Alkaline Phosphatase test (ALP test) is a standard quality and safety test that is mainly used in dairy industry to check whether milk and dairy products have been properly pasteurized. Alkaline phosphatase is an enzyme naturally present in raw milk and it is slightly more heat resistant than many harmful non spore forming pathogens that pasteurization aims to destroy such as Mycobacterium tuberculosis. Due to this it is used as a biological marker for pasteurization effectiveness.
This test works by measuring the residual activity of alkaline phosphatase enzyme in milk after heat treatment. When pasteurization is done properly with correct time and temperature the ALP enzyme is destroyed or inactivated. A negative ALP test (no or very low enzyme activity) indicates pasteurization was effective and the milk is considered safe. A positive test indicates active ALP is still present showing inadequate pasteurization or contamination of pasteurized milk with raw milk after pasteurization.
Principle of Alkaline Phosphatase test
Alkaline phosphatase (ALP) is an enzyme naturally present in raw milk and it shows slightly higher heat resistance than the non spore forming pathogens that are targeted during pasteurization. Due to this the complete destruction of ALP by heat is used as a reliable biological indicator to confirm that milk has been adequately pasteurized and it is safe for consumption.
This test works on the biochemical principle that active ALP hydrolyzes specific monophosphate esters in an alkaline condition. In this procedure the milk sample is incubated with a phosphate containing substrate. If active ALP is present in the sample (incomplete pasteurization or contamination with raw milk after pasteurization) the enzyme cleaves the phosphate group from the substrate.
Depending on method used this cleavage releases compounds that produce a visible colour change (blue or yellow) or it produces fluorescence or light. The intensity of colour fluorescence or light is proportional to the amount of active ALP present in the sample. If no colour or no signal is seen it indicates the enzyme has been destroyed and pasteurization was effective.
Objectives of Alkaline Phosphatase test
- To verify the effectiveness of pasteurization in milk and dairy products and to ensure adequate heat treatment for elimination of harmful non spore forming pathogens.
- To detect raw milk contamination in pasteurized dairy products after processing.
- To ensure food safety and protect public health by indicating pasteurization efficiency and reducing risk of milk borne illness.
- To verify compliance with legal regulations and public health standards required for commercial sale import and trading of dairy products.
- To monitor dairy plant quality control by identifying failures such as cracked pasteurizer plates operator errors or temperature fluctuations.
- To diagnose liver and organ diseases in clinical settings where increased ALP activity in blood indicates cellular suffering in organs or biliary tract.
- To detect bone growth dysfunction and related conditions such as osteomalacia rickets and hypophosphatasia.
- To assist in cancer diagnosis and disease monitoring as a biomarker for metastasis including bone liver prostate and breast cancer.
Requirement for Alkaline Phosphatase test
- Milk or dairy sample is required and it should be kept refrigerated at 0°C to 4.4°C (below 4.4°C) from collection till analysis.
- Testing should be done within 36 to 48 hours after sample collection.
- Liquid samples are mixed or homogenized properly before testing.
- Cheese samples should be free of visible mold and it is sampled before adding condiments peppers or spices as these can give false positive result.
- Glassware should be scrupulously clean and it should be protected from contamination during storage.
- Detergents used for glassware cleaning should be phenol free and closures or liners of test tubes should also be phenol free.
- Negative control sample is required (milk heated at high temperature such as 95°C for 1 minute) to destroy enzyme activity and to check reagents are free of contamination.
- Positive control sample is required (pasteurized milk spiked with small amount of raw milk such as 0.1% or 0.2%) to ensure reagents are working.
- Buffer control test is required to ensure colour development is due to ALP activity and not due to added colouring materials foreign substances or milk fat.
- Buffer and substrate are required to maintain alkaline condition and to react with ALP (example sodium carbonate-bicarbonate buffer and disodium p-nitrophenyl phosphate).
- Incubation system is required such as thermostatically controlled water bath or dry block incubator (35°C to 40°C depending on method).
- Measuring materials are required such as pipettes test tubes or cuvettes and result reading instrument such as colour comparator spectrophotometer fluorometer or chemiluminescent analyzer.
The procedure of Alkaline Phosphatase test
A. Procedure of Alkaline Phosphatase test (A&M method)
- 5 mL of buffer substrate (containing disodium p-nitrophenyl phosphate) is pipetted into a clean dry test tube.
- 1 mL of milk sample is added into the tube. It is closed with stopper and mixed thoroughly by inversion.
- A negative control is prepared in a second tube by mixing 5 mL of buffer substrate with 1 mL of boiled and cooled milk (enzyme is destroyed).
- Both test tube and control tube are kept in a water bath maintained at 37°C.
- Development of yellow colour is checked after 30 minutes and final reading is taken after 2 hours incubation.
- The intensity of yellow colour in test is compared with boiled control usually using standard colour disc comparator to determine enzyme activity.
B. Procedure of Scharer Rapid Phosphatase test
- 0.5 mL of milk sample is added into a test tube containing 5.0 mL of buffered substrate.
- The tube is incubated in water bath at 40°C for 15 minutes.
