Kinetic and thermodynamic parameters for thermal denaturation of ovine milk lactoferrin determined by its loss of immunoreactivity

Abstract

Lactoferrin is a protein with important biological functions that can be obtained from milk and by-products derived from the dairy industry, such as whey. Although bovine lactoferrin has been extensively studied, ovine lactoferrin is not quite as well known. In the present study, the effect of several heat treatments in 3 different media, over a temperature range from 66 to 75°C, has been studied on lactoferrin isolated from sheep milk. Denaturation of lactoferrin was determined by measuring its immunoreactivity with specific polyclonal antibodies. Kinetic and thermodynamic parameters obtained indicate that lactoferrin denatures by heat more rapidly in whey than in phosphate buffer or milk. The value of activation energy found for the denaturation process of lactoferrin when treated in whey is higher (390kJ/mol) than that obtained in milk (194kJ/mol) or phosphate buffer (179kJ/mol). This indicates that a great amount of energy is necessary to start denaturation of ovine lactoferrin, probably due to the interaction of this protein with other whey proteins. The changes in the hydrophobicity of lactoferrin after heat treatments were determined by fluorescence measurement using acrylamide. The decrease in the hydrophobicity constant was very small for the treatments from 66 to 75°C, up to 20min, which indicates that lactoferrin conformation did not experienced a great change. The results obtained in this study permit the prediction of behavior of ovine lactoferrin under several heat treatments and show that high-temperature, short-time pasteurization (72°C, 15 s) does not cause loss of its immunoreactivity and, consequently, would not affect its conformation and biological activity.

Stability-Indicating Analytical Approach for Stability Evaluation of Lactoferrin

“…The degradation of Lf at elevated temperatures was also quantitatively evaluated using the RP-HPLC method. The degradation of Lf in aqueous solution at 50-80 • C best fitted the zero-order kinetic model (Table 6) This is not in line with literature data that report either first or 1.5 order kinetics [34][35][36]. However, the determination coefficients (R 2 ) obtained in our study for the zero-order model were higher compared to those found in other research articles.…”

“…The activation energy calculated from the Arrhenius plot was found to be 78.4 kJ/mol, which is lower than values found in the literature. However, a completely different analytical technique was used in these studies [ 34 , 35 , 36 ]. The differences could also be due to different iron saturation and glycosylation patterns of Lf and/or media used in stability studies [ 12 ].…”

Effect of Processing Intensity on Immunologically Active Bovine Milk Serum Proteins

“…Generally whey proteins are susceptible to heat treatment [ 5 , 6 ], particularly immunoactive proteins such as lactoferrin or lactadherin [ 7 ]. Heating of heat-labile proteins results in denaturation and aggregation processes [ 8 ] and thereby leads to a loss of biological functionality (e.g., lactoferrin [ 9 ]). Denatured and aggregated proteins can be extracted from the milk with a combination of pH reduction and ultracentrifugation [ 10 ], after which remaining levels of non-aggregated milk serum proteins can be determined [ 7 ].…”

Changes in the structure and functional properties of lactoferrin with different iron saturations before and after simulated high‐temperature short‐time heat treatments

“…A current study using a rheometer for heat-treating LF with an iron saturation of about 9-10% showed that treatment at temperatures of 72-95°C caused irreversible changes in the structural and physicochemical properties of LF (Goulding et al 2021). Although LF does not lose its immunoreactivity under pasteurisation (72°C, 15 s) conditions, inappropriate heat treatment (80°C, 10 min) caused a reduction of its regular secondary structure elements (Navarro et al 2015). Denaturation and aggregation of LF under heat treatment are thought to be influenced by many factors, such as iron saturation.…”