CN114621886A - Process for producing antibacterial peptide by fermenting bacillus subtilis and preparation method - Google Patents
Process for producing antibacterial peptide by fermenting bacillus subtilis and preparation method Download PDFInfo
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- CN114621886A CN114621886A CN202011435782.3A CN202011435782A CN114621886A CN 114621886 A CN114621886 A CN 114621886A CN 202011435782 A CN202011435782 A CN 202011435782A CN 114621886 A CN114621886 A CN 114621886A
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- antibacterial peptide
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- bacillus subtilis
- seed
- supernatant
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- UCRLQOPRDMGYOA-DFTDUNEMSA-L zinc;(4r)-4-[[(2s)-2-[[(4r)-2-[(1s,2s)-1-amino-2-methylbutyl]-4,5-dihydro-1,3-thiazole-4-carbonyl]amino]-4-methylpentanoyl]amino]-5-[[(2s,3s)-1-[[(3s,6r,9s,12r,15s,18r,21s)-3-(2-amino-2-oxoethyl)-18-(3-aminopropyl)-12-benzyl-15-[(2s)-butan-2-yl]-6-(carbox Chemical compound [Zn+2].C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC([O-])=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2NC=NC=2)C(=O)N[C@H](CC([O-])=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 UCRLQOPRDMGYOA-DFTDUNEMSA-L 0.000 description 1
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Abstract
The invention provides a process for producing antibacterial peptide by fermenting bacillus subtilis and a preparation method thereof, belonging to the technical field of biological engineering. The invention takes bacillus subtilis which is screened in a laboratory and can be metabolized to generate antibacterial peptide as a fermentation production strain, and prepares the bacillus subtilis antibacterial peptide by technical methods such as liquid seed preparation, fermentation liquor pretreatment, antibacterial peptide separation and purification solution concentration, antibacterial peptide concentrated solution vacuum freeze drying and the like. The invention has simple process, high antibacterial activity and high yield of the antibacterial peptide, has no toxic or side effect, can be used as food preservatives and feed additives, and has the advantages of no toxicity, no residue, no drug resistance, high safety and the like.
Description
Technical Field
The invention relates to the field of biological fermentation and antibacterial peptide, in particular to a process for producing antibacterial peptide by fermenting bacillus subtilis and a preparation method thereof.
Background
Due to the widespread use of antibiotics, pathogenic microorganisms develop resistance to antibiotics, leading to the emergence of superbacteria, slowing the development of new antibiotics in the last decade, further exacerbating the current global challenges. Due to the complex and various lengths, sequences and structures of the antibacterial peptide, the antibacterial peptide has novel and unique amphipathic structural properties, has the functions of wide antibacterial spectrum, low drug resistance, high thermal stability, small molecular weight and the like, and is considered as an excellent substitute of antibiotics. Bacillus subtilis is metabolized to produce peptide antibiotics, peptide chains of the antibiotics are usually in a closed ring shape, free amino groups and carboxyl groups are almost absent, protease cannot decompose, and the stability of active peptides is high.
The antibacterial peptide is a secondary metabolite of microbial cells, the molecular weight is about 2000-7000Da, and most of the active polypeptides have the characteristics of strong basicity, thermal stability, broad-spectrum antibacterial property and the like. Compared with antibiotics, the antibacterial peptide has high safety, is harmless to normal cells of human and animals, belongs to a biological preservative which has no toxic or side effect, no residue and no bacterial drug resistance, and has wide application prospect in the pharmaceutical industry, the food industry and the feed industry.
In the field of food preservation, China has a small gap in technology and popularization compared with some developed countries in the world. The traditional food preservation method mainly adopts chemical preservatives or physical means such as pickling (sugar, vinegar, salt and the like), smoking, drying and the like, but the problems of food safety, quality, flavor change and the like can be caused. The natural preservative has the advantages of small toxic and side effects, degradability, safe use and the like, and has a tendency of replacing the traditional chemical preservative. The development of natural preservatives has been paid attention by many researchers in China. Among natural preservatives, antibacterial peptide preservatives are widely concerned by people because of their safety, non-toxicity and even health-care effect on human bodies, and are becoming a new favorite in the field of food preservation and freshness preservation.
