CN103320370B - The preparation method of a kind of subtilis C3 and anti-Listeria monocytogenes bacteriocin thereof - Google Patents
The preparation method of a kind of subtilis C3 and anti-Listeria monocytogenes bacteriocin thereof Download PDFInfo
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Abstract
Name of the present invention is called the preparation method of a kind of subtilis C3 and anti-Listeria monocytogenes bacteriocin thereof, and this bacteriocin is applicable to low temperature meat quail, preserving fruit and vegetable utilizing as natural antiseptic agent, can improve the security of food.The present invention utilizes subtilis (Bacillus subtilis) C3CGMCC No.9713 to adopt ammonium sulfate precipitation, anion-exchange chromatography, semi-preparative reverse-phase liquid phase chromatography polishing purification, subtilis C3 bacteriocin is tired and is improve 32 times, and purity improves 42.80 times.This bacteriocin has stronger bacteriostatic activity to Listeria monocytogenes, and to heat and ph stability better, can, by proteinase K digestion, be a kind of natural safe biological preservative.The raw material sources that the invention provides bacteriocin is extensive, and extraction process is simple, and purification process is efficient, stable, is suitable for suitability for industrialized production.
Description
Technical field
The present invention relates to the preparation method of a kind of subtilis C3 and anti-Listeria monocytogenes bacteriocin thereof in microorganism field.This bacteriocin is applicable to low temperature meat quail, preserving fruit and vegetable utilizing as natural antiseptic agent, can improve the security of food.
Background technology
Food, in the processes such as processing, storage, transport, is vulnerable to harmful microbe and pollutes and cause putrid and deteriorated or cause food poisoning, adopts the method for adding sanitas to suppress microbial growth, becomes the important means of food fresh keeping gradually.Traditional meat is mostly fresh-keeping by Chemical Preservative, and have certain potential safety hazard, improper use has certain side effect, and the health of long-term Excess free enthalpy to human consumer causes certain infringement, and causes environmental pollution.Therefore, research and development efficient, safety, eco-friendly natural meat products sanitas be food safety in the urgent need to.Bacteriocin refers to that some bacterium has polypeptide or the Precursor Peptide of bacteriostatic activity in metabolic process by a class of Ribosome biogenesis.Nisin studies bacteriocin the most widely so far, but because of alkalescence and poor heat stability, limits its application in food.This project has filtered out a strain has obvious inhibition subtilis C3 to Listeria monocytogenes, the bacteriocin that this bacterium produces has satisfactory stability to heat and pH, and can by proteinase K digestion, as food preservative freshness retaining agent, there is higher-security and stability, possess good development prospect.Therefore, a kind of preparation method that is efficient, that stablize subtilis C3 bacteriocin is set up extremely important.Bacteriocin preparation method mainly contains ammonium sulfate precipitation, ion exchange chromatography, gel chromatography, high performance liquid chromatography etc.
Following three kinds of modes are taked in the application of bacteriocin in food usually: the bacteriocin after purifying directly adds in food as biological preservative by (1); (2) bacteriocin producing strains is produced leavened food as starter, can effectively prevent living contaminants in fermenting process; (3) in the raw material of preparation packaging material for food, add bacteriocin, exploitation has the food pack, preservative film etc. of fungistatic effect.
At present about the patent report of subtilis bacteriocin preparation method, as " extracellular antiseptic protein of a kind of subtilis and separation purification method thereof ", the patent No. is: 200610097847.1; " subtilis LFB112, its bacteriocin produced and application ", application number is: 201010560828.4; " preparation of antibacterial extracellular product of bacillus subtilis ", application number is: 201010577733.3; " bacillus subtilis and the application in fungal disease control thereof ", application number is: 201110093415.4.Preparation method about the subtilis bacteriocin of anti-Listeria monocytogenes in current domestic patent of invention and document there is not yet relevant report.
Summary of the invention
First object of the present invention is to provide the subtilis C3 that a strain has anti-Listeria monocytogenes activity.
