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

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.

Alkaline phosphatase activity in pasteurized milk: A quantitative comparison of Fluorophos and colourimetric procedures

“…For Fluorophos, these ranged between 0.9% and 7.5% and for the A&M procedure between 3.5% and 29%. These values compare favourably with reported data (Rocco 1990; Harding and Garry 2005).…”

“…At higher raw milk additions (500–2000 mU/L equivalent), although the results were not in the expected ranges, good repeatability was achieved with mean RSD r values between 4.4% and 8.8% (Table 1). This compares favourably with values in the literature for this procedure (Rocco 1990; Scintu et al. 2000).…”

“…500 mU/L: 1 μg phenol/mL based on 0.1% raw milk equivalent (Rocco 2004). However, this relationship was not always observed in other studies, where deviations and wide variability also existed (Rocco 1990; Angelino et al. 1999; Langridge 1999; Scintu et al.…”

“…New methods of analysis for ALP have been developed, which are faster and more sensitive. Fluorophos, a quantitative fluorimetric technique, uses the substrate benzothiazole orthophosphoric monoester, from which is liberated a fluorescent product that can be detected instrumentally in 3 min (Rocco 1990). It has been approved and is detailed in ISO 11816‐1/IDF‐155 (International Organisation for Standardisation 2006).…”

Validation of Milk Product Pasteurization by Alkaline Phosphatase Activity

“…Research has recently led to the reduction of the maximum residual limiting rade Aproducts to 350mU/L (Painter and Bradley, [39]; International Dairy Federation,1999) found less than 150mU/Lof ALP activity in under pasteurized low fat milk. In a collaborative study with a fluorometric assay, Rocco [87] found that milk contaminated with as much as 0.5% raw milk showed less than 265mU/L of ALP activity, there by stressing the need to reconsider current regulatory standards. An additional study (Klotz [56]) on cow, goat and sheep milks also suggests that the present limit of 350mU/L can be reduced further given that raw goat milk ALP levels were<350mU/L.…”