Micro-encapsulation of Probiotics

The World Health Organization (WHO) defines probiotics as“ live micro-organisms, which, when administered in adequate amounts confer a health benefit on the host.
Probiotic based products are associated with many health benefits. However, the main problem is the low survival of these microorganisms in food products and in gastrointestinal tract.
To produce these beneficial effects in health, probiotics have to be able to survive and multiply in the host. Probiotics should be metabolically stable and active in the product, survive passage through the stomach and reach the intestine in large amounts. Providing probiotics with a physical barrier is an efficient approach to protect microorganisms and to deliver them into the gut.
Microencapsulation of probiotic bacteria can be used to enhance the viability during processing, and also for the targeted delivery in gastrointestinal tract.

1. Microencapsulation of Probiotics Presented by: Shalini Department of Food science, Nutrition & Technology CSK HPKV, Palampur

2. Contents Introduction Microencapsulation A dvantages of Microencapsulation Materials used for Microencapsulation Encapsulation techniques Encapsulated probiotics in food products D evelopments in the Food industr y Pharmaceutical Applications of Encapsulated probiotics Conclusion

3. Introduction The World Health Organization (WHO) defines probiotics as“ live micro-organisms, which, when administered in adequate amounts confer a health benefit on the host. The concept of probiotics was first introduced in the 20 th century by Noble prize winner and father of modern immunology, Elie Metchnikoff (1845-1916), when he noticed that the long, healthy lives of Bulgarian peasants resulted f rom their consumption of fermented milk products . The term “ Probiotics ” was first introduced in 1965 by Lilly and Stillwell , when it was described as growth promoting factors produced by microorganisms. The most commonly used probiotic microorganisms are Lactobacillus and Bifidobacteria strains.

4. Probiotic products are important functional foods as they represent about 65% of the world functional food market. Probiotic bacteria have been incorporated into a wide range of foods, including dairy products (such as yogurt, cheese, ice cream, dairy desserts) but also in non-dairy dairy products (such as chocolate, cereals, juices) (Anal and Singh. 2007). The beneficial effects of probiotics on the human gut flora include antagonistic effects and immune effects. The use of probiotic bacterial cultures stimulates the growth of preferred microorganisms, crowds out potentially harmful bacteria and reinforces the body’s natural defense mechanisms. Probiotics have been reported to play a therapeutic role by lowering cholesterol, improving lactose tolerance and preventing some cancers (Kailasapathy and Chin. 2000).

5. Probiotic based products are associated with many health benefits. However, the main problem is the low survival of these microorganisms in food products and in gastrointestinal tract. To produce these beneficial effects in health, probiotics have to be able to survive and multiply in the host. P robiotics should be metabolically stable and active in the product, survive passage through the stomach and reach the intestine in large amounts. Providing probiotics with a physical barrier is an efficient approach to protect microorganisms and to deliver them into the gut. Microencapsulation of probiotic bacteria can be used to enhance the viability during processing, and also for the targeted delivery in gastrointestinal tract.

6. Microencapsulation can be defined as the process in which cells are retained within an encapsulating membrane to reduce cell injury or cell lost, in a way that result in appropriate microorganism release in the gut (Sultana et al. 2000). 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 & Stevens . 2005 ) . A technology to Protect Probiotic Bacteria A microcapsule consists of a semipermeable, spherical, thin, and strong membrane surrounding a solid/liquid core, with a diameter varying from a few microns to 1 mm. Microencapsulation

7. Principle of Encapsulation: Membrane barrier isolates cells from the host immune system while allowing transport of metabolites and extracellular nutrients. Membrane with size selective pores (30-70 kDa). Source: INOTECH Encapsulation.

8. In designing the encapsulation process, the following questions should be asked: What function must be encapsulated ingredient provide for the final product ? What kind of coating material should be selected ? What processing conditions must the encapsulated ingredient survive before releasing its content ? What optimum concentration of the active material in the microcapsule ? By which mechanism will the ingredient be released from the microcapsule ? Which particle size , density , and stability requirements for the encapsulated ingredients ? What are the cost constraints of the encapsulated ingredient ?

9. A dvantages of Micro-encapsulation The microcapsule is composed of a semipermeable, spherical, thin and strong membranous wall. Therefore the bacterial cells are retained within the microcapsules (Jankowski et al. 1997). More over, compared to an entrapment matrix, there is no solid or gelled core in the microcapsule and its small diameter helps to reduce mass transfer limitations. The nutrients and metabolites can diffuse through the semipermeable membrane easily. The membrane serves as a barrier to cell release and minimises contamination. 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 and Vasishtha . 1995).

