DETERMINATION OF ALKALINE PHOSPHATASE ACTIVITY IN MILK AND MILK PRODUCTS BY FLUORIMETRIC METHOD

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1 Bull Vet Inst Pulawy 54, , 21 DETERMINATION OF ALKALINE PHOSPHATASE ACTIVITY IN MILK AND MILK PRODUCTS BY FLUORIMETRIC METHOD JOLANTA GRAŻYNA ROLA AND MACIEJ SOSNOWSKI Department of Hygiene of Food of Animal Origin, National Veterinary Research Institute, 24-1 Pulawy, Poland Received for publication July 2, 21 Abstract Sixty-four samples of cow pasteurised milk, 65 samples of pasteurised goat milk, three samples of raw cow milk, two samples of raw goat milk, and 58 cheese samples from pasteurised milk and 11 from raw milk were used in the study. The precision of the applied method was determined. Alkaline phosphatase activity of the most pasteurised samples was below the limits. Parameters of the method, like repeatability and reproducibility, fitted the criteria established for interlaboratory comparisons, which are included in the official reference standard parts 1 and 2. Key words: milk, cheese, pasteurisation, alkaline phosphatase, fluorimetry. Alkaline phosphatase (ALP) is the most important enzyme in dairy industry. Activity of this enzyme is related to the quality of the process of pasteurisation. During the heat treatment, its activity decreases about 5-fold (14). ALP is a membrane bound glycoprotein, common in animal tissues and microorganisms. The appearance of the enzyme was first recognised and characterised in 1925 by F. Demuth. It was shown that proper time-temperature combination, slightly more severe than for killing Mycobacterium tuberculosis, caused inactivation of ALP. Because of that, ALP became the indicator of the effectiveness of pasteurisation or addition of raw milk in pasteurised products (6). The quality of dairy products depends on milk quality from which the products are made. Contamination or addition of raw milk to pasteurised milk can cause the risk of pathogen contamination of the product. Wrong process of pasteurisation can influence the final product by denaturation of whey proteins, inactivation of enzymes, destruction of beneficial bacteria, or chemical changes, which can affect an increase or decrease in substrate availability for bacteria and enzymes. Because of that, the heat treatment of milk for consumption and production of dairy products should be limited. The lowest limit of proper process of heat treatment is defined as pasteurisation, and the parameter of this is the activity of ALP, which is inactivated during the process, and its presence indicates that the process was ineffective (1, 15). Approximately 3%-4% of ALP in milk is associated with the fat globule membranes, the rest of the enzyme is bound to the lipoproteins in skimmed milk fraction (9). Legislation of the European Union adapts the level of 35 mu L -1 of ALP activity as safe for consumption of milk and milk-based drinks (2). For cheese, this limit has not been established yet. To avoid the danger of cheese poisoning, it is required that cheese is made only from pasteurised milk (11). However, it is necessary to establish the limit, to check the product and the process of manufacture. According to the Food Safety Agency (AFSSA), tentative limits for cheese from pasteurised milk range from 2 to 1 mu g -1 (3). There is a possibility of making a mistake when analysing the positive result of ALP in cheese, caused by cheese microflora. However, it is possible to discriminate native ALP, which is less heatstable from ALP produced by microorganisms by testing cheese after repasteurisation at 62.8 C for 3 min. Here, the positive result is caused by microflora (11). The methods of determination of ALP in milk and dairy products are based on colorimetric reactions in which phenyl phosphate (Kay and Graham Method, Scharrer Method), or p-nitrophenyl phosphate (Aschaffenburg and Mullen Method), or phenolphthalein monophosphate (Kleyn Method) are used as substrates. The amount of colour developed is proportional to the activity of ALP. There are either instrumental techniques, like chemiluminescence, or amperometric and fluorimetric methods (6, 7, 12, 15). Official reference method for the measurement of ALP is the fluorimetric method ( and EN ISO ). It is adequate for measurement of ALP in cow, ovine, and caprine milk, whole, semi-skimmed and skimmed milk, milk-based drinks, and cheeses. It is suitable for the determination of ALP activity in raw

