Collaborative Evaluation of a Fluorometric Method for Measuring Alkaline Phosphatase Activity in Cow s, Sheep s, and Goat s Milk
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1 Jounal of Food Potection, Vol., No., 00, Pages Copyight, Intenational Association fo Food Potection Collaboative Evaluation of a Fluoometic Method fo Measuing Alkaline Phosphatase Activity in Cow s, Sheep s, and Goat s Milk FANK HADING * AND EILEEN GAY Shaon Close, Bookham, Suey KTLB, UK; and Advanced Instuments, Inc., Technology Way, Nowood, Massachusetts 00, USA MS 0-: eceived August 00/Accepted Decembe 00 ABSTACT Pasteuization of aw milk was intoduced to extend poduct shelf life and destoy pathogens. The measuement of alkaline phosphatase (ALP) activity has been used as an indicato of pope pasteuization in daiy poducts fo moe than yeas. This study was undetaken to evaluate six diffeent fluid daiy poducts at lowe phosphatase levels than peviously veified using the Fluoophos Test System, a sensitive and pecise method fo ALP activity detection. Thiteen laboatoies paticipated in this collaboative, intenational study to evaluate the fluoometic test at 0,,,, and mu/lite and extend the scope of the method to include milk fom not only cows but also goats and sheep. Initially, the statutoy level of ALP measued fluoometically was set to equivalent levels of coloimetic test standads ( mu/lite). The Euopean Union ecently announced its intention of loweing the legal limit fom to mu/lite and, in addition, setting a taget value of mu/lite, which if exceeded would tigge an investigation into the pasteuize plant pefomance. At mu/lite of ALP, this tial geneated elative standad deviation of epeatability values of.,., and.% and elative standad deviation of epoducibility values of.,.0, and.% fo all cow s, sheep s, and goat s milk samples, espectively. Data fom this study ae compaable to data fom pevious studies and indicate the suitability of the Fluoophos Test System method fo measuing ALP activity in milk fom cows, sheep, and goats not only at the cuent Euopean statutoy level of mu/lite but also at much lowe levels. Alkaline phosphatase (ALP), an enzyme widely distibuted in natue and natually pesent in aw milk, is used as an indicato of pope milk pasteuization. The measuement of ALP activity has been used to assess coect milk pasteuization fo moe than yeas (). Initially, coloimetic assays wee developed to allow the daiy industy to monito milk quality (). In, a moe sensitive and pecise fluoometic method fo the quantitative measuement of ALP in milk (Fluoophos Test System) was intoduced (, ). Duing the past yeas, this method has been widely investigated and extensively used and haeceived intenational appoval by govening bodies, including the Association of Official Analytical Chemists (), the Inte- * Autho fo coespondence. Tel: 0 ; Fax: 0 ; fhading@globalnet.co.uk. national Daiy Fedeation (IDF) (), the Intenational Oganization fo Standadization (ISO) (), and the National Confeence on Intestate Milk Shipments. The Euopean Union has stated its intention to lowe the statutoy ALP level fo pasteuized milk fom to mu/lite and to set a maximum taget value of mu/lite. This has stimulated the need to subject the Fluoophos Test System to intenational, intelaboatoy evaluation at lowe ALP values and to widen the scope to include milk fom cows, goats, and sheep. This study was undetaken to validate the test at lowe ALP levels (0,,,, and mu/lite) than peviously epoted. The evaluation coveed six diffeent types of milk tested in laboatoies in Euope and Ameica. The data will be of impotance fo pactical applications in outine use, to legis- TABLE. Compaison of data fom this tial with pevious intenational tial data at mu/lite of alkaline phosphatase a Tial IDF () AOAC () FSA (00) Cuent tial cow s milk Cuent tial sheep s milk Cuent tial goat s milk a DS, elative pecentage standad deviation of epeatability;, elative pecentage standad deviation of epoducibility; IDF, Intenational Daiy Fedeation; AOAC, Association of Official Analytical Chemists; FSA, United Kingdom Food Standads Agency.