With the development of economy, the living standard of people is improved, the food safety problem is more and more concerned, antibiotics as harmful bacteria growth inhibitors are used in large quantities in animal feed and feed additives for a long time, the problems of antibiotic resistance and antibiotic residue are continuously exposed over time, the problems of side effects caused by the use of the antibiotics, such as drug residue, drug resistance, environmental pollution and the like are more and more concerned, the antibiotics face the situation of being eliminated or forbidden in the feed industry, and the European Union has completely prohibited the antibiotics as the feed additives to be used in the feed in 2006. Therefore, the development of a novel broad-spectrum, non-toxic, pollution-free and residue-free antibacterial agent as a feed additive instead of antibiotics becomes an important content of the current scientific research of domestic and foreign feeds. However, for reasons of various aspects, the feed antibiotics are not completely forbidden in China at present, and most of the antibiotic feed additives produced in China are products which are eliminated or are to be forbidden abroad, such as ChalOH, arsanilic acid, bacitracin zinc, tylosin, virginiamycin and the like, which seriously affect the export of livestock and poultry products in China. According to statistics of relevant departments, the annual loss of China reaches billions of dollars, huge economic loss is caused, the development of animal husbandry of China is seriously influenced, the international reputation of domestic animal products of China is seriously damaged, and researches on accelerating relevant aspects are imperative in the face of the flood tide of developing environment-friendly high-efficiency feed additives all over the world.
Because the antibacterial peptide produced by microorganisms has the obvious advantages of low cost, no toxicity, short period and the like, research on the antibacterial peptide produced by microorganisms starts to be started in recent years, but the current research mainly focuses on lactic acid bacteria, and the antibacterial peptide produced by the lactic acid bacteria has the problems of poor heat resistance, narrow antibacterial spectrum and the like, so how to solve the problems still needs to be further researched, the antibacterial peptide produced by bacillus and paenibacillus has wide antibacterial spectrum and high stability, and attracts wide attention.
Disclosure of Invention
In order to solve the problems in the prior art and the defects in the production of the antibacterial peptide by fermenting the bacillus subtilis, the invention provides a process for producing the antibacterial peptide by fermenting the bacillus subtilis and a preparation method thereof.
In order to achieve the aim, the bacillus subtilis strain capable of metabolically producing antibacterial peptide is prepared by carrying out long-term separation and purification experiment screening by a company research center and is named as QR-KJ-092.
The main biological characteristics of the strain are as follows: aerobic bacteria, which are rod-shaped bacteria with cell size of 0.6-0.8μm x (1.8-2.5 μm), exist singly, have no capsule, and are periphytic gram-positive bacteria with bristles and movement; the surface of the bacterial colony is rough and opaque; the colony on the culture medium is round or irregular, the surface color is dark, the colony is thickened and opaque and has folds, the colony is cream color or yellowish, and the fermentation culture can utilize glucose, arabinose, xylose, starch, plant protein and animal-derived protein and can hydrolyze starch and tryptophan; the growth temperature range is 35-55 ℃, and the optimal temperature is 40 ℃.
Meanwhile, the invention firstly provides a bacillus subtilis preservation culture medium and a fermentation culture medium.
The bacillus subtilis preservation culture medium comprises the following components in percentage by mass: 2% of sucrose, 0.5% of peptone, 1.5% of beef extract, 0.1% of NaCl and K2HPO40.5%, agar 2%, pH7.0.
The bacillus subtilis fermentation medium comprises the following components in percentage by mass: 10% of glucose, 0.75% of yeast extract, 5% of soybean meal, 1% of corn steep liquor dry powder, 0.15% of sodium chloride, 0.05% of magnesium sulfate, 0.05% of monopotassium phosphate and 0.02% of manganese sulfate, and adjusting the pH value of the fermentation liquor to 7.0.
