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Thermal Inactivation Characteristics of Alkaline Phosphatase in Ultrafiltered Milk1
Thermal Inactivation Characteristics of Alkaline Phosphatase in Ultrafiltered Milk, V. v. MISTRY Dairy Science Department
South DakotaState University Brookings 57007
ABSTRACT The objectives of this study were to determine the heat inactivation kinetics of alkaline phosphatase in UF milk and to study the effect of concentration of UF milk on beat inactivation of alkaline phosphatase. Equal quantities of alkaline phosphatase were added as raw milk to pasteurized skim milk and UF skim milk retentates (10.01 and 16.61% protein). Residual alkaline phosphatase was measured in all milk samples heated to 60"C for 20, 30, and 40 min and to 63°C for 10, 20, and 30 min. Heat inactivation of phosphatase was more rapid in the 16.61% protein retentate than in non UF skim milk and 10.01% protein retentate at both 60 and 63"C and for all holding periods. The inactivation constant for the 16.61% protein retentate was higher than for skim milk and 10.01% protein retentate. Addition of lactose to 16.61% protein retentate increased the heat resistance of alkaline phosphatase. Results suggest that the removal of lactose during UF alters the heat inactivation characteristics of alkaline phosphatase; thus, the use of this enzyme to measure the efficiency of pasteurization of highly concentrated UF milk is questionable.
INTRODUCTION Ultrafiltration technology is widely accepted as a preconcentrating step in cheese making (1, 6, 12, 15, 16) and is used commerciaUy for
Received September 6, 1988. Accepted November 11, 1988. iPublished with the approval of the Director of the South Dakota Agricuhural ExperimentStation as Publication Number 2350 in the Journal Series. 1989 J Dairy Sci 72:1112-1117
making many cheese varieties in Europe. In this country a cheese plant in Minnesota is currently setfing up for producfion of Cheddar cheese using the APV- CSIRO process (1) and expects to be in producuon within a year. UltrafiltraUon can thus be envisioned as a routine process in many dairy plants of the future. When milk is UF, all components are not concentrated in equal proportions (6, 7). Some components, such as soluble salts, vitamins, and lactose are løst into the permeate, whereas others (fat, protein, and associated minerals) are retained and concentrated. As a result, the physical, chemical, and microbiological properfles of UF milk are quite different from those of the original milk (4, 18, 20). Information on properties that may affect public health is vital to ensttre optimal product quality as well as safety. Pasteurization of milk has been recognized as a necessary step in the processing of dairy products for almost 60 yr (13) and was made mandatory to prøtect consumers from pathogens and to destroy spoilage bacteria. The efficiency of pasteurization of dairy products is commonly assessed by the alkaline phosphatase test, which was developed by Kay and Graham in 1935 (11). This test has since undergone many modifications (21), bur the basic principle of the test remains unchanged. Jenness and Patton (10) have stated that metallic compounds influence alkaline phosphatase activity, and in 1948 Sanders and Sager (22) reported that the beat inactivation of alkaline phosphatase depends on product composition. Nø information except a preliminary report (17) is currently available on the beat inactivation kinetics of alkaline phosphatase in UF milk. Such information is necessary for proper processing and for determining the efficiency of pasteurization of UF milk. The objectives of this study were to determine the heat inactivation kinetics of alkaline phosphatase in UF milk and to study the effect
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ALKALINE PHOSPHATASE IN ULTRAFILTEREDMILK of concentration of UF milk on heat inactivation of alkaline phosphatase. MATERIALS AND METHODS Pmparation of Milk and Ultrafiltered MIIk Samples
Ultrafiltration. Approximately 140 kg of pasteurized skim milk (alkaline phosphatase content of <1 ktg phenol/5 ml) prepared by the South Dakota State University Dairy Plant was UF at 54*C to approximately 5:1 volumetric concentration ratio (VCR) in an Abcor Spiral wound UF model 1/1 sanitary pilot plant unit (Wilmington, MA). Portions of the 5:1 UF skim milk were diluted to 3:1 with permeate. Part of the original skim milk and the UF milks were maintained at approximately 250C until required for experiments. Addition of Alkaline Phosphatase. Equal quantities of alkaline phosphatase (106 ktg phenol/5 ml) were added to milk samples described, by way of raw milk. Approximately 6.5 mi of raw milk/100 ml milk were required to attain the above concentration of phosphatase. Heat inactivation experiments were conducted within a day of addition of phosphatase. Effects of Lactose. A series of experiments were conducted to study the effect of lactose on heat inactivation of alkaline phosphatase. Five different samples were prepared: 1) 5:1 UF skim milk, 2) 5:1 UF skim milk diluted to the volume of the original skim milk with permeate, 3) 5:1 UF skim milk diluted to the volume of the original skim milk with distilled water, 4) 5:1 skim milk diluted to the volume of original skim milk with 5% lactose solution prepared in distilled water, 5) 5:1 UF skim milk plus 20% lactose. Alkaline phosphatase was added as described. Hemt Trøatmønt
One hundred grams of milk and UF skim milk samples were filled into dilution blank bottles and heated in a boiling water bath to 60°C in 1.5 min. The bottles were then submerged in water maintained at 60"C in a water bath and held for 20, 30, and 40 min. Samples were then cooled to 25°C in 2 min under running cold water.