2 HADING AND GAY J. Food Pot., Vol., No. TABLE. Compaison of epeatability and epoducibility values fo intelaboatoy tials evaluating Fluoophos acoss seven levels of alkaline phosphatase activity a Alkaline phosphatase Tial FSA tial 00 0 Petial Cuent tial 00, cow Cuent tial 00, sheep Cuent tial 00, goat a FSA, United Kingdom Food Standads Agency;, epeatability;, epoducibility. TABLE. Five levels of alkaline phosphatase esults fom paticipants using blind duplicates of whole cow s milk samples a.0.. b b b. b b.. b b.. b b.. b c.. b b b.0. b b. b b.. b b.. b..... b Identified as staggles by Cochan s test fo cell standad deviations o Gubbs test fo one and two outlying cell means. c Identified as outlying cell means by Cochan s test fo cell standad deviations fo one and two outlying cell means. TABLE. and epoducibility values fo each of the five levels of alkaline phosphatase esults using whole cow s milk samples a a, epeatability standad deviation;, elative pecentage standad deviation of epeatability;, epoducibility standad deviation;
3 J. Food Pot., Vol., No. FLUOOMETIC METHOD FO MEASUING ALKALINE PHOSPHATASE IN MILK TABLE. Five levels of alkaline phosphatase esults fom paticipants using blind duplicates of semiskimmed cow s milk samples a b b b b c c b Identified as staggles by Cochan s test fo cell standad deviations o Gubb s test fo one and two outlying cell means. c Identified as outlying cell means by Cochan s test fo cell standad deviations fo one and two outlying cell means. TABLE. and epoducibility values fo each of the five levels of alkaline phosphatase esults using semiskimmed cow s milk samples a a, epeatability standad deviation;, elative pecentage standad deviation of epeatability;, epoducibility standad deviation; TABLE. Five levels of alkaline phosphatase esults fom paticipants using blind duplicates of skimmed cow s milk samples 0 a No esult c b b.0. c b b c c b Identified as staggles by Cochan s test fo cell standad deviations o Gubbs test fo one and two outlying cell means. c Identified as outlying cell means by Cochan s test fo cell standad deviations fo one and two outlying cell means.
4 0 HADING AND GAY J. Food Pot., Vol., No. TABLE. and epoducibility values fo each of the five levels of alkaline phosphatase esults using skimmed cow s milk samples a a, epeatability standad deviation;, elative pecentage standad deviation of epeatability;, epoducibility standad deviation; TABLE. Five levels of alkaline phosphatase esults fom paticipants using blind duplicates of stawbey flavoed cow s milk samples a..0. b b.. b b b. b b. b b. b b. b b Identified as staggles by Cochan s test fo cell standad deviations o Gubbs test fo one and two outlying cell means. TABLE. and epoducibility values fo each of the five levels of alkaline phosphatase esults using stawbey flavoed cow s milk samples a a, epeatability standad deviation,, elative pecentage standad deviation of epeatability;, epoducibility standad deviation; latos and egulatoy bodies, and fo appoval by intenational standadization agencies. MATEIALS AND METHODS Sample pepaation. The following samples wee heated to C fo min to poduce ALP-fee milk: pasteuized cow s milk, including whole, semiskimmed, skimmed, and stawbey flavoed; aw sheep s milk; and aw goat s milk. The fat content of vaious cow s milks ae set by legislation in Euope. Whole cow s milk contains appoximately.% fat, semiskimmed cow s milk contains. to.% fat, and skimmed cow s milk contains 0.% fat. Samples wee then cooled and spiked with vaying amounts of the elevant specieaw milk to give samples with ALP values close to 0,,,, and mu/lite of ALP. These values ae of geatest commecial inteest, being the cuent legal level, and being the values to be used by the Euopean Union, and 0 to being the values most likely to be obseved in outine testing of popely pasteuized milk. Bulk samples wee caefully mixed and tested fo ALP using the Fluoophos Test System befoe being subsampled into tubes fo efigeation. andom testing of subsamples confimed homogeneity of ALP befoe sample dispatch.
