阅读说明：本技术 一种抗菌肽的发酵制备方法、抗菌肽水凝胶及应用 (Fermentation preparation method of antibacterial peptide, antibacterial peptide hydrogel and application ) 是由 陈刚 于 2021-03-08 设计创作，主要内容包括：本发明公开了一种抗菌肽的发酵制备方法、抗菌肽水凝胶及应用,该制备方法包括以下步骤：S1、将枯草芽孢杆菌种于LB液体培养基中,得到培养液；S2、将培养液以1％的接种量接种于发酵培养基中进行发酵；S3、将步骤S2获得的培养液进行离心,然后经过质量浓度为390g/L的硫酸铵进行沉淀,沉淀物即为得到的抗菌肽。抗菌肽从乳酸杆菌和解淀粉芽孢杆菌中分离获得,而抗菌肽在对GV等致病菌杀伤的同时,又可以促进乳酸杆菌的生长,这种药物药效学效果对于细菌性阴道炎的治疗是十分有利的,且可用于治疗HPV人乳头瘤病毒。(The invention discloses a fermentation preparation method of antibacterial peptide, antibacterial peptide hydrogel and application, wherein the preparation method comprises the following steps: s1, putting the bacillus subtilis into an LB liquid culture medium to obtain a culture solution; s2, inoculating the culture solution into a fermentation medium by an inoculation amount of 1% for fermentation; s3, centrifuging the culture solution obtained in the step S2, and then precipitating the culture solution by ammonium sulfate with the mass concentration of 390g/L, wherein the precipitate is the obtained antibacterial peptide. The antibacterial peptide is obtained by separating from lactobacillus and bacillus amyloliquefaciens, can promote the growth of the lactobacillus while killing pathogenic bacteria such as GV, and the like, has favorable pharmacodynamic effect on the treatment of bacterial vaginitis, and can be used for treating HPV (human papilloma virus).)

1. The fermentation preparation method of the antibacterial peptide is characterized by comprising the following steps:

s1, putting the bacillus subtilis into an LB liquid culture medium to obtain a culture solution;

s2, inoculating the culture solution into a fermentation medium by an inoculation amount of 1% for fermentation;

s3, centrifuging the culture solution obtained in the step S2, and then precipitating the culture solution by ammonium sulfate with the mass concentration of 390g/L, wherein the precipitate is the obtained antibacterial peptide.

2. The method for preparing antibacterial peptide by fermentation according to claim 1, wherein the fermentation medium comprises the following components by weight percent: 16-22 parts of potato starch, 16-22 parts of glucose, 20-26 parts of raffinose, 1-3 parts of yeast powder, 4-6 parts of sodium acetate, 1-3 parts of diamine citrate, 2-4 parts of dipotassium hydrogen phosphate, 0.1-0.4 part of magnesium sulfate, 0.01-0.05 part of manganese sulfate and 0.1-0.2 part of tween 80.

3. The method for preparing antibacterial peptide by fermentation according to claim 1, wherein the fermentation temperature in step S2 is 36-38 ℃, and the fermentation time is 12-14 hours.

4. The preparation method of the antibacterial peptide hydrogel is characterized by comprising the following steps:

q1, degumming, dissolving and dialyzing the antibacterial peptide prepared by the method for preparing the antibacterial peptide by fermentation according to any one of claims 1 to 3 to form an antibacterial peptide water solution with the mass fraction of 40%; according to the mass ratio of lactide: preparing a polylactic acid-polyethylene glycol-polylactic acid copolymer by a ring-opening reaction, wherein the ratio of polyethylene glycol is 5: 1-1: 5;

q2, adding the polylactic acid-polyethylene glycol-polylactic acid copolymer into the antibacterial peptide aqueous solution, and standing for 20 minutes at 10-25 ℃ to form the antibacterial peptide hydrogel.

5. An antibacterial peptide hydrogel according to claim 4 for use in the treatment of vaginitis.

6. An application of the antibacterial peptide hydrogel of claim 4 in treating HPV human papilloma virus.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a fermentation preparation method of antibacterial peptide, antibacterial peptide hydrogel and application.

