CN108410941A - The method that antibacterial peptide is extracted from zymotic fluid - Google Patents
The method that antibacterial peptide is extracted from zymotic fluid Download PDFInfo
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Abstract
The invention discloses the methods that antibacterial peptide is extracted from zymotic fluid, including：It prepares seed liquor, prepare fermentation medium, fermentation, ultrafiltration, separation, culture prepares bacillus subtilis, S. cervisiae and aspergillus oryzae seed liquor respectively first, then seed liquor is inoculated in the fermentation medium that bean cake powder, wheatfeed, corn flour are formulated and is fermented, zymotic fluid obtains the polypeptide liquid of four sections of different molecular weights after removal of impurities, ultrafiltration, and polypeptide liquid successively detaches to obtain antibacterial peptide with liquid chromatography through gel permeation chromatography separation.It has the beneficial effect that：The method of the present invention prepares antibacterial peptide using mixed fermentation, the antibacterial peptide of four sections of different molecular weights is obtained after isolating and purifying, not only avoid the waste of active peptides in zymotic fluid, it can also more refine, study to differentiated the biological activity of segment polypeptide between each molecular weight area, while the method for the present invention improves accuracy, accuracy and the separative efficiency of antibacterial peptide separation.
Description
Technical field
The present invention relates to the extraction fields of antibacterial peptide, more particularly, to the method for extracting antibacterial peptide from zymotic fluid.
Technical background
Currently, all there are the pathogenic strain of corresponding drug resistance, the resistance problems of pathogenic bacteria in all Conventional antibiotics
Threaten people's health to getting worse.The antibiotic for finding brand new class is that one of solution resistance problems is effective
Approach.Antibacterial peptide is because antibacterial activity height, has a broad antifungal spectrum, type are more, alternative range is wide, target bacterial strain is not easy to produce resistance
The reasons such as mutation, and be considered to have broad application prospects on medical industry.Currently, having multiple polypeptides antibiotic
Preclinical feasibility study is being carried out,
Microbial fermentation refers under appropriate conditions being converted raw material to by specific metabolic pathway using microorganism
The process for the product that necessary for human is wanted.Microbial fermentation production level depends primarily on the hereditary capacity and culture item of strain itself
Part.The application range medical industry of Fermentation Engineering, food industry, energy industry, chemical industry, agricultural：Plant modification gene；It is raw
Object fixed nitrogen；Engineering bacterium for killing insect biological pesticide；Microorganism nutriment.Environmental protection etc..
Invention content
The purpose of the present invention is to provide the method for extracting antibacterial peptide from zymotic fluid, the method for the present invention is sent out using Mixed Microbes
Ferment prepares antibacterial peptide, and the antibacterial peptide of four sections of different molecular weights is obtained after isolating and purifying, and not only avoids active peptides in zymotic fluid
Waste, can also more refine, study to differentiated the biological activity of segment polypeptide between each molecular weight area, while this hair
Bright method improves accuracy, accuracy and the separative efficiency of antibacterial peptide separation.
Microorganism used in the method for the present invention and its bought enterprise are：Bacillus subtilis, the macro animal of Cangzhou City benefit
Health products Co., Ltd；S. cervisiae, Yantai Man Sen commerce and trade Co., Ltd；Aspergillus oryzae, the micro- agriculture biotechnology in Baoding are limited
Company.
The present invention is directed to the problem of being mentioned in background technology, and the technical solution taken is：Antibacterial peptide is extracted from zymotic fluid
Method, including：It prepares seed liquor, prepare fermentation medium, fermentation, ultrafiltration, separation, specifically include following steps：
Prepare seed liquor：Bacillus subtilis culture uses nutrient broth medium, in 140~160r/min, 35~37 DEG C of items
24~48h is cultivated under part；S. cervisiae culture uses nutrient broth medium, in 120~150r/min, 36~38 DEG C of conditions
24~36h of lower culture；Aspergillus oryzae culture uses Czapek's medium, in 160~180r/min, 28~30 DEG C of 48~72h of culture；Point
It is 1.0~1.2 × 10 not adjust bacterium number with sterile saline8cfu/mL；Under different condition of culture, it is withered to expand culture respectively
Careless bacillus, S. cervisiae and aspergillus oryzae utilize the battalion such as sufficient nitrogen source, carbon source and trace element in nutrient medium
Foster substance makes each strain be rapidly reached the eugonic stage, prepares for further mixed fungus fermentation；
Prepare fermentation medium：Using 100~120 parts of bean cake powders as major ingredient, with 12~15 parts of wheat bran powder, 10~15 parts of corns
Powder is auxiliary material, separately adds 0.8~1.0 part of honey, 1~1.5 portion of sucrose, addition distilled water to water content is 45~48%, after sterilizing
0.3~0.5 part of fermentation synergist is added up to fermentation medium；Higher moisture content is microorganism panning nutrition in fermentation medium
