CN103320370A - Bacillus subtilis C3 and its anti-Listeria monocytogenes bacteriocin preparation method - Google Patents
Bacillus subtilis C3 and its anti-Listeria monocytogenes bacteriocin preparation method Download PDFInfo
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Abstract
The name of the invention is a bacillus subtilis C3 and its anti-Listeria monocytogenes bacteriocin preparation method. The bacteriocin, used as a natural antiseptic, is suitable for low temperature meat and meat product and fruit and vegetable fresh-keeping, and can be used to raise safety of food. According to the invention, Bacillus subtilis C3 CGMCC No.9713 is utilized by ammonium sulfate precipitation, anion exchange chromatography and semi-preparative reversed phase liquid chromatography for fine purification. Thus, titer of the bacillus subtilis C3 bacteriocin is raised by 32 times, and purity is raised by 42.80 times. The bacteriocin has a very strong bacteriostatic activity of Listeria monocytogenes and good heat and pH stability, can be degraded by protease K, and is a natural safe biological preservative. The bacteriocin provided by the invention has advantages of wide sources of raw materials for production, simple extraction technology and efficient and stable purification method. The preparation method provided by the invention is suitable for industrial production.
Description
Technical field
The present invention relates to the preparation method of a kind of subtilis C3 in the microorganism field and anti-Listeria monocytogenes bacteriocin thereof.This bacteriocin is applicable to low temperature meat and meat product, preserving fruit and vegetable utilizing, the security that can improve food as natural antiseptic agent.
Background technology
Food is vulnerable to harmful microbe and pollutes and cause putrid and deteriorated or cause food poisoning in the process such as processing, storage, transportation, adopts the method for adding sanitas to suppress microbial growth, becomes gradually the important means of food fresh keeping.Traditional meat is mostly fresh-keeping by Chemical Preservative, has certain potential safety hazard, and improper use has certain side effect, and long-term excessive absorption causes certain infringement to human consumer's health, and causes environmental pollution.Therefore, efficient, the safety of research and development, the agent of eco-friendly natural meat product anticorrosion be food safety in the urgent need to.Bacteriocin refers to that some bacterium has polypeptide or the Precursor Peptide of bacteriostatic activity by a class of Ribosome biogenesis in metabolic process.Nisin studies the most widely bacteriocin so far, but because of alkalescence and poor heat stability, has limited its Applications in food.This project has filtered out a strain has obvious inhibition to Listeria monocytogenes subtilis C3, the bacteriocin that this bacterium produces has satisfactory stability to heat and pH, and can be degraded by Proteinase K, have higher-security and stability as food preservative freshness retaining agent, possess preferably development prospect.Therefore, set up a kind of preparation method efficient, that stablize subtilis C3 bacteriocin extremely important.The bacteriocin preparation method mainly contains ammonium sulfate precipitation, ion exchange chromatography, gel chromatography, high performance liquid chromatography etc.
The bacteriocin Applications in food is taked following three kinds of modes usually: (1) directly adds in food as biological preservative the bacteriocin behind the purifying; (2) bacteriocin is produced bacterium and produce leavened food as starter, can effectively prevent living contaminants in the fermenting process; (3) add bacteriocin in the raw material of preparation packaging material for food, exploitation has the food pack, preservative film of fungistatic effect etc.
At present relevant subtilis bacteriocin preparation method's patent report, such as " extracellular antiseptic protein of a kind of subtilis and separation purification method thereof ", the patent No. is: 200610097847.1; " bacteriocin of subtilis LFB112, its generation 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 the fungal disease control thereof ", application number is: 201110093415.4.Preparation method about the subtilis bacteriocin of anti-Listeria monocytogenes in present domestic patent of invention and the document there is not yet relevant report.
Summary of the invention
First purpose of the present invention provides 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 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, and the short chain shape is arranged, 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 Listeria monocytogenes is had stronger bacteriostatic activity, antibacterial circle diameter is 19.46mm.
