海洋动物肠道中抗氧化活性乳酸菌的分离及多样性分析
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摘要
【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.
【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.
引文
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[2]CHIN F L,TZU M P.In vitro effects of lactic acid bacteria on cancer cell viability and antioxidant activity[J].Journal of Food and Drug Analysis,2010,18(2):77-86.
[3]熊双丽,卢飞,史敏娟,等.DPPH自由基清除活性评价方法在抗氧化剂筛选中的研究进展[J].食品工业科技,2012,33(8):380-383.
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[7]吴祖芳,洪松虎,沈锡权,等.乳酸菌高抗氧化活性菌株的筛选及鉴定[J].中国食品学报2010,10(1):73-78.
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[9]王悦齐,李来好,蔡秋杏,等.分离自腌干鱼的抗氧化发酵菌株的筛选及鉴定[J].南方水产科学,2016,12(3):74-83.
[10]朱雯娟,安俊莹,张雪梅,等.梅香鱼发酵菌株的筛选及对品质的影响[J].食品科学,2015,36(23):162-166.
[11]钟正丹,何义国,赵兴秀,等.白酒酒槽中抗氧化乳酸菌的筛选及培养基优化[J].2016,44(7):533-536.
[12]张江魏,曹郁生.乳酸菌抗氧化活性的研究进展[J].中国乳品工业,2005,33(1):34-36.
[13]TAKASHI K,MIHO K,MAKI N,et al.In vitro antioxidant and anti-inflammation properties of lactic acid bacteria isolated from fish intestines and fermented fish from the Sanriku Satoumi region in Japan[J].Food Research International,2014,(64):248-255.
[14]黄玉军,刘冬,赵兰凤,等.6株人源乳酸菌体外抗氧化活性的比较[J].现代食品科技,2013,29(7):1518-1552.
[15]BUDANAN R E,GIBBONR N E.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984:136-357.
[16]凌代文.乳酸细菌分类鉴定及实验方法[M].北京:中国轻工业出版社,1999:1-129.
[17]NUGENTR P,Krohn M A,HILLIER SL,et al.Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation[J].Journal of Clinical Microbiology,1991,29(2):297-301.
[18]LU Q F,HU H Q,MO J,et al.Enhanced amplification of bacterial and fungal DNA using a new type of DNApolymerase[J].Australasian Plant Pathology,2012,41(6):661-663.
[19]黄苛,张丽,刘祝祥,等.硇洲岛海胆可培养细菌的多样性[J].微生物学报,2009,49(11):1424-1429.
[20]STACKBRANDT E,GOEBEL B M.Taxonomic note:a place for DNA-DNA reassociation and 16S rRNAsequence analysis in the present species definition in bacteriology[J].International Journal of Systematic and Evolutionary Microbiology,1994,44(4):846-849.
[21]张凤敏,田丰伟,陈卫,等.具抗氧化活性乳酸菌的筛选[J].中国乳品工业,2007,35(2):4-7.
[22]刘颖,时威,安俊莹,等.硇洲岛褐蓝子鱼肠道抗菌活性菌株的分离及多样性[J].微生物学报,2014,54(05):582-588.
[23]李晓军,马跃英,龚红,等.具有抗氧化活性的乳酸菌菌株的筛选与鉴定[J].中国微生态学杂志,2018,30(6):663-668.
[24]蔡英杰.第四届亚洲乳酸菌研讨会暨第三届乳酸菌与健康国际研讨会[R].上海:中国国际科技会议中心,2007.
[25]兰道亮,王永,张诚民,等.藏绵羊肠道乳酸菌的分离鉴定及系统进化分析[J].西南民族大学学报,2014,40(04):481-484.(责任编辑:刘朏)
[2]CHIN F L,TZU M P.In vitro effects of lactic acid bacteria on cancer cell viability and antioxidant activity[J].Journal of Food and Drug Analysis,2010,18(2):77-86.
[3]熊双丽,卢飞,史敏娟,等.DPPH自由基清除活性评价方法在抗氧化剂筛选中的研究进展[J].食品工业科技,2012,33(8):380-383.
