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Development of a colorimetric test kit to determine enzymatically onions
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Development of a colorimetric test kit to determine enzymatically produced pyruvic acid in sweet onions Presented by: Dawn C. Merrill December 9, 2009 History of Onions • Allium cepa • Many varietals, types • Manufacturing • Grown in ≈175 countries • US production • Consumption • Medicinal properties • Heart • Gastrointestinal • Anti-cancer • Flavor Dawn C. Merrill Oregon State University 12/9/2009 2 What is a Sweet Onion? • Originated in 1898 • Varietals include: • Walla Walla • Vidalia • Texas 1015 • Higher price in the market • Sweetness is inversely proportional to pungency • Indicators • Sulfur compounds, sugars, pyruvic acid Dawn C. Merrill Oregon State University 12/9/2009 3 Flavor Development • Many sources for flavor development • Plant metabolic pathways • Cellular disruption • Enzymatic process • Thermal processing • Fermentation • Enzymatic • Bacterial Dawn C. Merrill Oregon State University 12/9/2009 4 The Source of Flavor • Derived from the uptake of SO42- from soil SO42Photosynthesis Cysteine Glutathione 1-Propenyl cysteine sulfoxide Methyl cysteine sulfoxide Propyl cysteine sulfoxide Dawn C. Merrill Oregon State University 12/9/2009 5 Once an onion is cut… • Enzymatic activity by alliinase • Hydrolysis of alk(en)yl cysteine sulfoxides (ACSOs) • Products: • Thiopropanol S-oxide • Pyruvic acid • Ammonia O RSCH2-CH(NH2)COOH (Alliinase) CH2CH2C=S=O Dawn C. Merrill Oregon State University 12/9/2009 RSOH + CH3COCOOH + NH3 O R-S-S-R 6 Sensory Correlation Relationship between flavor perception and onion pungency1 Four studies: r = 0.92, 0.84, 0.95, and 0.79 R2 = 0.84, 0.71, 0.91, and 0.62 Dawn C. Merrill Oregon State University 12/9/2009 Wall, M. and Corgan, J. 1992. HortScience. 27(9):1029-1030. 1 7 Analyzing Pyruvic Acid • Standard industry practice • Definition of pungency determined by [pyruvic acid] • According to the following table: 1 – 4 μmol/g Very mild pungency (sweet) 5 – 7 μmol/g Mild pungency 8 – 10 μmol/g Intermediate pungency > 15 μmol/g Strong pungency (sharp) Dawn C. Merrill Oregon State University 12/9/2009 8 Method of Analysis • Current industry method: • Texas AgriLife Research & Extension • $60/sample analysis • Must have cooperative membership • Results take 1 week or longer • Shipping cost • Samples sent to Texas Dawn C. Merrill Oregon State University 12/9/2009 9 Purpose of Research • Objective 1 • Determine a colorimetric method for the determination of pyruvic acid in onions Dawn C. Merrill Oregon State University 12/9/2009 • Objective 2 • Develop a fast, field applicable, test kit for analyzing sweetness in onions 10 Literature Review • Method designed based on Schwimmer & Weston (1961) • 2, 4 – dinitrophenylhydrazine (DNPH) produces colored adduct • Measured spectrophotometrically • Wavelength at 420 nm • Standard in all rapid onion analyses Dawn C. Merrill Oregon State University 12/9/2009 11 DNPH Reaction • 2, 4 – dinitrophenylhydrazine (DNPH) • Used to chemically bind to ketone or aldehyde • Pyruvic acid is a ketone • DNPH-Pyruvic acid adduct is detected by spectrophotometry Dawn C. Merrill Oregon State University 12/9/2009 12 Spectrophotometer • Employed to measure the amount of light that a sample absorbs • Based on wavelength (λ) • Visible range: 350 – 750 nm • Data is used to calculate two quantities • Transmittance (T) • T = P/Po • Absorbance (A) Po P • A = -log10(T) Dawn C. Merrill Oregon State University 12/9/2009 13 Spectrophotometry • Beer-Lambert Law: • Measure of energy a sample absorbs • A = εbC • Back calculate [pyruvic acid] in sample • If Beer’s Law applies: • A plot of A vs. C will indicate a linear fit Dawn C. Merrill Oregon State University 12/9/2009 14 Pyruvic Acid Standard Curve 1.20 1.00 Beer’s Law: A = εbC Absorbance 0.80 0.60 0.40 Linear Fit, R2 = 0.9999 0.20 0.00 