Background

Women with bacterial vaginosis are often accompanied by a drop in vaginal acidity, rendering them susceptible to viruses such as HIV. The current research is to realize the controlled release of lactic acid by degradable hydrogel based on PEG nano-carrier. The PEG-la nano carrier and the lactic acid are covalently combined through PEG-SH. PEG-LA has 4 copies of the lactic acid molecule and can achieve controlled release of lactic acid such that the maximum release ranges in phosphate buffered saline and acetate buffer are 23% and 47%, respectively. When the elastic coefficient is greater than 100 times the viscosity coefficient, the hydrogel can be formed within 20 min. The maximum release rate is generally between 10% and 14% due to steric hindrance of the polymer chains. In contrast, the hydrogel with entrapped lactic acid achieved complete release within 30 min. Lactic acid showed bacteriostatic (original bv pathogen Gardnerella vagianalis) activity at a MIC of 3.6 mg/mL. The hydrogel containing entrapped lactic acid completely inhibited the growth of g. These studies all show the great potential of PEG nanocarrier based hydrogels (intravaginal administration) for the prevention and treatment of BV.

Therefore, a fermentation preparation method of the antibacterial peptide, the antibacterial peptide hydrogel and the application thereof are in urgent need to be provided.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a fermentation preparation method of antibacterial peptide, antibacterial peptide hydrogel and application.

In order to solve the technical problems, the invention provides the following technical scheme:

the invention provides a fermentation preparation method of antibacterial peptide, which comprises the following steps:

s1, putting the bacillus subtilis into an LB liquid culture medium to obtain a culture solution;

s2, inoculating the culture solution into a fermentation medium by an inoculation amount of 1% for fermentation;

s3, centrifuging the culture solution obtained in the step S2, and then precipitating the culture solution by ammonium sulfate with the mass concentration of 390g/L, wherein the precipitate is the obtained antibacterial peptide.

As a preferred technical scheme of the invention, the fermentation medium comprises the following components in percentage by weight: 16-22 parts of potato starch, 16-22 parts of glucose, 20-26 parts of raffinose, 1-3 parts of yeast powder, 4-6 parts of sodium acetate, 1-3 parts of diamine citrate, 2-4 parts of dipotassium hydrogen phosphate, 0.1-0.4 part of magnesium sulfate, 0.01-0.05 part of manganese sulfate and 0.1-0.2 part of tween 80.

As a preferred technical scheme of the invention, the fermentation temperature in the step S2 is 36-38 ℃, and the fermentation time is 12-14 hours.

As a preferred technical scheme, the invention also provides a preparation method of the antibacterial peptide hydrogel, which comprises the following steps:

q1, degumming, dissolving and dialyzing the antibacterial peptide prepared by the antibacterial peptide fermentation preparation method to form an antibacterial peptide water solution with the mass fraction of 40%; according to the mass ratio of lactide: preparing a polylactic acid-polyethylene glycol-polylactic acid copolymer by a ring-opening reaction, wherein the ratio of polyethylene glycol is 5: 1-1: 5;

q2, adding the polylactic acid-polyethylene glycol-polylactic acid copolymer into the antibacterial peptide aqueous solution, and standing for 20 minutes at 10-25 ℃ to form the antibacterial peptide hydrogel.

As a preferred technical scheme, the invention also provides an antibacterial peptide hydrogel for treating vaginitis.

As a preferable technical scheme, the antibacterial peptide hydrogel is applied to treatment of HPV (human papilloma virus).

Compared with the prior art, the invention has the following beneficial effects:

first, the antimicrobial peptides are isolated from lactobacilli and bacillus amyloliquefaciens, which are found in fermented dairy products. These bacteria are naturally present in human consumer products and are therefore by definition safe. The subtilosin A obtained from Bacillus amyloliquefaciens KATMIRA1933 has been shown to be safe for normal human tissue, have a certain spermicidal activity, and are effective in killing food-borne and vaginal pathogens.

