CN115851498B - Bacillus subtilis producing leader peptide-free bacteriocin and preparation method and application thereof - Google Patents
Bacillus subtilis producing leader peptide-free bacteriocin and preparation method and application thereof Download PDFInfo
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- 108010062877 Bacteriocins Proteins 0.000 title claims abstract description 66
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 27
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 244000052616 bacterial pathogen Species 0.000 claims abstract description 11
- 238000004321 preservation Methods 0.000 claims abstract description 10
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 6
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 11
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 4
- 241001660259 Cereus <cactus> Species 0.000 claims description 2
- 241000194017 Streptococcus Species 0.000 claims 2
- 241000193403 Clostridium Species 0.000 claims 1
- 241000186781 Listeria Species 0.000 claims 1
- 238000000855 fermentation Methods 0.000 abstract description 14
- 230000004151 fermentation Effects 0.000 abstract description 14
- 239000000047 product Substances 0.000 abstract description 12
- 239000006228 supernatant Substances 0.000 abstract description 9
- 230000001580 bacterial effect Effects 0.000 abstract description 6
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 abstract description 4
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- 229940124350 antibacterial drug Drugs 0.000 abstract description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 16
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- 241000194021 Streptococcus suis Species 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 4
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- CTBBEXWJRAPJIZ-VHPBLNRZSA-N (1S,2S,3S,6R,8R,9S,10R)-2-benzoyl-1,3,8,10-tetrahydroxy-9-(4-methoxy-6-oxopyran-2-yl)-5-oxatricyclo[4.3.1.03,8]decan-4-one Chemical compound O1C(=O)C=C(OC)C=C1[C@H]1[C@]([C@@H]2O)(O)[C@H](C(=O)C=3C=CC=CC=3)[C@@]3(O)C(=O)O[C@@H]2C[C@]31O CTBBEXWJRAPJIZ-VHPBLNRZSA-N 0.000 description 3
- CTBBEXWJRAPJIZ-UHFFFAOYSA-N Enterocin Natural products O1C(=O)C=C(OC)C=C1C1C(C2O)(O)C(C(=O)C=3C=CC=CC=3)C3(O)C(=O)OC2CC31O CTBBEXWJRAPJIZ-UHFFFAOYSA-N 0.000 description 3
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- 241000193755 Bacillus cereus Species 0.000 description 2
- PYUSHNKNPOHWEZ-YFKPBYRVSA-N N-formyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC=O PYUSHNKNPOHWEZ-YFKPBYRVSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
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- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
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- 241000193155 Clostridium botulinum Species 0.000 description 1
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- 241000186660 Lactobacillus Species 0.000 description 1
- 241000186606 Lactobacillus gasseri Species 0.000 description 1
- 241000194035 Lactococcus lactis Species 0.000 description 1
- 241000186779 Listeria monocytogenes Species 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000191963 Staphylococcus epidermidis Species 0.000 description 1
- 235000014897 Streptococcus lactis Nutrition 0.000 description 1
- 230000009471 action Effects 0.000 description 1
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- 108010050826 aureocin A70 Proteins 0.000 description 1
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- GEWHMNRKXIVZJF-UHFFFAOYSA-N enterocin l50 Chemical compound Cl.C=1C=CC=CC=1C(C=1C=CC=CC=1)(C(=O)N(C)C)CCN(CC1)CCC1(O)C1=CC=C(Cl)C(C(F)(F)F)=C1 GEWHMNRKXIVZJF-UHFFFAOYSA-N 0.000 description 1
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- 238000002955 isolation Methods 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The application discloses bacillus subtilis producing leader peptide-free bacteriocin, a preparation method and application thereof, and belongs to the field of medical application. The preservation number of the bacillus subtilis disclosed by the application is CGMCC No.25552. Fermenting and culturing by using the bacillus subtilis to obtain fermentation liquor; separating the fermentation liquor, collecting supernatant, and separating and purifying the supernatant by using ion exchange resin and high-phase liquid chromatography to obtain a bacteriocin product. Experiments prove that the obtained bacterial product has better effect of inhibiting various pathogenic bacteria, and has wide application prospect in the fields of animal cultivation, food preservation, medical health, preparation of microbial inoculum, antibacterial drugs and the like.
Description
Technical Field
The application relates to the field of medical application, in particular to bacillus subtilis producing leader peptide-free bacteriocin, and a preparation method and application thereof.