The subtilis C3 with anti-Listeria monocytogenes activity that aforesaid method obtains belongs to protection domain of the present invention.
Second purpose of the present invention provides a kind of subtilis C3 bacteriocin preparation method with anti-Listeria monocytogenes activity.
Extracting method with anti-Listeria monocytogenes activated bacterial element provided by the present invention is the bacteriocin that is obtained by subtilis C3 fermentation.
(1) subtilis C3 fermented liquid is 4 ℃, and the centrifugal 15～20min of 10000～11000r/min removes thalline.
(2) adopt 55%～65% (W/V, mass volume ratio concentration) the ammonium sulfate precipitation subtilis C3 fermented supernatant fluid of saturation ratio, 4 ℃ leave standstill 12h, in 4 ℃, the centrifugal 15-20min of 10000～11000r/min, the collecting precipitation thing, the amount redissolution precipitation of pressing fermented liquid 1/10 volume with deionized water.
(3) will redissolve liquid and be distributed in the pretreated dialysis tubing, and place deionized water 4C dialysis 48h desalination, dialyzate is the bacteriocin crude extract.
(4) the bacteriocin crude extract filters with 0.21 μ m sterile filters, adopts DEAE-Sepharose Fast Flow anion exchange chromatography stepwise elution purification of bacterial element, and the 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/ pipe is collected the elutriant that 95～105min has bacteriostatic activity.
(5) activated collection liquid is distributed in the pretreated dialysis tubing dialysis 24h desalination in 4 ℃ of deionized waters.
(6) the ion-exchange dialyzate is concentrated under 10mbar, 50 ℃ of conditions with centrifugal vacuum concentration instrument, concentration ratio is 10: 1.
(7) the ion-exchange concentrated solution filters through 0.2 μ m sterile filters, adopts half preparation type reverse phase liquid chromatography linear gradient elution purification of bacterial element, and anti-phase preparative column model is Agilent ZORBAX300SB-C18, and the post specification is 9.4mm * 250mm; Moving phase: A liquid is acetonitrile+0.1% trifluoroacetic acid (V/V), and 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, collects the elutriant that 19～20min has bacteriostatic activity, and traditional vacuum is concentrated, removes acetonitrile, again with its lyophilize, the dried bacteriocin sterling pulverulence that is white in color.
The preparation method of the above-mentioned subtilis C3 bacteriocin with anti-Listeria monocytogenes activity belongs to protection domain of the present invention.
Subtilis (Bacillus subtilis) C3 separation screening is from Beijing Agricultural College's orchard soil, and the bacteriocin of its generation can be degraded by Proteinase K.The present invention utilizes ammonium sulfate precipitation, anion-exchange chromatography, half preparation type reversed-phase liquid chromatography method to prepare bacteriocin, finally tires and has improved 32 times, and purity has improved 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 protection scope of the present invention for following embodiment.
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the 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%, the pH nature;
The seed culture condition: will activate good subtilis and move in the seed culture medium by 1% inoculum size, 37 ℃ of culture temperature, 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 soak powder 1.5%, KH
2PO
40.1%, MgSO
47H
2O0.15%, NaCl0.5%, pH7.0;
Fermentation condition: seed liquor is moved in the fermention medium 37 ℃ of leavening temperatures, liquid amount 150mL/500mL, 180r/min shaking table oscillation and fermentation 48h by 2% inoculum size.
2, the preparation of subtilis C3 bacteriocin crude extract
(1) fermented liquid is with 10000～11000r/min, 15～20min, 4 ℃ of centrifugal removal thalline, supernatant liquor places the 1000mL beaker, takes by weighing ammonium sulfate after the grinding by saturation ratio 60% (390g/1000mL), slowly stirs at the uniform velocity in the same way with glass stick, add while stirring ammonium sulfate, complete operation within the 15min, standing over night in 4 ℃ of refrigerators, the amount redissolution precipitation of pressing 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 ratios of cup-plate method comparative determination, 70%, the fungistatic effect of 80%1 throw outs redissolution liquid, the result shows that 60% saturation ratio sedimentation effect is best, almost bacteriocin is all precipitated, and reduced as far as possible the difficulty that causes separation and purification because of the precipitation of medium component.
