CN106754584A - For the Lactobacillus plantarum SWUN5815 of bacteriocinogeny high and its application - Google Patents
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Abstract
The invention discloses a kind of for the Lactobacillus plantarum SWUN5815 of bacteriocinogeny high and its application, the bacterial strain was preserved in China typical culture collection center (CCTCC) on 08 02nd, 2016, and preserving number is CCTCC NO:M2016419；The strain isolation is in yak excrement.The bacteriocin that the bacterial strain is produced can be easily degraded by proteases, and have very strong inhibitory action to salmonella and Escherichia coli, and the bacterial strain has good acidproof and bile tolerance ability acid resistance stronger, and survival rate is 93.36 ± 4.06% after 3h under the simulated gastric fluid of pH3.0；Can slowly be grown under 1.0% concentration cholate, growth efficiency reaches without cholate culture 15.26 ± 1.19%.The bacteriocin can effectively suppress salmonella, the growth of Escherichia coli and staphylococcus aureus, therefore, a kind of bacteriocinogeny Lactobacillus plantarum high of the invention has the potentiality that yak enteron aisle is applied to as probiotics.
Description
Technical field
The invention belongs to microbial technology field, specifically, it is related to a kind of Lactobacillus plantarum for bacteriocinogeny high
SWUN5815 and its application.
Background technology
Lactic acid bacteria is the superior microorganism flora in humans and animals enteron aisle, and probiotic lactic acid bacteria has regulation people in body
With animal body microecological balance, suppress and kill pathogenic bacteria, strengthen the effect of immunity of organisms.
Lactobacillus plantarum is a kind of extensively to utilize vegetalitas carbon in pickles, Yoghourt and the medium discovery of animal gastrointestinal tract
Source, tolerance cholate and low Bacillus acidi lactici.Some Lactobacillus plantarums can produce class bacteriocin, to coliform, salmonella, will
The pathogenic entero becterias such as Hayes bacterium have inhibitory action.The class bacteriocin that Lactobacillus plantarum is produced is made up of natural amino acid, after transcription
Do not modify, be heat-staple hydrophobic peptide, the synthesis of such bacteriocin is adjusted control by intervention school-based.
Common yak diarrhoea enteric pathogenic bacteria such as Escherichia coli, salmonella, campylobacter jejuni, proteus mirabilis
Deng because unreasonable use of antibiotic causes yak diarrhoea problem to be difficult to control to.Lactobacillus bacteria procatarxis its it is safe and nontoxic and
There is stronger bacteriostatic activity, the maximally effective substitute of antibiotic is generally considered so far.And have high-efficiency antimicrobial active,
Can be degraded by body, be paid close attention to the advantages of drug resistance is not produced and by people.Yak source bacteriocin-producing lactic acid bacteria is not only to height
Former environment and yak intestinal environment have stronger adaptive faculty, suppress yak enteric pathogenic bacteria and mitigate yak diarrhoea problem, may be used also
To promote Yak Growth development, regulation body to exempt from, promote intestinal growth and local resistance, also have ten in food security aspect
Divide important meaning.
The antimicrobial spectrum of most of lactic acid bacteria bacteriocinogeny is narrow in the prior art, can only individually suppress gram-positive bacteria or
Gram-negative bacteria；Due to species specificity, most of lactic acid bacteria does not adapt to the geographical environment of Qinghai-Tibet sternness, it is impossible to suitable
For yak.
The content of the invention
In view of this, the present invention is directed to above-mentioned problem, there is provided a kind of Lactobacillus plantarum for bacteriocinogeny high
SWUN5815 and its application.
In order to solve the above-mentioned technical problem, the invention discloses a kind of Lactobacillus plantarum for bacteriocinogeny high
SWUN5815, the bacterial strain was preserved in China typical culture collection center (CCTCC) on 08 02nd, 2016, protected
Tibetan number is CCTCC NO:M2016419.
Further, the morphological feature of the Lactobacillus plantarum is specially：
Thalline feature：In Gram-positive, cell is shaft-like, and about 0.5-1.0 μm of thalline is wide, and 1.5-4 μm is long, Cheng Dan,
Paired or chaining, does not form gemma, and two ends are circular；
Colony characteristicses：Obvious bacterium colony is formed on MRS culture mediums, diameter is circular between 0.5-2.0mm, and edge is whole
Together, milky, transparent, surface wettability is smooth, not chromogenesis.