Background
Bacteriocins are a class of polypeptide substances synthesized by bacteria during metabolism by ribosomes and having antibacterial activity against producing bacteria. Bacteriocins are markedly different from antibiotics in terms of biosynthesis, mode of action, resistance mechanism and antibacterial activity, and are considered to be the best substitutes for antibiotics, with the most intensive research being conducted at present on lanthiobacterins. Bacteriocins are widely used in food preservation, animal breeding, medical treatment and the like. However, the large-scale production of bacteriocins is limited by factors such as lower yield, difficult mass production, high cost and the like. Unlike most bacteriocins, the bacteriocins without leader peptide are active bacteriocins which are synthesized by ribosomes and do not undergo any modification after translation, have simple genetic structure, are easy to express in other microbial cells, are convenient for large-scale production through bioengineering, have unique antibacterial mechanisms and have huge commercial application potential. However, few such bacteriocins have been identified and reported, and only 19 leader peptide-free bacteriocins from different species of bacteria have been identified so far.
The first reported leader peptide-free bacteriocins were from strains in 1998E.faeciumL50 produces the bacteriocin Enterocin L50 (EntL 50) containing two peptide chains. In addition, there are EntQ, enterocin K1 (EntK 1), enterocin EJ97 (EntEJ 97), enterocin 7 (Ent 7, and reported from enterococcus faeciumEnterocin MR10 is the same), aureocin A53 (Aur) from Staphylococcus aureus, aureocin A70 (Aur), epidermicin NI01 (EpiNI 01) from Staphylococcus epidermidis, lacticin Q (LnqQ) from lactococcus lactis, lacticin Z ((LnqZ), lsbB, lactobacillus BU (LlibU), BHT-B from Streptococcus suis, weisselicin Y (WelY) from Weisselicarkii, weisselicin M (WelM), garKS from Lactobacillus gasseri, cerucin X (CerX), cerucin H (CerH), cerucin V (CerV) from Bacillus cereus. Because different leader-free bacteriocins have different antibacterial mechanisms and antibacterial spectra, the screening of novel leader-free bacteriocin-producing bacteria has important significance for the development and application of novel bacteriocins.
Disclosure of Invention
The application aims to provide bacillus subtilis producing leader peptide-free bacteriocin, a preparation method and application thereof, so as to solve the problems in the prior art, and the bacillus subtilis can produce leader peptide-free bacteriocin which has a good effect of inhibiting various pathogenic bacteria and has a wide application prospect in the fields of animal cultivation, food preservation, medical health, preparation of bactericides, antibacterial medicines and the like.
In order to achieve the above object, the present application provides the following solutions:
the application provides a bacillus subtilisBacillus subtilis) The preservation number of the bacillus subtilis is CGMCC No.25552; the preservation time is 2022, 8 and 19 days; the preservation unit is China general microbiological culture Collection center (CGMCC); the preservation address is North Chen Silu No. 1 and No. 3 in the Chaoyang area of Beijing city.
The application also provides bacteriocins produced by the bacillus subtilis.
Preferably, the bacteriocin is a leader peptide-free bacteriocin. More preferably, the leader-free bacteriocin is bacteriocin subticin A1, bacteriocin subticin A2, bacteriocin subticin A3, and the amino acid sequence of the bacteriocin subticin A1 is MIAFLRIVAQLGARAARWAWANKDRVLGWIRDGMAIDWIINKINDMVS; the amino acid sequence of subticin A2 is MITFLRIVAQLGARAAKWAWANKDRVLNWIKNGVAIDWIIDKINDMVN; the amino acid sequence of subtticin A3 was MVTFLRIVAQLGARAARWAWANKDRILGWIRDGMAIDWIINKINDMVN. More preferably, the first amino acid in the amino acid sequence of bacteriocin subticin A1, bacteriocin subticin A2, bacteriocin subticin A3 is formylated to formylmethionine.
The application also provides a fermentation culture method of the bacillus subtilis, which comprises the steps of inoculating the bacillus subtilis into an LB liquid culture medium, and carrying out shaking culture at 37 ℃ and 220rpm for 8 hours to obtain fermentation liquor.
The application also provides a production method of the bacteriocin, which comprises the following steps:
(1) Fermenting and culturing by using the bacillus subtilis to obtain fermentation liquor;
(2) Separating the fermentation liquor, collecting supernatant, and separating and purifying the supernatant by using ion exchange resin and high-phase liquid chromatography to obtain a bacteriocin product.
