., 2003; Chandramouli et al., 2004; Gouin, 2004): Easily form gel matrices around bacterial cells, they are not poisonous to the body (is safe or biocompatible), they are cheap, mild process conditions (such as temperature) are needed for their performance, can be easily prepared and performed (simplicity and ease of handling) and properly resolve in the intestine and release entrapped cells. Alginate gel matrix appropriately surrounds the bacterial cells with a diameter of 1-3 mm and the pores sizes formed at the surface of alginate beads do not exceed 7 nm (Klien et al., 1983). However, some disadvantages are attributed to alginate beads. For example they are susceptible to acidic environments and their crackling and loss of mechanical stability in the lactic acid-containing environments have been verified (Eikmeier and Rehm, 1987; Roy et al., 1987; Audet et al., 1988; Ellenton, 1998). Also, because alginate gel is formed in the presence of calcium ions, its integrity is deteriorated when subjected to monovalent ions or chelating agents which absorb calcium ions such as phosphates, lactates and citrates (Roy et al., 1987; Smidsrod and Skjak-Braek, 1990; Ellenton, 1998). Other disadvantages include difficulties in industrial scale applications due to their high expenses and weak ability of scaling up as well as formation of crackled and porous bead surfaces (Gouin, 2004). Latter specification leads to the relatively fast diffusion of moisture and other fluids through the capsules which reduce their barrier properties against unfavorable environmental factors (Gouin, 2004). The mentioned defects can be efficiently compensated by blending of alginate with other polymer compounds, coating other compounds on its capsules and structural modification of the alginate by using various additives (Krasaekoopt et al., 2003). Blending alginate with starch is a common practice and it has been shown that encapsulation effectiveness of different bacterial cells especially lactic acid bacteria were improved by applying this method (Jankowski et al., 1997; Sultana et al., 2000; Sun and Griffiths, 2000; Truelstrup-Hansen et al., 2002; Krasaekoopt et al., 2003). Besides good protection from bacterial cells, alginate-starch blends render the advantage of micronutrients and metabolites diffusing through the capsules, inside and outside of the entrapped cell(s). As a result, beads would contain metabolically active cells (Jankowski et al., 1997). Blending calcium alginate with Hi-maite starch produces capsules with high cell viability due to formation of capsules with a good integrated structure as well as prebiotic effect of the latter compound (Sultana et al., 2000). Alginate-glycerol blend improved survivability of the cells deep frozen with liquid nitrogen and kept at -20ºC. This has been attributed to the cryogenic effect of glycerol (Truelstrup-Hansen et al., 2002). Formation of a coat/shell around the alginate capsule has been verified to considerably improve its physicochemical characteristics. It has been reported that by coating semipermeable layers of chitosan polymer (as a polycationic compound) around the alginate capsules (which have negative charges), beads with improved physical and chemical stability were produced. This structure was tolerant against the deteriorative effects of calcium chelating and antigelling agents. Also structurally, the beads were denser and much stronger, thus avoiding breaking and cell(s) release (Smidsrod and Skjak-Braek, 1990; Zhou et al., 1998; Krasaekoopt et al., 2003). In fact low-molecular-weight chitosan diffuses faster into the alginate matrix compared with the high-molecular-weight one, resulting in the formation of capsules with higher density and strength. Coating of calcium chloride on the alginate capsules has also been investigated (Chandramouli et al. 2004). Regarding the function of calcium ions in alginate gel formation, this coating causes generation of more stable beads with a higher protective effect on the probiotic cells, and as a result, higher viability. Poly-amino acids such as poly-L-lysine (PLL) are from other poly-cationic polymers coated on the alginate capsules. Similar to chitosan, these polymers make strong complexes with alginate matrix and give it the advantages as mentioned for chitosan (Smidsrod and Skjak-Braek, 1990; Champagne et al., 1992a; Larisch et al., 1994). Generation of multilayer shells of PLL on the alginate capsules has also been investigated: the first layer of PLL on the capsule surface produces positive charge, then the second alginate coat gives the beads surface negative charge. This trend can be repeated several times. As a result, layers of alginate and PLL would be formed alternatively (Champagne et al., 1992a; Larisch et al., 1994; Marx, 1989). Coatings of Polyetylenamine and glutaraldehyde (as other types of polycationic polymers) on the alginate capsules has also been reported. Cross-linked alginate matrix (produced at low pHs) is obtained from modified alginate structures applied to probiotics encapsulation. Although this kind of matrix has more density and strength compared with the alginate matrix alone, it is able to successfully release the bacterial cells into the intestine (Marx, 1989).  

