CN107937319A - The bacillus subtilis strain SX3411 of one plant of production antibacterial peptide - Google Patents
The bacillus subtilis strain SX3411 of one plant of production antibacterial peptide Download PDFInfo
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- CN107937319A CN107937319A CN201810000464.0A CN201810000464A CN107937319A CN 107937319 A CN107937319 A CN 107937319A CN 201810000464 A CN201810000464 A CN 201810000464A CN 107937319 A CN107937319 A CN 107937319A
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- bacillus subtilis
- antibacterial peptide
- bacteriostatic activity
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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Abstract
The invention belongs to feed additive field, and in particular to the bacillus subtilis of one plant of production antibacterial peptide is separated near a pig farm（Bacillus subtilis）SX3411, the culture presevation is in China General Microbiological culture presevation administrative center, culture presevation CGMCC NO.14396.After the zymotic fluid of the bacillus subtilis SX3411 is centrifuged by ultrafiltration, it, which retains liquid, preferable bacteriostatic activity, and composition is mainly peptide material, and shows that Substance is mainly polypeptides matter substantially without bacteriostatic activity in filtrate, i.e. antibacterial peptide.The bacterial strain produces antibacterial peptide still has bacteriostatic activity under 80 DEG C of high temperature and in the range of the soda acid of pH2 11, shows that the bacterial strain is adapted as beneficial bacteria of intestinal tract and is used in feed addictive, or probiotics is made and is used.
Description
Technical field
The present invention relates to feed addictive --- a kind of microbial preparation field, and in particular to withered grass gemma for producing antibacterial peptide
Bacillus strain SX3411.
Background technology
34 are defined in No. 2045 bulletin catalogue of feed additive varieties of The Ministry of Agriculture of the People's Republic of China, MOA in 2013
Strain can be used as animal microorganism feed and feed addictive, wherein just containing bacillus subtilis.
Bacillus subtilis is more many by force than the bacterium of other classes to the tolerance degree of high temperature under normal circumstances, therefore very suitable
Together in high temperature granulating feed applications；Bacillus subtilis can produce and secrete a variety of materials beneficial to animal intestinal tract, including disappear
Change enzyme, B family vitamin and a variety of antibacterial substances.In addition, bacillus subtilis stimulating animal enteron aisle, activating immune system, carries
The high resistivity to disease.Data show that bacillus subtilis can have preferable acidproof with the attack of resistance to saliva and bile
Alkali characteristic, can keep activity in acidic stomach environment, be the viable bacteria of intestine and small intestine of can going directly in microorganism.
Antibacterial peptide is usually made of 12~100 amino acid residues, more containing hydrophobic amino acid, majority be it is amphipathic sun from
Sub- peptide.Natural antibacterial peptide has a broad antifungal spectrum, has heat endurance and preferable water solubility, almost non-toxic to higher mammal normal cell
Evil effect, and it is not likely to produce drug resistance.Research to different plant species antibacterial peptide becomes hot spot, and antibacterial peptide is possible as new one
The antibacterials in generation substitute conventional antibiotic.
Antibacterial peptide from bacterium is also referred to as bacteriocin（bacteriocin）.Bacteriocin is in generation by some bacteriums
The one kind produced during thanking by Ribosome biogenesis mechanism has the polypeptide or Precursor Peptide of bacteriostatic activity, and usual bacteriocin is all
It is some cationic peptides, there is hydrophobicity or amphipathic.Bacteriocin is divided into 4 classes according to their biochemical property：I class is thin
Rhzomorph, is referred to as lantibiotic, and molecular mass is less than 5 kDa, is made of 19~38 amino acid, active and resistance to mucous membrane
Hot, its molecule contains rare thioether amino acid, such as lanthionine（lanthionine）With Beta-methyl lanthionine
( β-methyllanthionine).The molecular mass of II class bacteriocins is smaller (4~6 kDa), has heat endurance, again
Three groups can be divided into：（1）IIa classes（bacteriocin IIa）For Listeria active peptides（Listeria-active
peptides）, in N-terminal band consensus-Y-G-N-G-V-X-C-；（2）IIb classes（bacteriocin IIb）For dipeptides bacterium
Element（two-peptide bacteriocins）, its antibacterial activity two compositions of requirement；（3）IIc classes（bacteriocin
IIc）For sulphydryl activity peptide, Active pharmaceutical has reproducibility cysteine residues.III class bacteriocin molecular mass is more than 30 kDa,
For thermally labile protein.IV class bacteriocins are complex polypeptides, and Active pharmaceutical includes lipid or carbohydrate group.