Substance provides channel, can promote microorganism growth, the progress of enzymatic reaction and distributing for fermentation heat production, fermentation synergist
The growth and breeding that microorganism species can be remarkably promoted, to improve fermenting speed and fermentation yield；
Fermentation：According to weight ratio 1:0.3~0.5:0.3~0.5 ratio takes bacillus subtilis seed liquor, saccharomyces cerevisiae strain
Mixed bacteria liquid is inoculated with by sub- liquid and aspergillus oryzae seed liquor according to 10.5~12.5% ratio of fermentation medium dry matter content
Enter in fermentation medium, 72~84h is left to ferment in 28~30 DEG C of constant incubators；Oscillation water logging extraction 45 after fermentation
~60min, rear quiescent settling, 2~3 times slagging-off of filtered through gauze obtain fermented bean dregs stoste；It can be played using mixed fungus fermentation more
Synergistic effect between kind of strain, can reduce the disadvantage of single bacterium fermentation, and there may be synbiosis, metabolites between three kinds of strains
Complementary effect, the unfavorable factor that intermediate product can be overcome excessively to be generated to antibacterial peptide occurs, therefore is conducive to antibacterial peptide yield
With the raising of yield；
Ultrafiltration：Zymotic fluid sequentially adds activated carbon, DA201-C macroporous resin adsorptions grease, impurity particle and desalination, afterwards with 400
~500 mesh sock filtrations remove adsorbent, add florisil silica combination plate filter and are removed by filtration activated carbon and extra
Grease obtains more clear zymotic fluid；With the ultrafiltration membrane classified filtering zymotic fluid that molecular cut off is 10KDa, 3KDa, 1kDa,
Obtain the polypeptide mixed liquor component T1 of four sections of different molecular weights（>10kDa）、T2（10~3kDa）、T3（3~1kDa）With T4（<
1kDa）, it is freeze-dried 48~60h after concentration in the case where temperature is -10~-20 DEG C, vacuum degree is 50~100Pa respectively；It will fermentation
Liquid carries out ultrafiltration by three kinds of ultrafiltration membranes, obtains the polypeptide mixed liquor of four sections of different molecular weights, not only avoids in zymotic fluid
The waste of active peptides can also be refined more, study to differentiated the biological activity of segment polypeptide between each molecular weight area；
Separation：0.12~0.15g/mL concentrated solutions are made with ultrapure water dissolution respectively in T1, T2, T3 and T4 component, successively with solidifying
The separation of glue penetration chromatography is detached with liquid chromatography, collects each eluting peak component respectively, be freeze-dried up to antibacterial peptide；
Through gel permeation chromatography and the isolated antibacterial peptide components of liquid chromatogram to Escherichia coli, deformation in T1, T2, T3 and T4 component
The Glanzs negative bacterium such as bacillus, Brucella has stronger inhibiting effect.
Preferably, preparing the substance that the fermentation synergist in fermentation medium step includes following content：70~80% urine
Element, 3~5% α-naphthaleneacidsodiums, 15~18% calcium oxide, 3~5% magnesium sulfate, 1~5% dimethyl phosphate；Ammonification work can occur for urea
With dregs of beans part, wheatfeed and the corn flour porosity after ammoniated treatment increase, and increase with the contact area of enzyme, are conducive to send out
The progress of ferment；The α-naphthaleneacidsodium Yu dimethyl phosphate of special proportioning have synergistic effect, the synergistic effect in fermentation synergist
Bacillus subtilis can be promoted to enhance the secretion to acetyl coenzyme A, and then promote its condensation reaction with oxaloacetic acid, accelerated
The synthesis of citric acid can increase substantially the speed of bacillus subtilis tricarboxylic acid cycle, accelerate the life of bacillus subtilis
Long breeding and fermentating metabolism promote bacillus subtilis to be metabolized out more enzymes and are acted on fermentation substrate, and then improve antibacterial peptide
Yield and yield.
Preferably, the chromatographic condition of gel permeation chromatography separation is：φ 7.8mm × 300mm, mobile phase A are ultrapure
Water, Mobile phase B are acetonitrile；Condition of gradient elution：0~10min 5%B, 10~15min 5%B, 15~20min 10%B；Flow velocity：
0.8~1.0mL/min；Column temperature：28~30 DEG C；Wavelength：220nm；Collect each eluting peak component；When gel permeation chromatography detaches,
The antibacterial peptide of larger molecular weight can only pass through from the gap between gel particles, and flow velocity is very fast, and the antibacterial peptide of relatively small molecular weight enters
Through-hole in gel particles, flow velocity is slower, then selects 220nm as absorbing wavelength, can guarantee the sensitivity and response of detection
It is worth highest, gel permeation chromatography separating rate is very fast, separation accuracy is higher, while will not destroy the structure and property of antibacterial peptide again
Matter；
Preferably, the chromatographic condition of liquid chromatography separation is：φ 4.6mm × 250mm, 100A；Mobile phase A is ultra-pure water,
Mobile phase B is acetonitrile；Condition of gradient elution：0~5min 2.5%B, 5~18min 80%B；Flow velocity：0.8~1.0mL/min；Column
Temperature：28~30 DEG C；Wavelength：220nm；Collect each eluting peak component；Liquid chromatography be the difference based on substance suction-operated and
It realizes its separation, has the advantages that quick, sensitive, selectivity is good, and will not be right during isolating and purifying antibacterial peptide
Antibacterial peptide pollutes, degrades.
