2. 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

3. PROBLEM STATEMENT To produce a freeze-dried synbiotic supplement with optimal cell viability, consisting of Lactobacillus plantarum and enzyme-treated purple rice bran fiber, and evaluate its maintained viability during variant storage conditions.

4. OVERVIEW  Background  Design Constraints  Measureable Objectives  Product Design Specification  Concept Evaluation and Selection  Testing Plans  Design Tools  Budget  Summary

5. 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

6. PROBIOTICS  Live microorganisms found in  Intestines  Dairy products  Provides beneficial digestive health  Prevents the growth of harmful bacteria  Maintains healthy balance for homeostasis

7. 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

8. MEASUREABLE OBJECTIVES  74 % Dietary fiber in Enzyme-treated Rice Fiber  Recover 8.78 +/- 0.10 log CFU L. plantarum per mL of synbiotic after growth  Maintained viability of 10 6 CFU/g after 30 days of storage  Model doubling time of probiotic during growth, and reduction rate during storage Synbiotic Synthesis

9. PRODUCT DESIGN SPECIFICATION (PDS)

10. PDS CONTINUED

12. CONCEPT EVALUATION & SELECTION PREBIOTIC 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

13. ENZYME-TREATMENT PROCESS

14. ENZYME ADDITION α-Amylase Alcalase Hemicellulase

15. ENZYME ADDITION

16. ENZYME ADDITION & DRYING

17. CONCEPT EVALUATION & SELECTION PROBIOTIC SOURCE: L. PLANTARUM  Nutrition Requirements and Growth Rate  Tolerate pH of stomach

18. GROWTH OF PROBIOTIC ON FIBER  Better survives harsh condition of stomach  Scanning Electron Microscopy (SEM) to confirm growth

19. INOCULATION OF L. PLANTARUM

20. ADHERENCE AND GROWTH RATE

21. VIABILITY

22. MEASURING CFU/ML  Serial Dilutions  Triplicate Plating  Accept 30-300 cell plate  Before/During/After Multiple Stages

23. METHOD OF PRESERVATION FREEZE 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

24. FREEZE-DRYING PROCESS AIR-BLAST FREEZER

25. 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

26. MODELING  Bacteria grown on Fiber  Bacteria added to Fiber  Bacteria separate from Fiber

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

28. 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

29. 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

30. DESIGN TOOLS GANTT CHART

31. BUDGET Synbiotic Synthesis Budget Item:Quantity:Unit Price:Total Price: Purple rice bran50 lbs$120.00$120.00 - Provided Hemicellulase100 g$41.00$41.00 - Provided Alcolase50 ml$144.00 Heat-Stable α -Amylase 10 ml$47.00$47.00 - Provided L. plantarum1 ml$200.00$200.00 - Provided Storage bags200 pk$10.00 Total: $154.00 Rodent Study Budget Item:Quantity:Unit Price:Total Price: Rodent RQ 18-6 Lab diet 50 lb$0.94 / lb$46.95 Sprague Dawley Rats38$15.00 Each$570.00 Rodent Housing21 days$0.95 / day / rat$758.10 Dextran Sodium Sulfate: 50 g$2.96/gram$148.00 Microbial Classification (PCR Analysis) 50 samples$24.00 / sample$1200.00 Short Chain Fatty Acid (SCFA) analysis 100 samples$6.00 / sample$600.00 GLP-1 Elisa kit38 samples$15.00 / sample$570.00 PYY RIA Cytokine kit100 samples$6.50 / sample$650.00 Total: $4553.05

32. 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

33. ACKNOWLEDGEMENTS  Dr. Subramaniam Sathivel  Arranee Chotiko  Dr. Diana Coulon  Dr. Daniel Hayes

34. QUESTIONS?

35. APPENDIX

36. DESIGN TOOLS PHYSICAL DECOMPOSITION Rat’s DietFoodProteinCarbsFatsSupplementProbioticL. plantarumPrebioticPRFEnzymeshemicellulaseamylaseproteaseWater

37. PICTURE REFERENCES  Slide 2:  http://3.bp.blogspot.com/-t7sZZGyVIC4/T-KwDSNvrHI/AAAAAAAAAvI/GzSOXz-Hw-s/s1600/209569,xcitefun-black-rice- 2.jpg 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 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 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://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 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 http://classes.midlandstech.com/carterp/Courses/bio225/chap06/06-15_PlateCounts_1.jpg  Slide 27:  http://www.sageresearchmodels.com/files/cox2_rat.jpg http://www.sageresearchmodels.com/files/cox2_rat.jpg

38. INFLAMMATORY BOWEL DISORDER ULCERATIVE COLITIS  Inflammation of the colon  Overly aggressive immune response of nonpathogenic bacteria  Causes dysentery, abnormal bowel movements, pain, cramping  No current cure

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

40. EXPERIMENTAL DESIGN SYNBIOTIC SYNTHESIS Raw Purple Rice Bran 300 g Enzyme Treatment Refined PRF Suspend L. plantarum + PRF Synbiotic Air Blast FreezePour Plate MethodDetermine CFU of Synbiotic Lactobacillus plantarum Plate and Incubate L. plantarum Centrifuge into Pellets PROCESS MATERIAL Key

41. DESIGN TOOLS MORPHOLOGICAL CHART