1 / 40

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

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. Subramaniam Sathivel.

yagil
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related