Subtilis provided by the present invention is: subtilis (Bacillus subtilis) C3, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on July 12nd, 2013, preserving number is: CGMCC No.9713.
Subtilis (Bacillus subtilis) C3, separation screening is from Beijing Agricultural College's orchard soil.
On beef-protein medium, the bacterium colony size of subtilis (Bacillus subtilis) C3 is 3 ~ 5mm, and canescence, opaque, surface drying, edge are irregular, and somatic cells is elongated rod shape, short catenation, central spore, G
+aerobic bacteria.
In order to determine the antagonistic property of subtilis, the bacteriostasis property of its fermented supernatant fluid is measured.Test strain: Listeria monocytogenes (abbreviation Listeria monocytogenes), enterococcus faecalis, streptococcus aureus, intestinal bacteria, Salmonellas, Shigellae, aspergillus niger, white edge mould etc.Test-results shows that subtilis C3 fermented supernatant fluid has restraining effect to Listeria monocytogenes, enterococcus faecalis, streptococcus aureus, aspergillus niger, the growth of white edge mould, wherein have stronger bacteriostatic activity to Listeria monocytogenes, antibacterial circle diameter is 19.46mm.
The subtilis C3 of anti-Listeria monocytogenes activity that what aforesaid method obtained have belongs to scope.
Second object of the present invention is to provide a kind of subtilis C3 bacteriocin preparation method with anti-Listeria monocytogenes activity.
The extracting method with anti-Listeria monocytogenes activated bacterial element provided by the present invention to be fermented the bacteriocin obtained by subtilis C3.
(1) subtilis C3 fermented liquid 4 DEG C, the centrifugal 15 ~ 20min of 10000 ~ 11000r/min, removes thalline.
(2) 55% ~ 65% (W/V is adopted, mass volume ratio concentration) the ammonium sulfate precipitation subtilis C3 fermented supernatant fluid of saturation ratio, 4 DEG C of standing 12h, in 4 DEG C, the centrifugal 15-20min of 10000 ~ 11000r/min, collecting precipitation thing, presses the amount redissolution precipitation of fermented liquid 1/10 volume with deionized water.
(3) be distributed in pretreated dialysis tubing by redissolution liquid, be placed in deionized water 4C dialysis 48h desalination, dialyzate is bacteriocin crude extract.
(4) bacteriocin crude extract 0.21 μm of sterile filters is filtered, and adopt DEAE-Sepharose Fast Flow anion exchange chromatography stepwise elution purification of bacterial element, chromatography column specification is 10mm × 200mm; Damping fluid: A liquid 0.025mol/L pH7.5Tric-HCl, B liquid contains the 0.025mol/L pH7.5Tric-HCl of 0.4mol/L NaCl; Stepwise elution step (V/V, volume fraction): 50min A liquid → 90min70%A liquid+30%B liquid → 40min50%A liquid+50%B liquid → 40min100%B liquid; Flow velocity is 1mL/min, applied sample amount 5mL, and automatic collector 5mL/ manages, and collects the elutriant that 95 ~ 105min has bacteriostatic activity.
(5) activated collection liquid is distributed in pretreated dialysis tubing, 24h desalination of dialysing in 4 DEG C of deionized waters.
(6) ion-exchange dialyzate is concentrated under 10mbar, 50 DEG C of conditions with centrifugal vacuum concentrating instrument, concentration ratio is 10: 1.
