CN108192841B - Bacillus psychrophile and bacteriocin for producing bacteriocin, and extraction method and application thereof - Google Patents
Bacillus psychrophile and bacteriocin for producing bacteriocin, and extraction method and application thereof Download PDFInfo
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- CN108192841B CN108192841B CN201810040095.8A CN201810040095A CN108192841B CN 108192841 B CN108192841 B CN 108192841B CN 201810040095 A CN201810040095 A CN 201810040095A CN 108192841 B CN108192841 B CN 108192841B
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Abstract
The invention belongs to the fields of biotechnology and treatment and medicine of bacterial infectious diseases, and particularly relates to a psychrophile for producing bacteriocin, a bacteriocin extraction method and bacteriocin of the psychrophile and application of the bacteriocin. The preservation number of the psychrophile is CCTCC NO: m2017480. The bacteriocin produced by the psychrophile has obvious antibacterial activity, wider antibacterial spectrum, better thermal stability and strong acid and alkali resistance.
Description
Technical Field
The invention belongs to the fields of biotechnology and treatment and medicine of bacterial infectious diseases, and particularly relates to a psychrophile for producing bacteriocin, a bacteriocin extraction method and bacteriocin of the psychrophile and application of the bacteriocin.
Background
Due to the discovery of antibiotics and the successful development of antibacterial drugs, human beings have a favorable score in the aspect of treating infectious diseases, and many infectious diseases of human beings and animals which are difficult to cure in the past are well treated, but in recent years, due to the wide use of the antibacterial drugs, the service level of basic hospitals and veterinary staff is different from that of professional literacy, particularly, the phenomenon of abuse of the antibacterial drugs in livestock and poultry breeding is severe day by day, so that the problem of drug resistance of common pathogenic bacteria to various antibacterial drugs is more serious at present, multiple drug resistance of common pathogenic bacteria is developed to different degrees, and super bacteria which are not available for drugs are developed. The development speed of new drugs at present cannot keep up with the drug resistance of bacteria. In view of this, extensive researchers are struggling to find antibacterial drug substitutes.
Bacteriocins are proteins or peptides which are synthesized by intracellular nucleoproteins of bacteria in the metabolic process of the bacteria and secreted outside the bacterial cells and have antibacterial activity, can inhibit or prevent the growth of certain microorganisms, are not easy to generate drug resistance, can be degraded by enzymes, do not have pathogenic effects on human bodies or animals, and have the potential of replacing antibacterial drugs, and the bacteriocins are different from antibiotics and belong to protein substances.
Disclosure of Invention
The invention aims to provide a psychrophile for producing bacteriocin, the bacteriocin, and an extraction method and application thereof. The bacteriocin produced by the psychrophile has obvious antibacterial activity, wider antibacterial spectrum, better thermal stability and strong acid and alkali resistance.
The first aspect of the present invention provides a bacteriocin-producing psychrophile (SLJ) with a collection number of CCTCC NO: m2017480. The preservation information of the strain is as follows: the preservation date is as follows: 9 month 8 in 2017; the preservation unit: china Center for Type Culture Collection (CCTCC); the address of the depository: china, wuhan university; the preservation number is: CCTCC NO: m2017480; and (3) classification and naming: psychrophile (psychrophile.sp). The psychrophile has the nucleotide sequence shown in SEQ ID NO: 1.
The inventors of the present invention have occasionally found on a nutrient agar plate for culturing Staphylococcus aureus bacteria that contaminated an off-white colony around which neither Staphylococcus aureus grew (as shown in FIG. 1). The strain was subjected to isolation culture and culture characteristic observation, gram staining morphology observation (as shown in FIG. 4), biochemical test, preliminary bacteriostatic test (as shown in FIGS. 5A and 5B), DNA template was prepared, and 16SrRNA gene PCR amplification (as shown in FIG. 6, the length of the target product was 1400bp), sequence determination, and homology analysis were performed. The results show that: the bacterium is a gram-positive catenulobacterium, can ferment glucose, sucrose and maltose to produce acid only, cannot utilize lactose, and is negative in indole formation and methyl red tests and VP tests, and homology analysis shows that: the homology of the bacillus subtilis and the psychrophile with the accession number of JX196613.1 in GenBank is as high as 99.9 percent, so the bacillus subtilis is the psychrophile. The results of the initial in vitro bacteriostatic test show that: the bacteriocin of the strain has the effect of inhibiting the growth of various bacteria.
The second aspect of the present invention provides the method for extracting bacteriocin from psychrophile, which comprises:
and (3) bacterial culture: selecting a single bacterial colony of the strain, inoculating the single bacterial colony into a culture medium, carrying out shake culture at 37 ℃, centrifuging culture solution, removing thalli, and keeping supernatant;
acid precipitation: adjusting the pH value of the supernatant to 1.5-2.5, cooling and precipitating, centrifuging and removing the supernatant, and dissolving the precipitate with phosphate buffer solution to obtain crude bacteriocin.
