灰树花多糖纳米乳的制备及其对H9亚型禽流感病毒与常见细菌抑制效果的评估  

Preparation of nanoemulsion from polysaccharides of Grifola frondosa and evaluation of its inhibition effect on H9 subtype avian influenza virus and common bacteria

作　　者：刘博 毕研丽 杨丽梅[2] 冯宗玲[4] 李佳 苏海歌 孙红武 张艳 王文秀[2,6] LIU Bo;BI Yan-li;YANG Li-mei;FENG Zong-ling;LI Jia;SU Hai-ge;SUN Hong-wu;ZHANG Yan;WANG Wen-xiu(Hainan Provincial Key Laboratory of Tropical Animal Breeding and Disease Research,Institute of Animal Science and Veterinary Medicine,Hainan Academy of Agricultural Sciences,Haikou 571100,China;Shandong Binzhou Animal Science&Veterinary Medicine Academy,Binzhou 256600,China;Shandong Lidu Bio-Sciences&Technology Co.,Ltd,Binzhou 256600,China;Binzhou Science and Technology Innovation Development Research Institute,Binzhou 256600,China;Army Medical University,Chongqing 400038,China;Binzhou Golden Touch Technology Transfer Centre,Binzhou 256600,China)

机构地区：[1]海南省农业科学院畜牧兽医研究所海南省热带动物繁育与疫病研究重点实验室,海南海口571100 [2]山东省滨州畜牧兽医研究院,山东滨州256600 [3]山东绿都生物科技有限公司,山东滨州256600 [4]滨州市科技创新发展研究院,山东滨州256600 [5]中国人民解放军陆军军医大学,重庆400038 [6]滨州点石成金技术转移与成果转化服务中心,山东滨州256600

出　　处：《中国预防兽医学报》2024年第8期856-862,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：2023年海南省热带动物繁育与疫病研究重点实验室开放课题资助项目(HKL202302);滨州市农社领域科技创新政策引导计划项目(2022KTPY011、2022KTPY025)。

摘　　要：为制备灰树花多糖(GFP)纳米乳,并观察其理化及形态学特征,评估其对H9亚型禽流感病毒(AIV)及3种常见细菌的抑制效果,本研究采用低能乳化法制备GFP纳米乳,经不同方式处理后观察GFP纳米乳的稳定性,分别采用透射电镜和扫描电镜观察其形态特征,使用激光粒度仪测定GFP纳米乳的粒径与多分散系数(PDI),使用电位仪测定其Zeta电位。结果显示:该GFP纳米乳外观澄清、透明,8000 r/min离心20 min和室温静止7 d后不分层、不破乳,表明制备的GFP纳米乳性状稳定。电镜观察可见许多单一分布的均匀圆球,其周围包裹着网格状凝胶基质;GFP纳米乳的粒径为10 nm~100 nm,平均粒径24.12 nm,PDI为0.096,Zeta电位为-18.6±4.3 m V,表明GFP纳米乳稳定性较好。将不同浓度的GFP纳米乳及免手洗凝胶消毒剂分别于MDCK细胞中与H9亚型AIV NJ02株作用,通过计算各药物对H9亚型AIV的抑制率比较GFP纳米乳对H9亚型AIV的抑制效果。将不同稀释度的GFP纳米乳、GFP溶液及免手洗凝胶和75%乙醇分别与AIV NJ02株作用后,采用荧光定量PCR(q PCR)检测H9亚型AIV基因的拷贝数,并计算各药物对H9亚型AIV的抑制率。采用药敏纸片法检测GFP纳米乳、GFP原液、免手洗凝胶对禽舍3种常见细菌的抑菌效果。对H9亚型AIV的抑制率检测结果显示,当浓度低于10μL/mL时,GFP纳米乳及免手洗凝胶消毒剂对H9亚型AIV的抑制率均小于55%,效果不明显;当浓度大于40μL/mL时,GFP纳米乳对H9亚型AIV的抑制率极显著高于同浓度的免手洗凝胶(P<0.01);当浓度为80μL/mL时,GFP纳米乳可以100%抑制病毒增殖,且对细胞无毒性。qPCR结果显示,10倍稀释的各药物对H9亚型AIV的抑制率均明显高于100倍稀释的各药物,且10倍稀释的GFP纳米乳对H9亚型AIV的抑制率均极显著高于免手洗凝胶、GFP原液和75%乙醇(P<0.01)。纸片扩散法结果显示,GFP纳米乳对金黄色葡萄球菌的抑菌效果较差,对枯草芽孢杆菌To develop the nanoemulsions from polysaccharide of Grifola frondosa(GFP)and assess its physicochemical and morphological characteristics,as well as their potential to inhibit avian influenza viruses(AIV)and three common bacteria,we employed a low-energy emulsification method to prepare stable GFP nanoemulsions,which were then subjected to various treatments to evaluate their stability.Morphological characteristics were observed by transmission electron microscopy(TEM)and scanning electron microscopy(SEM),which particle size and polydispersity index(PDI)were determined with a laser particle size analyzer.The Zeta potential was measured using potentiometry.The results showed that the GFP nanoemulsion presented a clear and transparent appearance,and it remained stable without any signs of layering or emulsion breakdown after centrifugation at 8000r/min for 20 minutes or being left room temperature for 7 days.Electron microscope observation shows that the presence of uniformly distributed spherical particles,enveloped by a grid-like gel matrix.The particle size of GFP nanoemulsion ranges from 10nm to 100nm,with an average particle size of 24.12nm,a PDI of 0.096,and a Zeta potential of-18.6±4.3 mV,all of which are indicative of good stability.The GFP nanoemulsion and a hand wash-free gel disinfectant were tested for their inhibitory effects on the H9 subtype AIV NJ02 strain in MDCK cells.The virus inhibition rate were calculated for each treatment,and it was observed that the GFP nanoemulsion demonstrated a significantly higher virus inhibition rate compared to the hand wash-free gel at concentrations above 40μL/mL(P<0.01).Notably,at a concentration of 80μL/mL,the GFP nanoemulsion was able to completely inhibit virus growth without any cytotoxic effects.Further,the GFP nanoemulsion was tested at various dilutions,along with GFP solution,hand wash-free gel,and 75%ethanol,against the AIV NJ02 strain.The copy number of the viral gene was quantified using fluorescence quantitative PCR(qPCR),and the inhibition rate of

关 键 词：灰树花多糖 纳米乳 H9亚型禽流感病毒 抑菌效果 

分 类 号：S855.3[农业科学—临床兽医学]