Second, the antimicrobial peptide has a negative charge. It is well known that one of the clinical symptoms that bacterial vaginosis presents is an increase in the pH of vaginal secretions. The negative charge carried by the antimicrobial peptide has a unique advantage in maintaining the acidic microenvironment of the vagina.

Thirdly, the antimicrobial peptide can be obtained from lactobacillus, which is a product of lactobacillus, and is useful for the growth of lactobacillus. Lactobacilli are present as a self-protective probiotic in the vagina. In the vagina of a patient with bacterial vaginitis, the proportion of lactobacillus is disordered due to abnormal growth of pathogenic bacteria (Gardner GV, anaerobic bacteria (mainly campylobacter), mycoplasma, escherichia coli, enterococcus faecalis, streptococcus and the like), while the antibacterial peptide can promote the growth of the lactobacillus while killing the pathogenic bacteria such as GV and the like, the pharmacodynamic effect of the medicine is very favorable for treating the bacterial vaginitis, and the medicine can be used for treating HPV (human papilloma virus).

Fourthly, the fermentation preparation method of the invention adopts ammonium sulfate precipitation distribution to remove part of impurities and reduce the pressure of a purification column.

Drawings

FIG. 1 is a diagram showing the form of the antibacterial peptide of the present invention.

FIG. 2 is a graph showing the results of the test of the bacteriostatic activity of the antimicrobial peptide of the present invention.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Example 1

In order to achieve the object of the present invention, in one embodiment of the present invention, there is provided a method for preparing an antimicrobial peptide by fermentation, comprising the steps of:

s1, putting the bacillus subtilis into an LB liquid culture medium to obtain a culture solution.

S2, inoculating the culture solution into a fermentation medium by an inoculation amount of 1% for fermentation; wherein the fermentation temperature is 38 ℃, and the fermentation time is 14 hours; the fermentation medium comprises the following components in percentage by weight: 22 parts of potato starch, 22 parts of glucose, 26 parts of raffinose, 3 parts of yeast powder, 6 parts of sodium acetate, 3 parts of diamine citrate, 4 parts of dipotassium hydrogen phosphate, 0.4 part of magnesium sulfate, 0.05 part of manganese sulfate and 0.2 part of tween 80.

S3, centrifuging the culture solution obtained in the step S2, and then precipitating the culture solution by ammonium sulfate with the mass concentration of 390g/L, wherein the precipitate is the obtained antibacterial peptide.

Example 2

As a second embodiment, this example provides a method for preparing antibacterial peptide by fermentation, comprising the following steps:

s1, putting the bacillus subtilis into an LB liquid culture medium to obtain a culture solution.

S2, inoculating the culture solution into a fermentation medium by an inoculation amount of 1% for fermentation; wherein the fermentation temperature is 36 ℃, and the fermentation time is 12 hours; the fermentation medium comprises the following components in percentage by weight: 16 parts of potato starch, 16 parts of glucose, 20 parts of raffinose, 1 part of yeast powder, 4 parts of sodium acetate, 1 part of diamine citrate, 2 parts of dipotassium hydrogen phosphate, 0.1 part of magnesium sulfate, 0.01 part of manganese sulfate and 0.1 part of Tween 80.

S3, centrifuging the culture solution obtained in the step S2, and then precipitating the culture solution by ammonium sulfate with the mass concentration of 390g/L, wherein the precipitate is the obtained antibacterial peptide.

Example 3

As a third embodiment, this example provides a method for preparing antibacterial peptide by fermentation, comprising the following steps:

s1, putting the bacillus subtilis into an LB liquid culture medium to obtain a culture solution.

S2, inoculating the culture solution into a fermentation medium by an inoculation amount of 1% for fermentation; wherein the fermentation temperature is 37 ℃, and the fermentation time is 13 hours; the fermentation medium comprises the following components in percentage by weight: 20 parts of potato starch, 20 parts of glucose, 25 parts of raffinose, 2.5 parts of yeast powder, 5 parts of sodium acetate, 2 parts of diamine citrate, 3 parts of dipotassium hydrogen phosphate, 0.2 part of magnesium sulfate, 0.03 part of manganese sulfate and 0.15 part of Tween 80.

