The Establishment Of Microencapsulation Technology Based On Sodium Alginate And Its Application In Lactobacillus Sp.in Dry State

With the development of economy and the improving of people's living standard, great interest has been focused on developing functional foods that exert a beneficial effect on the host health. Probiotic agents have gained great attention in the food technology field. Probiotics are defined as"live microorganisms which when administered in adequate amounts confer a health benefit on the host". Lactic acid bacteria, which are among the most important probiotic microorganisms, have many beneficial effects on the human including maintenance of a healthy gut microflora, reducing risk factors for colon cancer, improving the organism immunity, and lowering serum cholesterol. However, studies indicate that the bacteria may not survive in sufficient numbers when incorporated into dairy products as well as during their passage through the gastro-intestinal tract. The low pH of the stomach and bile salts commonly encountered in the GIT are detrimental to their survival. Microencapsulation techniques have been widely utilized to protect probiotics against adverse environmental conditions (drying, gastric acid, bile salts, pancreatic enzyme, etc.). Among the available techniques for immobilizing living cells, the extrusion method has been frequently used, because the preparation process of microcapsules is under mild conditions compared to freeze drying and spray drying.In this paper, four kinds of Lactobacillus sp. loaded microcapsules are prepared with the extrusion method, using alginate, gelatin, chitosan and carboxymethyl chitosan as wall material, fibre and microporous starch as carrier. The characteristics of microcapsules are detected. Factors affecting the survival of dried Lactobacillus sp. are also studied.Lactobacillus sp. loaded microcapsules are prepared by extrusion technology with fibre and alginate. The survival condition of Lactobacillus sp. and the release profile of encapsulated cells in gastro-intestinal tract exhibit slight difference when the alginate concentration altered from 2% to 5% (w/v) and 5% is used in the succeeding experiments. Drying at 4℃is beneficial to the survival of Lactobacillus sp. compared with 25℃. The immobilization technique and the addition of fibre are much beneficial to the survival of encapsulated Lactobacillus sp.. Trehalose, as a reducing disaccharide, can slightly reduce the acid production of Lactobacillus sp. and has a negative effect on its proliferation. Trehalose can improve the survival ability of Lactobacillus sp. after drying and maintain the live cell numbers over 107 cfu/g after 8 weeks storage at 4℃. The survival rate of microencapsulated Lactobacillus sp. reaches 89% after they are exposed to simulated gastric fluids (pH 1.2) for 2 h. The numbers of viable Lactobacillus sp. decrease with the relative humidity increased from 33% to 97%.Lactobacillus sp. loaded microcapsules based on alginate and gelatin have been prepared by extrusion technology. The size of microcapsules is 1.1±0.2 mm and the product can increase the live cell number of Lactobacillus sp. to 107 cfu/g in the dry state after storage at 4℃for 1 week. The surface of microcapsules treated with sodium citrate presents as the form of sheets, while the one without treatment exhibits some rumple. The live cell number of Lactobacillus sp. decreases with the storage time prolonged and microcapsules treated sodium citrate is much beneficial to the survival of Lactobacillus sp.. The moisture absorption rate of microcapsules increases significantly with the relative humidity altered from 33% to 97%. The pH values and ion intensity of solution affect the swelling behavior of alginate/gelatin microcapsules in the same way. The microcapsules swell rapidly and reach its equilibrium at pH 2.4. When the pH is higher than 5.2, the microcapsules become unstable and disintegrate much rapidly. The swelling ratio of microcapsules decreases with the ion intensity increased from 0.01 to 1 mol/l. Cells of Lactobacillus sp. can be continuously released from the microcapsules during gastro-intestinal tract, and the release amounts and speeds of Lactobacillus sp. in simulated intestinal fluid are much higher and faster than that in simulated gastric fluid.Chitosans with different molecular weight (120 kDa, 338 kDa, 540 kDa and 1360 kDa) and degree of deacetylation (91.26%,83.34% and 65.41%) are prepared using acid degradation and acetylation