Upload
dokiet
View
223
Download
0
Embed Size (px)
Citation preview
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
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
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
Beleidsdomein Landbouw en Visserij
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
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
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
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
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)
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
Validation: influence purity color reagent and influence purification
0.1200.1140.1250.0815Purity ≥ 99% and purified
0.1200.1170.1220.0810Purity ≥ 99%
0.1210.1140.1270.0912Purity ≥ 98% and purified
0.1190.1140.1230.1095Purity ≥ 98%
MeanResult 2Result 1Blanc/air
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
Validation: influence purity color reagent and influence purification
0.1190.1130.1240.0299Purity ≥ 99% and purified
0.1200.1170.1220.0297Purity ≥ 99%
0.1200.1140.1260.0402Purity ≥ 98% and purified
0.1190.1140.1240.0575Purity ≥ 98%
MeanResult 2Result 1Blanc/H2O
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
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!!!
ILVO – Technologie & Voedinghttp://www.ilvo.vlaanderen.be
Beleidsdomein Landbouw en Visserij
Thank You for your attention!!!