Further, tolerance is characterized as in Lactobacillus plantarum body：
The bacterial strain acid resistance of Lactobacillus plantarum is stronger, after 3h under the simulated gastric fluid of pH3.0 survival rate be 92.46 ±
4.06%；Can slowly be grown under 1.0% concentration cholate, growth efficiency reaches without cholate culture 17.36 ± 1.19%.
The invention also discloses a kind of applications of Lactobacillus plantarum SWUN5815 in Lactobacillus plantarum bacteriocin is prepared.
Further, above-mentioned Lactobacillus plantarum SWUN5815 is inoculated in MRS culture mediums and is cultivated, obtain plant breast bar
Bacterium fermented liquid.
Further, the method for preparing Lactobacillus plantarum bacteriocin is specially：
1) bacterial strain SWUN5815 is preserved liquid to be inoculated into the MRS fluid nutrient mediums of 5mL, concussion is mixed, 37 DEG C of constant temperature trainings
24h is supported, three generations is continuously activated with 2% inoculum concentration, taken the zymotic fluid of the third generation and 10min is centrifuged with 4000 × g, take supernatant, on
Clear liquid is filtered through 0.22 μm of mocromembrane, and -20 DEG C of Refrigerator stores are standby, prepares thick Lactobacillus plantarum MRS fermented supernatant fluids；
2) the Lactobacillus plantarum MRS fermented supernatant fluids 500mL of exponential phase of growth, 4 DEG C, 12000 × g centrifugations after collecting
20min collect supernatant, with 0.22 μm of membrane filtration supernatant with remove remnants cell fragment；Add three chloroethenes of precooling
To final concentration of the 12.5% of trichloroacetic acid, -20 DEG C stand 8h and albumen is fully precipitated acid/acetone soln, 4 DEG C, 15000 × g
Centrifugation 20min collects albumen precipitation, to the 3-5 trichlorine to remove residual of pre- cold acetone cyclic washing that 80% is added in precipitation
Acetic acid, room temperature places 4-5min makes acetone volatilize completely, adds 100 μ L ultra-pure waters soluble proteins precipitation, prepares after purification
Lactobacillus plantarum MRS fermented supernatant fluids, as Lactobacillus plantarum bacteriocin.
Further, MRS medium components are as follows：Peptone 10.0g, beef extract 10.0g, dusty yeast 5g, glucose
20.0g, Tween 80 1.1g, dipotassium hydrogen phosphate 2.0g, sodium acetate 3.0g, lemon acid diamine 2.0g, magnesium sulfate 0.6g, manganese sulfate
0.25g and distilled water 1000mL.
Compared with prior art, the present invention can be obtained including following technique effect：
1) Lactobacillus plantarum of the present invention can simultaneously suppress gram-positive bacteria and negative bacterium；
2) Lactobacillus plantarum of the present invention contains various bacteria plain gene, including plnA, plnB, plnD, plnEF, plnJ,
PlnK, plnI, plnL and plnN；
3) Lactobacillus plantarum of the present invention adapts to the interior environment in the severe environments and yak body of Qinghai-Tibet Platean.
Certainly, implement any product of the invention to it is not absolutely required to while reaching all the above technique effect.
Brief description of the drawings
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes a part of the invention, this hair
Bright schematic description and description does not constitute inappropriate limitation of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 is influence of the different condition of the present invention to bacteriocin activity；Wherein, 1. Lactobacillus plantarum MRS fermented supernatant fluids
(fermenation raw liquid), 2. excludes the Lactobacillus plantarum MRS fermented supernatant fluids after acid, 3. the MRS broth bouillon (blank pair of identical PH
According to), 4. Proteinase K treatment；
Fig. 2 is influence of the hydrogen peroxide ferment treatment of the present invention to bacteriocin activity；Wherein, 1. Lactobacillus plantarum MRS fermentations
Supernatant (fermenation raw liquid), 2. Lactobacillus plantarum MRS fermented supernatant fluids after hydrogen peroxide ferment treatment；
Fig. 3 is SWUN5815 synthesis Related Bacteria plain gene PCR primer electrophoretogram of the present invention；Wherein, M.DNA standards
mark1；1.plnA；2.plnB；3.plnD；4.plnEF；5.plnJ；6.plnK.；7.plnI；8.plnL；9.plnN.
Specific embodiment
Describe embodiments of the present invention in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section can fully understand and implement according to this to solve technical problem and reach the implementation process of technology effect.
