microencapsulation of probiotic bacteria of

Agri. Review, 35 (4): 287 - 294, 2014 doi:10.5958/0976-0741.2014.00916.7

AGRICULTURAL RESEARCH COMMUNICATION CENTRE

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MICROENCAPSULATION OF PROBIOTIC BACTERIA OF AVAILABLE TECHNIQUES, FOCUSING ON BIOMATERIALS - A REVIEW Santosh Chopde* , Nilkanth Pawar, Vijay Kele and Sudhakar Changade College of Dairy Technology, Udgir, Latur-413 517, India

Received: 16-05-2014

Accepted: 30-08-2014

ABSTRACT In the recent past, there has been a rapid increase in the popularity of probiotic health-based products. Benefits of probiotics to human and animal health have been proven in many of scientific research. Survival and stability of probiotic organisms in functional foods have been a major concern, because a high number of organisms are needed to confer health benefitsto consumers. Unfortunately, many processing (heating, freezing) and storage conditions (redox level, pH) used in dairy technology are detrimental to the viability of the probiotic bacteria. Providing probiotic living cells with a physical barrier against adverse environmental conditions is therefore an approach currently receiving considerable interest. Microencapsulation of the probiotic cells is one of the highly efficient methods, which is now under the special consideration and is being developed by various researchers. This increase has been the result of several factors, but the primary factor has been the increased awareness on the part of the food industry of the real advantages offered by encapsulated ingredients. This review focuses on probiotics microencapsulation, its methods and biomaterials utilized for encapsulation.

Key words: Biomaterial, Microencapsulation, Probiotics The term probiotic is derived from two Greek words which literally means “for life”. Probiotics are microorganisms which settle in the intestine 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 (Gismondo et al. 1999). The most commonly used definition of probiotics is “probiotics are live microbial feed supplements that beneficially affect the host by improving its intestinal microbial balance” (Fuller 1989). Food and Agriculture Organization (FAO) of the United Nations and the World Health Organization (WHO) define probiotics as ‘‘Live microorganisms (bacteria or yeasts), which when ingested or locally applied in sufficient numbers confer one or more specified demonstrated health benefits for the host’’ (FAO/WHO, 2001). The probiotic microorganisms consist mostly of the strains of the genera Lactobaci llus and Bifidobacterium, but strains of Bacillus, Pediococcus *Corresponding author’e-mail: [email protected]

and some yeasts have also been found as suitable candidates. Because of their perceived health benefits, probiotic bacteria have been increasingly included in fermented dairy products, including yoghurts, soft, semi-hard and hard cheeses, ice cream and frozen fermented dairy desserts (Stanton et al., 2005). Food, particularly dairy products are considered as an ideal vehicle for delivering probiotic bacteria to the human gastrointestinal tract (Ross et al., 2002). Various health benefits have been attributed to probiotics such as anti-mutagenic and anticarcinogenic properties, anti-infection properties immune system stimulation, serum cholesterol reduction, alleviation of lactose intolerance and nutritional enhancement (Mombelli et al., 2000). The efficiency of added probiotic bacteria depends on dose level and their viability must be maintained throughout storage, products shelf-life and they must survive the gut environment (Kailasapathy et al., 2000). In order to provide health benefits for probiotic

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bacteria (Shah, 2007) it has been recommended that they must be present at a minimum level of 106 CFU/ g of food product or 107 CFU/g at point of delivery or be eaten in sufficient amounts to yield a daily intake of 108 CFU (Lopez-Rubio et al., 2006).

