Phenolphthalein Phosphate As a Reagent For. Alkaline

Phenolphthalein phosphate as a reagent for alkaline phosphatase estimation in milk

Item Type text; Thesis-Reproduction (electronic)

Authors De souza, Marciano José, 1936-

Publisher The University of Arizona.

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Link to Item http://hdl.handle.net/10150/318096 PHENOLPHTHALEIN PHOSPHATE AS A REAGENT

FOR. ALKALINE PHOSPHATASE ESTIMATION IN MILK

by

Marciano Jose de Souza

Y - . ' ‘

A Thesis Submitted to the Faculty of the

DEPARTMENT OF AGRICULTURAL BIOCHEMISTRY

In Partial Fulfillment of the Requirements For the Degree of

MASTER OF SCIENCE

In the Graduate College

THE UNIVERSITY OF ARIZONA

19 6 8 STATEMENT BY AUTHOR

i This thesis has been submitted in partial fulfillment of re­ quirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judg­ ment the proposed use of the material is in the interests of scholar­ ship. In all other instances, however, permission must be obtained from the author.

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

"J. W. Stull essor of Dairy Science ACKNOWLEDGMENTS

The author wishes to express his sincere gratitude to the

following faculty and staff members of the University of Arizona,

Tucson, Arizona: Dr. J. W. Stull, Professor of Dairy Science, under whose supervision this study was planned and carried out; Dr. A. R.

Kemmerer, Head of the Department of Agricultural Biochemistry;

Dr. W. H. Brown, Professor of Dairy Science; and Mr. R. R. Taylor,

Research Associate in Dairy Science.

Acknowledgments are also extended to Mrs. C. H. Gardner, whose generosity made my education in this country possible, to the

Miller Foundation, and to the Gulbenkian Foundation. To the General

Diagnostics Division of Warner-Chilcott, thanks are extended for having supplied the phenolphthalein monophosphate substrate. Finally^ my appreciation is expressed to my wife, Augusta, for her understand­

ing and patience and for having typed this thesis.

iii TABLE OF CONTENTS

• Page

LIST OF T A B L E S ...... v

LIST OF ILLUSTRATIONS ...... vi

ABSTRACT ...... '. , . . , . . . . vii

INTRODUCTION...... 1

REVIEW OF LITERATURE ...... ' 3

EXPERIMENTAL PROCEDURE ...... 12

Obtaining a Spectrophotometrically Clear Milk Filtrate’ . ;. . . 12 Incubation of Milk with PhenoIphtha1ein Monophosphate Substrate ...... A ...... 1 2 Comparative Analysis ...... 13

RESULTS AND DISCUSSION ...... 15.

Determination of Concentration of PhenoIphthalein Required for Measurement of Color in Milk Filtrate at 550 my .... 15 Color Development in Raw Milk from PhenoIphthalein Hydro­ lyzed from Various Concentrations of PhenoIphthalein ; Monophosphate Substrate . , . . . . . > . . i ...... 16 Color Development from Phenolphthalein Monophosphate Hydrolysate in Milk Heated for 30 Minutesat Various Temperatures . . . • ...... 20 Color Development from Phenolphthalein Monophosphate : Hydrolysate in Milk Heated at 61.6 C for Various Time Intervals . . ^ ...... 22 Color Development from Phenolphthalein Monophosphate Hydrolysate in Heated Milk Contaminated with Raw Milk . . . 23

REFERENCES ...... 2.5

iv LIST OF TABLES

Table Page

1. Serial Concentrations of Substrate and Adjustments for Final p H ...... 14

2. Color Intensity Developed by Various Concentrations of Phenolphthalein in Milk ...... 16

3. Color Intensity Developed in Raw Milk from Various Concentrations of Hydrolyzed Phenolphthalein Monophosphate ...... 18

4. Color Intensity in Raw Milk Heated for 30 Minutes at Various Temperatures as Determined by the Phenol­ phthalein Monophosphate and A.O.A.C. Methods ...... 21

5 . Color Development in Raw Milk, Heated at 61.6 C for Various Time Intervals as Determined by the Phenol­ phthalein Monophosphate and A.O.A.C. Methods ...... 23

6. Color Development in Boiled Milk Contaminated with Various Levels of Raw Milk as Determined by the Phenolphthalein Monophosphate and A.O.A.C. Methods ...... 24

v LIST OF ILLUSTRATIONS

Figure. Page

1. Color Intensity Developed by Various Concentrations of Phenolph.thalein in Milk ...... 17

2. Color Intensity Developed in Raw Milk from Various Concentrations of Hydrolyzed Phenolphthalein Monophosphate ...... > ... 19 ABSTRACT

Alkaline phosphatase activity is widely used as a criterion

in the evaluation of the effectiveness with which milk is pasteurized.