0.0 2.0 4.0 6.0 8.0 10.0 12.0 Concentration (umol/mL) Dawn C. Merrill Oregon State University 12/9/2009 15 Identifying Wavelength • Based on absorbance spectra of Anthon & Barrett (2003) • Reagent concentrations • Wavelength at 515 nm • Wavelength selection • Three wavelengths investigated • 420 nm, 445 nm, 515 nm • Ran standard curves at each wavelength • Based on sensitivity to adduct Dawn C. Merrill Oregon State University 12/9/2009 16 Standard Curve at 420 nm 1.40 1.20 Absorbance 1.00 0.80 0.60 0.40 420 nm R2 = 0.9625 0.20 0.00 0 2 4 6 8 10 Concentration (umol/mL) Dawn C. Merrill Oregon State University 12/9/2009 17 Standard Curve at 445 nm 1.40 1.20 Absorbance 1.00 0.80 0.60 0.40 420 nm R2 = 0.9625 445 nm R2 = 0.9924 0.20 0.00 0 2 4 6 8 10 Concentration (umol/mL) Dawn C. Merrill Oregon State University 12/9/2009 18 Standard Curve at 515 nm 1.40 1.20 Absorbance 1.00 0.80 0.60 0.40 420 nm R2 = 0.9625 445 nm R2 = 0.9924 515 nm R2 = 0.9998 0.20 0.00 0 2 4 6 8 10 Concentration (umol/mL) Dawn C. Merrill Oregon State University 12/9/2009 19 Reaction Time • Preliminary Data • Variability between onion samples • Hypothesis: • Formation of pyruvic acid product from ACSOs requires a specific amount of time • Test for optimum concentration • Expectation • Bell shaped curve showing formation and degradation of pyruvic acid Dawn C. Merrill Oregon State University 12/9/2009 20 Reaction Time Concentration Pyruvic acid (umol/g) 6.2 6.0 5.8 5.6 5.4 5.2 0 10 20 30 40 50 Time (minutes) Dawn C. Merrill Oregon State University 12/9/2009 21 Onion Sensory Scale • Observation • Not a sensory experiment • Expectation • Replication of Wall & Corgan (1992) • Disadvantages • Based on aroma • One analyst vs. trained panel • Bias Dawn C. Merrill Oregon State University 12/9/2009 Score: Evaluation: 6 Extremely strong 5 Very strong 4 Strong 3 Distinct 2 Slight 1 Very slight 0 Neutral (no intensity present) 22 Intensity vs. Concentration 6 Sweet White Yellow Boiler Intensity Score 5 4 3 2 1 0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 Concentration P.A. (umol/g) Dawn C. Merrill Oregon State University 12/9/2009 23 Laboratory Procedure • 25 μL filtered onion juice • Add reagents • 1 mL 18 mΩ H2O • 1 mL 2, 4-DNPH in 1 N HCl • Water Bath • 10 minutes at 37˚C ± 2˚C • Add reagent • 1 mL 1.5 N NaOH • Absorbance • 515 nm Dawn C. Merrill Oregon State University 12/9/2009 24 Background Levels • Heat denaturation of alliinase • Microwave (W = 1450) • Procedure • Processed exactly the same as the uncooked sample • Absorbance at 515 nm • Data provides: • TPA – BPA = EPA Dawn C. Merrill Oregon State University 12/9/2009 25 Onion Results Table • Triplicate analysis • n=5 Varietal Average TPA (μmol/g) Average BPA (μmol/g) Average EPA (μmol/g) Sweet 5.90 ± 0.24 1.88 ± 0.43 4.03 ± 0.49 White 6.59 ± 0.39 1.77 ± 0.35 4.83 ± 0.35 Yellow 7.44 ± 0.31 1.58 ± 0.38 5.86 ± 0.08 Boiler 6.46 ± 0.32 1.58 ± 0.29 4.88 ± 0.39 Key: TPA: Total Pyruvic Acid BPA: Background Pyruvic Acid EPA: Enzymatically Produced Pyruvic Acid Dawn C. Merrill Oregon State University 12/9/2009 26 Onion Results (continued) • Average all three trials • n = 15 Varietal Average Pyruvic acid (umol/g) Sweet 4.05 ± 0.48 White 4.85 ± 0.09 Yellow 5.85 ± 0.06 Boiler 5.02 ± 0.09 • Percent recovery • Over-spiked with known concentration P.A. Average (3 replicates) 104% S.D. (3 replicates) 10.4% Dawn C. Merrill Oregon State University 12/9/2009 27 Literature Results (TPA)1 Varietal Schwimmer & Weston Anthon & (1961) Barrett (2003) (μmol/g) (μmol/g) Merrill, et. al. (2009)* (μmol/g) Sweet (Vidalia) 4.47 ± 0.24 3.61 ± 0.24 - Sweet (Melody) 4.30 ± 0.15 3.43 ± 0.14 - Sweet** - - 5.90 ± 0.24 White 6.54 ± 0.65 6.98 ± 0.69 6.59 ± 0.39 Yellow 12.34 ± 1.17 10.81 ± 1.11 7.44 ± 0.31 Boiler - - 6.46 ± 0.32 1 Anthon & Barrett (2003) J Sci Food Agric 83:1213 Dawn C. Merrill Oregon State University 12/9/2009 * Results not published ** Unknown origin 28 Literature Results (BPA)1 Varietal Schwimmer & Weston (1961) (μmol/g) Sweet (Vidalia) 0.76 ± 0.03 0.19 ± 0.01 - Sweet (Melody) 0.78 ± 0.06 0.16 ± 0.02 - Sweet** - - 1.88 ± 0.43 White 0.26 ± 0.02 0.21 ± 0.01 1.77 ± 0.35 Yellow 1.27 ± 0.27 0.32 ± 0.13 1.58 ± 0.38 Boiler - - 1.58 ± 0.29 1 Anthon & Barrett (2003) J Sci Food Agric 83:1213 Dawn C. Merrill Oregon State University 12/9/2009 Anthon & Merrill, et. al. Barrett (2003) (2009)* (μmol/g) (μmol/g) * Results not published ** Unknown origin 29 Objective 1: Summary • Method determination • Combined Schwimmer & Weston (1961) & Anthon & Barrett (2003) • 515 nm wavelength • 20 minutes for full formation of pyruvic acid • Color = [pyruvic acid] • Results compare to previous literature • Method appropriate for test kit Dawn C. Merrill Oregon State University 12/9/2009 30 Objective 2: Test Kit • Criteria • Materials • Ease of preparation • Stability • Temperature • Reactivity • Non-hazardous • Disposal down the drain • Method • Detection in the visible range • % Recovery • 80 – 120 Dawn C. Merrill Oregon State University 12/9/2009 31 Criteria (continued) • Materials for onion processing facility • No spectrophotometer • Reduce time • Current wait time: >1 week for results • Reduce cost • • • • Current cost is approximately $60 p/ sample Co-op membership required for this price Shipping expense Labor Dawn C. Merrill Oregon State University 12/9/2009 32 Results: Test Kit • Test kit formulation: • Use data from objective 1 • Pyruvic acid indicator • Color denotes concentration • Reduced total turn around time • Time of analysis is reduced to ≈ 66 minutes • Video instruction of method Dawn C. Merrill Oregon State University 12/9/2009 33 Video Demonstration Dawn C. Merrill Oregon State University 12/9/2009 34 Materials • Onion Processor needs: • • • • • Knife Test tubes Analytical balance Cheese cloth Water bath • Capable of sustaining 37˚C temperature • Blender/Homogenizer • Distilled water • Pipettes • 25 μL & 1 mL Dawn C. Merrill Oregon State University 12/9/2009 35 Materials (continued) • Provided in test kit : • • • • 1 - 20 mL vial 2, 4-DNPH in 1 N HCl 1 - 20 mL vial 1.5 N NaOH 1 - Color chart with reference concentrations Instruction manual & video demo Dawn C. Merrill Oregon State University 12/9/2009 36 Color Chart • Included in test kit • Compares color in sample to varying concentrations of pyruvic acid 1 2 3 4 5 6 7 8 9 10 Concentration of Pyruvic acid: in μmol/g Dawn C. Merrill Oregon State University 12/9/2009 37 Prototype – Test Kit Dawn C. Merrill Oregon State University 12/9/2009 38 Cost Analysis Cost Per Sample Current Industry Analysis $60.00 Shipping $4.90 - $25.15 Labor - Total: $75.00 Analysis $0.34 Shipping - Labor $1.13 Total: $1.47 Test Kit Dawn C. Merrill Oregon State University 12/9/2009 39 Results: Criteria Criteria Expectation Ease of Prep Quick (< 1 day) YES Stability Stable at T, mp, bp, vp, reactivity YES Non-Hazardous Disposal in general waste YES Temperature 25˚C YES Visible Range 420 – 515 YES % Recovery 80 – 120 YES Lab Materials No Spectrophotometer needed YES Time Same day analysis YES Cost Less than or same as current method YES Dawn C. Merrill Oregon State University 12/9/2009 Goal Met? 40 Future Research • Method validation • Robustness studies • Reduction of background pyruvic acid • More replications for greater statistical analysis • Development of the final test kit • Market and sell to onion industry Dawn C. Merrill Oregon State University 12/9/2009 41 Conclusions • Objective 1 • Established laboratory method • SOP in progress • Objective 2 • Create a prototype for a field test kit • Proof-of-concept Dawn C. Merrill Oregon State University 12/9/2009 42 Acknowledgements • Kim Anderson • Lab Crew • Bioresource Research: • Kate Field • Wanda Crannell • Margaret Corvi • FS&T Department: • Faculty • Students (“FSTers”) • Michael Penner • Additional Support • Holli Kalaleh • Nicolle Merrill • Anne Ryan Questions?
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