Alkaline Phosphatase (ALP) is an enzyme that is naturally found within all the raw milks that is utilized to determine the quality of pasteurization of milk. A complete pasteurization process can deactivate the enzyme below levels that can be detected by traditional methods. Because the stability of the heat of ALP is higher than the stability of pathogens that could be found in milk, it serves as a metric of security. However, failure for detection of ALP activity is not a mean an item is safe from pathogens.

Alkaline Phosphatase

Alkaline phosphatase is one of the proteins which was first discovered by Suzuki. It is abundantly found in nature and can be found in numerous tissues of the human body, including kidneys, a sliver of blood cells and bone. It is also found in milk as well as other bodily fluids from a variety of species at different levels. Alkaline Phosphatase is a membrane bound glycol protein that has sialic acid being the sugar moiety.

It is a phosphomonoesterase enzyme which catalyzes the hydrolysis monoesters of the phosphoric acid (atalkaline pH) which produces alcohol and phosphate. The abundance of the enzyme in the nature and significance for this enzyme within biological processes has resulted in ALP activities tests among the top frequently tests for leper-formed enzymatics. Alkaline phosphatase is one of the more than 60 enzymes that are found in bovine milk that is in raw form.

Principle of Alkaline Phosphatase test

Pasteurization is a crucial step for the production of milk that has been proven safe and is free of pathogens. Alkaline Phosphatase is an enzyme naturally found in milk, but it is destroyed at a temperature that is close to the temperature of pasteurization. The Alkaline Phosphatase test can be utilized to identify whether milk has been properly pasteurized or if it was affected by raw milk following pasteurization. This test is based upon the idea that the enzyme known as alkaline-phosphatase in raw milk releases phenol from a disodium-para-nitro the phenyl phosphate. It forms the yellow-colored complex with alkaline pH (Scharer 1943). The intensity of the yellow color produced is proportional to level of activity produced by the enzyme. The intensity of the colour is determined through direct comparison with conventional colour discs inside the Lovibond comparator. This test does not apply to milk that is sour or milk preserved using chemical preservatives.

Purpose of Alkaline Phosphatase test

• To validate the Pasteurization of Milk and Milk product

Requirement for Alkaline Phosphatase test

• Water-Bath – is maintained at 37+°C, thermostatically controlled.
• Comparator – with special discs of standard colour glasses calibrated in µg p-nitrophenol per ml milk, and 2 x 25 mm cells.
• Test Tubes – of size 16 x 1.50 mm and rubber stoppers to fit.
• Pipettes – 1, 5, and 10 ml.
• Filter Paper – Whatman No. 2 or equivalent.
• Litmus Paper

Reagents

• Sodium Carbonate-Bicarbonate Buffer – Dissolve 3.5 g of anhydrous sodium carbonate and 1.5 g of sodium bicarbonate in one liter of distilled water.
• Buffer Substrate – Dissolve 1.5 g of disodium p-nitrophenyl phosphate in one liter of sodium carbonate-bicarbonate buffer. This solution will be stable if it is stored in a fridge at 4°C or less for a month, however a colour control test should be performed on the stored solution.

The procedure of Alkaline Phosphatase test

1. Pipette 5ml buffer substance into a dry, clean test tube. Then, add 1 ml of milk to be tested. Stop this tube. Mix via inversion, and then place it in the water-bath
2. In addition, place a control tube within the water bath which contains 5 ml of buffer substrate and 1 ml of boiled dairy of a similar type as the milk under test, which is homogenized pasteurized low fat.
3. After two hours, take out the tubs from the water bath, flip each one and then read the color that developed using comparator and special disc, with the tube that contains the control for boiling milk is placed on the left side of the stand, and the tube that contains the sample being tested is placed on the opposite side. Take readings that are in between standard discs of color by adding either a plus (+) or plus (-) sign to the image of the closest standard.

NOTE – If artificial lighting is required when taking these measurements, an approved “daylight” source of light must be utilized.

Other Methods for Alkaline Phosphatase Measurement

1. Scharer Rapid Phosphatase Test

Scharer’s rapid Phosphatase test is based on the Kay as well as the Graham method, but provides an impressive reduction in testing duration (~75min). In short, ALP enzymatically cleaves a phosphate group from added di sodium phenyl phosphate substrate. The released phenol group is extracted with butanol and then reacted with 2,6-dichloroquinonechlorimidetoformindophenol, a blue color compound. In the controlled conditions of this test, increased ALP activation in the sample of milk causes the creation of vivid blue color. The intensity of blue is visually compared to the normal assets and that it can be read with an spectrophotometer. The results are reported as micrograms of phenol per milliliter milk.