methods. Lactobacillus sp. loaded microcapsules based on alginate, chitosan and microporous starch have been prepared by three different methods. All the microcapsules appear as a spherical structure about 2.1±0.2 mm in diameter. The water content of microcapsules is lower than 10%. The moisture absorption rate of microcapsules increases significantly with the relative humidity altered from 33% to 97%. The swelling profile of microcapsules in simulated gastro-intestinal fluid is not affected by preparation methods. The external crosslinking method is chosen for the following experiment because it can protect Lactobacillus sp. against the gastric acidity efficiently and provide the best protection of Lactobacillus sp. against desiccation. The survival condition of microencapsulated Lactobacillus sp. improves with the level of microporous starch increased from 0.2 to 0.4 g/ml. In the process of storage the survival of bacteria has no obvious difference when the microporous starch is added between 0.3 and 0.4 g/ml, the concentration of 0.3 g/ml is selected to use in the succeeding experiments. The chitosan molecular weight of 120 kDa, deacetylation degree of 91.26% and concentration of 1% (w/v) are the optimal condition to prepare Lactobacillus sp. microcapsules.Carboxymethyl chitosan with different molecular weight (172.7 kDa,490.2 kDa,720.9 kDa and 1771.5 kDa) are prepared with chitosan and chloroacetic acid in alkaline condition and all the samples are light yellow powder and water soluble. Microcapsules containing Lactobacillus sp. are prepared by extrusion technology with microporous starch, alginate, chitosan and carboxymethyl chitosan. All the microcapsules appear as a spherical structure about 2.2±0.1 mm in diameter. The water content of microcapsules is lower than 10%. With the relative humidity altered from 33% to 97%, the moisture absorption rate of microcapsules increases significantly while the live cell number decreases. The membrane of microcapsules shows permselectivity. The effect of carboxymethyl chitosan molecular weight on the permeability of microcapsules membrane has no regularity. The permeability of microcapsule membrane decreases with the increase of carboxymethyl chitosan concentration. The molecular weight and concentration of carboxymethyl chitosan has little effect on the swelling behavior of microcapsules in different pH and ion intensity condition. The carboxymethyl chitosan molecular weight of 721 kDa and concentration of 1% (w/v) are the optimal condition to prepare microcapsules. Lactobacillus sp. loaded in the microcapsules has the same growth profile (S-type) with the free cells. Results indicate that Lactobacillus sp. immobilized in the microcapsules survive much better under low pH and bile conditions compared with the free cells.Factors that influencing the survival of dried Lactobacillus sp. are studied. With the concentration of 0.5, 1.5, 2.0% (w/v), inulin is beneficial to the growth of Lactobacillus sp. while fructo oligosaccharides has no positive effect. Inulin with concentration of 0.5% is found to be the most effective of the prebiotic in retaining the viability of immobilized Lactobacillus sp. under drying conditions. Chitosan oligosaccharide can be developed and utilized as a micro-ecological regulator. As a component of growth medium, chitosan oligosaccharide can improve the live cell number to 5.6×10~7 cfu/g after storage at 4℃for 4 weeks. As the protective additive, chitosan oligosaccharide (0.4%, w/v) can improve the live cell number to 3.6×10~8 cfu/g after storage at 4℃for 6 weeks. The presence of buffer salts (disodium hydrogen phosphate, sodium acetate and sodium citrate) in the growth medium improves the viability of Lactobacillus sp. after dried at 25℃and its resistance to osmolarity. Trehalose and skimmed milk are found to be the most suitable rehydration medium in retaining the viability of microencapsulated Lactobacillus sp. in dry state. With the rehydration time increasing, the concentration of the rehydration medium (trehalose and skimmed milk) has no obvious effect on the recovery of dried Lactobacillus sp..In summary, immobilized Lactobacillus sp. with alginate, gelatin, chitosan and carboxymethyl chitosan proves to be very efficient in improving the viability of Lactobacillus sp. in dry state. The microencapsulation method reported in this paper also improves the resistance of Lactobacillus sp. to gastric acid and bile salts and it provides a basis for the broad application of probiotics.