(2) fermented liquid is after precipitation with 10000～11000r/min, 15～20min, and 4 ℃ are 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 ℃ of deionized water dialysis 48h, and dialysate namely gets subtilis C3 bacteriocin crude extract.
Antagonistic property and the physico-chemical property of embodiment 2, subtilis C3 bacteriocin crude extract
Measure antagonistic property and the physico-chemical property of C3 bacteriocin crude extract in the process of the test with cup-plate method.
Cup-plate method: first the indicator bacterial strain is diluted to 10
7CFU/mL, behind the corresponding solid medium mixing of heating and melting, pour into approximately 15mL in plate, place gently aseptic Oxford cup after solidifying, get 100 μ L subtilis C3 crude extracts and add in the cup of Oxford, cultivate 12h for 37 ℃, observe inhibition zone, and measure 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
Annotate: Oxford cup diameter is 7.50mm.
By as seen from Table 1, subtilis C3 bacteriocin crude extract has restraining effect to the growth of Listeria monocytogenes, enterococcus faecalis, streptococcus aureus, aspergillus niger, white edge mould, wherein Listeria monocytogenes there is stronger bacteriostatic activity, 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 indicator strain in the physics and chemistry test.
2, the enzyme sensitivity test of subtilis C3 bacteriocin crude extract
Subtilis C3 bacteriocin crude extract is with 0.2 μ m sterile filters filtration sterilization.Process with catalase, with the hydrogen peroxide whether of the antibacterial substance in the checking C3 bacteriocin crude extract.3 kinds of common proteolytic enzyme are mixed with respectively 20mg/mL solution, and regulate the optimal pH of various enzyme liquid effect, join in the C3 bacteriocin crude extract, making its final concentration is 1mg/mL, 37 ℃ of water-bath 4h, the unified pH6.0 (pH is 6.0 after the fermentation) that recalls to uses the C3 bacteriocin crude extract of not enzyme-added processing as blank again, to verify that whether antibacterial substance is by proteasome degradation.Adopt cup-plate method to do the bacteriostatic test of Listeria monocytogenes, the results are shown in Table 2.
The enzyme susceptibility result of table 2 subtilis C3 bacteriocin crude extract
Annotate: Oxford cup diameter is 7.50mm.
By as seen from Table 2, after the hydrogen oxide enzyme is processed subtilis C3 bacteriocin, compare fungistatic effect with blank still stronger, illustrate that the C3 bacteriocin is not hydrogen peroxide.Proteinase K has the ability of degraded natural protein, after the Proteinase K processing, the anti-Listeria monocytogenes of C3 bacteriocin is active significantly to be reduced, and stomach en-and trypsinase are 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, as blank, measure bacteriocin crude extract after processing to the bacteriostatic activity of Listeria monocytogenes with the C3 bacteriocin of heat treated not, the results are shown in Table 3.
The thermostability result of table 3 subtilis C3 bacteriocin crude extract
Annotate: Oxford cup diameter is 7.50mm.
As shown in Table 3, compare with the blank group, 60～80 ℃ of heat treated 30～120min, antibacterial circle diameter all more than 18mm, illustrate that bacteriocin C3 bacteriostatic activity is still stronger, show that it is 60～80 ℃ of better heat stability; Process 30～60min for 100 ℃, antibacterial circle diameter illustrates that bacteriocin C3 still has better stability at 100 ℃ between 14.28～17.78mm; Antibacterial circle diameter is 12.22mm behind 121 ℃ of processing 15min, and its bacteriostatic activity has kept 63.1%, further specifies this bacteriocin thermostability stronger.Therefore this bacteriocin is applied in the food, and the food processing technology can not affect its bacteriostatic activity.