Preferably, gel permeation chromatography separation with liquid chromatography lock out operation in mobile phase A and Mobile phase B in
The Dimethylsilanediol of the trifluoroacetic acid containing 550~580ppm, 35~38ppm；Gel permeation chromatography is solid with liquid chromatogram
The dielectric surface remaining for determining phase has a small amount of uncapped hydroxyl, this part of hydroxyl to be easy dissociation in mobile phase, lead to chromatogram
Deformation is bad or separation fails, and trifluoroacetic acid is a kind of strong ion-pairing agent, inhibits hydroxyl by dissociateing hydrogen ion
Dissociation, it is acidity to make the pH value of system, is also prevented from the long bacterium of mobile phase, is conducive to the separation at peak；In addition, in stationary phase in addition to
The silicone hydroxyl of remnants when also having filler bonding outside carbon long-chain, and silicone hydroxyl is easier to cause the secondary reserve effects of sample molecule generation
Hangover, Dimethylsilanediol can react away silicone hydroxyl, make the hydrogen in silicone hydroxyl become one of Dimethylsilanediol it is lazy
Property group, reacts away many silicone hydroxyls, while also having shielding action, reagentia to be cooperateed with shielding action remaining silicone hydroxyl
Silicone hydroxyl this action site is eliminated, the chance for effectively preventing sample to be contacted with silicone hydroxyl avoids secondary reserve effects
Generation, also just effectively avoid trailing phenomenon, improve accuracy and the accuracy of separation, improve separative efficiency.
Compared with the prior art, the advantages of the present invention are as follows：
1）In the method for the present invention, the synergistic effect between a variety of strains can be played using mixed fungus fermentation, single bacterium fermentation can be reduced
Disadvantage, there may be synbiosis, metabolite, and complementary effect occurs, intermediate product can be overcome excessively multipair between three kinds of strains
The unfavorable factor that antibacterial peptide generates, therefore be conducive to the raising of antibacterial peptide yield and yield；
2）The α-naphthaleneacidsodium Yu dimethyl phosphate of special proportioning have synergistic effect, the synergistic effect can be in fermentation synergist
Promote bacillus subtilis to enhance the secretion to acetyl coenzyme A, and then promote its condensation reaction with oxaloacetic acid, accelerates lemon
The synthesis of acid, can increase substantially the speed of bacillus subtilis tricarboxylic acid cycle, the growth for accelerating bacillus subtilis is numerous
It grows and fermentating metabolism, promotes bacillus subtilis to be metabolized out more enzymes and acted on fermentation substrate, and then improve the production of antibacterial peptide
Rate and yield；
3）Dimethylsilanediol can react away silicone hydroxyl, make the hydrogen in silicone hydroxyl become one of Dimethylsilanediol it is lazy
Property group, reacts away many silicone hydroxyls, while also having shielding action, reagentia to be cooperateed with shielding action remaining silicone hydroxyl
Silicone hydroxyl this action site is eliminated, the chance for effectively preventing sample to be contacted with silicone hydroxyl avoids secondary reserve effects
Generation, also just effectively avoid trailing phenomenon, improve accuracy and the accuracy of separation, improve separative efficiency；
4）The method of the present invention prepares antibacterial by using bacillus subtilis, S. cervisiae and aspergillus oryzae mixed fermentation
Zymotic fluid is carried out ultrafiltration by peptide by three kinds of ultrafiltration membranes, obtains detach after the polypeptide mixed liquor of four sections of different molecular weights pure
Change, not only avoids the waste of active peptides in zymotic fluid, can also more refine, study to differentiated each molecular weight
The biological activity of section segment polypeptide is a kind of method for efficiently, accurately extracting antibacterial peptide from zymotic fluid.