S3, centrifuging the culture solution obtained in the step S2, and then precipitating the culture solution by ammonium sulfate with the mass concentration of 390g/L, wherein the precipitate is the obtained antibacterial peptide.

Example 4

As a fourth embodiment, this embodiment further provides a method for preparing an antimicrobial peptide hydrogel, comprising the following steps:

q1, degumming, dissolving and dialyzing the antibacterial peptide prepared by the antibacterial peptide fermentation preparation method to form an antibacterial peptide water solution with the mass fraction of 40%; according to the mass ratio of lactide: preparing a polylactic acid-polyethylene glycol-polylactic acid copolymer by a ring-opening reaction, wherein the ratio of polyethylene glycol to polyethylene glycol is 1: 5;

q2, adding the polylactic acid-polyethylene glycol-polylactic acid copolymer into the antibacterial peptide aqueous solution, and standing for 20 minutes at 25 ℃ to form the antibacterial peptide hydrogel.

Example 5

As a fifth embodiment, this embodiment further provides a method for preparing an antimicrobial peptide hydrogel, comprising the following steps:

q1, degumming, dissolving and dialyzing the antibacterial peptide prepared by the antibacterial peptide fermentation preparation method to form an antibacterial peptide water solution with the mass fraction of 40%; according to the mass ratio of lactide: preparing a polylactic acid-polyethylene glycol-polylactic acid copolymer by a ring-opening reaction, wherein the ratio of polyethylene glycol to polyethylene glycol is 5: 1;

q2, adding the polylactic acid-polyethylene glycol-polylactic acid copolymer into the antibacterial peptide aqueous solution, and standing for 20 minutes at 10 ℃ to form the antibacterial peptide hydrogel.

Example 6

As a sixth embodiment, this embodiment further provides a method for preparing an antimicrobial peptide hydrogel, including the following steps:

q1, degumming, dissolving and dialyzing the antibacterial peptide prepared by the antibacterial peptide fermentation preparation method to form an antibacterial peptide water solution with the mass fraction of 40%; according to the mass ratio of lactide: preparing a polylactic acid-polyethylene glycol-polylactic acid copolymer by a ring-opening reaction, wherein polyethylene glycol is 2: 1;

q2, adding the polylactic acid-polyethylene glycol-polylactic acid copolymer into the antibacterial peptide aqueous solution, and standing for 20 minutes at 20 ℃ to form the antibacterial peptide hydrogel.

The molecular weight of the antibacterial peptide is 3.4kDa, and the antibacterial peptide plays the greatest role in inhibiting bacteria at the temperature of 4 ℃ and the concentration of 9-12 mu g/mL. The storage condition is pH 4-7, the temperature is 4-18 ℃, and the storage can be stably carried out for at least 1.5-2 years. The pH is maintained at the highest activity of the neutral antimicrobial peptide subtilosin.

The embodiment also provides an antibacterial peptide hydrogel applied to treating vaginitis.

The inhibitory effect of the antimicrobial peptide hydrogel on pathogens of vaginal inflammation is tested below.

(1) The experimental principle is as follows:

the microbial flora in vagina is a complex environment formed by a plurality of microorganisms, and clinically, vaginitis can be divided into four types according to pathogenic pathogens: trichomonas Vaginitis (TV), candida vaginitis (VVC), Bacterial Vaginitis (BV), and Aerobic Vaginitis (AV). The four types of vaginitis are different in pathogens, namely trichomonas vaginalis, candida albicans, gardnerella vaginalis, other aerobic bacteria and the like. In addition, lactobacillus is used as normal flora in vagina, such as vaginitis caused by vaginal environment change caused by quantity reduction. Therefore, trichomonas vaginalis, candida albicans, escherichia coli and lactobacillus (two strains) are planned and selected as experimental objects in the subsequent bacteriostatic experiments.