2 538 milk and milk after heat treatment with ALP activity over 2, mu L -1 after dilution of the sample. The Advanced Fluorophos assay is based on a fluorimetric substrate called Fluorophos, which when activated by ALP, is converted to a highly fluorescent product. The rate of fluorophore formation is monitored for 3 min in a fluorometer. The enzyme activity is calculated automatically in mu L -1 (for milk and milk-based drinks) or mu kg -1 (for cheese) (1). The study was undertaken because there are no full scale studies on the determination of ALP in dairy products by this new method in Poland. The subject of this study was therefore the monitoring of pasteurisation by the measurement of ALP in dairy products from the retail outlets of various producers and various types. Moreover, the repeatability and reproducibility of the method were determined. Material and Methods The samples of milk and cheese used in the study were from retail outlets. They were collected from 28 to 21. The samples of milk had different fat content, and were pasteurised under various conditions. There were 64 samples of cow milk and 65 samples of goat milk. For comparison, three samples of raw cow milk and two samples of raw goat milk were used. The cheese samples, made from cow and goat milk, were of various types: hard, soft, and cottage. Fifty-eight cheese samples were made from pasteurised milk and 11 from raw milk. Preparation of milk sample. Milk sample was prepared as described in (4). Preparation of cheese sample. Cheese sample was prepared as described in (5). Because of the absence of ALP activity in the centre part of cheese, the portion was cut between the 2 nd and 3 rd cm from outside. To avoid sample contamination with microbial phosphatase, the knife and other equipment were cleaned with ethanol. Cheese portion was mixed by blender. Sample of cheese weighing.5 g was transferred into the beaker. The 1 ml of ALP free milk was added to the sample and homogenised with Ultraturrax (Heidolph, Germany). ALP free milk was obtained by heating of whole pasteurised milk in water bath at 95 C by 5 min (5). Testing the samples for ALP activity. The cheese samples were examined on channel, which was calibrated before for cheese. The test portion of.75 ml was added to glass cuvettes containing 2 ml of Fluorophos substrate (Advanced Instruments Inc, USA), which was incubated before at 38 C for 1 min. The content of the cuvette was immediately mixed on the vortex. The samples were tested on Fluorophos FLM 2 (Advanced Instruments Inc., USA). Each sample was examined in two repetitions and the final result of ALP activity was the mean of two repetitions (4, 5). Scaling of results. For milk, the s obtained in apparatus in mu L -1 were rating of ALP activity in a sample. For cheese, the s indicated in apparatus were in mu kg -1 or mu L -1. To get result in mu g -1, the result in mu kg -1 or mu L -1 was divided by 1,. If the activity of ALP in the sample was over 2, mu kg -1, the sample was diluted (by dilution factor II) to obtain the below 2, mu kg -1. The obtained from apparatus was multiplied by dilution factor I (.5 g of cheese in 1 ml of milk without ALP, so dilution factor I = 2) and then by dilution factor II. Determination of parameter of the precision. The precision of the methods was determined by calculating repeatability and reproducibility. Repeatability was determined by multiplying the standard deviation (root of the total of variance of individual results for 3 analysts divided by 3) by 2.8. Reproducibility was determined by multiplying the standard deviation of all of the results by 2.8. The s of repeatability and reproducibility obtained in the study were compared with criteria established for interlaboratory comparisons, which are included in the standards and (4, 5). Results The results of ALP activity in pasteurised milk in comparison with the limit of 35 mu L -1 are presented in Fig. 1. The s of ALP activity of most samples fitted the limit of 35 mu L -1, and were below 1 mu L -1. One sample had the activity equal to 35 mu L -1 and the activity of two samples was over this limit: about 5 and 7 mu L -1. For comparison, the ALP activity of raw cow and goat milk are presented in Fig. 2. The activity was higher for cow milk and ranged between 6, 8, and even 1,, mu L -1, ALP activity of goat raw milk was about 2, mu L -1. The s of the precision of the method, like repeatability and reproducibility in each level of ALP activity fitted the criteria established for interlaboratory comparisons, which are included in the standard (4). The s obtained in the study with reference to the s from the standard are presented in Tables 1 and 2. Parameter of the precision of the method was determined for cow milk at the levels of 4, 1, and 35 mu L -1, and for goat milk at the levels of 2, 4, 35, and 5 mu L -1. The best reproducibility was obtained on the level of 1 mu L -1 for cow milk with fat content of 3.2% and equaled to mu L -1, for cow milk with fat content of 2% and for goat milk was obtained on the lowest level, and equaled to 8.4 and 6.97 mu L -1, respectively. Along with growth of ALP activity the reproducibility diminished. The s of reproducibility for cow milk with fat content of 3.2% ranged 3.23, 14.77, and mu L -1, respectively, for cow milk with fat content of 2% ranged 8.4, 11.47, and mu L -1, respectively. In the standard the limit of reproducibility, the s for cow milk equaled: 31.8, 51., and For goat milk the s of reproducibility were 6.97, 8.38, 18.68, 49.24, and mu L -1, respectively. The s of reproducibility in the standard equalled 1.96, 2.55, 28.71, 127.9, and Similarly, the repeatability was the best on the lowest level for each kind of milk, and diminished along with growth of ALP activity.

