CN104232520A - Preparation method and application of lactobacillus plantarum and bacteriocin of lactobacillus plantarum - Google Patents
Preparation method and application of lactobacillus plantarum and bacteriocin of lactobacillus plantarum Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms and discloses lactobacillus plantarum, bacteriocin which is generated by lactobacillus plantarum as well as a preparation method and application of lactobacillus plantarum and bacteriocin. The strain is preserved in CGMCC on 29th, March, 2013 with the preservation number of CGMCCNO.7390 and the preservation address of Institute of Microbiology, CAS, #3, Yard 1, West Beichen Road, Chaoyang District, Beijing. The new strain of lactobacillus plantarum and the bacteriocin which is generated by lactobacillus plantarum are wide in antimicrobial spectrum and high in antimicrobial activity and can be used for inhibiting various food-borne pathogenic bacteria and Gram-positive and Gram-negative bacteria inducing food spoilage. The method for preparing bacteriocin by using lactobacillus plantarum is simple and low in cost.
Description
Technical field
The present invention relates to microbial technology field, particularly relate to a lactobacillus plantarum and bacteriocin of producing thereof and preparation method thereof and application.
Background technology
The source of bacteriocin mainly bacterium in specific pathways metabolism, the protein produced by rrna, polypeptide or protein mixture.Bacteriostatic activity is only limitted to the bacterium suppressing sibship nearer, but it does not possess any inhibit activities to producing strains.Although bacteriocin is the antiseptic-germicide suppressing the food pathogens such as Salmonellas (salmonella), differ greatly with conventional antibiotic; Traditional peptide antibiotics, its main source is formed through the catalysis of cell multienzyme complex, and not by specific genes encoding, but bacteriocin is then coded by concrete structure gene, therefore can utilize molecular biology method transformation.The antibiosis of the synthesis of these bacteriocins and binding mode and Clinical practice have a great difference.
Bacteriocin generally defines: a kind of protein antimicrobial substance acting on other bacterial strain of of the same race with producing strains or that sibship is nearer kind by bacteriogenic usual.It is the mixture of a peptide species or polypeptide and sugar and fat, but along with the discovery of more broad-spectrum bacteriocins, makes its concept not only just be confined to polypeptide or polypeptide composite.The kind of bacteriocin is a lot, and the mechanism of action is also diversified.Bacteriocin has security, not easily produces the advantage that other sanitass such as resistance, thermostability cannot reach, and therefore researches and develops novel broad-spectrum bacteriocins significant as foodstuff additive.
Still phenylformic acid and salt, Sorbic Acid and the chemosynthesis such as salt, parabens sanitas thereof that current China uses in a large number.But research finds, this class sanitas has certain toxic action to human body, long-term eating has disadvantageous effect to health.Therefore, Recent study person invests sight the exploitation of natural antiseptic agent.Relative to synthetic preservative, sphere of action is wide because having for natural antiseptic agent, the feature of nontoxicity, strong germ resistance.Through studying intensively for many years, microbialpreservatives has developed the safe sanitass such as streptococcus acidi lactici bacteriocin (Nisin), N,O-Diacetylmuramidase.Bacteriocin, meets the future developing trend of food preservatives because himself is safe and efficient, become now one of field of food science research direction burning the hotest already.But in actual application, also there is a lot of problem.The existing cost acquisition source that is very high, bacteriocin of such as preparing bacteriocin is not enough, not deep enough for the antimicrobial spectrum research of bacteriocin self, the mechanism of action of bacteriocin is clear and definite not, and above problem will the development and utilization of restricting bacterial element significantly.
Summary of the invention
The object of this invention is to provide a lactobacillus plantarum new strains and institute bacteriocinogeny thereof and preparation method, this plant lactobacillus new strains and institute bacteriocinogeny thereof have that antimicrobial spectrum is wide, bacteriostatic activity is high, can suppress multiple food-borne pathogens and cause Gram-positive and the gram negative bacterium of food spoilage.
Another object of the present invention is to provide bacteriocin prepared by described plant lactobacillus and preparation method thereof and application.