The invention also provides a fermentation production method of the antibacterial peptide, which mainly comprises the following steps:
(1) activating the bacillus subtilis strain for producing the antibacterial peptide;
(2) preparing a 50L first-level seed tank and a 500L second-level seed tank;
(3) and (3) fermenting and culturing in a 5T fermentation tank, and culturing the metabolism of the bacillus subtilis by using the bacillus subtilis fermentation culture medium to produce the antibacterial peptide.
The number of the antibacterial peptide-producing bacillus subtilis strain in the step (1) is QR-KJ-092.
Inoculating the prepared slant mother seeds under the aseptic condition, selecting two rings by using an inoculating ring, culturing in a 150mL triangular flask mother seed culture medium at 40 ℃ at 150r/min for 1.5 days, and preparing a primary triangular flask seed solution;
inoculating the primary seed solution into 5L seed liquid culture medium at an inoculum size (volume percentage) of 2.5%, and culturing at 40 deg.C and 150r/min for 1.5d to obtain secondary triangular flask seed solution;
inoculating the second-stage triangular flask seed solution into 50L liquid seed tank culture medium at 42 deg.C under 0.05MPa and with 2.5m ventilation3Culturing for 35h under the condition of/h;
inoculating 50L of liquid seed tank seed liquid into 500L of liquid seed tank at 2.5% (volume percentage), at 42 deg.C, 0.05MPa, and 30m of ventilation3Culturing for 35h under the condition of/h;
inoculating 500L of seed liquid fermented in the liquid seed tank into a 5T fermentation tank according to the inoculation amount of 2.5% (volume percentage), and performing inoculation at 42 ℃, the tank pressure of 0.05MPa and the ventilation volume of 50m3Culturing for 35h under the condition of/h.
(4) The invention also provides a preparation method of the antibacterial peptide, which comprises the following steps:
a. pretreating the fermentation liquor, namely centrifuging the fermentation liquor obtained in the step (3) by adopting a GQ105 tubular centrifuge at a set rotation speed of 16000r/min, removing bacterial sludge, and collecting supernatant to obtain supernatant containing the antibacterial peptide;
b. b, adding the antibacterial peptide-containing supernatant obtained in the step a into 0.1mol/L HCl solution, adjusting the pH of the solution until a large amount of precipitate appears in the supernatant, collecting the precipitate, and removing the supernatant;
c. dissolving the antibacterial peptide-containing precipitate obtained in the step b by using 0.02mol/L phosphate buffer solution to obtain a primary treated crude antibacterial peptide solution;
d. concentrating the crude antibacterial peptide solution prepared in the step c, concentrating the crude antibacterial peptide solution by using an ultrafiltration membrane with the molecular weight cutoff of 1000-;
e.slowly adding ammonium sulfate into the concentrated solution at 25 ℃ to 10% saturation, standing at 4 ℃ for 2h, then centrifuging at 8500r/min for 10min, collecting supernatant, adding ammonium sulfate into the collected supernatant to 60% saturation, standing at 4 ℃ for 4h, centrifuging at 12000r/min for 20min, collecting precipitate, and then dissolving with 0.02mol/L Tris-HCl buffer solution;
f. d, further dialyzing the antibacterial peptide solution prepared in the step e by adopting a 1000Da dialysis bag, and replacing the deionized water once every 4 hours to obtain a completely dialyzed antibacterial peptide solution;
g. f, performing gel column chromatography separation and purification on the antimicrobial peptide SephadexG50, performing SephadexG50 gel column chromatography on the antimicrobial peptide solution obtained by dialysis in the step f, controlling the flow rate to be 0.5mL/min, taking 0.02mol/L phosphate buffer solution as eluent, and collecting chromatographic solution to obtain further purified antimicrobial peptide solution;
h. and g, preparing antibacterial peptide pure powder, namely concentrating the antibacterial peptide solution prepared in the step g to 1/3 times of the original volume under the conditions of-0.15 pa and 45 ℃, then removing water in the antibacterial peptide solution by adopting a vacuum freeze-drying method, setting the freezing temperature to-40 ℃, carrying out vacuum 0.24Mpa, heating according to a program, and drying for 44 hours to obtain the antibacterial peptide powder product.