(7) ion-exchange concentrated solution filters through 0.2 μm of sterile filters, and adopt semi-preparative reverse-phase liquid phase chromatography linear gradient elution purification of bacterial element, reverse phase preparative column model is Agilent ZORBAX300SB-C18, and post specification is 9.4mm × 250mm; Moving phase: A liquid is acetonitrile+0.1% trifluoroacetic acid (V/V), B liquid is water+0.1% trifluoroacetic acid (V/V); Linear gradient elution condition (V/V, volume fraction): 0 ~ 5min, 10% ~ 30%A liquid → 5 ~ 10min, 30% ~ 50%A liquid → 10 ~ 55min, 50% ~ 90%A → 55 ~ 60min90%A; Flow velocity 4.0mL/min, applied sample amount 1mL, 1 pipe/min collects automatically, and collect the elutriant that 19 ~ 20min has bacteriostatic activity, traditional vacuum concentrates, and remove acetonitrile, then by its lyophilize, dried bacteriocin sterling is white powder state.
The above-mentioned preparation method with the subtilis C3 bacteriocin of anti-Listeria monocytogenes activity belongs to scope.
Subtilis (Bacillus subtilis) C3 separation screening is from Beijing Agricultural College's orchard soil, and its bacteriocin produced can by proteinase K digestion.The present invention utilizes ammonium sulfate precipitation, anion-exchange chromatography, semi-preparative reverse-phase liquid-phase chromatography method to prepare bacteriocin, and finally tire and improve 32 times, purity improves 42.80 times.The raw material sources of this bacteriocin is extensive, and extraction process is simple, and purification process is efficient, stable, is suitable for suitability for industrialized production.
Embodiment
The present invention will be further described and do not limit the scope of the invention for following embodiment.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Percentage composition in following embodiment, if no special instructions, is volume fraction.
The preparation of embodiment 1, subtilis C3 bacteriocin crude extract
1, the preparation of subtilis C3 fermented liquid
(1) preparation of seed liquor
Seed culture based formulas (W/V, mass volume ratio concentration): glucose 1%, peptone 1%, NaCl1%, KH
2pO
40.15%, MgS0
47H
2o0.15%, pH nature;
Seed culture condition: moved in seed culture medium by 1% inoculum size by the subtilis activated, culture temperature 37 DEG C, liquid amount 75mL/250mL, 150r/min shaking table shaking culture 18h, viable count is about 10
9cFU/mL.
(2) preparation of fermented liquid
Fermentative medium formula (W/V, mass volume ratio concentration): glucose 1%, peptone 1.5%, yeast leaching powder 1.5%, KH
2pO
40.1%, MgSO
47H
2o0.15%, NaCl0.5%, pH7.0;
Fermentation condition: seed liquor is moved in fermention medium by 2% inoculum size, leavening temperature 37 DEG C, liquid amount 150mL/500mL, 180r/min shaking table oscillation and fermentation 48h.
2, the preparation of subtilis C3 bacteriocin crude extract
(1) fermented liquid is with 10000 ~ 11000r/min, 15 ~ 20min, 4 DEG C of centrifugal segregation thalline, supernatant liquor is placed in 1000mL beaker, takes the ammonium sulfate after grinding by saturation ratio 60% (390g/1000mL), slowly stirs at the uniform velocity in the same way with glass stick, add ammonium sulfate while stirring, complete operation within 15min, hold over night in 4 DEG C of refrigerators, presses the amount redissolution precipitation of fermented liquid 1/10 volume with deionized water.(mass volume ratio concentration is 40%, 50%, 55%, 60%, 65% to adopt the different ammonium sulfate saturation ratio of cup-plate method comparative determination, 70%, the fungistatic effect of 80%1 throw outs redissolution liquid, result shows that 60% saturation ratio sedimentation effect is best, almost bacteriocin is all precipitated, and reduce the difficulty causing separation and purification because of the precipitation of medium component as far as possible.
(2) fermented liquid is after precipitation with 10000 ~ 11000r/min, 15 ~ 20min, 4 DEG C centrifugal, throw out is redissolved in 100mL water, being distributed into molecular weight cut-off is in the pre-treatment dialysis tubing of 3500D, 4 DEG C of deionized water dialysis 48h, namely dialysate obtains subtilis C3 bacteriocin crude extract.