Specific implementation mode
The present invention program is described further below by embodiment：
Embodiment 1：
The method that antibacterial peptide is extracted from zymotic fluid, includes the following steps：
1）Bacillus subtilis culture uses nutrient broth medium, is cultivated for 24 hours under the conditions of 140r/min, 35 DEG C；Wine brewing ferment
Female bacterium culture uses nutrient broth medium, is cultivated for 24 hours under the conditions of 120r/min, 36 DEG C；Aspergillus oryzae culture is trained using Cha Shi
Base is supported, in 160r/min, 28 DEG C of culture 48h；It is 1.0 × 10 to adjust bacterium number with sterile saline respectively8cfu/mL；2）With
100 parts of bean cake powders are major ingredient, using 12 parts of wheat bran powder, 10 portions of corn flour as auxiliary material, separately add 0.8 part of honey, 1 portion of sucrose,
It is 45% to add distilled water to water content, and 0.3 part of fermentation synergist is added after sterilizing up to fermentation medium；3）According to weight ratio
1:0.3:0.3 ratio takes bacillus subtilis seed liquor, S. cervisiae seed liquor and aspergillus oryzae seed liquor, by Mixed Microbes
Liquid is inoculated with according to 10.5% ratio of fermentation medium dry matter content in fermentation medium, quiet in 28 DEG C of constant incubators
Set fermentation 72h；45min, rear quiescent settling are extracted in oscillation water logging after fermentation, and 2 times slagging-off of filtered through gauze obtain fermented bean dregs
Stoste；4）Zymotic fluid sequentially adds activated carbon, DA201-C macroporous resin adsorptions grease, impurity particle and desalination, rear 400 mesh
Sock filtration removes adsorbent, adds florisil silica combination plate filter and is removed by filtration activated carbon and excess oil, obtains
To more clear zymotic fluid；With the ultrafiltration membrane classified filtering zymotic fluid that molecular cut off is 10KDa, 3KDa, 1kDa, four sections are obtained
The polypeptide mixed liquor component T1 of different molecular weight（>10kDa）、T2（10~3kDa）、T3（3~1kDa）With T4（<1kDa）, concentration
Afterwards 48h is freeze-dried in the case where temperature is -10 DEG C, vacuum degree is 50Pa respectively；5）By T1, T2, T3 and T4 component respectively with ultrapure
0.12g/mL concentrated solutions are made in water dissolution, are successively detached with liquid chromatography with gel permeation chromatography separation, collect respectively each
Eluting peak component be freeze-dried up to antibacterial peptide.
Embodiment 2：
The method that antibacterial peptide is extracted from zymotic fluid, includes the following steps：
1）Prepare seed liquor：Bacillus subtilis culture uses nutrient broth medium, is cultivated under the conditions of 160r/min, 37 DEG C
48h；S. cervisiae culture uses nutrient broth medium, and 36h is cultivated under the conditions of 150r/min, 38 DEG C；Aspergillus oryzae culture
Using Czapek's medium, in 180r/min, 30 DEG C of culture 72h；Respectively with sterile saline adjust bacterium number be 1.2 ×
108cfu/mL；Under different condition of culture, expands culture bacillus subtilis, S. cervisiae and aspergillus oryzae respectively, utilize
The nutriments such as sufficient nitrogen source, carbon source and trace element in nutrient medium make each strain be rapidly reached eugonic rank
Section is prepared for further mixed fungus fermentation；
2）Prepare fermentation medium：Using 120 parts of bean cake powders as major ingredient, using 15 parts of wheat bran powder, 15 portions of corn flour as auxiliary material, separately
Add 1.0 parts of honey, 1.5 portions of sucrose, addition distilled water to water content is 48%, 0.5 part of fermentation synergist is added after sterilizing to obtain the final product
Fermentation medium, fermentation synergist include the substance of following content：70% urea, 5% α-naphthaleneacidsodium, 15% calcium oxide, 5% sulfuric acid
Magnesium, 5% dimethyl phosphate；Higher moisture content provides channel for microorganism panning nutriment in fermentation medium, can promote
Microorganism growth, the progress of enzymatic reaction and distributing for fermentation heat production, fermentation synergist can also remarkably promote microorganism species
Growth and breeding, to improve fermenting speed and fermentation yield；
3）Fermentation：According to weight ratio 1:0.5:0.5 ratio takes bacillus subtilis seed liquor, S. cervisiae seed liquor and rice
Mixed bacteria liquid is inoculated with according to 12.5% ratio of fermentation medium dry matter content in fermentation medium by Aspergillus seed liquor,
