CN115851498A - Bacillus subtilis for producing leader-free peptide bacteriocin, preparation method and application thereof - Google Patents
Bacillus subtilis for producing leader-free peptide bacteriocin, preparation method and application thereof Download PDFInfo
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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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Abstract
The invention discloses bacillus subtilis for producing leader-free peptide 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 invention is CGMCC No.25552. Fermenting and culturing 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 the bacteriocin product. Experiments prove that the obtained bacterial product has a good effect of inhibiting various pathogenic bacteria, and has wide application prospects in the fields of animal breeding, food preservation, medicine health, preparation of microbial agents, antibacterial drugs and the like.
Description
Technical Field
The invention relates to the field of medical application, in particular to bacillus subtilis for producing non-leader peptide bacteriocin, a preparation method and application thereof.
Background
Bacteriocins are a class of polypeptides synthesized by ribosomes during the metabolic process of bacteria that have antibacterial activity against the producing bacteria. Bacteriocins differ significantly from antibiotics in biosynthesis, mode of action, resistance mechanism and antibacterial activity and are considered to be the best alternatives to antibiotics, with lantibiotics being the most extensively studied at present. The bacteriocin can be widely applied to food preservation, animal culture, medical treatment and the like. However, the large-scale application of bacteriocin is restricted by factors such as low yield, difficult large-scale production, high cost and the like. Unlike most bacteriocins, the leader-free peptide bacteriocins are active bacteriocins which are synthesized by ribosomes and translated without any modification, have simple genetic structures, 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 bacteriocins of this type have been identified and reported, and only 19 kinds of non-leader peptide bacteriocins from bacteria of different species have been identified so far.
The first reported leader-free peptide bacteriocin was the bacteriocin Enterocin L50 (EntL 50) containing two peptide chains produced by strain e.faecium L50 in 1998. Besides, there are EntQ, enterocin K1 (EntK 1) from enterococcus faecium, enterocin EJ97 (EntEJ 97), enterocin 7 (Ent 7, same as reported Enterocin MR 10), aureocin a53 (Aur 53), aureocin a70 (Aur 70) from staphylococcus aureus, epidermicin NI01 (epic 01) from staphylococcus epidermidis, lactin Q (LnqQ), lactin Z ((LnqZ), lsbB, lactoristerin BU (lilibu), BHT-B from streptococcus muris, weissecin Y (WelY), weisseicin M (WelM), garKS from lactococcus garvieae, cereucin X (ceruca), cerebellin K (ceresin), cesensin (ceresin), new antibacterial peptides (ceresin), and non-bacterial peptides with different mechanisms for development and for the development of new antibacterial agents.
Disclosure of Invention
The invention aims to provide bacillus subtilis for producing non-leader peptide bacteriocin and a preparation method and application thereof, and aims to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a Bacillus subtilis (Bacillus subtilis), wherein the preservation number of the Bacillus subtilis is CGMCC No.25552; the preservation time is 2022 years, 8 months and 19 days; the preservation unit is China general microbiological culture Collection center (CGMCC); the preservation address is No. 3 Xilu No. 1 of Beijing, chaoyang, the district of rising Yang.
The invention also provides bacteriocins produced by the Bacillus subtilis.
Preferably, the bacteriocin is a leader-free bacteriocin. More preferably, the leader-free peptide bacteriocin is bacteriocin A1, bacteriocin A2 and bacteriocin A3, and the amino acid sequence of the bacteriocin A1 is MIAFLR IVA LGARWANKVLGWIIRDGMAIDWIINKINDMVS; the amino acid sequence of subticin A2 is MITFLRIVAQGARAAKWANKDRVLNWIKNGVIDAIDWIDKINDMVN; the amino acid sequence of subticin A3 is MVTFLR VAQARWAWANKDRILGWIRDGMIDWIINKINDMVN. More preferably, the first amino acid in the amino acid sequence of bacteriocin 1, bacteriocin A2 and bacteriocin A3 is formylmethionine.
