CN109652482B

CN109652482B - Antibacterial peptide and preparation method and application thereof

Info

Publication number: CN109652482B
Application number: CN201811314937.0A
Authority: CN
Inventors: 方祥; 陈海彬; 王洁; 廖振林; 卢华敏; 梁嘉仪
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2018-11-06
Filing date: 2018-11-06
Publication date: 2021-12-17
Anticipated expiration: 2038-11-06
Also published as: CN109652482A

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to an antibacterial peptide, and a preparation method and application thereof. The antibacterial peptide is prepared by fermenting milk through a mixed strain of lactobacillus paracasei and kluyveromyces marxianus and mainly comprises the following steps: (1) preparing a mixed strain fermentation mother liquor; (2) inoculating fermentation mother liquor, and fermenting at low temperature for a long time to prepare whey; (3) separating whey by ultrafiltration to obtain crude extract containing antibacterial peptide; (4) the antibacterial peptide of interest is purified by gel chromatography. The antibacterial peptide prepared by the invention has good thermal stability, no stimulation to human bodies and wide antibacterial spectrum, particularly has obvious inhibition effect on propionibacterium acnes, can replace antibiotics, effectively reduces the use of the antibiotics, and can be used for preventing and treating acne.

Description

Antibacterial peptide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an antibacterial peptide, and a preparation method and application thereof.

Background

Acne, commonly known as whelk, is a chronic inflammatory skin disease associated with gonadal endocrine dysfunction. It is common in adolescence and can extrude yellowish lipid plugs, which are manifested as skin lesions such as black and white acne, papule, pustule, nodule, cyst and scar. Such skin diseases can affect the beauty and even cause disfigurement in severe cases, thereby bringing great psychological burden and mental stress to patients. The etiology and pathogenesis of acne are complex, and are mainly related to four main reasons of propionibacterium acnes infection, androgen, increased sebum secretion and hyperkeratosis at the opening of pilo sebaceous glands, and the propionibacterium acnes becomes one of the most main inducing factors of acne.

Propionibacterium acnes belongs to the order of Actinomycetales, the family, genus, Propionibacterium acnes, and is a gram-positive, anaerobic, immotile brevibacterium. It is widely found in the air, soil, water bodies and animal bodies, especially human skin. Propionibacterium acnes secrete esterases that break down the cutin and lipid components of the skin, and metabolites such as triacylglycerols that enhance the inflammatory response, leading to the production of acne. It often invades into deep part of hair follicle and epidermis, is anaerobic, grows slowly and has physicochemical properties, so that it is not easy to be removed, and becomes conditioned pathogen.

At present, antibiotics are used as an important method for treating acne, and main antibacterial drugs comprise tetracyclines, macrolides, clindamycin, compound sulfamethoxazole, quinolones and the like. However, in recent years, the use of antibiotics is not standardized, and the drug resistance of propionibacterium acnes is stronger, so that the safety of the antibiotics is more and more questioned. If a substance could be developed that could inhibit propionibacterium acnes instead of antibiotics, this would be a boon for many acne patients.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the invention aims to provide the preparation method of the antibacterial peptide, the method is simple and convenient to operate, large-scale industrial production is easy to realize, the raw materials are edible materials, and the preparation method is green, healthy and extremely high in safety.

The invention also aims to provide the antibacterial peptide prepared by the preparation method, which has good thermal stability, no stimulation to normal animals and obvious inhibition to propionibacterium acnes.

Still another object of the present invention is to provide the use of the above antibacterial peptide.

