海洋动物肠道中抗氧化活性乳酸菌的分离及多样性分析

Screening and Diversity Analysis of Marine Lactic Acid Bacteria with Antioxidant Activity

【目的】分析海洋动物肠道中抗氧化活性乳酸菌的多样性,并对活性菌株胞外与胞内代谢物清除1,1-二苯基-2-三硝基苯肼(DPPH)自由基能力进行测定。【方法】采用溶钙圈分离培养技术和DPPH自由基清除法,从湛江海域6种海洋动物肠道中分离活性乳酸;通过构建16S rDNA基因序列系统发育树,分析其多样性;利用DPPH自由基清除法,测定其抗氧化活性。【结果】共分离得到16株抗氧化活性乳酸菌,分属于细菌域厚壁菌门(Firmicutes)的4个科(Leuconostocaceae,Streptococcaceae,Lactobacillaceae,Enterococcaceae)、6个属(Weissella,Pediococcus,Leuconostoc,Lactococcus,Lactobacillus,Enterococcus),其中除菌株zy-3、LY-87、ZH-54的16S r DNA基因序列与数据库EzBioCloud中的模式菌相似性100%外,其余13株活性菌株与模式菌存在一定的遗传差异(97.42%～99.93%),菌株zy-I与模式菌株Weissella cibaria(KACC11862T)的相似性仅为97.42%,推测为Weissella cibaria属中的潜在新种;16株活性菌株胞外代谢物对DPPH的抗氧化活性均大于胞内代谢物,且5株乳酸菌株(zy-4、zy-s6、YM-49、ZH-53、ZH-54)的胞外的DPPH清除能力均在45%以上,高于其它11株菌株。【结论】湛江海域动物肠道中存在较为丰富的抗氧化活性乳酸菌,并蕴藏潜在乳酸菌新种。

【Objective】The diversity of antioxidant lactic acid bacteria in the intestinal tract of marine animals was analyzed, and the free radical scavenging ability of extracellular and intracellular metabolites from the active strains against 1, 1-diphenyl-2-trinitrophenylhydrazine(DPPH) was determined.【Methods】In this paper, the dissolving calcium circle method was used to screen lactic acid bacteria strains from the intestinal tracts of 6 marine animals in Zhanjiang sea area. The 16 S rDNAs of isolates were sequenced and the phylogenetic tree was constructed for diversity analysis. The antioxidant activity of isolates was determined by measuring DPPH scavenging ability.【Results】According to the morphological observation, hydrogen peroxide contact enzyme experiment and molecular biological identification, a total of 16 strains of antioxidant active lactic acid bacteria were isolated, they could be divided into 4 families(Leuconostocaceae, Streptococcaceae, Lactobacillaceae,Enterococcaceae) and 6 genera(Weissella, Pediococcus, Leuconostoc, Lactococcus, Lactobacillus,Enterococcus), Except for the 16 S rDNA gene sequences of strains zy-3, LY-87 and ZH-54 that were100% similar to the model bacteria in the EzBioCloud database, there were certain genetic differences among the other 13 active strains and the model bacteria(97.42%-99.93%).The similarity between zy-I strain and model strain Weissella cibaria(KACC11862 T) was only 97.42%, which was speculated to be a new species of the genus Weissella cibaria. The antioxidant activity of extracellular metabolites from 16 active strains against DPPH was greater than that of their intracellular metabolites, and the DPPH scavenging rate of 5 strains of lactic acid bacteria(zy-4, zy-s6, YM-49, ZH-53 and ZH-54) was higher than 45% and above that of 11 other strains.【Conclusion】The study shows that there are abundant lactic acid bacteria with antioxidant activity in the intestinal tract of animals in Zhanjiang sea area, which contains potential new species of lactic acid bacteria. They may provide a basis for the development of lactic acid bacteria with antioxidant substance.