In order to solve the problems of the technologies described above, the present invention is solved by following technical proposals:
One lactobacillus plantarum (
lactobacillus plantarum) ZJ5, it is characterized in that: this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 29th, 2013, its preserving number is CGMCC NO.7390, and preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
As preferably, described plant lactobacillus (
lactobacillus plantarum) ZJ5 can be used for preparing bacteriocin PZJ5.
As preferably, described plant lactobacillus (
lactobacillus plantarum) ZJ5 can be used for preparing food or feed anticorrosion agent.
As preferably, described plant lactobacillus (
lactobacillus plantarum) ZJ5 prepares the method for bacteriocin PZJ5, comprise the steps:
Prepare fermented liquid: by plant lactobacillus (
lactobacillus plantarum) ZJ5 is inoculated in MRS liquid nutrient medium by the inoculum size of 3%, 30 DEG C of quiescent culture 24 h; The fermented liquid obtained is at 10000 rpm, and under the condition of 4 DEG C, centrifugal 15 min, get supernatant liquor, places 4 DEG C and saves backup;
Prepare bacteriocin: get supernatant liquor by after centrifugal 15 min of fermentation liquor 10000 r/ m of preparation, in supernatant liquor, slowly add ammonium sulfate solids powder saltout, limit edged stirs, until saturation ratio reaches 40, then under 4-5 DEG C of condition, 2 h are left standstill, again under 4-5 DEG C of condition, 12000 r/ m, centrifugal 30 min, discard precipitation.In supernatant liquor, slowly add ammonium sulfate solids powder again saltout, limit edged stir, until saturation ratio reaches 80, then under 4-5 DEG C of condition leave standstill 12 h, then under 4-5 DEG C of condition, 12000 r/ m, centrifugal 30 min, abandoning supernatant obtains bacteriocin; Bacteriocin PZJ5 is obtained finally by after cation-exchange chromatography, hydrophobic chromatography and molecular sieving.
The present invention, owing to have employed above technical scheme, has significant technique effect:
Plant lactobacillus new strains of the present invention and institute bacteriocinogeny thereof have that antimicrobial spectrum is wide, bacteriostatic activity is high, multiple food-borne pathogens can be suppressed and cause Gram-positive and the gram negative bacterium of food spoilage, this plant lactobacillus is utilized to prepare bacteriocin method simple, with low cost.
Accompanying drawing explanation
Fig. 1 is colonial morphology and the thalli morphology figure of plant lactobacillus ZJ5 bacterial strain of the present invention; Wherein, A: colonial morphology; B: thalli morphology;
Fig. 2 is plant lactobacillus pcr amplification result figure of the present invention; M:Marker; 1:CK; 2:PZJ5;
Fig. 3 is that the 16s rDNA phylogenetic analysis of plant lactobacillus ZJ5 of the present invention and other milk-acid bacterias illustrates;
Fig. 4 is SP-Sepharose purifying PZJ5 color atlas of the present invention;
Fig. 5 is Resource ETH purifying PZJ5 color atlas of the present invention;
Fig. 6 is Superdex PP purifying PZJ5 color atlas of the present invention;
Fig. 7 is that the N of PZJ5 of the present invention holds sequencing result.
Embodiment
Below in conjunction with accompanying drawing 1 to accompanying drawing 7 and embodiment, the present invention is described in further detail:
embodiment 1
One lactobacillus plantarum (
lactobacillus plantarum) ZJ5, it is characterized in that: this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 29th, 2013, its preserving number is CGMCC NO.7390, and preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Identification of strains:
Bacteriocinogeny bacterial strain: plant lactobacillus (
lactobacillus plantarum) ZJ5.By plant lactobacillus ZJ5 bacterial classification in the line of MRS solid medium upper flat plate, 30 oC cultivate 24 h.Picking list bacterium colony, in the test tube that 10 ml MRS liquid nutrient mediums are housed, after quiescent culture 24 h, then is inoculated in 100 ml liquid nutrient mediums by the inoculum size of 3%, quiescent culture 24 h, and 4 oC preserve.This bacterium colonial morphology is circle, moistening, surface condition is protruding or smooth (Figure 1A), opaque, color be white, do not produce pigment, neat in edge.These common features are identical with the colony characteristics of typical milk-acid bacteria.Through gramstaining and microscopic examination, the individual morphology characteristic results of bacterial strain is shown in Figure 1B.Microscopy is Gram-positive Bacterium lacticum, and thalline is tyrothricin shape, meets the form of Bacterium lacticum.