10. Materials used for Microencapsulation Various types of encapsulating materials are used for the process. Namely : Alginat e Gellan gum and xanthan gu m k -Carrageena n Cellulose acetate phthalat Chitosa n Starc h Pectin Gelati n Whey proteins Chickpea protein The selection of any material depends on its capsule forming capability, its strength , its enhancing viability of probiotics, its cheapness, its availability , biocompatibility.

11. Encapsulation of probiotics in k-carrageenan Carrageenan is a natural polysaccharide that is extracted from marine macroalgae and is commonly used as a food additive. Gelation of k-carrageenan is generally dependent on a change in temperature. The cell slurry is added to the heat-sterilized carrageenan solution at 40 - 45 ℃ and gelation occurs by cooling to room temperature. The beads are formed after dropping the mixture of polymer and cells into a potassium chloride (KCl) solution.

12. The conventional encapsulation methods, with sodium alginate in calcium chloride (CaCl 2 ) has been used to encapsulate L. acidophilus to protect this organism from the harsh acidic conditions in gastric fluid. Studies have shown that calcium-alginate immobilized cell cultures are better protected, shown by as increase in the survival of bacteria under different conditions, than the non-encapsulated state.

13. Encapsulation of probiotics in alginate systems Alginate is a naturally derived polysaccharide extracted from various species of algae and composed of β -D-mannuronic and α - L-guluronic acids. The composition of the polymer chain varies in amount and in sequential distribution according to the source of the alginate and this influences functional properties of alginate as supporting material. Alginate hydrogels are extensively used in cell encapsulation and calcium alginate is preferred for encapsulating probiotics because of its simplicity, non-toxicity, biocompatibility and low cost . Sodium alginate is also used for encapsulating probiotics

14. The success of the use of alginate in microencapsulation of probiotics is due to the basic protection against acidity it provides to the cells Alginates with a high content of guluronic acid blocks (G blocks) are preferable for capsule formation because of their high mechanical stability, high porosity and tolerance to salts and chelating agents (Nicetic et al. 1999). Chen et al. 2005 used prebiotics (fructooligosaccharides or isomaltooligosaccharides), a growth promoter (peptide) and sodium alginate as coating materials to microencapsulate different probiotics such as L. acidophilus , L. casei , B. bifidum and B. longum . A mixture containing sodium alginate (1% w/v) mixed with peptide (1% w/w) and fructooligosaccharides (3% w/w) as coating materials produced the highest survival in terms of probiotic count.

15. Encapsulation of probiotics in cellulose acetate phthalate (CAP) Because of its ionizable phthalate groups, this cellulose derivative polymer is insoluble in acid media at pH 5 and lower but is soluble at pH higher than 6. In addition, CAP is physiologically inert when administered in vivo , and is, therefore, widely used as an enteric coating material for the release of core substances for intestinal targeted delivery systems. Rao et al. 1989 reported the encapsulation of B. pseudolongum in CAP using an emulsion technique. Microencapsulated bacteria survived in larger numbers (10 9 cfu/mL) in an acidic environment than non-encapsulated organisms, which did not retain any viability when exposed to a simulated gastric environment for 1 h.

16. Encapsulation of probiotics in proteins and polysaccharide mixtures Gelatin is useful as a thermally reversible gelling agent for encapsulation. Because of its amphoteric nature, it is also an excellent candidate for incorporating with anionic-gelforming polysaccharides, such as gellan gum. These hydrocolloids are miscible at pH >6, because they both carry net negative charges and repel one another. However, the net charge of gelatin becomes positive when the pH is adjusted below its isoelectric point and causes a strong interaction with the negatively charged gellan gum (King . 1995).

17. Hyndman et al. 1993 used high concentrations of gelatin (24% w/v) to encapsulate Lactobacillus lactis by cross-linking with toluene-2,4-diisocyanate for biomass production. In a recent study, Guerin et al. 2003 encapsulated Bifidobacterium cells in a mixed gel composed of alginate, pectin and whey proteins.

18. Encapsulation of probiotics in chitosan The biopolymer chitosan, the N -deacetylated product of the polysaccharide chitin, is gaining importance in the food and pharmaceutical field because of its unique polymeric cationic character, good biocompatibility, non-toxicity and biodegradability. Chitosan can be isolated from crustacean shells, insect cuticles and the membranes of fungi. The properties of chitosan vary with its source. In order to achieve sufficient stability, chitosan gel beads and microspheres can be ionically cross-linked with polyphosphates and sodium alginate.