The invention also provides a fermentation culture method of the bacillus subtilis, which comprises the steps of inoculating the bacillus subtilis to an LB liquid culture medium, and shaking-culturing at 37 ℃ and 220rpm for 8 hours to obtain fermentation liquor.
The invention also provides a production method of the bacteriocin, which comprises the following steps:
(1) Fermenting and culturing 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 the bacteriocin product.
Preferably, in step (1), the Bacillus subtilis is fermentatively cultured by the method described above.
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 subjected to low-temperature rotary evaporation and concentration and then is separated and purified by the high-phase liquid chromatography; wherein, the chromatographic conditions for separation and purification are as follows: a chromatographic column TC-C18; and (3) mobile phase acetonitrile with the flow rate of 1mL/min, performing gradient elution by increasing the acetonitrile concentration from 10% to 80% for 55min, and respectively collecting fractions with the retention time of 49min, 50min and 51min to obtain the bacteriocin product.
The invention also provides a product comprising the bacillus subtilis or the bacteriocin.
The invention also provides an application of the bacillus subtilis or the bacteriocin in preparation of a microbial inoculum or an antibacterial drug.
The invention discloses the following technical effects:
according to the invention, a bacillus subtilis capable of producing the leader peptide-free bacteriocin is obtained by separating and purifying in soil, three bacteriocins are obtained by separating the bacillus subtilis fermentation liquor, and the amino acid sequences of the three bacteriocins are different from those of the existing bacteriocin through mass spectrometry, so that the three bacteriocins are all novel bacteriocins. Experiments prove that the leader-free peptide bacteriocin subtilin A1 has good antibacterial activity on various food-borne pathogenic bacteria such as clostridium botulinum and the like; the leader-free peptide bacteriocins subtilin A2 and subtilin A3 have good antibacterial activity on streptococcus suis and streptococcus hemolyticus B. Therefore, the newly separated bacillus subtilis and the newly separated bacteriocin have wide application prospects in the fields of animal breeding, food preservation, medicine health, preparation of microbial agents, antibacterial drugs and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings 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 invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a first-order mass spectrometric analysis of bacteriocin produced by Bacillus subtilis ZXF 04; a: subticin A1; b: subticin A2; c: subticin A3;
FIG. 2 shows the HPLC separation and purification results of bacteriocin subtilin A1, A2, A3.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but rather as a more detailed description of certain aspects, features and embodiments of the invention.
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 invention. In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, 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 invention 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 invention. All documents mentioned in this specification are incorporated by reference herein 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 present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1 screening and identification of Bacillus subtilis ZXF04
1. The soil is collected from a certain cultivated land in Lankou county, city, henan province, and is air-dried, crushed and sieved by a 40-mesh sieve, 1g of the soil is taken and placed in a triangular flask filled with 100mL of sterile water for shaking for 20min, and water bath at 80 ℃ for 15min, then the soil is coated on an LB solid plate by a coating method, and the soil is cultured for 15h at 30 ℃, and a single colony is selected.
2. Culture characteristics
Colony morphology: colonies were pale, crude opaque, indicating wrinkles and irregular edges (LB plates).
Bacterial cells: after culturing at 37 ℃ and 220rpm for 20h in LB liquid medium, the generation of spores is obviously observed by a microscope.
Characteristics of the culture in LB liquid Medium: after inoculation, the culture is carried out at 37 ℃ and 220rpm in a shaking way, antibacterial substances are generated after 8 hours, and the fermentation supernatant has antibacterial activity on pathogenic bacteria such as bacillus cereus, listeria monocytogenes, streptococcus suis and the like.
