optimized Synthesis of a synbiotic from Enzyme-Treated Purple Rice Bran Fiber and Lactobacillus Plantarum

1. optimized Synthesis of a synbiotic from Enzyme-Treated Purple Rice Bran Fiber and Lactobacillus Plantarum Daniel Bergeron Kristi Harrison Christina Juneau Jason Newton Laura DeLatin Advisor: Dr. SubramaniamSathivel

2. To produce a freeze-dried synbiotic supplement with optimal cell viability, consisting of Lactobacillus plantarumand enzyme-treated purple rice bran fiber, and evaluate its maintained viability during variant storage conditions. Prebiotics Problem Statement Probiotics

3. Overview • Background • Design Constraints • Measureable Objectives • Product Design Specification • Concept Evaluation and Selection • Testing Plans • Design Tools • Budget • Summary

4. Prebiotics • Non-digestible oligosaccharides • In Intestines: • Stimulates the growth and activity of healthy bacterial flora • Carbon and energy source for flora • In vivo: protective fiber matrix increases stability and viability of probiotics when freeze-dried

5. Probiotics • Live microorganisms found in • Intestines • Dairy products • Provides beneficial digestive health • Prevents the growth of harmful bacteria • Maintains healthy balance for homeostasis

6. Design Constraints • Time • Money • Access to equipment • Minimum effective dose of Synbiotic (CFU/g) • IACUC (Institutional Animal Care and Use Committee) • Process of Approval in progress: #13-100

7. Measureable Objectives Synbiotic Synthesis • 74 % Dietary fiber in Enzyme-treated Rice Fiber • Recover 8.78 +/- 0.10 log CFUL. plantarum per mL of synbiotic after growth • Maintained viability of 106 CFU/g after 30 days of storage • Model doubling time of probiotic during growth, and reduction rate during storage

8. Product Design Specification (PDS)

9. PDS continued

10. PDS Continued

11. Concept Evaluation & selectionPrebiotic Source: Enzyme-Treated Purple Rice Fiber • Blanca Isabel Purple Rice • Bran is by-product of Rice • Locally grown and milled • High Antioxidant content • Cryoprotectant • Enzyme-Treatment • Purifies Bran from 24% to 74% Dietary Fiber

12. Enzyme-treatment Process

13. Enzyme Addition • α-Amylase • Alcalase • Hemicellulase

14. Enzyme Addition

15. Enzyme Addition & drying

16. Concept evaluation & SelectionProbiotic Source: l. plantarum • Nutrition Requirements and Growth Rate • Tolerate pH of stomach

17. Growth of Probiotic on Fiber • Better survives harsh condition of stomach • Scanning Electron Microscopy (SEM) to confirm growth

18. Inoculation of L. Plantarum

19. Adherence and Growth Rate

20. Viability

21. Measuring CFU/ml • Serial Dilutions • Triplicate Plating • Accept 30-300 cell plate • Before/During/After Multiple Stages

22. Method of PreservationFreeze drying • Decreases surrounding pressure until frozen water sublimates • Fiber serves as cryoprotectant for probiotic • Less cell damage and cell reduction • Improves stability and viability during storage • Prolongs shelf-life • Air blast freezer can be used for a variety of irregular shapes • Particularly small sizes

23. Freeze-Drying ProcessAir-Blast Freezer

24. Testing Plans • Modeling • Growth of Bacteria on Fiber • Viability during Storage over One Month • Storage • To determine the optimal temperature necessary to keep our supplement viable • Animal Study • To test effectiveness of synbiotic in intestinal environment for future marketability • Ulcerative Colitis Model • Form of IBD • Past studies of treating Colitis with probiotics

25. Modeling • Bacteria grown on Fiber • Bacteria added to Fiber • Bacteria separate from Fiber Doubling Time • Exponential Increase • X = cell concentration • X0 = initial cell concentration • μ= specific growth rate, units of time-1 • t = time • Concentration at t = td is 2X0 • td = doubling time

26. Storage • Storage Conditions • At Room Temperature (25°C) vs. Refrigerator (4°C) • Storage duration for one month • Bi weekly viability count (CFU/mL)

27. Animal Study • Foundational Study • Feed supplemented diets • Induce Colitis with DSS • Post-mortem evaluations • Test groups based on respective supplemental diets • Control (no supplement) • Prebiotic supplement only (ERF) • Probiotic supplement only (L. plantarum) • Synbiotic supplement (ERF + L. plantarum) • Process of Approval in progress

28. Animal Study • Terminal Measurements after Dissection • Weight and length of colon to analyze the effects of inflammation • Cecal sample • Microbiota characterization determined by RT-qPCR • Short Chain Fatty Acid content • Elisa kit for Cytokine analysis [IL-6 and TNF-α] • H&E stain of colonic mucosal tissue to visually assess mucosal damage • Measurable Objectives (Relative to the Control) • Longer colon length • Decreased cytokine levels • Lower mucosal damage score • Abundance of microbial strains

29. Design toolsGantt Chart

30. BUDGET

31. Summary • Prebiotics + Probiotics = Synbiotic • Probiotic grown on prebiotic fiber • Freeze-dried • Altering storage conditions to increase viability after storage • Planning and Testing • Decision Matrices, Flowcharts, and PDS • Tests and Modeling • Animal Study

32. Acknowledgements • Dr. SubramaniamSathivel • ArraneeChotiko • Dr. Diana Coulon • Dr. Daniel Hayes

33. Questions?

34. Appendix

35. Design ToolsPhysical Decomposition

36. Picture references • Slide 2: • http://3.bp.blogspot.com/-t7sZZGyVIC4/T-KwDSNvrHI/AAAAAAAAAvI/GzSOXz-Hw-s/s1600/209569,xcitefun-black-rice-2.jpg • http://www.probiotics-summit.eu/images/plantarum2.jpg • Slide 4: • http://www.letstalkmagazine.com/home/wp-content/uploads/2012/06/Whole-Grain-Group-Pic.jpg • Slide 5: • http://www.nutritionaloutlook.com/sites/nutritionaloutlook.com/files/imagecache/Image_Default/images/probiotics.jpg • http://static.caloriecount.about.com/images/medium/roundys-probiotic-yogurt-nonfat-4511.jpg • Slide 6: • Slide 21: • http://classes.midlandstech.com/carterp/Courses/bio225/chap06/06-15_PlateCounts_1.jpg • Slide 27: • http://www.sageresearchmodels.com/files/cox2_rat.jpg

37. Inflammatory Bowel DisorderUlcerative Colitis • Inflammation of the colon • Overly aggressive immune response of nonpathogenic bacteria • Causes dysentery, abnormal bowel movements, pain, cramping • No current cure

38. timeline Laboratory acclimation feeding • Week 1 Specific supplemental diet introduced • Week 2 Induce Colitis with Dextran Sodium Sulfate • Week 3 Euthanasia, dissection, and analysis • Week 4

39. MATERIAL Experimental DesignSynbiotic Synthesis • PROCESS Key

40. Design toolsMorphological chart