The physiological and biochemical property test-results of milk-acid bacteria
As shown in Table 1, this bacterium is after Starch Hydrolysis experiment, urea experiment and fat hydrolysis experiment, and reaction is feminine gender.This bacterium can not secrete and produce amylase, urease, lipase and gelatinase to macromolecular substance of degrading, as starch, fat and gelatin etc. in metabolic process.After M.R test, V.P test and hydrogen sulfide production test, reaction is also feminine gender, and sugar fermentating test then great majority is positive.This result and the feature comparison of Bacterium lacticum in " milk-acid bacteria taxonomic identification and test method ", meet the physicochemical property of Bacterium lacticum substantially.
The physiological and biochemical property test-results of table 1 milk-acid bacteria
Note: "+" represents that result is positive; "-" represents that result is negative.
Note：“+”indicates that result is positive；“-”indicates that result is negative.
The PCR qualification of milk-acid bacteria
Pcr amplification adopts amplification milk-acid bacteria 16S rDNA universal primer, and synthesized by Shanghai Sheng Gong company limited, preserving concentration is 10 μm of ol/L.
27 F (5’-AGAGTTTGATCCTGGCTCAG-3’)
1492 R (5’-GGTTACCTTGTTACGACTT-3’)
PCR reaction system (50 μ L system):
| Volume | Concentration | |
| 10×PCR buffer | 5 μL | 1× |
| dNTP Mixture 2.5 mmol/L | 4 μL | 0.2 mmol/L |
| taq(5 U/μL) | 2 μL | 0.05 U/μL |
| 5’ primer 10 μmol/L | 2 μL | 0.4 μmol/L |
| 3’ primer 10 μmol/L | 2 μL | 0.4 μmol/L |
| DNA | 5 μL | |
| ddH 2O | 30 μL | |
| Total volume | 50 μL |
After trial test result optimizing, select amplification condition: 94 oC denaturation 5 min; 94 oC sex change 30 s, 51 oC anneal 30 s, and 72 oC extend 40 s, 35 circulations; Last at 72 oC downward-extension 5 min, termination reaction.Hai Shenggong order-checking is served after being reclaimed by gained object band.By checking order, the DNA sequence dna obtained is submitted to GenBank, adopts Blast n program and known array to carry out similarity analysis.
(5) 16S rDNA sequencing result
With plant lactobacillus ZJ5 genomic dna for template, employing be 50 μ L reaction systems, have chosen milk-acid bacteria Auele Specific Primer (
l.plantarum, 996 kb), pcr amplification plant lactobacillus 16 S rDNA specific fragment, and check order.Pcr amplification result is as Fig. 2.
In Fig. 2, plant lactobacillus Auele Specific Primer (
l.plantarum, 996 kb) PCR primer have an obvious band at 1000 bp places.Through order-checking (Shanghai Sheng Gong bio-engineering corporation), obtaining ordered sequence is 948 bp.This sequence is delivered to comparison on NCBI (Genbank sequence number KF032707), result shows, and plant lactobacillus homology is the highest.Comprehensive morphological is observed and physio-biochemical characteristics research, and this Strain Designation is plant lactobacillus ZJ5 by we.
Gene order is as follows:
CCATTTGGGATGTGTGAGTACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAATGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGG
Plant lactobacillus (
lactobacillus plantarum) to prepare the concrete grammar of bacteriocin PZJ5 as follows for ZJ5
:
(1) ammonium sulphate gradient precipitation
1, milk-acid bacteria fermentation culture 24 h in MRS liquid nutrient medium, fermented liquid 10000 rpm, 4 oC are centrifugal, and 30 min remove thalline.
2, get 2 L lactobacillus-fermented supernatant liquors, with ultrafiltration in the ultrafiltration cup of nitrogen pressurization, employing molecular weight cut-off is the ultra-filtration membrane of 3000 Da, collects film fermentation liquid, to remove large molecular weight protein.
3, be positioned on magnetic stirring apparatus by the fermented liquid after ultrafiltration and slowly stir, it is 40 % that supernatant liquor adds solid ammonium sulfate to saturation ratio, and 4 oC vibrate 2 h, and albumen is fully precipitated.