3.2. Starch: As mentioned previously (section 3.1), starch has been used as a material for coating of alginate capsules. High-amylose corn starch (HACS) can be applied for enhancing functions of capsule- or shell/coat formation (Dimantov et al., 2003). Lyophilized corn starch (LCS) has been reported to be used as capsule-forming material, however, its decomposes after being subjected to pancreatic enzymes (Fanta et al., 2001). Resistant starch (RS) is not degraded by the pancreatic amylase enters the intestine in the indigestible form. This specification apart from giving the microbeads good enteric delivery characteristic (good release of bacterial cells in the large intestine), also gives them prebiotic functionality as they can be used by the probiotic bacteria in the intestine (Kritchevsky, 1995; Muir et al., 1995; Phillips et al., 1995; Silvester et al., 1995;  Haralampu, 2000; Thompson, 2000). HACS with 20% RS has been recognized to be suitable for the enteric delivery purpose. By applying hydro-thermal and retrogradation processes on the native high-amylose corn starch (NHACS), RS-rich fractions which are suitable for encapsulation can be prepared (Dimantov et al., 2004). It has been reported that fermentation of starch by microorganisms such as bifidobacteria, Lactobacilli, Streptococci and Entrobacteriaceae reduces the pH of the intestine via formation of short chain fatty acids (Macfarlane and Gummings, 1991; Kleessen et al., 1997; Le Blay et al., 1999). Also, consumption of resistant starch reduces the risk of intestinal cancer because of having dietary fiber functionality (Dimantov et al., 2004). 

3.3. Mixture of xanthan-gelan: A mixture of xanthan-gelan gum has been used for the microencapsulation of probiotics (Paquin et al., 1990; Sanderson, 1990; Sultana et al., 2000; Sun and Griffiths, 2000. The optimum mixing proportion was 1:0.75 for xanthan: gelan (Sun and Griffiths, 2000). In contrary with alginate, this mixture is resistant to acidic conditions. Also, as apposed to from carrageenan which needs potassium ions for structural stabilization (it is harmful for the body in high concentrations), this gum can be stabilized with calcium ions (Klein and Vorlop, 1985; Sanderson, 1990). It should be noted that although gelan gum is able to generate gel-bead structure for microencapsulation, it is not used on its own for this purpose because of having a high gel-setting temperature (80-90°C for about 1 h) which results in heat injuries to the probiotic cells (Sun and Griffiths, 2000).

3.4. Carrageenan and its mixtures: K-carrageenan is a neutral polysaccharide which requires high temperatures (60-90°C) for dissolution especially when applied at high concentrations such as 2-5% (Klein and Vorlop, 1985). When cell slurry containing probiotics is added to the sterilized and cooled (40-45ºC) solution of this polymer, subsequent cooling down to room temperature results in its gelatinization. Adding monovalent ions such as potassium in the form of KCl leads to the establishment of gel-beads (Krasaekoopt et al., 2003). However, KCl has been reported to have an inhibitory effect on some lactic acid bacteria such as Streptococcus salivarius ssp. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus (traditional yogurt bacteria) (Audet et al., 1988). As replacements, Rb+, Cs+ and NH4+ ions have been recommended. These ions, regardless of resolving the above mentioned problem, produce stronger gel beads compared with potassium ion. Mixture of k-carrageenan-locust bean renders good efficiency in lactic-fermented products (such as yogurt) due to its lower susceptibility to the organic acids. This mixture has been widely used for microencapsulation of probiotics in fermented products (Audet et al., 1988; Arnauld, 1992). However, gel formation of k-carrageenan-locust bean mixture is dependent on calcium ions, which have adverse effects on both viability of Bifidobacterium spp. and the human body. The latter property arises from its undesirable effect on the electrolyte equilibrium of liquids in the body (Paquin et al., 1990; Sun and Griffiths, 2000). It has been reported that the proportion of 1:2 for carrageenan-locust been gives a strong gel for microencapsulation (Miles et al., 1984; Takata et al., 1977).

