Abstract
Milk with different κ-casein (CN) phenotypes has previously been found to influence its gastric digestion rate. Therefore, the aim of the present study is to disentangle contributions of genetic variation and its related sialylation on the in vitro digestion process of κ-CN. Accordingly, κ-CN was purified from milk representing homozygous cows with κ-CN phenotypes AA, BB, or EE and used as substrate molecules in model studies using the INFOGEST 2.0 in vitro static digestion model. Furthermore, the effect of removal of the terminal sialic acids present on the O-linked oligosaccharides of the purified κ-CN A, B, and E protein variants were studied by desialylation enzymatic assays. The κ-CN proteins were purified by reducing anion exchange chromatography with purities of variants A, B, and E of 93.0, 97.1, and 90.0%, respectively. Protein degradations of native and desialylated κ-CN isolates in gastric and intestinal phases were investigated by sodium dodecyl sulfate-PAGE, degree of hydrolysis (DH), and liquid chromatography electrospray ionization mass spectrometry. It was shown that after purification, the κ-CN molecules reassembled into multimer states, which then constituted the basis for the digestion studies. As assessed by DH, purified variants A and E were found to exhibit faster in vitro digestion rates in both gastric and intestinal phases compared with variant B. Desialylation increased both gastric and intestinal digestion rates for all variants, as measured by DH. In the gastric phase, desialylation promoted digestion of variant B at a rate comparable with native variants A and E, whereas in the intestinal phase, desialylation of variant B promoted better digestion than native A or E. Taken together, the results confirm that low glycosylation degree of purified κ-CN promotes faster in vitro digestion rates, and that desialylation of the O-linked oligosaccharides further promotes digestion. This finding could be applied to produce dairy products with enhanced digestibility.
Highlights
Kappa-casein genetic variants A and B are the most common variants among different breeds (Caroli et al, 2009), including Nordic breeds (Poulsen et al, 2016)
Apart from glycosylation, bovine κ-CN is known to be modified through disulfide bonds, with its 2 cysteine residues per κ-CN molecule (Cys11 and Cys88) being involved in intra- and intermolecular disulfide bonds (Mercier, Brignon, and Ribadeau-Dumas, 1973; Rasmussen et al, 1992). κ-CN is present in raw bovine milk mainly as oligomers with 2 or more monomers linked by disulfide bonds, and only 10% of total κ-CN has been reported to be present as monomer (Huppertz, 2013)
Bovine κ-CN variants A, B, and E were purified from pooled skim milk from homozygous cows by reducing anion exchange chromatography, which, after purification, was thoroughly dialyzed at nonreducing conditions
Summary
Kappa-casein genetic variants A and B are the most common variants among different breeds (Caroli et al, 2009), including Nordic breeds (Poulsen et al, 2016). The glycosylation degree (GD), the ratio of glycosylated κ-CN to total κ-CN, and the glycan combinations all show high degrees of heterogeneity between genetic variants A, B, and E (Nilsson et al, 2020; Sheng et al, 2022). Κ-CN variant B is known to have a higher GD than variant A, and recent reports indicate that variant E has a GD around or slightly higher than that of B (Jensen et al, 2015; Poulsen et al, 2016; Sheng et al, 2022). Apart from glycosylation, bovine κ-CN is known to be modified through disulfide bonds, with its 2 cysteine residues per κ-CN molecule (Cys and Cys88) being involved in intra- and intermolecular disulfide bonds (Mercier, Brignon, and Ribadeau-Dumas, 1973; Rasmussen et al, 1992). κ-CN is present in raw bovine milk mainly as oligomers with 2 or more monomers linked by disulfide bonds, and only 10% of total κ-CN has been reported to be present as monomer (Huppertz, 2013)
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