4, centrifugal 30 min of 10000 rpm, 4 oC, get supernatant liquor.Adding solid ammonium sulfate again makes its saturation ratio rise to 80 % from 40 %, and 4 oC slowly vibrate 12 h.
Centrifugal 30 min of 10000 rpm, 4 oC, abandon supernatant.Be precipitated and dissolved in 0.1 M PBS damping fluid, 4 oC preserve stand-by.Load in 100 Da dialysis tubings by albumen after dissolving, 4oC dialyses 12 h, and period changes dialyzate 2-3 time.Lyophilize concentrates sample in dialysis tubing, and measures protein content by Bradford method.
(2) ion-exchange chromatography
1, with pure water cleaning AKTApurifier protein purification system and chromatography column until baseline balance;
2, with 50mmol/L Tris-HCl damping fluid (pH6.0) the balance cation displacement chromatography post (SP-sepharose Fast Flow) of 10 times of column volumes (10 CV);
3, the bacteriocin sample (ammonium sulfate precipitated protein or fermented supernatant fluid) of suction filtration sterilizing is entered cationic exchange coloum by systems pumps;
4,5 times of column volumes (5 CV) 50mmol/LTris-HCl buffer solution elution is first used in conjunction with foreign protein, to be eluted to baseline values;
5, then use 1mol/L NaCl solution gradient elution of bound target protein, speed is l ml/min, 3ml/ pipe fraction collection, and 215 nm ultraviolet detection collect protein peak, detects protein content and anti-microbial activity respectively;
(3) hydrophobic chromatography separating lactic acid bacterium bacteriocin
1, chromatography media process: RESOURCE ETH hydrophobic chromatoghaphy medium is kept in 20% ethanol, is linked into AKTApurifier protein purification system by hydrophobic chromatography post to specifications;
2, column equilibration: with 10 mmol/L Tris-HCl damping fluid (pH 5.0) balances, 5 column volumes containing 1 mol/L NaCl, remain that chromatography media is in solution.
3, loading: sample thief adds the chromatography column balanced, and collect chromatography column end opening outflow component, flow velocity is 1 ml/min, and often 3 ml collected by pipe;
4, rinse: wash away not absorbed component with 10 mmol/L Tris-HCl damping fluid (pH 5.0) 1-2 column volume containing 1 mol/L NaCl;
5, wash-out and collection: 1 mol/L NaCl solution is according to 100%-0% 1 mol/L NaCl gradient elution, and 215 nm places measure uv-absorbing, collects elutriant, detects protein content and anti-microbial activity respectively;
6, hydrophobic medium cleaning and preservation: with 10 mmol/L Tris-HCl damping fluid (pH 5.0) wash-outs, then wash with water and be kept in 20% ethanol to neutrality, 4 oC save backup.
(4) molecular sieving bacteriocin lab
1, with pure water cleaning AKTApurifier protein purification system and Superdex PP chromatography column until baseline balance.
2, the bacteriocin sample after hydrophobic chromatography is entered gel column exchange column by systems pumps.
3, first use 10 times of column volumes (10 CV) deionized water eluted protein, be eluted to baseline values.Speed is 0.8 ml/min, 5 ml/ pipe fraction collections, and 215 nm ultraviolet detection collect protein peak, detects protein content and anti-microbial activity respectively.
4, AKTA system and ion column are kept in the ethanolic soln of 20%.
5, collect required component, vacuum concentration is preserved, for subsequent use.
The qualification of lactobacillus-fermented supernatant liquor bacteriostatic activity of the present invention:
Cup-plate method is adopted to measure the bacteriostatic activity of bacteriocin in fermented supernatant fluid.Employing micrococcus luteuses etc. are as indicator, and in test, the concentration of indicator all selects 10
6cfu/mL.
The titration step of fermented supernatant fluid: get 9 cm disposable plastic culture dish, evenly put into 4 Oxford cups, pour the LB semisolid medium (65 oC incubation) that 20 mL contain 300 μ L micrococcus luteus bacterium liquid into.Shake even gently, after to be solidified, extract Oxford cup, in hole, add 200 μ L fermented supernatant fluids, under 4 oC, leave standstill 30 min, 37 oC quiescent culture 24 h, measure antibacterial circle diameter, and be converted into and tire.
