被膜态与浮游态植物乳杆菌LR⁃39对比格犬血清免疫指标及肠道菌群结构的影响

Effects of Biofilm and Planktonic Lactobacillus plantarum LR⁃39 on Serum Immune Indexes and Intestinal Microbiota Structure of Beagle Dogs

本试验旨在比较被膜态与浮游态植物乳杆菌LR⁃39对比格犬血清免疫指标及肠道菌群结构的影响。制备被膜态与浮游态植物乳杆菌LR⁃39,采用平板计数法比较不同生物状态的植物乳杆菌LR⁃39经酸、胆盐环境处理后的存活率。选取15只3.5月龄比格犬,随机分为被膜组、浮游组及对照组3个组,每组5只。被膜组、浮游组每日早上采食犬粮后分别灌饲被膜态与浮游态植物乳杆菌LR⁃39菌液1 mL(1×109 CFU/mL),对照组灌饲等体积磷酸盐缓冲液(PBS),连续灌饲28 d。检测血清免疫球蛋白G(IgG)、分泌型免疫球蛋白A(sIgA)、白细胞介素-6(IL⁃6)、肿瘤坏死因子-α(TNF⁃α)含量,并对肠道菌群进行16S rDNA高通量测序分析。结果表明:1)经酸性和胆盐环境处理后,被膜态植物乳杆菌LR⁃39存活率显著或极显著高于浮游态植物乳杆菌LR⁃39(P<0.05或P<0.01)。2)与对照组相比,被膜组血清IgG和sIgA含量极显著提高(P<0.01);与浮游组相比,被膜组血清sIgA含量极显著提高(P<0.01),血清IL⁃6含量有降低趋势(0.05≤P<0.10);与对照组相比,浮游组血清IgG、sIgA、IL⁃6、TNF⁃α含量无显著差异(P>0.05)。3)α多样性分析显示,与对照组相比,被膜组和浮游组Observed species指数、Chao指数、Ace指数均升高,被膜组Shannon指数升高,Simpson指数下降;浮游组Shannon指数下降,Simpson指数升高。主坐标分析(PCoA)结果显示,3组样本间有明显聚类现象。4)在门水平上,与对照组相比,被膜组肠道厚壁菌门(Firmicutes)相对丰度升高,肠道拟杆菌门(Bacte⁃roidetes)、梭杆菌门(Fusobacteria)、变形菌门(Proteobacteria)、放线菌门(Actinobacteria)相对丰度降低;浮游组肠道Bacteroidetes、Fusobacteria相对丰度升高,Firmicutes、Actinobacteria相对丰度降低。5)在属水平上,与对照组相比,被膜组肠道双歧杆菌属(Bifidobacterium)、乳杆菌属(Lac⁃tobacillus)、Allobaculum、消化链球菌属(Peptostreptococcus)相对丰度升高;浮游组肠道Lactoba⁃cillus、Peptostreptococcus、梭杆菌属(Fusobacterium)、普雷沃菌属(Prevotella)、萨特氏菌属(Sut⁃terella)相对丰度升高。6)LEfSe分析表明,被膜组优势菌属是Lactobacillus,浮游组优势菌属是Fusobacterium,对照组优势菌属包括柯林斯氏菌属(Collinsella)、链型杆菌属(Catenibacterium)、真杆菌属(Eubacterium)。7)Spearman相关性分析显示,血清IgG、sIgA、IL⁃6、TNF⁃α含量与肠道富集菌群相对丰度无显著相关性(P>0.05)。由此可见,被膜态与浮游态植物乳杆菌LR⁃39对犬肠道菌群结构影响不同,被膜态植物乳杆菌LR⁃39具有更强的耐酸及胆盐能力,可有效调整肠道菌群结构并能提高机体免疫水平。

This experiment was to compare the effects of biofilm and planktonic Lactobacillus plantarum LR⁃39 on serum immune indexes and intestinal microbiota structure of beagle dogs.The planktonic and biofilm Lacto⁃bacillus plantarum LR⁃39 were prepared,and the survival rates of two different biological states of Lactobacil⁃lus plantarum LR⁃39 treated with acid and bile salt environment were compared by colony counting method.Fifteen 3.5⁃month⁃old beagle dogs were selected and randomly divided into biofilm group,planktonic group and control group,each group contained 5 dogs.The biofilm group and the planktonic group were gavaged with 1 mL(1×109 CFU/mL)biofilm and planktonic Lactobacillus plantarum LR⁃39 after feeding dog food every morning,respectively,and the control group was gavaged with equal volume phosphate buffered saline(PBS),continuous gavage 28 days.The contents of immunoglobulin G(IgG),secretory immunoglobulin A(sIgA),interleukin⁃6(IL⁃6),tumor necrosis factor⁃α(TNF⁃α)in serum were detected,and intestinal mi⁃crobiota were detected by 16S rDNA high throughput sequencing analysis.The results showed as follows:1)after treated with acid and bile salt environment,the survival rate of biofilm Lactobacillus plantarum LR⁃39 was significantly higher than planktonic Lactobacillus plantarum LR⁃39(P<0.05 or P<0.01).2)Compared with the control group,the contents of IgG and sIgA in serum of biofilm group were significantly increased(P<0.01);compared with the planktonic group,the serum sIgA content of biofilm group was significantly in⁃creased(P<0.01),and the serum IL⁃6 content was tend to decrease(0.05≤P<0.10);compared with the control group,the contents of IgG,sIgA,IL⁃6 and TNF⁃αin serum of planktonic group were no significant difference(P>0.05).3)Theαdiversity analysis showed that compared with the control group,the Observed species index,Chao index and Ace index of biofilm group and planktonic group were increased,the Shannon index of biofilm group was increased,and the Simpson index was decreased;the Shannon index of planktonic group was decreased,and the Simpson index was increased.The principal coordinate analysis(PCoA)results showed that there was obvious clustering among three groups of samples.4)At the phylum level,compared with the control group,the relative abundances of Firmicutes in intestinal tract of biofilm group were increased,and the relative abundances of Bacteroidetes,Fusobacteria,Proteobacteria and Actinobacteria in intestinal tract were decreased;the relative abundances of Bacteroidetes and Fusobacteria in intestinal tract of planktonic group were increased,and the relative abundances of Firmicutes and Actinobacteria in intestinal tract were de⁃creased.5)At the genus level,compared with the control group,the relative abundances of Bifidobacterium,Lactobacillus,Allobaculum and Peptostreptococcus in intestinal tract of biofilm group were increased;the rela⁃tive abundances of Lactobacillus,Peptostreptococcus,Fusobacterium,Prevotella,Sutterella in intestinal tract of planktonic group were increased.6)LEfSe analysis showed that the dominant genus of biofilm group was Lactobacillus,the dominant genus of planktonic group was Fusobacterium,and the dominant genera of control group included Collinsella,Catenibacterium and Eubacterium.7)Spearman correlation analysis showed that there was no significant correlation between the contents of IgG,sIgA,IL⁃6 and TNF⁃αand the relative abun⁃dance of intestinal enriched flora(P>0.05).In conclusion,biofilm and planktonic Lactobacillus plantarum LR⁃39 have different effects on intestinal microbiota structure of dogs,the biofilm Lactobacillus plantarum LR⁃39 has stronger acid and bile salt resistance,and can effectively adjust the intestinal microbiota structure and improve the body immune level.