Determination of Whey Protein Nitrogen Index in Milk Powder

Abstract: Whey protein nitrogen index (WPNI) is a well-known method of classifying non-fat dry milk powder (NFDM) based on its heat treatment. This present work was aimed at the quantitative determination of WPNI in heat treated milk powder samples. The analysis has been carried out spectrophotometric ally by determining turbidity of soluble whey proteins at 420 nm using a standard curve of known WPNI value. Five different samples of skimmed milk powder were analyzed and it was observed that WPNI values varied from 1.22 to 7.41mg/g. Based on the WPNI values, the milk powders were classified as Low Heat Powder, Medium Heat Powder and High heat Powder.

Keywords: WPNI, Milk Powder


Prior to drying, skim milk or skim milk concentrates can be given an additional heat treatment to alter the functional properties such as solubility, water holding, emulsification, and viscosity of the resulting dried powder. In the native state, whey proteins have a definite conformation, which when exposed to heat above a certain critical level, is disrupted, and characteristic properties of the protein are altered. Because the extent of whey protein denaturation has important consequences on the functional properties of many milk products, a number of techniques have been developed to measure this phenomenon.

It has been shown that this heat treatment is related to the extent of whey protein denatured. The above relationship was used to establish the widely practiced heat classification system based on the Whey Protein Nitrogen Index (WPNI) analytical method.  This classification scheme provides one criterion for the selection of NFDM for food applications.

 Class  WPN
 High heat powder  < 1.5
 Medium heat powder  > 1.5 – < 6.0
 Low heat powder  > 6.0

Materials and Methods:

Samples of low heat, medium heat and standardized SMP were analyzed for WPNI by American Dairy Products Institute (ADPI) method. This turbid metric method is based on removal of casein and denatured whey protein from unfeatured whey protein by precipitation with saturated NaCl solution. Then, 2 drops of 10% HCl is added to a test tube containing 1 mL sample of filtrate and 10 mL of saturated NaCl solution. Then, the developed turbidity of the sample is measured at 420 nm and compared against a standard curve generated from analysis of “standard” milk powders of WPNI value obtained from ADPI low heat NFDM (WPNI of 7.32mgN·g−1) and high heat NFDM (WPNI of 0.32 mgN·g−1) using the above method. It is assumed that developed turbidity corresponds to the amount of UWPN in the original milk powders. Hence, results are reported as mg soluble whey protein nitrogen per g of powder.

Results and Discussion:

5 different milk powder samples were analyzed for WPNI. The results were incorporated in table 1. The analyzed samples were classified as HHP (high Heat Powder), MHP (Medium Heat Powder) and LHP (Low Heat Powder) as per the WPNI values.

Table 1: Whey Protein Nitrogen Index (WPNI) of Milk powders in mg/g of Milk Powder –

Milk Powder Sample 1 Sample 2 Sample 3 Sample 4 Sample 5
1.35 1.22 4.58 6.59 7.41


Heat treatment of the original product or the concentrate can cause denaturation of serum proteins. The extent of denaturation is an important quality mark in relation to the use of milk powder. For instance, if the powder is to be used in cheese making, practically no serum protein should have been denatured in view of the rennet ability; in infant formulas, on the other hand, the rennet ability should be poor. The whey protein nitrogen index (WPNI) is generally used to classify milk powders according to the intensity of the heat treatment(s) applied during manufacture.

Contributed by: Dr. Amartya Kumar Gupta