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The same procedure was repeated at 63°C with holding times of 10, 20, and 30 min. Heat up and cool down times were 1.75 and 2 min, respectively. The five samples for the lactose study were heated to 60°C, held for 20 min, and then cooled to 25°C. Milk Analysis Milk samples to which raw milk was added were analyzed for total protein by the macroKjeldahl method using 6.38 as the factor for converting percentage N to percentage total protein. Residual phosphatase was determined in heated and cooled samples using the dialysis phosphatase method (14). A test was also conducted to determine if the recovery of phosphatase by the dialysis test was equal for skim milk and UF skim milk. This was accomplished by conducting the test on a sample of skim milk and UF skim milk to which equal quantities of raw milk were added. Statistical Analysis
The experiments at 60 and 63°C were replicated three times, and the experiments on the effects of lactose were replicated twice. All measurements of phosphatase were in duplicate. StaUstical analysis was conducted using the GLM procedure of SAS. RESULTS AND DISCUSSION Composition of Milk and Ultrafilterød Milk
As fat sometimes oils off when frozen milk or UF whole milk is thawed, skim milk was used for experiments in this study. The total protein content of skim milk and of 3:1 and 5:1 UF skim milk with added raw milk is shown in Table 1. The respective protein contents were 3.5, 10.1, and 16.61%, giving protein concentration factors of 1:1, 3.08:1, and 5.11:1. Heat Inactivation of Alkallnø Phosphatase
Recovery of Phosphatase by the Dialysis Test. Alkaline phosphatase content of raw milk Journal of Dairy Science Vol. 72, Nø. 5, 1989
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TABLE 1. Total protein content of skim milk and ultratiltered skim milk used in assessing heat inactivation of alkaline phosphatase. Milk typet 1:1 3:1 5:1
Total protein (%) 3.25 10.01 16.61
SE .009 .280 .230
~1:1 = Skim miik; 3:1 and 5:1 = UF skim milk. is approximately 1500 to 2000 I.tg phenol/5 g. When raw milk is UF, the high temperature used during UF lowers the phosphatase content of milk considerably; hence, for these experiments phosphatase was added to milk samples to attain a low phosphatase level (approximately 100 lxg phenol/5 g). Table 2 shows the amount of alkaline phosphatase recovered from skim milk and 5:1 UF skim milk to which equal quantities of raw milk were added. The quantities, 106.5 ~tg phenol/5 g for skim milk and 109.1 ~tg phenol/5 g for 5:1 UF skim milk, are not significantly different (P<.05) from each other. It was therefore concluded that the dialysis phosphatase test could be used to make valid comparisons between skim milk and UF skim milk. A problem encountered in conducting the phosphatase test on retentate was the difficulty in volumetrically measuring 5 ml with a pipet. This difficulty arose due to the high viscosity of retentate, especially the 5:1 UF skim milk. Five grams were therefore used for all experiment and for all milks including skim (1:1). Results were expressed as micrograms phenol/5 g. Heat lnactivation at 60 and 63 °C. The Residual phosphatase values in skim milk (1:1) and in 3:1, and 5:1 UF skim milks heated to 60°C and held for 20, 30, and 40 min are shown in Table 3. For each milk type the residual phosphatase values decreased with increasing holding time, e.g., from 24.19 ~tg phenol/5 g at 20 min to 12.29 Ixg/5 g at 30 min to 10.56 ~tg at 40 min for skim milk. This decrease in residual phosphatase with increasing holding time is due to denaturation of phospharase (22), which was expected, since phosphatase is a heat-sensitive enzyme. Except at 40-min holding, there was also a decrease in residual phosphatase value with increasing protein content for all milk types. Journal of Dairy Science Vol. 72, Nø. 5, 1989
The values for main effect of milk type were 15.68 ~tg phenol/5 g for skim milk, 12.81 I.tg phenol/5 g for 3:1 UF milk, and 8.93 ~tg/5 g for 5:1 UF milk. The value for 5:1 was significantly lower (P<.05) than that for skim milk and 3:1, indicating a faster rate of denaturation in the former than in the later two. There was nø significant difference (P<.05) between skim milk and 3:1 UF skim milk. The constant of inacUvafion, k value, (micrograms phenol/5 g per min), and the Dvalue (minutes for one log reduction) were calculated for the three milk types for the first 30 min of holding (Table 4). The inactivation constant for 5:1 UF skim milk (.102 I.tg phenol/ 5 g per min) was significantly higher (P<.05) than that for skim milk and 3:1 UF skim milk. The D value for 5:1 UF skim milk (22.5 min) was significantly lower (P<.05) than that for skim milk (27.2 min) and 3:1 retentate (26.5 min). As the low temperature, long time pasteurization of milk is conducted at 62.8°C for 30 min, inactivation studies were also conducted at 63°C. Table 5 shows residual phosphatase values for milks heated to 63°C and held for 10, 20, and 30 min. As at 60°C, the phosphatase values decreased with increased holding time and with increasing protein content except for the 30-min holding period. The value for main effect of milk type decreased with increasing protein content (1.56 ~tg phenol/5 g for skim milk, 1.3 ~tg phenol/5 g for 3:1, and .82 ~tg phenol for 5:1). The variability of measurements was high at the low phenol concentrations; hence, the differences observed were not statistically significant. The k value for skim milk, 3:1 UF milk, and 5:1 UF milk during the first 20 min were .277, .254, and .300 I,tg phenol/5 g per min, respectively. The D values for the three milk types
TABLE 2. Recovery of alkaline phosphatase in skim milk and ultrafiltered skim milk by the dialysis method. Milk type~ 1:1 5:1
Alkaline phosphatase (~tg phenol/5 g) 106.5 109.1
SE 8.8 7.1
tl:l = Skim milk; 5:1 = UF skim milk.