And h, carrying out temperature programming, wherein the specific process is as follows: (1) heating at the temperature of 40-0 ℃ for 5 hours; (2) programming at 0-5 ℃ for 12 h; (3) programming to raise the temperature at 5-10 ℃ for 10 hours; (4) heating at the temperature of 10-20 ℃ for 3 h; and (3) analysis and drying stage: the temperature is increased by a program at 20-30 ℃ for 6 hours.
QR-KJ-092 antibacterial peptide titer determination adopts an agar perforation diffusion method to determine the antibacterial peptide titer of fermentation liquor, and takes escherichia coli as an indicator bacterium to prepare a titer plate. Then, the cells were punched with a punch (diameter: 2.7 mm), and 10. mu.L of the QR-KJ-092 strain antimicrobial peptide:
the method for measuring the potency of the antibacterial peptide comprises the following steps:
antibacterial potency =2xX 1000 x diluteMultiple of release
In the formula, X = (the diameter of a bacteriostatic ring is-2.7)/2.1 (Liangjie, Penzhong bond, Liangshuwa and the like; research on the production of antibacterial peptide by deep fermentation of transgenic yeast [ J ]. modern food technology, 2009,25(6): 639-.
The invention has the beneficial effects that:
the invention provides a preparation method for producing antibacterial peptide by fermenting bacillus subtilis, the strain provided by the invention is a specific active strain screened by the company, and the fermentation mass production of the strain is realized through a large number of experiments.
The invention prepares the finished product of the high-purity antibacterial peptide powder by the technical methods of liquid seed preparation, fermentation liquor pretreatment, antibacterial peptide extraction and refining, antibacterial peptide dry powder preparation and the like, thereby greatly improving the purification efficiency.
The invention improves the preparation efficiency of the antibacterial peptide by combining ultrafiltration, salting out and gel chromatography.
Drawings
FIG. 1 is a diagram of the chromatographic separation and purification of the antibacterial peptide of Bacillus subtilis by SephadexG50 gel column.
FIG. 2 is a diagram showing the identification of Bacillus subtilis antimicrobial peptide by SephadexG50 gel column chromatography.
FIG. 3 chromatogram of standard protein. Note: 1: insulin, 2: yeast ribonucleic acid, 3: bacitracin, 4: and (3) reducing glutathione.
FIG. 4 chromatogram of antibacterial peptide from Bacillus subtilis.
Detailed Description
The invention discloses a process for producing antibacterial peptide by fermenting bacillus subtilis and a preparation method thereof, and a technical formula meeting the actual equipment conditions of the technical formula can be obtained by properly improving process parameters by taking the contents of the process as reference. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention.
The process for producing antibacterial peptide by fermenting bacillus subtilis and the preparation method thereof are described by specific examples, and it should be noted that the following description is only for explaining the invention and does not limit the content thereof.
Example 1
The invention relates to a bacillus subtilis strain preservation medium for producing antibacterial peptide by metabolism, which comprises the following steps: 2% of sucrose, 0.5% of peptone, 1.5% of beef extract, 0.1% of NaCl and K2HPO40.5%, agar 2%, pH7.0.
The bacillus subtilis fermentation culture medium comprises the following components in percentage by mass: 10% of glucose, 0.75% of yeast extract, 5% of soybean meal, 1% of corn steep liquor dry powder, 0.15% of sodium chloride, 0.05% of magnesium sulfate, 0.05% of monopotassium phosphate and 0.02% of manganese sulfate, and adjusting the pH value of the fermentation liquor to 7.0.