The content of the invention
The technical problem to be solved in the present invention is：A kind of production antibacterial peptide bacillus subtilis strain SX3411, its energy are provided
Effectively suppress gram-positive bacteria and Gram-negative bacteria under the conditions of high temperature and pH2-11, improve animal intestinal tract health.For solution
Certainly above-mentioned technical problem, the technical solution taken are as follows：
Production antibacterial peptide bacterial strain provided by the invention is specially bacillus subtilis（Bacillus subtilis）SX3411, the bacterium
Kind is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, culture presevation CGMCC NO.14396.
The bacillus subtilis strain（Bacillus subtilis）The colonial morphology of SX3411 is：Bacterium colony surface is thick
Rough irregular and edge is irregular, and bacterium colony is opaque, and canescence is presented or shows slightly yellow.
The bacillus subtilis strain SX3411 screened from one pig farm of Nanchang City, Jiangxi Province Ma Qiu towns near, can produce anti-
Bacterium peptide, has the characteristic for suppressing gram-positive bacteria and Gram-negative bacteria.
The bacillus subtilis strain SX3411 after fermented and cultured, its zymotic fluid by super filter tube ultrafiltration with
Afterwards, the reservation liquid obtained has a good bacteriostatic activity, and the few bacteriostatic activities of filtrate that ultrafiltration is later.This shows its suppression
Fungus matter is not small-molecule substance, and mainly polypeptides matter.
The bacillus subtilis strain SX3411, under conditions of discontinuous culture, starts when the 18-20 of culture is small
Antibacterial peptide is produced, its zymotic fluid has bacteriostatic activity.
Antibacterial peptide caused by the bacillus subtilis strain SX3411 can have preferably in the range of pH2-11
Bacteriostatic activity, show that antibacterial peptide produced by bacillus subtilis strain SX3411 has the characteristic for well adapting to soda acid, be adapted to
Bacteriostasis is played in enteron aisle.
Antibacterial peptide caused by the bacillus subtilis strain SX3411 has the characteristic for well adapting to high temperature, Ke Yi
Still there is bacteriostatic activity under 80 DEG C of high temperature.
Beneficial effect：Characteristic possessed by bacillus subtilis strain SX3411 provided by the invention is highly suitable for moving
The improvement of thing intestinal health.Its viable bacteria has resistance high temperature, suitable for the processing of feed；It produces the bacteriostatic activity of antibacterial peptide can be with
It is still active when temperature reaches 80 DEG C of high temperature, suitable for industrialized production and processing；It, which produces antibacterial peptide, has preferable soda acid
Accommodation, can be still active in the case of strong acid, suitable for being played function in enteron aisle, significantly improves nutriment in enteron aisle
In digest and assimilate.
Brief description of the drawings
The Phylogenetic analysis result of Fig. 1 bacterial strain SX3411 16S rDNA sequences
Bacteriostatic test of Fig. 2 SX3411 bacterial strain fermentation liquors after ultrafiltration（Note：8 mm of filter paper diameter）
Fig. 3 SX3411 bacterial strain fermentation liquors retain the SDS-PAGE analyses of liquid after ultrafiltration
The bacteriostatic activity of Fig. 4 SX3411 bacterial strain fermentation liquors（Note：8 mm of filter paper diameter）
Influence of Fig. 5 pH value to antibacterial substance bacteriostatic activity in bacterial strain SX3411 zymotic fluids（Note：8 mm of filter paper diameter）
Influence of Fig. 6 pH value to antibacterial substance bacteriostatic activity in bacterial strain SX3411 zymotic fluids（Note：8 mm of filter paper diameter）.
Embodiment
Embodiment 1
The screening and identification of bacterial strain：
Step 1. sample collection is near the slurry gutte on several family pig farms periphery in Nanchang City, Jiangxi Province Ma Qiu towns, collection
Point includes pig manure, neighbouring soil and water body, collects each sample with the centrifuge tube of 10 mL, gathers 30~50 samples every time, point
Gather three times.