The separation of the bacterium of embodiment 1 and identification
First, the separation of bacterium
Weigh in 2g fresh yaks fecal specimens and 20ml sterilized waters, concussion is serially diluted after mixing with 10 times, takes respectively
Different dilution factors aseptically, in plate, plate, 37 DEG C of culture 48h are down flat with MRS (containing 0.5% calcium carbonate) culture medium.
Picking has molten calcium circle, and variform single bacterium colony carries out Gram's staining, chooses gram-positive bacteria, is inoculated in respectively
MRS fluid nutrient mediums, 37 DEG C of cultures will be screened the lactic acid bacteria for obtaining and be numbered after setting-out purification storage on MRS solid mediums
It is stand-by；
33 strains of lactic acid bacteria are filtered out from 90 strains of lactic acid bacteria using Odontothrips loti obvious inhibitory action to indicator strain,
Still having 9 strains of lactic acid bacteria after the influence for excluding acid has obvious inhibitory action to indicator bacteria, screens the wherein best suppression of inhibition
Lactic acid producing bacteria is named as SWUN5815.
2nd, molecular biology identification
(1) morphological feature, cultural character and physio-biochemical characteristics identification：
According to " the outstanding Bacteria Identification handbook of uncle " (the 8th edition) and " common bacteria system identification handbook " (east show pearl, Cai Miaoying
Etc. writing, Beijing：Science Press, 2001.2) described in method, morphological feature, culture are carried out to bacterial strain SWUN5815 special
Property, concrete outcome is as follows：
Thalline feature：In Gram-positive, cell is shaft-like, and about 0.5-1.0 μm of thalline is wide, and 1.5-4 μm is long, Cheng Dan,
Paired or chaining, does not form gemma, and two ends are circular.
Colony characteristicses：Obvious bacterium colony is formed on MRS culture mediums, diameter is circular between 0.5-2.0mm, and edge is whole
Together, milky, transparent, surface wettability is smooth, not chromogenesis.
The bacterial strain acid resistance of Lactobacillus plantarum is stronger, after 3h under the simulated gastric fluid of pH3.0 survival rate be 92.46 ±
4.06%；Can slowly be grown under 1.0% concentration cholate, growth efficiency reaches without cholate culture 17.36 ± 1.19%.
(2) 16S rDNA experiments
The strains tested of freezen protective is inoculated in MRS fluid nutrient mediums, 30 DEG C of incubated 24h, is passed on through MRS and trained
After supporting for 2~3 generations, the thalline culture for taking 2mL logarithmic growth latter stages is placed in centrifugation in aseptic EP pipes, and 3min is centrifuged through 8000 × g
(4 DEG C) collects thalline afterwards, abandons supernatant, and the genomic DNA of bacterial strain is extracted using the special CTAB freeze-thaw methods of lactic acid bacteria.Drawn using general
Thing, forward primer is 27f (corresponding to Escherichia coil 8-27 bit bases)：5′-AGAGTTTGATCCTGGCTCAG-
3 ' (nucleotide sequence is as shown in SEQ ID NO.28)；Reverse primer is：5 '-CTACGGCTACCTTGTTACGA-3 ' (nucleotides
Sequence is as shown in SEQ ID NO.29), it is amplification template PCR amplifications 16S rRNA gene regions, amplified production warp with thallus DNA
Carry out 16S rRNA gene sequencings after purification, the sequence for measuring as shown in SEQ ID No.1, then by gene order ratio
It is right.Compared according to Gen-Bank sequence homologies, bacterial strain SWUN5815 and Lactobacillus plantarum WCFS1
(GenBank accession number NC_004567.2) homology is 99%；Obtained strains show and Lactobacillus plantarum highest molecule
Affiliation in systematics, preliminary judgement bacterium is Lactobacillus plantarum；
Based on features above, bacterial strain SWUN5815 is named as Lactobacillus plantarum (Lactobacillus plantarum)
SWUN5815.The bacterial strain was preserved in China typical culture collection center (CCTCC) on 08 02nd, 2016, protected
Tibetan number is CCTCC NO:M2016419.