It is left to ferment 84h in 30 DEG C of constant incubators；60min, rear quiescent settling, filtered through gauze are extracted in oscillation water logging after fermentation
3 times slagging-off, obtain fermented bean dregs stoste；Synergistic effect between a variety of strains can be played using mixed fungus fermentation, list can be reduced
The disadvantage of bacterium fermentation, there may be synbiosis, metabolite, and complementary effect occurs, can overcome intermediate product between three kinds of strains
The unfavorable factor that excessively antibacterial peptide is generated, therefore be conducive to the raising of antibacterial peptide yield and yield；
4）Ultrafiltration：Zymotic fluid sequentially adds activated carbon, DA201-C macroporous resin adsorptions grease, impurity particle and desalination, rear to use
500 mesh sock filtrations remove adsorbent, add florisil silica combination plate filter and are removed by filtration activated carbon and extra oil
Fat obtains more clear zymotic fluid；With the ultrafiltration membrane classified filtering zymotic fluid that molecular cut off is 10KDa, 3KDa, 1kDa, obtain
To the polypeptide mixed liquor component T1 of four sections of different molecular weights（>10kDa）、T2（10~3kDa）、T3（3~1kDa）With T4（<
1kDa）, after concentration 60h is freeze-dried in the case where temperature is -20 DEG C, vacuum degree is 100Pa respectively；Zymotic fluid is passed through into three kinds of ultrafiltration
Film carries out ultrafiltration, obtains the polypeptide mixed liquor of four sections of different molecular weights, not only avoids the waste of active peptides in zymotic fluid,
It can also more refine, study to differentiated the biological activity of segment polypeptide between each molecular weight area；
5）Separation：0.15g/mL concentrated solutions are made with ultrapure water dissolution respectively in T1, T2, T3 and T4 component, are successively oozed with gel
Saturating chromatography separation is detached with liquid chromatography, collects each eluting peak component respectively, be freeze-dried up to antibacterial peptide；T1、
Through gel permeation chromatography and the isolated antibacterial peptide components of liquid chromatogram to Escherichia coli, deformed rod in T2, T3 and T4 component
The Glanzs negative bacterium such as bacterium, Brucella has stronger inhibiting effect.
Gel permeation chromatography separation chromatographic condition be：φ 7.8mm × 300mm, mobile phase A are ultra-pure water, Mobile phase B
For acetonitrile；Condition of gradient elution：0~10min 5%B, 10~15min 5%B, 15~20min 10%B；Flow velocity：1.0mL/min；
Column temperature：30℃；Wavelength：220nm；Collect each eluting peak component；Liquid chromatography separation chromatographic condition be：φ4.6mm×
250mm, 100A；Mobile phase A is ultra-pure water, and Mobile phase B is acetonitrile；Condition of gradient elution：0~5min 2.5%B, 5~18min
80%B；Flow velocity：0.8~1.0mL/min；Column temperature：30℃；Wavelength：220nm；Collect each eluting peak component.
Gel permeation chromatography separation contains with the mobile phase A and Mobile phase B in liquid chromatography lock out operation
The trifluoroacetic acid of 580ppm, the Dimethylsilanediol of 38ppm；The dielectric surface of gel permeation chromatography and Stationary Phase for HPLC is residual
There is a small amount of uncapped hydroxyl, this part of hydroxyl is easy dissociation in mobile phase, chromatomap is caused to degenerate or detach mistake
It loses, and trifluoroacetic acid is a kind of strong ion-pairing agent, inhibits the dissociation of hydroxyl by dissociateing hydrogen ion, makes the pH value of system
For acidity, it is also prevented from the long bacterium of mobile phase, is conducive to the separation at peak；In addition, there is filler in stationary phase other than carbon long-chain
Remaining silicone hydroxyl when bonding, and silicone hydroxyl is easier to cause to trail to the secondary reserve effects of sample molecule generation, dimethyl-silicon two
Alcohol can react away silicone hydroxyl, so that the hydrogen in silicone hydroxyl is become an inertia group of Dimethylsilanediol, react away very
More silicone hydroxyls, while also having shielding action to remaining silicone hydroxyl, reagentia cooperateed with shielding action eliminate silicone hydroxyl this
A action site, the chance for effectively preventing sample to be contacted with silicone hydroxyl avoid the generation of secondary reserve effects, also just effectively
Ground avoids trailing phenomenon, improves accuracy and the accuracy of separation, improves separative efficiency.