After step 2. sample collection, sterile water suspended sample is added in the centrifuge tube of each dress sample, is existed immediately
10 min of sample is handled in 100 DEG C of boiling water, each sample of after treatment takes 100uL to be coated on LB solid mediums, 37 DEG C
24 h are cultivated, collect possible Bacillus colonies, are for scalping.
The bacterium colony of step 3. scalping is seeded in the centrifuge tube of 1.5 mL with 0.5 mL LB fluid nutrient mediums respectively,
37 DEG C of 24~30 h of culture, are then collected by centrifugation supernatant, filter paper method dips supernatant with the filter paper of fixed size, with golden yellow
Color staphylococcus is antibacterial object, and screening has the bacterial strain of bacteriostatic activity, obtains the bacterial strain SX3411 of one plant of tool bacteriostatic activity.
The method that the identification of step 4. strain is sequenced using 16S rDNA.After PCR amplification, the 16S rDNA sequences of acquisition
Arrange and upload GenBank databases, its sequence number（Accession No）For KY848340.1, BLAST ratios are carried out in NCBI
It is right（See Fig. 1）, it is bacillus subtilis to determine strain, i.e.,Bacillus subtilis SX3411。
Embodiment 2
Antibacterial peptide in ultrafiltration crude separation zymotic fluid
The ultra-filtration centrifuge tube bought in 3 kD of Millipore companies of the U.S. is placed in 0.2mo1/L NaOH solution by step 1.
In, 30 more than min are soaked, is taken out, is soaked in 20% alcohol and preserves.
After step 2 aseptic double-distilled water cleaning super filter tube, 500 μ L fermented supernatant fluids are taken in 3 KD super filter tubes, 5,000
- 10,000 r/min is centrifuged, and middle centrifugal speed incrementally increases, and it is about 40 μ L that liquid product is retained in final super filter tube, is received
Collection retains liquid and filtrate.
Step 3. respectively takes the reservation liquid of 20 μ L and filtrate to carry out disc diffusion method and do bacteriostatic activity test（Filter paper is straight
8 mm of footpath）.Using staphylococcus aureus as object bacteria, the results showed that liquid of only withing a hook at the end is there are preferable bacteriostatic activity, and filtrate
Without bacteriostatic activity（See Fig. 2）.This shows the material of small molecule without bacteriostatic activity, and play bacteriostatic activity is mainly polypeptides matter.
Ultrafiltration is centrifuged later reservation liquid and carries out SDS-PAGE electrophoretic analysis by step 4., tentatively judges that antibacterial material is
Antibacterial peptide, its molecular weight is smaller, about 5 kDa sizes（See Fig. 3）.
Embodiment 3
Bacteriostatic activity of the SX3411 bacterial strain fermentation liquors antibacterial substance to bacterium
The activation of step 1. SX3411 bacterial strains
The SX3411 strains of -70 DEG C of preservations take out to be diluted with fresh LB fluid nutrient mediums, and LB solid cultures are seeded to oese
Base, 37 DEG C of constant incubator culture 12h.Picking single bacterium drops down onto fresh LB culture mediums, and 37 DEG C carry out in 200 rpm/min of shaking table
Expand culture.
The activation of step 2. indicator strain
The gram-positive bacteria used：Staphylococcus aureus（Staphylococcus aureus）, bacillus subtilis
（Bacillus subtilis）（Note：Non- bacterium）And Streptococcus suis（Streptococcus suis）；Gram-negative
Strain：Salmonella (SalmonellaSP), Escherichia coli (Escherichia coli) and thermophilic aqueous vapor pseudomonas bacillus
(Aeromonas.hydrophila).These bacterium are used as the bacteriostatic activity of indicator strain measure SX3411 zymotic fluids.
Respectively by above-mentioned bacterial strains after dilution, LB solid mediums, 37 DEG C of constant incubator trainings are seeded to using oese
Support 12 h.Picking single bacterium drops down onto fresh LB culture mediums, and 37 DEG C expand culture in 200 rpm/min of shaking table progress, and use is ultraviolet
Spectrophotometer measurement bacterium solutionOD 600, culture is extremelyOD 600=1 or so, stop culture.