The bacteriostatic activity of the thalline of embodiment 2
1. Lactobacillus plantarum MRS fermented supernatant fluids are prepared：
Preservative fluid in cryopreservation tube is inoculated into the MRS fluid nutrient mediums of 5mL, concussion is mixed, and 37 DEG C incubated
24h, three generations is continuously activated with 2% inoculum concentration, takes the zymotic fluid of the third generation and 10min is centrifuged with 4000 × g, takes supernatant, supernatant
Liquid is filtered through 0.22 μm of mocromembrane, and -20 DEG C of Refrigerator stores are standby, prepares Lactobacillus plantarum MRS fermented supernatant fluids.
2. the exclusion of sour inhibitory action
Inhibition of the Lactobacillus plantarum MRS fermented supernatant fluids to indicator bacteria, it may be possible to the acid product of lactic acid bacteria secretion
Such as acetic acid, lactic acid effect.To remove the interference of acidic materials, Lactobacillus plantarum MRS fermented supernatant fluids pH is adjusted to from 4.0
6.0, the Lactobacillus plantarum MRS fermented supernatant fluids after excluding acid are prepared, and with the MRS broth bouillon conducts of identical pH
Blank.
Experimental result is shown in Fig. 1, and Lactobacillus plantarum MRS supernatants have inhibitory action to indicator bacteria, after eliminating the influence of acid,
Inhibition zone reduces.Illustrate that acid has certain inhibitory action to indicator bacteria, but exclude after acid still with the presence of antibacterial substance.
3. the exclusion of hydrogen peroxide effect
Catalase is dissolved in distilled water and is made into mother liquor, be added to the Lactobacillus plantarum MRS excluded through acid and ferment
The ultimate density of catalase is reached 1mg/mL in supernatant, taken out after 37 DEG C of water-bath 2h, detect hydrogen peroxide ferment treatment
The bacteriostatic activity of Lactobacillus plantarum MRS fermented supernatant fluids afterwards.
The interference of hydrogen peroxide is excluded using can the degrade characteristic of hydrogen peroxide of catalase.As shown in Fig. 2 through
Cross the Lactobacillus plantarum MRS fermented supernatant fluids and Lactobacillus plantarum MRS fermented supernatant fluids (fermenation raw liquid) of hydrogen peroxide ferment treatment
Do not reduced compared to its bacteriostatic activity, it is believed that bacteriostatic active ingredients are not hydrogen peroxide.
4. the sensitiveness of protease
From fig. 1, it can be seen that after fermented supernatant fluid is processed through Proteinase K, its bacteriostatic activity completely loses.Illustrate this antibacterial substance
It is higher to pepsin, Proteinase K and Trypsin sensitivities.Primarily determine that in fermented supernatant fluid there is protein-based bacteriostatic activity
Material.
The antimicrobial spectrum of embodiment 3 is determined
The SWUN5815 institutes bacteriocinogeny antimicrobial spectrum of table 1
Note：Antibacterial circle diameter (mm)：+++, 19~24；++, 15~18；+, 10~15；-, without fungistatic effect.
Choosing common yak source causes diarrhoea enteric pathogenic bacteria Bacterium enteritidis, salmonella typhimurium, Dublin husky
Door Salmonella, produce will congratulate Escherichia coli, as indicator bacteria, carry out Oxford cup bacteriostatic experiment with cell free fermentation supernatant.
SWUN5815 bacteriocin antimicrobial spectrums the results are shown in Table 1.Be can see from table, SWUN5815 institutes bacteriocinogeny has wide
Spectrum bacteriostatic activity, its cause of disease indicator bacteria separates and yak.In addition to there is good inhibiting effect with indicator bacteria to this research institute,
Also there is good inhibitory action to some common pathogenic microorganisms in Yak product.
Tolerance is characterized as in the lactobacillus fermenti body：
The bacterial strain acid resistance of lactobacillus fermenti is stronger, after 3h under the simulated gastric fluid of pH3.0 survival rate be 93.36 ±
4.06%；Can slowly be grown under 1.0% concentration cholate, growth efficiency reaches without cholate culture 15.26 ± 1.19%；
(1) probiotics tolerates the screening of pH3.0 simulated gastric fluids.
The preparation of simulated gastric fluid：To be used after sodium chloride (0.2g/100mL) and pepsin (0.35g/100mL) dissolving
1mol/L hydrochloric acid adjustment pH value be 3.0 after, in aseptic operating platform with vavuum pump suction filtration it is degerming rear standby.