The culture conditions of the bacillus subtilis are as follows:
inoculating a prepared slant mother seed under an aseptic condition, selecting two rings by using an inoculating ring, culturing in a 150mL triangular flask mother seed culture medium at 40 ℃ at 150r/min for 1.5d to prepare a primary triangular flask seed solution;
inoculating the primary seed solution into 5L seed liquid culture medium at an inoculum size (volume percentage) of 2.5%, and culturing at 40 deg.C and 150r/min for 1.5d to obtain secondary triangular flask seed solution;
inoculating the second-stage triangular flask seed solution into 50L liquid seed tank culture medium at 42 deg.C under 0.05MPa and with 2.5m ventilation3Culturing for 35h under the condition of/h;
inoculating 50L of liquid seed tank seed liquid into 500L of liquid seed tank at 2.5% (volume percentage), at 42 deg.C, 0.05MPa, and ventilation amount of 30m3Culturing for 35h under the condition of/h;
inoculating 500L of seed liquid fermented in the liquid seed tank into a 5T fermentation tank according to the inoculation amount of 2.5% (volume percentage), and performing inoculation at 42 ℃, the tank pressure of 0.05MPa and the ventilation volume of 50m3Culturing for 35h under the condition of/h.
Example 2
The invention provides a process for producing antibacterial peptide by fermenting bacillus subtilis and a preparation method thereof, wherein the process for treating the antibacterial peptide-containing fermentation broth comprises the following steps:
a. pretreating the fermentation liquor, namely centrifuging the fermentation liquor obtained in the step (3) in a GQ105 tube type, removing bacterial sludge, and collecting supernatant to obtain supernatant containing the antibacterial peptide;
the fermentation liquor centrifugation conditions of the step a are as follows: the rotating speed is 16000r/min, the centrifugal temperature is controlled to be 20 ℃ by adopting a circulating water cooling mode, and the flow rate is 5L/min.
b. B, adding the antibacterial peptide-containing supernatant obtained in the step a into 0.1mol/L HCl solution, adjusting the pH of the solution until a large amount of precipitate appears in the supernatant, collecting the precipitate, and removing the supernatant;
c. and (c) dissolving the antibacterial peptide-containing precipitate obtained in the step (b) by using 0.02mol/L phosphate buffer solution to obtain a crude antibacterial peptide solution subjected to primary treatment.
Example 3
The invention provides a separation and purification process of bacillus subtilis antimicrobial peptide, which comprises the following steps:
a. concentrating the crude antibacterial peptide solution, concentrating the crude antibacterial peptide solution by using an ultrafiltration membrane with the cut-off molecular weight of 1000-;
slowly adding ammonium sulfate into the concentrated solution at 25 ℃ to 10% saturation, standing at 4 ℃ for 2h, centrifuging at 8500r/min for 10min, collecting supernatant, adding ammonium sulfate into the collected supernatant to 60% saturation, standing at 4 ℃ for 4h, centrifuging at 12000r/min for 20min, collecting precipitate, and dissolving with 0.02mol/L Tris-HCl buffer solution;
c. the prepared antibacterial peptide solution is further dialyzed by a 1000Da dialysis bag, and deionized water is replaced once every 4 hours to obtain a completely dialyzed antibacterial peptide solution;
d. and (3) carrying out SephadexG50 gel column chromatography on the antibacterial peptide solution obtained by dialysis, controlling the flow rate to be 0.5mL/min, taking 0.02mol/L phosphate buffer solution as eluent, and collecting the chromatographic solution to obtain the further purified antibacterial peptide solution.