(2) Experimental Material

Trichomonas vaginalis

Culture medium: adding serum into the liver extract culture medium

The culture method comprises the following steps: anaerobic culture at 37 ℃

Experimental reagent: metronidazole

Other experimental materials: 96-well plates (Nunc), triangular flasks, Tips, etc

Candida albicans

Culture medium: sandcastle culture medium

The culture method comprises the following steps: culturing at 37 deg.C

Experimental reagent: amphotericin B

Other experimental materials: 96-well plates (Nunc), triangular flasks, Tips, etc

Escherichia coli

Culture medium: LB Medium

The culture method comprises the following steps: culturing at 37 deg.C

Experimental reagent: ceftriaxone

Other experimental materials: 96-well plates (Nunc), triangular flasks, Tips, etc

Lactobacillus strain

Culture medium: lactobacillus broth culture medium

The culture method comprises the following steps: culturing at 37 deg.C

Other experimental materials: 96-well plates (Nunc), triangular flasks, Tips, etc

(3) Experimental methods

1. Experiment on inhibition effect of antibacterial peptide on trichomonas vaginalis

The trichomonas was inoculated into 5ml of the liver extract medium and incubated overnight at 37 ℃ in an incubator.

Fresh liquid medium was prepared and the broth was placed in a 15ml tube. Serial dilution of the antibacterial peptide is carried out, and a metronidazole positive control group is arranged. There were 12 tubes in total, two replicates per group.

Negative control group: PBS (Poly Butylene succinate)

Positive control group: metronidazole 60 microgram/ml

Experimental group 1: 440 mu g/ml antibacterial peptide

Experimental group 2: 220 mu g/ml antibacterial peptide

Experimental group 3: 110 mu g/ml antibacterial peptide

Experimental group 4: 55 mu g/ml antibacterial peptide

0.05ml of trichomonas suspension is taken, added into each tube and mixed evenly. The test tube is placed in an incubator at 37 ℃ for culturing for 48h, and a drop of culture solution is sucked from the bottom of the tube and placed on a glass slide for observation under a microscope. The concentration of the minimum concentration tube in which the trichomonas does not grow is the minimum inhibitory concentration.

2. Experiment of inhibitory effect of antibacterial peptide on candida albicans

Colonies were picked and inoculated into 5ml of sandcastle medium and incubated overnight at 37 ℃ in an incubator.

Transferring the strain to a fresh sandcastle culture medium at a ratio of 1:100, and putting the strain liquid into a 96-well plate. Divided into 7 groups, each group is provided with eight complex holes.

Negative control group: DMSO (amphotericin B preparation solvent)

Negative control group: PBS (solvent for antibacterial peptide)

Positive control group: 10 ug/ml amphotericin B

Experimental group 1: 440 mu g/ml antibacterial peptide

Experimental group 2: 220 mu g/ml antibacterial peptide

Experimental group 3: 110 mu g/ml antibacterial peptide

Experimental group 4: 55 mu g/ml antibacterial peptide

The 96-well plate was incubated at 37 ℃ in an incubator, and the bacterial OD600 values were monitored at 24h and 48h, respectively.

3. Experiment of inhibitory effect of antibacterial peptide on Escherichia coli

Colonies were picked and inoculated in 5ml of LB medium and incubated overnight at 37 ℃ in an incubator.

1:100 was transferred to fresh LB medium and the bacterial solution was placed in a 96-well plate. Divided into 6 groups, each group is provided with eight complex holes.

Negative control group: PBS (Poly Butylene succinate)

Positive control group: 10 mug/ml ceftriaxone

Experimental group 1: 440 mu g/ml antibacterial peptide

Experimental group 2: 220 mu g/ml antibacterial peptide

Experimental group 3: 110 mu g/ml antibacterial peptide

Experimental group 4: 55 mu g/ml antibacterial peptide

The 96-well plate was incubated at 37 ℃ in an incubator, and the bacterial OD600 values were monitored at 24h and 48h, respectively.