PZJ5 antimicrobial spectrum:
To select in food 8 kinds of common gram positive bacteriums, 7 kinds of gram negative bacteriums and 1 Yeasts as indicator, observe the antibacterial situation of bacteriocin PZJ5.
Table 2 indicator used medium and culture condition
a ATCC, American Type Culture Collection, Rockville, MD; CGMCC, China General Microbiological Culture Collection Center, Peking, China;
Adopt agar hole diffusion process to detect antimicrobial substance that plant lactobacillus ZJ5 bacterial strain produces is to the inhibit activities of various Gram-positive and gram negative bacterium.Found that the fermentation supernatant of this bacterial strain effectively can suppress numerous food corruption and pathogenic gram-positive bacterium.Its antimicrobial spectrum is very wide, wherein to the restraining effect of pseudomonas putida clearly, to bacillus subtilis, singly increase the pathogenic bacterium such as listeria spp, shigella dysenteriae and streptococcus aureus and also have stronger restraining effect, antimicrobial spectrum comprises most of Gram-negative bacteria and part gram-positive microorganism, belongs to broad spectrum bacteriocin.Plantaricin ZJ5 is all inhibited to the selected G-bacterium of experiment, and this and the plant lactobacillus reported in the past have relatively big difference.
The antimicrobial spectrum of table 3 Plantaricin by L. plantarum PZJ5
Antibacterial circle diameter (mm): +++, 19-24; ++, 15-18; +, 10-15;-, without fungistatic effect
Inhibition zone in diameter (mm)：+++，19-24；++，15-18；+，10-15；-， no inhibition activity.
Ammonium sulphate gradient precipitates
24 h fermentation cultures are done ammonium sulphate gradient precipitation, have selected saturation ratio gradient is 40%-80%, extracts supernatant liquor and precipitation respectively, and surveys its bacteriostatic activity, in table 4.
The inhibition zone of table 4 ammonium sulphate gradient precipitation extract is tired
| Ammonium sulphate gradient precipitation extract | Inhibition zone is tired (IU/mL) |
| The supernatant liquor of 40 % | 458 |
| The precipitation of 40 % | 0 |
| The supernatant liquor of 80 % | 94 |
| The precipitation of 80 % | 711 |
As shown in Table 4, the supernatant liquor of 40 % and the precipitation of 80 % detect obvious bacteriostatic activity, and the precipitation of 40 % is without bacteriostatic activity, the supernatant slightly bacteriostatic activity of 80 %, this shows that ammonium sulfate concentrations is containing a large amount of bacteriocins in the precipitation of 80 %, only containing a small amount of bacteriocin in its supernatant liquor, the separated extraction of most bacteriocin.Bacteriocin PZJ5 purifying 5.1 times after ammonium sulfate precipitation, ult rec is 31%.
Ion-exchange chromatography
Belong to cationic polypeptide due to most of bacteriocin and stablize in acid condition, so adopt cation-exchange chromatography.The Tris-HCl damping fluid of pH4.5 is selected to carry out exchanging and wash-out.This experiment adopts fermentation broth coarse extract 20 ml, carries out loading with the flow velocity of 1 ml/min, and carry out gradient elution with the Tris-HCl damping fluid containing 1 mol/L NaCl after loading, often about 10 mL collected by pipe.Absorb-elute curve as shown in Figure 4.As can be known from Fig. 4,1 mol/L NaCl pH4.5Tris-HCl damping fluid can good bacteriocin under wash-out, has occurred an independent Peak Activity, and substantially can separate with other foreign proteins.Through ion exchange chromatography bacteriocin PZJ5 purifying 17.3 times, ult rec is 8.5%.
Hydrophobic chromatography
Have strong hydrophobic property according to general bacteriocin, the further separation and purification of PZJ5 sample utilizing hydrophobic chromatography post Resource ETH to obtain SP-Sepharose Fast Flow, the results are shown in Figure shown in 5.PZJ5 obtains good separation and purification through RESOURCE ETH hydrophobic chromatography post, has occurred an independent vigor peak, purification and reclaim yield and reach 33.2 and 4.1% respectively.
