1. Presenter : Kim Sun Yeong Food Microbiology Laboratory Survival of Double-Microencapsulated Bifidobacteria breve in Milk in Simulated Gastric and Small Intestinal Conditions

3. Contents Introduction Objective Materials and methods Results Discussion

4. Introduction Bifidobacterium - Anaerobic, rod shaped Gram-positive bacteria - inhabitants of the gastrointestinal tract of mammals - One of the major genera of bacteria that make up the colon flora in mammals - Some are used as probiotic

5. Introduction Beneficial effect - Improvement of intestinal flora - Activation of the immune system - Increased protein digestion - Improvement of diarrhea or constipation Therefore, used in foods as a probiotic adjunct

6. Introduction Population level of LAB and Bifidobacteria in initially contained commercial dairy products - LAB(lactic acid bacteria) : 10^7~10^8 CFU/ml or more - Bifidobacteria : 10^5~10^6 CFU/ml or less Bifidobacteria are very sensitive and intolerant of acid, oxygen, temperature, bile salt and the presence of other cultures

7. Introduction Microencapsulation techniques ff - Utilized to protect microorganisms - To protect them against adverse effects of gastric and small intestinal conditions

8. Introduction Double-microencapsulation - More protective effect than single-microencapsulation - The outer layer can be resistant to gastric acid and bile salts - The inner layer can be broken down by lipase in the small intestine to reach the colon

9. Introduction Inulin - P lant-derived carbohydrate with benefits of soluble dietary fiber - Not digested or absorbed in the small intestine - Used to promote the proliferation of benefical bacteria in the gastrointestinal system Chicory - D erived inulin - Prebiotic effects from the proximal to distal colon

10. Introduction Prebiotic - Indigestible food ingredients - Beneficially affect the host by selectively stimuating growth and activity of one or a number of health-promoting colon bacteria - Ingredients to help Intestinal microbial growth or activity

11. Introduction Supplement of inulin - Increases colonic Ca, Mg, Fe absorption - Enhances bone calcium stores - Influences blood glucose levels - Reduces the levels of cholesterol and serum lipids

12. Objective Manufacture of MBM(Microencapsulated Bifidobacteria supplemented Milk) - To determine the survival of the double- microencapsulated Bifidobacteria in milk under human gastrointestinal conditions. * Double-microencapsulated Bifidobacterium breve CBG-C2 as probiotic * chicory, Inulin as prebiotic

13. Objective The Outer layer : sweet potato starch and gelatin - for protection in the stomach and small intestine, respectively. The inner layer : hard oil (corn oil) - was decomposed by lipase and fully digested at the end of small intestine

14. Materials Food Emulsifiers : polyglycerol polyricinoleate(HLB 0.6), glycerol monooleate(HLB 2.8), propyleneglycol monoester(HLB 3.4), glycerol monolaurate(HLB 4.0), sorbitan monooleate(HLB 4.3), sorbitan, monopalmitate(HLB 6.7) etc., hydrogenated corn oil, sweet potato starch, gelatin, Bifidobacteria breve, distilled water, inulin, chicory

15. Methods Output of Emulsion Stability Index - Emulsion Stability Index (ESI, %) = [1- the volume of isolated (LAB layer / Corn layer) / the total volume of (LAB layer / Corn layer) in the Emulsion] X 100

16. Methods Core material in the W/O type emulsions - homogenizing Bifidobacterium + distilled water + primary emulsifier at 8000rpm - adding the hydrogenated corn oils (dyed to oil red O, the melting point was 30 ℃ ), homogenization - manufacture Core material in the W/O type emulsions

17. Methods Secondary coating materials - Mixture of sweet potato starch: gelatin = 1 : 4 (w / w) - Manufacture Secondary coating materials after gelatinizating mixture at 65 ℃ A composite material usage was proved more effecient than a single material usage

18. Methods Manufacture of W/O/W type emulsion - Maintained distilled water in a water bath at 50 ℃ - Strongly stirring mixture of the secondary core material (W/O type emulsion) and the emulsifier at 8000rpm for 50sec using Ultra-turrax - Stirred for 60sec after the addition of a secondary coating materials - Formed emulsion for the manufacture of W/O/W type double microencapsule

19. Methods Dispersion fluid Manufacture - Added emulsifier(HLB value = 9.6) at a concentration of 0.025% in distilled water - Strorage at 10 ℃ after Strongly stirring and dissolved * HLB(hydrophile-lipophile balance) : Ratio of the Hydrophilic and Lipophilic, Indicator of hydrophilic and lipophilic balance of the surfactant

20. Methods Double microencapsulation - Sprayed W/O/W type emulsion into the dispersion fluid - Manufacture Double Microcapsule of diameter of around 10 ㎛

21. Methods Microencapsulation yield measurements - Added Non-polar organic solvent n-hexane 50ml - Mix suspensions containing microcapsules and n-hexane layer at 1:1 (v/v) - After stirring at low speed of 50rpm, precipitating for 30 min - Measure the certain amount of n-hexane layer at absorbance 510nm * Yield of microencapsulation (YM, %) = {(B-A)B} X 100 ( A: absorbance of n-hexane layer, B: absorbance of sample extracted with n-hexane )

22. Methods Observation of the form of capsules and cells - SEM(Scanning electron microscopy) : Dry capsule for 24hr at room temperature and fixed, for 20 min in Critical Point Dryer(CPD) and gold-coated capsule for 7min at 80 ℃ : Observation the external membrane of Capsule - CLSM(Confocal laser scanning microscopy) : Staining the cells and capsule and Observating optically section reconfigured continuous image : Can be observed without Cell damage in three-dimensional space

23. Methods Survival test – simulated gastric conditions - 1ml sample was add to 9ml simulated gastric fluid - Vortexed for 20 sec, samples taken at 0, 1, 2, 3hr

24. Methods Survival test – simulated small intestinal conditions - 1ml each of remainders of the mixtures after incubating 0, 1, 2, 3hr were added to 9ml simulated small intestinal fluids, respectively - Vortexed for 20 sec, sample taken at 0, 2, 4, 6 hr

25. Methods Enumeration – Bifidobacteria and LAB - BS( Bifidobacteria selective) medium - BCP(Brom Cresol Purple) medium : Samples taken at each time point → diluted with sterilized saline solution → inoculated on BS(anaerobic package) and BCP agar medium → counted after incubation at 37 ℃ for 72 hr

26. Result Survival of the bacteria in MBM and CP(comercial product) in simulated gastric conditions

27. Survival of the bacteria in MBM and CP in simulated small intestinal conditions

30. Discussion Microencapsulated Bifidobacteria in MBM apperar to have better resistance against adverse conditions of the stomach and small intestine. Good efficacy as a probiotic in foods, with high survival rate. First product developed that gives both milk and yogurt functions. The inner layer of the double-microencapsulation was designed to be broken in the end of small intestine so that it can be colonized in the colon. When the Bifidobacteria reach the colon, the inulin(chicory) in milk was able to help the colonization and growth of Bifidobacteria as a prebiotic.