The purpose of the invention is realized by the following technical scheme:

a preparation method of antibacterial peptide comprises the following steps:

(1) preparing a mixed strain fermentation mother liquor: mixing milk powder and water to obtain 8-20% of reduced milk by mass percent, emulsifying and homogenizing the reduced milk, sterilizing and cooling; inoculating lactobacillus paracasei suspension and Kluyveromyces lactis suspension into the cooled reduced milk under aseptic conditions, and performing static culture at a constant temperature of 25-42 ℃ for 2-4 days to obtain a fermentation mother liquor;

(2) preparing whey from fermentation mother liquor: mixing milk powder and water to obtain 5-20% of reduced milk by mass percent, emulsifying and homogenizing the reduced milk, sterilizing and cooling; inoculating the fermentation mother liquor prepared in the step (1) into cooled reduced milk under an aseptic condition, and then carrying out fermentation culture at 25-42 ℃ for 2-30 days to obtain a fermentation liquor containing whey;

(3) and (3) performing ultrafiltration separation on whey to obtain the antibacterial peptide: filtering the fermentation liquor containing whey prepared in the step (2), collecting filtrate, and performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 3000-150000 daltons to obtain a crude extract of the antibacterial peptide;

(4) purifying the target antibacterial peptide by gel chromatography: purifying the crude extract of the antibacterial peptide prepared in the step (3) by using sephadex G-25 as a matrix and phosphate buffer solution as an eluent to obtain the antibacterial peptide;

the mass percentage of the reduced milk in the step (1) is preferably 8-15%;

the Lactobacillus paracasei (SLBC 16-4) in the step (1) is preserved in the China general microbiological culture Collection center at 2016, 12 and 26 days, with the preservation number of CGMCC NO. 13498;

kluyveromyces marxianus (SYKM 16-1) in step (1) is preserved in China general microbiological culture Collection center (CGMCC) at 26 months 12 in 2016 with the preservation number of CGMCC NO. 13499;

the initial concentration of the Lactobacillus paracasei suspension in step (1) is preferably 10^-7～10^-10cfu/ml；

The initial concentration of the Lactobacillus paracasei suspension in step (1) is more preferably 10^-9～10^-10cfu/ml；

The inoculation amount of the lactobacillus paracasei suspension in the step (1) is preferably 2-10% by volume;

the inoculation amount of the lactobacillus paracasei suspension in the step (1) is further preferably 5-10% by volume;

the initial concentration of the Kluyveromyces marxianus suspension in the step (1) is preferably 10^-7～10^-10cfu/ml；

The initial concentration of the Kluyveromyces marxianus suspension in the step (1) is more preferably 10^-9～10^- ¹⁰cfu/ml；

The inoculation amount of the Kluyveromyces marxianus suspension in the step (1) is preferably 2-10% by volume;

the inoculation amount of the Kluyveromyces marxianus suspension in the step (1) is further preferably 5-10% by volume;

the preferred sterilization in the step (1) is pasteurization at 60-82 ℃ for 20-30 min;

the cooling in the step (1) is preferably to be carried out to 25-42 ℃;

the temperature of the standing culture in the step (1) is preferably 25-37 ℃;

the time of the standing culture in the step (1) is preferably 2-3 days;

the mass percentage of the reduced milk in the step (2) is preferably 8-15%;

the inoculation amount of the fermentation mother liquor in the step (2) is preferably 1-15% by volume;

the inoculation amount of the fermentation mother liquor in the step (2) is further preferably 5-10% by volume;

the preferred sterilization in the step (2) is pasteurization at 60-82 ℃ for 20-30 min;

the cooling in the step (2) is preferably to be carried out to 25-42 ℃;

the temperature of the fermentation culture in the step (2) is preferably 25-37 ℃;

the time of fermentation culture in the step (2) is preferably 7-28 days;

filtering in the step (3) preferably by using a sterilized cloth bag to filter larger particles in the fermentation liquor;

the cutoff molecular weight of the ultrafiltration membrane in the step (3) is preferably 3000-10000 daltons;

the pH of the phosphate buffer solution in the step (3) is preferably 7.0;

the flow rate of the upper column purification in the step (4) is preferably 0.5-10 ml/min;

the flow rate of the upper column purification in the step (4) is further preferably 1-5 ml/min;

preferably, a nucleic acid protein detector is adopted for detection in the purification process in the step (4), and effluent liquid of the maximum peak is collected;

the detection wavelength is 254-280 nm;

the detection wavelength is preferably 260-280 nm;

an antibacterial peptide is prepared by the preparation method;

the application of the antibacterial peptide in preparing products for preventing and treating acne;

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

(1) the invention can obtain the relatively pure antibacterial peptide through ultrafiltration and one-step purification, and the method has simple and convenient operation and is easy for industrial production.

