Presented by: Dawn C. Merrill December 9, 2009 Development of a colorimetric test kit to determine enzymatically produced pyruvic acid in sweet onions

Presented by:

Dawn C. Merrill

December 9, 2009

Development of a colorimetric test kit to determine enzymatically

produced pyruvic acid in sweet onions

• Allium cepa

• Many varietals, types

• Manufacturing• Grown in ≈175 countries• US production

• Consumption• Medicinal properties

• Heart • Gastrointestinal • Anti-cancer

• Flavor

History of Onions

Dawn C. Merrill Oregon State University 12/9/2009 2

• 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

What is a Sweet Onion?


• Many sources for flavor development• Plant metabolic pathways• Cellular disruption

• Enzymatic process

• Thermal processing• Fermentation

• Enzymatic• Bacterial

Flavor Development


• Derived from the uptake of SO42- from soil

SO42-

Photosynthesis

Cysteine

Glutathione

1-Propenyl cysteine sulfoxide

Methyl cysteine sulfoxide

Propyl cysteine sulfoxide

The Source of Flavor


• Enzymatic activity by alliinase • Hydrolysis of alk(en)yl cysteine sulfoxides (ACSOs) • Products:

• Thiopropanol S-oxide • Pyruvic acid • Ammonia

O (Alliinase)

RSCH2-CH(NH2)COOH RSOH + CH3COCOOH + NH3

O

CH2CH2C=S=O R-S-S-R

Once an onion is cut…


Relationship between flavor perception and onion pungency1

Four studies:

1 Wall, M. and Corgan, J. 1992.HortScience. 27(9):1029-1030.

Sensory Correlation

r = 0.92, 0.84, 0.95, and 0.79

R2 = 0.84, 0.71, 0.91, and 0.62


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)


• 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

Method of Analysis


• Objective 1

• Determine a colorimetric method for the determination of pyruvic acid in onions

• Objective 2

• Develop a fast, field applicable, test kit for analyzing sweetness in onions

Purpose of Research


• 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

Literature Review


• 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

DNPH Reaction


• 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)• A = -log10(T)

Spectrophotometer

Po P


• 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

Spectrophotometry


Pyruvic Acid Standard Curve

Concentration (umol/mL)

0.0 2.0 4.0 6.0 8.0 10.0 12.0

Absorb

ance

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Linear Fit, R2 = 0.9999

Beer’s Law: A = εbC


• 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

Identifying Wavelength


Standard Curve at 420 nm



0 2 4 6 8 10

Abs

orba

nce

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

420 nm R2 = 0.9625




0 2 4 6 8 10

Abs

orba

nce

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

420 nm445 nm

R2 = 0.9625R2 = 0.9924




0 2 4 6 8 10

Abs

orba

nce

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

420 nm445 nm515 nm

R2 = 0.9625R2 = 0.9924R2 = 0.9998

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


Reaction Time

Time (minutes)

0 10 20 30 40 50

Concentr

ation P

yruvi

c a

cid

(um

ol/g)

5.2

5.4

5.6

5.8

6.0

6.2


• Observation• Not a sensory

experiment

• Expectation• Replication of Wall

& Corgan (1992)

• Disadvantages• Based on aroma• One analyst vs.

trained panel• Bias

Score: Evaluation:

6 Extremely strong

5 Very strong

4 Strong

3 Distinct

2 Slight

1 Very slight

0Neutral (no intensity present)

Onion Sensory Scale


Concentration P.A. (umol/g)

0.0 1.0 2.0 3.0 4.0 5.0 6.0

Inte

nsity

Sco

re

0

1

2

3

4

5

6

SweetWhiteYellowBoiler

Intensity vs. Concentration


• 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

Laboratory Procedure


• 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

Background Levels


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

Onion Results Table• Triplicate analysis

• n = 5

Key:TPA: Total Pyruvic AcidBPA: Background Pyruvic AcidEPA: Enzymatically Produced Pyruvic Acid


• Average all three trials• n = 15

• Percent recovery • Over-spiked with known concentration P.A.

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

Onion Results (continued)


Average (3 replicates) 104%

S.D. (3 replicates) 10.4%

Literature Results (TPA)1

Varietal

Schwimmer & Weston (1961)

(μmol/g)

Anthon & Barrett (2003)

(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

* Results not published** Unknown origin

1 Anthon & Barrett (2003) J Sci Food Agric 83:1213


Literature Results (BPA)1

Varietal

Schwimmer & Weston

(1961)(μmol/g)

Anthon & Barrett (2003)

(μmol/g)

Merrill, et. al. (2009)*

(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 * Results not published** Unknown origin


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


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


• 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

Criteria (continued)


• 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

Results: Test Kit


Video Demonstration


Materials• Onion Processor needs:

• Knife• Test tubes• Analytical balance • Cheese cloth• Water bath

• Capable of sustaining37˚C temperature

• Blender/Homogenizer• Distilled water• Pipettes

• 25 μL & 1 mL


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


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


Prototype – Test Kit


Cost AnalysisCost Per Sample

Current Industry

Analysis $60.00

Shipping $4.90 - $25.15

Labor -

Total: $75.00

Test Kit

Analysis $0.34

Shipping -

Labor $1.13

Total: $1.47


Results: CriteriaCriteria Expectation Goal Met?

Ease of Prep Quick (< 1 day)

StabilityStable at T, mp, bp, vp, reactivity

Non-Hazardous Disposal in general waste

Temperature 25˚C

Visible Range 420 – 515

% Recovery 80 – 120

Lab Materials No Spectrophotometer needed

Time Same day analysis

CostLess than or same as current method

YES

YES

YES

YES

YES

YES

YES

YES

YES


• 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

Future Research


• Objective 1• Established laboratory

method• SOP in progress

• Objective 2• Create a prototype for a

field test kit• Proof-of-concept

Conclusions


• Kim Anderson • Lab Crew

• Michael Penner

• Bioresource Research:• Kate Field• Wanda Crannell• Margaret Corvi

• FS&T Department:• Faculty • Students (“FSTers”)

• Additional Support • Holli Kalaleh• Nicolle Merrill• Anne Ryan

Acknowledgements

http://mail.google.com/mail/?ui=2&ik=1095594fd5&view=att&th=11f53e637d4fbe1b&attid=0.1&disp=inline&zw

http://mail.google.com/mail/?ui=2&ik=1095594fd5&view=att&th=11f53e637d4fbe1b&attid=0.1&disp=inline&zw

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Presented by: Dawn C. Merrill December 9, 2009 Development of a colorimetric test kit to determine enzymatically produced pyruvic acid in sweet onions