- The tube is removed and CQC solution (with copper catalyst) is added and mixed by inversion and it is re incubated for exactly 5 minutes.
- The tube is cooled in ice water bath below 10°C. Then butyl alcohol is added and tube is gently inverted several times to extract blue colour (indophenol) into alcohol layer.
- The tubes are kept upright and intensity of blue colour is compared with known colour standards or absorbance is measured by spectrophotometer.
Results of Alkaline Phosphatase test
Negative result– It indicates alkaline phosphatase enzyme is undetectable or it is reduced to very low acceptable level. It confirms pasteurization process was effective and dairy product is safe for consumption. Under current US and European regulation cow’s milk is considered negative when ALP activity is at or below 350 mU/L.
Positive result– It indicates active alkaline phosphatase is present in milk. It is a warning of pasteurization failure and milk may contain harmful pathogenic microorganism due to inadequate heating. It can also indicate post pasteurization contamination where pasteurized milk is mixed with raw milk. Sometimes it can be due to enzyme reactivation after high temperature treatment causing confusion in interpretation. In some cases positive result can be due to microbial ALP produced by heat resistant bacteria that survives pasteurization and not the native enzyme.
Uses of Alkaline Phosphatase Test
- It is used to verify pasteurization of milk cheese butter cream and other dairy products by confirming adequate heat treatment to destroy harmful non spore forming pathogens.
- It is used to detect raw milk contamination in pasteurized dairy products after heating process.
- It is used for monitoring dairy processing equipment and to detect processing errors such as temperature drop or cracked pasteurizer plate.
- It is used to ensure regulatory compliance and food safety as a standard quality control test required by health agencies.
- In human medicine it is used to measure blood ALP level for diagnosis and monitoring of liver diseases (hepatitis cirrhosis cholestasis) and bone conditions (rickets osteomalacia Paget’s disease bone metastasis).
- It is used as a biomarker for other physiological functions such as kidney and intestinal health stem cell activity and placental function during pregnancy.
- It is used in environmental monitoring for evaluating phosphate level in seafloor and monitoring phosphate removal from wastewater.
Limitation of Alkaline Phosphatase
- It does not guarantee pathogen free product. A negative test confirms ALP is destroyed by adequate heating but it cannot fully confirm that product is free from all pathogens or post pasteurization contamination.
- Enzyme reactivation can occur. In UHT or HTST treated products ALP can regain activity during storage and it can give false positive result. This is accelerated at storage temperature above 30°C or in presence of magnesium salts.
- Interference from microbial ALP can occur. Some heat resistant bacteria and molds can produce ALP that survives pasteurization and it can give false positive result which is difficult to differentiate from native ALP without further tests.
- It is not effective for some non bovine milks. Camel milk ALP is very heat resistant and it is not destroyed at standard pasteurization temperature. In equine (horse) milk ALP level is very low so it is not measured properly.
- It has limitation in highly acidic products. In cultured or high acid products like yogurt and buttermilk the acidic condition can inactivate enzyme giving false negative result.
- Chemical and flavouring interferences are seen. Vanillin can oxidize and cause false blue colour in colorimetric tests. Chocolate or cocoa can inhibit enzyme and give false negative. Unwashed glassware pesticides or antibiotics can also affect the result.
- It is affected by fat content. ALP binds with milk fat globules so fat content changes enzyme concentration and high fat products like cream require different baseline than skim milk.
Advantages of Alkaline Phosphatase
- It is a reliable indicator for food safety. ALP is naturally present in raw milk and it is slightly more heat resistant than harmful non spore forming pathogens. Its complete inactivation indicates sufficient heat treatment for safe consumption.
- It is easy for analytical detection. The enzyme activity can be measured rapidly by standard methods such as colorimetric fluorometric or chemiluminescent assays and it makes routine quality control practical in dairy industry.
- It is a valuable clinical biomarker. In human medicine ALP level in blood is used for diagnosis and monitoring of liver damage biliary tract diseases bone growth dysfunction and cancer metastasis.
- It has health and probiotic benefits. ALP supplementation is considered safe and it shows probiotic and prebiotic effect. It can help in protecting intestines of infants and it has potential in allergy intestinal diseases hepatitis acute kidney injury and diabetes.
References
- Agriculture Institute. (2026). Assessing milk pasteurization effectiveness.
- Albillos, S. M., Reddy, R., & Salter, R. S. (2011). Evaluation of alkaline phosphatase detection in dairy products using a modified rapid chemiluminescent method and official methods. Journal of Food Protection, 74(7), 1144–1154. https://doi.org/10.4315/0362-028X.JFP-10-422
- Angelino, P., Christen, G. L., Penfield, M., & Beattie, S. (1999). Residual alkaline phosphatase activity in pasteurized milk heated at various temperatures-measurement with the Fluorophos and Scharer rapid phosphatase tests. Journal of Food Protection, 62(1), 81-85.
- CDR FoodLab. (n.d.). Alkaline phosphatase (ALP) determination in cow milk.
- Comprehensive technical analysis of the alkaline phosphatase test in dairy quality assurance: Biochemical principles, laboratory methodologies, regulatory frameworks, and industry applications. (n.d.).