Molecular sieving bacteriocin lab
The antibacterial substance crude extract produced after hydrophobic chromatography, through Superdex PP chromatography column chromatography, collects elutriant with l ml/ pipe, measures anti-microbial activity.Find that 12 to 14 pipes have bacteriostatic activity (Fig. 6).Bacteriocin PZJ5 purifying 51 times after sieve chromatography, ult rec is 2.9%.
PZJ5 N terminal Sequence Analysis
Proteins and peptides N-holds sequencing technologies based on Edman chemical degradation method, its ultimate principle comprises the coupling of being held residue by the N-of isothiocyanic acid benzene fat and proteins and peptides, phenylamino formyl sulfide phthalein (PTC-peptide) cyclisation cracking, benzene different sulphur urine amino acid (PTH-amino acid) three main chemical steps are converted into thiazole purine ketone phenylamino (ATZ), each circulation is from protein and polypeptide cleavage amino-acid residue, expose the free amino acid made new advances simultaneously and carry out next Edman degraded, PTH-amino acid identities finally by transfer realizes the mensuration of protein sequence.
Step: (1) SDS-PAGE electrophoresis; (2) PVDF transferring film N holds automatic amino acid sequence analysis.PZJ5 through molecular sieving and collect after, through amino acid automatic sequence analyser result as shown in Figure 4.Analyze from the sequence map obtained, the N-terminal sequence of PZJ5 is: KTKQQFLIKAQTQLFKVFGYTL, and sequence display PZJ5 belongs to ClassII c bacterioid element.
In a word, the foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to the covering scope of patent of the present invention.
sequence table
<110> Zhejiang Prov Industrial And Commercial University
<160> 1
<210> 1
<211> 948
<212> DNA
<213> plant lactobacillus (
lactobacillus plantarum)
<400> 1
CCATTTGGGATGTGTGAGTACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAATGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGG
Claims (6)
1. a lactobacillus plantarum (
lactobacillus plantarum) ZJ5, it is characterized in that: this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 29th, 2013, its preserving number is CGMCC NO.7390, and preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
2. utilize plant lactobacillus as claimed in claim 1 (
lactobacillus plantarum) ZJ5 prepares application in bacteriocin.
3. utilize plant lactobacillus as claimed in claim 1 (
lactobacillus plantarum) ZJ5 prepares the method for bacteriocin PZJ5, it is characterized in that, comprise the steps:
Prepare fermented liquid: by plant lactobacillus (
lactobacillus plantarum) ZJ5 is inoculated in MRS liquid nutrient medium by the inoculum size of 3%, 30 DEG C of quiescent culture 24 h; The fermented liquid obtained is at 10000 rpm, and under the condition of 4 DEG C, centrifugal 15 min, get supernatant liquor, places 4 DEG C and saves backup;
Prepare bacteriocin: get supernatant liquor by after centrifugal 15 min of fermentation liquor 10000 r/ m of preparation, in supernatant liquor, slowly add ammonium sulfate solids powder saltout, limit edged stirs, until saturation ratio reaches 40, then under 4-5 DEG C of condition, 2 h are left standstill, again under 4-5 DEG C of condition, 12000 r/ m, centrifugal 30 min, discard precipitation; In supernatant liquor, slowly add ammonium sulfate solids powder again saltout, limit edged stir, until saturation ratio reaches 80, then under 4-5 DEG C of condition leave standstill 12 h, then under 4-5 DEG C of condition, 12000 r/ m, centrifugal 30 min, abandoning supernatant obtains bacteriocin; Bacteriocin PZJ5 is obtained finally by after cation-exchange chromatography, hydrophobic chromatography and molecular sieving.
4. plant lactobacillus as claimed in claim 1 (
lactobacillus plantarum) ZJ5 preparing the application in food or feed anticorrosion agent.
5. plant lactobacillus as claimed in claim 1 (
lactobacillus plantarum) ZJ5 preparing the application in bacterial inhibitor;
Described bacterium be micrococcus luteus (
micrococcus luteus), streptococcus aureus (
staphylococcus aureus), plant lactobacillus (
lactobacillus plantarum), Lactococcus lactis (
lactococcus lactis), subtilis (
bacillus subtilis), singly increase listeria spp (
listeria monocytogenes), enterococcus faecalis (
enterococcus faecalis), shigella dysenteriae (
shigella dysenteriae), Shigellae (
shigella. flexneri), pseudomonas putida (
pseudomonas putida), Salmonellas (
salmonella sp.), intestinal bacteria (
escherich. coli) or yeast saccharomyces cerevisiae (
saccharomyces cerevisiae).