In the acid precipitation step, the pH of the supernatant is preferably adjusted with concentrated hydrochloric acid.
According to a specific embodiment of the invention, a single colony of the strain is picked by an inoculating loop and inoculated in an LB liquid culture medium, shaking culture is carried out at 37 ℃ for 48h, a culture solution is centrifuged at 3500r/min and 4 ℃ for 10min, the strain body is removed, and a supernatant is kept to be a fermentation supernatant of the strain. The crude bacteriocin of the strain is prepared by an acid precipitation method. Adjusting pH of the fermented supernatant to 2.0 with concentrated hydrochloric acid, precipitating at 4 deg.C overnight at 10000r/min, centrifuging at 4 deg.C for 20min, discarding supernatant, dissolving the precipitate with phosphate buffer solution with pH of 6.5 to obtain crude bacteriocin, and storing at-20 deg.C.
The third aspect of the present invention provides a bacteriocin obtained by the above-mentioned bacteriocin extraction method.
In a fourth aspect, the present invention provides the use of a psychrophile as defined above and/or a bacteriocin as defined above for the preparation of a bacteriostatic agent.
The bacteriostatic agent can inhibit gram-positive bacteria and gram-negative bacteria. Specifically, the compound can inhibit salmonella paratyphi, staphylococcus aureus, duck proteus mirabilis, goose proteus mirabilis 1, goose proteus mirabilis 2, chicken staphylococcus albus, chicken escherichia coli, chicken pathogenic escherichia coli 1, chicken pathogenic escherichia coli 2, goose salmonella 1, piglet yellow dysentery escherichia coli 1, pigeon salmonella, corynebacterium glutamicum, pseudomonas and goose enterococcus faecalis.
Experiments prove that the bacteria in the invention are nontoxic, the produced bacteriocin has obvious antibacterial activity, good inhibition effect on staphylococcus aureus, escherichia coli and the like, wide antibacterial spectrum, good thermal stability and strong acid and alkali resistance, can be used for feed additives, food bacteriostats and the like, and has a prospect of further development and utilization.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
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The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
FIG. 1 shows that the growth of Staphylococcus aureus around colonies of the psychrophiles of the present invention was inhibited. As indicated by the arrows in the figure, the growth of Staphylococcus aureus around the colonies of the bacteria was inhibited.
FIG. 2A shows the growth performance of psychrophiles of the present invention on a common nutrient agar plate, and FIG. 2B shows the growth performance of psychrophiles of the present invention on a chicken blood agar plate.
FIG. 3 shows the growth performance of psychrophile of the present invention in LB liquid medium with shaking culture at 37 ℃ for 48 hours.
FIG. 4 shows the results of gram staining of psychrophile of the present invention.
FIGS. 5A and 5B show the partial inhibition results of crude bacteriocin by psychrophiles of the present invention. Wherein, FIG. 5A shows the bacteriostatic effect of the crude bacteriocin of psychrophile of the present invention on Bacillus subtilis. FIG. 5B shows the bacteriostatic effect of crude bacteriocin of psychrophile of the present invention on Staphylococcus aureus.
FIG. 6 shows the result of PCR amplification of psychrophile of the present invention. Wherein, M is DLmarker2000, and SLJ is the electrophoresis pattern of PCR products of the strain.
FIGS. 7A and 7B show the homology alignment of the 16SrRNA gene of the psychrophile SLJ of the present invention and 9 other strains of the psychrophile, and the gene clade.
Microbial deposits for patent procedures:
the preservation date is as follows: 9 month 8 in 2017;
the preservation unit: china Center for Type Culture Collection (CCTCC);
the address of the depository: china, wuhan university;
the preservation number is: CCTCC NO: m2017480;
and (3) classification and naming: psychrophile (psychrophile.sp).
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the invention, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein.
Strain verification
The present invention was discovered by chance from the microbiological laboratory. The growth performance of the strain on various culture media is shown in table 1: neither growth on Columbra nor MacConkey agar. White approximately round opaque and flat rough dry large or medium sized colonies were obtained on plain nutrient agar plates (as shown in FIG. 2A). The insoluble off-white approximately round, jagged edges, opaque, flat, dry, coarse, medium-sized colonies were present on chicken blood agar (as shown in FIG. 2B). In LB liquid, there was a biofilm formed on the surface, the upper layer of the culture was slightly turbid, the lower layer was slightly transparent, and there was a flocculent precipitate at the bottom after shaking (as shown in FIG. 3).
