Antioxidant Nature of Bovine Milk β-Lactoglobulin

Primary structure of β-LG. β-Lactoglobulin comprises 162 AA, including 5 Cys residues. Two disulfide linkages are located at residues Cys-106 to Cys-119 and Cys-66 to Cys-160. One free Cys is at position 121.

Antioxidant activity of β-LG and other antioxidants. The antioxidant activity was estimated by the degree of inhibition of Cu²⁺-induced formation of thiobarbituric acid-reactive substances. The assay was conducted using 100μg of low-density lipoprotein in a final 100μL of reaction mixture (see the Materials and Methods section). Each point represents the mean of duplicate determinations.

Changes in β-LG in Cu²⁺-induced low-density lipoprotein (LDL) oxidation over time. The antioxidant activity was determined in the absence or presence of 60μM of β-LG (A). The assay was conducted using 60μg of LDL in a final 100mL of reaction mixture. Notably, the formation of thiobarbituric acid-reactive substances was about half of that depicted in Figure 2. Each point represents the mean of duplicate determinations. β-Lactoglobulin was gradually oxidized, and some formed β-LG dimers, as analyzed by a 15% SDS-PAGE (B) and a Western blot (C). Lane M: molecular weight marker.

Effect of carboxymethylation on the antioxidant activity of β-LG. Carboxymethylation was conducted to explore the role of disulfide linkages in β-LG for their antioxidant activity. The overall inhibitory activity against low-density lipoprotein (60μg/mL) oxidation of native β-LG was significantly greater than that of carboxy-methylated (CM) β-LG (P<0.001). Each bar represents the mean±SD of triplicates.

Characterization of heated β-LG using PAGE and Western blot. β-Lactoglobulin in PBS (1 mg/mL) was heated at 100°C for 2min. Left: 15% SDS-PAGE. Right: Western blot analysis. Lane M: molecular weight markers; lane A: native β-LG; lane B: heated β-LG. Notably, dimers covalently linked through disulfide linkages were reversible via the addition of β-mercaptoethanol (data not shown).

Sodium dodecyl sulfate-PAGE of skim raw milk with and without β-LG depletion and the effects on antioxidant activity. (A) β-Lactoglobulin-depleted milk was obtained from a β-LG rabbit polyclonal antibody affinity column by collecting the pass-through fraction without further manipulation. Lane M: molecular weight markers; lane 1: β-LG-depleted raw milk; lane 2: raw milk. (B) The antioxidant assay was conducted using 60μg of low-density lipoprotein in a final 100-mL reaction mixture. Each point represents the mean±SD of triplicates.

Antioxidant activity of skim raw milk with and without heat. Heated milk was prepared by heating at 100°C for 2min. (A) The antioxidant assay was conducted using 60μg of low-density lipoprotein in a final 100-mL reaction mixture. Each point represents the mean±SD of triplicates. (B) Native PAGE of raw milk with and without heating. β-Lactoglobulin was substantially lost in heated raw milk by forming β-LG polymers or large aggregates with milk proteins.