猪肠道产多肽类抗生素活性菌株的筛选与鉴定
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摘要
为了筛选产多肽类抗菌物质的活性菌株,为生产猪用益生素及新型抗菌活性物质提供菌源,试验分别从猪的十二指肠、空肠、回肠、盲肠、结肠和直肠中分离肠道内生菌2 317株,利用临床分离的致病性大肠杆菌作为指示菌,通过点种法进行对峙培养,分析其抑菌活性。结果表明:复筛所得50株肠道内生菌株均对指示菌具有明显抑菌活性,筛选得率为2.2%。经过胰蛋白酶、蛋白酶K水解处理,将发酵上清液抑菌活性明显降低或丧失的结肠57号和盲肠2号菌确定为可产蛋白或多肽类抗生素的活性菌株。经16S rDNA的序列检测及比对,两菌株均属于枯草芽孢杆菌。
The aim of the present study was to screen some functional strains producing antimicrobial polypeptide which could provide bacterial sources for producing probiotics and novel antibiotic active substance in pigs. A total of 2 317 strains were isolated from porcine duodenum,jejunum,ileum,caecum,colon and rectum. All the isolates were cultured against a clinical isolated pathogenic Escherichia coli indicator using a seeding method. The 50 strains of enteric strains obtained from repeated screening showed obvious antibacterial activity against the indicator,and the ratio of screening was 2. 2%. After the trypsin and proteinase K degradation examination,two strains( caecum No. 2 and colon No. 57)were recognized as antimicrobial peptide producers due to their significantly reduce or lost antibacterial activity in fermentation supernatant. According to their 16 S rDNA sequence detection and alignment,both strains were identified as Bacillus subtilis.
The aim of the present study was to screen some functional strains producing antimicrobial polypeptide which could provide bacterial sources for producing probiotics and novel antibiotic active substance in pigs. A total of 2 317 strains were isolated from porcine duodenum,jejunum,ileum,caecum,colon and rectum. All the isolates were cultured against a clinical isolated pathogenic Escherichia coli indicator using a seeding method. The 50 strains of enteric strains obtained from repeated screening showed obvious antibacterial activity against the indicator,and the ratio of screening was 2. 2%. After the trypsin and proteinase K degradation examination,two strains( caecum No. 2 and colon No. 57)were recognized as antimicrobial peptide producers due to their significantly reduce or lost antibacterial activity in fermentation supernatant. According to their 16 S rDNA sequence detection and alignment,both strains were identified as Bacillus subtilis.
引文
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[4]陈世琼,李平兰,张篪.猪肠道中抑制大肠杆菌的乳酸菌的分离与筛选[J].中国农业大学学报,2002,7(5):117-120.
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[8]LI G,LIU B,SHANG Y,et al.Novel activity evaluation and subsequent partial purification of antimicrobial peptides produced by Bacillus subtilis LFB112[J].Ann Microbiol,2012,62(2):667-674.
[9]MORGULIS A,COULOURIS G,RAYTSELIS Y,et al.Database indexing for production Mega BLAST searches[J].Bioinformatics,2008,24(16):1757-1764.
[10]袁书林,孔庆新,金锋,等.猪源益生乳酸杆菌的分离选育[J].农业科技与装备,2011,209(11):27-29.
[11]刘志林.益生菌对猪肠道菌群的调控研究进展[J].山东畜牧兽医,2010,31(3):71-73.
[12]ELSHAGHABEE F M F,ROKANA N,GULHANE R D,et al.Bacillus as potential probiotics:status,concerns,and future perspectives[J].Front Microbiol,2017,10(8):1490.
[13]DOMINGUES P M,ALMEIDA A,LEAL L S,et al.Bacterial production of biosurfactants under microaerobic and anaerobic conditions[J].Rev Environ Sci Biotechnol,2017,16(2):239-272.
[14]WILLENBACHER J,RAU J T,ROGALLA J,et al.Foam-free production of Surfactin via anaerobic fermentation of Bacillus subtilis DSM 10T[J].AMB Express,2015,5(1):21.
[2]DUBIN A,MAK P,DUBIN G,et al.New generation of peptide antibiotics[J].Acta Biochim Pol,2005,52(3):633-638.
[3]谢琴,赵晓瑞,张学礼.猪肠道芽孢杆菌的分离鉴定、小鼠毒性以及体外颉颃试验[J].黑龙江畜牧兽医,2010(12上):109-111.
[4]陈世琼,李平兰,张篪.猪肠道中抑制大肠杆菌的乳酸菌的分离与筛选[J].中国农业大学学报,2002,7(5):117-120.
[5]MAJEED H,GILLOR O,KERR B,et al.Competitive interactions in Escherichia coli populations:the role of bacteriocins[J].ISME J,2010,5(1):71-81.
[6]GILLOR O,GILADI I,RILEY M A.Persistence of colicinogenic Escherichia coli in the mouse gastrointestinal tract[J].BMC Microbiol,2009,9(1):165.
[7]GILLOR O,ETZION A,RILEY M A.The dual role of bacteriocins as anti-and probiotics[J].Appl Microbiol Biotechnol,2008,81(4):591-606.
[8]LI G,LIU B,SHANG Y,et al.Novel activity evaluation and subsequent partial purification of antimicrobial peptides produced by Bacillus subtilis LFB112[J].Ann Microbiol,2012,62(2):667-674.
[9]MORGULIS A,COULOURIS G,RAYTSELIS Y,et al.Database indexing for production Mega BLAST searches[J].Bioinformatics,2008,24(16):1757-1764.
[10]袁书林,孔庆新,金锋,等.猪源益生乳酸杆菌的分离选育[J].农业科技与装备,2011,209(11):27-29.
[11]刘志林.益生菌对猪肠道菌群的调控研究进展[J].山东畜牧兽医,2010,31(3):71-73.
[12]ELSHAGHABEE F M F,ROKANA N,GULHANE R D,et al.Bacillus as potential probiotics:status,concerns,and future perspectives[J].Front Microbiol,2017,10(8):1490.
[13]DOMINGUES P M,ALMEIDA A,LEAL L S,et al.Bacterial production of biosurfactants under microaerobic and anaerobic conditions[J].Rev Environ Sci Biotechnol,2017,16(2):239-272.
[14]WILLENBACHER J,RAU J T,ROGALLA J,et al.Foam-free production of Surfactin via anaerobic fermentation of Bacillus subtilis DSM 10T[J].AMB Express,2015,5(1):21.