ALKALINE PHOSPHATASE IN ULTRAFILTERED MILK
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TABLE 3. lnactivation of alkaline phosphatase in skim milk and in ultrafiltered skim milk heated to 60°C. Milk typet Holding time
1:1
3:1
(min) 20 30 40 Main effects of milk ~pe
5:1 (~tg phenol/5 g)
SE 1.33 .92 1.25
24.19 12.29 10.56
SE 1.32 .50 .67
19.79 11.97 6.67 12.8P
15.60.
12.10 7.83 6.85
SE 2.09 1.47 .93
8.93b
'-bMcans with same sup¢rscript are not significandy different flom each other at P_<.05. tl:l = Skim milk; 3:1 and 5:1 = UF skim milk.
were 8.3, 9.0, and 7.6 min, respectively (Table 4). Results of studies at 60 and 63°C show that the thermal inactivation of alkaline phosphatase occurs faster in 5:1 UF skim milk than in nonU F milk or in UF skim milk of lower concentration. The current standard for pasteurization by the dialysis phosphatase test for milk and other fluid dairy products is <1.0 [tg phenol/5 ml, i.e., milk containing <1.0 ~tg phenol/5 ml is considered to be pasteurized at or above the m i n i m u m pasteurization temperature and time. The presence of >1.0 I.tg phenol indicates inadequate pasteurization. Because thermal inactivation of phosphatase occurs at a greater rate in 5:1 UF skim milk than in standard n o n - U F milk, 1.0 ~tg phenol is attained at lower than standard pasteurization temperature. The phosphatase test in its present form, therefore, does
not help determine the efficiency of pasteurizatjon of UF milk (5:1 and above).
Effect of Lactose on Heat Inactivation of Alkaline Phosphatase. During UF, lactose content of milk is reduced (6, 7). To understand why phosphatase is inactivated in UF milk more quicldy than in n o n - U F milk, this change in lactose content during UF was taken into account. When lactose was added to 5:1 UF skim milk either by way of permeate, as a solution in disfilled water, or as lactose powder, the resistance of phosphatase to heat inactivation increased (Table 6). Residual phosphatase in 5:1 UF skim milk and 5:1 UF skim milk diluted with water and heated to 60°C for 20 min were 3.59 and 3.64 ~tg phenol, respectively. When lactose was added to 5:1 UF skim milk as permeate the value was 5.92 ~tg phenol. A 5:1 U F skim milk diluted with 5% lactose solution had a phosphatase value of 6.36. The
TABLE 4. Inactivation kinetics of alkaline phosphatase in skim milk and ultrafiltered skim milk at 60 and 63"C. Milk type~ 60"C k2 D-Value, min 63"C k D-Value, min
1:1
3:1
5:1
.084' 27.2'
.087' 26.5"
.102b 22.5b
.277' 8.3'
.254' 9.0'
.300. 7.6'
"bMeans with same superscript in the same row are not significantly different from each other at P-<.05. q:l = Skim milk; 3:1 and 5:1 = UF skim milk. 2k = micrograms phenol/5 g per min. Joul'nal of Dairy Science Vol. 72, Nø. 5, 1989
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TABLE 5. lnactivation of alkaline phosphatase in skim milk and in ultrafiltered skim milk at 63"C. Milk typel Holding time
1:1
3:1
10 20 30 Main effects of milk type
5:1 (gg phenoF5 g)
(min)
3.62 .59 .47
SE .78 .16 .38
2.68 .92 .29
1.56'
SE .49 .20 .29
1.82 .34 .31
1.30"
SE .41 .07 .10
.82"
"-bMeans with same superscript are not significantly different from ¢ach othcr at P<_.05. 11:1 = Skim milk; 3:1 and 5:1 = UF skim milk.
residual phosphatase value was very high when 20% lactose was added to retentate (34.23 lag phenol). A 20% lactose retentate was prepared to simulate the composition of milk evaporated down to approximately one-fifth the original volume. These results suggest that the reduction in lactose during UF may be the cause for the increased thermal denaturauon of phosphatase in UF milk. This reasoning is supported by other studies, e.g., Daemen (5) studied the effects of spray drying milk on enzymes and stated that the thermoresistance of enzymes increased with an increase in total solids content of milk. Beilinsson (2), Pierre et ai. (19), and Van de Beek and Gerlsma (24) have stated that sugars protect enzymes and proteins against heat denaturation, whereas Sood and Kosikowski (23) concluded that the removal of lactose out of retentate during diafiltration accelerates the beat denaturation of whey proŒins. Blackwell and Kosikowski (3) studied the concentration phenomenon of alkaline phospha-
tase in milk by UF and observed that when skim milk was UF, activity increased threefold during the first 90 min, followed by a decline up to 150 min with an abrupt reducuon to zero after 180 min. An explanation was not provided for this abrupt reduction, but it may be due to the increasing reduction in lactose during continued UF at 52"C. This study shows that the alkaline phosphatase test taust be used with caution, particularly in dairy products with altered composition. As nø pasteurization standards for UF milk are currently available and as its phosphatase inacUvation characteristics are different from those of milk the area of heat destrucUon of bacteria in UF milk taust also be addressed. Some studies on the thermal death of bacteria in UF milk have been conducted (8, 9) but only with low retentate concentrations (_<2:1). These studies have shown either nø or very little effect of UF on thermal death times of bacteria. The present study shows that at low retentate concentraUons the change in composiUon is too
