A Brief Review on Alkaline Phosphatase Methodology

Enzymes are organic catalysts which occur naturally in most raw foods. When milk is pasteurized most of the enzymes are inactivated or their activity is greatly diminished. The first reliable enzymatic test for determining efficiency of pasteurization was developed by Kay and Graham in England in 1933. It was based upon the inactivation of alkaline phosphatase.

The phosphatase test is applied to dairy products to determine whether pasteurization was done properly and also to detect the possible addition of raw milk to pasteurized milk. The thermal resistance of alkaline phosphatase has been considered to be greater than that of any nonsporeforming pathogens that might be found in milk. However, the recent outbreaks of disease traced to Listeria monocytogenes in pasteurized milk lead one to begin to question this conclusion.

Alkaline phosphatase is a monesterase that catalyzes the hydrolysis of monoesters.

Studies have shown that the amount of alkaline phosphatase in raw milk is variable. The activity of phosphatase per unit of milk seems to be inversely correlated to milk yield, reaching a minimum in 1 or 2 weeks after calving and rising gradually to a maximum in about 25 weeks. Breed, feed of the cow, or fat content of the milk do not appear to influence phosphatase activity. Alkaline phosphatase is associated with the fat globule of milk, i.e., it is adsorbed to the fat globule membrane surface.

Phosphatase tests currently described in Standard Methods for the Examination of Dairy Products are based on the principle that the alkaline phosphatase enzyme in raw milk liberates phenol from a disodium phenyl phosphate substrate (Scharer Method) or phenolphthalein from a phenolphthalein monophosphate substrate (Rutgers Method) when tests are conducted at suitable temperature and pH. The amount of phenol or phenolphthalein liberated from the substrate is proportional to the activity of the enzyme. Phenol is measured calorimetrically after its reaction with 2,6 dichloroquinone-chloroimide (CQC) to form indophenol. Phenolphthalein is detected by addition of sodium hydroxide.

While the Scharer rapid method is relatively simple and quick, it must be recognized that it does possess some inherent weaknesses. There is a constant hazard of phenol contamination from reagents, glassware, and stoppers. Reagents are unstable as is the color formed by the reaction of phenol with the dye. Visual measurement of color is sometimes difficult, particularly with borderline cases; and emulsification frequently occurs during the extraction of the phenol with butanol.

Phenolphthalein monophosphate is a very stable substrate which is easily hydrolyzed by alkaline phosphatase to yield free phenolphthalein. Our studies have revealed that the use of this substrate provides greater sensitivity than disodium phenyl phosphate. The high sensitivity is due to several factors, namely, the ease of color comparison, the high rate of hydrolysis, the elimination of variations because of specific color reaction and extraction, and the slight contribution of yellow color of milk fat to the pink color of phenolphthalein.

It is necessary to run both positive and negative controls when conducting a phosphatase procedure. A negative control is prepared by heating a product to 90°C for 1 minute followed by rapid cooling. Any color developing when a test is run on the control indicates contamination of reagents or presence of interfering coloring materials or both. A positive control is run as a check on the proper functioning of reagents. It is conducted by adding 0.2 ml of fresh, raw mixed-herd milk to 100 ml of raw milk which has been heated at 90°C for 1 minute, followed by rapid cooling to room temperature. One should obtain a positive result on this test.

Another control test should be run on samples which yield positive results in the initial analysis. This test is conducted in order to distinguish residual alkaline phosphatase from microbial alkaline phosphatase. Microbial phosphatases are considerably more heat resistant than is alkaline milk phosphatase. Therefore, it is possible to differentiate these enzymes by pasteurization of the sample in question and retesting. If there is no significant difference in the results of the test, one then concludes that the original positive result was due to microbial phosphatase.

Reactivated phosphatase sometimes occurs in high fat dairy products which have been ultrapasteurized, such reactivation occurring quickly when samples are stored at non-refrigerated (70-90°F) temperatures. A test has been developed which permits one to distinguish residual from reactivated alkaline phosphatase.

Alkaline phosphatase methodology is applicable to cheese. However, consideration must be given to the possibility of obtaining false-positive tests due to the possible presence of mold in the cheese. In the early 1940's, Scharer reported as follows: "Our recent work has indicated that yeast and some molds (a culture of Oidium lactis and Penicillium notatum) which grown on cheese under certain conditions will produce appreciable amounts of phosphatase, but that if the mold growth is removed before the cheese sample is prepared for testing purpose, no difficulty or false positive is encountered." Thus, sampling of cheese is a very important consideration, i.e., one must be certain that no mold is evident. In addition, it is highly recommended that cheese be sampled before the addition of condiments such as peppers or spices as these materials may also be responsible for false positive tests. Ideally, cheese samples should be placed in clean containers, refrigerated, and tested within 36 hours in order to be certain that no development of microbial phosphatase has occurred. Finally, if cheese is not properly stored in the marketplace there is a possibility that microbial growth may occur which could result in false positive results for the alkaline phosphatase test. Thus, sampling of cheese for phosphatase analysis should be conducted before cheese enters marketing channels.

ALKALINE PHOSPHATASE METHODOLOGY
Dick H. Kleyn, Ph.D., 
Dept. of Food Science 
Rutgers University 
New Brunswick, NJ 08903

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