The antagonistic property of embodiment 2, subtilis C3 bacteriocin crude extract and physico-chemical property
Measure antagonistic property and the physico-chemical property of C3 bacteriocin crude extract by cup-plate method in process of the test.
Cup-plate method: first indicator bacterial strain is diluted to 10
7cFU/mL, after mixing to the corresponding solid medium of heating and melting, pour into about 15mL in plate, aseptic Oxford cup is placed gently after solidifying, getting 100 μ L subtilis C3 crude extracts adds in the cup of Oxford, cultivates 12h for 37 DEG C, observes inhibition zone, and measuring antibacterial circle diameter with vernier callipers, reading is accurate to 0.01mm.
1, the antagonistic property of subtilis C3 bacteriocin crude extract
The fungistatic effect of table 1 subtilis C3 bacteriocin crude extract
Note: Oxford cup diameter is 7.50mm.
From table 1, the growth of subtilis C3 bacteriocin crude extract to Listeria monocytogenes, enterococcus faecalis, streptococcus aureus, aspergillus niger, white edge mould has restraining effect, wherein there is stronger bacteriostatic activity to Listeria monocytogenes, aspergillus niger, white edge mould, enterococcus faecalis bacteriostatic action are taken second place.Because this bacteriocin is the strongest to the restraining effect of Listeria monocytogenes, therefore determine that it is the indicator strain in Physicochemical test.
2, the enzyme sensitivity test of subtilis C3 bacteriocin crude extract
The 0.2 μm of sterile filters filtration sterilization of subtilis C3 bacteriocin crude extract.Use hydrogen peroxide ferment treatment, to verify whether the antibacterial substance in C3 bacteriocin crude extract is hydrogen peroxide.Common 3 kinds of proteolytic enzyme are mixed with 20mg/mL solution respectively, and regulate the optimal pH of various enzyme liquid effect, join in C3 bacteriocin crude extract, its final concentration is made to be 1mg/mL, 37 DEG C of water-bath 4h, unification recalls to pH6.0 (after fermentation, pH is 6.0), with not adding the C3 bacteriocin crude extract of ferment treatment as blank, to verify whether antibacterial substance is easily degraded by proteases again.Adopt cup-plate method to do the bacteriostatic test of Listeria monocytogenes, the results are shown in Table 2.
The enzyme susceptibility results of table 2 subtilis C3 bacteriocin crude extract
Note: Oxford cup diameter is 7.50mm.
From table 2, after hydrogen oxide ferment treatment subtilis C3 bacteriocin, fungistatic effect is still comparatively strong compared with blank, illustrates that C3 bacteriocin is not hydrogen peroxide.Proteinase K has the ability of degraded natural protein, after Proteinase K process, the anti-Listeria monocytogenes of C3 bacteriocin is active significantly to be reduced, and stomach en-and trypsinase less to its Bacteriostatic Effect, illustrate that this bacteriocin contains the effect binding site of Proteinase K, show that this bacteriocin is protein matter.
3, the thermostability of subtilis C3 bacteriocin crude extract
Subtilis C3 bacteriocin crude extract is heat-treated by table 3 respectively, using the C3 bacteriocin of non-heat treated as blank, measures the bacteriocin crude extract after process to the bacteriostatic activity of Listeria monocytogenes, the results are shown in Table 3.
The thermal stability results of table 3 subtilis C3 bacteriocin crude extract
Note: Oxford cup diameter is 7.50mm.
As shown in Table 3, compared with blank group, 60 ~ 80 DEG C of heat treated 30 ~ 120min, antibacterial circle diameter, all at more than 18mm, illustrates that bacteriocin C3 bacteriostatic activity is still comparatively strong, shows that it is 60 ~ 80 DEG C of better heat stability; 100 DEG C of process 30 ~ 60min, antibacterial circle diameter, between 14.28 ~ 17.78mm, illustrates that bacteriocin C3 still has better stability at 100 DEG C; After 121 DEG C of process 15min, antibacterial circle diameter is 12.22mm, and its bacteriostatic activity remains 63.1%, further illustrates this bacteriocin thermostability stronger.Therefore this bacteriocin is applied in food, and food processing technology can not affect its bacteriostatic activity.
