乳杆菌S-层蛋白自组装机制及介导乳源功能肽肠道吸收机理研究

The main aim of this project is to identify and characterize functional domain of S-layer protein (SLP) of Lactobacillus and ascertain the self-assembly and intestinal adhesion mechanism of SLP. The intestinal transport, efflux and internalization mechanism of milk-derived bioactive peptide embedded with SLP coated-liposome is also researched. The SLPs of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus crispatus, Lactobacillus brevis and Lactobacillus johnsonii will be purified and characterized. The properties including self-assembly ability, intestinal adhesion ability, resistance to gastric intestinal protease ability of SLPs are going to be compared to choose the proper SLP as the research object. The Lactobacillus SLP gene sequence will be cloned to characterize the self-assembly domain, intestinal adhesion domain and cell wall anchoring domain. The intestinal receptor of SLP is also clarified. Caco-2 cell is used as intestinal absorption model to research intestinal absorption mechanism of milk-derived bioactive peptide embedded with SLP-coated-liposome. The intestinal transport, efflux and internalization mechanism is going to be clarified. The biological safety of peptide embedded with SLP-coated-liposome is evaluated to ensure the safety of its application in food industry. This project provides basic theory research for SLP of Lactobacillus and milk-derived bioactive peptide used in functional food market.

本项目主要研究目标为表征乳杆菌S-层蛋白（SLP）功能结构域，探明S-层蛋白自组装机制和肠道黏附机制；并研究S-层蛋白修饰脂质体介导乳源功能肽肠道转运、外排和内化机理。本项目拟分离纯化嗜酸乳杆菌、瑞士乳杆菌、卷曲乳杆菌、短乳杆菌、约氏乳杆菌等乳杆菌的S-层蛋白，筛选具有体外自组装性能、肠道黏附性能、耐胃肠道蛋白酶水解性能的乳杆菌S-层蛋白；克隆乳杆菌S-层蛋白基因序列，表征S-层蛋白自组装结构域、肠道黏附结构域及细胞壁锚定结构域，同时探明S-层蛋白肠道黏附相关受体；利用Caco-2细胞为肠道吸收模型，研究S-层蛋白修饰脂质体介导乳源功能肽的肠道吸收机理，探明S-层蛋白修饰乳肽脂质体肠道转运机制、外排机制和内化机制；评价S-层蛋白修饰乳肽脂质体的生物安全性，以保证其在食品工业中的安全应用。本项目为乳杆菌S-层蛋白、乳源功能肽在功能性食品市场的应用提供了理论研究依据。

乳源功能肽在口服后，容易被胃肠道水解失去活性，因此功能肽的包埋是目前提高其生物利用度的途径之一，也是生物活性肽开发的关键问题之一。本项目通过对嗜酸乳杆菌、瑞士乳杆菌、短乳杆菌、植物乳杆菌等表面疏水性、凝聚性、黏附性能的比较，选择具有较强肠道黏附性能的嗜酸乳杆菌CICC6074为目标菌株，分离纯化并鉴定了嗜酸乳杆菌CICC6074的S-层蛋白，分子量约为46KDa；探究了嗜酸乳杆菌CICC6074S-层蛋白的功能结构域，确定了细胞壁锚定结构域为S-层蛋白的C端，和菌体磷壁酸发生锚定作用，N端序列为自组装功能域；利用嗜酸乳杆菌CICC6074S-层蛋白的自组装性能修饰大豆卵磷脂和DPPC（二棕榈酰磷脂酰胆碱）脂质体，发现S-层蛋白修饰DPPC脂质体效果较好，而且DPPC脂质体经过S-层蛋白修饰后，可以显著提高脂质体的稳定性和胃肠道耐受性；以嗜酸乳杆菌CICC6074的S-层蛋白修饰DPPC脂质体包埋降胆固醇肽Leu-Qln-Pro-Glu（LQPE），以Caco-2细胞为肠道吸收模型，研究发现了S-层蛋白修饰DPPC脂质体可以提高乳源降胆固醇肽的肠道吸收。因此，本项目主要利用嗜酸乳杆菌CICC6074 的S-层蛋白自组装性能，制备了一种提高功能肽肠道吸收的口服载体，为功能肽的市场化应用提供了研究基础；另外乳杆菌S-层蛋白功能目前研究主要集中于维持细胞形态、抑制病原菌黏附、黏附特性等，对于其自组装性能的报导还较少，本项目也为乳杆菌S-层蛋白的自组装应用及拓展研究提供了基础。