4, the ph stability of subtilis C3 bacteriocin crude extract
It is 2.0,3.0,4.0,5.0,7.0,8.0,9.0,10.0,11.0 that subtilis C3 bacteriocin crude extract is regulated pH with 1mol/L NaOH and 1mol/L HCl, 37 ℃ of water-bath 3h, pH6.0 is recalled in unification again, bacteriocin crude extract after mensuration is processed the results are shown in Table 4 to the bacteriostatic activity of Listeria monocytogenes.
The ph stability result of table 4 subtilis C3 bacteriocin crude extract
Annotate: Oxford cup diameter is 7.50mm.
As shown in Table 4, subtilis C3 bacteriocin crude extract is behind effect 3h under pH2.0～4.0 conditions, and antibacterial circle diameter is between 15.36～16.32mm, and bacteriostatic activity slightly reduces, and illustrates that this bacteriocin acid resistance is better; Under pH5.0～8.0 conditions, antibacterial circle diameter shows that its suitableeest action pH is 5.0～8.0 more than 18.0mm; Under pH9.0～11.0 conditions, 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, so this bacteriocin as aseptic applications in food, but the tolerance acid-base environment.
Embodiment 3, subtilis C3 bacteriocin purification process
1, the purifying of subtilis C3 bacteriocin crude extract
(1) adopts anion exchange chromatography stepwise elution purifying
Subtilis C3 bacteriocin crude extract with 0.2 μ m sterile filters filtration sterilization, is adopted DEAE-Sepharose Fast Flow anion exchange chromatography stepwise elution purifying, and the 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/ pipe adopts cup-plate method to detect and collects elutriant to the bacteriostatic activity of Listeria monocytogenes, and test shows that the elutriant of 95～105min has bacteriostatic activity.To have the collection elutriant of bacteriostatic activity to put into pretreated dialysis tubing, in 4 ℃ of deionized water dialysis 24h desalinations, dialyzate is the purified product of bacteriocin stepwise elution.
(2) adopt half preparation type reverse phase liquid chromatography linear gradient elution to be further purified
The ion-exchange dialyzate is concentrated under 10mbar, 50 ℃ of conditions with centrifugal vacuum concentration instrument, and concentration ratio is 10: 1.The ion-exchange concentrated solution filters through 0.21 μ m sterile filters, adopts half preparation type reverse phase liquid chromatography linear gradient elution purification of bacterial element, and anti-phase preparative column model is Agilent ZORBAX300SB-C18, and the post specification is 9.4mm * 250mm; Moving phase: A liquid is acetonitrile+0.1% trifluoroacetic acid, and 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 collecting is concentrated into clean doing, in redissolution to 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 collections have the elutriant of bacteriostatic activity to be the purified product of linear gradient elution.
2, subtilis C3 bacteriocin purification effect is estimated
(1) bacteriocin Potency Analysis
Determining of bacteriocin valence value: with the continuous doubling dilution of testing sample, adopt the Antibacterial Activity of bacteriocin in the cup-plate method working sample liquid.Vigor represents with every milliliter unit of activity (AU).The definition of AU is the inverse that can see the high dilution (n) of obvious inhibition zone.
(2) adopt Xylene Brilliant Cyanine G G-250 Determination Staining total protein content
Xylene Brilliant Cyanine G G-250100mg is dissolved in the 50mL95% ethanol, adds 100mL85% phosphoric acid, with distilled water diluting to 1000mL, filter paper filtering.Contain 0.01% (W/V) Xylene Brilliant Cyanine G G-250,4.7% (W/V) ethanol in the final reagent; The crystallization bovine serum albumin is measured protein nitrogen content through micro-Kjeldahl in advance, is mixed with 1mg/mL, the 0.1mg/mL protein solution according to its purity with 0.15% (W/V) NaCl; Get 14 test tubes, divide two groups to press table 5 operation.With A
595nm(595nm place light absorption value) is ordinate zou, and standard protein content is X-coordinate, the 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 the A that measures
595nmValue is found the amount that it is equivalent to standard protein at typical curve, thereby is calculated the protein concn (mg/mL) of unknown sample, and the typical curve parameter list sees Table 5.