Limitations of Scharer Rapid Phosphatase Test

Scharer method has many weaknesses that hinder its usage and its wide acceptance.

• The potential for contamination of phenols from poorly cleaned glassware, or from flavorants like vanillin cause false-positive evaluations. To deal with this problem, Murthy published several specific ways to clean glassware as well as utensils cleaning and selection.
• The essential reagents along with the created do phenol chromagen are unstable over time, leading to an increase in negatives Scharer.
• Although it is quick, costly, and easy to use, Scharer’s fast Phosphatase test is based on a subjective assessment of color development. It is not reliable at the point of no detection, or when dealing with products that are colored.

2. Aschaffenburg and Mullen Test

• In this experiment, p-nitro phenyl phosphate serves as the substrate for the reaction.
• The formation of a yellow-colored can be used to determine the amount of the nitro phenol that has been liberated. 
• This method is described as being a little less sensitive than Kayand Graham technique. 
• It also does not require extraction or prolonged incubation time. 

3. Fluoro Metric Assays

The analytical techniques that are based on the properties of fluorescence of specific compounds are widely employed in the biological and chemical sciences, particularly in enzymatic experiments where greater sensitivities are required. Fernley and Walker published one of the first assays using fluorometry to measure bovine ALP.

The fluorometric assays are based on the ALP-mediated release of a phosphate radical from a self-indicating substrate, such as 4-methylum belliferyl phosphate, to form a highly fluorescent product, 7-hydroxy-4- methyl-coumarin (4-methylumbelliferone), which has optimal fluorescence at pH 14 Yoshitomi. A mono-phosphorylated derivative of benzothiazole as a self-indicating substrate can be used in this reaction.

At present, a fluoro-genicaromatic orthophosphoric monoester (Fluorophos, Advanced Instruments Inc., Needham Heights, MA)is utilized to perform an ALP assays in milk and milk-based products. In this test it is a fluorescent compound called “Fluoro yellow,” is created by hydrolysis of the Fluorophos substrate, and is then analysed with an instrument called a fluorometer. The measure of fluorescence that is generated over time is linked to the enzymatic activity or the amount of ALP.

The specific test is said to be faster (reported as 3 min) easy to use and up 1000 times more accurate than colorimetric tests like the Scharer technique Fox as well as Kelly,. This assay can be used to detect small variations in milk processing Rocco.

An alternative assay for fluorometry was presented by Fenoll form, which can be used to determine the remaining ALP activities in broad range of liquid and solid dairy products. The technique is based on the ALP that hydrolyzes an L-type fluorescent substance, trifluoromethyl-b-um be lliferone phosphate into its highly fluorescent phenolate. The assay is performed in a reverse micellar medium composed of mixed buffer (2-amino-2-methyl-1-propanol buffer, pH 9.0, and borate buffer, pH 9.0) in dioctyl sodium sulfosuccinate/isooctane, at a temperature of 38degC. The duration of the test is 450s , and the limits of detection include 0.04, 0.4, and 0.22 percent (vol/vol) for whole milk, raw chocolate milk, milk and butter, respectively. Fenoll.

The sensitivity, precision, and reliability of the Fluoro Phos system has been compared to those of the A Schaffenburg as well as Mullen as well as Scharer colorimetric methods used for ALP tests in milk using raw bovine ALP additions, as well as different methods of heat treatment. The Fluorophos method has been found to be highly precise as well as repeatability, and is more efficient and less complicated to carry out than more traditional tests using color Payne or Wilbey.

A number of international regulatory and trade agencies have accepted fluorometric assays because they are reliable tests of the effectiveness of pasteurization in a wide range of dairy products. Recent reports have confirmed that the sensitiveness of fluorometric assays is within an area of 0.003 to 0.006 percent raw milk contamination, or approximately 25-50mU/L of ALP Black this is a huge improvement over the colorimetric method, with sensitivity ranging between 0.1 to 0.5 percentage of Claeys.

A fluorometric method has been recognized from FDA, NCIMS, AOAC International and The International Association for Standardization, and the International Dairy Federation, and is included within the PMO.

4. Chemiluminescence Method

Chemiluminescence provides another method of assessing ALP activity. Chemiluminescent assays for ALP analysis are based on the ALP-mediated dephosphorylation of adamantly1,2-dioxetansubstrates (e.g.,adamantyl-1,2-dioxetane phenyl phosphate). This kind of substrate is specifically identified and degraded by ALP to produce the phenoxide intermediate that is broken down and emits an extended light, which can be detected by aluminometer or refrigerated device for photon counting.