This study was undertaken to determine the feasability of using the

color development in phenolphthalein enzymatically hydrolyzed from

phenoIphthalein monophosphate as a means of assessing alkaline phospha­

tase activity.

A linear relationship was obtained when the concentration of

phenolphthalein in processed milk was plotted against the per cent

transmission at 550 mp on semilog paper. Similarly> a linear relation­

ship was also observed between the concentration of phenolphthalein

monophosphate in raw milk and the per cent transmission at 550 my. A

series of tests using phenolphthalein monophosphate as a substrate were

conducted in parallel with the A.O.A.C. method on samples of milk sub­

jected to variation in temperature, holding time, and raw milk

contamination. It was found that phenolphthalein monophosphate when

used as a substrate under the conditions of the test was not sensitive

to critical differences in temperature, holding time, and levels of

raw milk contamination, whereas these same differences were more readily

detectable by the A.O.A.C. method. Extending the incubation time and

reducing dilution effects during coagulation and subsequent pH adjust­ ment are proposed as possible means of increasing the method’s

sensitivity.

' ' v i i ■ : ’ ' INTRODUCTION

A considerable number of enzymes have been partially or com­

pletely identified in normal bovine milk. These enzymes appear to be

constituents or products of the mammary tissue or blood and find their

way into milk either accidentally or unavoidably, pursuant to the nor­

mal secretory process. The enzymes present in milk do not, as far as is

known, contribute to the principal function of the miIk--the nutrition

of the young. One class of enzymes found in milk are the phosphatases,

which have the ability to catalyze the hydrolysis of phosphate esters.

These phosphatases differ from one another with regard to their speci­

ficity of substrates, optimum pH, and/or temperature of activity. Among

the important classes of phosphatases are the monoesterases, the dies­

terases, and the pyrophosphatases, each group catalyzing, respectively,

the hydrolysis of monoesters, diesters, and pyrophosphates. Milk alka­

line phosphatase, a monoesterase, has an optimum pH of 9.65 and is

inactivated when subjected to a "temperature of 62 C for 30 minutes. .

Alkaline phosphatase activity is Widely used as a criterion in

the evaluation of the effectiveness with which milk is pasteurized.

This is possible because the time-temperature conditions required for

heat inactivation of alkaline phosphatase are quite similar to those

needed for the destruction of such heat resistant pathogens as

. M. tuberculosis and yet do not appreciably change the physical or chem­

ical properties of milk. Qualitative or quantitative determination of

■ ' 1 ■' ' ' . ' ' ■ ' '■ 2

phenol liberated by the hydrolysis of disodium phenylphosphate is cur­

rently used as a method to estimate alkaline phosphatase activity.

However, possible contamination by a number of phenolic compounds can

cause misinterpretation of the results.

This study was initiated to determine the feasability of using

the color development of phenoIphthalein hydrolyzed from phenolphthalefn monophosphate by alkaline phosphatase in milk.as an analytical method

for estimating that enzyme's activity. Since the method incorporated

the use of colorimetric analysis of the phenolphthalein hydrolyzed from

the substrate, consideration was first given to obtaining a spectro- photometrically clear milk filtrate. Following this, quantitative

determinations were made of phosphatase activity in milk subjected to

various heat treatments as well as combinations of heated and unheated milk. REVIEW OF LITERATURE

The phosphatases are a group of enzymes which catalyze the hydrolysis of phosphate esters. These phosphatases differ from one another with, regard to their specificity of substrates as well as in their activity under optimum pH and temperature conditions. Among the important classes of phosphatases are the monoesterases, the diester­ ases, and the pyrophosphatases, each group catalyzing, respectively, the hydrolysis of monoesters, diesters, and pyrophosphates. Milk alka­ line phosphatase activity is optimum at pH 9.65, and, it is inactivated when subjected to a temperature of 62 C for 30 minutes (5).

The basic procedure followed in determining the alkaline phos­ phatase activity of pasteurized milk comprises the following steps:

(a) the addition of a large excess of a phosphate ester to a relatively small sample of the milk being tested, (b) a period of incubation under standard conditions during which residual phosphatase remaining after pasteurization, if present, will hydrolyze the ester which is otherwise quite stable, and (c) the stopping of the hydrolysis after incubation and colorimetric determination of the end products. The colorimetric values thus obtained aid in determing the effectiveness of pasteur­ ization. The most important factors in the technique summarized above are the correct integration of buffer concentration, pH, and incubation temperature (10).