(Borgogna et al., 2010). Microencapsulation is defined as a technology of packaging solids, liquids or gaseous materials in miniature, sealed capsules that can release their contents at controlled rates under the influences of specific conditions (Anal et Probiotic survival in products is affected by al., 2006). From a microbiological approach, a range of factors including pH, post -acidification microencapsulation can be defined as the process (during storage) in fermented products, hydrogen of entrapment/enclosure of microorganisms cells by peroxide production, oxygen toxicity (aerobic means of coating them with proper hydrocolloid(s) conditions), storage temperatures, stability in dried in order to isolate the cells from the surrounding or frozen form, poor growth in milk, lack of proteases environment; in a way that results in appropriate to break down milk protein to simpler nitrogenous cell release in the intestinal medium ( Picot et al., substances and compatibility with traditional starter 2003a). culture during fermentation (Shah, 2000). This has The technology of microencapsulation of encouraged researchers to find new efficient probiotic bacterial cells has evolved from the methods of viability improvement. Different immobilized cell culture technology. Its importance, approaches that increase the resistance of these as an efficient manner for increasing probiotics sensitive microorganisms against adverse conditions viability, justifies reviewing the achievements of this have been proposed, including appropriate selection technology. The present article reviews principles and of acid- and bile-resistant strains, use of oxygen methods of probiotics microencapsulation including impermeable containers, two-step fermentation, discussions of microcapsule structure and stress adaptation, incorporation of micronutrients components used for microencapsulation. such as peptides and amino acids, and From immobilization to microencapsulation: The microencapsulation (Gismondo et al., 1999). technology of microencapsulation of probiotic Microencapsulation is a powerful technology bacterial cells has evolved from the immobilized cell which has been developed for use in the food industry culture technology used in the biotechnological and allows the protection of bacterial cells (Fig. 1) industry to sophisticated and precise micro capsule formation. While encapsulation is the process of forming a continuous coating around an inner matrix that is wholly contained within the capsule wall as a core of encapsulated material, immobilisation refers ANTIBODIES to the trapping of material within or throughout a matrix. Entrapment of cells in a gel matrix of alginates is the most popular system of immobilisation reported (Champagne et al., 1994). Compared to immobilisation/entrapment techniques, microencapsulation has many advantages. NUTRIENT IMMUNE CELLS - The bacterial cells are retained within the microcapsules. - There is no solid or gelled core in the microcapsule and its small diameter helps to reduce mass transfer limitations. GROWTHFACTORS - The nutrients and metabolites can diffuse through the semi permeable membrane easily. - The membrane serves as a barrier to cell release METABOLITES and minimizes contamination. FIG 1: Principle of encapsulation: Membrane barrier isolates Benefits of microencapsulation: Microencapsulation cells from the host immune system while allowing transport has proven one of the most potent methods for of metabolites and extracellular nutrients. Membrane with maintaining high viability and stability of probiotic size selective pores (30-70kDa) (Adhikari et al., 2002).

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bacteria. The encapsulated core material is released by several mechanisms such as mechanical rupture of the cell wall, dissolution of the wall, melting of the wall and diffusion through the wall (Franjione et al., 1999). It will be used as tools to encapsulate both prebiotic ingredients and probiotic bacteria within the same capsule to enhance growth and multiplication of these bacteria through symbiotic effects when they are released in the gastro-intestinal tract. Micro-encapsulation confers protection to sensitive probiotic lactic acid bacteria from oxygen (Sunohara et al., 1995), freezing (Shah and Rarula, 2000) and acidic conditions during manufacture and storage (Adhikari et al., 2000) and gastrointestinal transit (Lee and Heo, 2000) its efficacy is established by pH, composition and texture of food matrix (Kailasapathy, 2003), strains of culture employed (Lian et al., 2002), initial cell population (Lee et al. 2000), method of encapsulation and wall material used (Muthukumarasamy et al., 2006). In dairy industry, microencapsulation has been applied to improve survival and delivery of bacterial cultures.

acidity it provides to the cells (Gbassi et al., 2009). It is a linear polymer of heterogeneous structure composed of two monosaccharide units: acid a-Lguluronic (G) and acid -D-mannuronic (M) linked by (1–4) glycosidic bonds (Dong et al., 2006). The M/G ratio determines the technological functionality of alginate. The gel strength is particularly important that the proportion of block G is high.

Alginate: Alginate gels are known to be insoluble in acidic media (Harnsilawat et al., 2006). The success of the use of alginate in microencapsulation of probiotics is due to the basic protection against

hydrolysis of collagen of animal origin. Gelatin is a heterogeneous mixture of single or multi-stranded polypeptides, protein gum, whi ch makes a thermoreversible gel and was used for probiotic

Carrageenan: The use of carrageenan in microencapsulation of probiotics is due to its capacity to form gel that can entrap the cells. Carrageenan is a natural polysaccharide that is extracted from marine macro-algae and is commonly used as a food additive. Three types of carrageenan are known: kappa (  ) carrageenan, iota (i) carrageenan and lambda ( ) carrageenan.  – carrageenan (monosulfated) and é carrageenan (bisulfated) have an oxygen bridge between carbons 3 and 6 of the D-galactose. This bridge is responsible for conformational transitions. The  -carrageenan (trisulfated) that does not have this bridge is unable to gel. Carrageenan gelation is induced by Biomaterial for microencapsulation: Biomaterial temperature changes. A rise in temperature (60 to is “any natural material or not, which is in direct 80 °C) is required to dissolve it and gelation occurs contact with a living cells and is intended to act with by cooling to room temperature (Mangione et al., biological systems”. The biomaterials used for 2003). The encapsulation of probiotic cells in  probiotics encapsulation include natural polymers carrageenan beads keeps the bacteria in a viable and synthetic polymers (Gentile et al., 1995). state but the produced gels are brittle and are not Coating material stabilizes core ingredients, they are able to withstand stresses (Chen et al., 2007). The ê inert toward active ingredients, they control release -carrageenan beads for probiotic encapsulation can under specific condition, they are economical, be produced using extrusion as well as emulsion flexible, non hygroscopic, tasteless, stable and soluble techniques. in aqueous media or solvent (Campos et al., 2011). Whey proteins: Whey proteins are usually used Various biopolymers have been utilized for coating because of their amphoteric character. Milk proteins probiotic cells. Issues involved when selecting are natural vehicles for probiotics cells and owing to biomaterials for probiotics encapsulation are: their structural and physico-chemical properties, they physicochemical properties (chemical composition, can be used for delivery system (Livney, 2010). ). morphology, mechanical strength, stability in gastric For example, the proteins have excellent gelation and intesti nal flui ds); toxicology assay; properties and this specificity has been recently manufacturing and sterilization processes. Typical exploited to encapsulate probiotic cells. The results biomaterials used for the purpose of probiotic of these studies are promising and using milk encapsulation include: alginate, carrageenan, proteins is an interesting way because of their gelatin, chitosan and cellulose acetate phthalate biocompatibility (Livney, 2010). (Gbassi et al., 2012). Gelatin: Gelatin is a protein derived by partial