4, the ph stability of subtilis C3 bacteriocin crude extract
PH is regulated to be 2.0,3.0,4.0,5.0,7.0,8.0,9.0,10.0,11.0 subtilis C3 bacteriocin crude extract 1mol/L NaOH and 1mol/L HCl, 37 DEG C of water-bath 3h, pH6.0 is recalled in unification again, measure the bacteriocin crude extract after process to the bacteriostatic activity of Listeria monocytogenes, the results are shown in Table 4.
The ph stability result of table 4 subtilis C3 bacteriocin crude extract
Note: Oxford cup diameter is 7.50mm.
As shown in Table 4, after subtilis C3 bacteriocin crude extract acts on 3h under the condition of pH2.0 ~ 4.0, antibacterial circle diameter is between 15.36 ~ 16.32mm, and bacteriostatic activity slightly reduces, and illustrates that this bacteriocin acid resistance is better; Under the condition of pH5.0 ~ 8.0, antibacterial circle diameter, at more than 18.0mm, shows that its suitableeest action pH is 5.0 ~ 8.0; Under the condition of pH9.0 ~ 11.0, antibacterial circle diameter is between 15.78 ~ 17.88mm, and bacteriostatic activity slightly reduces, and illustrates that this bacteriocin alkali resistance is also better.The pH of most food between 5.0 ~ 6.5, therefore this bacteriocin as aseptic applications in food, can tolerance acid-base environment.
Embodiment 3, subtilis C3 bacteriocin purification process
1, the purifying of subtilis C3 bacteriocin crude extract
(1) anion exchange chromatography stepwise elution purifying is adopted
By the 0.2 μm of sterile filters filtration sterilization of subtilis C3 bacteriocin crude extract, adopt DEAE-Sepharose Fast Flow anion exchange chromatography stepwise elution purifying, chromatography column specification is 10mm × 200mm; Damping fluid: A liquid 0.025mol/L pH7.5Tric-HCl, B liquid contains the 0.025mol/L pH7.5Tric-HCl of 0.4mol/L NaCl; Stepwise elution step: 50min100%A liquid → 90min70%A liquid+30%B liquid → 40min50%A liquid+50%B liquid → 40min100%B liquid; Flow velocity is 1mL/min, applied sample amount 5mL, and automatic collector 5mL/ manages, and adopt cup-plate method to detect and collect elutriant to the bacteriostatic activity of Listeria monocytogenes, test shows that the elutriant of 95 ~ 105min has bacteriostatic activity.To the collection elutriant of bacteriostatic activity be had to put into pretreated dialysis tubing, in 4 DEG C of deionized water dialysis 24h desalinations, dialyzate be the purified product of bacteriocin stepwise elution.
(2) semi-preparative reverse-phase liquid phase chromatography linear gradient elution is adopted to be further purified
Ion-exchange dialyzate is concentrated under 10mbar, 50 DEG C of conditions with centrifugal vacuum concentrating instrument, and concentration ratio is 10: 1.Ion-exchange concentrated solution filters through 0.21 μm of sterile filters, and adopt semi-preparative reverse-phase liquid phase chromatography linear gradient elution purification of bacterial element, reverse phase preparative column model is Agilent ZORBAX300SB-C18, and post specification is 9.4mm × 250mm; Moving phase: A liquid is acetonitrile+0.1% trifluoroacetic acid, B liquid is water+0.1% trifluoroacetic acid; Linear gradient elution condition: 0 ~ 5min, 10% ~ 30%A liquid → 5 ~ 10min, 30% ~ 50%A liquid → 10 ~ 55min, 50% ~ 90%A → 55 ~ 60min90%A; Flow velocity 4.0mL/min, applied sample amount 1mL, 1 pipe/min collects automatically, respectively the elutriant traditional vacuum of collection is concentrated into clean dry, redissolve in 100 ~ 200 μ L deionized waters, adopt cup-plate method to detect and collect liquid to the bacteriostatic activity of Listeria monocytogenes, test shows that the elutriant of 19 ~ 20 μ in has bacteriostatic activity.A large amount of collection has the elutriant of bacteriostatic activity to be the purified product of linear gradient elution.