19. Encapsulation of probiotics in starch Starch is a dietary component that has an important role in colonic physiology and functions and a potential protective role against colorectal cancer (Cassidy et al. 1994). Resistant starch is the starch that is not digested by pancreatic amylases in the small intestine and reaches the colon, where it can be fermented by human and animal gut microflora. The fermentation of carbohydrates by anaerobic bacteria produces short chain fatty acids and lowers the pH in the lumen Resistant starch can be used to ensure the viability of probiotic populations from the food to the large intestine.

20. Resistant starch also offers an ideal surface for adherence of the probiotics to the starch granule during processing, storage and transit through the upper gastrointestinal tract, providing robustness and resilience to environmental stresses. Bacterial adhesion to starch may also provide advantages in new probiotic technologies to enhance delivery of viable and metabolically active probiotics to the intestinal tract . Talwalkar and Kailasapathy (2003) produced alginatee starch gel beads by dropping a mixture of alginate starch bacteria into a CaCl 2 coagulation bath. The probiotic bacteria used for this study were L. acidophilus and B. lactis . They found that encapsulation prevented cell death from oxygen toxicity. It is known that alginate gel beads restrict the diffusion of oxygen through the gel, creating anoxic regions in the centre of the beads.

21. Encapsulation T echniques

22. E ncapsulation systems (a) reservoir type, (b) matrix type, and (c) coated matrix type. D ifferent types of encapsulates can be found, the reservoir type and the matrix type. The reservoir type has a shell around the core material and this is why it can also be called a capsule. In the case of matrix type, the active agent is dispersed over the carrier material and can also be found on the surface. A combination of these two types gives a third type of capsule: the matrix where the active agent is recovered by a coating

23. S ize range provided by each technique

24. General plan describing steps to produce microcapsules E ncapsulation technology is usually held in three stages.

25. Spray Drying T he most commonly used microencapsulation method in the food industry, is economical and flexible, and produces a good quality product The process involves : the dispersion of the core material into a polymer solution, forming an emulsion or dispersion, followed by homogenisation of the liquid, then atomisation of the mixture into the drying chamber. This leads to evaporation of the solvent (water) and hence the formation of matrix type micro capsules. A dvantage D isadvantage I t can be operated on a continuous basis the high temperature used in the process may not be suitable for encapsulating probiotic bacterial cultures P roper adjustment and control of the processing conditions such as the inlet and the outlet temperatures can achieve viable encapsulated cultures of desired particle size distribution.

26. At an inlet temperature of 100 ℃ and low outlet temperature of 45 ℃ , Bifidobacterium cells were encapsulated satisfactorily to produce micro spheres with gelatinised modified starch as a coating material (O’Riordan et al. 2001). A previous report indicated that survival of probiotic bacteria during spray drying decreased with increasing inlet temperatures (Mauriello et al. 1999) Inlet temperatures of above 60 ℃ resulted in poor drying and the sticky product often accumulated in the cyclone and sometimes in the receiving flask. Higher inlet temperatures (>120 ℃ resulted in higher outlet temperatures (>60 ℃ ) and significantly reduced the viability of encapsulated bifidobacteria (O’Riordan et al. 2001).

27. S pray-drying P rocedure The solution is pressured and then atomized to form a ‘‘mist’’ into the drying chamber. The hot gas (air or nitrogen) is blown in the drying chamber too. This hot gas allows the evaporation of the solvent. The capsules are then transported to a cyclone separator for recovery.

28. Extrusion technique Extrusion is a physical technique to encapsulate probiotic living cells and uses hydrocolloids (alginate and carrageenan) as encapsulating materials. The Micro-encapsulation of probiotic cells by extrusion involves projecting the solution containing the cells through a nozzle at high pressure. Extrusion of polymer solutions through nozzles to produce capsules is mainly reported on a laboratory scale, where simple devices such as syringes are applied. If the droplet formation occurs in a controlled manner (contrary to spraying) the technique is known as prilling . This is preferably done by the pulsation or vibration of the jet nozzle.

29. P rinciple of the technique S imple needle droplet-generator that usually is air driven (a) and pinning disk device (b). The probiotic cells are added to the hydrocolloid solution and dripped through a syringe needle or a nozzle spray machine in the form of droplets which are allowed to free-fall into a hardening solution such as calcium chloride. Extrusion technologies

30. Emulsion technique T he discontinuous phase ( small volume of the cell polymer suspension) is added to a large volume of oil (continuous phase). The mixture is homogenized to form water-in-oil emulsion. Once the water-in-oil emulsion is formed, the water soluble polymer is insolubilized (cross-linked) to form the particles within the oil phase . The beads are harvested later by filtration.

31. The size of the beads is controlled by the speed of agitation, and can vary between 25 μm and 2 mm. For food applications, vegetable oils are used as the continuous phase. Some studies have used white light paraffin oil and mineral oil. Emulsifiers are also added to form a better emulsion, because the emulsifiers lower the surface tension, resulting in smaller particles (Krasaekoopt et al. 2003).

