研究论文 RESEARCHPAPER

丙基菊粉纳米粒子作为益生元对植物乳杆菌抑菌性能的影响

  • 刘曦宇 ,
  • 杜丽欣 ,
  • 万入汇 ,
  • 邹雪 ,
  • 张建斌 ,
  • 蒲蕾 ,
  • 马吉飞 ,
  • 胡德宝 ,
  • 洪亮
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  • 天津农学院动物科学与动物医学学院, 天津市农业动物繁育与健康养殖重点实验室, 天津 300392
刘曦宇(1999—),女,天津人,本科生,研究方向为益生菌改良。E-mail:1500840040@qq.com

收稿日期: 2021-11-04

  网络出版日期: 2022-06-14

基金资助

天津市大学生创新训练计划项目(202010061044);河北省重点研发计划项目(19227523D);天津市生猪产业技术体系创新团队(ITTPRS2021006);青海省重点研发与转化计划-科技援青合作专项(2021-QY-204)

Effects of Propyl-Inulin Nanoparticles as Prebiotics on Antibacterial Properties of Lactobacillus plantarum

  • LIU Xiyu ,
  • DU Lixin ,
  • WAN Ruhui ,
  • ZOU Xue ,
  • ZHANG Jianbin ,
  • PU Lei ,
  • MA Jifei ,
  • HU Debao ,
  • HONG Liang
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  • Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300392, China

Received date: 2021-11-04

  Online published: 2022-06-14

摘要

为提高植物乳杆菌的抑菌能力,本试验先通过酯化反应合成了丙基菊粉(PI),丙基菊粉通过透析以自组装的形式合成了丙基菊粉纳米粒子(IPrN)。采用600 MHz 1H-核磁共振(NMR)光谱检验丙基在菊粉的取代率,利用扫描电子显微镜(SEM)及动态光散射(DLS)观察纳米粒子的特征。用4’,6-二脒基-2-苯基吲哚(DAPI)和异硫氰酸荧光素(FITC)对植物乳杆菌和IPrN进行染色,在共聚焦激光扫描显微镜(CLSM)下观察到植物乳杆菌对IPrN的内化结果。通过共培养法中病原体的活细胞数和琼脂扩散试验的抑菌圈大小测定IPrN内化后的植物乳杆菌对大肠杆菌K88和鸡沙门氏菌的抑菌能力。测定菊粉或IPrN处理后植物乳杆菌的生长曲线和pH的变化,在培养基中加入蛋白酶K后进行抑菌圈试验测定植物乳杆菌是否以分泌细菌素发挥其抑菌能力,最后通过荧光定量PCR技术来评估编辑细菌素的基因planS的基因表达水平。结果显示:丙基在菊粉的取代率为76.88 mol%,可观察到IPrN为球形粒子,直径为366.6 nm;通过Z-截面模式可观察到FITC的荧光在植物乳杆菌的中心处最强,表明IPrN已被内化;IPrN的内化过程提高了植物乳杆菌的抑菌能力;通过生长曲线和pH曲线可知IPrN的内化并没有影响植物乳杆菌的生长及乳酸的分泌;蛋白酶K的添加消除了由IPrN提升的抑菌性;planS的基因表达水平证明了IPrN内化使植物乳杆菌的细菌素表达增多。综上所述,本研究通过丙酸修饰菊粉多糖合成了IPrN,IPrN的内化可提高植物乳杆菌的细菌素的分泌,进而提高了植物乳杆菌对大肠杆菌K88和鸡沙门氏菌的抑菌能力。

本文引用格式

刘曦宇 , 杜丽欣 , 万入汇 , 邹雪 , 张建斌 , 蒲蕾 , 马吉飞 , 胡德宝 , 洪亮 . 丙基菊粉纳米粒子作为益生元对植物乳杆菌抑菌性能的影响[J]. 动物营养学报, 2022 , 34(6) : 3953 -3961 . DOI: 10.3969/j.issn.1006-267x.2022.06.054

Abstract

In order to improve the antimicrobial ability of Lactobacillus plantarum, the propyl-inulin (PI) was synthesized through esterification reaction, the propyl-inulin nanoparticles (IPrN) were synthesized by self-assembly mechanism of propyl-inulin. The replacement rate of propyl in inulin was detected by 600MHz 1H-nuclear magnetic resonance (NMR) spectroscopy, and the characteristics of nanoparticles were observed by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The Lactobacillus plantarum and IPrN were stained by 4',6-diamidino-2-phenylindole (DAPI) and fluorescin isothiocyanate (FITC), and the internalization result of IPrN by Lactobacillus plantarum was observed under confocal laser scanning microscope (CLSM). The antimicrobial activity of Lactobacillus plantarum internalized by IPrN against Escherichia coli K88 and Salmonella galinarum was determined by the number of viable cells of the pathogen co-culture assay and the size of inhibition zone in agar diffusion test. The growth curve and pH of Lactobacillus plantarum treated with inulin or IPrN were measured. After adding protease K into the culture medium, the agar diffusion test was conducted to test whether Lactobacillus plantarum exerted its antimicrobial ability by secreting bacteriocin. Finally, the expression level of planS of editing bacteriocin was evaluated by quantitative PCR. The results showed that the replacement rate of propyl in inulin was 76.88 mol%, and the IPrN was spherical with a diameter of 366.6 nm. The fluorescence of FITC was strongest in the center of Lactobacillus plantarum by Z-section pattern, indicating that IPrN had been internalized. Internalized stimulation of IPrN improved the antimicrobial ability of Lactobacillus plantarum. According to the growth curve and pH, the internalization of IPrN did not affect the growth and lactic acid secretion of Lactobacillus plantarum. The addition of protease K eliminated the antibacterial activity promoted by IPrN. The expression level of planS proved that the internalization of IPrN increased the expression of bacteriocin in Lactobacillus plantarum. In conclusion, propyl-inulin nanoparticles are synthesized by modifying inulin polysaccharide with propionic acid, the internalization of IPrN can improve the secretion of bacteriocin of Lactobacillus plantarum, and then improve the antibacterial ability of Lactobacillus plantarum against Escherichia coli K88 and Salmonella gallinarum.

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