3 In 1933, Kay and Graham (6) ascertained that phosphatase was sufficiently thermolabile to be destroyed completely by pasteurization if this process was properly carried out. It was further reported that at any temperature from 60-75 C in which 96% of the phosphatase was destroyed, there was complete destruction of M. tuberculosis.

In 1935, Kay and Graham (7) published their "Phosphatase Test for Pasteurized Milk." There had been a previous test designed by them in 1933, but by their own evaluation they found the later technique to be superior to the previous one.

Kay and Graham used disodium phenylphosphate as a substrate for their test. This substrate when buffered with sodium veronal in water saturated with chloroform constituted their buffer-substrate. Neither the pH of the buffer-substrate nor that of the reaction mixture was indicated in their paper.

The samples to be tested were incubated at 37-38 C for 24 hours

The hydrolyzed phenol was compared to a previously established color standard consisting of 2.3 Lovibond units of blue on the tintometer scale. If the color from the test samples exceeded the 2.3 Lovibond units, one could conclude that the milk was improperly pasteurized either as a result of (a) inadequate temperature of pasteurization,

(b) too short a holding time at the pasteurization temperature, or

(c) contamination by raw milk.

The test was sensitive enough to detect a difference of 1.4 C below the temperature of pasteurization. Differences of 5 minutes in the duration of pasteurization and 0.2% contamination by raw milk were also detectable by the test. The requirements for pasteurization con­ sidered as criteria for the Kay-Graham test were that the milk be held at a temperature of 62.8-65.6 C for 30 minutes.and then immediately

cooled to 12.8 C.

In 1938, Scharer (12) proposed a rapid phosphomonoesterase test.

•Scharer also used disodium phenylphosphate as a substrate for his test.

The substrate was buffered with a solution of sodium borate in sodium hydroxide and trichlorqmethane. The pH of the buffer-substrate solution was approximately 9.6. The pH of the reaction mixture was not indi­ cated.

The test samples were incubated at 37.5 C for 1 hour, and basic lead acetate was used as a protein precipitant. The phenol liberated by enzymatic hydrolysis was allowed to react with 2,6-dibromoquinone- chloroimide (BQC) to form an indophenol blue coloration. This blue color was compared to phenol color standards. It was, established that in no instance did properly pasteurized milk yield a color indicative . of more than 2.5 phosphomonoesterase units. (A phosphomonoesterase unit was defined as the amount of enzyme which under conditions of the test produced the color equivalent of 1 g of phenol.)

It was observed that the sensitivity of BQC to phenol was one part to twenty million, and the optimum pH for color development with

BQC was found to be 9.6. BQC was also found to be more specific for hydrolyzed phenol than the Folin-Ciocalteu reagent used by Kay and

Graham (7). . ' V ■ ; ' ■ . .. : : ' ' • . ' - 6

The test was found to be capable of detecting differences of

0.6 C below the level required by. the New York City sanitary code which prescribed that the milk be heated to at least 61.7 C and maintained at that temperature for at least 30 minutes, after which it was to be rapidly cooled. Differences of 5 minutes in the duration of pasteuriza­ tion and 0.5% contamination.with raw milk were also detectable.

In 1946, Sanders and Sager (11) published their version of the phosphatase test. Their method today constitutes the official test for pasteurization of the Association of Official Agricultural Chemists.

The substrate used in this test was disodium phenyIphosphate, • and barium borate hydroxide was used as a buffer. The pH of the barium borate hydroxide buffer was approximately 10.6. It was observed that the optimum pH of hydrolysis for the production of phenol under the con­ ditions of the test was 10.0-10.05.

The test samples were incubated at 37-38 C for 1 hour, after which the reaction was stopped and the protein precipitated out with a zinc-copper protein precipitant. To enhance color development, a color development buffer consisting of sodium metaborate and sodium chloride was added, thereby providing an optimum pH of 9.3-9.4 before the BQC was added.

Measurement of the hydrolyzed product was accomplished by colorimetric evaluation at a wavelength of 610 my. The colorimetric values obtained were then compared to the standard curve of phenol standards. ■ v ! 7 It was established that phenol values greater than 4 yg/ml indi­

cated improper pasteurization in cow's milk. The test was found to be sensitive enough to detect 0.05% raw milk contamination. No mention was made of the capacity of the test to detect variations in temperature or duration of holding.