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encapsulation, alone or in combination with other compounds. It is an excellent candidate for cooperation with anionic polysaccharides such as gellan gum due to amphoteric nature. This material is useful to obtain beads using extrusion technologies or form a w/o emulsion by cooling, but to stabilize the gel the beads may need to be crosslinked using glutaraldehyde or salts of Chrome. These hydrocolloids are miscible at a pH higher than 6, because they both carry net negatives charges and repel each other. However, the net charge of gelatin becomes positive when the pH is adjusted below the isoelectric point and this causes the formation of a strong interaction with the negatively charged gellan gum (Anal et al., 2007).

membrane. Each microcapsule consi sts of hydrocolloids coated around the bacterial cell(s). The coating serves as a barrier to cell release and minimizes contamination. The coating may also be designed to open in the specific areas of the body. It can therefore be used that is able to withstand acidic conditions in the stomach acids and allows those active ingredients to pass through the stomach (Anal et al., 2005).

Structure of microcapsule: A microcapsule consists of a semi-permeable, spherical, thin and strong membrane surrounding a solid or liquid core, with a diameter varying from a few microns to 1 mm (Anal et al., 2007). The material inside the microcapsule is referred to as the core, internal phase, or fill, whereas the wall is sometimes called shell, coating, wall material, biomaterial or

and Gharsallaoui et al. 2007). During this process a film is formed at the droplet surface, thereby retarding the larger active molecules while the smaller water molecules are evaporated. Finally, a porous, dry particle is formed. The minimum air inlet temperature reported in the literature for probiotic encapsulation is 100 °C while the maximum is 170 °C. Generally the droplet dries at wet bulb temperature of drying

Common techniques for microencapsulation of probiotics: Probiotic microencapsulation requires techniques that are gentle and non-aggressive towards the cells. Most of the reported literature on probiotic microencapsulation is based on small-scale laboratory procedures. The first encapsulation Chitosan: Chitosan is a positively charged techniques developed to improve the shelf-life of polysaccharide formed by deacetylation of chitin. probiotics were to transform cells cultures into Its solubility is pH-dependent. It is water insoluble at concentrated dry powder. The techniques of spraya pH higher than 5.4. This insolubility presents the drying, freeze-drying or fluidized bed drying have drawback of preventing the complete release of this shown their limitations because the cells biomaterial into the gut which pH is greater than encapsulated by these techniques are completely 5.4 (Huguet et al., 1996). However, studies have released into the product. However, probiotics in reported the effectiveness of chitosan as a coating dried or freeze-dried form exhibit compatibility with agent of alginate gel beads (Krasaekoopt et al., traditional starter culture such as milk or cheese and 2004). The encapsulation of probiotic bacteria with have a longer shelf-life compared to their cell slurry alginate and a chitosan coating provides protection form (Rokka et al., 2010). In general, three in simulated GI conditions and therefore, it is a good precautions need to be considered for developing way of delivery of viable bacterial cells to the colon microcapsules: formation of the wall around the material, ensuring that leakage does not occur and (Chavarri et al., 2010). ensuring that undesired materials are kept out. Cellulose acetate phthalate (CAP): CAP is widely The most common techniques used in used as a coating agent. It is used for controlling microencapsulation of probiotics are spray drying, drug release in the intestine due to its safety nature emulsion and extrusion. and being physically inert (Mortazavian et al., 2008). Spray drying: Because of its low cost, rapidity and The advantage of this component is that it is not available equipment spray drying is a commonly soluble at acidic pH (less than 5) but it is soluble at used method of encapsulation in the food industry pH higher than 6 because of its ionizable phthalate (Picot et al., 2004). Spray-drying of active agent is groups. The encapsulation of probiotic bacteria using commonly achieved by dissolving, emulsifying, or CAP provides good protection for microorganisms dispersing the active in an aqueous solution of in simulated GI conditions (Favaro-Trindade et al., carrier material, followed by atomization and 2002). spraying of the mixture into a hot chamber (Fig. 2