3 539 ALP activity in pasteurised cow and goat milk 8 7 ALP activity (mu/l) Fig. 1. ALP activity in pasteurised cow and goat milk. Samples 1-36: cow milk with fat content of 3.2%, 37-67: cow milk with fat content of 2.%, goat milk. ALP activity in cow and goat raw milk ALP activity (mu/l) Fig. 2. ALP activity in cow and goat raw milk. Samples 1-3: cow milk, 4-5: goat milk.

4 54 ALP activity in cheese from pasteurised cow and goat milk ALP activity (mu/g) Fig. 3. ALP activity in cheese from pasteurised cow and goat milk. Sample 1-17: hard cheese from cow milk, 18-28: soft cheese from cow milk, 29-43: cottage cheese from cow milk, 44-46: mozzarella cheese from cow milk, 47-54: cottage cheese from goat milk, 55-6: hard cheese from goat milk. ALP activity in cheese from raw cow milk ALP activity (mu/g) Fig. 4. ALP activity in cheese from raw cow milk.

5 541 Table 1 The s of repeatability and reproducibility with reference to the s of the standard for cow and goat milk (4) LEVEL OF ALP ACTIVITY (mu L -1 ) COW MILK WITH 3.2 % OF FAT REPEATABILITY REPRODUCIBILITY COW MILK WITH 2. % OF FAT REPEATABILITY REPRODUCIBILITY WHOLE GOAT MILK REPEATABILITY REPRODUCIBILITY Table 2 The s of repeatability and reproducibility with reference to the s of the standard for cheese (5) LEVEL OF ALP ACTIVITY (mu g -1 ) ,5 REPEATABILITY REPRODUCIBILITY The results of ALP activity for cheese of pasteurised cow and goat milk in comparison with the limit of 1 mu g -1 are presented in Fig. 3. For comparison, the s of ALP activity in raw cow milk cheese are presented in Fig. 4. Among 69 cheese samples, 46 were of cow pasteurised milk cheese (17 samples of hard cheese, 15 samples of cottage cheese, 11 samples of soft cheese, and three samples of mozzarella cheese), nine of raw cow milk hard cheese, and 14 of goat pasteurised cheese (five samples of hard cheese and nine of cottage cheese). ALP activity of most samples of pasteurised milk cheese fitted the tentative limit of 1 mu g -1. Two samples of hard cheese and two samples of soft cheese camembert had ALP activity above the limit and ranged over consecutively 19, 9 and 34 mu g -1. Among the samples of hard cheeses, which fitted the limit, the mean of ALP activity equaled 2.36 mu g -1, for soft cheeses 1.45 mu g -1, for cottage cheeses -.83 mu g -1, and for mozzarella cheeses.39 mu g -1. Among the samples of goat milk cheeses, the mean ALP activity for cottage cheeses was 1.75 mu g -1 and for hard cheeses 2.55 mu g -1. For cheeses from raw cow milk, the ALP activity ranged from about 1,9 to 2,2 mu g -1, and the mean equalled 1, mu g -1. The s of the precision of the method, like repeatability and reproducibility in each level of ALP activity fitted criteria established for interlaboratory comparisons, which are included in the standard (5). The parameters of precision of the method were determined on the levels of 2, 27, 9, and 2,5 mu g -1. The best repeatability was obtained in the lowest level and equalled.37 mu g -1. Along with growth of ALP activity, the repeatability diminished, and equalled for respective levels consequently 26.2, 8.82, and 213 mu g -1. Similarly, the reproducibility was the best on the level of 2 mu g -1, amounted to.48, and diminished along with growth of ALP activity to mu g -1, 18.6 mu g -1, and mu g -1 for respective levels.