TABLE 6. Eff¢ct of lactos¢ on ina¢fivation of alkaline phosphatase in 20 min at 60"C. Milk type
Rcsidual phosphat¢ acfivity (gg phcnol/5 g)
5:1 Skim milk œtcmatc + Pcrmcat¢ + Distilled water
+ 5% Lactose solution + 20% Lactos¢
3.59 5.92 3.64
SE .23 1.09
6.36
.70 .58
34.23
1.50
Journal of Dairy Science VoL 72, No. 5, 1989
ALKALINE PHOSPHATASE IN ULTRAFILTERED MILK
minor to warrant a change in the thermal properties of enzymes. A similar conclusion may therefore be also drawn for bacteria. Thermal studies on bacteria are required for higher concentrations of retentates (>3:1) in which there is a significant reduction in lactose. Information derived from such studies coupled with information on the thermal inactivation of alkaline phosphatase in UF milk will aid in the development of proper pasteurization parameters and control tests for UF milk. ACKNOWLEDGMENTS
The author is grateful to D. Casper for providing assistance in protein determination and to L. Tucker for assistance in staustical analysis. REFERENCES 1 APV-Crepaco, Inc. 1986. The worlds first continuous Cheddar cheese process. Caseus (Dec.):l 1. 2 Beilinsson, A. 1 9 2 9 . Thermostabilization der eiwiblostmgen mit ronrzuck¢r und glyøerine. Biochem. Z. 213:399. 3 Blackwell, J. H., and F. V. Kosikowski. 1979. Concentration phcnomenon of alkaline phosphatase ha goat's and cow's milk by ultrafiltration. J. Dairy Sci. 62(Suppl. 1): 59. (Abstr.) 4 Brule, G., J.-L. Maubois, and J. Faquant. 1974. Etude de la teneur en elements minerattx des produits obtenus lors de l'ultrafiltration du lait sur membrane. Le Lait 539-540: 600. 5 Daemen, A.L.H. 1981. The destruction of enzymes and bacteria during spray-drying of milk and whey. I. The thermoresistance of some enzymes and bacteria in milk and whey with various total solids contents. Neth. Milk Dairy J. 35:133. 6 Glover, F. A. 1985. Ultrafiltration and reverse osmosis for the dairy industry. Tech. Bull. 5, Natl. Inst. Res. Dairying, Reading, Engl. 7 Green, M. L., K. John Scott, M. Anderson, M.C.A. Griffha, and F. A. Glover. 1984. Chemical characterizatjon of milk concentrated by ultrafiltration. J. Dairy Res.
11 17
51:267. 8 Grieme, L. E., and D. M. Baxbano. 1983. Method for use of a differential scanning colorimeter for determination of bacterial thermal death times. J. Food Prot. 46:797. 9 Haggerty, P., and N. N. Porter. 1986. Growth and death of selected microorganisms in ultrafiltered milk. J. Food Prot. 49:233. 10 Jenness, R., and S. Patton. 1959. Principles of dairy chemistry. John Wiley and Sons, Inc., New York, NY. 11 Kay, H. D., and W. R. Graham, Jr. 1935. The phosphatase test for pasteurized milk. J. Dairy Res. 6:191. 12 Kealey, K. S., and F. V. Kosikowski. 1986. Cottage cheese from ultrafiltered skim milk retentates in indusu'ial cheese making. J. Dairy Sci. 69:1479. 13 Knutson, K. M., E. H. Marth, and M. K. Wagner. 1987. Important milestones in the history of milk pasteurizatjon. Dairy Food Sanit. 7:459. 14 Kosikowski, F. V. 1964. Dialysis phosphatase method for milk and all dairy products. J. Dairy Sei. 47:748. 15 Kosikowski, F. V., A. R. Masters, and V. V. Mistry. 1985. Cottage cheese from retentate-supplemented skim milk. J. Dalry Sci. 68:541. 16 Mattews, M. E., S. E. Co, C. H. Amtmdson, and C. G. Hill, Jr. 1976. Cottage cheese from ultrafiltered milk. J. Food Sci. 41:619. 17 Mistry, V. V. 1988. Heat inactivation of alkaline phøsphatase in ultrafiltered milk. J. Dairy Sci. 7 l(Suppl. 1):73. (Abstr.) 18 Mistry, V. V., and F. V. Kosikowski. 1985. Growth of lactic acid bacteria in highly concentrated ultrafiltered skim milk œtentates. J. Dairy Sci. 68:2536. 19 Pierre, A., G. Brule, J. Faquant, and M. Piot. 1977. Effect of heat treatment on whey soluble proteins contained in cow's and goat's milk UF retentate. Le Lait 569-570:646. 20 Rash, K. E., and F. V. Kosikowski. 1982. Behaviour of enteropathogenic Escherichia coli ba Camembert cheese made from ultratiltered milk. phosphatase. Food Sci. 47: 728. 21 Richardson, G. H., ed. 1985. Standard methods for the examinaåon of dairy products. 15th ed. Are. Publ. Health Assoc., Washington, DC. 22 Sanders, G. P., and D. S. Sager. 1948. Heat inactivation of phosphatase ha dairy products. J. Dairy Sci. 31:845. 23 Sood, V. K., and F. V. Kosikowski. 1979. Ultrafiltration of skim milk at high temperature. J. Food Prot. 42:958. 24 Van de Beek, M. J., and S. Y. Gerlsma. 1969. Preservatjon of the enzymatic activity of rennin during spray drying and during storage, and the effect of sugars and certain other additives. Neth. Milk Dairy J. 23:46.