Measure of the probiotics to artificial gastric juice resistance's property：The activated good strain cultured solutions of 5mL are taken, in aseptic operating platform
In pour into the 10mL centrifuge tubes that sterilized, 10min collects thallines are centrifuged through 3000r/min, add 5mL sterile salines to mix
Bacteria suspension is made, 1mL bacteria suspensions is taken and is mixed with the simulated gastric fluid of 9mL pH3.0, shaken up, be placed in and (37 are cultivated in constant temperature oscillator
DEG C, 300r/min), and pour into 37 DEG C of culture 48h with MRS agar mediums in 0h and 3h samplings respectively.Surveyed with colony counting method
Determine viable count, calculate its survival rate (%)：Viable count × 100% of the viable count/0h of survival rate (%)=3h.
(2) probiotics tolerates the measure of cholate.
The growth that strain of the survival rate more than 10% is done under different gallbladder salinities in pH3.0 simulated gastric fluids is selected to survey
Examination.The strain 5mL that will have been activated is inoculated in (i.e. empty containing 0.0% bovine bile respectively by 2% inoculum concentration (100 μ L) with liquid-transfering gun
In vain), 0.3% bovine bile, 0.5% bovine bile, l.0% bovine bile (W/V) MRS-THIO culture mediums (in MRS culture mediums plus
0.2% sodium thioglycolate).It is (nonvaccinated to compare with blank cultures after 37 DEG C of culture 24h in constant temperature oscillator
MRS-THIO culture mediums), the OD of above-mentioned various concentrations culture medium is determined respectively600nmValue, calculates tolerance of the bacterial strain to cholate.Courage
The OD of salt tolerance=culture medium containing cholate600nmThe OD of value/blank cultures600nmValue × 100%.
The Lactobacillus plantarum SWUN5815 acid and bile salt tolerance results of table 2
As shown in Table 2, bacterial strain SWUN5815 survival rates in the environment of pH3.0 are very high, there is stronger tolerance to acid.It is logical
Crossing the thalline survived after stomach will contact with the cholate in small intestine, and this experiment is used for lactic acid bacteria to the resistivity of cholate as diving
In a selection standard of probiotics.The result of the preferable SWUN5815 bacterial strains tolerance various concentrations cholate of acid resistance is right
0.3% cholate has certain tolerance, and when gallbladder salinity is increased to 0.5% and 1.0%, SWUN5815 is to cholate tolerance
Decline.
The method that embodiment 4 produces Lactobacillus plantarum bacteriocin
Preservative fluid in cryopreservation tube is inoculated into the MRS fluid nutrient mediums of 5mL, concussion is mixed, and 37 DEG C incubated
24h, three generations is continuously activated with 2% inoculum concentration, takes the zymotic fluid of the third generation and 10min is centrifuged with 4000 × g, takes supernatant, supernatant
Liquid is filtered through 0.22 μm of mocromembrane, and -20 DEG C of Refrigerator stores are standby, prepares thick Lactobacillus plantarum MRS fermented supernatant fluids.
The bacterium solution 500mL of exponential phase of growth after collection, 4 DEG C, 12000 × g centrifugations 20min collects supernatant, with 0.22 μm
Membrane filtration supernatant with remove remnants cell fragment；Trichloroacetic acid/the acetone soln of precooling is added to trichloroacetic acid
Final concentration of 12.5% (the trichloroacetic acid concentration can make most of albumen obtain preferable sedimentation effect), -20 DEG C of standing 8h
Albumen is set fully to precipitate, 4 DEG C, 15000 × g centrifugations 20min collects albumen precipitation, and 80% pre- cold acetone is anti-to being added in precipitation
The 3-5 trichloroacetic acid to remove residual is washed in after backwashing, and room temperature places 4-5min makes acetone volatilize completely, adds 100 μ L ultra-pure waters
Soluble protein is precipitated, and prepares Lactobacillus plantarum MRS fermented supernatant fluids after purification, as Lactobacillus plantarum bacteriocin.
Wherein, MRS medium components are as follows：Peptone 10.0g, beef extract 10.0g, dusty yeast 5g, glucose 20.0g,
Tween 80 1.1g, dipotassium hydrogen phosphate 2.0g, sodium acetate 3.0g, lemon acid diamine 2.0g, magnesium sulfate 0.6g, manganese sulfate 0.25g with
And distilled water 1000mL.
Embodiment 5PCR expands known plants lactobacillin gene
The PCR of table 3 expands known plants lactobacillin gene
In order to determine whether selected bacterial strain carries the gene for producing known Plantaricin by L. plantarum, PCR is used
Analysis uses the primer to single Plantaricin by L. plantarum gene specific.