3.5. Gelatin: Gelatin gum has been used for the microencapsulation of probiotics, alone or in mixture with other gums (Hyndman et al., 1993). It is a protein gum which makes a thermoreversible gel. Its amphoteric nature gives the ability of having synergistic effects with anionic polysaccharides such as gelan gum. The two mentioned polymers are miscible at  pHs > 6, because of having negative charges. When pH of the solution drops below the isoelectric pH of gelatin, this gum obtaines positive charge to interact with the gelan gum (King, 1995). Mixture of gelain-toluene diisocyanate makes strong capsules which are tolerant against crackling and breaking, especially at higher concentrations. This can be attributed to the cross-link formation between these polymers. Mentioned mixture has been used for the encapsulation of Lactobacillus lactis ssp. cremoris (Hyndman et al., 1993). Mixture of gelatin-arabic gum has also been applied in the coating of soybean-oil capsules (Truelstrup-Hansen et al., 2002).

3.6. Cellulose acetate phethalate: This component contains negative-charge groups of phethalate. It is soluble at pHs ³ 6, but insoluble at pHs £ 5 (Malm et al., 1951). Because of having a safe nature for purpose human ingestions, it is being widely used for drug capsulation in pharmacy (Rao et al., 1989; Krasaekoopt et al., 2003). Also, freeze dried Bifidobacterium pseudolangum capsulated with this compound and coated by wax has been reported to have  considerably higher survivability after passing through  gasteric juice (Rao et al., 1989).

3.7. Chitosan: Chitosan is a linear polysaccharide with negative charge arising from its amine groups which are obtained by deacetylation of chitin. It is soluble at pHs < 6 and like alginate, makes a gel structure by ionotropic gelation. Chitosan polymers can further polymerize by means of cross-link formation in the presence of anions and polyanions (Klien et al., 1983). As mentioned before (section 3.5), chitosan has been used for coating of gelatin capsules. Because its efficiency for increasing viability of probiotic cells is not satisfactory, it is most often used as a coat/shell, but not capsule. Usually, low-concentration chitosan solution (e.g. 0.4%) is applied for shell-making on capsules such as gelatin (Zhou et al., 1998). It has reported that mixture of chitosan and hexamethylene diisocyanate or chitosan and glutaraldehyde make stronger coats compared with chitosan alone (Groboillot et al., 1993). In order to coat chitosan on alginate capsules, solutions of microbeads with alginate capsules should be dripped into a chitosan-calcium chloride mixture. Presence of calcium ions is necessary for proper coating (Krasaekoopt et al., 2003).

3.8. Miscellaneous compounds: Components such as whey proteins used as capsule materials (Picot and Lacroix 2003a,b; Picot and Lacroix, 2004), soybean oil as capsule coated by a mixture of Arabic and gelatin gums (Truelstrup-Hansen et al., 2002), wax for coating different types of capsules (Rao et al., 1989) and calcium chloride for coating alginate capsules (Chandramouli et al., 2004) have also been used to encapsulate probiotics. Apart from the main materials which directly form capsule and/or coat structure, additives such as SDS, tween 80 (as emulsifiers) and cryo-protectants (e.g. glycerol) are usually added to the solution for the encapsulation process (Kearney et al., 1990).

4. Applications and Advantages of probiotics microencapsulation
Applications and advantages of probiotics encapsulation can be discussed from different angles including production of starter cultures, production of food products from the aspects of probiotic cells viability in the products, their sensory properties of them and probiotic cells immobilization in the products, viability of probiotics cells in the gastrointestinal tract (GT) and usage in fermentors. These aspects are discussed below:

4.1. Production of starter cultures: Microencapsulation can be used efficiently for preparation of bacterial starter cultures with higher viability. It has been shown that the shelf life of encapsulated Lactobacillus rhamnosus VTT E-97800 which is kept under room temperature and relatively high relative humidity is at least 6 months. This shelf life was successfully increased to at least 18 months when the encapsulated cells were deep frozen in liquid nitrogen. Encapsulated cells can be directly ingested in the products and consumed. Only 10% deterioration of such beads was observed after passing through simulated gastrointestinal conditions (Mattila-Sandholm et al., 2002). Picot and Lacroix (2003b) encapsulated starter cells by using whey protein fragments within a milk fat medium. By applying this method, production of starter culture powder with minimum of heat damage during spray drying was achieved. It has been understood that encapsulation of starter cells with the mixture of alginate-glycerol can significantly increase their survivability after the deep freezing process (Sultana et al., 2000).