Preferably, in the step (1), the bacillus subtilis is cultivated by fermentation by the above method.
Preferably, in the step (2), the supernatant is exchanged with the ion exchange resin Amberlite XAD7HP, and eluted with distilled water, 30% ethanol, and 80% ethanol, respectively, to obtain an active component.
Preferably, the active component is separated and purified by the high-phase liquid chromatography after being concentrated by low-temperature rotary evaporation; wherein, the chromatographic conditions of separation and purification are as follows: chromatographic column TC-C18; the acetonitrile of the mobile phase, the flow rate is 1mL/min, the gradient elution program is that the acetonitrile concentration is increased from 10% to 80%, the elution is carried out for 55min, and fractions with the retention time of 49min, 50min and 51min are respectively collected, so that the bacteriocin product is obtained.
The application also provides a product comprising the bacillus subtilis or the bacteriocin.
The application also provides an application of the bacillus subtilis or the bacteriocin in preparing a microbial inoculum or an antibacterial drug.
The application discloses the following technical effects:
according to the application, the bacillus subtilis capable of producing the leader peptide-free bacteriocin is obtained through separation and purification in soil, three bacteriocins are obtained through separation of bacillus subtilis fermentation liquor, and the amino acid sequences of the three bacteriocins are different from those of the existing bacteriocins through mass spectrometry, so that the three bacteriocins are novel bacteriocins. Experiments prove that the leader-free bacteriocin A1 has better antibacterial activity on various food-borne pathogenic bacteria such as clostridium botulinum and the like; the leader peptide-free bacteriocins subticin A2 and subticin A3 have better antibacterial activity on streptococcus suis and streptococcus suis hemolyticus. Therefore, the newly separated bacillus subtilis and the newly separated bacteriocin have wide application prospects in the fields of animal cultivation, food preservation, medical health, preparation of bactericides, antibacterial medicines and the like.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows Bacillus subtilisBacillus subtilis ZXF bacteriocin produced by ZXF first-order mass spectrometry; a: subtticin A1; b: subtticin A2; c: subtticin A3;
FIG. 2 shows the HPLC separation and purification results of bacteriocins A1, A2 and A3.
Detailed Description
Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
EXAMPLE 1 Bacillus subtilisBacillus subtilisScreening and identification of ZXF04
1. Soil is collected from a certain cultivated land in Lancole county in Henan province, air-dried, crushed and sieved by a 40-mesh sieve, 1g of the obtained product is placed in a triangular flask filled with 100 mL sterile water and shaken for 20min, water bath is carried out at 80 ℃ for 15min, the obtained product is coated on an LB solid flat plate by adopting a coating method, and the obtained product is cultured at 30 ℃ for 15h, so that single bacterial colonies are selected.
2. Culture characteristics
Colony morphology: colonies were grey, crude opaque, indicating wrinkles, and uneven edges (LB plates).
Somatic cells: after 20h incubation at 37℃and 220rpm in LB liquid medium, apparent sporulation was observed under microscope.
Culturing characteristics in LB liquid medium: after inoculation, the culture is carried out at 37 ℃ and 220rpm in an oscillating way, antibacterial substances are generated after 8 hours, and fermentation supernatant has antibacterial activity on pathogenic bacteria such as bacillus cereus, listeria monocytogenes, streptococcus suis and the like.
3. Gene identification
The genome of the strain is used as a template, and the universal primers 27F and 1541R for identifying bacteria are adopted for PCR amplification, wherein 27F is 5'-AGAGTTTGATCCTGGCTCAG-3',1541R is 5'-AAGGAGGTGATCCAGCCGCA-3'; the amplification system and amplification procedure are shown in Table 1:
table 1: PCR amplification system and amplification program
The PCR amplified product was sequenced by biosequencing company, and the result showed that the 16srDNA sequence (SEQ ID NO: 4) of the strain was:
cgagcggacagatgggagcttgctccctgatgttagcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccggatggttgtttgaaccgcatggttcaaacataaaaggtggcttcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctcaccaaggcaacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaagaacaagtaccgttcgaatagggcggtaccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaattattgggcgtaaagggctcgcaggcggtttcttaagtctgatgtgaaagcccccggctcaaccggggagggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctggtctgtaactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaacgcattaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggacagaacaaagggcagcgaaaccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtgaggtaa。
sequence alignment shows that the strain 16srDNA sequence and bacillus subtilis model strainBacillus subtilis The 16srDNA sequence identity of NCIB 3610 (T) (NCBI accession number ABQL 01000001) is 99.86%. The bacterial strain is confirmed to be bacillus subtilis according to colony morphology, cell morphology and 16srDNA sequence comparison.