(2) The invention utilizes probiotics such as lactobacillus paracasei and kluyveromyces marxianus and the like to ferment milk to prepare the antibacterial peptide, the used bacterial strains are edible bacterial strains, the used raw materials are edible materials, and the antibacterial peptide has no toxic action on human bodies and extremely high safety.

(3) The antibacterial peptide prepared by the invention can resist the high temperature of 121 ℃, has good thermal stability, has no stimulation to human body and wide antibacterial spectrum, particularly has obvious inhibition effect on propionibacterium acnes, can replace antibiotics, effectively reduces the use of the antibiotics, and can be used for preventing and treating acne.

(4) The antibacterial peptide prepared by the invention consists of 12-100 amino acid residues, contains hydrophobic amino acid, is mostly amphiphilic cation, and has various biological activities of resisting bacteria, fungi, viruses and parasites, selectively inducing tumor cells to apoptosis and the like. Because of the characteristics of wide antibacterial spectrum, good stability, almost no toxic action on normal animal cells, difficult generation of drug resistance and the like, the antibacterial peptide is likely to become a substitute of the traditional antibiotics.

Drawings

Fig. 1 is a gram stain and colony morphology map of lactobacillus paracasei, wherein a: gram stain (10 × 100), B: colony morphology.

FIG. 2 is a chart of methylene blue staining and colony morphology of Kluyveromyces marxianus, wherein A: methylene blue staining (10 × 40), B: colony morphology.

FIG. 3 is a diagram showing real-time monitoring of OD of a sample during SephadexG-25 gel purification of example 8₂₈₀Variation diagram, wherein the abscissa is time and the ordinate is OD₂₈₀The value is obtained.

FIG. 4 is a graph showing the results of the inhibition zone of the antibacterial peptide prepared in example 8 against Propionibacterium acnes ATCC 6919.

FIG. 5 is a graph showing the evaluation of the effect of the antibacterial peptide prepared in example 8 on acne patients.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

EXAMPLE 1 isolation and characterization of Lactobacillus paracasei strains

(1) The lactobacillus paracasei is isolated from kefir grains. The specific separation method is as follows:

1) 100 mul kefir grains fermentation liquor is inoculated in a liquid MRS culture medium and is statically cultured for 48 hours at the constant temperature of 37 ℃. 2) And (3) carrying out gradient dilution on the bacterial liquid, coating 100 mu l of diluted bacterial liquid on an MRS plate, and carrying out static culture at a constant temperature of 37 ℃ for 48 h. 3) Picking out single colony with larger colony according to colony morphology, streaking on a new MPS plate, standing and culturing at 37 ℃ for 48h, performing gram staining and microscopic examination, and observing cell morphology. 4) Inoculating the pure strain into MRS liquid culture medium after microscopic examination to obtain a pure strain, standing at 37 deg.C for 48 hr, adding sterilized glycerol 30 vol%, mixing, and storing at-80 deg.C.

(2) Identification of strains

The separated lactobacillus paracasei is purple in gram stain, long rod-shaped and arranged singly or in pairs, and can be seen in a figure 1A.

Secondly, the colony form of the separated lactobacillus paracasei is circular, the surface is smooth, raised and opaque, the edge is neat, and the color is milky, which can be seen in fig. 1B.

③ the physiological and biochemical identification results of the separated lactobacillus paracasei are shown in the following table:

TABLE 1 physiological and biochemical identification results of Lactobacillus paracasei

Note "+" indicates a positive result; "one" indicates a negative result.

