天然抗氧化肽对肠肽酶的耐受性及其跨膜转运机制

The physiological effects of bioactive peptides depend on their ability to reach their target sites in intact forms after oral intake. The intestinal absorption barrier and the susceptibility to action of enzymes in intestinal epithelium are the key biological barrier for bioactive peptides from oral intake into the blood stream. In this proposal, antioxidative peptides were chosen as a model of bioactive peptides. The casein was hydrolyzed by proteases to obtain antioxidative hydrolysates. Peptides showing strong antioxidant activities were isolated from the hydrolysate using chromatographic methods. A three-stage in vitro model system of simulated gastric juice - intestinal juice and Caco-2 Cell monolayers, was used to simulate the process of human gastrointestinal (GI) digestion and absorption of peptides. The study focuses on the processes governing the digestion, absorption and transport of peptide molecules across the intestinal epithelium. Insight into resistance to intestinal peptidases and transepithelial transport pathways of antioxidative peptides affected by peptide structural parameters, such as molecular masses, charge and electronic properties, hydrophobicity, are studied. The peptides absorbed intact are identified by comparing mass spectrum of peptides before and after transepithelial transport across the Caco-2 cell epithelium. The structural characteristics of peptides absorbed intact are analyzed and the antioxidative peptides resisting digestion are further identified and verified. Metabonomics methodology is innovatively used to study degradation by intestinal peptidases and the pathways for transepithelial absorption of peptides with different structural parameters by group indexes. The research will illuminate the bioavailability of antioxidative peptides after oral intake and the structural characteristics of peptides resisting digestion. This research will also contribute to understand the underlying factors affecting the bioavailability and absorption of bioactive peptides, enrich the oligopeptide absorption theory. The results will highlight the antioxidative peptides with "real efficacy" and adds further support to the feasibility applied in the formulation of functional foods.

活性肽潜在的生理功效在于经过口服后能否到达靶器官。小肠上皮细胞吸收屏障及其酶系统是活性肽口服进入机体的关键障碍所在。本研究以抗氧化肽为研究对象，以酪蛋白为原料酶法制备多肽混合物，采用色谱技术分离出高活性组分；采用胃液-肠液- Caco-2 细胞的三段式模型模拟体外连续消化吸收。着重研究抗氧化肽在小肠上皮细胞的代谢和吸收规律；研究分子量、荷电性和疏水性等结构性质与肽的耐消化性及其跨膜转运路径之间的内在联系。通过质谱图比对识别并鉴定完整转运的抗消化肽，研究抗消化肽的结构特征，进一步鉴定高活性抗消化肽。本课题创新地运用"组学"的研究方法，通过组群指标分析，揭示抗氧化肽的降解和跨膜转运机制及其影响因素，阐明口服抗氧化肽的生物可达性以及抗消化肽的结构特征。该研究有助于人们了解生物活性肽以及影响活性肽的生物利用度和吸收的因素，丰富寡肽吸收理论，并为生产具有"真正功效"的抗氧化肽功能食品提供理论依据。

活性肽潜在的生理功效在于口服后能否到达靶器官。小肠上皮细胞吸收屏障及其酶系统是活性肽口服进入机体的关键障碍。系统地研究活性肽的结构性质与肽转运路径的关联性以及对肠肽酶耐受性，可深入理解生物活性肽的跨膜转运和消化吸收机制。本项目着重研究抗氧化肽在小肠上皮细胞的代谢和吸收规律，研究分子量、荷电性和疏水性等结构性质与肽的耐消化性及其跨膜转运路径之间的内在联系以及抗消化肽的结构特征。研究结果表明：. 1、肽组分的生物利用度在4%-16%之间，肽的分子量、电荷性和疏水性对其生物利用度有较大影响。带有负电荷性，弱疏水性时，肽组分的吸收率和吸收物的抗氧化活性保留率较高，具有优异的生物利用度；. 2、当分子量<500 Da时，肽组分主要通过PepT1载体调节通路进行吸收转运；二肽和三肽C端的带电荷氨基酸在一定程度上会促进其吸收，在转运过程中肽的水解基本全部来自于BBPs，其最大水解速率为11.4×10-3 mM Gly/min；. 3、亲水性的肽段或带有较多的负电荷（≥2），通过细胞旁路进行吸收。在细胞旁路中，水解活性肽的酶主要为BBPs，最大水解速率为6.91×10-3 mM Gly/min；同时还有其他的肽酶参与水解，最大水解速率为5.59×10-3 mM Gly/min；. 4、转胞吞作用对带有较多正电荷（≥2）的肽段以及强疏水性的肽段具有较强的选择性，肽的疏水性与转胞吞作用总体上呈现正相关关系；. 5、不同氨基酸组成的抗氧化肽对应的胃肠消化肽对肠肽酶的耐受性不同。酸性组分的酪蛋白抗氧化肽具有较高的生物有效率；. 6、抗消化肽的结构特征与组成其肽序列的氨基酸的疏水性与亲水性有关。抗消化肽在氨基酸组成上具有疏水性氨基酸含量多，亲水性氨基酸含量少的特点，并且在抗消化肽的肽序列中酸性氨基酸的含量比碱性氨基酸的含量多。. 该研究有助于人们了解生物活性肽以及影响活性肽的生物利用度和吸收的因素，丰富寡肽吸收理论，并为生产具有"真正功效"的抗氧化肽功能食品提供理论依据。