Rapid Colorimetric Test for Alkaline Phosphatase in Dairy Products

1979 ◽  
Vol 42 (10) ◽  
pp. 794-799 ◽  
Author(s):  
G. K. MURTHY ◽  
R. MARTIN ◽  
U. S. RHEA ◽  
J. T. PEELER
Keyword(s):  
Alkaline Phosphatase ◽  
Dairy Products ◽  
Rapid Test ◽  
Raw Milk ◽  
Visual Observation ◽  
Visual Methods ◽  
Visual Examination ◽  
Colorimetric Test ◽  
Photoelectric Colorimeter ◽  
Hydrolysis Of

The Scharer rapid test for measuring alkaline phosphatase activity in milk and milk products was modified to include a photoelectric colorimeter in place of visual observation of color. Dairy products containing various amounts of added enzyme (3–15 μg phenol/ml or g) were prepared for analysis as per standard methods and analyzed by the rapid colorimetric test. A linear relationship was found between the percent of raw milk added and the enzyme activity with a correlation coefficient (r) range of 0.963 to 1.000. Hydrolysis of the substrate (disodium phenyl phosphate) by the surviving enzyme in different dairy products was similar. Recovery of the added enzyme varied, depending on the nature of the product. The method is quantitative, reproducible, and can be used as a rapid confirmatory procedure. Similarly, this method was applied to differentiating residual and reactivated enzyme in milk, cream and buttermilk. Compared to the Scharer rapid and the Rutgers visual methods, this test was more reliable in borderline cases because it eliminated the bias encountered in visual examination. A method was developed to analyze alkaline phosphatase in casein.

Collaborative Study of Rapid Methods for Alkaline Phosphatase in Milk and Cream

1979 ◽  
Vol 42 (10) ◽  
pp. 800-803 ◽  
Author(s):  
G. K. MURTHY ◽  
J. T. PEELER
Keyword(s):  
Alkaline Phosphatase ◽  
Limit Of Detection ◽  
Collaborative Study ◽  
Colorimetric Method ◽  
Skim Milk ◽  
Rapid Test ◽  
Raw Milk ◽  
Colorimetric Test ◽  
Photoelectric Colorimeter

Determination of alkaline phosphatase activity in milk and cream by the modified Scharer rapid test with use of photoelectric colorimeter for measuring absorbance was collaboratively studied. Milk samples (skim milk. milk and cream) with and without added raw milk were sent to 12 collaborators to be tested by (a) the modified Scharer rapid test using commercial standards and phenol standards for comparing colors, (b) the rapid colorimetric test and (c) the Rutgers method. The latter method was used for comparison only. In the modified Scharer rapid test, based on the category of standards, 73.3% of the samples using the commercial standards and 71.6% of the samples using phenol standards were correctly diagnosed. When the scoring was based on positive or negative, 98.4 and 92.6% of the samples were correctly diagnosed. Results with the phenol standards were significantly lower than those observed with the commercial standards. There were no false-positive results, as all incorrect readings were below limit of detection. Most of the errors occurred when the expected value was 1.0 μg phenol/ml. Results were 100% correct for the Rutgers method, but there are only two choices for this method, and they correspond to positive or negative. Compared to the theoretical values, data obtained by the colorimetric method ranged from 1.5 to 18.1% high, with a coefficient of variation of 4.4 to 13.4%. These variations were assumed satisfactory considering the levels at which phosphatase was tested.

Rapid Methods for Differentiating Reactivated from Residual Phosphatase in Milk and Cream: Collaborative Study

1979 ◽  
Vol 62 (4) ◽  
pp. 822-827
Author(s):  
Gopala K Murthy ◽  
James T Peeler
Keyword(s):  
Magnesium Chloride ◽  
Collaborative Study ◽  
Control Sample ◽  
Rapid Test ◽  
Magnesium Acetate ◽  
Visual Methods ◽  
Test Results ◽  
Milk Samples ◽  
Colorimetric Test ◽  
Photoelectric Colorimeter

Abstract Three methods for differentiating reactivated from residual phosphatase in milk and cream were collaboratively tested using both magnesium acetate and magnesium chloride for reactivating phosphatase. The methods evaluated were the modified Scharer rapid test, the rapid colorimetric test, and the Rutgers method. Nine collaborators tested 6 unknown milk samples containing reactivated and/or residual phosphatase, and 16 collaborators tested 6 unknown cream samples containing reactivated and/or residual phosphatase. Results indicated that use of magnesium acetate in place of magnesium chloride for reactivating phosphatase improved test results. Visual tests (modified Scharer rapid and Rutgers) predicted correct results when the samples contained high levels of reactivated or residual phosphatase. In borderline cases where the reactivated phosphatase contents of the undiluted control sample and the diluted sample containing Mg were very close, the test results of the visual methods were significantly different from 100% correct results at the α = 0.05 level. Use of a photoelectric colorimeter or its equivalent for measuring the absorbance in conjunction with the modified Scharer rapid test improved results considerably. The modified Scharer rapid test was adopted official first action.