5 J. Food Pot., Vol., No. FLUOOMETIC METHOD FO MEASUING ALKALINE PHOSPHATASE IN MILK TABLE. Summay of mean esults fo whole, semiskimmed, skimmed, and stawbey flavoed cow s milk samples fom paticipants a Alkaline phosphatase a, elative pecentage standad deviation of epeatability; Sample tanspotation and laboatoy paticipation. Samples wee pepaed on a Monday and distibuted, using intenational pacel distibution companies, to laboatoies, fou in the United States, fou in the United Kingdom, and one each in Fance, The Nethelands, Noway, Italy, and Switzeland. The sample boxes used wee designed to maintain tempeatues below C duing a -day peiod. Paticipating laboatoies wee asked to ecod the tempeatue and time of sample eceipt. All wee asked to test samples on Wednesday moning in an attempt to balance any sample deteioation effect. The ISO/IDF daft fluoometic method (ISO/CD -/IDF -) was followed by each laboatoy. A petial un was completed to familiaize analysts with the test method. Data analysis. Samples wee supplied as blind duplicates, yielding 0 samples pe laboatoy. Single deteminations wee made on the 0 samples (0 samples in blind duplicate). esults wee etuned on epot sheets povided. Data collected fom the collaboative tial wee collated and analyzed following the statistical pocedues defined in ISO - (). This compised the following steps. Fist, data fom each laboatoy wee collated into the appopiate pais. Second, cell means and spead fo each pai of samples wee calculated. Thid, Cochan s test was used to identify outlying values fo cell diffeences fo each test mateial type. Sample pais identified as outlies wee excluded fom futhe statistical analysis, and the test was applied iteatively to each set of data until all outlies wee identified. Fouth, fo each test mateial type, Gubbs test was used to identify outlying individual values fo cell means. Sample pais identified as outlies wee excluded fom futhe statistical analysis, and the test was applied iteatively to each set of data until all outlies wee identified. Whee no futhe outlies wee identified, Gubbs test fo two outlying values was applied, and pais of esults identified as outlies wee excluded fom futhe statistical analysis. Fifth, pecision values fo each type of test mateial wee calculated. ESULTS AND DISCUSSION Sample matix selection. Although it is moe difficult to tanspot and maintain the integity of liquid milk samples than feeze-died milk samples, liquid milk samples TABLE. Five levels of alkaline phosphatase esults fom paticipants using blind duplicates of goat s milk samples a.... b. b a.... b. b a. a a. a a 0. b b.0 b a. b b.0 b. b Identified as staggles by Cochan s test fo cell standad deviations o Gubbs test fo one and two outlying cell means. TABLE. and epoducibility values fo each of the five levels of alkaline phosphatase esults using goat s milk samples a a, epeatability standad deviation;, elative pecentage standad deviation of epeatability;, epoducibility standad deviation;
6 HADING AND GAY J. Food Pot., Vol., No. TABLE. Five levels of alkaline phosphatase esults fom paticipants using blind duplicates of sheep s milk samples a a wee used because they bette eflect the samples that will be tested in outine pactice by the method unde investigation. Consideation was given to the use of feeze-died samples, because this would have minimized sample deteioation duing tansit. Compaative data fom cuent study to pevious fluoometic ALP studies in fluid daiy poducts. Table compaes the epeatability and epoducibility of the Fluoophos Test System at mu/lite fo cow s, sheep s, and goat s milks with peviouepoted studies by the IDF, the Association of Official Analytical Chemists, and the United Kingdom Food Standads Agency () on cow s milk. data geneally ageed with ealie studies, wheeaepoducibility values, with the exception of goat s milk, wee somewhat highe, possibly due to changes in ALP duing extended sample tanspotation times. Table compaes the epeatability and epoducibility findings of the cuent study ove a wide ange of ALP values fo cow s, sheep s, and goat s milk with those epoted on cow s milk fom a petial intenational study and a study undetaken by the United Kingdom Food Standads Agency. and epoducibility values, although somewhat highe than pevious studies, wee consideed to be suitable fo the contol of ALP in pasteuized milk. ALP esults fom paticipants using milk fom cows, goats, and sheep that cove a ange of 0 to mu/lite. The pefomance of the Fluoophos Test System method was studied ove a ange of cow s milk of commecial impotance (Tables though ). Tables,,, and detail individual paticipant test esults on blind duplicate samples. Tables,,, and pesent calculated epeatability and epoducibility values fo each categoy of cow s milk unde study, and Table summaizes the epeatability and epoducibility values fo all cow s milk samples tested. Tables and epot individual paticipant test esults on blind duplicate samples fo goat s