By designing and synthesizing the specific primer that 11 pairs of bacteriocins synthesize and regulate and control related gene, with SWUN5815 thalline
Genomic DNA enters performing PCR amplification for masterplate, and agarose gel electrophoresis result is as shown in figure 3, wherein with plnA-F/R, plnB-F/
R, plnD-F/R, plnEF-F/R, plnJ-F/R, plnK-F/R, plnN-F/R, plnNC8-F/R, for the PCR primer of primer it is big
It is small identical with expected fragment length.
Described above has shown and described some preferred embodiments of invention, but as previously described, it should be understood that invention is not
Form disclosed herein is confined to, the exclusion to other embodiment is not to be taken as, and can be used for various other combinations, modification
And environment, and can be carried out by the technology or knowledge of above-mentioned teaching or association area in invention contemplated scope described herein
Change.And the change and change that those skilled in the art are carried out do not depart from the spirit and scope of invention, then all should be in the appended power of invention
In the protection domain that profit is required.
SEQUENCE LISTING
<110>Southwest University for Nationalities
<120>For the Lactobacillus plantarum Lactobacillus plantarum of bacteriocinogeny high and its application
<130> 2017
<160> 29
<170> PatentIn version 3.3
<210> 1
<211> 1455
<212> DNA
<213>Lactobacillus plantarum Lactobacillus plantarum
<400> 1
taacccttag gcggctggtt cctaaaaggt taccccaccg actttgggtg ttacaaactc 60
tcatggtgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg gcatgctgat 120
ccgcgattac tagcgattcc gacttcatgt aggcgagttg cagcctacaa tccgaactga 180
gaatggcttt aagagattag cttactctcg cgagttcgca actcgttgta ccatccattg 240
tagcacgtgt gtagcccagg tcataagggg catgatgatt tgacgtcatc cccaccttcc 300
tccggtttgt caccggcagt ctcaccagag tgcccaactt aatgctggca actgataata 360
agggttgcgc tcgttgcggg acttaaccca acatctcacg acacgagctg acgacaacca 420
tgcaccacct gtatccatgt ccccgaaggg aacgtctaat ctcttagatt tgcatagtat 480
gtcaagacct ggtaaggttc ttcgcgtagc ttcgaattaa accacatgct ccaccgcttg 540
tgcgggcccc cgtcaattcc tttgagtttc agccttgcgg ccgtactccc caggcggaat 600
gcttaatgcg ttagctgcag cactgaaggg cggaaaccct ccaacactta gcattcatcg 660
tttacggtat ggactaccag ggtatctaat cctgtttgct acccatactt tcgagcctca 720
gcgtcagtta cagaccagac agccgccttc gccactggtg ttcttccata tatctacgca 780
tttcaccgct acacatggag ttccactgtc ctcttctgca ctcaagtttc ccagtttccg 840
atgcacttct tcggttgagc cgaaggcttt cacatcagac ttaaaaaacc gcctgcgctc 900
gctttacgcc caataaatcc ggacaacgct tgccacctac gtattaccgc ggctgctggc 960
acgtagttag ccgtggcttt ctggttaaat accgtcaata cctgaacagt tactctcaga 1020
tatgttcttc tttaacaaca gagttttacg agccgaaacc cttcttcact cacgcggcgt 1080
tgctccatca gactttcgtc cattgtggaa gattccctac tgctgcctcc cgtaggagtt 1140
tgggccgtgt ctcagtccca atgtggccga ttaccctctc aggtcggcta cgtatcattg 1200
ccatggtgag ccgttacccc accatctagc taatacgccg cgggaccatc caaaagtgat 1260
agccgaagcc atctttcaag ctcggaccat gcggtccaag ttgttatgcg gtattagcat 1320
ctgtttccag gtgttatccc ccgcttctgg gcaggtttcc cacgtgttac tcaccagttc 1380
gccactcact caaatgtaaa tcatgatgca agcaccaatc aataccagag ttcgttcgac 1440
tgcatgtata gcatg 1455
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
gtacagtact aatgggag 18
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<211> 18
<212> DNA
<213>Artificial sequence
<400> 3
cttacgccaa tctatacg 18
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
ttcagagcaa gcctaaatga c 21
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence
<400> 5
gccactgtaa caccatgac 19
<210> 6
<211> 19
<212> DNA
<213>Artificial sequence
