ALP FAQs

Q1. What is Alkaline Phosphatase and why is it of value to the Dairy Industry?

A1. Alkaline Phosphatase (ALP) is an enzyme, which is naturally present in blood and the milk of all mammals. Enzymes are destroyed at specific time temperature conditions. ALP is destroyed at just below the time temperature conditions used to pasteurise milk (71.6 C/ 15seconds). As ALP is slightly less heat labile than most pathogenic bacteria, measurement of its destruction confirms proper pasteurisation.
This makes ALP an ideal test to demonstrate that milk has been correctly pasteurised.

Q2. How is ALP measured and how does the Fluorophos correlate with other tests?

A2. Enzymes are chemical keys, which unlock specific chemical bonds. When the first tests were introduced (in the 1930's) ALP was measured by inducing it to unlock chemical bonds, which released chemicals (phenol or paranitrophenol) that were measured by colour reaction.
In 1990 Advanced Instruments developed the Fluorophos ALP assay. This was based on the same chemistry as historic colorimetric tests with the difference that ALP (unlocking the same bonds) liberated a chemical, which was, measured fluorometrically.
As the chemistry of the two tests is basically the same one would expect the Fluorophos test to correlate well with historic tests. A very strong correlation has been verified by numerous comparative trials.
Ref: 1) Fluorometric determination of Alkaline Phosphatase in fluid dairy products. Rocco, RM Collaborative Study J Assoc. Off. Anal. Chem (Vol 73, No6, 1990).
2) Black RG et. Al. Evaluation of a fluorometric assay for Alkaline Phosphatase in fluid dairy products. The Australian J of Dairy Technology; 1992, 47:1 64-67.

Q3. What advantages does the Fluorophos offer over historic colorimetric tests?

A3. Colorimetric tests were developed in the 1930's. These tests are known to have a limit of detection of about 0.1% raw milk in the final pasteurised product and can only record a "pass" or a "fail" at this limit.
The Fluorophos on the other hand is much more sensitive and can measure as little as 0.003% raw milk (25 mU/L) in pasteurised milk. The Fluorophos provides clear differentiation between a "pass or fail" at a level of r 500 mU/L, this being the current level set to equate to about 0.1% raw milk in the pasteurised product. This "pass or fail" at a level of 0.1% raw milk was set simply to bring the Fluorophos "in line" with the historic colorimetric tests which are in the legislation. In the USA, the cut-off level has been set to 350 mU/L as an added measure of stringency to assure the proper pasteurization of milk.
The Fluoropohos does however offer a much greater reliability than colorimetric tests and it quantitatively measures residual ALP below these legal levels. This greater sensitivity is valuable for quality control purposes and an intrinsic part of of sound quality assurance (HACCP) programmes.
Consider a situation where raw milk is allowed to find access to the pasteurised milk. The residual ALP level will rise, due to the contribution from the unpasteurised milk. This increase will show in the Fluorophos test but not in colorimetric tests (unless over 0.1% raw milk is present). Raw milk is likely to contain pathogens. Thus regular monitoring of the Fluorophos base line level, which is usually around 25 to 75 mU/L, should form part of a Good Manufacturing Practice monitoring program in order to minimise any ingress of raw milk in the pasteurised milk.
In summary the greatly improved sensitivity of the Fluorophos makes it more reliable as a pass/fail test for pasteurised milk. In addition monitoring levels of ALP well below legal pass/fail standards provide valuable information on the hourly performance of the pasteurisation plant.

Q4. Is the Fluorophos test for ALP approved as a reference method?