The strain is named as bacillus subtilisBacillus subtilisZXF04 the preservation number is CGMCC No.25552; the preservation time is 2022, 8 and 19 days; the preservation unit is China general microbiological culture Collection center (CGMCC); the preservation address is North Chen Silu No. 1 and No. 3 in the Chaoyang area of Beijing city.
The bacterial strain contains bacteriocin biosynthesis gene cluster, and the sequence of the bacterial strain is shown as SEQ ID NO:5, as follows:
ttaatttaag atgatgtcgt actttctctt ctaattgagg tccatattga tgaacccaac
gcataatggt ggtgtgagcc attgataatc ctctttcctc catcatttct accaggttac
ggaagctcaa attgtaccgt aggtaccacc ttaccgttaa taagattagt tcaggctgat
aatgtttcca tttaaacaaa ttttcctttt ccatactgat cacacctttt ttagattcat
actatcagta tgtccaagtt taggagatta cttgcaagag ccttgaattt tttgcaccag
aacccgaata tttgttcatg taatggttat actaagaatt tgagaacgta tcaaatatca
acactacact ttaacagagt ttaatctttt aaataaaatt aaaataaaaa tcaattttaa
ttaaaataaa gttgattttt attttaatta tgttaaaatt caattaaagg agctgaagtt
tatgaatttt aaagacattc cgaattggct attaaattta gagagtgaag acattgagtt
tattaaaaat tttattttac actctggatc tttgaaagaa atagcaaaaa tttatgatgt
ttcttatcct acagtgaggg tacgggtaga taagctaatt caaaaaatta aagtgaatga
tactgatgaa gatgaagagt tcattacttt tattaaaaaa ctctccatcg aagatcgtat
taatttagaa gatgcaaaat tgataattga aaaatataaa aatgaaagag gggataatta
atgtttcgtc tatacggatt tttaattgca cttttattaa ttgggcttca atactttttg
tcaagaagag aaaatgttta tttgggaatg ggtttacctg ttttatatgt ggtaacacta
ctatatctat ggatgtctga gactttaatt gtcaaaggga atacacttat cttcattatt
attattttag gtggattagc tattttatta ggaatttgga ttaaaggaag agaagaacta
aaaaataaac agaaaaaaga attagaaaaa atgaaaacca aggatatgaa gtaatcgatt
agagataaaa agcattagct tctatcgact tgtgactgag tttgagtaca atactcattg
atttttaaat ttttgttatg aagggaatat atatcataca cattgcctta taataattgg
gatgaacttt taaaaatata attattgctc tatatgttct ttttttaaat aaggaaggat
gttataatgt ctctaatgct aatgggcttt tagaggatgg tgtaagagta aacggtttat
agaagtaagg tatgaaaatt aataatcatt atataaaaaa ggatagaact gcagatccta
tccttttaga attaattggt ttagattatg taattacacc aattagctta ccatgtcatt
aattttattg ataatccagt caatagccat accatctcta atccagccta aaactctatc
tttgttagcc caagcccatc tagcagctct agctcctaat tgagccacga ttcttaaaaa
tgcaatcatg ttatatacct cctaatatag taattgtaaa attaaaaatt cttagtttac
catgtcatta attttatcaa taatccagtc gattgccaca ccgtttttaa tccaatttaa
aactctatct ttgttagccc aagcccattt agcagctcta gctcctagtt gagctacgat
tcttaaaaat gtaatcatgt tatatacctc ctaatatagt aattgtaaaa ttaaaaattc
ttagtttacc atgtcattaa ttttattaat aatccagtca atagccatac catctctaat
ccagcctaaa attctatctt tgttagccca agcccatcta gcagctctag ctcctagttg
agctacgatt cttaaaaatg taaccataat atacctcctt ttgaattttt tggtgaatgc
ttttgcgatt ataaatatat ctgaatcttc aaattgtgtc aacttaattt ttagaaaaaa
atttatatta agttttttaa tattttattc caaaaaaatc tatacatgga tatattttta
agtataatta ttaagaggat ctttattgaa aatttagaat tgatagattg ttaatatgat
ttaattatca aaaaagggga ttgagattga aatgataaaa gaaccacaac aaaaaatctc