Fluorometric Analysis of Alkaline Phosphatase in Fluid Dairy Products

1990 ◽  
Vol 53 (7) ◽  
pp. 588-591 ◽  
Author(s):  
RICHARD M. ROCCO
Keyword(s):  
Alkaline Phosphatase ◽  
Dairy Products ◽  
Raw Milk ◽  
Test Sample ◽  
Reaction Rates ◽  
Product Formation ◽  
Test Time ◽  
Whole Milk ◽  
Repeated Analysis ◽  
Total Test Time

A new quantitative assay has been developed for measuring residual alkaline phosphatase (ALP) activity in a wide variety of dairy products including whole milk, low fat and skim milks, chocolate milk, and creams. ALP in the test sample hydrolyzes a nonfluorescent substrate, FluorophosR, to a highly fluorescent product. Product formation is monitored continuously during a short incubation period and enzyme activity is calculated from the rate of fluorescence increase. Total test time is 3 min. Reaction rates are linear up to 0.5% raw milk (equivalent to 5 μg phenol/ml/15 min) with a detection limit of 0.006% raw milk. Within and between run precision of the fluorometric method was assessed by repeated analysis of a pasteurized milk sample containing added mixed herd raw milk. The within run (N=10) mean was 190.4 mU/L, standard deviation (SD) 3.2, and a coefficient of variance (CV) of 1.7%. The procedure provides a rapid, sensitive, precise, and easy-to-use ALP assay, applicable to a wide variety of dairy products.

Fluorometric Determination of Alkaline Phosphatase in Fluid Dairy Products: Collaborative Study

1990 ◽  
Vol 73 (6) ◽  
pp. 842-849 ◽  
Author(s):  
Richard M Rocco
Keyword(s):  
Alkaline Phosphatase ◽  
Dairy Products ◽  
Collaborative Study ◽  
Skim Milk ◽  
Raw Milk ◽  
Chocolate Milk ◽  
Phenyl Phosphate ◽  
Whole Milk ◽  
Standard Deviations

Abstract Official methods for the measurement of alkaline phosphatase (ALP) in dairy products use either phenyl phosphate or phenolphthaleln monophosphate as substrate. Quantitation of results requires butanol extraction of the Indophenol (Scharer) or 3-h dialysis of the liberated phenolphthaleln (Rutgers). The Advanced Fluorophos® assay Is based on a self-indicating substrate which, when acted upon by ALP, loses a phosphate radical and becomes a highly fluorescent compound. The rate of fluorophore formation Is monitored for 3 mln In a fluorometer and the enzyme activity In mU/L Is calculated. Eight laboratories participated in a collaborative study to evaluate the Fluorophos® assay for determining ALP activity In whole milk, skim milk, chocolate milk, and cream (half and half). The comparative method was the AOAC quantitative phenyl phosphate method, 16.121-16.122 (14th Ed.). Mixed herd raw milk was added to pasteurized samples at 0.05, 0.1, and 0.2% (v/v). Method performance at 0.1% (v/v) added raw milk as measured by repeatability and reproducibility standard deviations (sr and sR) and relative standard deviations (RSDr and RSDR), respectively, were: whole milk, sr = 21.7%, sR = 34.6%, RSDr = 4.4%, RSDR = 7.0%; skim milk, sr = 19.2%, sR = 31.4%, RSDr = 3.8%, RSDR = 6.2%; chocolate milk, sr = 27.6%, sR = 45.8%, RSDr = 5.3%, RSDR = 8.8%. The method has been adopted official first action by AOAC for determination of alkaline phosphatase in whole milk, skim milk, and chocolate milk.