32. S pray coating technology A liquid coating material is sprayed over the core material and solidifies to form a layer at the surface. The liquid coating material can be injected from many angles over the core material: fluid-bed top spray coating (a), fluid-bed bottom spray coating with the Wurster device (b), and fluid bed tangential spray coating (c)

33. Encapsulated P robiotics in F ood P roducts

34. Cheese Many studies have reported the use of encapsulated probiotic cells and particularly in Cheddar cheese.

35. Yogurt The incorporation of probiotic living cells in yogurt enhances its therapeutic value. However, there is poor level of probiotic viability in yogurt because of the low pH (from 4.2 to 4.6). Studies have shown that the use of encapsulated probiotic bacteria was better for their survival. T he incorporation of probiotic cells into yogurts could be carried out without making many modifications from the traditional process (Kailasapathy . 2009).

36. Kailasapathy 2005 studied the survival and effect of free and calcium-induced alginate–starch encapsulated probiotic bacteria ( Lactobacillus acidophilus and Bifidobacterium lactis ) pH, exopolysaccharide productionand their influence on the sensory properties of yoghurt. The results showed that Addition of probiotic bacteria (free or encapsulated) reduced acid development in yogurt during storage. There was an increased survival of 2 and 1 log cell numbers of L. acidophilus and B. lactis , respectively due to protection of cells by microencapsulation. This study has shown that incorporation of free and encapsulated probiotic bacteria do not substantially alter the overall sensory characteristics of yogurts and microencapsulation helps to enhance the survival of probiotic bacteria in yogurts during storage.

37. Frozen dairy dessert s It is not easy to incorporate probiotic microorganisms into frozen desserts because of high acidity in the product, high osmotic pressure, freeze injury and exposure to the incorporated air during freezing. The introduction of probiotic bacteria in an encapsulated form into frozen desserts may overcome these difficulties and could produce useful markets and health benefits (Chen and Chen . 2007). Godward and Kailasapathy (2003) studied the incorporation of probiotic cells in ice cream in different states.The results have shown that free cells survive better than encapsulated cells. Freshly encapsulated probiotic cells had greater survival than those which were freeze-dried after encapsulation and co-encapsulation of L. acidophilus and B. bifidum enhances the survival of both strains. Finally, addition of probiotics does not affect air incorporation into ice cream.

38. Other food products Most of the products containing probiotic cells are dairy products and it is necessary to develop other food carrier for probiotics owing to lactose intolerance in certain populations . Efforts have been made to identify new food carriers. For example, good quality mayonnaise was obtained when incorporating encapsulated bifidobacteria. Calcium alginate provides protection for bifidobacteria against the bactericidal effects of vinegar.

39. D evelopments in the Food industr y During the past few years, food products containing encapsulated probiotic cells have been introduced on the market . In 2007, Barry Callebaut developed a process to produce chocolate containing encapsulated probiotic cells with the Probiocap technology in partnership with Lal’food. According to Barry Callebaut, the addition of encapsulated probiotic cells has no influence on chocolate taste, texture and mouth feel. A consumption of 13.5 g per day of probiotic chocolate seems to be sufficient to ensure the balance of the intestinal microflora. In Korea, yogurts containing encapsulated LAB are available on the market under the brand name Doctor-Capsule

40. Pharmaceutical Applications of Encapsulated probiotics Probiotic supplements are available in different forms . T he two most popular forms are: C apsules and F reeze dried powders. Probiotic strains of L. acidophilus 50 ME are sold as micro-encapsulated by Institut Rosell/ Lallemand The Americas, Montreal, Canada . Probiocap (micro encapsulated L. acidophilus 50 ME in an hydrophobic matrix) marketed by this company claims to have increased tolerance to gastric juices, improved survival during tableting, enhanced temperature resistance during food processing and extended shelf life at room temperatures.

41. Conclusion Microencapsulation has been proven to be one of the most efficient methods for maintaining viability and stability of probiotics, as it protects probiotics during food processing and storage, as well as in gastric conditions. Microencapsulation can achieve a wide variety of functionalities according to the development of the technology and nowadays, encapsulated probiotic cells can be incorporated in many types of food products.

42. P robiotics can be found not only in dairy products, but also in chocolate or cereals too. In the future multiple-devivery may be developed, such as co-encapsulating prebiotics and probiotics as well as nutraceuticals, thus a new area of more complex nutritional matrices will need to be investigated. More in vivo studies should be conducted using human subjects to confirm the efficacy of micro or nano encapsulation in delivering probiotic bacteria and their controlled release in the gastro-intestinal system.