New Symbiotic for treatment of IBD

optimized Synthesis of a synbiotic from Enzyme-Treated Purple Rice Bran Fiber and Lactobacillus PlantarumDaniel Bergeron Kristi HarrisonChristina JuneauJason NewtonLaura DeLatinAdvisor: Dr. Subramaniam Sathivel

LD1Problem StatementTo 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.

Prebiotics Probiotics

LD2OverviewBackground Design ConstraintsMeasureable ObjectivesProduct Design SpecificationConcept Evaluation and SelectionTesting PlansDesign ToolsBudgetSummary

LD3PrebioticsNon-digestible oligosaccharidesIn Intestines:Stimulates the growth and activity of healthy bacterial floraCarbon and energy source for floraIn vivo: protective fiber matrix increases stability and viability of probiotics when freeze-dried

KHPrebiotics are non-digestible oligosaccharides, meaning a type of carbohydrate not broken down by digestive enzymes.To be considered to have prebiotic activity, it must stimulate the growth of intestinal microflora, and be a food source for the microflora to ferment.Food sources with prebiotic activity include rice bran, whole grains, onions, and legumes.In application to our project, research has shown that prebiotics are considered a cryoprotectant, because the fiber serves as a protective matrix for probiotics when freeze-dried

4ProbioticsLive microorganisms found in IntestinesDairy products Provides beneficial digestive healthPrevents the growth of harmful bacteriaMaintains healthy balance for homeostasis

KHProbiotics are live microorganisms that naturally live in your intestines along with other microflora, and are also used in several dairy products. Having a maintained level of probiotics in your intestines greatly supports the healthy microflora present, and prevents growth of harmful bacteria, which together promotes general digestive health. So the potency of probiotics are constantly being improved.

Body: http://www.nutritionaloutlook.com/sites/nutritionaloutlook.com/files/imagecache/Image_Default/images/probiotics.jpg5Design ConstraintsTimeMoneyAccess to equipmentMinimum effective dose of Synbiotic (CFU/g)IACUC (Institutional Animal Care and Use Committee)Process of Approval in progress: #13-100

JN6Measureable Objectives74 % Dietary fiber in Enzyme-treated Rice Fiber Recover 8.78 +/- 0.10 log CFU L. plantarum per mL of synbiotic after growthMaintained viability of 106 CFU/g after 30 days of storageModel doubling time of probiotic during growth, and reduction rate during storage

Synbiotic Synthesis JN7Product Design Specification (PDS)

LD

Note: PDS is just an exercise. These are estimates as the team saw fit, though they may not be practical

http://www.naturalproductsinsider.com/articles/2013/07/the-best-of-both-worlds-synergetic-marketing.aspx8PDS continued

LD9PDS Continued

LD10Concept Evaluation & selectionPrebiotic Source: Enzyme-Treated Purple Rice FiberBlanca Isabel Purple RiceBran is by-product of RiceLocally grown and milledHigh Antioxidant contentCryoprotectant

Enzyme-TreatmentPurifies Bran from 24% to 74% Dietary Fiber

KHAfter researching prebiotic sources and evaluating with a decision matrix, we decided to use enzyme-treated purple rice bran fiber. The prebiotic is retrieved from bran, which is a by-product from the rice milling. Isabel Blanca purple rice is naturally enhanced black rice. It is locally grown and milled here at LSU, making it readily available and cheap for us to use. It is advantageous because of its high antioxidant content, anti-tumor capabilities, and ability to serve as a cryoprotectant.

11Enzyme-treatment Process

CJKnow the justification for this process why the order, why the duration of each enzyme, why the temps12Enzyme Addition

-AmylaseAlcalaseHemicellulase

CJ13Enzyme Addition

CJ14Enzyme Addition & drying

CJ15Concept evaluation & SelectionProbiotic Source: l. plantarumNutrition Requirements and Growth RateTolerate pH of stomach

CJ16Growth of Probiotic on FiberBetter survives harsh condition of stomachScanning Electron Microscopy (SEM) to confirm growth

KHState how we are using this process in comparison to (Arrannees) Alternate process of centrifuging bacteria into pellets, then adhering ERF. 17Inoculation of L. Plantarum

KH18Adherence and Growth Rate

KH19Viability

KH20Measuring CFU/ml Serial DilutionsTriplicate PlatingAccept 30-300 cell plateBefore/During/After Multiple Stages