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FIG 2: The encapsulation process of probiotics by spray drying

gas/air. Besides polysaccharides, proteins can also be used as carriers; skim milk has proved to be a better wall material than gelatin, soluble starch and gum arabic, for instance (Hsiao et al., 2004). The spray drying process is controlled by means of the product feed, gas flow and temperature. One disadvantage of spray drying is that the use of high temperature which is not compatible with the survival of bacteria. In order to improve probiotic survival, protectants can be added to the media prior to drying. For example, granular starch improves culture viability during drying and storage, soluble fibre increase probiotic viability during storage and trehalose is a thermo-protectant. The technique is highly reproducible and the most important is that it is suitable for industrial applications. Conventional spray-dried encapsulates release their active agent immediately upon addition to water (which may also depend on the porosity of the particles). Recent introductions of more hydrophobic and/or crosslinked carrier materials may provide a more gradual release upon dilution in water. Examples of these are denatured proteins, cross-linked proteins or crosslinked biopolymers.

FIG 3: The encapsulation process of probiotics by emulsion technique

emulsifiers, might prevent the separation of nonmiscible fluids (Appelqvist et al. 2007). The emulsion technique involves the dispersion of an aqueous phase containing the bacterial cells and polymer suspension into an organic phase, such as oil, resulting in water in oil emulsion. The dispersed aqueous droplets are hardened by cooling or by addition of a gelling agent or a crosslinking agent in the case of polyacrylamide gels. Following gelation, the beads are partitioned into water and washed to remove oil. The emulsion technique results in smaller diameter beads, and is better suited to scale up applications. This type of probiotics has been successfully applied to yoghurt cheddar cheese, ice cream (Adhikari et al., 2002).

Extrusion: It is a simple and cheap method that uses a gentle operation which causes no damage to probiotic cells and gives high probiotic viability. The technique involves preparing a hydrocolloid solution, adding the probiotics ingredient to the solution and dripping the cell suspension through a nozzle spray machine in the form of droplets which are allowed Emulsion: Emulsions are kinetically rather than to fall freely into a hardening solution (Fig. 4). thermodynamically stable two-phase systems and Hardening solution consists of multivalent cations ultimately, both the continuous and dispersed phase (usually calcium in the form of calcium chloride). will separate. It is a chemical technique to After dripping, alginate polymers immediately encapsulate probiotic living cells and use surround the added cells and form three-dimensional hydrocolloids (alginate, carrageenan and pectin) as lattices by cross linkages of calcium ions. It is encapsulating materials (Fig. 3). The principle of this common to apply concentration ranges of 1-2% and emulsion technique is based on the relationship 0.05-1.5 M for alginate and calcium chloride, between the discontinuous and continuous phase. respectively. Most of the generated beads size is in Proper formulation design of both phases and the the range of 2-3 mm in diameter (Krasaekoopt interface, including choice of ingredients like et al., 2003). This parameter is strongly influenced

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technique (Heinzen, 2002). Most of the literature reported on the encapsulation of probiotic bacteria has used the emulsion technique to produce small amount of capsules. CONCLUSION The technology of microencapsulation has developed from a simple immobilization or entrapment to sophisticated and precise micro capsule formation. The use of microencapsulated probiotics for controlled release applications is a promi si ng alternati ve to solving the major FIG 4: Flow diagram of extrusion process. The bacterial problems of these organisms that are faced by culture with alginate is dropped using a nozzle into hardening solution. The produced microcapsules are added food industries. The delivery of viable micro into a second coating solution, if required. The process ends encapsulated probiotic bacteria will become at * if only a single encapsulation is required (Krasaekoopt et important in the near future. In the future multipleal., 2003). delivery technique may be developed, such as coby the factors such as type of alginate, its encapsulating prebiotics and probiotics as well as concentration and as a result, viscosity of alginate nutraceuticals, thus a new area of more complex solution, distance between the syringe and setting nutritional matrices will need to be explored. New batch and particularly diameter of the extruder orifice food regulations may specify labelling including (needle) (Smidsrod et al., 1990). Mass production the strains and the number of viable probiotic of beads can either be achieved by multi-nozzle bacteria at the end of shelf life of a food or systems, rotating disc atomizers or by the jet cutting supplement claimed to be probiotic. REFERENCES

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