TABLE 1 growth performance of the bacterium on various media
The 16SrRNA sequence of the strain is subjected to PCR amplification agarose gel electrophoresis
The bacterial universal primers are adopted as follows: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2), and its downstream primer: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 3), the synthesis was performed by general biosystems (Anhui) Inc.
Reaction system: DNA template 5. mu. L, Taq polymerase (5U/. mu.L) 0.25. mu.L, 10 XTaq buffer 5. mu. L, dNTPs (2.5. mu. mol/. mu.L) 4.0. mu.L, and each 1. mu.L of upstream and downstream primers at a concentration of 10. mu. mol/. mu.L were made up to 50. mu.L with ultrapure water.
And (3) PCR reaction conditions: denaturation at 95 deg.C for 5 min; annealing at 60 ℃ for 30 s; the extension is carried out for 2min at 72 ℃ for 30 cycles; finally, extension is carried out for 5min at 72 ℃.
The PCR product was electrophoresed using 1% agarose, voltage 110V, time 30min, and observed and photographed in a gel imaging system. The results are shown in FIG. 6.
The PCR end product sequence of the strain is determined and the homology is analyzed
Sequencing was performed by the Anhui Universal biosystems, Inc. And (5) carrying out homology analysis on the sequencing result.
The sequencing result of the strain is SEQ ID NO: 1, submitted in NCBI for alignment, this strain has 99.9% homology with the psychrophilic JX 196613.1. 9 strains of bacteria were selected from the psychrophiles, DNASTAR software was used to convert each strain sequence into a form that can be recognized by the software, genetic distances were calculated for each strain sequence using a program, and a comparison graph of gene homology and a phylogenetic tree of genes with the psychrophile SLJ were constructed (see FIGS. 7A and 7B). It can be seen that: the homology of the bacillus subtilis and 9 psychrophiles selected from gene Genbank is more than 99%.
Bacteriocin extraction
The psychrophile of the invention has low requirement on nutrition and is easy to culture. Meanwhile, the bacteriocin extraction step of the strain is not complex. Selecting single colony of the strain by using an inoculating loop, inoculating the single colony in an LB liquid culture medium, performing shake culture at 37 ℃ for 48h, centrifuging the culture solution at 3500r/min and 4 ℃ for 10min, removing the strain body, and reserving supernatant, namely fermentation supernatant of the strain. The crude bacteriocin of the strain is prepared by an acid precipitation method. Adjusting pH of the fermented supernatant to 2.0 with 36% concentrated hydrochloric acid, precipitating at 4 deg.C overnight, 10000r/min, centrifuging at 4 deg.C for 20min, discarding supernatant, dissolving the precipitate with a little phosphate buffer solution with pH of 6.5 to obtain crude bacteriocin, and storing at-20 deg.C for use.
Verification of bacteriostatic effect
The results of the bacteriostasis test of the crude bacteriocin of the psychrophile of the invention show that: the crude bacteriocin of the strain has good bacteriostatic effect.
The crude bacteriocin of this strain of psychrophile had a broad antibacterial spectrum, as shown in table 2: can inhibit the growth and the propagation of staphylococcus aureus, staphylococcus albus, enterococcus, duck, goose proteus mirabilis, chicken, pigeon, goose salmonella, chicken escherichia coli and other bacteria, namely has stronger bacteriostatic action on gram-negative bacteria such as gram-positive bacteria and escherichia coli, has further development and utilization prospect and has good popularization and application values.