The preparation of step 3.SX3411 bacterial strain fermentation liquors
10 000r/min of bacillus subtilis SX3411 zymotic fluids centrifuges 15min, abandons thalline and takes supernatant, that is, obtains antibacterial
Matter crude extract.
Step 4. indicates the preparation of bacteria suspension
It will cultivate extremelyOD 600Indicator bacteria when=1 carries out gradient dilution with the LB fluid nutrient mediums of fresh sterilizing respectively, obtains 10-1、10-2、10-3、10-4Instruction bacteria suspension.
Step 5. rubbing method prepares antibacterial tablet
LB solid mediums are prepared, 121 DEG C of 20 min of sterilizing, when being cooled to about 55 DEG C or so, each culture dish is toppled over
About 30 mL；After solidification, 100 uL instruction bacteria suspension even spreads in tablet are added, bacterium solution can not be excessive, with nothing on tablet
Liquid trickling is advisable, 37 DEG C of 10 min of culture.
The bacteriostatic activity of the antibacterial substance of step 6. filter paper enzyme measure SX3411 bacterial strain fermentation liquors
Using sterile fresh LB fluid nutrient mediums as control, 2 layers of filter paper are gently sticked to the antibacterial tablet prepared
On, press lightly on.Take the zymotic fluid of 25 μ L SX3411 bacterial strains slowly to drip to the circular filter paper on piece of a diameter of 8 mm, press
According to being tested clockwise.After adding sample, being placed on 4 DEG C of 4 more than h of refrigerator makes zymotic fluid abundant on agarose plate
Diffusion, be then placed within 37 DEG C of 12 h of constant incubator culture or so, periodically check inhibition zone occur situation, make a record and
Measure antibacterial circle diameter.After 60 h of fermented and cultured, the bacteriostatic activity of SX3411 bacterial strain fermentation liquors is embodied.Study table
Bright, SX3411 bacterial strain fermentation liquors can inhibit all gram-positive bacterias, i.e. staphylococcus aureus, bacillus subtilis and pig
Streptococcus, bacteriostatic activity is begun with 18 h of fermentation, has best bacteriostatic activity in 36-42 h（See Fig. 4）；It is and blue to leather
Family name's negative bacterium Salmonella and Escherichia coli have preferable bacteriostatic activity to thermophilic aqueous vapor pseudomonas bacillus, are sending out without bacteriostatic activity
Bacteriostatic activity is begun with during 16 h of ferment, there is highest bacteriostatic activity in 42 h（See Fig. 4）.
Embodiment 4
Bacterial strain SX3411 zymotic fluid antibacterial substance analysis of physical and chemical property
Step 1. selects the research of zymotic fluid progress physicochemical properties of the bacterial strain SX3411 under most suitable fermentation time, with golden yellow
Staphylococcus is indicator strain, and the fermented and cultured of bacterial strain SX3411 is carried out according to the experimental procedure in embodiment 3, is obtained with antibacterial
The zymotic fluid of material.
Influence of step 2. pH value to antibacterial substance bacteriostatic activity in bacterial strain SX3411 zymotic fluids
SX3411 bacterial strain fermentation liquors obtained above are taken respectively, respectively with 1mol/L HCl solution and 1mol/L NaOH solution
Adjust to pH=2, pH=4, pH=5, pH=7, pH=8, pH=9, pH=10, pH=11, pH=12 and bacterial strain fermentation liquor without processing and do sun
Property control (CK ﹢), handle 1 h at 25 DEG C.After treatment is blank control with the fresh LB fluid nutrient mediums of sterilizing
(CK ﹣), detects the different lower zymotic fluids of pH processing and suppresses staphylococcus aureus activity.The result shows that in the range of pH2~11,
The antibacterial composition of SX3411 bacterial strain fermentation liquors still has bacteriostatic activity, and stablizes（See Fig. 5）.
