voor Landbouw en Visserijonderzoek Eenheid Technologie amp Voeding wwwilvovlaanderenbe Alkaline Phosphatase activity in cheese and its detection problems Jan De Block ID: 365228
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Instituut voor Landbouw- en VisserijonderzoekEenheid Technologie & Voedingwww.ilvo.vlaanderen.be
Alkaline Phosphatase activity in cheese and its detection problems
Jan De Block
18 december / décembre 2014Opleiding / FormationCommunicatiegroep / Groupe de Communication:NRL Milk and Milk ProductsSlide2
Nationale Referentie Laboratoria - Melk en MelkproductenLaboratoire National de référence – Lait et produits laitiers4/07/20142Slide3
What is Alkaline Phosphatase?Dimeric enzyme (EC 3.1.3.1)Hydrolase enzyme responsible for removing phosphate groups (dephosphorylation) from many types of molecules (e.g. nucleotides, proteins, alkaloids,…)Most effective in an alkaline environmentNaturally produced during the normal metabolic process in animalsThis enzyme is then passed through the animal and is found in raw milkIndicator of successful pasteurization
Batch Pasteurization: 68°C during 30 minHTST Pasteurization: 72°C during 15 secWhen milk is pasteurized most of the enzymes are inactivated or their activity is greatly diminishedSlide4
Where is the Alkaline Phosphatase test used for?Was the pasteurization successful?Used in dairy industry as indicator for succesful pasteurizationThe most heat stable bacterium found in milk, Mycobacterium paratuberculosis, is destroyed by temperatures than those required to denature ALP (however: bacterial spores can remain intact)Indicator for raw milk contamination
As little as 0,01% or less raw milk contamination (fluorimetric method)As little as 1°C drop in processing temperatureSlide5
False positive results?They can occurAcidified productHigh fat contentAddition of raw milk after pasteurizationMicrobial contaminationReactivation of phosphataseHigh fat dairy products after UHT (stored at ambient temperature)Residual alkaline phosphataseHeat stable microbial alkaline phosphataseSlide6
Applicable to cheese?Yes, but possibility of false-positive test resultsMoldsAdded culturesAcidified productSlide7
Colorimetric methodp-nitrophenyl phosphate + OH- p-nitrophenol (yellow complex)Alkaline phosphatase induces the hydrolysis of p-nitrophenyl phosphate in an alkaline solution, and forms a yellow colored complex whose intensity, measured at 405 nm, is directly proportional to the concentration of ALP in the sample.
ALP
raw heated (1% raw) heatedSlide8
The enzyme photo-activated substrates method (EPAS) ISO 22160 / IDF 209:2007Hydrolysis of monophosphate esters at the proper pH and temperature:R-O-PO3H2 + H2O ® R-OH + PO4H3EPAS
Substrate-PO3H2 ® Detectable Light Output + PO4H3Marina NicolasWorkshop on heat treatment of milk6/5/206Slide9
9EU COMMUNITY REFERENCE LABORATORY FOR MILK & MILK PRODUCTS Marina Nicolas
Workshop on heat treatment of milk6/5/206Marina NicolasWorkshop on heat treatment of milk6/5/206Slide10
MethodDRAFT INTERNATIONAL STANDARD ISO/DIS 11816-2 IDF 155-2Milk and milk products – Determination of alkaline phosphatase activityPart 2:Fluorimetric method for cheeseVoting terminated on 2014-12-10Slide11
DRAFT INTERNATIONAL STANDARD ISO/DIS 11816-2 IDF 155-2Scope The method specifies a fluorimetric method for the determination of alkaline phosphatase (ALP, EC 3.1.3.1) activity in cheeseThe method is applicable to: soft cheeses (e.g. Brie, Camembert) semi-hard (e.g. Emmental, Gouda) and hard
cheeses (e.g. Parmesan) mold only on surface and not in the inner part (e.g. blue veined cheeses, e.g. Roquefort, Gorgonzola) Slide12
DRAFT INTERNATIONAL STANDARD ISO/DIS 11816-2 IDF 155-2NoteIn large hard cheeses where the whey curd mixture is “cooked” at temperatures above 50°C, high temperatures remain for a relatively long time, especially in the centre of those cheeses, promoting phosphatase inactivation; special sampling is needed
Piave (Italy)Gruyere (Switzerland)Slide13
PrincipleThe alkaline phosphatase activity is expressed as milliunits of enzyme activity per gram of sample (mU/g)Definition: 1 Unit of alkaline phosphatase activity is the amount of enzyme that catalyses the transformation of 1 µmol of substrate per minuteThe activity is measured by a continuous fluorimetric direct kinetic assayA non-fluorescent substrate (*) undergoes hydrolysis of its phosphate radical producing a highly fluorescent product
(*) 2’-[2-benzothiazolyl]- 6’-hydroxybenzothiazole phosphateSlide14
Principle
Advanced Instruments Fluorophos ALP test systemSlide15
Sample preparation
Remove rind or surface with a clean knifeSurface should not be contaminated with surface microflore (soft cheese with moulded surface!)Slide16
Sample preparationSlide17
Sample preparationSlide18