2, subtilis C3 bacteriocin purification effect is evaluated
(1) bacteriocin Potency Analysis
The determination of bacteriocin valence value: by continuous for testing sample doubling dilution, adopts the Antibacterial Activity of bacteriocin in cup-plate method working sample liquid.The vigor unit of activity (AU) of every milliliter represents.The definition of AU is the inverse of the most high dilution (n) seeing obvious inhibition zone.
(2) Coomassie brilliant G-250 Determination Staining total protein content is adopted
Coomassie brilliant G-250 100mg is dissolved in 50mL95% ethanol, adds 100mL85% phosphoric acid, with distilled water diluting to 1000mL, and filter paper filtering.Containing 0.01% (W/V) Coomassie brilliant G-250 in final reagent, 4.7% (W/V) ethanol; Crystallization bovine serum albumin, measures protein nitrogen content through micro-Kjeldahl in advance, is mixed with 1mg/mL, 0.1mg/mL protein solution according to its purity 0.15% (W/V) NaCl; Get 14 test tubes, divide two groups and operate by table 5.With A
595nm(595nm place light absorption value) is ordinate zou, and standard protein content is X-coordinate, drawing standard curve; Measuring method is the same, gets suitable unknown sample volume, makes its measured value in the linear extent of typical curve.According to measured A
595nmvalue, typical curve is found the amount that it is equivalent to standard protein, thus calculates the protein concn (mg/mL) of unknown sample, typical curve parameter list is in table 5.
The formula of bovine serum albumin (BSA) the protein standard curve gained adopting Coomassie Brilliant Blue to record is: y=12.111x+0.0173, R
2=0.9938, illustrate that the linear degree of this standard curve is higher, can be used as the typical curve of determination of protein concentration.
Table 5 Coomassie Brilliant Blue surveys protein content typical curve parameter list
The evaluation of table 6 subtilis C3 bacteriocin purification effect
From table 6, after purifying, the Rate activity of bacteriocin is increased to 4131.11AU/mg, and purification is 42.80 times, tires and improves 32 times, shows thus and adopts simple and quick purification step can effectively reach purifying object.
Project 1 belonging to this patent: the Department of Science and Technology " 12 " national science and technology supporting plan " animal-derived food HACCP System Construction and pathogenic bacterium high throughput testing technology " sub-problem " detects the Study and appliance of streptococcus aureus and Listeria monocytogenes " based on immune colloid gold
Item number: 2012BAD28B02-01
The project beginning and ending time: 2012.01-2015.12
Project leader: Liu Hui
Project 2 belonging to this patent: Department of Science and Technology's national science and technology key special subjects " disease-resistant transgenic sheep rearing new variety " sub-problem " foundation that disease-resistant transgenic sheep expansion traditional font is/disease-resistant transgenic goat-anti disease, production performance and safety evaluation "
Item number: 2013ZX08008-005
The project beginning and ending time: 2013.01-2013.12
Project leader: Liu Hui
Project 3 belonging to this patent: agricultural-food harmful microorganism and the residual safety detection of agriculture with control key lab 2012 of Beijing ladder planning item
Item number: Z121106002812039
The project beginning and ending time: 2012.08-2013.08
Project leader: Zhang Hongxing
Claims (1)