Evaluation of Alkaline Phosphatase Detection in Dairy Products Using a Modified Rapid Chemiluminescent Method and Official Methods

2011 ◽  
Vol 74 (7) ◽  
pp. 1144-1154 ◽  
Author(s):  
S. M. ALBILLOS ◽  
R. REDDY ◽  
R. SALTER
Keyword(s):  
Public Health ◽  
Alkaline Phosphatase ◽  
Dairy Products ◽  
Limit Of Detection ◽  
Raw Milk ◽  
Modified Method ◽  
European Public ◽  
Single Laboratory ◽  
Chemiluminescent Method ◽  
The U.S

Alkaline phosphatase is a ubiquitous milk enzyme that historically has been used to verify adequate pasteurization of milk for public health purposes. Current approved methods for detection of alkaline phosphatase in milk include the use of enzyme photoactivated substrates to give readings in milliunits per liter. The U.S. and European public health limit for alkaline phosphatase in pasteurized drinks is 350 mU/liter. A modified chemiluminescent method, fast alkaline phosphatase, was compared with the approved fluorometric and chemiluminescent alkaline phosphatase methods to determine whether the modified method was equivalent to the approved methods and suitable for detecting alkaline phosphatase in milk. Alkaline phosphatase concentrations in cow's, goat's, and sheep's milk and in flavored drinks and cream were determined by three methods. Evaluations in each matrix were conducted with pasteurized samples spiked with raw milk to produce alkaline phosphatase concentrations of 2 to 5,000 mU/liter. The tests were performed by the method developer and then reproduced at a laboratory at the National Center for Food Safety and Technology following the criteria for a single laboratory validation. The results indicated that the fast alkaline phosphatase method was not significantly different from the approved chemiluminescent method, with a limit of detection of 20 to 50 mU/liter in all the studied matrices. This modified chemiluminescent method detects alkaline phosphatase in the 350 mU/liter range with absolute differences from triplicate data that are lower and within the range of the allowed intralaboratory repeatability values published for the approved chemiluminescent method.

Method for Predicting Minimum Detectable Residual Alkaline Phosphatase in High-Temperature, Short-Time Processed Dairy Products

1980 ◽  
Vol 43 (1) ◽  
pp. 46-48 ◽  
Author(s):  
G. K. MURTHY ◽  
J. T. PEELER
Keyword(s):  
Alkaline Phosphatase ◽  
High Temperature ◽  
Control Sample ◽  
Rank Correlation ◽  
Raw Milk ◽  
Milk Product ◽  
Colorimetric Test ◽  
High Temperature Short Time ◽  
Highly Correlated ◽  
Short Time

High-temperature, short-time (HTST) processed milk, cream and buttermilk were mixed with small portions (0 to 0.6%) of the raw milk product to obtain desired levels of residual alkaline phosphatase. Samples were subjected to the differential test to discern reactivation and analyzed for phosphatase activity by the rapid colorimetric test. The experimental data were fitted to a linear statistical model to determine the minimum detectable residual phosphatase (Eo) in the product. These observed values and the computed expected values were highly correlated, with a rank correlation coefficient of 0.956, which was significant at a = 0.05 level. The values of [Eo] varied depending upon the extent of phosphatase reactivation in the HTST product when the residual phosphatase was zero. As the differential values of reactivation (reactivated [E] of the control sample minus the reactivated [E] of diluted sample containing magnesium) increased, the [Eo] increased also. In general, the [Eo] in cream was greater than that in milk. A method is proposed for predicting [Eo] in liquid HTST products.

Development of a monoclonal antibody-based immunoassay for specific quantification of bovine milk alkaline phosphatase

2007 ◽  
Vol 74 (3) ◽  
pp. 290-295 ◽  
Author(s):  
Nathalie Geneix ◽  
Eric Dufour ◽  
Annie Venien ◽  
Didier Levieux
Keyword(s):  
Monoclonal Antibody ◽  
Alkaline Phosphatase ◽  
Monoclonal Antibodies ◽  
Dairy Products ◽  
Indirect Elisa ◽  
Bovine Milk ◽  
Raw Milk ◽  
Heat Stable ◽  
Nitrophenyl Phosphate ◽  
Enzymatic Methods

The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0·02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.

Bacteriological Standards for Perishable Foods

1952 ◽  
Vol 72 (4) ◽  
pp. 404-410
Author(s):  
Arthur Rowlands
Keyword(s):  
Heat Treatment ◽  
Dairy Products ◽  
Rapid Test ◽  
Raw Milk ◽  
Human Consumption ◽  
Treatment Processes ◽  
Keeping Quality ◽  
Methylene Blue Test ◽  
General Compliance