KH30-300 cell count is accepted because it is an established standard for the most accurate approximation without error. 21Method of PreservationFreeze drying Decreases surrounding pressure until frozen water sublimatesFiber serves as cryoprotectant for probioticLess cell damage and cell reductionImproves stability and viability during storageProlongs shelf-lifeAir blast freezer can be used for a variety of irregular shapes Particularly small sizes

DBFreeze then drying process decreasing surrounding pressure until frozen water sublimates

Fiber serves as cryoprotectant for probioticLess cell damage and cell reductionImproves stability and viability during storageProlongs shelf-lifeAir blast freezer can be used for a variety of irregular shapes Particularly small sizes

Normally used to preserve perishable materialsProcess: freeze material then reduce pressures enough to provide conditions for the sublimation of frozen water 22Freeze-Drying ProcessAir-Blast Freezer

DB23Testing PlansModelingGrowth of Bacteria on FiberViability during Storage over One MonthStorageTo determine the optimal temperature necessary to keep our supplement viableAnimal StudyTo test effectiveness of synbiotic in intestinal environment for future marketabilityUlcerative Colitis Model Form of IBDPast studies of treating Colitis with probiotics

LD24ModelingBacteria grown on FiberBacteria added to FiberBacteria separate from Fiber

CJ25StorageStorage ConditionsAt Room Temperature (25C) vs. Refrigerator (4C)Storage duration for one monthBi weekly viability count (CFU/mL)

JN26Animal StudyFoundational StudyFeed supplemented dietsInduce Colitis with DSSPost-mortem evaluationsTest groups based on respective supplemental dietsControl (no supplement) Prebiotic supplement only (ERF)Probiotic supplement only (L. plantarum) Synbiotic supplement (ERF + L. plantarum) Process of Approval in progress

DB27Animal StudyTerminal Measurements after DissectionWeight and length of colon to analyze the effects of inflammationCecal sampleMicrobiota characterization determined by RT-qPCRShort Chain Fatty Acid contentElisa kit for Cytokine analysis [IL-6 and TNF-]H&E stain of colonic mucosal tissue to visually assess mucosal damage Measurable Objectives (Relative to the Control)Longer colon lengthDecreased cytokine levelsLower mucosal damage scoreAbundance of microbial strains

DB28Design toolsGantt Chart

LD29BUDGET Synbiotic Synthesis BudgetItem:Quantity:Unit Price:Total Price:Purple rice bran50 lbs$120.00$120.00 - ProvidedHemicellulase100 g$41.00$41.00 - ProvidedAlcolase50 ml$144.00$144.00Heat-Stable -Amylase10 ml$47.00$47.00 - ProvidedL. plantarum1 ml$200.00$200.00 - ProvidedStorage bags200 pk$10.00$10.00Total: $154.00Rodent Study BudgetItem:Quantity:Unit Price:Total Price:Rodent RQ 18-6 Lab diet50 lb$0.94 / lb$46.95Sprague Dawley Rats38$15.00 Each$570.00Rodent Housing21 days$0.95 / day / rat$758.10Dextran Sodium Sulfate:50 g$2.96/gram$148.00Microbial Classification (PCR Analysis)50 samples$24.00 / sample$1200.00Short Chain Fatty Acid (SCFA) analysis100 samples$6.00 / sample$600.00GLP-1 Elisa kit38 samples$15.00 / sample$570.00PYY RIA Cytokine kit100 samples$6.50 / sample$650.00Total: $4553.05

LD

Note that costs can be cut by scaling back number of analytical tests performed for animal study 30SummaryPrebiotics + Probiotics = SynbioticProbiotic grown on prebiotic fiber Freeze-dried Altering storage conditions to increase viability after storagePlanning and Testing Decision Matrices, Flowcharts, and PDS Tests and Modeling Animal Study

CJChange this to be shorter and quicker. Restate the problem statement31AcknowledgementsDr. Subramaniam SathivelArranee Chotiko Dr. Diana CoulonDr. Daniel Hayes

LD32Questions?LD33AppendixDesign Tools

Physical Decomposition

Laura35Picture referencesSlide 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.jpghttp://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

Inflammatory Bowel DisorderUlcerative ColitisInflammation of the colonOverly aggressive immune response of nonpathogenic bacteriaCauses dysentery, abnormal bowel movements, pain, cramping No current cure

Christina37timelineWeek 1 Week 2Week 3Week 4Laboratory acclimation feeding Specific supplemental diet introducedInduce Colitis with Dextran Sodium SulfateEuthanasia, dissection, and analysisDaniel38Experimental Design

Synbiotic SynthesisPROCESSMATERIALKeyArrannees method 39Design toolsMorphological chart

Laura40