<400> 6
agcagatgaa attcggcag 19
<210> 7
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<213>Artificial sequence
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ataatccaac ggtgcaatcc 20
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<212> DNA
<213>Artificial sequence
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tgaggacaaa cagactggac 20
<210> 9
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<212> DNA
<213>Artificial sequence
<400> 9
gcatcggaaa aattgcggat ac 22
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<211> 21
<212> DNA
<213>Artificial sequence
<400> 10
ggcatagtta aaattccccc c 21
<210> 11
<211> 19
<212> DNA
<213>Artificial sequence
<400> 11
caggttgccg caaaaaaag 19
<210> 12
<211> 24
<212> DNA
<213>Artificial sequence
<400> 12
ctcgacggtg aaattaggtg taag 24
<210> 13
<211> 26
<212> DNA
<213>Artificial sequence
<400> 13
cgtttatcct atcctctaag cattgg 26
<210> 14
<211> 18
<212> DNA
<213>Artificial sequence
<400> 14
taacgacgga ttgctctg 18
<210> 15
<211> 24
<212> DNA
<213>Artificial sequence
<400> 15
aatcaaggaa ttatcacatt agtc 24
<210> 16
<211> 22
<212> DNA
<213>Artificial sequence
<400> 16
ctgtaagcat tgctaaccaa tc 22
<210> 17
<211> 18
<212> DNA
<213>Artificial sequence
<400> 17
actgctgacg ctgaaaag 18
<210> 18
<211> 22
<212> DNA
<213>Artificial sequence
<400> 18
tgcggttatc agtatgtcaa ag 22
<210> 19
<211> 19
<212> DNA
<213>Artificial sequence
<400> 19
cctcgaaaca atttccccc 19
<210> 20
<211> 20
<212> DNA
<213>Artificial sequence
<400> 20
ggtctgcgta taagcatcgc 20
<210> 21
<211> 19
<212> DNA
<213>Artificial sequence
<400> 21
cctaaaccat gccatgcac 19
<210> 22
<211> 20
<212> DNA
<213>Artificial sequence
<400> 22
ggtctgcgta taagcatcgc 20
<210> 23
<211> 29
<212> DNA
<213>Artificial sequence
<400> 23
aaattgaaca tatgggtgct ttaaattcc 29
<210> 24
<211> 20
<212> DNA
<213>Artificial sequence
<400> 24
gccttaccag cgtaatgccc 20
<210> 25
<211> 22
<212> DNA
<213>Artificial sequence
<400> 25
ctggtgatgc aatcgttagt tt 22
<210> 26
<211> 17
<212> DNA
<213>Artificial sequence
<400> 26
tcacacgaaa tattcca 17
<210> 27
<211> 23
<212> DNA
<213>Artificial sequence
<400> 27
ggcaagcgta agaaataaat gag 23
<210> 28
<211> 20
<212> DNA
<213>Artificial sequence
<400> 28
agagtttgat cctggctcag 20
<210> 29
<211> 20
<212> DNA
<213>Artificial sequence
<400> 29
ctacggctac cttgttacga 20
Claims (7)
1. a kind of Lactobacillus plantarum SWUN5815 for bacteriocinogeny high, it is characterised in that the bacterial strain is in 08 month 2016
China typical culture collection center (CCTCC) is preserved within 02nd, preserving number is CCTCC NO:M2016419.
2. the plant breast bar SWUN5815 for bacteriocinogeny high according to claim 1, it is characterised in that the plant
The morphological feature of lactobacillus is specially：
Thalline feature：In Gram-positive, cell is shaft-like, and about 0.5-1.0 μm of thalline is wide, and 1.5-4 μm is long, Cheng Dan, in pairs
Or chaining, gemma is not formed, two ends are circular；
Colony characteristicses：Obvious bacterium colony is formed on MRS culture mediums, diameter is circular between 0.5-2.0mm, neat in edge, breast
White, transparent, surface wettability is smooth, not chromogenesis.
3. the Lactobacillus plantarum SWUN5815 for bacteriocinogeny high according to claim 1, it is characterised in that the plant
Tolerance is characterized as in thing lactobacillus body：
The bacterial strain acid resistance of Lactobacillus plantarum is stronger, and survival rate is 92.46 ± 4.06% after 3h under the simulated gastric fluid of pH3.0；
Can slowly be grown under 1.0% concentration cholate, growth efficiency reaches without cholate culture 17.36 ± 1.19%.