This technique provides a basic one-reagent test with the limitation of detection to 1zmol (603molecules) of the enzyme Kricka. A chemiluminescent assay that has sensitiveness and assay times comparable to fluorescent methods was devised to determine the amount of ALP in milk by Girotti. The results were consistent with the results obtained using quantitative and qualitative absorption spectrometry of ALP as well as the chemiluminescent technique gave precise results without the hassle of procedure and preparation of the sample.

A chemiluminescent test (Paslite, Charm Sciences Inc., Lawrence, MA) has been reviewed and approved by FDAand NCIMS. NCIMS as well as the International Association for Standardization/ International Dairy Federation (ISO22160/IDF209) and is in tandem assay that uses the fluorometric method. one of the two methods specifically mentioned within the 2009 PMO as being suitable for ALP testing on Grade A products made from milk.

A different chemiluminescent technique that is rapid is also approved by NCIMS in 2009 and is now an approved ALP test. It is known by the Fast Alkaline Phosphatase test (Charm Sciences Inc., Lawrence, MA) and is believed to have a limit for 20mU/L for ALP activity in milk HTST. This test is quick and requires only a small amount of sample and preparation. It is said to have the ability to respond in 45 seconds.

Limitation of Alkaline Phosphatase 

The ALP assay can be affected by a variety of variables, including;

• the composition of the product being tested,
• the ability of ALP to regain activity, 
• the presence of microbial ALP. 

The following topics are discussed to provide a detailed description of the issues to be considered when applying or conducting ALP results. ALP assay.

Compositional Factors

Painter and Bradley conducted ALP tests on cream and milk treated at various times and temperature settings to assess ALP stability by employing a fluorometric technique. The study showed that the increase in fat levels led to a higher remaining ALP activity.

Products that have high milk fat levels can have higher initial ALP values. The residual ALP levels in nonfat milk are estimated to be around half that of whole milk, when pasteurized at the HTST temperature and time at a minimum specifications.

Interfering Compounds and Conditions

• Dairy products that are colored like strawberry milk pose an obvious interfering issue when used in colorimetric ALP tests. Other non-pigmented food additives that contain reactive phenolic compounds, like vanillin (when converted to vanillic acid) and r-hydroxy benzoic acid and salicylic acid can affect the ALP assay’s substrate and give False positive results.
• Oxytetracycline-based antibiotics and penicillin containing phenolic moieties have been found to provide false positive results using colorimetric tests Manolkidis.
• Similar findings have been observed when using pesticides with polychlorinated biphenyls. This is due to the reactivity the phenyl groups Kumar.
• The alkaline Phosphatase function has been proven to be reduced by flavonoids, saccharides Kuzuya ascorbic acid, and flavonoids.
• Certain polyphenolic compounds found in cocoa may also be thought to exert an inhibitory impact on ALP.
• The sodium chloride (0.25M) is proven to decrease the ALP’s thermal stability by around 50 percent Linden.
• In contrast, a higher concentration of lactose improves the thermal stability of ALP Sanders. The ALP enzyme is susceptible to an irreparable loss of function at pH levels that are acidic that are typical of cultured milk and yogurt.

Microbial Alkaline Phosphatase

ALK Phosphatase produces by numerous bacteria and often has a higher thermal stability than bovine ALP, possibly increasing the likelihood of ales positive results.

References

• https://foodsafety.foodscience.cornell.edu/sites/foodsafety.foodscience.cornell.edu/files/shared/documents/CU-DFScience-Notes-Milk-Alk-Phosphatase-11-07.pdf
• https://www.cdrfoodlab.com/foods-beverages-analysis/alkaline-phosphatase-cow-milk/
• https://lupinepublishers.com/dairy-veterinary-science-journal/fulltext/validation-of-milk-product-pasteurization-by-alkaline-phosphatase-activity.ID.000113.php
• https://www.idbiotech.com/en/alkaline-phophatase-activitity-alp-in-milk-and-cheese/
• https://www.aicompanies.com/education-training/dairy-analysis/confirming-pasteurization/
• https://watermark.silverchair.com/0362-028x-42_10_794.pdf
• https://www.journalofdairyscience.org/article/S0022-0302(66)88122-5/fulltext
• https://www.dairyknowledge.in/content/alkaline-phosphatase-test-pasteurized-milk