Embodiment 3：
The method that antibacterial peptide is extracted from zymotic fluid, including：It prepares seed liquor, prepare fermentation medium, fermentation, ultrafiltration, separation,
Specifically include following steps：
Prepare seed liquor：Bacillus subtilis culture uses nutrient broth medium, is cultivated under the conditions of 150r/min, 36 DEG C
36h；S. cervisiae culture uses nutrient broth medium, and 30h is cultivated under the conditions of 150r/min, 37 DEG C；Aspergillus oryzae culture
Using Czapek's medium, in 170r/min, 28 DEG C of culture 48h；Respectively with sterile saline adjust bacterium number be 1.2 ×
108cfu/mL；Under different condition of culture, expands culture bacillus subtilis, S. cervisiae and aspergillus oryzae respectively, utilize
The nutriments such as sufficient nitrogen source, carbon source and trace element in nutrient medium make each strain be rapidly reached eugonic rank
Section is prepared for further mixed fungus fermentation；
Prepare fermentation medium：Using 115 parts of bean cake powders as major ingredient, using 15 parts of wheat bran powder, 10 portions of corn flour as auxiliary material, separately add
Add 1 part of honey, 1 portion of sucrose, addition distilled water to water content is 45%, and 0.4 part of fermentation synergist is added after sterilizing and ferments to obtain the final product training
Support base；Higher moisture content provides channel for microorganism panning nutriment in fermentation medium, microorganism can be promoted to grow,
The progress of enzymatic reaction and distributing for fermentation heat production, fermentation synergist can also remarkably promote the growth and breeding of microorganism species,
To improve fermenting speed and fermentation yield；
Fermentation：According to weight ratio 1:0.4:0.4 ratio takes bacillus subtilis seed liquor, S. cervisiae seed liquor and meter Qu
Mixed bacteria liquid is inoculated with according to 12% ratio of fermentation medium dry matter content in fermentation medium, 28 by mould seed liquor
It is left to ferment 72h in DEG C constant incubator；60min, rear quiescent settling, filtered through gauze 3 times are extracted in oscillation water logging after fermentation
Slagging-off, obtains fermented bean dregs stoste；Synergistic effect between a variety of strains can be played using mixed fungus fermentation, single bacterium hair can be reduced
The disadvantage of ferment, there may be synbiosis, metabolite, and complementary effect occurs, intermediate product can be overcome excessive between three kinds of strains
To the unfavorable factor that antibacterial peptide generates, therefore be conducive to the raising of antibacterial peptide yield and yield；
Ultrafiltration：Zymotic fluid sequentially adds activated carbon, DA201-C macroporous resin adsorptions grease, impurity particle and desalination, afterwards with 400
Mesh sock filtration removes adsorbent, adds florisil silica combination plate filter and is removed by filtration activated carbon and excess oil,
Obtain more clear zymotic fluid；With the ultrafiltration membrane classified filtering zymotic fluid that molecular cut off is 10KDa, 3KDa, 1kDa, four are obtained
The polypeptide mixed liquor component T1 of section different molecular weight（>10kDa）、T2（10~3kDa）、T3（3~1kDa）With T4（<1kDa）, dense
After contracting 48h is freeze-dried in the case where temperature is -15 DEG C, vacuum degree is 80Pa respectively；Zymotic fluid is surpassed by three kinds of ultrafiltration membranes
Filter, obtains the polypeptide mixed liquor of four sections of different molecular weights, not only avoids the waste of active peptides in zymotic fluid, can also be more
The biological activity for adding fining, studying to differentiated segment polypeptide between each molecular weight area；
Separation：0.14g/mL concentrated solutions are made with ultrapure water dissolution respectively in T1, T2, T3 and T4 component, successively with gel infiltration
Chromatography separation is detached with liquid chromatography, collects each eluting peak component respectively, be freeze-dried up to antibacterial peptide；T1、T2、
In T3 and T4 components through gel permeation chromatography and the isolated antibacterial peptide components of liquid chromatogram to Escherichia coli, proteus,
The Glanzs negative bacterium such as Brucella has stronger inhibiting effect.
Prepare the substance that the fermentation synergist in fermentation medium step includes following content：73% urea, 3% α-naphthylacetic acid
Sodium, 16% calcium oxide, 4% magnesium sulfate, 4% dimethyl phosphate；Ammoniation can occur for urea, and dregs of beans part, wheatfeed and corn flour pass through
Porosity increases after ammoniated treatment, increases with the contact area of enzyme, is conducive to the progress of fermentation；It is special in fermentation synergist to match
The α-naphthaleneacidsodium of ratio with dimethyl phosphate there is synergistic effect, the synergistic effect bacillus subtilis enhancing can be promoted to second
The secretion of acyl coenzyme A, and then promote its condensation reaction with oxaloacetic acid, accelerate the synthesis of citric acid, can increase substantially
The speed of bacillus subtilis tricarboxylic acid cycle accelerates the growth and breeding and fermentating metabolism of bacillus subtilis, promotes withered grass bud
Spore bacillus is metabolized out more enzymes and is acted on fermentation substrate, and then improves the yield and yield of antibacterial peptide.
