卵黄高磷蛋白磷酸肽在Caco-2细胞单层模型中的促钙转运作用  

Pro-calcium Transport by Phosvitin Phosphopeptides in a Monolayer Model of Caco-2 Cells

作　　者：李松明 赵梦蝶 刘伟 马美湖[1] 金永国[1] 黄茜[1] Li Songming;Zhao Mengdie;Liu Wei;Ma Meihu;Jin Yongguo;Huang Xi(National Research and Development Center for Egg Processing,College of Food Science and Technology,Huazhong Agricultural University,Wuhan 430070;Yibin Forestry and Bamboo Industry Research Institute,Yibin 644000,Sichuan)

机构地区：[1]华中农业大学食品科学技术学院、蛋品加工技术国家地方联合工程中心农业农村部蛋品加工重点实验室,武汉430070 [2]宜宾林竹产业研究院,四川宜宾644000

出　　处：《中国食品学报》2023年第6期8-17,共10页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金面上项目(32072237);湖北省自然科学基金项目(2020CFB583)。

摘　　要：结合高温低压处理、胰蛋白酶和嗜热菌蛋白酶复合酶解制备具有高钙结合能力的卵黄高磷蛋白磷酸肽(Phosvitin phosphopeptides,PPP)。通过优化确定制备PPP-Ca的最佳条件为pH 9.5,多肽与钙质量比7∶1,此条件下钙螯合率达到97%,PPP-Ca在模拟胃肠道消化中显示了极高的稳定性。使用Caco-2细胞单层模型验证PPP-Ca在体外的钙转运作用,通过细胞形态、电阻值和荧光素钠透过率等验证Caco-2细胞模型,毒性实验(MTT)结果显示PPP-Ca对Caco-2细胞呈现低毒性作用。研究PPP-Ca和PPP对Caco-2细胞转运钙离子质量浓度和碱性磷酸酶(alkaline phosphatase,AKP)活性的影响,采用RT-PCR测定Calbindin D9K mRNA的相对表达量。结果表明,PPP和PPP-Ca能够显著提高AKP活性,钙转运量从(3.06±0.08)μg增到(5.66±0.62)μg,Calbindin D9K mRNA的表达量增加2倍,进而增强人体肠道中钙的转运吸收。研究结果为开发新的钙补充剂和卵黄高磷蛋白提供了科学证据。Phosvitin phosphopeptides(PPP)with high calcium binding capacity were prepared by a combination of high-temperature and low-pressure treatment,trypsin and thermophilic protease complex digestion.The optimum conditions for the preparation of PPP-Ca were determined to be pH 9.5 and a peptide-to-calcium mass ratio of 7:1.97%calcium chelation was achieved under these conditions and PPP-Ca showed excellent stability in simulated gastrointestinal diges tion.The monolayer model of Caco-2 cells was used to verify the calcium transport of PPP-Ca in vitro.The Caco-2 cell model was validated by cell morphology,electrical resistance and sodium fluorescein permeation,and MTT results showed that PPP-Ca exhibited low toxic effects on Caco-2 cells.The effects of PPP-Ca and PPP on the mass concentration of calcium ions transported and the activity of alkaline phosphatase(AKP)in Caco-2 cells were investigated,and the relative expression of CalbindinD9K m RNA was determined by RT-PCR.The results showed that PPP and PPP-Ca significantly increased AKP activity,increased calcium transport from(3.06±0.08)μg to(5.66±0.62)μg,and increased CalbindinD9K m RNA expression by 2-fold,thus enhancing calcium transport and absorption in the human intestine.The findings provide scientific evidence for the development of new calcium supplements and phosvitin phosphopeptides.

关 键 词：卵黄高磷蛋白磷酸肽 肽钙螯合物 稳定性 CACO-2细胞 转运吸收 

分 类 号：TS201.4[轻工技术与工程—食品科学]