[4]秦曼曼,张雪梅,王晓杰,等.产抗氧化活性物质的海洋放线菌筛选与鉴定[J].食品工业科技,2016,5(37):167-170.
[5]邓元源,刘芸,刘欣,等.福建省腌菜中乳酸菌的分离与鉴定[J].食品安全质量检测学报,2018,9(3):481-490.
[6]SHOBHARANI P,SACHINDRA N.Potential of marine lactic acid bacteria to ferment Sargassum sp.for enhanced anticoagulant and antioxidant properties[J].Journal of Applied Microbiology,2012,114(1):96-107.
[7]吴祖芳,洪松虎,沈锡权,等.乳酸菌高抗氧化活性菌株的筛选及鉴定[J].中国食品学报2010,10(1):73-78.
[8]代永刚,田志刚,南喜平.等.乳酸菌及其生理功能研究的进展[J].农产品加工,2009(7):24-26.
[9]王悦齐,李来好,蔡秋杏,等.分离自腌干鱼的抗氧化发酵菌株的筛选及鉴定[J].南方水产科学,2016,12(3):74-83.
[10]朱雯娟,安俊莹,张雪梅,等.梅香鱼发酵菌株的筛选及对品质的影响[J].食品科学,2015,36(23):162-166.
[11]钟正丹,何义国,赵兴秀,等.白酒酒槽中抗氧化乳酸菌的筛选及培养基优化[J].2016,44(7):533-536.
[12]张江魏,曹郁生.乳酸菌抗氧化活性的研究进展[J].中国乳品工业,2005,33(1):34-36.
[13]TAKASHI K,MIHO K,MAKI N,et al.In vitro antioxidant and anti-inflammation properties of lactic acid bacteria isolated from fish intestines and fermented fish from the Sanriku Satoumi region in Japan[J].Food Research International,2014,(64):248-255.
[14]黄玉军,刘冬,赵兰凤,等.6株人源乳酸菌体外抗氧化活性的比较[J].现代食品科技,2013,29(7):1518-1552.
[15]BUDANAN R E,GIBBONR N E.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984:136-357.
[16]凌代文.乳酸细菌分类鉴定及实验方法[M].北京:中国轻工业出版社,1999:1-129.
[17]NUGENTR P,Krohn M A,HILLIER SL,et al.Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation[J].Journal of Clinical Microbiology,1991,29(2):297-301.
[18]LU Q F,HU H Q,MO J,et al.Enhanced amplification of bacterial and fungal DNA using a new type of DNApolymerase[J].Australasian Plant Pathology,2012,41(6):661-663.
[19]黄苛,张丽,刘祝祥,等.硇洲岛海胆可培养细菌的多样性[J].微生物学报,2009,49(11):1424-1429.
[20]STACKBRANDT E,GOEBEL B M.Taxonomic note:a place for DNA-DNA reassociation and 16S rRNAsequence analysis in the present species definition in bacteriology[J].International Journal of Systematic and Evolutionary Microbiology,1994,44(4):846-849.
[21]张凤敏,田丰伟,陈卫,等.具抗氧化活性乳酸菌的筛选[J].中国乳品工业,2007,35(2):4-7.
[22]刘颖,时威,安俊莹,等.硇洲岛褐蓝子鱼肠道抗菌活性菌株的分离及多样性[J].微生物学报,2014,54(05):582-588.
[23]李晓军,马跃英,龚红,等.具有抗氧化活性的乳酸菌菌株的筛选与鉴定[J].中国微生态学杂志,2018,30(6):663-668.
[24]蔡英杰.第四届亚洲乳酸菌研讨会暨第三届乳酸菌与健康国际研讨会[R].上海:中国国际科技会议中心,2007.
[25]兰道亮,王永,张诚民,等.藏绵羊肠道乳酸菌的分离鉴定及系统进化分析[J].西南民族大学学报,2014,40(04):481-484.(责任编辑:刘朏)
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