3. Gene identification
Using the genome of the strain as a template, and adopting universal primers 27F and 1541R for identifying bacteria to perform PCR amplification, wherein 27F; the amplification system and the amplification procedure are shown in table 1:
table 1: PCR amplification system and amplification program
The PCR amplification product is sent to a biological sequencing company for sequencing, and the result shows that the 16srDNA sequence (SEQ ID NO: 4) of the strain is as follows:
cgagcggacagatgggagcttgctccctgatgttagcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccggatggttgtttgaaccgcatggttcaaacataaaaggtggcttcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctcaccaaggcaacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaagaacaagtaccgttcgaatagggcggtaccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaattattgggcgtaaagggctcgcaggcggtttcttaagtctgatgtgaaagcccccggctcaaccggggagggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctggtctgtaactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaacgcattaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggacagaacaaagggcagcgaaaccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccgaagtcggtgaggtaa。
sequence alignment shows that the 16srDNA sequence of the strain has 99.86% of consistency with the 16srDNA sequence of Bacillus subtilis NCIB 3610 (T) (NCBI accession number is ABQL 01000001). According to the colony morphology, the cell morphology and the 16srDNA sequence alignment condition, the strain is confirmed to be bacillus subtilis.
The strain is named as Bacillus subtilis ZXF04, and the preservation number is CGMCC No.25552; the preservation time is 2022 years, 8 months and 19 days; the preservation unit is China general microbiological culture Collection center (CGMCC); the preservation address is No. 3 Xilu No. 1 of Beijing, chaoyang, the district of rising Yang.
Through identification, the bacterial strain contains a bacteriocin biosynthesis gene cluster, and the sequence of the bacteriocin biosynthesis gene cluster is as follows: and 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 identification of Bacillus subtilis ZXF04 fermentation product
The Bacillus subtilis ZXF04 separated and purified in the example 1 is inoculated into an LB culture medium according to the inoculation amount of 5 percent, and is cultured for 8 hours at 30 ℃ and 220rpm with shaking, and an LB fermentation supernatant of the strain is obtained.
The LB fermentation supernatant can be detected into three bacteriocins, namely, subtilin A1, subtilin A2 and subtilin A3 by a high-resolution liquid chromatograph-Mass analyzer Agilent Technologies 6540UDH Accurate-Mass Q-TOF LC/MS.
Wherein, the mass spectrum detection conditions are as follows: capillary voltage: 3500V; spraying pressure: 35lb/in2; Q-TOF Scan Range: 500-2,000m/z; flow rate of drying gas: 9 litters/min; temperature: 300 ℃; data acquisition rate: 1spectrum/s.
Using a targeted MS/MS mode, the target ions are further fragmented at a voltage of 40V and detailed sequence information of the antibacterial substance can be determined by analysis of the fragment ions.
The primary mass spectrum of the three bacteriocins is shown in FIG. 1.
Secondary mass spectrometry analysis shows that the amino acid sequence (SEQ ID NO: 1) of bacteriocin A1 is MIAFLR VIGARAARWAWANKDRVLGWIRDGMAIDWINDINKINDMVS;
the amino acid sequence of subticin A2 (SEQ ID NO: 2) is MITFLR IVAQLGARAKWANKDRVLNWIKNGVIDAIWIIDKINDMVN;
the amino acid sequence of subticin A3 (SEQ ID NO: 3) is MVTFLR VAQARRAWANKDRILGWIDGADGMIDWIINKINDMVN.
The first amino acids of bacteriocins A1, A2 and A3 are identified as formylmethionine. BlastP analysis showed: the amino acid sequences of the subtilin A1, A2 and A3 are different from the amino acid sequences of bacteriocins reported and identified at present, which indicates that the bacteriocins are novel leader-free peptide bacteriocins and are researched, identified and reported by the inventor for the first time.
Example 3 isolation and identification of Bacillus subtilis ZXF04 fermentation product
The LB fermentation supernatant was obtained according to the method of example 2, exchanged with ion exchange resin Amberlite XAD7HP, eluted with distilled water, 30% and 80% ethanol (pH = 2), respectively, bacillus cereus ATCC 14579 was used as indicator bacteria, and its antibacterial activity was determined by agar diffusion method, and the active fraction (80% ethanol eluate) was concentrated by low temperature rotary evaporation to obtain crude bacteriocin.
