Determination of Vegetal Proteins in Milk Powder by Enzyme-Linked Immunosorbent Assay: Interlaboratory Study

Abstract

Eight laboratories participated in a collaborative study to evaluate an enzyme-linked immunosorbent assay (ELISA) to determine soy, pea, and wheat proteins in pasteurized or ultra-high temperature (UHT) milk powders. To perform this assay, polyclonal antibodies for soy, pea, and wheat proteins were obtained from rabbit sera. Collaborators received calibration standards composed of milk powder containing 0–8% (w/w) vegetal protein in total protein and blind test samples containing approximately 1, 2, and 5% (w/w) vegetal protein. An indirect competitive ELISA was performed with a kit prepared by a participating laboratory; the kit contained plates coated with soy, pea, or wheat proteins, the corresponding specific antisera, enzyme-labeled second antibody, and substrate solution. Test samples and calibrants were extracted with phosphate-buffered saline, pH 7.4, containing 0.05% Tween and assayed with the ELISA kits. The degree of adulteration was affected by the type of heat treatment applied to the samples. The estimated percentage of vegetal protein addition was close to the theoretical value for pasteurized samples but much lower for UHT samples. For pasteurized samples, intralaboratory relative standard deviations ranged from 5 to 22% and interlaboratory relative standard deviations ranged from 14 to 34%.

Distinction Between Dry and Raw Milk Using Monoclonal Antibodies Prepared Against Dry Milk Proteins

“…Particularly, such supplementation happens when the supply of raw milk is not sufficient in the summer as consumer demand increases and cow milk production decreases. Because the ultra-heat treatment procedure has been widely used in preparing milk powder, efforts using heat-denatured milk proteins as bioindicators have been extended to detect false practices (Relkin, 1996;Sanchez et al, 2002;Steffensen et al, 2002). For example, Recio and Olieman (1996) showed that the amount of heat-denatured proteins could be estimated by analyzing the casein fraction using capillary zone electrophoresis.…”

Milk Processing and Quality Management

“…Proteins of plant or animal origin could be added to milk, a practice that is common in countries if they are cheaper than milk proteins. A number of electrophoretic (Kanning et al, 1993;Cattaneo et al, 1994;López-Tapia et al, 1999;Manso et al, 2002a,b), chromatographic (Cattaneo et al, 1994;Espeja et al, 2001) and immunochemical (Turin & Bonomi, 1994;Sánchez et al, 2002) methods have been used to detect the addition of non-dairy protein in milk products.…”

Detection of plant protein adulterated in fluid milk using two-dimensional gel electrophoresis combined with mass spectrometry

“…Added soy, pea, and soluble wheat protein in milk powder could be detected through immunological tests. Development of polyclonal antibodies in immunoassays has made it possible to detect these adulterants in a range of 1-5% of plant protein in the total milk protein content, although the results seem to be significantly affected by ultra-high temperature (UHT) treatment applied to the samples (Haasnoot et al 2001;Sanchez et al 2002). Compared with Enzyme-Linked Immunosorbent Assay (ELISA), the results of vegetable protein percentages obtained from Sodium Dodecyl Sulfate-Capillary Electrophoresis (SDS-CE) had a higher rate of accuracy.…”

“…Compared with Enzyme-Linked Immunosorbent Assay (ELISA), the results of vegetable protein percentages obtained from Sodium Dodecyl Sulfate-Capillary Electrophoresis (SDS-CE) had a higher rate of accuracy. However, due to its poor reproducibility, SDS-CE requires improvement in terms of instrument and operation standardisation before large-scale use is feasible (Sanchez et al 2002). The application of mass spectroscopy (MS) allowed the identification of plant protein added to milk powder.…”