In vitro digestion of purified β-casein variants A1, A2, B, and I: Effects on antioxidant and angiotensin-converting enzyme inhibitory capacity

Abstract

Genetic polymorphisms of bovine milk proteins affect the protein profile of the milk and, hence, certain technological properties, such as casein (CN) number and cheese yield. However, reports show that such polymorphisms may also affect the health-related properties of milk. Therefore, to gain insight into their digestion pattern and bioactive potential, β-CN was purified from bovine milk originating from cows homozygous for the variants A(1), A(2), B, and I by a combination of cold storage, ultracentrifugation, and acid precipitation. The purity of the isolated β-CN was determined by HPLC, variants were verified by mass spectrometry, and molar extinction coefficients at λ=280nm were determined. β-Casein from each of the variants was subjected to in vitro digestion using pepsin and pancreatic enzymes. Antioxidant and angiotensin-converting enzyme (ACE) inhibitory capacities of the hydrolysates were assessed at 3 stages of digestion and related to that of the undigested samples. Neither molar extinction coefficients nor overall digestibility varied significantly between these 4 variants; however, clear differences in digestion pattern were indicated by gel electrophoresis. In particular, after 60min of pepsin followed by 5min of pancreatic enzyme digestion, one ≈4kDa peptide with the N-terminal sequence (106)H-K-E-M-P-F-P-K- was absent from β-CN variant B. This is likely a result of the (122)Ser to (122)Arg substitution in variant B introducing a novel trypsin cleavage site, leading to the changed digestion pattern. All investigated β-CN variants exhibited a significant increase in antioxidant capacity upon digestion, as measured by the Trolox-equivalent antioxidant capacity assay. After 60min of pepsin + 120min of pancreatic enzyme digestion, the accumulated increase in antioxidant capacity was ≈1.7-fold for the 4 β-CN variants. The ACE inhibitory capacity was also significantly increased by digestion, with the B variant reaching the highest inhibitory capacity at the end of digestion (60min of pepsin + 120min of pancreatic enzymes), possibly because of the observed alternative digestion pattern. These results demonstrate that genetic polymorphisms affect the digestion pattern and bioactivity of milk proteins. Moreover, their capacity for radical scavenging and ACE inhibition is affected by digestion.

Hydrolytic degree and antioxidant activity of purified casein characterised by different haplotypes (αs1‐, β‐ and k‐casein) after enzymatic hydrolysis with pepsin and enzymatic extract from Pleurotus eryngii

“…Whereas, Pleurotus eryngii possesses a pool of acid and basic proteases (Inácio et al , ) belonging to the families of aspartyl (Wang & Ng, ), serine (Cha et al , ) and metalloproteases (Shen et al , ), characterised by different active sites that hydrolyse the protein in several sites simultaneously, thus leading with a consequent increase in DH value. As shown in Table , DH values were significantly influenced by the CN haplotype ( P < 0.001), in agreement with Petrat‐Melin et al () who reported that the haplotype influences positively or negatively the presence of zones resistant to enzymatic hydrolysis. In fact, changes in the casein primary structure may alter enzyme cleavage sites, modifying both the structure of proteins and the behaviour of liberated peptides (Kamiński et al , ; Caroli et al , ).…”

Separation of angiotensin I‐converting enzyme inhibitory peptides from bovine connective tissue and their stability towards temperature, pH and digestive enzymes

“…The method was performed according Petrat‐Melin et al . (). The factor of lysine residues in the collagen is 0.028, and the average molecular weight of amino acids in collagen is 99 g mol −1 .…”

“…The DH of collagen hydrolysates was quantified by N-terminal amines with fluorescamine (Udenfriend et al, 1972). The method was performed according Petrat-Melin et al (2015). The factor of lysine residues in the collagen is 0.028, and the average molecular weight of amino acids in collagen is 99 g mol À1 .…”

In vitro digestibility and immunoreactivity of bovine milk proteins

“…Thus, the ability of food allergens to reach the jejunal mucosa is considered a prerequisite for allergenicity. While other studies have demonstrated the digestive stability of purified milk proteins, b-lactoglobulin and casein (Benedé et al, 2014;Peram, Loveday, Ye, & Singh, 2013;Petrat-Melin et al, 2015;Peyron, Mouécoucou, Frémont, Sanchez, & Gontard, 2006;Wal, 2001), few studies have examined the pepsin and/or pancreatin digestive stability of total bovine whole milk proteins. Moreover, limited digestibility studies have examined the immunoreactivity and potential allergenicity of digestive stable total bovine whole milk proteins, using allergen specific rabbit IgG antibodies and IgE antibodies from the sera of milk-allergic patients.…”