and sheep s milk, and Tables and summaize the epeatability and epoducibility values fo goat s and sheep s milk. Paticipating laboatoies and had a numbe of esults that wee high in ALP. Thee was no evidence that they had incoectly calibated thei test instument; hence, it was consideed possible that sample deteioation may have contibuted to the elevated ALP values. Also, a poo pipetting technique might have contibuted to these highe than expected esults. Whee appopiate, statistical outlies wee eliminated fom calculated values. Little diffeence existed between the elative standad TABLE. and epoducibility values fo each of the five levels of alkaline phosphatase esults using sheep s milk samples a a, epeatability standad deviation;, elative pecentage standad deviation of epeatability;, epoducibility standad deviation;
7 J. Food Pot., Vol., No. FLUOOMETIC METHOD FO MEASUING ALKALINE PHOSPHATASE IN MILK deviation of epeatability ( ) and the elative standad deviation of epoducibility ( ) values fo cow s milk types, except that the value fo whole cow s milk at mu/lite gave a high epoducibility value. This was due to the fact that some esults used fell just inside the outlie values. The fo whole milk at mu/lite was.% (Table ) compaed with.00% (Table ) epoted by the Association of Official Analytical Chemists collaboative study fom. Statistical staggles fom laboatoies and contibuted to this high value, because they wee included in calculations and significantly affected the epoducibility value due to the small sample population. Although the Fluoophos Test System validly ecoded one o both esults as mu/lite, no statistical pocedue exists fo dealing with such esults. Based on this collaboative tial, the fluoometic method has been submitted to the ISO and IDF fo appoval as a final intenational standad method. This tial demonstates the applicability of the method fo outine contol and legislative suveillance. Data geneated in this study wee geneally compaable to data fom pevious studies, with the exception of epoducibility data at mu/lite fo whole milk. This intenational tial povideesults indicating that the Fluoophos Test System method is a satisfactoy contol test fo detecting ALP activity in pasteuized milk fom cows, sheep, and goats down to 0 mu/ lite. ACKNOWLEDGMENTS We gatefully acknowledge Quada Chem Laboatoies (Foest ow, East Sussex, UK) fo sample pepaation and distibution, L. Coveney (Savant Technologies, Powys, UK) fo expetly undetaking all of the statistical analyses, and Linda Snyde fo he technical assistance in completing the manuscipt fo submission. We thank Advanced Instuments, Inc., fo supplying all eagents and consumables fo this study. We also thank the following fo paticipating in this collaboative intenational intelaboatoy study: Maina Nicolas (Agence Fancaise de Sanitaie Des Aliments, Pais, Fance), Tony Woods and Mak Whatton (Quada Chem Laboatoies), Fea Woltjes (Centale Oganisatie voo de Kwaliteitsaangelegenheden in de Zuivel, Leusden, The Nethelands), Jaques Meye (Eidg. Foschungsanstalt fu Milchwitschaft, Liebefeld, Switzeland), Luisa Pellegino (Depatment of Food Science and Technology, Milan, Italy), Kevin Williamson (Lancashie NHS Tust, Lancashie, UK), Melody Geenwood (Health Potection Agency, South East Laboatoy, Southampton, UK), May Bulthaus (Daiy Quality Contol Institute, Mounds View, Minn.), obet Badley (Wisconsin Cente fo Daiy eseach, Madison), Doug Lake (Expess Daiy Company, East Sussex, UK), Kal Eckne (The Nowegian Institute fo Food & Envionmental Analysis, Oslo), and Kut Mangione (New Yok State Depatment of Agicultue, Albany). EFEENCES. Intenational Daiy Fedeation (IDF). July. Standad :: milk and milk-based dinks detemination of alkaline phosphatase activity fluoometic method. IDF, Bussels.. Intenational Oganization fo Standadization (ISO).. Intenational standad ISO -: accuacy (tueness and pecision) of measuement methods and esults pat : basic method fo the detemination of epeatability and epoducibility of a standad measuement. ISO, Geneva.. Intenational Oganization fo Standadization (ISO).. Intenational standad ISO -: milk and milk poducts detemination of alkaline phosphatase activity using a fluoometic method pat : milk and milk-based dinks. ISO, Geneva.. Kay, H. D., and W.. Gaham.. The phosphatase test fo pasteuised milk. J. Daiy es. : 0.. Mashall,. T. (ed.).. Standad methods fo the examination of daiy poducts. th ed. Ameican Public Health Association, Washington, D.C.. occo,. M.. Fluoometic analysis of alkaline phosphatase in fluid daiy poducts. J. Food Pot. :.. occo,. M.. Fluoometic detemination of alkaline phosphatase in fluid daiy poducts, collaboative study. J. Assoc. Off. Anal. Chem. :.. Schae, H.. A apid phosphomonoestease test fo contol of daiy pasteuization. J. Daiy Sci. :.. Wood,., A. Damant, and F. Hading. 00. Method to estimate alkaline phosphatase in pasteuised milk. Submitted fo publication.
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