核桃青皮提取物对肉用仔鸡机体抗氧化性能的影响及抗菌机理研究

Research and development on antibiotic alternatives are hotspot issues in recent 20 years. Studies have shown that, under in vitro conditions, walnut (Juglans regia L.) green husk extracts present favorable antibacterial property and antioxidant activity. Up to now, however, there is lack of related researches about the roles of walnut green husk extracts in human and animal in vivo. Therefore, based on the existing achievements and our laboratory research foundation, this project will be carried out with animal feeding experiments to elucidate the effects of the extracts on growth, slaughter performance and meat quality of broilers. Biochemistry and enzymology analysis will by adopted to verify the extracts on antioxidant function in broilers in vivo. Furthermore, PCR-DGGE and 16S rDNA high-throughput sequencing will be employed to depth analyze of the extracts on bacterial community diversity, and the relative abundance of beneficial bacteria and pathogens. By using of meta-transcriptome sequencing technology (RNA-seq), the effects of the extracts on gene expression of gut microbial will be cleared, and the network of differentially expressed genes will be constructed. And the major genes of gut microflora affected by the extracts will be identified. Meanwhile, the molecular biology mechanisms of antibacterial property of the walnut green husk extracts will be revealed by combining with intestinal microbial cell function, and morphological change and meta RNA-seq results. This will provide important information to establish the theoretical foundation for walnut green husk extracts as a feed antibiotic alternative.

饲用抗生素替代品的研发是近20年来的研究热点问题。研究表明，核桃青皮提取物在体外条件下具有良好的抑菌和抗氧化作用。而有关核桃青皮提取物在人和动物体内的作用还未见相关研究。因此，本项目根据现有研究成果和本实验室的研究基础，以动物饲养试验为基础，阐明添加核桃青皮提取物对肉鸡生产性能、屠宰性能和肉品质的影响；结合生物化学和酶学分析探明核桃青皮提取物在活体条件下对肉鸡机体抗氧化机能的影响；借助PCR-DGGE和16S rDNA高通量测序技术深度解析核桃青皮提取物对肠道细菌群落多样性以及有益菌和病原菌相对丰度的影响；利用宏转录组测序技术(Meta RNA-seq)明确核桃青皮提取物对肠道微生物基因表达的影响，构建差异表达基因网络，筛选核桃青皮提取物影响肠道微生物的差异表达基因；基于宏转录组测序、微生物细胞功能和形态学变化揭示核桃青皮提取物的抑菌机理。为核桃青皮提取物作为饲用抗生素替代品奠定理论基础。

植物提取物在替代饲用抗生素方面具有潜在的价值。本项目根据现有研究成果和本实验室的研究基础，以动物饲养试验为基础，阐明添加核桃青皮提取物对肉鸡生产性能、屠宰性能和肉品质的影响；结合生物化学和酶学分析探明核桃青皮提取物在活体条件下对肉鸡机体抗氧化机能的影响；借助16S rDNA高通量测序技术深度解析核桃青皮提取物对肠道细菌群落多样性以及有益菌和病原菌相对丰度的影响；利用PICRUSt和FAPROTAX对肠道微生物功能的影响；基于宏转录组测序、微生物细胞功能和形态学变化揭示核桃青皮提取物的抑菌机理。通过研究发现（1）在以蒸馏水及25%、50%、75%和100%（v/v）的甲醇、乙醇，丙酮为溶剂获得的核桃青皮提取物中，25%乙醇提取物的抑菌作用、抗氧化特性最好；（2）对核桃青皮中主要的7种化合物进行抑菌试验，发现鞣花酸对大肠杆菌和枯草芽孢杆菌有较强的抑制作用，胡桃醌对大肠杆菌和金黄色葡萄球菌具有较强的抑制作用，这两种分子在核桃青皮提取物的抑菌作用中可能起重要作用；（3）添加1.0%核桃青皮25%乙醇提取物具有提高黄羽肉仔鸡料重比、降低屠体脂肪率、改善肌肉嫩度、降低十二指肠和空肠隐窝深度，提高钙、磷表观代谢率的作用；但是对生长性能、屠宰性能、肉色、肌肉pH、免疫器官发育均无影响；添加核桃青皮提取物后，十二指肠中有益菌如Lactobacillus属、Halomonas属丰度增加，有害菌如Proteobacteria菌门、Escherichia-Shigella属丰度降低；分析显示添加核桃青皮提取物后，肠道微生物氨基酸代谢、外源物降解和代谢、信号传导、能量代谢、辅因子和维生素代谢功能显著下降，而核酸代谢、翻译、复制与修复、碳水化合物代谢等功能显著增强。（4）添加0.05%核桃青皮90%乙醇提取物通过提高42日龄肝脏谷胱甘肽还原酶和过氧化氢酶活性以提高机体抗氧化能力。目前关于核桃青皮提取物抑菌机理正在研究中。通过项目的实施，截至目前累计培养研究生3名，发表4篇论文，申请发明专利1项。