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?