accaaatgca gtaaaagtat ggaggataag tgatgcaatt acttatacta ctgcactttg
tgtattagga attcttttgt ttttacagca ttattatgat tggaaaagct ggataagtat
tatttgttat atcattattg ctttactcat tatttcttcc atattcgaac tatccataat
acctatttat cgacagcgaa catggcgtta tgaaattgat gaaaattata ttcaattaaa
acatggagga gcattaatga gaaagcatct aataataccg atgacgaaag tacaatatgt
aaatacaaat caaggaccaa tattacgaaa atttggtttg tctacgttaa caataggtac
aatggcttca gaacatgaaa taccggctat ttctgaaaag aaagcaacag aattaagagc
gaacattgca tatttagctg gaatcaatga aataaatgaa taaaatagga gggctatcat
acaatgaagc aagaattagc atatgaccaa atagctcagc gaatgcatcc gttatggatg
ctcttttcca tggttaaatc tataaaagaa cttattcttt ggattgtttt ttttgctatt
tttataagtt caaattctaa tcctattttt ctgattgtag gtgtagtagc aggagttgtt
tatctaattt ataattttgt ttcaatattt cttgattgga aacattttaa gtatgtattt
actgataaag aaatgtacat ttatgaggga cgttttatta aagaaaatcg ttttattcca
ttagaacgta tccaaggtat tagtcaaaat acacactttt ttcatcgctt atttggacta
acatcacttt tattagaaac tggttccaat gaaaaaaaat cctctattaa acttgaaatg
ataacttatg aagaagctgc acggatacaa gaacatttag gacatattgg ttttattcca
aaaaaagaga atgaatcaga tatacagcaa gtagagttaa tagaatctaa agatatttca
agggacaaac attataaaat ggcgcctaaa gaaatattgg ttaaatcact tatgtcactt
aaattacttt tattgatccc attaattcaa gagatttatt ctaatataaa tgattttatt
tccatagacg gttatataaa tgaaattacc tctttttttc aaaaatcatg gtttctaatg
actcttggca ttcttattct tttaataatg tcattagctt atgggataat taagacttat
attcaatatg ggaactttga agtcacttct gaccaacatc gaatttttgt tagaacaggt
gtatttaatg aaactgaatt ttcaattcca aaggagaaaa tccaagctat taatattaat
actaatctat taaaaagatg gtttggttta gtacaagtaa aaattataag tattactgat
atggaagata aggaaatgag aactgctaaa gtactctttc cacttataga taaaaataga
gctttatcac taattcctga gattctacca acatttaaaa ttgatacaag aatgattagt
attccaaaat atacaatttt tataaagatg gtacgattaa gttatatttg gatcataagt
acgatactta cttattattt ttggtcagag ttttggtata tcccaataat attattcgta
cttgttaaca catcacaaat attaaactgt ttttatagtg gttacaaact taatggggaa
ttcattcaat tgcaacgggg aggattttca acaaatttat tcataacaaa ccgaaagaat
attgaagagt tgaaaataat agaatcaggg atacaaagtc catttggcct tgttactctt
caaacttcaa cacgtgctaa accagttaag aaaacaaaga ttttagatat cccaaaggat
gtagctgtac attattatca ttggtatgga agtaacaaga aacatataaa agcaaaaaaa
atctagtatt aaagaaatga gatgcaatca catatttgat taaataagta tttgtattca
aactttattt tatatttttg aatcctttaa attcgaaata aagactatgc acaaaaagga
gaacaatttc catgctaaca aatttagata caaaaaaaag gaaaacaaaa aaatggatta
tcctcggggt tattgcgtta attgctatcg ttgcagctat taatattttc gtgatgcaag
ggaagaagaa agaaacggcg aaaacagatg ctgtaagttt tgagaaggta acagagcgta
agctaaataa tacaaaatta atttcgggac aagtaaaacc aggaaatatt gaaagcttct
actcagatcc gactaaagga aaagtgaaag atattgcggt gaaagaagga caagaggtag
aaaaaggaac aaaattattc tcttatgata atgaagaagt caatttacag ataaagcaag
ctgatcttga tcaaaagatg gcagatatgc gttacgatca aggaaaaaag aaaatagatt
cattgaaaac agaaattaag aaggcgaaag atagcggtgc tacgaaagaa gtaatagatt