Journal of Dairy Science Vol. 72, No. 5, 1989
South DakotaState University Brookings 57007
ABSTRACT The objectives of this study were to determine the heat inactivation kinetics of alkaline phosphatase in UF milk and to study the effect of concentration of UF milk on beat inactivation of alkaline phosphatase. Equal quantities of alkaline phosphatase were added as raw milk to pasteurized skim milk and UF skim milk retentates (10.01 and 16.61% protein). Residual alkaline phosphatase was measured in all milk samples heated to 60"C for 20, 30, and 40 min and to 63°C for 10, 20, and 30 min. Heat inactivation of phosphatase was more rapid in the 16.61% protein retentate than in non UF skim milk and 10.01% protein retentate at both 60 and 63"C and for all holding periods. The inactivation constant for the 16.61% protein retentate was higher than for skim milk and 10.01% protein retentate. Addition of lactose to 16.61% protein retentate increased the heat resistance of alkaline phosphatase. Results suggest that the removal of lactose during UF alters the heat inactivation characteristics of alkaline phosphatase; thus, the use of this enzyme to measure the efficiency of pasteurization of highly concentrated UF milk is questionable.
INTRODUCTION Ultrafiltration technology is widely accepted as a preconcentrating step in cheese making (1, 6, 12, 15, 16) and is used commerciaUy for
Received September 6, 1988. Accepted November 11, 1988. iPublished with the approval of the Director of the South Dakota Agricuhural ExperimentStation as Publication Number 2350 in the Journal Series. 1989 J Dairy Sci 72:1112-1117
making many cheese varieties in Europe. In this country a cheese plant in Minnesota is currently setfing up for producfion of Cheddar cheese using the APV- CSIRO process (1) and expects to be in producuon within a year. UltrafiltraUon can thus be envisioned as a routine process in many dairy plants of the future. When milk is UF, all components are not concentrated in equal proportions (6, 7). Some components, such as soluble salts, vitamins, and lactose are løst into the permeate, whereas others (fat, protein, and associated minerals) are retained and concentrated. As a result, the physical, chemical, and microbiological properfles of UF milk are quite different from those of the original milk (4, 18, 20). Information on properties that may affect public health is vital to ensttre optimal product quality as well as safety. Pasteurization of milk has been recognized as a necessary step in the processing of dairy products for almost 60 yr (13) and was made mandatory to prøtect consumers from pathogens and to destroy spoilage bacteria. The efficiency of pasteurization of dairy products is commonly assessed by the alkaline phosphatase test, which was developed by Kay and Graham in 1935 (11). This test has since undergone many modifications (21), bur the basic principle of the test remains unchanged. Jenness and Patton (10) have stated that metallic compounds influence alkaline phosphatase activity, and in 1948 Sanders and Sager (22) reported that the beat inactivation of alkaline phosphatase depends on product composition. Nø information except a preliminary report (17) is currently available on the beat inactivation kinetics of alkaline phosphatase in UF milk. Such information is necessary for proper processing and for determining the efficiency of pasteurization of UF milk. The objectives of this study were to determine the heat inactivation kinetics of alkaline phosphatase in UF milk and to study the effect
1112
ALKALINE PHOSPHATASE IN ULTRAFILTEREDMILK of concentration of UF milk on heat inactivation of alkaline phosphatase. MATERIALS AND METHODS Pmparation of Milk and Ultrafiltered MIIk Samples
Ultrafiltration. Approximately 140 kg of pasteurized skim milk (alkaline phosphatase content of <1 ktg phenol/5 ml) prepared by the South Dakota State University Dairy Plant was UF at 54*C to approximately 5:1 volumetric concentration ratio (VCR) in an Abcor Spiral wound UF model 1/1 sanitary pilot plant unit (Wilmington, MA). Portions of the 5:1 UF skim milk were diluted to 3:1 with permeate. Part of the original skim milk and the UF milks were maintained at approximately 250C until required for experiments. Addition of Alkaline Phosphatase. Equal quantities of alkaline phosphatase (106 ktg phenol/5 ml) were added to milk samples described, by way of raw milk. Approximately 6.5 mi of raw milk/100 ml milk were required to attain the above concentration of phosphatase. Heat inactivation experiments were conducted within a day of addition of phosphatase. Effects of Lactose. A series of experiments were conducted to study the effect of lactose on heat inactivation of alkaline phosphatase. Five different samples were prepared: 1) 5:1 UF skim milk, 2) 5:1 UF skim milk diluted to the volume of the original skim milk with permeate, 3) 5:1 UF skim milk diluted to the volume of the original skim milk with distilled water, 4) 5:1 skim milk diluted to the volume of original skim milk with 5% lactose solution prepared in distilled water, 5) 5:1 UF skim milk plus 20% lactose. Alkaline phosphatase was added as described. Hemt Trøatmønt
One hundred grams of milk and UF skim milk samples were filled into dilution blank bottles and heated in a boiling water bath to 60°C in 1.5 min. The bottles were then submerged in water maintained at 60"C in a water bath and held for 20, 30, and 40 min. Samples were then cooled to 25°C in 2 min under running cold water.
