Transcript PowerPoint-presentatie

Alkaline Phosphatase activity in cheese
and its detection problems
Jan De Block
18 december / décembre 2014
Opleiding / Formation
Communicatiegroep / Groupe de Communication:
NRL Milk and Milk Products
Instituut voor Landbouw- en Visserijonderzoek
Eenheid Technologie & Voeding
www.ilvo.vlaanderen.be
Nationale Referentie Laboratoria - Melk en
Melkproducten
Laboratoire National de référence – Lait
et produits laitiers
2
4/07/2014
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 environment
 Naturally produced during the normal metabolic process in animals
 This enzyme is then passed through the animal and is found in raw milk
• Indicator of successful pasteurization
 Batch Pasteurization: 68°C during 30 min
 HTST Pasteurization: 72°C during 15 sec
 When milk is pasteurized most of the enzymes are inactivated or their
activity is greatly diminished
Where is the Alkaline Phosphatase test used for?
• Was the pasteurization successful?
 Used in dairy industry as indicator for succesful pasteurization
 The 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 temperature
False positive results?
• They can occur




Acidified product
High fat content
Addition of raw milk after pasteurization
Microbial contamination
• Reactivation of phosphatase
 High fat dairy products after UHT (stored at ambient temperature)
• Residual alkaline phosphatase
 Heat stable microbial alkaline phosphatase
Applicable to cheese?
• Yes, but possibility of false-positive test results
 Molds
 Added cultures
 Acidified product
Colorimetric method
• p-nitrophenyl phosphate + OH-
ALP
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.
raw
heated (1% raw)
heated
The enzyme photo-activated substrates
method (EPAS) ISO 22160 / IDF 209:2007
R-O-PO3H2 + H2O R-OH + PO4H3
EPAS
Substrate-PO3H2 Detectable Light Output + PO4H3
Marina Nicolas
Workshop on heat treatment of
milk
6/5/206
Marina Nicolas
Workshop on heat treatment of milk
6/5/206
Tracer
Phosphatase
Negative
Sample
9
EU COMMUNITY REFERENCE
LABORATORY FOR
MILK & MILK PRODUCTS
Positive
Sample
Marina Nicolas
Workshop on heat treatment of milk
6/5/206
Method
 DRAFT INTERNATIONAL STANDARD ISO/DIS 11816-2
IDF 155-2
Milk and milk products – Determination of alkaline
phosphatase activity
Part 2:
Fluorimetric method for cheese
Voting terminated on 2014-12-10
DRAFT INTERNATIONAL STANDARD ISO/DIS 11816-2
IDF 155-2
Scope
The method specifies a fluorimetric method for the determination of
alkaline phosphatase (ALP, EC 3.1.3.1) activity in cheese
The 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)
DRAFT INTERNATIONAL STANDARD ISO/DIS 11816-2
IDF 155-2
Note
In 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)
Principle
– The 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 minute
– The activity is measured by a continuous fluorimetric direct kinetic assay
– A non-fluorescent substrate (*) undergoes hydrolysis of its phosphate
radical producing a highly fluorescent product
– (*) 2’-[2-benzothiazolyl]- 6’-hydroxybenzothiazole phosphate
Principle
Advanced Instruments Fluorophos
ALP test system
Sample preparation
Remove rind or surface with a clean knife
Surface should not be contaminated with surface microflore (soft cheese
with moulded surface!)
Sample preparation
Sample preparation
Sample preparation: grinding
Keep sample in an airtight container
French Agency for Food, Environmental and Occupational Health Safety – Laboratory for Food Safety
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 mark
French Agency for Food, Environmental and Occupational Health Safety – Laboratory for Food Safety
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 fat
French Agency for Food, Environmental and Occupational Health Safety – Laboratory for Food Safety
EU-RL MMP
European Union Reference Laboratory for
Milk and Milk Products
Maisons-Alfort Laboratory for Food Safety
PROFICIENCY TESTINGS
Determination of Alkaline Phosphatase activity in cheese
Reporting PT 2013 & PT 2014
Anne-Cecile BOITELLE, Caroline DESBOURDES, Hanene GHEZZAL, Marina NICOLAS
17thWorkshop of the NRLs on Milk & Milk Products, October 2014
French Agency for Food, Environmental and Occupational Health Safety – Maisons-Alfort Laboratory for Food Safety
23, avenue du Général de Gaulle - F94706 Maisons-Alfort Cedex - Telephone: + 33 149 771 300 - Fax: + 33 143 689 762 www.anses.fr
COUNTRY (CITY)
LABORATORY NAME
PT1
PT2
Austria (Vienna)
Belgium (Melle)
Cyprus (Nicosia)
Czech Republic (Praha)
Finland (Helsinki)
France (Maisons-Alfort)
Germany (Kiel)
Hungary (Budapest)
2013
2013
2013
2013
2013
2013
-
2014
2014
2014
2014
2014
Ireland (Co. Kildare)
Italy (Brescia)
AGES - INZT
ILVO T&V
LCFAO - VETERINARY SERVICES
State Veterinary Institute Prague
Finnish Food Safety Authority Evira
Anses, Food safety laboratory of Maisons-Alfort
MRI, Max Rubner-Institute
Food Analytical National Reference laboratory, Food
and Feed Safety Directorate, NFCSO
Dairy Science Laboratory
IZSLER - Reference Centre for Bovine Milk Quality
2013
2013
2014
2014
Italy (Milan)
Netherlands (Wageningen)
State University of Milan - DeFENS
RIKILT
2013
2013
2014
2014
Poland (Pulawy)
National Veterinary Research Institute, department
of Hygiene of Food of Animal Origin
Instituto Nacional de Investigação Agrária e
Veterinária (INIAV), IP
University Of Ljubljana, Veterinary Faculty / National
Veterinary Institute Unit Kranj
Laboratorio Agroalimentario de Santander
Agroscope Liebefeld Posieux
UK NRL
2013
2014
-
2014
2013
2014
2013
2013
2013
2014
2014
2014
Portugal (Lisboa)
Slovenia (Ljubljana)
Spain (Santander)
Switzerland (Berne)
United Kingdom (Belfast)
15 participating laboratories
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)
Summary of collaborative trial statistical parameters
Alkaline Phosphatase in cream
 Often 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 approach
Alkaline Phosphatase in cream
 In 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 AP
1 liter cream (40% by volume) consists of:
600 ml skimmed milk containing 312500 mU AP
400 ml fat containing 5000000 mU AP
AP activity in cream (40%) is about 5,31 times AP
activity of milk
Alkaline Phosphatase in cream
AP activity in cream (40%) is about 5,31 times AP
activity of milk
Conclusion
For 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!
Thank you for your
attention!
Instituut voor Landbouw- en Visserijonderzoek
Eenheid Technologie & Voeding
Onderzoeksdomeinen 'Agrotechniek' en 'Productkwaliteit en -innovatie'
Burg. Van Gansberghelaan 115 bus 1
9820 Merelbeke
T +32 9 272 28 00
Onderzoeksdomeinen 'Voedselveiligheid' en 'Productkwaliteit en -innovatie'
Brusselsesteenweg 370
9090 Melle
T +32 9 272 30 00
www.ilvo.vlaanderen.be