caatggaaga gcaagtaaat gaactagaaa ttcagcaaaa gacaacggac cttgagaaag
aaaaaagcaa attacaagca gaagaattaa agaaaaaaca gaaagaactt acgatttata
gtaatttcgc tggtgttgta cagaagttag ataaagatgc agcacaaagt tcctcacaag
taataggtgg acaaggaaaa tcatttttac aaattgcttc taaagatcca ttccaaattc
aagggacatt aacggaactt cagaagtcac aaattcaaaa agaccaaaca tttactgtaa
ctgcaaaagc gaataataag aagaaatgga caggtaaaat tacagaagta agtgaattcc
caacgagcgc agaaatggat caaactgcag gtgcaggtgc aggtgaggga actcaaaata
tgtctcatta tacatataaa gcaagtcttg atgggcaaga aggtttatct ccaggctatc
atgtttcttt acaagtaaac ttagagaata agacgatgat tgctgttcct agtaagagca
ttgtagaaaa ggataatgat gcatttgttt acatagaagg aaatggtaaa cttcaaaaac
aaaatgtaaa aaaaggtgct actgatggag attggacaga gattgttgag ggtgtaacag
ttgggcaaaa ggttgtaaaa aatccttccg acaacgtgta tgatggaatg gaagtgaaag
aaaaatgatt acgttaaatc atatttctaa aacatattat caaggtaaac tagcagtgcc
aattttacac ggtattagtt taaagattcg aaatggtgag tttatttcca ttatgggacc
atctggttcg gggaaatcaa cgttaatgaa tattattggt tgcctagatc gtccaacaga
aggtgaatat acgttgaatg gtgtaaatat cttaacagca gacgaggcaa aacttgctct
tattcgtaat gaataccttg gttttgtatt ccagcacttt aatttattac cgcgactttc
agcggtggaa aacgttgaac ttccgctcat ctacggtggc gtgaagaaag cagagcgtcg
tcaaagagcg cttgaagcac ttagtaaagt tggattatcg gacagggttc atcatttacc
tagtgaatta tccggtgggc aaaagcagcg tgtagcgatt gcaagatcaa tcgccaataa
tccaacattt attatggcag atgagccgac aggcgctctt gatacgaagt ctggtgaaca
agttatgaat atcttcacga agttaaatgc agaaggtacg acaattgtta tggttacaca
tgaagaagaa gtagctgcct attcctcccg tcgtatcgtg ttacgagatg ggaaaattac
agaagataga aggtgtgcag tatgagttta ctagatagta taaaaattgc cttgtcttct
attttagctc ataaactgcg ctcagccctg acgatgcttg ggattgttat cggtgtagct
tctatcatta ccgttgttgc aattggacag ggtggggaag cgacgttaaa gtcattattt
gctggtaagg gaaataacgt tgttcctatt cattataccc cagatattaa tgattcattt
gatacagaaa accctaagct aactgaggag gacatttatg aagtacaaaa gattccagaa
gtagcatatg ttcttacaac aaactctagt atggagcctc ttgatatcga agataaaaaa
gaaatggtca gtattacagg attagataag gaatatttta aagttaatca agtgaatgta
ttaaaaggac ggtcattaca agagtctgat attatccaag gaaataacgt agtcatggtt
agtacaggga tgaaagagaa agtatttaaa aagcaaaatc ctattgataa aattattgaa
attagaggtc aaccaatgca aattattggt gtatataaat cagataatga ttttatggga
atggaatctt cagaggctct aattccaatt acattatggc caacgttata tggaaaagat
gaaattcaaa atatttctgt gcaagcaaaa aatgttgata acttagaagt agctggaaaa
aaagcagttg aagtattaaa tagccgcaaa tcaagcgagt ttacaggtaa atatgaagta
acgaacttaa aagaacttca agatggtatt tctcaaatga caaatatcat gactatgatt
atcggtggca tcgctggcat ttccttagtt gttggtggca ttggggttat gaatattatg
cttgtatctg taacagagcg cacacgcgaa attggagtac gtaaagcact tggagcaacg
cgcagtaaaa tattattaca attcttaatt gaagcggtta tggtaacgct tcttggtgga
ttaattggaa ttggtcttgg ttacgcgggc gcttatgttg tatctattta tgcgaaatgg
ccaccacttg tttcgtggga agttgttgtg ggaggcgtat tattctctat gacgcttggt
attatctttg gattaattcc tgcaaacaaa gcagcaaaat tggatccaat tgaagcactt
cgttatgaat aggtttcatg tatagtaaag atattctctt tactgtacgt gattctatta