4.2. Viability of probiotics in gastrointestinal tract: Various reports confirm that microencapsulation efficiently increases the probiotics viability through the passing from acidic-enzymatic-bile conditions of the gastrointestinal tract. For instance, Rao in 1989 understood that encapsulation of B. pseudolongum with cellulose acetate phethalate (CAP) increased its viability in the simulated conditions of the gastrointestinal tract (Groboillot et al., 1993). Experiments of Lee and Heo in 2000 showed that survivability of B. longum encapsulated with calcium alginate in the simulated conditions of gastric juice (pH 1.5) could be considerably increased. Experiments indicated that coating of the calcium chloride on sodium alginate capsules containing L. acidophilus increased tolerance of the mentioned bacteria against harsh acidic (pH 2) and bile (1%) conditions (Chandramouli et al., 2004). Simulated conditions of the stomach (pH 1.5) led to a dramatic loss in the viable counts of B. infantice (from 1.23 ´109 to <10 cfu/ml after 30 min), nevertheless, its viability loss under the same conditions after microencapsulation did not exceed the 0.67% of the first viable cell amount (Sun and Griffiths, 2000). Research results have revealed that resistant starch is an efficient component for the purpose of probiotics encapsulation, because it is not dissolved or decomposed in the gastric acid, neutral pH and by the enzymatic activity of pancreas, but releases its cells when the beads enter the intestine (Englyst et al., 1992; Sun and Griffiths, 2002). Microencapsulation with CAP has also been claimed to have a suitable effect on the viability of B. pseudolongum after being exposed to the simulated gastric conditions. According to the same research, the unencapsulated cells were completely destroyed after 1 h. It should be pointed out that apart from the type of capsulation materials; diameter of capsules or coats is also a determinable factor for improving the viability of probiotics. Excessive reduction in diameter can weaken or remove the protective function of encapsulation. For example, it has been reported that survivability of encapsulated probiotics with alginate capsules under the acidic-bile conditions showed no significant difference when the diameter of gel-beads were 20 and 70 mm compared with the bigger sizes (Sultana et al. 2000). Also, microencapsulation of Bifidobacterium spp. did not significantly increase their viability when the cells encountered the simulated gastric juice (Chandramouli et al. 2004).

4.3. Application in fermentors: It has been claimed that during biomass production, microencapsulation of probiotics can include the following advantages: increasing the tolerance of microorganisms against factors such as bacteriophage infection (Steenson et al., 1987), chemical poisoning agents, protecting microorganism cells against unwanted changes such as genetic mutations, reaching good productivity in metabolite production especially at high agitation rates (Arnauld et al., 1992) and producing more dense biomass (Champagne et al., 1992b).

4.4. Production of food products: Advantages of probiotic microencapsulation in food probiotic products can be discussed from four points of view: increasing viability of probiotics in products till the moment of consumption, achieving new methods in food manufacture, fixing and improving the sensory properties of probiotic products and immobilizing probiotic cells in the products. The above, mentioned sections are discussed separately below.

