Detection of Hydroxyproline in Chicken Meat - Van Royen... · Detection of Hydroxyproline in...

ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be

Beleidsdomein Landbouw en Visserij

Detection of Hydroxyproline in Chicken Meat

Geert Van RoyenMinistry of Flemish Community

Institute for Agricultural and Fisheries ResearchTechnology & Food Unit

Melle (Belgium)

OUTLINE

• ILVO• Introduction• Determination of HP• Validation of method• Conclusion

Institute for Agricultural and Fisheries Research

ILVO – Unit Technology and Food

Research and consultancy in following research domains:

– Agricultural Engineering– Food safety– Product quality and innovation– Business unit and service centre

Business unit and service centre• National reference lab milk- and milk products• Reference working and expertise on GMO

regulation and new EU-authorizations (EFSA, CRL)

• Accredited service for spray technology lab• Scientific guidance MCC-Vlaanderen and dairy

industrie• National reference lab controlling water content in

poultry meat and part of European Coordinating Board (together with Denmark and Germany)

INTRODUCTION (1)• Controlling water content: determination

ratio water/protein• Limits depend on part of chicken (with or

without skin) and chilling method (air, air-spray and immersion)

• Chicken pumped up with water→ not illegal if consumers are notified

• Addition of water retaining agents• Most frequently used agent is collagen

INTRODUCTION (2)• “Plastic” chicken: both factors of ratio

increase and ratio stays constant• Extra parameter necessary: determination of

hydroxyproline (HP)• HP can be used to determine amount of

collagen added in chicken• Collagen consists out of three helices and

each helix is built out of ± 1000 amino acids

INTRODUCTION (3)• Sequence amino acids is

very specific• Collagen contains as well

hydroxyproline ashydroxylysine (as one of the few proteins)

• Collagen contains ± 9%hydroxyproline (constant part, in contrary to other proteins)

DETERMINATION OF HP

• Following ISO 3496: Meat and meat products – Determination of hydroxyprolinecontent

• Principle: – hydrolysis test portion in sulfuric acid at 105°C– oxidation by chloramine T– formation of red compound– photometric measurement at 558nm

DETERMINATION OF HP• Reagents:

– sulfuric acid solution: concentration 3 mol/l– buffer solution (citric acid monohydrate, sodium

hydroxide and anhydrous sodium acetate): stable several weeks

– chloramine-T reagent (N-chloro-p-toluene-sulfonamidetrihydrate): dissolve in buffer solution and preparation immediately before use

– colour reagent (p-dimethylaminobenzaldehyde inperchloric acid solution and propan-2-ol): preparation on day of use

– hydroxyproline: standard solutions (0.5 µg/ml, 1 µg/ml, 1.5 µg/ml and 2 µg/ml), stock solution stable at least one month in fridge, solutions prepare on day of use

DETERMINATION OF HP• Equipment (1):

– meat mincer: horizontal blades, high speed– hydrolysis flasks– drying oven: operated at 105 ± 1°C– filter paper discs– pH-meter– aluminium foil or plastic caps

DETERMINATION OF HP• Equipment (2):

– water bath: maintained at 60 ± 0.5°C– spectrometer: use at 558 ± 2 nm– glass cells– analytical balance: accuracy of 1 mg– volumetric flasks– watch glasses

DETERMINATION OF HP• Sampling:

– proceed from representative sample of 200 g• Preparation of test sample:

– homogenize in meat mincer– store so no change in quality or composition is

possible– start analysis always within 24 h after

homogenization

DETERMINATION OF HP• Procedure of analysis

– weigh about 4 g test sample (accuracy of 1 mg)– hydrolysis in 30ml sulfuric acid in oven at

105°C for 16 hours– filtration of hot hydrolysate– washing of filter with hot sulfuric acid solution

(three times 10 ml)– take volume V of hydrolysate, so after dilution

to 250ml concentration HP is between 0.5 and 2 µg/ml

DETERMINATION OF HP• Procedure of analysis (2)

– mix 4ml of solution with 2 ml chloramine-T and leave 20 min at room temperature

– add 2 ml color reagent, mix and cap tube– leave 20 min in hot water bath of 60°C– cool tube under running water for 3 min and

leave 30 min at room temperature– measure absorbance at 558nm– subtract blank and read HP concentration from

calibration graph

DETERMINATION OF HP• Blank test

– replace 4 ml of solution by water

• Calibration graph– replace 4 ml of solution by in turn each of the four

standard HP solutions– plot measured absorbance values against concentrations

and construct best fitting

• Calculation HP concentration

Vmcw h

**25.6

VALIDATION OF METHOD

• Repeatability (r) = 0.013 + 0.032* wh• Reproducibility (R) = 0.019 + 0.052* wh• Use of internal reference: conservable

canned meat sample (comparison with reference laboratory Denmark)

• Use of certified meat reference material• Test influence purity color reagent and

influence purification

Validation: use of internal reference

0.0290.0120.1730.161WEEK 5

0.196REFERENCE VALUE

0.0160.0000.1800.180WEEK 4

0.0190.0040.1750.179WEEK 3

0.0130.0030.1840.181WEEK 2

0.0220.0030.1720.175WEEK 1

R (limit: 0.029)

r (limit: 0.019)

RESULT 2RESULT 1(g/100g)

Validation: use of certified meat reference material

0.0130.0080.1160.1244th analysis

0.0190.0010.1140.1131st analysis0.0210.0010.1140.1102nd analysis

0.0010.0030.1300.1335th analysis

0.0120.0020.1200.1223rd analysis

0.133REFERENCE VALUE

R (limit: 0.026)

r (limit: 0.017)

RESULT 2

RESULT 1

(g/100g)

Validation: influence purity color reagent and influence purification

0.1200.1140.1250.0815Purity ≥ 99% and purified

0.1200.1170.1220.0810Purity ≥ 99%

0.1190.1140.1230.1095Purity ≥ 98%

MeanResult 2Result 1Blanc/air

Validation: influence purity color reagent and influence purification

0.1200.1170.1220.0297Purity ≥ 99%

0.1190.1140.1240.0575Purity ≥ 98%

MeanResult 2Result 1Blanc/H2O

CONCLUSION• Besides the preparation steps (time consuming)

relative fast and simple method• No screening on proteins from other sources than

collagen• Method is not animal specific• After treatment of sample, hydrolysate could

already be coloured (→ blanco or filtration step)• Alternatives? (e.g. Foodscan – FOSS)• Determination of HP could be ideal solution for

detection of water injection!!!

Thank You for your attention!!!

Detection of Hydroxyproline in Chicken Meat - Van Royen... · Detection of Hydroxyproline in...

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