Effects of genetic variants and sialylation on in vitro digestibility of purified κ-casein

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 dodecylsulphate-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.

Processing-Induced Markers in Proteins of Commercial Plant-Based Drinks in Relation to Compositional Aspects

“…Samples of PBDs were electrophoresed, as described by Sheng et al [15], with some modifications. The samples were diluted in Milli-Q water to a final concentration of 2 µg/µL protein, according to the declared protein content provided on the product packages, and mixed 1:1 with the Laemmli sample buffer (20 mM Tris, 2% SDS, 20% Glycerol, bromophenol blue, 20 mM dithioerythritol) under reducing conditions.…”

“…The gels were run at 200 V for 30-40 min on a Criterion Dodeca Cell (Bio-Rad Laboratories, Hercules, CA, USA) with a Laemmli running buffer (250 mM Tris-Base, 1.89 M Glycine, 10% SDS). The gels were fixed in 50% ethanol, 8% phosphoric acid for at least 2 h and then stained with Coomassie Blue (5% in Milli-Q, w/v) [15]. The gels were photographed with ChemiDoc XRS+ and processed with ImageLab software version 6.0 (Bio-Rad Laboratories, Hercules, CA, USA).…”