The crude bacteriocin was further separated by High Performance Liquid Chromatography (HPLC) on TC-C18 column. The chromatographic conditions are as follows: the mobile phase acetonitrile with the loading amount of 10 mu L and the flow rate of 1mL/min, the gradient elution procedure is that the concentration of the acetonitrile is increased from 10 percent to 80 percent and is eluted for 55min, the detection wavelength is 210nm, fractions with the retention times of 49min, 50min and 51min (wherein the peak parts of the subtilin A2 and the subtilin A3 are collected) are collected for multiple times respectively, and then the bacteriocin products of subtilin A1, subtilin A2 and subtilin A3 with the purity of more than 95 percent can be obtained by rotary evaporation and freeze drying (as shown in figure 2).
Bacteriostatic experiments of bacteriocins against pathogenic bacteria: wherein, the bacteriostatic activity of the subtilin A2 and the subtilin A3 to pathogenic bacteria is similar.
TABLE 2 minimum inhibitory concentrations of bacteriocin subtilin A1 against various food-borne pathogenic bacteria
TABLE 3 minimum inhibitory concentration of bacteriocin A3 against Streptococcus suis and some pathogenic microorganisms
The injection strains Streptococcus suis strain JZH01, JZH03 and JZH04 are separated from piglet disease material and identified by 16srDNA sequence; ATCC denotes American Type Culture Collection; CMCC stands for China medical bacteria strain preservation management center, and other indicator bacteria come from laboratory preservation.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. The Bacillus subtilis is characterized in that the preservation number of the Bacillus subtilis is CGMCC No.25552.
2. A bacteriocin produced by the Bacillus subtilis of claim 1.
3. The bacteriocin of claim 2, wherein the bacteriocin is a leader-free bacteriocin.
4. The method for fermenting and culturing the bacillus subtilis as claimed in claim 1, wherein the bacillus subtilis is inoculated into an LB liquid culture medium and is shake-cultured at 37 ℃ and 220rpm for 8 hours to obtain a fermentation broth.
5. A method for producing bacteriocins, comprising the steps of:
(1) Fermenting and culturing the bacillus subtilis to obtain a fermentation broth;
(2) Separating the fermentation liquor, collecting supernatant, separating and purifying the supernatant by using ion exchange resin and high-phase liquid chromatography, and freeze-drying to obtain the bacteriocin product.
6. The production method according to claim 5, wherein the Bacillus subtilis is fermentatively cultured in step (1) by the method according to claim 4.
7. The production method according to claim 5, wherein in the step (2), the supernatant is subjected to exchange with the ion exchange resin Amberlite XAD7HP, and eluted with distilled water, 30% ethanol, and 80% ethanol, respectively, to obtain an active ingredient.
8. The process according to claim 7, wherein the active ingredient is concentrated by low temperature rotary evaporation and then purified by the high phase liquid chromatography; wherein, the chromatographic conditions for separation and purification are as follows: a chromatographic column TC-C18; and (3) mobile phase acetonitrile with the flow rate of 1mL/min, and performing gradient elution by increasing the acetonitrile concentration from 10% to 80% for 55min, and respectively collecting fractions with activity to obtain the bacteriocin preparation.
9. A product comprising the bacillus subtilis of claim 1 or the bacteriocin of any one of claims 2 to 3.
10. Use of the bacillus subtilis of claim 1 or the bacteriocin of any one of claims 2 to 3 for the preparation of a microbial preparation or an antibacterial medicament.