A4. Yes the Fluorophos has been thoroughly evaluated in a number of trials resulting in its approval and inclusion in a number of national standards. Additionally it has been approved, as a reference method, by Standard Authorities of international standing such as The International Dairy Federation (IDF), The International Standards Organisation (ISO) and Association of Official Analytical Chemists (AOAC). Reference methods are accepted in trade disputes and are used to check the validity of "routine" methods. Some of the more important approvals are referenced below:

· ISO/DIS 11816 :1997 Milk and Milk based drinks - Determination of alkaline phosphatase activity - Fluorimetric method.
· AOAC Official Methods of Analysis, 2000 17th Edition, method 991.24
· IDF Standard 155A: 1999
· FDA LQAB/NCIMS Phosphatase Method, Form 2400
· Standard Methods for the Examination of Dairy Products, 1992 16th Edition, pp. 427-430
· New Zealand Dairy Board, Method Acceptance Number 98-13-01

The significance of these approvals is that, having been subjected to a large number of thorough interlaboratory trials, the Fluorophos has been proven to have a very high level of repeatability and Reproducibility. This establishes its status, making the results of Fluorophos ALP tests accepted as a reference method in any cases involving legal disputes and trade.

Q5 Can I use the Fluorophos ALP tests for milk from species other than the cow?

A5. Yes. Milk from all mammals contains ALP although the amount varies from species to species. Most of the milk that is pasteurised comes from cows, however there is a growing consumer interest in pasteurised milk and milk products from sheep and goats. There is about 5 times as much ALP in raw ewes milk as there is in cows milk and goats milk has about one fifth of the ALP found in raw cows milk.
(Reference: Dairy Industries International December 2000).
The average Fluorophos values are:
Raw goat milk = 100,000 mU/L
Raw cows milk = 500,000 mU/L
Raw ewe milk = 2,500,000 mU/L
Currently the same maximum ALP value is applied for pasteurised milks from each of these species i.e. 500 mU/L. This is somewhat illogical as it allows up to 0.1% raw cows milk, 0.02% raw ewe milk and 0.5% raw goat milk in each of the species pasteurised milk supply. A number of recent publications have argued for a tightening of these standards in order to minimise public health risks.

Q6 What developments are taking place which might affect the position of the Fluorophos compared with other test methods?

A6. There has been a significant increase recently in public health awareness regarding food poisoning outbreaks. There have also been concerns about pathogens such as Salmonella enteroditis, Listeria monocytogenes, E. coli and Mycobacterium paratuberculosis in milk and milk products. These issues have created such an atmosphere of public concern that every effort needs to be made by the Dairy Industry to reduce to an absolute minimum any risk of carry over of pathogens from raw milk into pasteurised products, in order to protect the image of milk.
A recent spate of papers have questioned the current status of ALP tests used to assess whether or not milk has been correctly pasteurised. Langridge (Dairy Industries International April 2000) demonstrated that the colorimetric test used in the UK (Aschaffenburg Mullen) can falsely pass pasteurised milk containing greater than 0.1% raw milk.

Angelino (Journal of Food Protection, Vol. 62, No. 1, 1999 pp81 - 85) reported that "the Scharer test may be indicating that milk is correctly pasteurised when in fact it is not". Both papers show that whilst the Fluorophos is reliable at the current legal level colorimetric tests are not.

Since the Fluorophos is able to accurately measure much lower than 0.1% raw milk in pasteurised milk, both authors also argue for a tightening of the legal standard. A paper by Harding (Control of pasteurised milk - Milk Industry International December 2000) also argues for lowering the amount of raw milk allowed to be present in correctly pasteurised milk. This paper additionally recommends that public health inspectors regularly check the base line level of ALP routinely recorded by the Fluorophos.

Q7. As the cost of testing with the Fluorophos System is higher than that of colorimetric tests, what cost justifications can be sited?

A7. Colorimetric tests simply give a "pass or fail" result (see A6). The Fluorophos gives much more meaningful quantitative values. In view of recent publications health inspectors are more likely to want to view quantitative data when inspecting pasteurisation records.
In addition the Fluorophos can be used to measure ALP, not only in milk but also in many milk and dairy products. It can be used to measure ALP in coloured/flavoured pasteurised products whereas colourimetric tests cannot.
The Fluorophos has been approved for the measurement of ALP in whole milk, semi-skimmed milk, skimmed milk, flavoured milks and cheese.

Q8 In demonstrating the Fluorophos System, what are the critical points that need to be observed with regard to testing, calibration etc.

A8. Calibration: Be sure that product is thoroughly mixed with the calibrator (improper mixing can result in calibration complications). Ensure that calibrators (A, B, C) are allowed to equilibrate to the proper temperature (38°C) for at least ten minutes before attempting to calibrate the instrument.

