( 1907 ) , is one of over 60 endogenous enzymes present in raw bovine milk ( schlimme et al . ,
1997 ) . since alp is inactivated by thermal treatment , reduced levels of alp activity are an indicator of adequate milk pasteurization ( rankin et al . , 2010 ) . the alp assay is widely used and recognized as the most appropriate indirect method for verifying complete milk pasteurization due to its rapidity and sensitivity ( rankin et al . , 2010 ) .
the united states food and drug administration ( fda ) outlines quantitative criteria for alp levels in cheese products ( united states food and drugs administration , 2001 ) ; the levels can be measured with a spectrophotometer and are expressed as micrograms of phenol per gram of cheese ( united states food and drugs administration , 2001 ) .
in contrast , korea uses a qualitative standard for determining alp levels in pasteurized milk products ( korea food and drug administration , 2015 ) , and the results are compared visually against a set of standards ( korea food and drug administration , 2015 ) .
the results of such a visual colorimetric method can be easily misinterpreted , which poses a huge public health risk , since contaminated milk is an important vehicle for the foodborne transmission of numerous pathogens ( klotz et al . , 2008 ) .
therefore , the interpretation of the results of alp assays for milk samples should be improved .
the aim of this study was to establish quantitative standards for alp in pasteurized milk by using a spectrophotometric method .
raw bovine milk was kindly provided by a cow farm located in gyeonggi province and stored at 4 for 1 h. the raw milk was divided into conical tubes in 10 ml aliquots and subjected to heat treatment for 0 , 10 , 20 , 30 , and 40 min at 63 using a heat block .
the heat - treated milk samples were used to determine alp cut - off values in properly pasteurized milk .
the determined cut - off value was verified using seven low - temperature and five ultra - high temperature ( uht ; heat treated for 3 sec at 135 ) pasteurized milk products purchased from a retail market in seoul , korea . in addition , the raw milk was heat treated for 5 min at 95 , regarded as high - temperature short - time ( htst ) pasteurized milk .
alp levels in the milk samples were analyzed using a spectrophotometer ( multiskan fc ; thermo fisher scientific , usa ) following a method outlined in the fda s bacteriological analytical manual ( united states food and drugs administration , 2001 ) .
two 0.5 ml samples ( test and boiled control samples ) were mixed with 0.5 ml of distilled water .
controls were heated in a boiling water bath for 2 min and then rapidly cooled in an ice bath .
five milliliters of 2-amino-2-methyl-1-propanol ( amp ) buffer substrate were added to both the test and boiled control and mixed by vortexing .
test samples and boiled controls were then immediately incubated in a 40 water bath for 15 min .
subsequently , the test samples were mixed with 0.2 ml 2,6-dichloro - quinonechloroimide ( cqc ) catalyst solution .
the mixture was immediately placed back in the 40 water bath for 5 min and then cooled in an icewater bath for 5 min .
next , 3 ml of butanol was added to the samples and mixed by inverting the tubes six times .
the mixtures were chilled in an ice - water bath for 5 min and then centrifuged for 5 min at 2400 g. the butanol ( upper ) phase was transferred to a new test tube .
the alp standard curve was created using a phenol standard solution according to the bam method ( united states food and drugs administration , 2001 ) .
one gram of pure phenol was added to 1 l of 0.1 n hcl and gently mixed .
one hundred microliters of the solution were added to 100 ml of amp buffer , and gently mixed .
finally , 0 , 0.25 , 0.5 , 1 , 2.5 , and 5 ml of the solution were added to 5 ml of amp buffer and then 0.5 ml of water was added to each tube .
the phenol standard solutions were incubated in a water bath for 15 min at 40. subsequently , the test samples were mixed with 0.2 ml cqc catalyst solution .
the mixture was immediately placed back in the 40 water bath for 5 min then cooled in an ice - water bath for 5 min .
next , 3 ml of butanol were added to the samples and mixed by inverting the tubes six times .
the mixtures were chilled in an ice - water bath for 5 min and then centrifuged for 5 min at 2400 g. the butanol ( upper ) phase was removed to a new test tube .
the standard curve was plotted as g phenol against absorbance and a linear function equation was calculated .
the absorbance of milk samples measured with a spectrophotometer was converted into g phenol / ml values using the standard curve .
the g phenol / ml milk value of the boiled blank was subtracted from the corresponding sample providing the final g phenol / ml milk for the sample .
the g phenol / ml milk value of each sample was analyzed for statistical significance using a student s t - test .
raw bovine milk was kindly provided by a cow farm located in gyeonggi province and stored at 4 for 1 h. the raw milk was divided into conical tubes in 10 ml aliquots and subjected to heat treatment for 0 , 10 , 20 , 30 , and 40 min at 63 using a heat block .
the heat - treated milk samples were used to determine alp cut - off values in properly pasteurized milk .
the determined cut - off value was verified using seven low - temperature and five ultra - high temperature ( uht ; heat treated for 3 sec at 135 ) pasteurized milk products purchased from a retail market in seoul , korea . in addition , the raw milk was heat treated for 5 min at 95 , regarded as high - temperature short - time ( htst ) pasteurized milk .
alp levels in the milk samples were analyzed using a spectrophotometer ( multiskan fc ; thermo fisher scientific , usa ) following a method outlined in the fda s bacteriological analytical manual ( united states food and drugs administration , 2001 ) .
two 0.5 ml samples ( test and boiled control samples ) were mixed with 0.5 ml of distilled water .
controls were heated in a boiling water bath for 2 min and then rapidly cooled in an ice bath .
five milliliters of 2-amino-2-methyl-1-propanol ( amp ) buffer substrate were added to both the test and boiled control and mixed by vortexing .