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| CN116041453A (en) * | 2022-09-16 | 2023-05-02 | 河南牧业经济学院 | Leader peptide-free bacteriocin A1 for resisting various food-borne pathogenic bacteria and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090070975A (en) * | 2007-12-27 | 2009-07-01 | 조선대학교산학협력단 | Bacillus subtilis separated from meju and a antimicrobial composition comprising the same |
| CN102071162A (en) * | 2009-11-26 | 2011-05-25 | 中国农业大学 | Bacillus subtilis LFB112 as well as bacteriocin produced by same and application thereof |
| CN103013861A (en) * | 2012-11-30 | 2013-04-03 | 山西农业大学 | Preparation method of bacillus subtilis HJDA32 and bacteriocin generated by bacillus subtilis HJDA32 |
| KR20150126251A (en) * | 2014-05-03 | 2015-11-11 | 조선대학교산학협력단 | Bacillus subtilis separated from meju and a antibacterial composition comprising the same |
| CN109627299A (en) * | 2018-11-03 | 2019-04-16 | 北京工商大学 | A kind of bacteriocin Gr17 and its application with broad spectrum antibiotic activity |
| CN111961617A (en) * | 2020-08-13 | 2020-11-20 | 中国农业大学 | Multi-effect bacillus subtilis for high yield of immune polysaccharide and bacteriocin and application thereof |
| KR20210001106A (en) * | 2019-06-26 | 2021-01-06 | 전북대학교산학협력단 | Bacillus subtilis BSC35 strain having antimicrobial activity and uses thereof |
-
2022
- 2022-09-16 CN CN202211130248.0A patent/CN115851498B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090070975A (en) * | 2007-12-27 | 2009-07-01 | 조선대학교산학협력단 | Bacillus subtilis separated from meju and a antimicrobial composition comprising the same |
| CN102071162A (en) * | 2009-11-26 | 2011-05-25 | 中国农业大学 | Bacillus subtilis LFB112 as well as bacteriocin produced by same and application thereof |
| CN103013861A (en) * | 2012-11-30 | 2013-04-03 | 山西农业大学 | Preparation method of bacillus subtilis HJDA32 and bacteriocin generated by bacillus subtilis HJDA32 |
| KR20150126251A (en) * | 2014-05-03 | 2015-11-11 | 조선대학교산학협력단 | Bacillus subtilis separated from meju and a antibacterial composition comprising the same |
| CN109627299A (en) * | 2018-11-03 | 2019-04-16 | 北京工商大学 | A kind of bacteriocin Gr17 and its application with broad spectrum antibiotic activity |
| KR20210001106A (en) * | 2019-06-26 | 2021-01-06 | 전북대학교산학협력단 | Bacillus subtilis BSC35 strain having antimicrobial activity and uses thereof |
| CN111961617A (en) * | 2020-08-13 | 2020-11-20 | 中国农业大学 | Multi-effect bacillus subtilis for high yield of immune polysaccharide and bacteriocin and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| NOWAKOWSKI, M.ET AL: "ACCESSION NO.WP_017154623，Aureocin A54 family class IId bacteriocin[Bacillus bingmayongensis]", 《GENBANK》 * |
| NOWAKOWSKI, M.ET AL: "ACCESSION NO.WP_221781902，Aureocin A54 family class IId bacteriocin[Bacillus bingmayongensis]", 《GENBANK》 * |
| NOWAKOWSKI, M.ET AL: "ACCESSION NO.WP_221781903，Aureocin A54 family class IId bacteriocin[Bacillus bingmayongensis]", 《GENBANK》 * |
| SHU LIU ET AL: "Four Novel Leaderless Bacteriocins, Bacin A1, A2, A3, and A4 Exhibit Potent Antimicrobial and Antibiofilm Activities against Methicillin-Resistant Staphylococcus aureus", 《MICROBIOL SPECTR》, vol. 10, no. 5, pages 1 - 15 * |
| XIAOFENG ZHANG ET AL: "Three novel leaderless bacteriocins have antimicrobial activity against gram‑positive bacteria to serve as promising food biopreservative", 《MICROBIAL CELL FACTORIES》, pages 2 * |
Cited By (4)
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|---|---|---|---|---|
| CN115850409A (en) * | 2022-09-16 | 2023-03-28 | 河南牧业经济学院 | Leader-free peptide bacteriocin A3 for resisting various pathogenic bacteria, and preparation method and application thereof |
| CN116041453A (en) * | 2022-09-16 | 2023-05-02 | 河南牧业经济学院 | Leader peptide-free bacteriocin A1 for resisting various food-borne pathogenic bacteria and application thereof |
| CN115850409B (en) * | 2022-09-16 | 2023-12-19 | 河南牧业经济学院 | Leader-free bacteriocin A3 resistant to multiple pathogenic bacteria, and preparation method and application thereof |
| CN116041453B (en) * | 2022-09-16 | 2024-01-26 | 河南牧业经济学院 | Leader peptide-free bacteriocin A1 for resisting various food-borne pathogenic bacteria and application thereof |
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