In 1949, Kosikowsky (8) proposed a "Simple Universal Dairy

Products Phosphatase Test." This test subsequently came to be known as the Cornell University Test, This test, according to the author, dif­ fered from previous ones in that (a) the buffer and precipitating agent were different, (b) only one concentration of buffer and precipitating solutions was required for all dairy products, and (c) it was not neces­ sary to heat inactivate the phosphatase enzyme after incubation since the precipitating agent reduced the pH of the reaction mixture to pH 1-2.

Disodium phenyIphosphate was used as a substrate in this test, and it was buffered with sodium carbonate and sodium bicarbonate. The test samples were incubated at 32-37 C for 12-24 hours. After incuba­ tion, the solids were precipitated out with a mixture of 25% trichloro­ acetic acid and 18% hydrochloric acid.

Following filtration, a color developer consisting of a mixture of copper sulfate, sodium carbonate, and Calgon solution was added.

BQC was added next, and the color was allowed to develop for 15 minutes / at 37 C. The color from the test samples was then compared with suit­ able standards. 8 It was tentatively established for the.long incubation method

that a value exceeding 5 iag/0.5 ml milk or 5 pg/0.25 g cheese would

indicate improper pasteurization or raw milk products. Data from this paper indicate that the test was sensitive enough to detect 0.1% raw milk contamination. No data was presented on variations in time or

temperature of pasteurization. The conditions necessary for pasteuriza­ tion were that the milk be heated to at least 61.7 C for 30 minutes.

In 1949, Aschaffenburg and Mullen (2) in England developed a new test using disodium-p-nitrophenyl phosphate as a substrate. The sub­

strate was buffered with hydrochloric acid, sodium carbonate, and sodium

diethyl barbiturate. It was found that optimum hydrolysis occurred at a pH of 9.8-10.0.

The samples were incubated at a temperature of 37 C for 2 hours.

A solution of 28% trichloroacetic acid in water was used as a protein precipitant. A mixture of sodium hydroxide and sodium hexa-meta phos­ phate was used as a color developing reagent. The hydrolyzate p-nitro- phenyl yielded a yellow color which could then be eolorimetrically

analyzed at 445 mp. It was determined that values exceeding 4 gg p-nitrophenyl/ml of milk indicated improper pasteurization.

The conditions required for pasteurization were that the milk be held at 62.8 C for 30 minutes. The test was found to be capable of de­

tecting 0,1% raw milk contamination. Samples of milk held at 61.7 C

instead of 62.8 C for 30 minutes were found to be improperly pasteurized.

The test was found to be incapable of distinguishing with certainty . > . ■ . : : ; . ; : . ■.? between samples of milk held at 62.8 C for 20 minutes and samples of milk held at 62.8 C for 30 minutes.

In 1966, Babson et at. (3) used phenolphthalein monophosphate as a substrate in the determination of blood serum alkaline phosphatase ac­ tivity. The buffer used was 2-amino-2-methy1-1-propano1 as its pK (9.9) was found to be closest to the optimum pH of the reaction.

It was reported that the optimum pH for alkaline phosphatase varied with buffer and substrate species and substrate concentration.

However;, using the criteria of maximum enzymatic activity and minimum alteration in that activity with slight changes in condition, a pH of

10.15 at 25 C was selected as optimum for the conditions of the assay.

The actual pH of the reaction mixture at 37 C was 9.9.

A mixture of disodium hydrogen phosphate and trisodium phosphate was used as the color developer. The pH of this color developer was

11.2> and the color thus developed was found to be stable for an hour.

The reaction mixture was incubated at 37 C for 20 minutes., and the ab­ sorption was read at 550 mp against a reagent blank without serum.. The alkaline phosphatase activity was read from a standard curve. •

It was found that the substrate was readily hydrolyzed and produced a chromogen for colorimetric analysis.. The phenolphthalein hydrolyzed was found to bear a linear relationship with respect to both incubation time and enzyme concentration up to about 60 pg of phenol­ phthalein released. Phenolphthalein monophosphate was found to be more sensitive, as a substrate than phenolphthalein diphosphate and . . ' 10 p-nitrophenyl phosphate when the latter two were used in comparative procedures.

•In 1966, shortly after Babson et dl. (3) published their find­ ings on the use of phenoIphthalein monophosphate as a substrate in the determination of blood serum alkaline phosphatase activity, Wilkinson and Vodden (14) published the results of a similar study.

In their work, Wilkinson and Vodden also used phenoIphthalein monophosphate as a substrate in the determination of serum alkaline . phosphatase activity. The substrate was buffered with 2-amino-2-methyl-

1-propanol. The pH of the diluted buffer-substrate mixture was 10,15 at

25 C. Following the addition of serum, and after raising the tempera­ ture of the reaction mixture to 37 C, the pi I fell to approximately 9.9.