Example 4
The invention provides a production process of a bacillus subtilis antibacterial peptide powder, which comprises the following specific implementation steps:
a. concentrating the antibacterial peptide solution by low-pressure concentration equipment;
the low-pressure concentration conditions of the antibacterial peptide are as follows: and (3) starting a vacuum pump to adjust the vacuum degree of the low-pressure concentration equipment to-0.15 pa, slightly opening a steam valve to control the pressure of a pipeline to be 0.15mPa, maintaining the temperature of the antibacterial peptide solution at 45 ℃, and concentrating the volume of the antibacterial peptide solution to be 1/3 times of the original volume under the condition.
b. Removing water in the antibacterial peptide solution from the bacillus subtilis antibacterial peptide concentrated solution by adopting a vacuum freeze-drying method;
the vacuum freeze drying process conditions are as follows: freezing at-40 deg.C, vacuum at 0.24Mpa, heating according to program, and drying for 44 hr to obtain antibacterial peptide powder.
The temperature programming in the above steps is specifically carried out in a primary sublimation stage: (1) heating at 40-0 deg.C for 5 h; (2) programming at 0-5 ℃ for 12 h; (3) programming to raise the temperature at 5-10 ℃ for 10 hours; (4) heating at the temperature of 10-20 ℃ for 3 h; and (3) analysis and drying stage: the temperature is increased by a program at 20-30 ℃ for 6 hours.
Example 5
The invention provides a method for detecting the molecular weight of antibacterial peptide by bacillus subtilis antibacterial peptide liquid phase, which comprises the following determination conditions:
a. selection of mobile phase and wavelength
The test is carried out by respectively using methanol-water, acetonitrile-water and methanol-KH2PO4The mobile phase is prepared by adjusting pH of each mobile phase with acetic acid to 6.5 or so, setting column temperature at 30 deg.C, flow rate at 0.6mL/min, sample amount at 10 μ L, and different elution conditions of each mobile phase, performing full-wavelength scanning, measuring peak effect to obtain accurate chromatographic condition with good peak effect, and testing with methanol-KH2PO4Is mobile phase, methanol is KH2PO4=55:45, the peak effect is good when the wavelength is 254nm, so that the test selects the methanol: KH2PO4And (4) =55:45 as mobile phase and chromatographic condition with wavelength of 254 nm.
b. Selection of flow rate
At a wavelength of 254nm, the mobile phase is methanol KH2PO445, the column temperature is 30 ℃, the sample injection amount is 10 mu L, the set flow rates are 1.0mL/min, 0.8mL/min and 0.6mL/min respectively, and when the flow rate is 0.6mL/min, the chromatographic peak shape and the standard substance separation effect are good, so the flow rate of 0.6mL & ltion & gt is selected in the testmin。
c. Standard curve and regression equation
The retention time of each standard product of insulin, bacitracin, yeast ribonucleic acid and reductive glutathione is respectively determined by experiments as follows: 2.591min, 4.834min, 5.653min and 7.894min, and carrying out regression analysis on the protein to obtain a regression equation of the standard protein: log (mw) = -0.2548t + 4.3753, R2 = 0.9983. The regression equation determines the coefficient 0.9983, which indicates that the common logarithm of the relative molecular mass of the protein has a good linear relationship with the retention time of its chromatographic peak, and thus can be used as a standard curve for the determination of the relative molecular mass of the polypeptide.
d. Determination of samples
And (3) measuring the molecular weight of the separated bacillus subtilis antibacterial peptide by using an experimentally established method. The liquid chromatography determination is carried out on the bacillus subtilis antibacterial peptide under the same chromatographic condition, and the molecular weight range of the bacillus subtilis antibacterial peptide obtained by the determination method is 1290-8490 Da.
Example 6, the present invention provides a test for evaluating antibacterial potency of Bacillus subtilis antimicrobial peptides.
Dissolving 1g of freeze-dried antibacterial peptide powder with 0.2mol/L phosphate solution until the solution concentration is 0.01g/L, measuring the titer of the antibacterial peptide in the fermentation liquor by adopting an agar perforation diffusion method, and preparing a titer plate by using escherichia coli as an indicator bacterium. Then a hole puncher (diameter is 2.7 mm) is used for punching, 10 microliter of QR-KJ-092 strain antibacterial peptide is respectively added into each hole, the mixture is pasted in tryptone soybean agar culture medium coated with staphylococcus aureus, escherichia coli nutrient agar culture medium coated with escherichia coli nutrient agar and aspergillus flavus nutrient agar, the mixture is cultured for 20 hours in an incubator at 37 ℃ at constant temperature, and the diameter of a bacteriostatic circle is observed and measured by a vernier caliper.