6 542 Discussion The study confirmed the efficiency of the fluorimetric method for the determination of ALP in milk and cheese according to the standard and the new standard under laboratory conditions. Moreover, the competence of analysts and proficiency of the method in laboratory was evidenced in Proficiency Testing organised by LGC Standards in five rounds in The fluorimetric method for the determination of ALP activity in milk is commonly used in dairy industry to verifiy the completeness of pasteurisation process of milk, however, this method for the determination of ALP activity in cheese according to EN ISO is still on the stage of verification and validation in laboratories, so there are no examples of results for this method in the literature. The methods, which are applied for monitoring of ALP activity in cheese are colorimetric, for example Scharer s method, where ALP activity is expressed as micrograms of phenol equivalent/.25 g of cheese. A below 1 µg of phenol/.25 g of cheese indicates that the pasteurised process was proper (11). Other method is fluorimetric method, based on 4-methylumbelliferyl phosphate substrate, which releases a highly fluorescent product, similarly as in Fluorophos method, but with results expressed as units of fluorescence, which correspond to nanograms of fluorescent product and ALP activity in sample examined (15). The results of ALP activity in various type of cheese have been presented at the 11 th Workshop of the EU CRL for Milk and Milk Products. The Italian researchers illustrated that extra hard cheeses had the ALP activity on the level of 4 mu g -1, semi-hard cheeses at.8 mu g -1, and pressed cheeses at.6-.9 mu g -1 (8). In French studies of 39 samples of soft cheese Camembert from pasteurised milk, the mean result of the assay was 2.83 mu g -1, the single result ranged from 1 to 5 mu g -1. The Camembert from thermised milk had mean of ALP activity at the level of 729 mu g -1, and the Camembert from micro-filtrated milk had a higher mean activity than thermised, equaled 93 mu g -1. In 22 samples of cheese Brie from pasteurised milk, ALP activity ranged from <2 to 6, and the mean was Brie from thermised milk showed ALP activity on the level of 83 mu g -1. ALP activity in each type of products, both milk and cheese, was parallel to the s obtained in products examined in other countries presented during the Workshop. Most of the examined products had ALP activity fitted to the criteria from the standards (3-5). Our study supports the tentative limits of ALP activity in cheese and indicates that the limit for milk is justifiable and suitable for both cow and goat milk. Both methods for milk and cheese - in spite of initial stage of determination of ALP activity in cheese by reference fluorimetric method (this method was validated and verified in laboratory, and the requirements of precision of the method were included in part 1 and 2), are fitted. The comparison of results for ALP activity in our studies and in studies presented at Workshop showed that the majority of examined cheeses fitted to the tentative limits. In spite of all producers declaration to use only pasteurised milk for the production of cheeses, ALP activity of four samples significantly differed from the rest. It probably attested to improper process of pasteurisation of milk, addition of raw milk, or microfiltration applied. References 1. Ardo Y., Lindblad O., Qvist K.B.: Study of methods to routinely monitor heat load to cheese milk. Int Dairy J 1999, 9, Commission Regulation (EC) No. 1664/26 of 6 November 26 amending Regulation (EC) No. 274/25 as regards implementing measures for certain products of animal origin intended for human consumption and repealing certain implementing measures. 3. Desbourdes C., Nicolas M, Boitelle A.C.: Phosphatase Activity in Cheese. AFSSA, CRL Milk and Milk Products. 11 th Workshop of the EU CRL for Milk and Milk Products, Vienna, :26 Milk and milk products. Determination of alkaline phosphatase activity. Part 1: Fluorimetric method for milk and milk-based drinks. 5. :23 Milk and milk products. Determination of alkaline phosphatase activity. Part 2: Fluorometric method for cheese. 6. Fox P. F., Kelly A. L.: Indigenous enzymes in milk: overview and historical aspects-part 2. Int Dairy J 26, 16, Murthy G.K., Kleyn D.H., Richardson T., Rocco R.M.: Alkaline phosphatase methods. In: Standard Methods for the Examination of Dairy Products, edited by Marshall R. 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J AOAC Int 26, 4, Vamvakaki A.N., Zoidou E., Moatsou G., Bokari M., Anifantakis E.: Residual alkaline phosphatase activity after heat treatment of ovine and caprine milk. Small Rumin Res 26, 65, Wilińska A., Bryjak J., Illeova V., Polakovic M.: Kinetics of thermal inactivation of alkaline phosphatase in bovine and caprine milk and buffer. Int Dairy J 27, 17, Yoshitomi K.: Alkaline phosphatase activity in cheeses measured by fluorometry. Inter J Food Sci Technol 24, 39,