1. the method utilizing subtilis (Bacillus subtilis) C3CGMCC No.9713 to prepare anti-Listeria monocytogenes bacteriocin, it is characterized in that: (1) subtilis C3 fermented liquid 4 DEG C, centrifugal 15 ~ the 20min of 10000 ~ 11000r/min, removes thalline; (2) the ammonium sulfate precipitation subtilis C3 fermented supernatant fluid of 55% ~ 65% saturation ratio is adopted, 4 DEG C of standing 12h; In 4 DEG C, the centrifugal 15 ~ 20min of 10000 ~ 11000r/min, collecting precipitation thing, presses the amount redissolution precipitation of fermented liquid 1/10 volume with deionized water; (3) be distributed in pretreated dialysis tubing by redissolution liquid, be placed in deionized water 4 DEG C dialysis 48h desalination, dialyzate is bacteriocin crude extract; (4) bacteriocin crude extract 0.2 μm of sterile filters is filtered, and adopt DEAE-Sepharose Fast Flow anion exchange chromatography stepwise elution purification of bacterial element, chromatography column specification is 10mm × 200mm; Damping fluid: A liquid 0.025mol/L pH 7.5Tric-HCl, B liquid contains the 0.025mol/L pH 7.5Tric-HCl of 0.4mol/L NaCl; Stepwise elution step: 50min A liquid → 90min 70%A liquid+30%B liquid → 40min 50%A liquid+50%B liquid → 40min 100%B liquid; Flow velocity is 1mL/min, applied sample amount 5mL, and automatic collector 5mL/ manages, and collects the elutriant that 95 ~ 105min has bacteriostatic activity; (5) activated collection liquid is distributed in pretreated dialysis tubing, 24h desalination of dialysing in 4 DEG C of deionized waters; (6) ion-exchange dialyzate is concentrated under 10mbar, 50 DEG C of conditions with centrifugal vacuum concentrating instrument, concentration ratio is 10: 1; (7) ion-exchange concentrated solution filters through 0.2 μm of sterile filters, and adopt semi-preparative reverse-phase liquid phase chromatography linear gradient elution purification of bacterial element, reverse phase preparative column model is Agilent ZORBAX 300SB-C18, and post specification is 9.4mm × 250mm; Moving phase: A liquid is acetonitrile+0.1% trifluoroacetic acid, B liquid is water+0.1% trifluoroacetic acid; Linear gradient elution condition: 0 ~ 5min, 10% ~ 30%A liquid → 5 ~ 10min, 30% ~ 50%A liquid → 10 ~ 55min, 50% ~ 90%A → 55 ~ 60min 90%A; Flow velocity 4.0mL/min, applied sample amount 1mL, 1 pipe/min collects automatically, and collect the elutriant that 19 ~ 20min has bacteriostatic activity, traditional vacuum concentrates, and remove acetonitrile, then by its lyophilize, dried bacteriocin sterling is white powder state.
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| CN104480042A (en) * | 2014-12-11 | 2015-04-01 | 北京农学院 | Preparation method of bacillus subtilis C3 live bacteria preparation with resistance to listeria monocytogenes |
| CN104450577A (en) * | 2014-12-11 | 2015-03-25 | 北京农学院 | Fermentation method of bacillus subtilis C3 bacteriocin for producing anti-listeria monocytogenes |
| CN104774890B (en) * | 2015-04-13 | 2018-12-14 | 光明乳业股份有限公司 | One Bacillus species bacteriocin crude extract and its preparation method and application |
| CN105733008B (en) * | 2016-04-21 | 2019-02-12 | 北京农学院 | A kind of preparation method of antibacterial biological preservation film |
| CN107937319A (en) * | 2018-01-02 | 2018-04-20 | 江西天佳生物工程股份有限公司 | The bacillus subtilis strain SX3411 of one plant of production antibacterial peptide |
| CN108048373B (en) * | 2018-02-01 | 2021-02-09 | 安徽科技学院 | Bacillus subtilis AH1005 and application thereof |
| CN108715877A (en) * | 2018-04-27 | 2018-10-30 | 武汉华扬动物药业有限责任公司 | The method for preparing bacteriocin using bacillus subtilis |
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