- Cornell University, Department of Food Science. (n.d.). Alkaline phosphatase (ALP) testing for milk pasteurization. Dairy Foods Science Notes.
- Dairy Knowledge Portal. (n.d.). Alkaline phosphatase test for pasteurized milk. National Dairy Development Board.
- Egger, L., & Brügger, C. (2023, November 14). Milk and milk products — Determination of alkaline phosphatase activity — Fluorimetric microplate method [Webinar]. Agroscope.
- FMCG Industry Solutions. (n.d.). Alkaline phosphatase test strips.
- Hardy Diagnostics. (2020). Alkaline phosphatase kit: Instructions for use.
- Hygiena. (n.d.). Validation of ZymoSnap for alkaline phosphatase (ALP) detection in dairy products.
- ISO / IDF. (2024). ISO 11816-1 | IDF 155-1: 2024 – Determination of alkaline phosphatase activity — Part 1: Fluorimetric method for milk and milk-based drinks.
- Lifeasible. (n.d.). Determination of alkaline phosphatase activity.
- Lightbody, L. G. (1955). Results with the Aschaffenburg-Mullen phosphatase test for pasteurized milk. Queensland Journal of Agricultural Science, 12(4), 175-178.
- Murthy, G. K., Cox, S., & Kaylor, L. (1976). Reactivation of alkaline phosphatase in ultra high-temperature, short-time processed liquid milk products. Journal of Dairy Science, 59(10), 1699-1710. https://doi.org/10.3168/jds.S0022-0302(76)84427-X
- Murthy, G. K., & Peeler, J. T. (1979). Rapid methods for differentiating reactivated from residual phosphatase in milk and cream: Collaborative study. Journal of Association of Official Analytical Chemists, 62(4), 822–827. https://doi.org/10.1093/jaoac/62.4.822
- NANOLAB. (2020). ALP test in milk pasteurization: Quality and reliability.
- Payne, C., & Wilbey, R. A. (2009). Alkaline phosphatase activity in pasteurized milk: A quantitative comparison of Fluorophos and colourimetric procedures. International Journal of Dairy Technology, 62(3), 308-314. https://doi.org/10.1111/j.1471-0307.2009.00503.x
- Pinzón-Sánchez, C., & Reinemann, D. (n.d.). Milking in conventional systems vs. automated systems—What is the difference? Division of Extension.
- Policies and regulations governing milk and dairy testing: A Wisconsin overview. (2024).
- Punoo, H. A. (2018). Validation of milk product pasteurization by alkaline phosphatase activity. Concepts of Dairy & Veterinary Sciences, 1(3). https://doi.org/10.32474/CDVS.2018.01.000113
- Rankin, S. A., Christiansen, A., Lee, W., Banavara, D. S., & Lopez-Hernandez, A. (2010). The application of alkaline phosphatase assays for the validation of milk product pasteurization. Journal of Dairy Science, 93(12), 5538-5551.
- Rocco, R. M. (2004). Alkaline phosphatase methods. In Standard Methods for the Examination of Dairy Products (17th ed., pp. 341-362). American Public Health Association.
- Rola, J. G., & Sosnowski, M. (2010). Determination of alkaline phosphatase activity in milk and milk products by fluorimetric method. Bulletin of the Veterinary Institute in Pulawy, 54(4), 537-542.
- shashsrd. (n.d.). Alkaline phosphatase test for milk safety. Scribd.
- Soares, C. F., Fonseca, L., Leite, M. O., & Oliveira, M. C. P. P. (2013). Application of Scharer’s quantitative method for the determination of residual alkaline phosphatase activity in standard Minas cheese. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 65(4), 1223-1230. https://doi.org/10.1590/S0102-09352013000400039
- Thermo Fisher Scientific Inc. (2012). Alkaline phosphatase (ALP) reagent.
- Tzereme, A., Koureas, M., Manouras, A., Malissiova, E., Soultani, G., Poulianiti, K., & Gogou, E. (2025). A comparative kinetic study on alkaline phosphatase thermal inactivation in different milk types. Kinases and Phosphatases, 3(2), 11. https://doi.org/10.3390/kinasesphosphatases3020011
- U.S. Food and Drug Administration. (2020). M-a-98 (Revision 1). Grade A Milk Search.
- Wernery, U., Fischbach, S., Johnson, B., & Jose, S. (2008). Evaluation of alkaline phosphatase (ALP), γ-glutamyl transferase (GGT) and lactoperoxidase (LPO) activities for their suitability as markers of camel milk heat inactivation. Milchwissenschaft, 63(3), 265-267.
- Yoganandi, J., Jain, A. K., Mehta, B. M., Wadhwani, K. N., & Aparnathi, K. D. (2014). Comparative studies on selected enzymes activities of camel, cow and buffalo milk. Journal of Animal Research, 21(2), 249-252. https://doi.org/10.5958/2277-8934.2014.00044.7
- Ziobro, G. C. (2001). Bacteriological analytical manual chapter 27: Screening method for phosphatase in cheese (January 2001 ed.). U.S. Food and Drug Administration.