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The same procedure was repeated at 63°C with holding times of 10, 20, and 30 min. Heat up and cool down times were 1.75 and 2 min, respectively. The five samples for the lactose study were heated to 60°C, held for 20 min, and then cooled to 25°C. Milk Analysis Milk samples to which raw milk was added were analyzed for total protein by the macroKjeldahl method using 6.38 as the factor for converting percentage N to percentage total protein. Residual phosphatase was determined in heated and cooled samples using the dialysis phosphatase method (14). A test was also conducted to determine if the recovery of phosphatase by the dialysis test was equal for skim milk and UF skim milk. This was accomplished by conducting the test on a sample of skim milk and UF skim milk to which equal quantities of raw milk were added. Statistical Analysis
The experiments at 60 and 63°C were replicated three times, and the experiments on the effects of lactose were replicated twice. All measurements of phosphatase were in duplicate. StaUstical analysis was conducted using the GLM procedure of SAS. RESULTS AND DISCUSSION Composition of Milk and Ultrafilterød Milk
As fat sometimes oils off when frozen milk or UF whole milk is thawed, skim milk was used for experiments in this study. The total protein content of skim milk and of 3:1 and 5:1 UF skim milk with added raw milk is shown in Table 1. The respective protein contents were 3.5, 10.1, and 16.61%, giving protein concentration factors of 1:1, 3.08:1, and 5.11:1. Heat Inactivation of Alkallnø Phosphatase
Recovery of Phosphatase by the Dialysis Test. Alkaline phosphatase content of raw milk Journal of Dairy Science Vol. 72, Nø. 5, 1989
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TABLE 1. Total protein content of skim milk and ultratiltered skim milk used in assessing heat inactivation of alkaline phosphatase. Milk typet 1:1 3:1 5:1
Total protein (%) 3.25 10.01 16.61
SE .009 .280 .230
~1:1 = Skim miik; 3:1 and 5:1 = UF skim milk. is approximately 1500 to 2000 I.tg phenol/5 g. When raw milk is UF, the high temperature used during UF lowers the phosphatase content of milk considerably; hence, for these experiments phosphatase was added to milk samples to attain a low phosphatase level (approximately 100 lxg phenol/5 g). Table 2 shows the amount of alkaline phosphatase recovered from skim milk and 5:1 UF skim milk to which equal quantities of raw milk were added. The quantities, 106.5 ~tg phenol/5 g for skim milk and 109.1 ~tg phenol/5 g for 5:1 UF skim milk, are not significantly different (P<.05) from each other. It was therefore concluded that the dialysis phosphatase test could be used to make valid comparisons between skim milk and UF skim milk. A problem encountered in conducting the phosphatase test on retentate was the difficulty in volumetrically measuring 5 ml with a pipet. This difficulty arose due to the high viscosity of retentate, especially the 5:1 UF skim milk. Five grams were therefore used for all experiment and for all milks including skim (1:1). Results were expressed as micrograms phenol/5 g. Heat lnactivation at 60 and 63 °C. The Residual phosphatase values in skim milk (1:1) and in 3:1, and 5:1 UF skim milks heated to 60°C and held for 20, 30, and 40 min are shown in Table 3. For each milk type the residual phosphatase values decreased with increasing holding time, e.g., from 24.19 ~tg phenol/5 g at 20 min to 12.29 Ixg/5 g at 30 min to 10.56 ~tg at 40 min for skim milk. This decrease in residual phosphatase with increasing holding time is due to denaturation of phospharase (22), which was expected, since phosphatase is a heat-sensitive enzyme. Except at 40-min holding, there was also a decrease in residual phosphatase value with increasing protein content for all milk types. Journal of Dairy Science Vol. 72, Nø. 5, 1989
The values for main effect of milk type were 15.68 ~tg phenol/5 g for skim milk, 12.81 I.tg phenol/5 g for 3:1 UF milk, and 8.93 ~tg/5 g for 5:1 UF milk. The value for 5:1 was significantly lower (P<.05) than that for skim milk and 3:1, indicating a faster rate of denaturation in the former than in the later two. There was nø significant difference (P<.05) between skim milk and 3:1 UF skim milk. The constant of inacUvafion, k value, (micrograms phenol/5 g per min), and the Dvalue (minutes for one log reduction) were calculated for the three milk types for the first 30 min of holding (Table 4). The inactivation constant for 5:1 UF skim milk (.102 I.tg phenol/ 5 g per min) was significantly higher (P<.05) than that for skim milk and 3:1 UF skim milk. The D value for 5:1 UF skim milk (22.5 min) was significantly lower (P<.05) than that for skim milk (27.2 min) and 3:1 retentate (26.5 min). As the low temperature, long time pasteurization of milk is conducted at 62.8°C for 30 min, inactivation studies were also conducted at 63°C. Table 5 shows residual phosphatase values for milks heated to 63°C and held for 10, 20, and 30 min. As at 60°C, the phosphatase values decreased with increased holding time and with increasing protein content except for the 30-min holding period. The value for main effect of milk type decreased with increasing protein content (1.56 ~tg phenol/5 g for skim milk, 1.3 ~tg phenol/5 g for 3:1, and .82 ~tg phenol for 5:1). The variability of measurements was high at the low phenol concentrations; hence, the differences observed were not statistically significant. The k value for skim milk, 3:1 UF milk, and 5:1 UF milk during the first 20 min were .277, .254, and .300 I,tg phenol/5 g per min, respectively. The D values for the three milk types
TABLE 2. Recovery of alkaline phosphatase in skim milk and ultrafiltered skim milk by the dialysis method. Milk type~ 1:1 5:1
Alkaline phosphatase (~tg phenol/5 g) 106.5 109.1
SE 8.8 7.1
tl:l = Skim milk; 5:1 = UF skim milk.
