Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, 150030 Harbin, China.
College of Food Science, Southwest University, 400715 Chongqing, China.
J Dairy Sci. 2021 Feb;104(2):1351-1363. doi: 10.3168/jds.2020-19018. Epub 2020 Dec 11.
During the thermal processing of milk, Maillard reactions occur between proteins and lactose to generate glycated proteins. In this study, a lactose-glycated caseinate was hydrolyzed by trypsin. The obtained glycated caseinate (GCN) hydrolysate had a lactose content of 10.8 g/kg of protein. We identified its glycation sites and then assessed it for its protective effect against lipopolysaccharide-induced barrier injury using a rat intestinal epithelial cell line (IEC-6 cells) as a cell model and unglycated caseinate (CN) hydrolysate as a reference. Results from our liquid chromatography-mass spectrometry analysis of the GCN hydrolysate verified that lactose glycation occurred at the Lys residues in 3 casein components (α-casein, β-casein, and κ-casein), and this resulted in the formation of 5 peptides with the following amino acid sequences: EMPFPKYPKYPVEPF, HIQKEDVPSE, GSENSEKTTMPL, NQDKTEIPT, and EGIHAQQKEPM. The results from cell experiments showed that the 2 hydrolysates could promote cell growth and decrease lactate dehydrogenase release in the lipopolysaccharide-injured cells; more importantly, they could partially protect the damaged barrier function of the cells by increasing trans-epithelial electrical resistance, decreasing epithelial permeability, and upregulating the expression of the 3 tight junction proteins zonula occludens-1, occludin, and claudin-1. However, compared with CN hydrolysate, GCN hydrolysate showed lower efficacy in protecting against cellular barrier dysfunction. We propose that the different chemical characteristics of the CN hydrolysate and the GCN hydrolysate (i.e., amino acid loss and lactose conjugation) contributed to the lower barrier-protective efficacy of the GCN hydrolysate. During dairy processing, protein glycation of the Maillard type might have a non-negligible, unfavorable effect on dairy proteins, in view of the resulting protein glycation we found and the critical function of proteins for maintaining the integrity of the intestinal barrier.
在牛奶的热处理过程中，蛋白质和乳糖之间发生美拉德反应，生成糖化蛋白质。在这项研究中，胰蛋白酶水解乳糖糖化的酪蛋白。所得的糖化酪蛋白（GCN）水解产物的蛋白质中乳糖含量为 10.8 g/kg。我们鉴定了其糖化位点，并使用大鼠肠上皮细胞系（IEC-6 细胞）作为细胞模型，用未糖化的酪蛋白（CN）水解产物作为对照，评估其对脂多糖诱导的屏障损伤的保护作用。通过对 GCN 水解产物的液相色谱-质谱分析，结果证实乳糖糖化发生在 3 种酪蛋白成分（α-酪蛋白、β-酪蛋白和κ-酪蛋白）的赖氨酸残基上，由此形成 5 种具有以下氨基酸序列的肽：EMPFPKYPKYPVEPF、HIQKEDVPSE、GSENSEKTTMPL、NQDKTEIPT 和 EGIHAQQKEPM。细胞实验结果表明，两种水解产物均可促进细胞生长，减少脂多糖损伤细胞中乳酸脱氢酶的释放；更重要的是，它们可以通过增加跨上皮电阻、降低上皮通透性和上调 3 种紧密连接蛋白（闭合蛋白-1、闭合蛋白和克劳丁-1）来部分保护受损的细胞屏障功能。然而，与 CN 水解产物相比，GCN 水解产物在保护细胞屏障功能方面的效果较低。我们提出，CN 水解产物和 GCN 水解产物的不同化学特性（即氨基酸损失和乳糖结合）导致 GCN 水解产物的屏障保护效果较低。在乳制品加工过程中，美拉德型蛋白质糖化可能对乳制品蛋白质产生不可忽视的不利影响，鉴于我们发现的蛋白质糖化和蛋白质对维持肠道屏障完整性的关键功能。