6. application according to claim 5, is characterized in that: described bacterium be micrococcus luteus (
micrococcus luteus) CGMCC 1.193, streptococcus aureus (
staphylococcus aureus) CGMCC 1.879, streptococcus aureus (
staphylococcus aureus) CGMCC 1.128, plant lactobacillus (
lactobacillus plantarum) CGMCC 1.551, plant lactobacillus (
lactobacillus plantarum) CGMCC 1.556, Lactococcus lactis (
lactococcus lactis) ATCC 15577, subtilis (
bacillus subtilis) CGMCC 1.1627, singly increase listeria spp (
listeria monocytogenes) ATCC 7648, enterococcus faecalis (
enterococcus faecalis) CGMCC 1.125, shigella dysenteriae (
shigella dysenteriae) ATCC 9753, Shigellae (
shigella. flexneri) CGMCC 1.1868, pseudomonas putida (
pseudomonas putida) CGMCC 1.645, Salmonellas (
salmonella sp.) CGMCC 1.1552, intestinal bacteria (
escherich. coli) CGMCC 1.1580, intestinal bacteria (
escherich. coli) JM109 or yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) CGMCC 2.1643.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104818232A (en) * | 2015-05-08 | 2015-08-05 | 内蒙古农业大学 | Lactobacillus plantarum AB-3 having bacteria inhibition activity and application thereof |
| CN104830730A (en) * | 2015-05-08 | 2015-08-12 | 内蒙古农业大学 | Lactobacillus plantarum AB-4 with activity of resisting fusariwn oxysporum and phytophthora drechsleri and application of lactobacillus plantarum |
| CN106188252A (en) * | 2016-07-18 | 2016-12-07 | 北京农学院 | Polypeptide and Lactobacillus plantarum Extracellular metabolism and their application and the method for induction Lactobacillus plantarum bacteriocinogeny and authentication method |
| CN107488220A (en) * | 2017-09-08 | 2017-12-19 | 吉林省凯博生物技术有限公司 | A kind of Lactobacillus plantarum bacteriocin and its preparation method and application |
| CN108611299A (en) * | 2018-05-10 | 2018-10-02 | 南京师范大学 | One plant of lactobacillus plantarum for producing bacteriostatic peptide and its application |
| CN109666601A (en) * | 2018-12-03 | 2019-04-23 | 杭州娃哈哈科技有限公司 | One plant of lactobacillus plantarum with antagonistic property and its application in diarrhea prevention |
| CN111004743A (en) * | 2019-10-31 | 2020-04-14 | 山西大学 | Method for screening plant rhizosphere soil antagonistic bacteria |
| CN114874937A (en) * | 2022-04-30 | 2022-08-09 | 浙江工商大学 | Separation and purification of lactobacillus sake bacteriocin, antibacterial application and lactic acid bacteria used in separation and purification |
| CN116478256A (en) * | 2021-01-19 | 2023-07-25 | 西南大学 | Bacteriocin produced by lactobacillus fermentum and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101812414A (en) * | 2009-08-06 | 2010-08-25 | 东北农业大学 | Lactobacillus plantarum and bacteriocins produced by lactobacillus plantarum and capable of inhibiting Gram negative bacteria |
| CN102041238A (en) * | 2009-10-22 | 2011-05-04 | 中国农业大学 | Lactobacillus plantarum, method for fermenting and preparing bacteriocin of Lactobacillus plantarum, and application of Lactobacillus plantarum and bacteriocin |
| CN103667106A (en) * | 2013-10-10 | 2014-03-26 | 南京农业大学 | Lactobacillus plantarum, bacteriocins and culture, separation and purification method thereof |
-
2014
- 2014-08-29 CN CN201410430688.7A patent/CN104232520A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101812414A (en) * | 2009-08-06 | 2010-08-25 | 东北农业大学 | Lactobacillus plantarum and bacteriocins produced by lactobacillus plantarum and capable of inhibiting Gram negative bacteria |