gacaaaacgt tggtttttga gagataatac gaattgacta atagaacgta gtatataaac
acaagggggc tatcaaaatg gaattaaatg ttgataaaca agaggttatt ggagagaaac
cgtcattatt aggaatgatt acatctccaa gtttacaatt tgaaagaatg aagaatagta
atgtcatttg gagaggcttt ttgctattag caatcttaac gggtattgta tatctaatta
atacatatgc gtatgttctg tctccagaag gtaaaaaagc aaatgcagag tttgggttag
atgtctcttt aaattggcaa ttagggagtg gtttttttac aggagctatt ggttttatgg
taggtgcttt tattactgca gctttttata aaatattaat gatgtttatg aataacgata
cgtcttataa aaaattatta gcaatttctg tttatggaag tattattaca acacttggtt
tacttataaa tagtcttttg gctattatca ttgaaggaac cggacaagaa atgtatacag
gtttgggttc tttgttttct tcatctaatg atgttttgca tggtgttatg agatcatttg
aaatttttac aatttggtca ttagttatat ctgcgttagg attacatatt acagccggat
taagtaaaaa acaggcaaca gtggttgtaa tcatcttctt tattttatca ttagctcttg
gggcacttgg tggtatgatg ccaaaatttt aagtgtagat gctagtgtta aaaggctaga
tatttaagtt tttttacctt tttccagtag aaaatatctt ccttcaaaat gtgaaggtgt
ggaatctctc cattggctgg gagaagattg ttgataagta taaataaaca tatttaaaag
gatgtacctt aattaatatc ttgcaaaaat aacactaaca agtataaaga gaactggaat
gacccaatga aataagacaa ggaataaaaa cctccgtata ttcgtattta aattaaatac
gaatatacgg aggtttttat tccttgcttg atatttcaaa tttccccata tctaatgagg
attggagaat taaaagaatc tctttttcgg tggcatttaa attttttata tcatttataa
atttatcaac caactctttt atcatttttg tacgaagttc tttgattttt tcttctctat
tggttatata tcttcctatt cctctacgtg tcacactaac ctcttctttc tctaactctt
ggaatgtacg ttgaatcgta ttttgattta cttctaattc attagctaat gcacgtacag
taggaatttt atctcctatt gccaaacgac cggttgcgat ttctatttta ataaattcta
ttacttgaat atatattggt atattaggtg aaaaatcaat tctcatttcc ttacccctca
aaattaattt gttaactata taatatactg gttctggtgc aaaggtaaaa taggttctgt
tgcaaagatt tctaaactga gataacctgt agaaaaagtg tgagcgggag aatgatatat
ggggtatttt aaaggaaaac agtttaaaaa ggatattatt ttggtaaccg tcggctacta
ttgtcgtttt tctttaagtt atcgtgatgt atctgaaatc ttgaaagagc gtggtgtttc
ggttcaccca acaaccatca tgcgctgggt tcatgaatat ggaaatctta tctatcaaat
ttggaagaag aaaaataaaa acgttcaatt atcttggaaa ctagatgaaa cctatataaa
agtcaaagga aaatggtgtt atttatatcg tgcaattgat aaagacgggc acacattgga
tattcaactt cgtaaaaaac gggatcaaca ggctgcctat gcctttatga aaagattggt
caaaatcttt ggagaaccag cggttctcac tacagacaaa gcaccag。
EXAMPLE 2 Bacillus subtilisBacillus subtilisZXF04 identification of fermentation products
The bacillus subtilis isolated and purified in example 1Bacillus subtilisZXF 04A strain was inoculated in an inoculum size of 5% to LB medium, and cultured with shaking at 30℃and 220rpm for 8 hours to obtain LB fermentation supernatant of the strain.
Three bacteriocins, subticin A1, subticin A2 and subticin A3, were detected from LB fermentation supernatants by high resolution LC/MS Agilent Technologies 6540 UDH Accurate-Mass Q-TOF LC/MS.
Wherein, mass spectrum detection conditions are: capillary voltage: 3500 V, V; spray pressure: 35 lb/in2; Q-TOF scan range: 500-2,000 m/z; drying gas flow rate: 9 liters/min; temperature: 300 ℃; data acquisition rate: 1 spoke/s.