The method for measuring the potency of the antibacterial peptide comprises the following steps:
antibacterial potency =2xX 1000 x dilution factor
Wherein X = (diameter of inhibition zone-2.7)/2.1.
TABLE 1 comparison of bacteriostatic Effect of the strains (unit: mm)
| Staphylococcus aureus | Escherichia coli | Aspergillus flavus | |
| Diameter of zone of inhibition | 18.16±0.22 | 22.09±0.25 | 7.76±1.14 |
Example 7, bacillus subtilis antimicrobial peptide feeding assay.
A breeding base cooperated by a company nearby Jiaxing is selected as a feeding test base, 600 healthy 579 chicks (the weight difference is not significant, P is more than 0.05) with the age of 1 day are selected, a single-factor complete random test design is adopted, the chicks are randomly divided into 5 treatment groups which are respectively numbered A, B, C, D, E, each treatment group is provided with 6 repetitions, and each repetition is 20 chicks, and the test scheme is as shown in the following table.
TABLE 2 Bacillus subtilis antimicrobial peptide feeding test protocol
| Test grouping | Treatment protocol | Adding antibacterial peptide |
| A | Basal diet +5mg/kg AFB1 | 1g/kg |
| B | Basal diet | 1g/kg |
| C | Basal diet + spiramycin | 1g/kg |
| D | Basal diet + rickettsial +5mg/kg AFB1 | 1g/kg |
| E | Basal diet | 0g/kg |
The blank control group (E) is fed with basic daily ration (the basic daily ration does not contain antibiotics and antibacterial peptides), the antibiotic control group (AC) is added with 5mg/kg of flavomycotoxin in the basic daily ration, the experimental worker is added with 1g/kg of antibacterial peptides in the basic daily ration, after 20d of feeding, the average daily gain of the experimental group B is the highest and is increased by 4.85% compared with that of the AC group, the average daily feeding of the experimental group A, C, D also has a reduction trend compared with that of the group B, but the difference is not obvious, the material weight of the experimental group B is reduced by 3.16% compared with that of the group A, C, D, and the material weight of the experimental group B is reduced by 4.06% compared with that of the control group E.
Claims (5)
1. A process for producing antibacterial peptide by fermenting bacillus subtilis is characterized by comprising the following technical steps:
(1) the serial number of the antibacterial peptide-producing bacillus subtilis strain is QR-KJ-092;
(2) inoculating the prepared slant mother seeds under the aseptic condition, selecting two rings by using an inoculating ring, culturing in a 150mL triangular flask mother seed culture medium at 40 ℃ at 150r/min for 1.5d to prepare a first-stage triangular flask seed solution;
(3) inoculating the first-stage seed solution into 5L seed liquid culture medium at an inoculum size (volume percentage) of 2.5%, and culturing at 40 deg.C and 150r/min for 1.5d to obtain second-stage triangular flask seed solution;
(4) inoculating the second-stage triangular flask seed solution into 50L liquid seed tank culture medium at 42 deg.C under 0.05MPa and with 2.5m ventilation3Culturing for 35h under the condition of/h;
(5) inoculating 50L of liquid seed tank seed liquid into 500L of liquid seed tank at 2.5% (volume percentage), at 42 deg.C, 0.05MPa, and ventilation amount of 30m3Culturing for 35h under the condition of/h;
(6) inoculating 500L of seed liquid fermented in a liquid seed tank into a 5T fermentation tank according to the inoculation amount of 2.5% (volume percentage), and performing inoculation at 42 ℃, the tank pressure of 0.05MPa and the ventilation volume of 50m3Culturing for 35h under the condition of/h;
(7) pretreating fermentation liquor, namely centrifuging the fermentation liquor obtained in the step (3) by adopting a GQ105 tubular centrifuge at a set rotation speed of 16000r/min, removing bacterial sludge, and collecting supernatant to obtain supernatant containing antibacterial peptide;