4. Antibacterial peptide inhibiting vaginal normal bacteria lactobacillus

Two kinds of common lactobacillus in vagina, lactobacillus gasseri and lactobacillus crispatus, were selected in this experiment.

Colonies were picked and inoculated into 5ml of lactobacillus broth and cultured overnight in an anaerobic chamber at 37 ℃.

1:100 was transferred to fresh lactobacillus broth medium and the broth was placed in a 96-well plate. Is divided into 5 groups, and each group is provided with eight holes.

Negative control group: PBS (Poly Butylene succinate)

Experimental group 1: 440 mu g/ml antibacterial peptide

Experimental group 2: 220 mu g/ml antibacterial peptide

Experimental group 3: 110 mu g/ml antibacterial peptide

Experimental group 4: 55 mu g/ml antibacterial peptide

The 96-well plate was placed in an anaerobic chamber at 37 ℃ for culture, and the bacterial OD600 values were monitored at 24h and 48h, respectively.

Three strains of candida albicans are preserved in our laboratory, and are respectively highly sensitive, moderately sensitive and insensitive to antifungal drugs. Therefore, a total of 8 pathogens were used in this antimicrobial peptide experiment: trichomonas vaginalis, 3 strains of Candida albicans, Escherichia coli, Lactobacillus gasseri and Lactobacillus crispatus.

Estimating the using amount of the antibacterial peptide: the concentration of the antimicrobial peptide stored before is 4.4mg/ml, about 1.5ml of antimicrobial peptide is needed in one experiment of each pathogen, namely, the dosage of the antimicrobial peptide is 6.6mg, and the total amount of the antimicrobial peptide is 66mg considering that the loss caused by operation can be caused in the actual experiment.

The bacteriostatic activity of the antimicrobial peptides was tested as follows.

(1) Preparation of antibacterial peptide suspension

The antimicrobial peptide dry powder weighed 6.0mg, had a purity of 95%, and was dissolved in 1mL of PBS to a final concentration of 5.7 mg/mL.

(2) Antibacterial peptide bacteriostasis test

Colonies on a blood plate are picked up to be placed in a liquid culture medium, after 48 hours of culture, the colonies are transferred into a fresh culture medium, the initial OD600 is made to be 0.1, PBS, metronidazole and antibacterial peptide are respectively added in sequence, and the OD600 values are measured after 6, 12, 24, 36 and 48 hours of culture.

(3) The above-mentioned OD600 values were processed as follows: the A-H complex wells of each group were averaged, and the final OD600 value of each group was calculated by subtracting the average value of Blank groups, and a line graph was constructed with time (hour) as abscissa and OD600 as ordinate. The results are shown in FIG. 2.

From the test results, when the concentration of metronidazole is 60 mug/ml, the gardnerella vaginalis does not grow obviously, and when the concentration of metronidazole is 30 mug/ml, the bacteria partially grow in 48 hours. When the concentration of the antibacterial peptide is 35.6 mu g/ml, the bacteria grow normally, and when the concentration is 71.25 mu g/ml or more, the bacteria do not grow obviously.

Hydrogel structure: PEG-SH (8-arm) and PEG-NHS (4-arm) are covalently cross-linked by thioester linkages based on PEG.

Controlled release of lactic acid by hydrogel: the vaginal environment was reconstructed based on degradable hydrogels based on PEG-lactic acid bacteria nanocarriers.

PEG nanocarrier based hydrogels were used to achieve controlled release of lactic acid. Such nanocarrier-based hydrogels form rapidly (within 20 min), are elastic under physiologically acidic conditions, and can degrade. The process of release of lactic acid from the hydrogel may last for several days. The maximum release rate is about 14%. In contrast, the hydrogel with entrapped lactic acid can achieve a release rate of up to 90% in 30 min. Lactic acid showed bacteriostatic (original bv pathogen Gardnerella vagianalis) activity at a MIC of 3.6 mg/mL. The hydrogel containing entrapped lactic acid completely inhibited the growth of g. These studies all show the great potential of PEG nanocarrier based hydrogels (intravaginal administration) for the prevention and treatment of BV.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.