啟良重楼内生细菌的分离鉴定及其次级代谢产物研究

Isolation and Identification of Endophytic Bacteria in Paris qiliangiana and Study on Their Secondary Metabolites

对抗生素产生耐药性的细菌引起的感染及自由基积累导致的氧化损伤对人类健康造成重大威胁,由于重楼属植物内生菌中含有抗菌、抗氧化等活性物质,因此本试验采用形态学和16S rDNA鉴定技术对啟良重楼内生细菌的种类进行鉴定;通过滤纸片法和DPPH自由基清除试验研究其体外抑菌和抗氧化活性,并运用GC-MS鉴定活性组分。结果表明,从啟良重楼根茎中分离鉴定出3株内生细菌:巨大芽孢杆菌(Bacillus megaterium CLX1,G^(+))、Lelliottia aquatilis CLX2(G^(-))、居中克吕沃尔氏菌(Kluyvera intermedia CLX3,G^(-));其中B.megaterium(CLX1)具有最佳抑菌活性,对3株革兰氏阴性菌和7株革兰氏阳性菌的抑菌圈范围为(10.39±0.24)mm~(13.44±0.10)mm,MIC范围为0.33~2.61 mg/mL;L.aquatilis(CLX2)的DPPH自由基清除能力最强,EC50值为1.276 mg/mL。采用GC-MS从内生细菌B.megaterium的活性次级代谢产物中鉴定出7种抑菌活性化合物:二甲基三硫、甲基甲烷硫代磺酸盐、甲基硫代磺酸甲酯、苯甲酸、环(脯氨酸-亮氨酸)二肽、正二十一烷、2,5-二叔丁基酚,从L.aquatilis鉴定出2种抗氧化活性化合物:2,3-二氢-3,5二羟基-6-甲基-4(H)-吡喃-4-酮、十六烷酸(棕榈酸)。本研究结果为从药用植物啟良重楼内生细菌中发掘抑菌、抗氧化天然活性物质及开发新型抗生素、解决氧化损伤问题提供了科学参考。

The infection caused by bacteria tolerant to antibiotics and the oxidative damage caused by ac-cumulated free radicals pose serious threats to human health.The endophytic bacteria in the Paris plants contain antibacterial and antioxidant substances.Therefore,in this experiment,the microscopic morphology and 16S rDNA identification technology were used to identify the types of endophytic bacteria in P.qiliangiana,their in vitro antibacterial and antioxidant activities were studied using filter paper method and DPPH free radical scavenging test,and the active components were identified using GC-MS.The results showed that three endophytic bacteria were isolated from the rhizomes of P.qiliangiana:Bacillus megaterium CLX1(G^(+)),Lel-liottia aquatilis CLX2(G^(-)),and Kluyvera intermedia CLX3(G^(-)).Among them,B.megaterium(CLX1)had the best antibacterial activity with the inhibitory zone of(10.39±0.24)mm~(13.44±0.10)mm and the MIC value of 0.33~2.61 mg/mL against 3 strains of Gram negative bacteria and 7 strains of Gram positive bacteria;L.aquatilis(CLX2)had the strongest DPPH radical scavenging ability with the EC50 value of 1.276 mg/mL.Using GC-MS,seven antibacterial active compounds were identified from the secondary metabolites of B.megaterium,including dimethyl trisulfide,methyl methane thiosulfonate,methyl thiosulfonate,benzoic acid,cyclic(proline-leucine)dipeptide,n-tert butane,and 2,5-di-tert-butylphenol;identified from L.aquatilis two antioxidant active compounds were as 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one and palmitic acid.These results could provide scientific references for exploring natural antibacterial and antioxidant substances from the endophytic bacteria of P.qiliangiana,developing new antibiotics,and solving the problem of oxidative damage.