ALKALINE PHOSPHATASE IN ULTRAFILTERED MILK
1115
TABLE 3. lnactivation of alkaline phosphatase in skim milk and in ultrafiltered skim milk heated to 60°C. Milk typet Holding time
1:1
3:1
(min) 20 30 40 Main effects of milk ~pe
5:1 (~tg phenol/5 g)
SE 1.33 .92 1.25
24.19 12.29 10.56
SE 1.32 .50 .67
19.79 11.97 6.67 12.8P
15.60.
12.10 7.83 6.85
SE 2.09 1.47 .93
8.93b
'-bMcans with same sup¢rscript are not significandy different flom each other at P_<.05. tl:l = Skim milk; 3:1 and 5:1 = UF skim milk.
were 8.3, 9.0, and 7.6 min, respectively (Table 4). Results of studies at 60 and 63°C show that the thermal inactivation of alkaline phosphatase occurs faster in 5:1 UF skim milk than in nonU F milk or in UF skim milk of lower concentration. The current standard for pasteurization by the dialysis phosphatase test for milk and other fluid dairy products is <1.0 [tg phenol/5 ml, i.e., milk containing <1.0 ~tg phenol/5 ml is considered to be pasteurized at or above the m i n i m u m pasteurization temperature and time. The presence of >1.0 I.tg phenol indicates inadequate pasteurization. Because thermal inactivation of phosphatase occurs at a greater rate in 5:1 UF skim milk than in standard n o n - U F milk, 1.0 ~tg phenol is attained at lower than standard pasteurization temperature. The phosphatase test in its present form, therefore, does
not help determine the efficiency of pasteurizatjon of UF milk (5:1 and above).
Effect of Lactose on Heat Inactivation of Alkaline Phosphatase. During UF, lactose content of milk is reduced (6, 7). To understand why phosphatase is inactivated in UF milk more quicldy than in n o n - U F milk, this change in lactose content during UF was taken into account. When lactose was added to 5:1 UF skim milk either by way of permeate, as a solution in disfilled water, or as lactose powder, the resistance of phosphatase to heat inactivation increased (Table 6). Residual phosphatase in 5:1 UF skim milk and 5:1 UF skim milk diluted with water and heated to 60°C for 20 min were 3.59 and 3.64 ~tg phenol, respectively. When lactose was added to 5:1 UF skim milk as permeate the value was 5.92 ~tg phenol. A 5:1 U F skim milk diluted with 5% lactose solution had a phosphatase value of 6.36. The
TABLE 4. Inactivation kinetics of alkaline phosphatase in skim milk and ultrafiltered skim milk at 60 and 63"C. Milk type~ 60"C k2 D-Value, min 63"C k D-Value, min
1:1
3:1
5:1
.084' 27.2'
.087' 26.5"
.102b 22.5b
.277' 8.3'
.254' 9.0'
.300. 7.6'
"bMeans with same superscript in the same row are not significantly different from each other at P-<.05. q:l = Skim milk; 3:1 and 5:1 = UF skim milk. 2k = micrograms phenol/5 g per min. Joul'nal of Dairy Science Vol. 72, Nø. 5, 1989
1116
M1STRY
TABLE 5. lnactivation of alkaline phosphatase in skim milk and in ultrafiltered skim milk at 63"C. Milk typel Holding time
1:1
3:1
10 20 30 Main effects of milk type
5:1 (gg phenoF5 g)
(min)
3.62 .59 .47
SE .78 .16 .38
2.68 .92 .29
1.56'
SE .49 .20 .29
1.82 .34 .31
1.30"
SE .41 .07 .10
.82"
"-bMeans with same superscript are not significantly different from ¢ach othcr at P<_.05. 11:1 = Skim milk; 3:1 and 5:1 = UF skim milk.
residual phosphatase value was very high when 20% lactose was added to retentate (34.23 lag phenol). A 20% lactose retentate was prepared to simulate the composition of milk evaporated down to approximately one-fifth the original volume. These results suggest that the reduction in lactose during UF may be the cause for the increased thermal denaturauon of phosphatase in UF milk. This reasoning is supported by other studies, e.g., Daemen (5) studied the effects of spray drying milk on enzymes and stated that the thermoresistance of enzymes increased with an increase in total solids content of milk. Beilinsson (2), Pierre et ai. (19), and Van de Beek and Gerlsma (24) have stated that sugars protect enzymes and proteins against heat denaturation, whereas Sood and Kosikowski (23) concluded that the removal of lactose out of retentate during diafiltration accelerates the beat denaturation of whey proŒins. Blackwell and Kosikowski (3) studied the concentration phenomenon of alkaline phospha-
tase in milk by UF and observed that when skim milk was UF, activity increased threefold during the first 90 min, followed by a decline up to 150 min with an abrupt reducuon to zero after 180 min. An explanation was not provided for this abrupt reduction, but it may be due to the increasing reduction in lactose during continued UF at 52"C. This study shows that the alkaline phosphatase test taust be used with caution, particularly in dairy products with altered composition. As nø pasteurization standards for UF milk are currently available and as its phosphatase inacUvation characteristics are different from those of milk the area of heat destrucUon of bacteria in UF milk taust also be addressed. Some studies on the thermal death of bacteria in UF milk have been conducted (8, 9) but only with low retentate concentrations (_<2:1). These studies have shown either nø or very little effect of UF on thermal death times of bacteria. The present study shows that at low retentate concentraUons the change in composiUon is too