| CN102041238A (en) * | 2009-10-22 | 2011-05-04 | 中国农业大学 | Lactobacillus plantarum, method for fermenting and preparing bacteriocin of Lactobacillus plantarum, and application of Lactobacillus plantarum and bacteriocin |
| CN103667106A (en) * | 2013-10-10 | 2014-03-26 | 南京农业大学 | Lactobacillus plantarum, bacteriocins and culture, separation and purification method thereof |
Non-Patent Citations (4)
| Title |
|---|
| DA-FENG SONG 等: "Purification and Characterization of Plantaricin ZJ5, a New Bacteriocin Produced by Lactobacillus plantarum ZJ5", 《PLOS ONE》 * |
| 宋达峰 等: "AUG28214.1", 《GENBANK》 * |
| 宋达峰 等: "新型植物乳杆菌细菌素PZJ5的分离和鉴定", 《第8届乳酸菌与健康国际研讨会》 * |
| 李平兰 等: "产细菌素植物乳杆菌菌株的筛选及其细菌素生物学特征研究", 《食品与发酵工业》 * |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830730A (en) * | 2015-05-08 | 2015-08-12 | 内蒙古农业大学 | Lactobacillus plantarum AB-4 with activity of resisting fusariwn oxysporum and phytophthora drechsleri and application of lactobacillus plantarum |
| CN104818232B (en) * | 2015-05-08 | 2016-07-06 | 内蒙古农业大学 | One strain has Lactobacillus plantarum AB-3 and the application thereof of bacteriostatic activity |
| CN104818232A (en) * | 2015-05-08 | 2015-08-05 | 内蒙古农业大学 | Lactobacillus plantarum AB-3 having bacteria inhibition activity and application thereof |
| CN106188252A (en) * | 2016-07-18 | 2016-12-07 | 北京农学院 | Polypeptide and Lactobacillus plantarum Extracellular metabolism and their application and the method for induction Lactobacillus plantarum bacteriocinogeny and authentication method |
| CN106188252B (en) * | 2016-07-18 | 2019-07-19 | 北京农学院 | The method and identification method of polypeptide and lactobacillus plantarum Extracellular metabolism and their application and induction lactobacillus plantarum bacteriocinogeny |
| CN107488220A (en) * | 2017-09-08 | 2017-12-19 | 吉林省凯博生物技术有限公司 | A kind of Lactobacillus plantarum bacteriocin and its preparation method and application |
| CN107488220B (en) * | 2017-09-08 | 2020-10-23 | 吉林省凯博生物技术有限公司 | Lactobacillus plantarum bacteriocin, and preparation method and application thereof |
| CN108611299B (en) * | 2018-05-10 | 2022-03-08 | 南京师范大学 | Lactobacillus plantarum for producing antibacterial peptide and application thereof |
| CN108611299A (en) * | 2018-05-10 | 2018-10-02 | 南京师范大学 | One plant of lactobacillus plantarum for producing bacteriostatic peptide and its application |
| CN109666601A (en) * | 2018-12-03 | 2019-04-23 | 杭州娃哈哈科技有限公司 | One plant of lactobacillus plantarum with antagonistic property and its application in diarrhea prevention |
| CN109666601B (en) * | 2018-12-03 | 2022-01-18 | 杭州娃哈哈科技有限公司 | Lactobacillus plantarum with antibacterial property and application thereof in diarrhea prevention |
| CN111004743A (en) * | 2019-10-31 | 2020-04-14 | 山西大学 | Method for screening plant rhizosphere soil antagonistic bacteria |
| CN116478256A (en) * | 2021-01-19 | 2023-07-25 | 西南大学 | Bacteriocin produced by lactobacillus fermentum and application thereof |
| CN116478256B (en) * | 2021-01-19 | 2024-05-14 | 西南大学 | Bacteriocin produced by lactobacillus fermentum and application thereof |
| CN114874937A (en) * | 2022-04-30 | 2022-08-09 | 浙江工商大学 | Separation and purification of lactobacillus sake bacteriocin, antibacterial application and lactic acid bacteria used in separation and purification |
| CN114874937B (en) * | 2022-04-30 | 2024-02-20 | 浙江工商大学 | Separation and purification of bacteriocin produced by lactobacillus sake and antibacterial application and lactobacillus used by same |
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