刺参机体酵母菌组成及其拮抗活性的研究

采用PDA、YPD、MEA培养基从刺参Apostichopus japonicus的体表、肠道和呼吸树分离出23株酵母菌,使用玻璃珠破碎方法提取其DNA,然后用酵母菌通用引物NL-1/NL-4从DNA中成功扩增出26S rDNA片段,将PCR产物迸行测序,将各菌株序列在GenBank数据库中进行检索,从Blast比对结果中取相似性最高的序列,用Mega 4.0软件对所有序列进行聚类分析,采用邻接法构建系统发育树.结果表明:从大连柏岚子海域刺参中分离出的菌株分别属于红酵母属、梅奇酵母属和丝孢酵母属,从大连市黑石礁海域刺参中分离出的菌株分别属于红酵母属、假丝酵母属、德巴利氏酵母属、有孢汉逊酵母属和毕赤酵母属；以8株刺参病原菌作为指示菌,采用双层琼脂扩散法对分离菌株的拮抗活性进行测定,获得拮抗酵母菌17株,占总测试菌株的73.9％,从刺参机体的体表、肠道和呼吸树可以筛选出不同得率的活性菌株,其抗菌谱和活性强度各不相同,其中C11菌株的抗菌谱最广,抗菌活性最强；从肠道分离出的拮抗菌C11、C14和C21菌株的胞外产物经硫酸铵沉淀后也具有抗菌活性,C14菌株胞外产物经65％硫酸铵沉淀获得的粗提物对热和蛋白酶K敏感,表明其拮抗物质为蛋白质.

The yeast composition and antibacterial activities in sea cucumber Apostichopus japonicus

In this study,23 yeast strains were isolated from body surface,intestine and respiratory tree of sea cucumber Apostichopus japonicus using PDA,YPD,and MEA media.The genomic DNA was extracted from the yeasts by bead beating method and PCR amplification of the yeast 26S rDNA fragments was performed using primer pairs NL-1/NL-4.PCR products of 26S rDNA were sequenced and the most similar sequences were obtained by Blast in GenBank database.All the sequences were analyzed by the package Mega 4.0 statistics and cluster analysis.The phylogenetic tree generated by the Neighbor-joining method showed that the yeasts isolated from the sea cucumber collected at Bailanzi area in cost were identified as Rhodotorula spp.,Metschnikowia sp.,and Trichosporon spp.,and the yeasts in the sea cucumber collected in sea waters of Heishijiao were divided into Rhodotorula spp.,Candida spp.,Debaryomyces spp.,Hanseniaspora sp.and Pichia sp.Antagonistic activity tests performed by the indicators of eight strains pathogenic bacteria found in the sea cucumber using overlay technique revealed that about 73.9% of the isolated yeast strains showed antibacterial activity.The yeasts derived from body surface,intestine and respiratory tree of the sea cucumber had different bioactivity and various antibacterial spectra and strengths.The antibacterial activity of strain C11 was found to be the maximum,and the extracellular products of strains C11,C14 and C21 precipitated with ammonium sulfate also had antibacterial activity.Crude extract obtained by precipitation of the supernatant of C14 with 65% ammonium sulfate was sensitive to heat and proteinase K,indicating its antagonistic substance was protein.