TABLE 6. Eff¢ct of lactos¢ on ina¢fivation of alkaline phosphatase in 20 min at 60"C. Milk type
Rcsidual phosphat¢ acfivity (gg phcnol/5 g)
5:1 Skim milk œtcmatc + Pcrmcat¢ + Distilled water
+ 5% Lactose solution + 20% Lactos¢
3.59 5.92 3.64
SE .23 1.09
6.36
.70 .58
34.23
1.50
Journal of Dairy Science VoL 72, No. 5, 1989
ALKALINE PHOSPHATASE IN ULTRAFILTERED MILK
minor to warrant a change in the thermal properties of enzymes. A similar conclusion may therefore be also drawn for bacteria. Thermal studies on bacteria are required for higher concentrations of retentates (>3:1) in which there is a significant reduction in lactose. Information derived from such studies coupled with information on the thermal inactivation of alkaline phosphatase in UF milk will aid in the development of proper pasteurization parameters and control tests for UF milk. ACKNOWLEDGMENTS
The author is grateful to D. Casper for providing assistance in protein determination and to L. Tucker for assistance in staustical analysis. REFERENCES 1 APV-Crepaco, Inc. 1986. The worlds first continuous Cheddar cheese process. Caseus (Dec.):l 1. 2 Beilinsson, A. 1 9 2 9 . Thermostabilization der eiwiblostmgen mit ronrzuck¢r und glyøerine. Biochem. Z. 213:399. 3 Blackwell, J. H., and F. V. Kosikowski. 1979. Concentration phcnomenon of alkaline phosphatase ha goat's and cow's milk by ultrafiltration. J. Dairy Sci. 62(Suppl. 1): 59. (Abstr.) 4 Brule, G., J.-L. Maubois, and J. Faquant. 1974. Etude de la teneur en elements minerattx des produits obtenus lors de l'ultrafiltration du lait sur membrane. Le Lait 539-540: 600. 5 Daemen, A.L.H. 1981. The destruction of enzymes and bacteria during spray-drying of milk and whey. I. The thermoresistance of some enzymes and bacteria in milk and whey with various total solids contents. Neth. Milk Dairy J. 35:133. 6 Glover, F. A. 1985. Ultrafiltration and reverse osmosis for the dairy industry. Tech. Bull. 5, Natl. Inst. Res. Dairying, Reading, Engl. 7 Green, M. L., K. John Scott, M. Anderson, M.C.A. Griffha, and F. A. Glover. 1984. Chemical characterizatjon of milk concentrated by ultrafiltration. J. Dairy Res.
11 17
51:267. 8 Grieme, L. E., and D. M. Baxbano. 1983. Method for use of a differential scanning colorimeter for determination of bacterial thermal death times. J. Food Prot. 46:797. 9 Haggerty, P., and N. N. Porter. 1986. Growth and death of selected microorganisms in ultrafiltered milk. J. Food Prot. 49:233. 10 Jenness, R., and S. Patton. 1959. Principles of dairy chemistry. John Wiley and Sons, Inc., New York, NY. 11 Kay, H. D., and W. R. Graham, Jr. 1935. The phosphatase test for pasteurized milk. J. Dairy Res. 6:191. 12 Kealey, K. S., and F. V. Kosikowski. 1986. Cottage cheese from ultrafiltered skim milk retentates in indusu'ial cheese making. J. Dairy Sci. 69:1479. 13 Knutson, K. M., E. H. Marth, and M. K. Wagner. 1987. Important milestones in the history of milk pasteurizatjon. Dairy Food Sanit. 7:459. 14 Kosikowski, F. V. 1964. Dialysis phosphatase method for milk and all dairy products. J. Dairy Sei. 47:748. 15 Kosikowski, F. V., A. R. Masters, and V. V. Mistry. 1985. Cottage cheese from retentate-supplemented skim milk. J. Dalry Sci. 68:541. 16 Mattews, M. E., S. E. Co, C. H. Amtmdson, and C. G. Hill, Jr. 1976. Cottage cheese from ultrafiltered milk. J. Food Sci. 41:619. 17 Mistry, V. V. 1988. Heat inactivation of alkaline phøsphatase in ultrafiltered milk. J. Dairy Sci. 7 l(Suppl. 1):73. (Abstr.) 18 Mistry, V. V., and F. V. Kosikowski. 1985. Growth of lactic acid bacteria in highly concentrated ultrafiltered skim milk œtentates. J. Dairy Sci. 68:2536. 19 Pierre, A., G. Brule, J. Faquant, and M. Piot. 1977. Effect of heat treatment on whey soluble proteins contained in cow's and goat's milk UF retentate. Le Lait 569-570:646. 20 Rash, K. E., and F. V. Kosikowski. 1982. Behaviour of enteropathogenic Escherichia coli ba Camembert cheese made from ultratiltered milk. phosphatase. Food Sci. 47: 728. 21 Richardson, G. H., ed. 1985. Standard methods for the examinaåon of dairy products. 15th ed. Are. Publ. Health Assoc., Washington, DC. 22 Sanders, G. P., and D. S. Sager. 1948. Heat inactivation of phosphatase ha dairy products. J. Dairy Sci. 31:845. 23 Sood, V. K., and F. V. Kosikowski. 1979. Ultrafiltration of skim milk at high temperature. J. Food Prot. 42:958. 24 Van de Beek, M. J., and S. Y. Gerlsma. 1969. Preservatjon of the enzymatic activity of rennin during spray drying and during storage, and the effect of sugars and certain other additives. Neth. Milk Dairy J. 23:46.
Journal of Dairy Science Vol. 72, No. 5, 1989