Sample preparation: grinding
French Agency for Food, Environmental and Occupational Health Safety – Laboratory for Food Safety
Keep sample in an airtight containerSlide19
Sample preparation Weigh 0,3 to 0,5 g of prepared test sample in 10 ml beaker Add 5 ml cheese extraction buffer Wait 5-10 min (soft and semi hard cheese) or 15-20 min (hard cheese) turrax (35-50 s) transfer to 25 ml volumetric flask Add 5 ml cheese extraction buffer and rinse turrax and beaker; turrax 30 s transfer to 25 ml volumetric flask Add 5 ml cheese extraction buffer and rinse turrax and beaker transfer to 25 ml volumetric flask and adjust with extraction buffer to the 25 ml markSlide20
French Agency for Food, Environmental and Occupational Health Safety – Laboratory for Food SafetySlide21
Sample preparation Shake gently Intoduce 5ml in a centifugation tube centrifuge at 1000g at 4°C during 10 min to remove fat three layers are obtained: pellet, supernatant and surface fatSlide22
French Agency for Food, Environmental and Occupational Health Safety – Laboratory for Food SafetySlide23
Maisons-Alfort Laboratory for Food SafetyEU-RL MMPEuropean Union Reference Laboratory forMilk and Milk ProductsPROFICIENCY TESTINGSDetermination of Alkaline Phosphatase activity in cheeseReporting PT 2013 & PT 2014
Anne-Cecile BOITELLE, Caroline DESBOURDES, Hanene GHEZZAL, Marina NICOLAS17thWorkshop of the NRLs on Milk & Milk Products, October 2014French Agency for Food, Environmental and Occupational Health Safety – Maisons-Alfort Laboratory for Food Safety23, avenue du Général de Gaulle - F94706 Maisons-Alfort Cedex - Telephone: + 33 149 771 300 - Fax: + 33 143 689 762 - www.anses.frSlide24
COUNTRY (CITY)LABORATORY NAMEPT1PT2
Austria (Vienna)AGES - INZT2013
2014Belgium (Melle)ILVO T&V
20132014Cyprus (Nicosia)LCFAO - VETERINARY SERVICES2013-
Czech Republic (Praha)
State Veterinary Institute Prague
-
2014
Finland (Helsinki)
Finnish Food Safety Authority Evira
2013
-
France (Maisons-Alfort)
Anses, Food safety laboratory of Maisons-Alfort
2013
-
Germany (Kiel)
MRI, Max Rubner-Institute
2013
2014
Hungary (Budapest)
Food Analytical National Reference laboratory, Food and Feed Safety Directorate, NFCSO
-
2014
Ireland (Co. Kildare)
Dairy Science Laboratory
2013
2014
Italy (Brescia)
IZSLER - Reference Centre for Bovine Milk Quality
2013
2014
Italy (Milan)
State University of Milan - DeFENS
2013
2014
Netherlands (Wageningen)
RIKILT
2013
2014
Poland (Pulawy)
National Veterinary Research Institute, department of Hygiene of Food of Animal Origin
2013
2014
Portugal (Lisboa)
Instituto Nacional de Investigação Agrária e Veterinária (INIAV), IP
-
2014
Slovenia (Ljubljana)
University Of Ljubljana, Veterinary Faculty / National Veterinary Institute Unit Kranj
2013
2014
Spain (Santander)
Laboratorio Agroalimentario de Santander
2013
2014
Switzerland (Berne)
Agroscope Liebefeld Posieux
2013
2014
United Kingdom (Belfast)
UK NRL
2013
2014
15
participating
laboratoriesSlide25
PROFICIENCY TEST ITEMS Individual test samples in blind duplicates One determination per sample Pasteurized samples of each type for calibration different types and different heat treatments 2013 6 Hard Cheeses 4 portions (pasteurized and raw) 2 pots of grated cheese (raw) 2 Semi Hard Cheeses (thermized) 2014 4 Soft Cheeses 2 pots of grinded cheese (raw) 2 portions (thermized) 2 Semi Hard Cheeses (raw)Slide26Slide27
Summary of collaborative trial statistical parametersSlide28Slide29Slide30
Alkaline Phosphatase in creamOften samples pasteurized cream with > 350 mU/liter (= legal limit for milk)Are these samples positive?Inadequate pasteurization?Why such high values?Is limit for milk also applicable to cream? Looking for a pragmatic approachSlide31
Alkaline Phosphatase in creamIn milk about 50% of AP is attached to fat fraction (of the total activity of 1000000 mU/liter)1 liter milk consists of:960 ml skimmed milk containing 500000 mU AP 40 ml fat containing 500000 mU AP1 liter cream (40% by volume) consists of:600 ml skimmed milk containing 312500 mU AP
400 ml fat containing 5000000 mU APAP activity in cream (40%) is about 5,31 times AP activity of milkSlide32
Alkaline Phosphatase in creamAP activity in cream (40%) is about 5,31 times AP activity of milkConclusionFor a similar decimal reduction of AP in cream as in milk (corresponding to a correct pasteurization) a residual activity of 350 x 5,31 = 1859 mU per Liter could be tolerated!Slide33
Thank you for your attention!Instituut voor
Landbouw- en VisserijonderzoekEenheid Technologie & VoedingOnderzoeksdomeinen 'Agrotechniek' en 'Productkwaliteit en -innovatie' Burg. Van Gansberghelaan 115 bus 19820
MerelbekeT +32 9 272 28 00Onderzoeksdomeinen 'Voedselveiligheid' en 'Productkwaliteit en -innovatie' Brusselsesteenweg 3709090 MelleT +32 9 272 30 00www.ilvo.vlaanderen.be