Determination of the Heat Treatment Undergone by Milk by Following the Denaturation of α-Lactalbumin with a Biosensor

Abstract

Dairy industries are interested in knowing the heat treatment undergone by milk so as to control the quality of drinking milks or to control their heating systems. Among the different techniques available to characterize the heat treatment of milk, estimation of the denaturation of proteins has been widely used. However, because the concentration of the proteins in raw milk can fluctuate significantly, determining only the concentration of a native protein without knowing its concentration in the raw milk before undergoing heat treatment can lead to significant imprecision. The objective of this study was to develop, on Biacore 3000, a biosensor assay for determining the denaturation index of alpha-lactalbumin by quantifying separately the native and "heat-denatured" forms of alpha-lactalbumin with specific monoclonal antibodies. alpha-Lactalbumin denaturation index is independent of the concentration of alpha-lactalbumin in the original raw milk. The technique developed is discriminating, fast, repeatable, fully automated, and requires no pretreatment of the milk sample.

Authentication of dairy products by immunochemical methods: a review

“…Mild time/temperature treatments of milk, including pasteurization, can be evaluated by a specific immunoassay using a monoclonal antibody capable of monitoring heat-induced conformational changes in bovine milk alkaline phosphatase (Levieux et al 2007). Similar results have also been obtained by quantifying separately the native and "heat-denatured" forms of α-lactalbumin using monoclonal antibodies in different assay formats (Dupont et al 2004;Jeanson et al 1999). In contrast, thermal treatments which are more severe than pasteurization can be classified on the basis of lactoferrin denaturation (Indyk et al 2007).…”

“…Immunosensors are analytical devices composed of antibodies immobilized on a solid support and connected to transducers converting the binding of the antigen into a physical signal. Applications of immunosensors to the control of milk and dairy products have been reviewed (Dupont et al 2006), as regards the detection of antibiotics, drug residues, chemical contaminants, hormones, pathogens and toxins, single caseins and also monitoring of α-lactoglobulin denaturation to evaluate severity of heat treatment applied to milk (Dupont et al 2004). Immunosensors might be especially valuable tools for direct online analysis as they give a response in real time, but the development of portable low-cost devices is required for field measurements by immunosensor-based techniques.…”

Distinction of different heat-treated bovine milks by native-PAGE fingerprinting of their whey proteins

“…The heat load can be estimated by the quantitative analysis of native whey proteins, which remain soluble at pH 4.6 after heat treatment, and are easily distinguished from the other major proteins in milk such as caseins (Dupont, Rolet-Répécaud, & Muller-Renaud, 2004;Law & Leaver, 2000;Losito, Carbonara, Monaci, & Palmisano, 2007). The most common whey proteins are b-lactoglobulin (b-LG), a-lactalbumin (a-LA), blood serum albumin (BSA) and immunoglobulins (IG).…”

“…The most common whey proteins are b-lactoglobulin (b-LG), a-lactalbumin (a-LA), blood serum albumin (BSA) and immunoglobulins (IG). Various techniques that measure the denaturation of whey proteins such as b-LG (Rada-Mendoza, Villamiel, Molina, & Olano, 2006) or a-LA (Dupont et al, 2004) have been proposed for overall characterization of heat-treated milks.…”

Analysis of denaturation of bovine IgG by heat and high pressure using an optical biosensor

“…More recent developments have extended immunoassay through direct, label-free monitoring of this interaction by various transducer techniques (Gapper, Copestake, Otter, & Indyk, 2007). Of these methods, surface plasmon resonance (SPR)-based biosensor immunoassays are capable of producing rapid, automatable and reliable data for the quantitative analysis of components in complex food matrices with minimal sample preparation, and have been applied to the measurement of native IgG in bovine milk and colostrum (Indyk & Filonzi, 2003) and to monitoring heat-induced denaturation of bovine milk a-lactalbumin and lactoferrin (Dupont, Rolet-Repecaud, & Muller-Renaud, 2004;Indyk, McGrail, Watene, & Filonzi, 2007). Milk protein denaturation may induce modifications to conformational epitopes which may be exploited using specific antibodies and SPR-immunoassay (Dupont & Muller-Renaud, 2006).…”