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Instituut voor Landbouw- en Visserijonderzoek

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Title: Instituut voor Landbouw- en Visserijonderzoek


1
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 Eenh
eid Technologie Voeding www.ilvo.vlaanderen.be
2
Nationale Referentie Laboratoria - Melk en
MelkproductenLaboratoire National de référence
Lait et produits laitiers
4/07/2014
3
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 68C during 30 min
  • HTST Pasteurization 72C during 15 sec
  • When milk is pasteurized most of the enzymes are
    inactivated or their activity is greatly
    diminished

4
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 1C drop in processing temperature

5
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

6
Applicable to cheese?
  • Yes, but possibility of false-positive test
    results
  • Molds
  • Added cultures
  • Acidified product

7
Colorimetric method
ALP
  • p-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.

raw heated (1
raw) heated
8
The enzyme photo-activated substrates method
(EPAS) ISO 22160 / IDF 2092007
  • Hydrolysis of monophosphate esters at the proper
    pH and temperature

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
9
Marina Nicolas Workshop on heat treatment of
milk 6/5/206
Marina Nicolas Workshop on heat treatment of
milk 6/5/206
EU COMMUNITY REFERENCE LABORATORY FOR MILK
MILK PRODUCTS
10
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

11
  • 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)

12
  • 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 50C,
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)
13
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

14
Principle
Advanced Instruments Fluorophos ALP test system
15
Sample preparation
Remove rind or surface with a clean knife Surface
should not be contaminated with surface
microflore (soft cheese with moulded surface!)
16
Sample preparation
17
Sample preparation
18
Sample preparation grinding
Keep sample in an airtight container
French Agency for Food, Environmental and
Occupational Health Safety Laboratory for Food
Safety
19
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

20
French Agency for Food, Environmental and
Occupational Health Safety Laboratory for Food
Safety
21
Sample preparation
  • Shake gently
  • Intoduce 5ml in a centifugation tube
  • centrifuge at 1000g at 4C during 10 min to
    remove fat
  • three layers are obtained pellet, supernatant
    and surface fat

22
French Agency for Food, Environmental and
Occupational Health Safety Laboratory for Food
Safety
23
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
24
COUNTRY (CITY) LABORATORY NAME PT1 PT2
Austria (Vienna) AGES - INZT 2013 2014
Belgium (Melle) ILVO TV 2013 2014
Cyprus (Nicosia) LCFAO - VETERINARY SERVICES 2013 -
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 laboratories
25
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)

26
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27
Summary of collaborative trial statistical
parameters
28
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29
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30
Alkaline Phosphatase in cream
  • Often samples pasteurized cream with gt 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

31
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

32
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!

33
Thank you for your attention!
Instituut voor Landbouw- en Visserijonderzoek Eenh
eid Technologie Voeding Onderzoeksdomeinen
'Agrotechniek' en 'Productkwaliteit en
-innovatie' Burg. Van Gansberghelaan 115 bus
19820 MerelbekeT 32 9 272 28
00 Onderzoeksdomeinen 'Voedselveiligheid' en
'Productkwaliteit en -innovatie'
Brusselsesteenweg 3709090 MelleT 32 9 272 30
00 www.ilvo.vlaanderen.be
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