摘要
为研究细菌对鲤鱼(Cyprinus carpio)肠道的粘附特性、建立有效筛选鲤鱼肠道益生菌的方法,本试验采用体外固定鲤鱼前肠粘液于96孔细胞培养板,并结合同位素示踪的方法,研究了9株细菌的粘附特性和作用机理,以及有益细菌对病原菌粘附的抑制作用。
本试验从四川农业大学教学农场水产养殖基地养殖池、动物微生态实验室水族箱和鲤鱼肠道共分离出20株细菌(CM2、WP1、WP2、WP3、GL1、GL2、CI1、WL1、C3、Z2,D1、D2、D3、D4、E1、E2、E3、L1、L2、L3),以及四川农业大学动物微生态实验室保存的8株芽孢杆菌(JS01、D9401、ND1、ND2、ND3、ND4、ND5和ND6)作为本实验的试验菌株。
通过常规的耐热试验和双层平板法拮抗试验,筛选出具有一定耐热性并对致病性大肠杆菌和嗜水气单胞菌具有拮抗作用的5株细菌(Z3、C3、L2、E2、D2),通过16S rRNA测序,进行菌种的鉴定,最后确定为2株芽孢杆菌(Bacillus),2株肠球菌(Enterococcus)和1株柠檬酸杆菌(Citrobacter)。
本试验中,由于同位素标记物γ-~(32)P-ATP是粘附试验的示踪材料,因此做了γ-~(32)P-ATP对细菌生长影响的试验。实验结果表明:添加同位素标记物的细菌均出现活菌数降低的情况,但是差异不明显,甚至有些细菌出现活菌数增加的现象,说明所有细菌在培养过程中尽管添加了同位素γ-~(32)P-ATP,但是其活菌数均不受影响,能达到试验所需要细菌数量(10~8～10~9cfu/ml)。
对来源于鱼肠道的肠球菌和柠檬酸杆菌以及养殖环境中的芽孢杆菌的附着活性、细菌表面凝集素和粘液蛋白的受体的化学组成、其对病原菌附着鲤鱼前肠粘液的影响等进行了研究。试验结果表明:鱼源的肠球菌的相对粘附数量极显著高于异源的芽孢杆菌(p＜0.01),同源的柠檬酸杆菌与异源芽孢杆菌的相对粘附数量差异不显著(p＞0.05)。致病性强的嗜水气单胞菌AH2相对粘附数量极显著高于嗜水气单胞菌AH1、大肠杆菌CVCC2060和大肠杆菌ATCC25922(p＜0.01)。5种菌经高碘酸钠和蛋白水解酶修饰后的附着试验表明,高碘酸钠能极显著降低芽孢杆菌、肠球菌和柠檬酸杆菌的粘附率(p＜0.01),而蛋白水解酶修饰对多数菌株的影响不显著(p＞0.05)。粘液蛋白经蛋白水解酶处理后,部分菌对其的附着数量显著降低(p＜0.05),而粘液蛋白经高碘酸钠处理后,5株菌的粘附率显著上升(p＜0.05)。由于细菌表面的凝结素存在差异,导致其相对粘附数量的不同。因此,作为鲤鱼肠道的原籍菌,肠球菌L2的相对粘附数量极显著地高于其他细菌(p＜0.01)。部分细菌通过排斥、竞争、取代的方式能够显著降低部分病原菌的粘附率(p＜0.05),同时出现显著提高病原菌粘附率的现象(p＜0.05),这是由于细菌改变了粘液表面结构所致。9株菌均能粘附到粘液体外模型,细菌表面的凝集素主要为具糖蛋白性质的物质,粘液蛋白上存在的5种细菌的特异性受体物质可能为蛋白质。取代作用对于病原菌的粘附抑制效果最好,三种作用方式均具有菌株特异性。
To investigate the adhesion characteristics of bacteria to carp(Cyprinus carpio) intestine and established a efficacious screening method of carp intestinal probiotics. Used immobilized mucosal model in vitro,combined with the isotope tracing method,the adhesion characteristics and mechanisms of nine bacteria and the inhibition to the pathogens were investigated.
Twenty bacteria were isolated from the cultivation pool,aquarium of Animal Micro-ecology Lab and the gut of carp in teaching farm of Sichuan Agricultural University(CM2、WP1、WP2、WP3、GL1、GL2、CI1、WL1、C3、Z2, D1、D2、D3、D4、E1、E2、E3、L1、L2、L3),eight bacteria were stored byAnimal Micro-ecology Lab(JS01、D9401、ND1、ND2、ND3、ND4、ND5、ND6) were totally used in this test.
By inhibition to morbigenous Escherichia coli and Aeromonas hydrophila in vitro and heat treatment,five bacteria were selected which could resist heat somewhat(Z3、C3、L2、E2、D2).By 16S rRNA sequencing,they were identified as Bacillus,Enterococcus and Citrobacter.
In this study,the isotopeγ-~(32)p-ATP was used as the tracing material,so the influence on the growth of bacteria was invested.The result showed,after addedγ-~(32)p -ATP,the number of the bacteria were declined,but the difference is unnotable,even the number of some bacteria rised.Generally,all the bacteria were influenced byγ-~(32)P -ATP,but the number of them were between 10~8 to 10~9 cfu/ml.
The adhesion characteristics of Enterococci,Citrobacter isolated from carp intestinal and Bacillus isolated from water to the carp foregut mucus including the characteristics of mucus receptor,lectin and the interaction between pathogens and Bacillus,Enterococci and Citrobacter were investigated.
Results showed that the relative adhesion population of homologous Enterococci were higher than heterogenous bacteria'(p＜0.01).The relative adhesion population of homologous Citrobacter and the Bacillus differed unnotable(p＞0.05).The relative adhesion population of pathogens which has the stronger pathogenicity is higher than others(p＜0.01).After the modification with proteolytic enzymes or sodium metaperiodate,The relative adhesion percentages of five bacteria were declined extreme significantly after they were treated with sodium metaperiodate(p＜0.01),but it was affected less by the proteolytic enzymes treatment(p＞0.05).After the modification with proteolytic enzymes or sodium metaperiodate of mucus.The relative adhesion percentages of part of five bacteria were declined significantly after the mucus treated with proteolytic enzymes(p＜0.05),but it was affected significantly by the sodium metaperiodate treatment(p＜0.05).The relative adhesion population were different led by the different of lectins on the surface of bacteria.So to be the homologous bacteria of carp intestine,the relative adhesion population of Enterococci L2 was higher than the others extremely(p＜0.01).According to the interactions such as exclusion,competition,displacement,part of the five bacteria can reduce the relative adhesion percentages of pathogens significantly(p＜0.05),and the relative adhesion percentages of pathogens were improved sometimes(p＜0.05),this was because the bacteria changed the surface construction of the mucus.
In conclusion,all of the bacteria had the ability to adhere to carp foregut mucus immobilized on the cell culture plates.The main lectins existing on the cell surface of the five bacteria are glycoprotein compounds.The special receptors of the microorganisms are protein compounds.The inhibition of displacement to the pathogens is best,but the disparity was led by the different bacteria.
引文
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