TABLE 2 results of bacteriostatic test of crude bacteriocin of the bacterium
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Sequence listing
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<120> a psychrophile and bacteriocin for producing bacteriocin, and extraction method and application thereof
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<213> Psychrophile (Psychrophile)
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gccttcccga aggttaggct atccacttct ggtgcaatca actcccatgg tgtgacgggc 60
ggtgtgtaca aggcccggga acgtattcac cgcggcattc tgatccgcga ttactagcga 120
ttcctacttc atggagtcga gttgcagact ccaatctgga ctacgatagg ctttttgaga 180
ttcgcatcac atcgctgtgt agctgccctc tgtacctacc attgtagcac gtgtgtagcc 240
ctggtcgtaa gggccatgat gacttgacgt cgtccccgcc ttcctccagt ttgtcactgg 300
cagtatcctt agagttcccg gcttaacccg ctggtaacta aggacaaggg ttgcgctcgt 360
tgcgggactt aacccaacat ctcacgacac gagctgacga cagccatgca gcacctgtat 420
tctaattccc gaaggcactc ccgcatctct gcaggattct agatatgtca agaccaggta 480
aggttcttcg cgttgcatcg aattaaacca catgctccac cgcttgtgcg ggcccccgtc 540
aattcatttg agttttaacc ttgcggccgt actccccagg cggtctactt attgcgttag 600
ctgcgtcact aagtcctcaa gggacccaac gactagtaga catcgtttac ggcgtggact 660
accagggtat ctaatcctgt ttgctaccca cgctttcgaa cctcagtgtc aatatgatgc 720
cagaaggctg ccttcgccat cggtattcct ccagatctct acgcatttca ccgctacacc 780
tggaattcta ccttcctctc acctattcta gcctaacagt atcagatgca gttcccaggt 840
taagcccggg gatttcacat ctgacttatc aagccaccta cgctcgcttt acgcccagta 900
attccgatta acgcttgcac cctctgtatt accgcggctg ctggcacaga gttagccggt 960
gcttattctg cagctaatgt catcgtccat gggtattaac catggagtct tcttcactgc 1020
ttaaagtgct ttacaaccaa aaggccttct tcacacacgc ggcatggctg gatcagggtt 1080
gcccccattg tccaatattc cccactgctg cctcccgtag gagtccgggc cgtgtctcag 1140
tcccggtgtg gctgatcatc ctctcagacc agctacagat cgtcgccatg gtaggccttt 1200
accccaccat ctagctaatc cgacttaggc tcatctaata gcgagagcaa caagttgccc 1260
cctttctccc gtaggtcgta tgcggtatta attcgagttt ccccgagcta tcccccacta 1320
ctaggtagat tcctaagtat tactcacccg tccgccgctc gacgcctgat agcaagctat 1380
catcgtttcc gctcgactgc 1400
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agagtttgat cctggctcag 20
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<213> Artificial Sequence (Artificial Sequence)
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tacggctacc ttgttacgac tt 22
Claims (3)
1. A psychrophile for producing bacteriocin (A)Psychrophile.sp) The preservation number is CCTCC NO: m2017480, wherein the nucleotide sequence of 16S rRNA of the psychrophile is shown as SEQ ID NO: 1 is shown.
2. Use of the psychrophile of claim 1 for the production of bacteriocins.
3. The use of a psychrophile according to claim 2 for producing a bacteriocin, wherein the bacteriocin is extracted by a process comprising:
and (3) bacterial culture: selecting a single bacterial colony of the strain, inoculating the single bacterial colony into a culture medium, carrying out shake culture at 37 ℃, centrifuging a culture solution, removing thalli, and keeping a supernatant;
acid precipitation: adjusting pH of the supernatant to 1.5-2.5 with concentrated hydrochloric acid, cooling, centrifuging, removing supernatant, and dissolving the precipitate with phosphate buffer solution to obtain crude bacteriocin.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773725A (en) * | 2014-01-21 | 2014-05-07 | 石河子大学 | Low-temperature resistant lactic acid bacterium for producing bacteriocin and application thereof |
| CN104531561A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | (Lactobacillus planetarium subsp. plantarum)Zhang-LL bacteriocin and Nisin composite bacteriostatic agent and its use in chilled meat |
| EP3081636A1 (en) * | 2015-04-15 | 2016-10-19 | University of Copenhagen | Use of lactic acid bacteria for bioprotection |
| CN107513511A (en) * | 2017-09-04 | 2017-12-26 | 国家海洋局第海洋研究所 | One plant of arctic pseudomonad with antifungal activity |
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2018
- 2018-01-16 CN CN201810040095.8A patent/CN108192841B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773725A (en) * | 2014-01-21 | 2014-05-07 | 石河子大学 | Low-temperature resistant lactic acid bacterium for producing bacteriocin and application thereof |
| CN104531561A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | (Lactobacillus planetarium subsp. plantarum)Zhang-LL bacteriocin and Nisin composite bacteriostatic agent and its use in chilled meat |
| EP3081636A1 (en) * | 2015-04-15 | 2016-10-19 | University of Copenhagen | Use of lactic acid bacteria for bioprotection |
| CN107513511A (en) * | 2017-09-04 | 2017-12-26 | 国家海洋局第海洋研究所 | One plant of arctic pseudomonad with antifungal activity |
Non-Patent Citations (4)
| Title |
|---|
| "Bacterium strain SLJ 16S ribosomal RNA gene, partial sequence，Accession Number：MF568072.1";Lu,Z. et al.;《Genbank》;20170802;第1页 * |
| "Complete genome sequence of Pseudomonas antarctica PAMC 27494, a bacteriocin-producing psychrophile isolated from Antarctica";Jaejin Lee et al.;《Journal of Biotechnology》;20170814;第259卷;第15-18页 * |
| "不同提取剂对粗细菌素提取效果的影响";许亦峰等;《微生物学杂志》;20130228;第33卷(第1期);摘要、第36页第1栏第1.2.1节 * |
| Lu,Z. et al.."Bacterium strain SLJ 16S ribosomal RNA gene, partial sequence，Accession Number：MF568072.1".《Genbank》.2017, * |
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