Gel permeation chromatography separation chromatographic condition be：φ 7.8mm × 300mm, mobile phase A are ultra-pure water, Mobile phase B
For acetonitrile；Condition of gradient elution：0~10min 5%B, 10~15min 5%B, 15~20min 10%B；Flow velocity：1mL/min；Column
Temperature：28℃；Wavelength：220nm；Collect each eluting peak component；When gel permeation chromatography detaches, the antibacterial peptide of larger molecular weight can only
Pass through from the gap between gel particles, flow velocity is very fast, and the antibacterial peptide of relatively small molecular weight enters the through-hole in gel particles, flow velocity compared with
Slowly, it then selects 220nm as absorbing wavelength, can guarantee sensitivity and the response highest of detection, gel permeation chromatography point
It is higher from speed, separation accuracy, while the structure and property of antibacterial peptide will not be destroyed again；
Liquid chromatography separation chromatographic condition be：φ 4.6mm × 250mm, 100A；Mobile phase A is ultra-pure water, and Mobile phase B is
Acetonitrile；Condition of gradient elution：0~5min 2.5%B, 5~18min 80%B；Flow velocity：1mL/min；Column temperature：28℃；Wavelength：
220nm；Collect each eluting peak component；Liquid chromatography is the difference based on substance suction-operated and realizes what it was detached, is had
Quickly, advantage sensitive, selectivity is good, and antibacterial peptide will not be polluted, be dropped during isolating and purifying antibacterial peptide
Solution.
Gel permeation chromatography separation contains with the mobile phase A and Mobile phase B in liquid chromatography lock out operation
The trifluoroacetic acid of 564ppm, the Dimethylsilanediol of 36ppm；The dielectric surface of gel permeation chromatography and Stationary Phase for HPLC is residual
There is a small amount of uncapped hydroxyl, this part of hydroxyl is easy dissociation in mobile phase, chromatomap is caused to degenerate or detach mistake
It loses, and trifluoroacetic acid is a kind of strong ion-pairing agent, inhibits the dissociation of hydroxyl by dissociateing hydrogen ion, makes the pH value of system
For acidity, it is also prevented from the long bacterium of mobile phase, is conducive to the separation at peak；In addition, there is filler in stationary phase other than carbon long-chain
Remaining silicone hydroxyl when bonding, and silicone hydroxyl is easier to cause to trail to the secondary reserve effects of sample molecule generation, dimethyl-silicon two
Alcohol can react away silicone hydroxyl, so that the hydrogen in silicone hydroxyl is become an inertia group of Dimethylsilanediol, react away very
More silicone hydroxyls, while also having shielding action to remaining silicone hydroxyl, reagentia cooperateed with shielding action eliminate silicone hydroxyl this
A action site, the chance for effectively preventing sample to be contacted with silicone hydroxyl avoid the generation of secondary reserve effects, also just effectively
Ground avoids trailing phenomenon, improves accuracy and the accuracy of separation, improves separative efficiency.
Embodiment 4：
The biocidal property of antibacterial peptide is tested：
1）Filter paper is sterilized 12min under 121 DEG C, normal pressure, respectively by T1, T2, T3 and T4 component（Embodiment 3）It is configured to
0.1%, 0.2%, 0.3%, 0.4% and 0.5% peptide liquid, while control test is done, the filter paper after sterilizing is individually positioned in peptide liquid
In, impregnate 12h；
2）Using gradient dilution method, the culture plate that first generation activation staphylococcus aureus bacterium solution is diluted to 1000 times is prepared,
Culture for 24 hours, is distributed uniform bacterium colony；
3）Filter paper is placed on culture plate containing bacterium, cultivates 48h, the results are shown in Table 1 for observation.
Antibacterial circle diameter in the test of 1. biocidal property of table（mm）
The antibacterial peptide gone out as shown in Table 1 from broth extraction has the effect of preferably inhibiting staphylococcus aureus, and T3
（3~1 KDa）The fungistatic effect of molecule section is the most excellent, at the same can further be seen that antibacterial peptide fungistatic effect have dosage according to
Lai Xing.
Routine operation in operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. the method that antibacterial peptide is extracted from zymotic fluid, including：Seed liquor is prepared, fermentation medium is prepared, fermentation, ultrafiltration, divides
From, it is characterised in that：It is added in fermentation medium in the preparation fermentation medium step and includes the following hair containing quantity of material
Ferment synergist：0~80% urea, 3~5% α-naphthaleneacidsodiums, 15~18% calcium oxide, 3~5% magnesium sulfate, 1~5% phosphoric acid diformazan
Ester, total amount 100%.
2. the method according to claim 1 for extracting antibacterial peptide from zymotic fluid, it is characterised in that：The preparation fermentation training
Supporting base step is：Using 100~120 parts of bean cake powders as major ingredient, supplemented by 12~15 parts of wheat bran powder, 10~15 portions of corn flour
Material separately adds 0.8~1.0 part of honey, 1~1.5 portion of sucrose, and addition distilled water to water content is 45~48%, is added after sterilizing
0.3~0.5 part of fermentation synergist is up to fermentation medium.