A mixture of disodium hydrogen phosphate and trisodium phosphate with a pH of 11.2 was used as a color developer. The reaction mixture was incubated at 37 C for 20 minutes, and the absorption was read at

550 my against a water blank. The pink color produced was found to be stable for at least 1 hour, and a practically linear relationship was obtained when the volume of serum was plotted against the observed phos­ phatase activity. A linear relationship was also observed between the period of incubation and the amount of phenoIphthalein liberated when the duration of incubation was varied between 10 and 50 minutes.

; : It was ascertained that the addition of Mg as MgSO^ did not result in increased phosphatase activity. The addition of ethylene di- aminetetracetic acid, however, was found to result in a marked reduction of phosphatase activity. This inhibition was partially reversed by the ' 11

•f* “f* •'f' addition of Mg , and the addition of Ca as CaCl^ resulted in a fur­ ther increase in activity. But the maximum obtained was approximately

5% below the level of the control.

Phosphatase determinations of 167 samples of serum were carried out by the new method in parallel with the method of Shinowara et al.

(13) , and a close correlation (r - +0.92, P < 0.001) was observed be-r tween the two methods.

In 1967, Lin and Kleyn (9) used phenolphthalein monophosphate as a substrate in the determination of alkaline phosphatase activity in milk. It was reported that the hydrolyzed phenolphthalein produced a red color which was then compared to a standard prepared in the same milk. The alkaline phosphatase activity of 30 samples of commercial,: homogenized, pasteurized milk was determined by the above method, and the results were compared to the Scharer I (Rapid) Method (12). The sensitivity of the test to residual amounts of alkaline phosphatase in milk subjected to various levels of heat treatment and pasteurized milk contaminated with low levels of raw milk was studied. EXPERIMENTAL PROCEDURE

Obtaining a Spectrophotometrically Clear Milk Filtrate

In order to, obtain a spectrophotometrically clear milk filtrate, a means to precipitate out the milk solids was developed. It was con­ sidered that the method should be simple and not require manipulations such as centrifugation. A precipitant consisting of a mixture of 6% trichloroacetic acid and 4% metaphosphoric acid in aqueous solution was used (4). The precipitant was found to have a pH of 1.1.

Equal volumes of the precipitant and milk were mixed at room temperature. This mixture was found to have a pH of 1.4-1.65. The mix­ ture was then filtered through S .§ S. No. 595 filter paper to yield a

clear filtrate.

Incubation of Milk with Phenblphthalein Monophosphate Substrate

The method outlined here is the basic method followed throughout the study. Unless otherwise specified, all steps were carried out at room temperature. Forty ml of raw milk from a mixed herd was pipetted

into a 100 ml beaker. The phenolphthalein monophosphate substrate was

then added to the sample. This resulted in a variation of pH from

sample to sample in certain cases involving serial concentrations of

substrate. The pH's were adjusted to the highest pH in the samples by

the addition of appropriate amounts of 1 M NaOH solution. These samples

1. This substrate was supplied by the General Diagnostics Division of Warner-Chilcott, Los Angeles, California.

■■■ ■ 1 2 ■ : ' v - '■ 13

were further standardized with respect to final volume.by.the addition

of distilled water (Table 1).

The samples were then incubated at 37 C for 20 minutes in a water bath. Following incubation, each sample was coagulated by the ad­

dition of a precipitant consisting of a mixture of 6% trichloroacetic

acid and 4% metaphosphoric acid in aqueous solution. The samples were

filtered through S.§ S. No. 595 filter paper, and 20 ml of filtrate was

collected separately from each sample. Each sample now consisting of

20 ml of filtrate was adjusted to an average pH of 11.25 by the addition of 12 ml of 1 M NaOH solution. The pH was adjusted to permit optimum

color development of the enzymatically hydrolyzed substrate. The ele­ vated pH resulted in the formation of a suspension of unidentified char­

acter in solution. The samples were refiltered through S.§ S. No. 595

filter paper, and the resulting filtrate was colorimetrieally analyzed

in a Bausch and Lomb Spectronic 20 spectrophotometer at 550 mp. The blank used to calibrate the spectrophotometer consisted of 40 ml of boiled milk to which 32 drops of substrate had been added. In every

other respect the blank was subjected to the same treatment as the sam­ ples (Table 1).

Comparative Analysis

In that portion of the study requiring comparative analysis, the

A.O.A.C. method detailed in Standard Methods for the Examination of .