Influence of step 3. temperature to antibacterial substance bacteriostatic activity in bacterial strain SX3411 zymotic fluids
SX3411 bacterial strain fermentation liquors obtained above are taken respectively, respectively handle 30 min at 40 DEG C, 60 DEG C, 80 DEG C, 100 DEG C respectively
With 1 h, after treatment is with untreated zymotic fluid（CK ﹢）With the fresh LB fluid nutrient mediums (CK ﹣) that sterilize for control, inspection
Survey the influence to zymotic fluid activity under treatment of different temperature.The result shows that when handling 30 min, the suppression of bacterial strain SX3411 zymotic fluids
The bacteriostatic activity of fungus matter in the range of 40~80 DEG C of detection, decay slow by bacteriostatic activity（See Fig. 6）；And handle 60 min
When, its bacteriostatic activity is raised and decayed with temperature.The SX3411 bacterial strain fermentation liquors of both processing times are after 80 DEG C of processing
Still there is bacteriostatic activity（See Fig. 6）.
Sequence table
<110>Jiangxi Tian Jia bioengineering limited company
Jiangxi Normal University
<120>The bacillus subtilis strain SX3411 of one plant of production antibacterial peptide
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1434
<212> DNA
<213>Bacillus subtilis (Bacillus subtilis)
<400> 1
aatacatgca agtcgagcgg acagatggga gcttgctccc tgatgttagc ggcggacggg 60
tgagtaacac gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggggctaa 120
taccggatgg ttgtttgaac cgcatggttc aaacataaaa ggtggcttcg gctaccactt 180
acagatggac ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca aggcaacgat 240
gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct 300
acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc 360
gtgagtgatg aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtaccgtt 420
cgaatagggc ggtaccttga cggtacctaa ccagaargcc acgggctaac tacgtgccag 480
gcagccgcgg taataacgta ggtggcaaag cgttgtccgg aaattattgg gcgtaaaggg 540
ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg gagggtcatt 600
ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta gcggtgaaat 660
gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt aactgacgct 720
gaggagcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca cgccgtaaac 780
gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaac gcattaagca 840
ctccgcctgg ggagtacggt cgcaagactg aaactcaaag ggawttgacg ggggcccgca 900
caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaacctttac caggtcttga 960
catcctctga caatcctaga gataggacgt ccccttcsgg ggcagagtga caggtggtgc 1020
atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080
ttgatcttag ttgccagcat tcagttgggc actctaaggt gactgccggt gacaaaccgg 1140
aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct 1200
acaatggaca gaacaaaggg cagcgaaacc gcgaggttaa gccaatccca caaatctgtt 1260
ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagc tggaatcgct agtaatcgcg 1320
gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg 1380
agagtttgta acacccgaag tcggtgaggt aacctttagg agccagccgc cgaa 1434
Claims (3)
1. one plant of production antibacterial peptide bacillus subtilis strain, it is characterised in that：The bacterial strain is that bacillus subtilis isBacillus subtilisSX3411, culture presevation is in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart, culture presevation CGMCC NO.14396.
2. one plant of production antibacterial peptide bacillus subtilis strain as claimed in claim 1, it is characterised in that：Suppression in its zymotic fluid
Bacterium composition is mainly polypeptide or protein substance, i.e. antibacterial peptide or bacteriocin.
3. one plant of production antibacterial peptide bacillus subtilis strain as claimed in claim 1 is as feed addictive in animal feed
Application.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949872A (en) * | 2018-08-08 | 2018-12-07 | 江西天佳生物工程股份有限公司 | A kind of method of a small amount of fermenting and producing Lantibiotics Subtilomycin |
| CN113151057A (en) * | 2021-03-15 | 2021-07-23 | 中科康源(唐山)生物技术有限公司 | Bacillus subtilis |
| CN113215060A (en) * | 2021-06-09 | 2021-08-06 | 江西农业大学 | Disease-resistant probiotic bacillus for pigs and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949872A (en) * | 2018-08-08 | 2018-12-07 | 江西天佳生物工程股份有限公司 | A kind of method of a small amount of fermenting and producing Lantibiotics Subtilomycin |
| CN113151057A (en) * | 2021-03-15 | 2021-07-23 | 中科康源(唐山)生物技术有限公司 | Bacillus subtilis |
| CN113151057B (en) * | 2021-03-15 | 2024-03-12 | 中科康源(唐山)生物技术有限公司 | High-yield strain of antibacterial peptide and application thereof |
| CN113215060A (en) * | 2021-06-09 | 2021-08-06 | 江西农业大学 | Disease-resistant probiotic bacillus for pigs and application thereof |
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