The formula of bovine serum albumin (BSA) the protein typical curve gained that employing Xylene Brilliant Cyanine G method records is: y=12.111x+0.0173, R
2=0.9938, illustrate that the linear degree of this typical curve is higher, can be used as the typical curve of determination of protein concentration.
Table 5 Xylene Brilliant Cyanine G method is surveyed protein content typical curve parameter list
The evaluation of table 6 subtilis C3 bacteriocin purification effect
By as seen from Table 6, the ratio vigor of bacteriocin is increased to 4131.11AU/mg behind the purifying, and purification is 42.80 times, tires and has improved 32 times, shows thus and adopts the purification step of Simple fast can effectively reach the purifying purpose.
Project 1 under this patent: the sub-problem of 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 " " detects research and the application 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 under this patent: the sub-problem of the great special project of Department of Science and Technology's national science and technology " disease-resistant transgenic sheep rearing new variety " " foundation of disease-resistant transgenic sheep expansion traditional font system/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 under this patent: agricultural-food harmful microorganism and agricultural residual safety detection and control Beijing key lab 2012 ladder planning items
Item number: Z121106002812039
The project beginning and ending time: 2012.08-2013.08
Project leader: Zhang Hongxing
Claims (2)
1. subtilis (Bacillus subtilis) C3CGMCC No.9713.
2. one kind is utilized bacterial strain C3CGMCC No.9713 to be the anti-Listeria monocytogenes bacteriocin preparation method of subtilis (Bacillus subtilis), it is characterized in that: 4 ℃ of (1) subtilis C3 fermented liquids, centrifugal 15～the 20min of 10000～11000r/min removes thalline; (2) the ammonium sulfate precipitation subtilis C3 fermented supernatant fluid of employing 55%～65% saturation ratio, 4 ℃ leave standstill 12h; In 4 ℃, the centrifugal 15～20min of 10000～11000r/min, collecting precipitation thing, the amount redissolution precipitation of pressing fermented liquid 1/10 volume with deionized water; (3) will redissolve liquid and be distributed in the pretreated dialysis tubing, and place 4 ℃ of dialysis of deionized water 48h desalination, dialyzate is the bacteriocin crude extract; (4) the bacteriocin crude extract filters with 0.2 μ m sterile filters, adopts DEAE-Sepharose Fast Flow anion exchange chromatography stepwise elution purification of bacterial element, and the 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: 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/ pipe is collected the elutriant that 95～105min has bacteriostatic activity; (5) activated collection liquid is distributed in the pretreated dialysis tubing dialysis 24h desalination in 4 ℃ of deionized waters; (6) the ion-exchange dialyzate is concentrated under 10mbar, 50 ℃ of conditions with centrifugal vacuum concentration instrument, concentration ratio is 10: 1; (7) the ion-exchange concentrated solution filters through 0.21 μ m sterile filters, adopts half preparation type reverse phase liquid chromatography linear gradient elution purification of bacterial element, and anti-phase preparative column model is Agilent ZORBAX300SB-C18, and the post specification is 9.4mm * 250mm; Moving phase: A liquid is acetonitrile+0.1% trifluoroacetic acid, and 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, collects the elutriant that 19～20min has bacteriostatic activity, and traditional vacuum is concentrated, removes acetonitrile, again with its lyophilize, the dried bacteriocin sterling pulverulence that is white in color.
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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 |
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| 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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