3. the method according to claim 1 for extracting antibacterial peptide from zymotic fluid, it is characterised in that：The preparation seed liquor
Step is：Culture prepares bacillus subtilis, S. cervisiae and aspergillus oryzae seed liquor respectively, respectively with sterile saline tune
It is 1.0~1.2 × 10 to save bacterium number8cfu/mL。
4. the method according to claim 1 for extracting antibacterial peptide from zymotic fluid, it is characterised in that：The fermentation step
For：The seed mixture liquid for taking bacillus subtilis, S. cervisiae and aspergillus oryzae, according to fermentation medium dry matter content
10.5~12.5% ratio is inoculated in fermentation medium, oscillation water logging extraction, rear quiescent settling, yarn after constant temperature standing for fermentation
Cloth filtering and removing slag obtain fermented bean dregs stoste.
5. the method according to claim 4 for extracting antibacterial peptide from zymotic fluid, it is characterised in that：The fermentation step
In, the mass ratio of bacillus subtilis seed liquor, S. cervisiae seed liquor and aspergillus oryzae seed liquor is 1:0.3~0.5:
0.3~0.5.
6. the method according to claim 1 for extracting antibacterial peptide from zymotic fluid, it is characterised in that：The ultrafiltration step
For：Zymotic fluid sequentially adds activated carbon, macroporous resin adsorption grease, impurity particle and desalination, after removed and adsorb with sock filtration
Agent adds filter and is removed by filtration activated carbon and excess oil, obtains clarified broth；With molecular cut off be 10KDa,
The ultrafiltration membrane classified filtering zymotic fluid of 3KDa, 1kDa obtain the polypeptide mixed liquor component of four sections of different molecular weights, after concentration respectively
It is dried in vacuo.
7. the method according to claim 1 for extracting antibacterial peptide from zymotic fluid, it is characterised in that：The separating step
For：0.12~0.15g/mL concentrated solutions are made with ultrapure water dissolution respectively in the polypeptide fractions of four sections of different molecular weights, successively with
Gel permeation chromatography separation is detached with liquid chromatography, collects each eluting peak component respectively, is freeze-dried up to antibacterial peptide.
8. the method according to claim 7 for extracting antibacterial peptide from zymotic fluid, it is characterised in that：The separating step
In, the chromatographic condition of gel permeation chromatography separation is：φ 7.8mm × 300mm, mobile phase A are ultra-pure water, and Mobile phase B is second
Nitrile；Condition of gradient elution：0~10min 5%B, 10~15min 5%B, 15~20min 10%B, flow velocity are 0.8~1.0mL/
Min, column temperature are 28~30 DEG C, wavelength 220nm.
9. the method according to claim 7 for extracting antibacterial peptide from zymotic fluid, it is characterised in that：The separating step
In, the chromatographic condition of liquid chromatography separation is：φ 4.6mm × 250mm, 100A；Mobile phase A is ultra-pure water, and Mobile phase B is second
Nitrile；Condition of gradient elution：0~5min 2.5%B, 5~18min 80%B, flow velocity are 0.8~1.0mL/min, and column temperature is 28~30
DEG C, wavelength 220nm.
10. the method for extracting antibacterial peptide in the slave zymotic fluid according to claim 8,9, it is characterised in that：The separation step
In rapid, gel permeation chromatography separation with contain 550 in the mobile phase A and Mobile phase B in liquid chromatography lock out operation~
The trifluoroacetic acid of 580ppm, the Dimethylsilanediol of 35~38ppm.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406701A (en) * | 2018-09-26 | 2019-03-01 | 江南大学 | A method of the water-soluble polypeptide in separation identification spirit stillage |
| CN111607627A (en) * | 2020-05-12 | 2020-09-01 | 南京大方生物工程有限公司 | Fermentation process for producing antibacterial peptide bacillus subtilis and application of fermentation process in disease-resistant and yield-increasing of livestock and poultry |
| CN113354025A (en) * | 2021-06-07 | 2021-09-07 | 烟台金堃新材料科技有限公司 | Treatment method of oily wastewater |
| CN114097956A (en) * | 2021-11-16 | 2022-03-01 | 厦门嘉康饲料有限公司 | Functional feed for litopenaeus vannamei and preparation method thereof |
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2018
- 2018-04-03 CN CN201810290867.3A patent/CN108410941A/en not_active Withdrawn
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406701A (en) * | 2018-09-26 | 2019-03-01 | 江南大学 | A method of the water-soluble polypeptide in separation identification spirit stillage |
| CN111607627A (en) * | 2020-05-12 | 2020-09-01 | 南京大方生物工程有限公司 | Fermentation process for producing antibacterial peptide bacillus subtilis and application of fermentation process in disease-resistant and yield-increasing of livestock and poultry |
| CN113354025A (en) * | 2021-06-07 | 2021-09-07 | 烟台金堃新材料科技有限公司 | Treatment method of oily wastewater |
| CN114097956A (en) * | 2021-11-16 | 2022-03-01 | 厦门嘉康饲料有限公司 | Functional feed for litopenaeus vannamei and preparation method thereof |
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