Question

Which chemical is used as a substrate for alkaline phosphatase for evaluating the efficiency of pasteurization?

• A. Rosalic acid
• B. Sodium Bicarbonate
• C. Dimethyl aminobenzaldehye
• D. p-Nitrophenyl Phosphate

Hint:

Phosphatase Substrates:
Aschaffenburg-Mullen Rapid Method $\rightarrow$ p-NITROPHENYL PHOSPHATE (pNPP, yields yellow p-nitrophenol).
Kay-Graham Standard Method $\rightarrow$ Disodium phenyl phosphate (Folin-Ciocalteu blue).

Answer 1

The Correct Option is D

Step 1: Understanding the Concept:
Alkaline Phosphatase (ALP) pasteurization verification assay: the standard Aschaffenburg-Mullen and AOAC fluorometric/colorimetric tests employ p-nitrophenyl phosphate (PNPP) or disodium phenyl phosphate as synthetic chromogenic substrates cleaved to yellow p-nitrophenol at alkaline pH 9.8.
Key Formula or Approach:
\[ \mathbf{\textit{p}\text{-Nitrophenyl Phosphate (Colorless)}} + \text{H}_2\text{O} \xrightarrow[\text{pH } 9.8 - 10.2]{\mathbf{Alkaline \text{ } Phosphatase}} \mathbf{\textit{p}\text{-Nitrophenol (Yellow Chromophore, } 405\text{ nm)}} + \text{P}_i \]

Step 2: Detailed Explanation:
In dairy analytical quality control and pasteurization validation:
- Alkaline Phosphatase Test: The universal statutory test for verifying adequate thermal pasteurization of milk ($71.7^\circ ext{C}$ for 15 s).
- In the standard Aschaffenburg and Mullen Rapid Phosphatase Test (BS: 770 ISO 3356):
1. The synthetic chromogenic substrate utilized is Disodium p-Nitrophenyl Phosphate (pNPP) (D).
2. If alkaline phosphatase is incompletely inactivated (undercooked milk or $> 0.1\%$ raw milk contamination), the enzyme hydrolyzes colorless pNPP in alkaline carbonate buffer (pH 10.2) to liberate p-Nitrophenol, generating an intense yellow color measured photometrically at $405\text{ nm}$ or against Lovibond comparator discs.

Step 3: Final Answer:
Therefore, p-Nitrophenyl Phosphate is used as the substrate, corresponding to option (D).