General Testing: Do not mix samples by inverting the tube over your finger (human skin contains sufficient ALP to skew test results). When mixing by inversion, gently shake - be sure not to agitate violently or otherwise incorporate bubble in the sample. A vortex mixer is preferable, especially with high fat products.

Handling of Reagents, etc. Observe Good Laboratory Practice in the storage of the Fluorophos ALP reagents and calibrators. Clean the 2.0-ml bottle dispenser with deionized water after each bottle of Substrate Buffer is dispensed. Be sure to return the dispenser bottle to refrigerated storage immediately after dispensing.
Demos: Transport reagents for demo purposes in climate controlled environment (i.e. a cooler) when outdoor temperature exceeds 25°C or prolonged transport in a warm vehicle is anticipated.

Q9 What type of stability do the reagents and calibrators have? What storage conditions apply?

A9. Prior to reconstitution, Fluorophos ALP Substrate and Buffer kits are stable for two years at 4°C. The actual expiration date is printed on the kit packaging. After reconstitution, the Fluorophos Substrate Buffer is stable for 60 days at 4°C. After the Fluorophos Substrate Buffer is dispensed into the heating block at 38°C, it is stable for eight hours.
Fluorophos ALP Calibrator kits are stable for 1.5 years at 4°C. The actual expiration date is printed on the kit packaging. The stability of opened ALP Calibrators is heavily dependent upon good laboratory procedure and the minimisation of time that the bottle is open and un-refrigerated. With proper handling, it can be expected that the Calibrators would be stable for 60 days. After the Fluorophos ALP Calibrators are dispensed into the heating block at 38°C, they should be used with two hours.
Prior to reconstitution, PhosphaCheck ALP Controls are stable for 1.5 years at 4°C. The actual expiration date is printed on the kit packaging. After reconstitution, PhosphaCheck ALP Controls are stable for 72 hours at 4°C. If microbial contamination becomes evident, discard the reconstituted controls.

Q10 What type of laboratories are best candidates for this test?

A10. Quality control and quality assurance laboratories in mid-sized to large dairy processing plants responsible for pasteurising raw milk and processing related dairy products are your best prospect. Companies that are truly dedicated to developing and maintaining market share based on a standardised product of high quality are most receptive to the benefits of the test.

Other candidates include:
ü Research & Development Laboratories focusing on developing value added dairy products (ice creams, yoghurts, cheese and cheese spreads, etc.)
ü Goats & Ewes milk processing plants or facilities involved with developing or processing products made from goat & ewes milk
ü Contract Testing Laboratories that perform various services for the Dairy Industry (typically for small to mid-sized dairy processing companies)
ü Universities and institutions for higher learning specialising in educating and/or providing on-going training in Dairy Science.
ü Governmental agencies responsible for monitoring the safety of milk and milk products

Q11 What questions should I ask prospective customers to ensure that my presentation is geared to their needs.

A11.
ü How many gallons of milk a day are processed in your plant?
ü What type of pasteurizer(s) is used?
ü How do you currently confirm that fluid milk and other dairy products are properly processed?
ü Do you currently perform an Alkaline Phosphatase Test? (If so, what test are you using? How many tests do you do each day?)
ü Are you satisfied with the ALP test you are currently performing (Simple test protocol/user friendly? Rapid method? Accurate/reliable? Cost? Other comments?)
ü What products are you currently processing in your plant (fresh pasteurised fluid milk? Flavoured Milk? Ice cream? Yoghurt? Cheese? Etc.?
ü What brand names has your company developed? How important are they to your revenues?
ü Is any R&D done in the plant? If so, what is the overall role R&D plays (i.e. mission to develop 4 new novelties/value added products per year? Continuous improvement? Product extensions? Etc.?
ü Is there any specific area where we may be of assistance in addressing Critical Control Points, improvement in overall product consistency/standardisation or future development plans?
ü Have you heard about our Fluorophos ALP Test before? (If so, where and what do you recall about the product?)
ü What products would you be interested in testing with the Fluorophos ALP Test? How many tests would you anticipate doing per day?