4.4.1. Viability of probiotics: Microencapsulation can noticeably improve the viability of probiotic microorganisms due to its protective effects against detrimental environmental factors such as high acidity, low pH, molecular oxygen (in the case of obligatory anaerobic microorganisms), poisoning agents generated during the process (especially heat treatment), digestive enzymes, bacteriophages, hydrogen peroxide, short-chain fatty acids, carbonyl-aromatic compounds (three last cases are produced by starter cultures during fermentation) and heat processing (e.g. drying) (Mortazavian et al., 2006a). Increasing viability of probiotics will lead to the increase of products shelf life. Undoubtably, high acidity and low pH of fermented products are the main factors that cause viability loss of probiotics, especially during refrigerated storage (Shah et al., 1995; Dave and Shah and Lankaputhra, 1997; Mortazavian et al., 2006a,b,c). Microencapsulation of L. acidophilus and bifidobacteria with calcium alginate did not considerably increase their viability after being subjected to the intense acid (pH 2) and bile (2%) environment, vice versa, however, at mild acidic conditions (natural acidity of yogurt), throughout 8 weeks of refrigerated storage improving the probiotics survivability was noticeable. Mixture of alginate-HACS or alginate-RS compared with calcium alginate alone, improves the coherency and continuity of capsule structure (alginate and starch showed synergistic effect in gel formation) and as a result, viability of probiotic cells (Sultana et al., 2000). Experiments made by Kebary et al. (1998) showed that encapsulation of bifidobacteria with alginate could significantly increase their viability in frozen ice milk, whereas, using k-carrageenan for this reason was not as successful as the previous one. Encapsulated B. longum in milk medium showed higher viability compared with free cells during storage time (Truelstrup-Hansen et al., 2002). According to Kalil and Mansur investigation (1998), encapsulation of Bifidobacterium spp. with calcium alginate significantly improved their viability in mayonnaise with pH 4.4 (Khalil and Mansour, 1998). Higher survivability of B. infantis in yogurt during the refrigerated storage was reported when the cells were encapsulated by mixture of gelan-xanthan. The average size of the beads was 3 mm after the encapsulation process (Sun and Griffiths, 2000). Encapsulated probiotics with an alginate-starch mixture and a bead size range of 0.5 to 1.0 mm were considerably more viable in yogurt during the storage period (Sultana et al., 2000). Increase in the viability probiotics Lactobacilli in frozen ice milk after encapsulation with alginate (size range from 25 to 62 mm) has been reported (Sheu and Marshall, 1993). The same results were achieved in the case of fermented frozen dairy desserts. Coating of alginate beads with PLL considerably increased probiotics viability against severs process conditions (Shah and Rarula, 2000). Other research indicated that survivability of Bifidobacterium spp. and L. acidophilus noticeably increased in fermented frozen dairy desserts when alginate with SPS and tween 80 additives were used for encapsulation (Sultana et al., 2000). The improvement of B. bifidum viability in yogurt after encapsulation with calcium alginate was in a way similar that throughout the 3 weeks refrigerated storage at 4ºC, its viable counts did not fall below 107 cfu/ml. Also, no undesirable sensory properties were observed in the final product. The above mentioned results were also obtained after frozen storage of the product (Sultana et al., 2000). Good efficiency for encapsulation process after the encapsulation of B. infantis with xanthan-gelan mixture in yogurt with pH 4 during the 6 wks of storage period at 4ºC has been reported. Mentioned cells showed higher survivability during the pasteurization process (Sun and Griffiths, 2000). B. longum ATCC 15696 cells added to cheddar cheese at the stage of curd milling, were totally viable after 24 weeks of ripening period. The cells were completely metabolically inactive during this time (Dinakar and Mistry, 1994; Sun and Griffiths, 2000). It has been verified that the viability of Lactobacilli encapsulated with calcium alginate could be increased up to 40% in frozen products such as ice cream and frozen ice milk (Sheu and Marshall, 1993). Because microencapsulation of probiotic starter cultures considerably decreases their metabolic activity, viability of the cells would increase due to the slower acid production rate. For instance, it has been reported that incubation time for yogurt made with L. casei and L. acidophilus up to the end point of pH 5, increased from 6 h in the case of free cells to 30 h in the case of encapsulated cells (Sultana et al., 2000). This fact was also evident during the refrigerated storage period. Decrease in acidification rate of starter bacteria and as a result pH drop during this period leads to the considerable extension of product shelf life due to increasing probiotics viability within the storage time (Mortazavian et al., 2006b).

4.4.2. Achieving new methods in food manufacture: Nowadays, by applying encapsulated starter culture bacteria, new innovations have been achieved in the manufacture of dairy probiotic products such as yogurt. Specific encapsulation of probiotic (even traditional yogurt bacteria) cells can cause desirable rate of cellular metabolic activity. For example, new continuous method of yogurt production with encapsulated traditional yogurt bacteria (Streptococcus salivarius ssp. thermophilus and Streptococcus delbrueckii ssp. bulgaricus) has been proposed which has the following advantages compared with traditional metho

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