Dairy Products was used (1). Table 1

Serial Concentrations of Substrate and Adjustments for Final pH

Sample Number ■ Substrate pH of Milk 1 M NaOH . Adjusted pH - H2O Added for Concentration And Substrate Added Vol. Correction (mg/ml) ' (ml) (ml)

1 0.05 ' 7.69 1.2 9.97

2 0.10 • 8.95 1.1 9.95 • 0.1

3 0.21 9.50 0.9 9.95 0.3

4 0.41 ; 9.85 0.6 9.95 0.6

5 0.83 9,95 9.95 . 1.2 • RESULTS AND DISCUSSION

Determination of Concentration of Phenolphthalein Required for Measurement of Color in Milk Filtrate at 550 my

In order to determine the amount of phenolphthalein required to impart sufficient intensity of color for measurement at 550 my,, a series of dosed milk samples was prepared. In doing this, a stock solution of phenolphthalein in 95% ethanol was first prepared. Varying amounts of this stock solution were then added to a series of five samples of homogenized, pasteurized milk. In no instance did the volume of stock solution added exceed 5 ml per 100 ml of iniIk which had been heated to

61.6 for 30 minutes.

The phenolphthalein-dosed milk Was then coagulated and filtered as described in the "Experimental Procedure." Equal volumes of filtrate were collected separately from each sample, and the pH of these fil­ trates was adjusted to approximately 10.0 by the addition of appropriate amounts of 1 M NaOH solution. This resulted in the formation of a fine particulate suspension of unidentified character in the solution.

Refiltration through S .§ S. No. 595 filter paper, however, resulted in a filtrate suitable for colorimetric evaluation.

. Color measurement was made in a Bausch and Lomb Spectronic 20 spectrophotometer at 550 my. The samples were tested against a blank to which distilled water was added instead of the phenolphthalein stock solution. In all other respects the blank was standardized'by the same treatment as the samples being tested (Table 2 and Fig. 1). Table 2

Color Intensity Developed by Various Concentrations of Phenolphthalein in Milk

Sample Number Phenolphthalein Concentration Transmission (mg/ml) at 550 my (%)

1 0.15 57

■2 0.25 36

3 0.35 27

4 0.45 : 19

5 0.55 - 13

These results indicate that a straight line relationship is ob­ tained in color intensity developed in phenolphthalein concentrations in milk, ranging from 0.15 to 0.55 mg/ml when the measurement is made in a filtrate prepared as described. The possibility of absorbed losses of phenolphthalein in the precipitated coagulum appeared to be proportion­ ally uniform or insignificant in the concentrations studied.

Color Development in Raw Milk from Phenolphthalein Hydrolyzed From Various Concentrations of Phenolphthalein Monophosphate Substrate

Varying concentrations of phenolphthalein monophosphate sub­ strate were added to five samples of raw milk. This resulted in a vari­ ation of the pH from sample to sample. The pH of each sample was then adjusted to approximately 10.1 by the addition of appropriate amounts of

1 M NaOH solution. The samples were standardized with respect to final volume by the addition of distilled water.

The substrate-dosed samples were then incubated for 20 minutes at 37 C in a water bath following which they were coagulated and filtered TRANSMISSION AT 550 m p-PER C EN T 0 0 1 0 4 20 80 60 0.05 IUE CLR NEST DVLPD Y AIU CNETAIN OF CONCENTRATIONS VARIOUS BY DEVELOPED INTENSITY COLOR I FIGURE HNLHHLI I MILK IN PHENOLPHTHALEIN 0.25 HNLHHLI CNETAIN mgml) g/m (m CONCENTRATION PHENOLPHTHALEIN .504 .50.65 0.55 0.45 0.35

0.750.15 ' . 18

in the manner previously indicated. Aliquots of filtrate were collected

separately from each sample, and the pH was adjusted to 11.25 for color

development. One M NaOH solution was used to raise the pH of each

sample. The samples of filtrate were refiltered as a result.of the for­

mation of a suspension of unidentified nature in the solution..

The samples obtained after the second filtration were colori-

metrically assessed as previously described. A reference blank consist­

ing of the filtrate from boiled milk was used. In every other way the

treatment of the blank was similar to that of the milk samples being

tested. The pH of the blank was 11.35. The results are shown in Table

'3 and Fig. 2. •

Table 3

Color Intensity Developed in Raw Milk.from Various . Concentrations of Hydrolyzed Phenolphthaloin Monophosphate

Sample Number Substrate Concentration Transmission (mg/ml) at 550 mu (%)

1 0.05 88 .

2 0.10 . 75 .