(8) adding 0.1mol/L HCl solution into the antibacterial peptide supernatant, adjusting the pH of the solution until a large amount of precipitate appears in the supernatant, collecting the precipitate, and removing the supernatant;
(9) dissolving the obtained antibacterial peptide-containing precipitate substance by using 0.02mol/L phosphate buffer solution to obtain a primary treated crude antibacterial peptide solution;
(10) concentrating the crude antibacterial peptide solution by using an ultrafiltration membrane with the molecular weight cutoff of 1000-;
(11) slowly adding ammonium sulfate into the concentrated solution at 25 ℃ to 10% saturation, standing at 4 ℃ for 2h, centrifuging at 8500r/min for 10min, collecting supernatant, adding ammonium sulfate into the collected supernatant to 60% saturation, standing at 4 ℃ for 4h, centrifuging at 12000r/min for 20min, collecting precipitate, and dissolving with 0.02mol/L Tris-HCl buffer solution;
(12) further dialyzing the antibacterial peptide solution by adopting a 1000Da dialysis bag, and replacing the deionized water once every 4 hours to obtain a completely dialyzed antibacterial peptide solution;
(13) carrying out SephadexG50 gel column chromatography on the antibacterial peptide solution, controlling the flow rate to be 0.5mL/min, taking 0.02mol/L phosphate buffer solution as eluent, and collecting chromatographic solution to obtain further purified antibacterial peptide solution;
(14) concentrating the antibacterial peptide solution by low-pressure concentration equipment;
(15) and removing water in the antibacterial peptide solution by a vacuum freeze-drying method.
2. The process and preparation method for producing antibacterial peptide by fermenting bacillus subtilis according to claim 1, wherein the slant mother culture medium in the step (2) is: 2% of sucrose, 0.5% of peptone, 1.5% of beef extract, 0.1% of NaCl and K2HPO40.5%, agar 2%, pH7.0.
3. The process and preparation method for producing antibacterial peptide by fermenting bacillus subtilis according to claim 1, wherein the bacillus subtilis liquid fermentation culture medium comprises the following components by mass percent: 10% of glucose, 0.75% of yeast extract, 5% of soybean meal, 1% of corn steep liquor dry powder, 0.15% of sodium chloride, 0.05% of magnesium sulfate, 0.05% of monopotassium phosphate and 0.02% of manganese sulfate, and adjusting the pH value of the fermentation liquor to 7.0.
4. The process and preparation method for producing antibacterial peptide by fermenting bacillus subtilis according to claim 1, wherein the low-pressure concentration conditions in step (14) are as follows: and starting a vacuum pump to adjust the vacuum degree of the low-pressure concentration equipment to-0.15 pa, slightly opening a steam valve to control the pressure of a pipeline to be 0.15mPa, keeping the temperature of the antibacterial peptide solution at 45 ℃, and concentrating the volume of the antibacterial peptide solution to 1/3 times of the original volume under the condition.
5. The process for producing antibacterial peptide by fermenting bacillus subtilis and the preparation method thereof according to claim 1, wherein the vacuum freeze drying process conditions in the step (15) are as follows: freezing at-40 deg.C, vacuum at 0.24Mpa, heating according to program, and performing primary sublimation: (1) heating at 40-0 deg.C for 5 h; (2) programming at 0-5 ℃ for 12 h; (3) programming to raise the temperature at 5-10 ℃ for 10 hours; (4) heating at the temperature of 10-20 ℃ for 3 h; and (3) an analysis drying stage: the temperature is increased by a program at 20-30 ℃ for 6 hours.
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