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
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.
OVERVIEW
 Background
 Design Constraints
 Measureable Objectives
 Product Design Specification
 Concept Evaluation and
Selection
 Testing Plans
 Design Tools
 Budget
 Summary
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
PROBIOTICS
 Live microorganisms found in
 Intestines
 Dairy products
 Provides beneficial digestive health
 Prevents the growth of harmful bacteria
 Maintains healthy balance for homeostasis
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
MEASUREABLE OBJECTIVES
Synbiotic Synthesis
 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 106 CFU/g after 30 days of storage
 Model doubling time of probiotic during growth, and reduction
rate during storage
PRODUCT DESIGN SPECIFICATION (PDS)
PDS CONTINUED
PDS CONTINUED
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
ENZYME-TREATMENT PROCESS
ENZYME ADDITION
• α-Amylase
• Alcalase
• Hemicellulase
ENZYME ADDITION
ENZYME ADDITION & DRYING
CONCEPT EVALUATION & SELECTION
PROBIOTIC SOURCE: L. PLANTARUM
 Nutrition Requirements and Growth Rate
 Tolerate pH of stomach
GROWTH OF PROBIOTIC ON FIBER
 Better survives
harsh condition of
stomach
 Scanning Electron
Microscopy (SEM) to
confirm growth
INOCULATION OF L. PLANTARUM
ADHERENCE AND GROWTH RATE
VIABILITY
MEASURING CFU/ML
 Serial Dilutions
 Triplicate Plating
 Accept 30-300 cell plate
 Before/During/After
Multiple Stages
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
FREEZE-DRYING PROCESS
AIR-BLAST FREEZER
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
MODELING
 Bacteria grown on Fiber
Doubling Time
 Bacteria added to Fiber
 Exponential Increase
 Bacteria separate from Fiber
 𝑋 = 𝑋0 𝑒 𝜇𝑡
 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
 2 = 𝑒 𝜇𝑡
 𝑡𝑑 =
ln 2
𝜇
STORAGE
Storage Conditions
 At Room Temperature (25°C) vs. Refrigerator (4°C)
 Storage duration for one month
 Bi weekly viability count (CFU/mL)
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
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
DESIGN TOOLS
GANTT CHART
BUDGET
Synbiotic Synthesis Budget
Item:
Quantity:
Unit Price:
Total Price:
Purple rice bran
Hemicellulase
Alcolase
Heat-Stable α-Amylase
L. plantarum
Storage bags
50 lbs
100 g
50 ml
10 ml
1 ml
200 pk
$120.00
$41.00
$144.00
$47.00
$200.00
$10.00
$120.00 - Provided
$41.00 - Provided
$144.00
$47.00
- Provided
$200.00 - Provided
$10.00
Total: $154.00
Rodent Study Budget
Item:
Quantity:
Unit Price:
Total Price:
Rodent RQ 18-6 Lab
diet
Sprague Dawley Rats
50 lb
$0.94 / lb
$46.95
38
$15.00 Each
$570.00
Rodent Housing
Dextran Sodium
Sulfate:
Microbial
Classification (PCR
Analysis)
Short Chain Fatty
Acid (SCFA) analysis
GLP-1 Elisa kit
PYY RIA Cytokine kit
21 days
50 g
$0.95 / day / rat
$2.96/gram
$758.10
$148.00
50 samples
$24.00 / sample
$1200.00
100 samples
$6.00 / sample
$600.00
38 samples
100 samples
$15.00 / sample
$6.50 / sample
$570.00
$650.00
Total: $4553.05
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
ACKNOWLEDGEMENTS
 Dr. Subramaniam Sathivel
 Arranee Chotiko
 Dr. Diana Coulon
 Dr. Daniel Hayes
QUESTIONS?
APPENDIX
Rat’s Diet
Food
Protein
Carbs
DESIGN TOOLS
Water
Fats
PHYSICAL
DECOMPOSITION
Supplement
Probiotic
L. plantarum
Prebiotic
PRF
Enzymes
hemicellulase
amylase
protease
PICTURE REFERENCES
 Slide 2:
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http://3.bp.blogspot.com/-t7sZZGyVIC4/T-KwDSNvrHI/AAAAAAAAAvI/GzSOXz-Hw-s/s1600/209569,xcitefun-black-rice2.jpg

http://www.probiotics-summit.eu/images/plantarum2.jpg
 Slide 4:
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http://www.letstalkmagazine.com/home/wp-content/uploads/2012/06/Whole-Grain-Group-Pic.jpg
 Slide 5:
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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:
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http://classes.midlandstech.com/carterp/Courses/bio225/chap06/06-15_PlateCounts_1.jpg
 Slide 27:
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http://www.sageresearchmodels.com/files/cox2_rat.jpg
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
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
Raw Purple Rice Bran
300 g
Lactobacillus
plantarum
Enzyme Treatment
Refined PRF
Suspend L. plantarum + PRF
Plate and Incubate
L. plantarum
Centrifuge into
Pellets
EXPERIMENTAL
DESIGN
SYNBIOTIC SYNTHESIS
Synbiotic
Air Blast Freeze
Pour Plate Method
Key
MATERIAL
Determine CFU of Synbiotic
PROCESS
DESIGN TOOLS
MORPHOLOGICAL CHART