.3 0.21 63

4 0.41 . 4 0

■ 5 . ■ 0.83 ' :: 12

These data show that a straight line relationship is obtained in

the color developed from phenolphthalein which is hydrolyzed by alkaline

phosphatase in raw milk from phenolphthalein monophosphate substrate. TRANSMISSION AT 550 mjj - PERCENT 0 0 2 100 40 20 60 80 IUE CLR NEST DVLPD N A ML FO VROS OCNRTOS OF CONCENTRATIONS VARIOUS FROM MILK RAW IN DEVELOPED INTENSITY COLOR 2 FIGURE OCNRTO O PEOPTAEN OOHSHT ( ml) /m g (m MONOPHOSPHATE PHENOLPHTHALEIN OF CONCENTRATION YRLZD HNLHHLI MONOPHOSPHATE PHENOLPHTHALEIN HYDROLYZED .505 0.65 0.55 0.45 0.750.35 0.850.15 0250.05

20

This relationship is true in considering the action of raw milk alkaline phosphatase at substrate concentrations ranging from 0.05 to 0.83 mg/ml.

Color Development from Phenolphthalein Monophosphate Hydrolysate in Milk Heated for 30 Minutes at Various Temperatures

Four samples of raw milk were heated to various temperatures for

30 minutes in a water bath followed by rapid cooling by immersing in shaved ice. Approximately 30 minutes was required to raise the tempera­ ture to the final value. To each sample a constant amount of substrate was. added.such that the concentration of substrate was 0.41 mg/ml. This concentration was selected from the data in Table 3 as being that which would most likely give a measurable color development intensity. No pH or volumetric adjustment was necessary as the concentration of substrate in all the samples was constant, resulting in a uniform pH of 9.8.

Following incubation for 20 minutes at 37 C, the milk solids were precipitated and filtered out in the manner described previously.

A constant amount of filtrate was obtained from each sample, and the pH was raised as previously described to permit color development. The samples were refiltered and the color was spectrophotometrically assessed. A blank consisting of boiled milk was used to calibrate the spectrophotometer. The blank contained the same concentration of sub­ strate" as the other samples and was treated in a similar manner in all other respects. The results from this test were" compared with the results obtained from identical samples analyzed by the A.O.A.C. method (Table 4). 21

. Table 4

' Color Intensity in Raw Milk Heated for 30 Minutes at Various Temperatures As Determined by the Phenolphthalein Monophosphate and A.O.A.C. Methods

■ Temperature Transmission _■ c o ■■ ':;0 - ■; c o

' Phenolphthalein Monophosphate A.O.A.C;

60.5 98 ‘ 83

61.1 98 85

61.6 99 92

62.2 . 99 95

These results indicate that there was insufficient color devel­

opment in the phenolphthalein hydrolyzed from the substrate by the

residual phosphatase in milk heated for 30 minutes at temperatures rang­

ing from 60.5-62.2 C. The A;0.A.C. method indicated Adequate sensi­

tivity. Of the variables involved in the phenolphthalein monophosphate

procedure, the. following changes offer the possibility of improved

sensitivity.

1. Use of a more effective buffering system during the incubation

and. hydrolysis step.

2. An increase of the incubation time from 20 up to 30 or 60

~ . minutes.

3. Reduction of the dilution effect during filtrate preparation by

increasing the concentration of the coagulant reagents. This in

turn would reduce the Volume of coagulant required.

4. Further reduction of the dilution effect involved in the pH ' \ : ' ' . V ^ ' , . 22

adjustment of the filtrate required for final color development.

This can be accomplished by using a higher concentration of

NaOH. The use of another reagent as a color developer at this •

stage also offers the possibility of improved sensitivity.

Color Development from Phenolphthalein Monophosphate Hydrolysate in Milk Heated at 61.6 C for Various Time Intervals

Four samples of raw milk were heated at 61.6 C for various time intervals followed by rapid cooling by immersing in shaved ice. The time required to reach 61.6 C was approximately 30 minutes. A constant amount of substrate was added to each sample such that the concentration of substrate in each sample was 0.83 mg/ml. The pH of each sample was

9.95.

The samples were incubated for 20 minutes at 37 C. The solids from each sample were next precipitated and filtered out.. Equal amounts of filtrate from each sample were alkalized, refiltered, and the color spectrophotometrically measured. The blank consisted of boiled milk and contained a substrate concentration equivalent in amount to the other samples. In all other respects the blank was treated in the same manner as the samples. A comparison between the results.of this test and results obtained from identical samples by the A.O.A.C. method are shown in Table 5. - 23

Table 5

■ Color Development in Raw Milk Heated at 61.6 C for Various Time Intervals As Determined by the Phenolphthalein Monophosphate and A.O.A.C. Methods.

