Detection of Hydroxyproline in Chicken Meat Geert Van Royen Ministry of Flemish Community Institute for Agricultural and Fisheries Research Technology & Food Unit Melle (Belgium)
OUTLINE ILVO Introduction Determination of HP Validation of method Conclusion 2
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, airspray 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 as hydroxylysine (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 hydroxyproline content 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-sulfonamide trihydrate): dissolve in buffer solution and preparation immediately before use colour reagent (p-dimethylaminobenzaldehyde in perchloric 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 w h = 6.25 m * * V c
VALIDATION OF METHOD Repeatability (r) = 0.013 + 0.032* w h Reproducibility (R) = 0.019 + 0.052* w h 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 (g/100g) RESULT 1 RESULT 2 r (limit: 0.019) R (limit: 0.029) WEEK 1 0.175 0.172 0.003 0.022 WEEK 2 0.181 0.184 0.003 0.013 WEEK 3 0.179 0.175 0.004 0.019 WEEK 4 0.180 0.180 0.000 0.016 WEEK 5 0.161 0.173 0.012 0.029 REFERENCE VALUE 0.196
Validation: use of certified meat reference material (g/100g) 1st analysis 2nd analysis 3rd analysis 4th analysis 5th analysis RESULT 1 0.113 0.110 0.122 0.124 0.133 REFERENCE VALUE RESULT 2 0.114 0.114 0.120 0.116 0.130 r (limit: 0.017) 0.001 0.001 0.002 0.008 0.003 0.133 R (limit: 0.026) 0.019 0.021 0.012 0.013 0.001
Validation: influence purity color reagent and influence purification Blanc/air Result 1 Result 2 Mean Purity 98% 0.1095 0.123 0.114 0.119 Purity 98% and purified 0.0912 0.127 0.114 0.121 Purity 99% 0.0810 0.122 0.117 0.120 Purity 99% and purified 0.0815 0.125 0.114 0.120
Validation: influence purity color reagent and influence purification Blanc/H 2 O Result 1 Result 2 Mean Purity 98% 0.0575 0.124 0.114 0.119 Purity 98% and purified 0.0402 0.126 0.114 0.120 Purity 99% 0.0297 0.122 0.117 0.120 Purity 99% and purified 0.0299 0.124 0.113 0.119
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!!!