For milk and dairy products complete freedom from pathogens is the only accept able standard. To achieve this, pasteurisation or some other form of heat treatment is necessary, adequate precautions being taken to prevent subsequent reinfection of the milk. Physical checks of plant coupled with the use of the phosphatase test, rather than bacteriological methods, serve to ensure the bactericidal efficiency of heat treatment processes and the safety of milk supplies. Bacteriological standards are desirable also to ensure milk which will keep sweet until it is utilized. For this purpose a keeping quality of twenty-four hours after delivery to the consumer may be considered a satisfactory minimum standard. At present, different modifications of the methylene blue test are in use officially for raw and pasteurised milk. In general, compliance with the standards imposed ensures a satisfactory keeping quality, but there is much to be said for a common test and standard for all milk sampled during distribution to the consumer. Direct measurement of keeping quality, using clot-on-boiling to determine the end-point, deserves serious consideration as a method for routine control purposes. For milk to be used for processing or manufacture, a rapid test to be applied to all milk as received is the first consideration. The I0-minute reszaurin test, despite its limitations, is now in general use for this purpose. In addition, however, it is desirable to carry out tests on samples from all producers at intervals, in order to maintain their interest in milk quality and to encourage improvement where necessary. There are no official bacteriological standards for dairy products. There is, in fact, little call for such standards, although manufacturers must, in their own interest, maintain strict bacteriological control over the quality of the raw milk and manufac turing processes. Failure to do so may lead to faults in the products which seriously impair their marketable value or make them entirely unsuitable for human consumption.

Extracellular enzymatic activities in the aquatic ecosystems of the Danube Delta. 2. Alkaline phosphatase activity

2021 ◽  
Vol 26 (1) ◽  
pp. 2269-2274
Author(s):  
IOAN PĂCEŞILĂ ◽  
EMILIA RADU
Keyword(s):  
Alkaline Phosphatase ◽  
Water Column ◽  
Aquatic Ecosystems ◽  
Temporal Dynamics ◽  
Extracellular Enzyme ◽  
Danube Delta ◽  
Phytoplankton Communities ◽  
Extracellular Enzymatic Activities ◽  
Adjacent Channels ◽  
Hydrolysis Of

Phosphorus is one of the most important inorganic nutrients in aquatic ecosystems, the development and functioning of the phytoplankton communities being often correlated with the degree of availability in assimilable forms of this element. Alkaline phosphatase (AP) is an extracellular enzyme with nonspecific activity that catalyses the hydrolysis of a large variety of organic phosphate esters and release orthophosphates. During 2011-2013, AP Activity (APA) was assessed in the water column and sediments of several aquatic ecosystems from Danube Delta: Roșu Lake, Mândra Lake and their adjacent channels – Roșu-Împuțita and Roșu-Puiu. The intensity of APA widely fluctuated, ranging between 230-2578 nmol p-nitrophenol L-1h-1 in the water column and 2104-15631 nmol p-nitrophenol g-1h-1 in sediment. Along the entire period of the study, APA was the most intense in Roșu-Împuțita channel, for both water and sediment samples. Temporal dynamics revealed its highest values in summer for the water column and in autumn for sediment. Statistical analysis showed significant seasonal diferences of the APA dynamics in spring vs. summer and autumn for the water column, and any relevant diferences for sediment.

Estimation of Aflatoxin M1 Levels in Some Dairy Products Manufactured from Raw Milk Experimentally Inoculated with Toxin

2019 ◽  
Vol 43 (1) ◽  
pp. 50-58
Author(s):  
H. S. Alnaemi
Keyword(s):  
Dairy Products ◽  
Raw Milk ◽  
Aflatoxin M1 ◽  
Consumer Health ◽  
Goat’S Milk ◽  
White Cheese ◽  
Elisa Technique ◽  
Permissible Levels ◽  
Goat's Milk

     Fate of AflatoxinM1 in soft white cheese and its by-product (whey) and in yogurt locally made from raw sheep's and goat's milk experimentally inoculated with 0.05 and 0.5 µg/l AflatoxinM1 were investigated using ELISA technique. Results reported that AflatoxinM1 was concentrated in cheese at levels significantly higher than that recorded in the raw milk that used for its processing, with a significant decrease in AflatoxinM1 levels in its by-product (whey) comparable to the raw milk used in manufacturing at both inoculated levels. Yogurt produced from raw sheep's milk at second inoculated level exerted AflatoxinM1concentration significantly lower than that present in the milk. Significant differences in AflatoxinM1distribution in cheese and whey produced from sheep's milk comparable to their counterparts produced from goat's milk were recorded. Finally, results revealed the efficacious role of the various dairy manufacturing processes in AflatoxinM1 distribution and the necessity to issue of local legislations concerning the maximum permissible limits for AflatoxinM1 in milk in order to stay within the universal permissible levels for AflatoxinM1 in dairy products to provide greater protection for consumer health. 

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