ILVO – Technologie & Voeding http://www.ilvo.vlaanderen.be Beleidsdomein Landbouw en Visserij Detection of Hydroxyproline in Chicken Meat Geert Van Royen Ministry of Flemish Community Institute for Agricultural and Fisheries Research Technology & Food Unit Melle (Belgium)
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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)
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
OUTLINE
• ILVO• Introduction• Determination of HP• Validation of method• Conclusion
2
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
Institute for Agricultural and Fisheries Research
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
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
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Beleidsdomein Landbouw en Visserij
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)
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
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
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
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
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
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)
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
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
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
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
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
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
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
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
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
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
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
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
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
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
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Beleidsdomein Landbouw en Visserij
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
=
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
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
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
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)
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
Validation: use of certified meat reference material