test samples and boiled controls were then immediately incubated in a 40 water bath for 15 min .
subsequently , the test samples were mixed with 0.2 ml 2,6-dichloro - quinonechloroimide ( cqc ) catalyst solution .
the mixture was immediately placed back in the 40 water bath for 5 min and then cooled in an icewater bath for 5 min .
next , 3 ml of butanol was added to the samples and mixed by inverting the tubes six times .
the mixtures were chilled in an ice - water bath for 5 min and then centrifuged for 5 min at 2400 g. the butanol ( upper ) phase was transferred to a new test tube .
the alp standard curve was created using a phenol standard solution according to the bam method ( united states food and drugs administration , 2001 ) .
one gram of pure phenol was added to 1 l of 0.1 n hcl and gently mixed .
one hundred microliters of the solution were added to 100 ml of amp buffer , and gently mixed .
finally , 0 , 0.25 , 0.5 , 1 , 2.5 , and 5 ml of the solution were added to 5 ml of amp buffer and then 0.5 ml of water was added to each tube .
the phenol standard solutions were incubated in a water bath for 15 min at 40. subsequently , the test samples were mixed with 0.2 ml cqc catalyst solution .
the mixture was immediately placed back in the 40 water bath for 5 min then cooled in an ice - water bath for 5 min .
next , 3 ml of butanol were added to the samples and mixed by inverting the tubes six times .
the mixtures were chilled in an ice - water bath for 5 min and then centrifuged for 5 min at 2400 g. the butanol ( upper ) phase was removed to a new test tube .
the standard curve was plotted as g phenol against absorbance and a linear function equation was calculated .
the absorbance of milk samples measured with a spectrophotometer was converted into g phenol / ml values using the standard curve . the g phenol / ml milk value of the boiled blank
was subtracted from the corresponding sample providing the final g phenol / ml milk for the sample .
the g phenol / ml milk value of each sample was analyzed for statistical significance using a student s t - test .
raw milk , a highly perishable , potentially hazardous food source , has been recognized as a significant vehicle for the foodborne transmission of many pathogens ( rankin et al . , 2010 ) .
many regulatory bodies have consequently mandated that milk be subjected to thermal treatment ( klotz et al . , 2008 ) .
different letters in the same column indicate statistical differences ( p<0.05 , student s t - test ) .
colorimetric , fluorometric , chemiluminescent , and immunochemical methods have been employed to detect residual alp activity for decades ( rankin et al . , 2010 ) . in dairy industry ,
the colorimetric assay is the most commonly and broadly adopted to verify pasteurization adequacy ( rankin et al . , 2010 ) . despite many advantages of being rapid , inexpensive , and convenient
, colorimetric alp assays might be unreliable especially in samples with alp levels of near the detection limits , because the interpretation depends on subjective visual assessments of color development ( murthy et al . , 1992 ) .
consequently , it is needed to develop a more objective methodology to confirm the results of colorimetric alp assays ( murthy et al . , 1992 ) .
in order to quantify the residual alp level in milk samples , a standard curve was generated using a phenol standard solution ( fig .
the linear function equation for the alp standard curve was y = 0.0047x + 0.0844 ( x , g phenol / ml milk ; y , absorbance at 650 nm ) and the correlation coefficient ( r ) was 0.9916 .
significant differences were apparent among the levels of residual alp in the milk samples heat - treated for 0 , 10 , 20 , 30 , and 40 min .
since the legal definition for pasteurization is 63 for 30 min , the cut - off value was set at 2 g phenol / ml of milk , which is just above the alp value of milk samples heat - treated for 30 min .
in addition , the mean value of residual alp in htst - pasteurized milk was 0 g phenol / ml milk .
the residual alp level of seven commercial low - temperature and five uht - pasteurized milk products was tested using the determined cut - off value .
the mean value of residual alp in commercial pasteurized milk products was 0 g phenol / ml milk ; none of the tested milk samples demonstrated levels higher than the cut - off value , suggesting that all tested milk products were properly pasteurized . according to the bam ,
the quantitative criteria for residual alp in cheese products differ by cheese type ( united states food and drugs administration , 2001 ) ; the maximum levels range from 12 to 20 g phenol / g cheese
. this cut - off value is higher than the value determined in the present study .
this might be due to cheese containing bacterial starter cultures , which could provide a source of alp ( fanni , 1983 ; hammer and olson , 1941 ) .
in addition , according to the aoac official methods , the criterion for residual alp in milk is 4 g phenol / ml of cow milk ( aoac international , 2000 ) .
this is less stringent than the 2 g phenol / ml of milk value determined in this study , suggesting that quantitative criteria could improve the safety of milk products .
the results of the present study provide basic information regarding the relationship between the duration of heat - treatment and residual alp levels , which could aid in establishing quantitative criteria for residual alp in milk products , thereby facilitating microbiological quality control of dairy products and improving public health .
 the alkaline phosphatase ( alp ) assay is a rapid and convenient method for verifying milk pasteurization . 
 since colorimetric alp assays rely on subjective visual assessments , their results are especially unreliable near the detection limits . in this study 
 , we attempted to establish quantitative criteria for residual alp in milk by using a more objective method based on spectrophotometric measurements . 
 raw milk was heat - treated for 0 , 10 , 20 , 30 , and 40 min and then subjected to alp assays . 
 the quantitative criteria for residual alp in the milk was determined as 2 g phenol / ml of milk , which is just above the alp value of milk samples heat - treated for 30 min . 
 these newly proposed methodology and criteria could facilitate the microbiological quality control of milk . 
Introduction
Materials and Methods
Milk samples
ALP assay
Standard curves
Quantification of ALP activity
Data analysis
Results and Discussion