4. applications of the Lactobacillus plantarum SWUN5815 described in claim 1 in Lactobacillus plantarum bacteriocin is prepared.
5. application according to claim 4, it is characterised in that comprise the following steps：By the plant breast described in claim 1
Bacillus SWUN5815 is cultivated in being inoculated in MRS culture mediums, obtains Lactobacillus plantarum fermented liquid.
6. application according to claim 5, it is characterised in that the method for preparing Lactobacillus plantarum bacteriocin is specially：
1) bacterial strain SWUN5815 is preserved liquid to be inoculated into the MRS fluid nutrient mediums of 5mL, concussion is mixed, and 37 DEG C incubated
24h, three generations is continuously activated with 2% inoculum concentration, takes the zymotic fluid of the third generation and 10min is centrifuged with 4000 × g, takes supernatant, supernatant
Liquid is filtered through 0.22 μm of mocromembrane, and -20 DEG C of Refrigerator stores are standby, prepares thick Lactobacillus plantarum MRS fermented supernatant fluids；
2) the Lactobacillus plantarum MRS fermented supernatant fluids 500mL of exponential phase of growth after collecting, 4 DEG C, 12000 × g centrifugations 20min is received
Collection supernatant, with 0.22 μm of membrane filtration supernatant with remove remnants cell fragment；Add the trichloroacetic acid/acetone of precooling
To final concentration of the 12.5% of trichloroacetic acid, -20 DEG C stand 8h and albumen is fully precipitated solution, 4 DEG C, 15000 × g centrifugations
20min collects albumen precipitation, to 3-5 three chloroethenes to remove residual of pre- cold acetone cyclic washing that 80% is added in precipitation
Acid, room temperature places 4-5min makes acetone volatilize completely, adds 100 μ L ultra-pure waters soluble proteins precipitation, prepares after purification
Lactobacillus plantarum MRS fermented supernatant fluids, as Lactobacillus plantarum bacteriocin.
7. application according to claim 5, it is characterised in that MRS medium components are as follows：Peptone 10.0g, beef extract
10.0g, dusty yeast 5g, glucose 20.0g, Tween 80 1.1g, dipotassium hydrogen phosphate 2.0g, sodium acetate 3.0g, lemon acid diamine
2.0g, magnesium sulfate 0.6g, manganese sulfate 0.25g and distilled water 1000mL.
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| CN109234181A (en) * | 2017-12-22 | 2019-01-18 | 浙江大学 | Lactobacillus plantarum ZJUF HN9 and its application |
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| CN111979153A (en) * | 2020-08-26 | 2020-11-24 | 北京中农创研生物科技有限公司 | Lactobacillus plantarum E-1 and application thereof |
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| CN109234182A (en) * | 2017-12-22 | 2019-01-18 | 浙江大学 | Lactobacillus plantarum ZJUF T34 and its application |
| CN109234181A (en) * | 2017-12-22 | 2019-01-18 | 浙江大学 | Lactobacillus plantarum ZJUF HN9 and its application |
| CN109234181B (en) * | 2017-12-22 | 2020-06-09 | 浙江大学 | Lactobacillus plantarum ZJUF HN9 and application thereof |
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| CN110205267A (en) * | 2019-05-31 | 2019-09-06 | 上海应用技术大学 | The lactobacillus plantarum of one plant of bacteriocinogeny and its application |
| CN111979153A (en) * | 2020-08-26 | 2020-11-24 | 北京中农创研生物科技有限公司 | Lactobacillus plantarum E-1 and application thereof |
| CN111979153B (en) * | 2020-08-26 | 2022-05-10 | 北京中农创研生物科技有限公司 | Lactobacillus plantarum E-1 and application thereof |
| CN112779188A (en) * | 2021-01-19 | 2021-05-11 | 自然资源部第二海洋研究所 | Medium-temperature bacterium for producing high-salinity tolerance ester hydrolase and application thereof |
| CN112779188B (en) * | 2021-01-19 | 2022-06-17 | 自然资源部第二海洋研究所 | Medium-temperature bacterium for producing high-salinity tolerance ester hydrolase and application thereof |
| CN113249252A (en) * | 2021-05-10 | 2021-08-13 | 合肥工业大学 | Method for preventing harmful bacteria and biofilm formation on surface of stainless steel product |
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