’Time Held at 61.6 C Transmission (minutes) (%)

Phenolphthalein Monophosphate A.O.A.C.

0 93 55 ,

10 94 79

20 99 : 86

30 100 98

These data show that the same probable limiting factors as were proposed in the previous portion of the study (Table 4) were operative in this case. There was insufficient color development in the substrate hydrolysate under these conditions for adequate sensitivity in measure­ ment. The A.O.A.C. method, however, indicated adequate sensitivity.

Color Development from.Phenolphthalein Monophosphate Hydrolysate in Heated Milk Contaminated with Raw Milk

Each of five samples of boiled milk was contaminated with a different concentration of raw milk. A uniform concentration of

0.83 mg/ml substrate was added to each•sample. All the samples were in­ cubated at 37 C for 20 minutes. The solids were precipitated and fil­ tered out as previously described. Following this, equivalent amounts of filtrate from each sample were alkalized to a constant pH suitable for color development. The samples were refiltered and the color spec- trophotometrically evaluated against a blank consisting of boiled milk 24 .■

to which a concentration of substrate equivalent in amount to that of

the samples had been added. In all other respects the blank received

the same;treatment as the samples. Results from this test were compared

"with those obtained from identical samples by the A.O.A.C. method

(Table 6). .

Table 6

Color Development in Boiled Milk Contaminated with Various Levels of Raw Milk As Determined by the PhenoIphthalein Monophosphate and A.O.A.C. Methods

Raw Milk Contamination Transmission (%) (%)

•Phenolphthalein Monophosphate A.O.A.C.

0.05 100 95

0.10 100 96

0.20 1 100 95

0.40 100 85

0.80 - 100 71

Under the conditions of this trial, the color intensity de­

veloped from the phenolphthalein monophosphate hydrolysate at levels

ranging from 0.05-0.80% raw milk contamination was insufficient for

definitive measurement. The A.O.A.C. method proved to be sensitive to

contamination at the 0.4% level and above. REFERENCES

(1) American Public Health Association, Inc. 1960. Standard'Methods for the Examination of Dairy Products. 11th ed. New York.

(2) ■ Aschaffenburg, R., and Mullen, J. E. C. 1949. A Rapid and Simple Phosphatase Test for Milk, J. Dairy Res., 16: 58.

(3) Babson, A. L., Greeley, S., Coleman, C. M., and Phillips, G. E. 1966. Phenolphthalein Monophosphate as a Substrate for Serum Alkaline Phosphatase. Clinical Chemistry, 12: 482.

(4) Doan, .F. J., and Josephson, I). V. 1943. Observations on the Ascorbic Acid Content of Evaporated Milk. J. Dairy Sci., 26: 1031.

(5) Jenness, R., and Patton, S. 1959. Principles of Dairy Chemistry. John Wiley § Sons, Inc., New York.

- (6) Kay, H. D., and Graham, W. R., Jr. 1933. Phosphorous Compounds of Milk. J. Dairy Res., 5: 63.

(7) Kay, H. D., and Graham, W. R., Jr. 1935. The Phosphatase Test for Pasteurized Milk. J . Dairy Res., 6: 191.

. (8) Kosikowsky, F. V. 1949. A Simple Universal Dairy Products Phos­ phatase Test. Science, 110: 480.

(9) Lin, S. H. C.and Kleyn, D. H. 1967. New Alkaline Phosphatase Activity Assay System as Applied to Milk. (Abs.) J. Dairy Sci., 50: 942. V ,

(10) Sager, 0. S., Sanders, G. P',y and Hupfer, J. A., Jr. 1951. In­ fluences of Buffer Concentrations, pH Values and Incubation Temperatures on Sensitivity and Accuracy of Phosphatase Tests. (Abs.) J. Dairy Sci., 34: 306.

(11) Sanders, G. P., and Sager, 0. S. 1946. Modification of the Phos­ phatase Test as Applied to Cheddar Cheese and Application of the Test to Fluid Milk. J. Dairy Sci., 20: 737.

(12) Scharer, H. 1938. A Rapid Phosphomonoesterase Test for Control of Dairy Pasteurization. J. Dairy Sci., 20: 21.

25 26

(13) Shinowara, F. Y., Jones, L.M., and Reinhart, H. L. 1942. Esti­ mation of Serum Inorganic Phosphate and "Acid" and "Alkaline" Phosphatase Activity. J, Biol. Ghem., 142: 921.

(14) Wilkinson, J. H., and Vodden, A, 1966. Phenolphthalein Monophos- . phate as a Substrate for Serum Alkaline Phosphatase. Clinical Chemistry, 12: 701.