Using the targeted MS/MS mode, the target ions were further fragmented at a voltage of 40V, and detailed sequence information of the antibacterial substance was determined by analysis of the fragment ions.
A primary mass spectrum analysis chart of the three bacteriocins is shown in FIG. 1.
Secondary mass spectrometry showed that the amino acid sequence of bacteriocin A1 (SEQ ID NO: 1) was MIAFLRIVAQLGARAARWAWANKDRVLGWIRDGMAIDWIINKINDMVS;
the amino acid sequence of subticin A2 (SEQ ID NO: 2) was MITFLRIVAQLGARAAKWAWANKDRVLNWIKNGVAIDWIIDKINDMVN;
the amino acid sequence of subtticin A3 (SEQ ID NO: 3) is MVTFLRIVAQLGARAARWAWANKDRILGWIRDGMAIDWIINKINDMVN.
The first amino acid of bacteriocins A1, A2 and A3 is identified to be formylated into formylmethionine. BlastP analysis showed that: the amino acid sequences of the subticin A1, A2 and A3 are different from those of the bacteriocins reported and identified at present, so that the bacteriocins are novel leader peptide-free bacteriocins, and are researched, identified and reported for the first time by the inventor.
EXAMPLE 3 isolation and identification of Bacillus subtilisBacillus subtilisZXF04 fermentation product
LB fermentation supernatant was obtained as in example 2, and subjected to ion exchange resin Amberlite XAD7HP exchange, eluting with distilled water, 30% and 80% ethanol (pH=2), respectively, to giveBacillus cereus ATCC 14579 is indicator bacteria, the antibacterial activity of which is measured by an agar diffusion method, and the active component (80% ethanol eluent) is concentrated by low-temperature rotary evaporation to obtain a bacteriocin crude product.
The bacteriocin crude product was further separated by High Performance Liquid Chromatography (HPLC) TC-C18 column. The chromatographic conditions are as follows: the acetonitrile in the mobile phase, the loading amount is 10 mu L, the flow rate is 1mL/min, the gradient elution program is that the acetonitrile concentration is increased from 10% to 80%, the elution is carried out for 55min, the detection wavelength is 210nm, fractions with retention time of 49, 50 and 51min are respectively collected for a plurality of times (wherein the peaks and the tips of the subtticin A2 and the subtticin A3 are collected), and then rotary evaporation and freeze drying are carried out, so that bacteriocin products subtticin A1, subtticin A2 and subtticin A3 with purity of more than 95% can be obtained (shown in figure 2).
Bacteriostasis experiment of bacteriocin on pathogenic bacteria: wherein the subtticin A2 and the subtticin A3 have similar antibacterial activity on pathogenic bacteria.
TABLE 2 minimum inhibitory concentration of bacteriocin Subticin A1 against various food borne pathogenic bacteria
TABLE 3 minimum inhibitory concentration of bacteriocin A3 on Streptococcus suis and part of pathogenic microorganisms
Strain injectionStreptococcus suis strainJZH01, JZH03 and JZH04 are separated from piglet disease feed and identified by 16srDNA sequences; ATCC means American Type Culture Collection; CMCC represents the chinese medical bacterial strain collection management center, other indicator bacteria are derived from laboratory collection.
The above embodiments are only illustrative of the preferred embodiments of the present application and are not intended to limit the scope of the present application, and various modifications and improvements made by those skilled in the art to the technical solutions of the present application should fall within the protection scope defined by the claims of the present application without departing from the design spirit of the present application.
Claims (4)
1. Bacillus subtilis producing leader peptide-free bacteriocinBacillus subtilis) The bacillus subtilis is characterized by having a preservation number of CGMCC No.25552.
2. The use of the bacillus subtilis of claim 1 in the preparation of a microbial inoculum.
3. The use of a bacteriocin produced by bacillus subtilis according to claim 1 for the preparation of a medicament for inhibiting pathogenic bacteria, wherein the bacteriocin has an amino acid sequence as set forth in SEQ ID NO:1, wherein the pathogenic bacteria is clostridium botulinumC.Botulinum) Bacillus cereusBacillus cereus) Or Listeria monocytogenesListeria monocytogenes)。
4. The use of a bacteriocin produced by bacillus subtilis according to claim 1 for the preparation of a medicament for inhibiting pathogenic bacteria, wherein the bacteriocin has an amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:3, the pathogenic bacteria is streptococcus suisstreptococcus suis) Or beta-hemolytic streptococcusβ-Hemolytic streptococcus）。
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