Fourthly, the rDNA-ITS full-length basic sequence of the separated lactobacillus paracasei is as follows:

GTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCGCTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGCCGACAACAGTTACTCTGCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAAGCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCC

the result shows that the strain is a new strain of Lactobacillus paracasei, the strain is named as Lactobacillus paracasei (Lactobacillus paracasei) SLBC16-4, and is preserved in China general microbiological culture Collection Center (CCM) 26 th 2016, 12 th and 26 th, wherein the preservation number is CGMCC NO.13498, and the microbial research institute of China academy of sciences No. 3, North Cheng West Lu No.1 institute of south China, the Naja area, Beijing.

The Lactobacillus paracasei (Lactobacillus paracasei) SLBC16-4 separated and preserved in the example was activated and cultured to obtain a concentration of 10^-7cfu/ml、10^-9cfu/ml and 10^-10cfu/ml of Lactobacillus paracasei suspension was used in the following examples.

Example 2 isolation and characterization of Kluyveromyces marxianus

(1) Kluyveromyces marxianus is obtained by separating from kefir grains. The specific separation method is as follows:

1) 100 mul kefir grains of fermentation broth were inoculated into YPD liquid medium and incubated at 28 ℃ for 72 h. 2) The bacterial liquid was diluted in a gradient, 100. mu.l of the diluted bacterial liquid was applied to YPD plates and cultured at 28 ℃ for 72 hours. 3) A single large colony is selected according to the colony morphology, streaked on a new YPD plate, cultured at 28 ℃ for 72h, and then subjected to blue-blue staining and microscopic examination to observe the cell morphology. 4) Inoculating pure strain to YPD liquid culture medium, culturing at 28 deg.C for 72 hr, adding sterilized glycerol 30 vol%, mixing, and storing at-80 deg.C.

(2) Identification of strains

The separated Kluyveromyces marxianus is found to be bluish purple through methylene blue staining, and is round or oval in shape, and one end of the Kluyveromyces marxianus buds, which is shown in FIG. 2A.

The colony form of the separated Kluyveromyces marxianus is round, the surface is smooth and wet, the color is milky white, the surface is convex, the edge is neat and opaque, and a figure 2B can be seen.

③ the physiological and biochemical identification results of the separated kluyveromyces marxianus are as follows:

note that "+" indicates that the gas production result is positive; "one" indicates that the gas production result is negative.

The rDNA-ITS full-length base sequence of the isolated Kluyveromyces marxianus is as follows:

AAGGAGACAAACACCAGCGAGTCTTTATAACACCTATGAGTCTCTATGACCCAAGCTTACCACGAATTGGCGCAAACCTAAGACGTAGATGTGCAAGAGTCGAGTCCATAGACTTGACACGCAGCCCTGCTCACGCAGATGGCAACGGCTAGCCACTTTCAAGTTAACCCGAGACGAGTATCACTCACTACCAAACCCAAAGGTTTGAGAGAGAAATGACGCTCAAACAGGCATGCCCCCTGGAATACCAGAGGGCGCAATGTGCGTTCAAAGATTTGATGATTCACGAAAATCTGCAATTCACAATACATATCGCAATTCGCTGCGTTCTTCATCGATGCGAGAACCAAGAGATCCGTTGTTGAAAGTTTTGAATATTAAATTTTATAGTATAATAATAGTTTTTCATAATACAAAATATTGTTTGTGTTTATGTCCACTGGAGAGACGAGCTCTCCAGGGAAGTAGTTCATAGAGAAAAAACTCCATTGTGTTTAGGATGAGAAATAGAAAACTGATAGCAGAGAATCAAGAACTGGCCGCGCAATTAAGCGCAGGCCTTGTTCAGACGATTCCCCCAGCAATCTATTCATTCATAATCTTTAATGATCCTTGCGCAGGTCA

the result shows that the strain is a new strain of Kluyveromyces marxianus (Kluyveromyces marxianus) SYKM16-1, is preserved in China general microbiological culture Collection center (CGMCC NO. 13499) at 26 th of 2016, and has the preservation number of CGMCC NO.13499, and the microbial research institute of China academy of sciences No. 3 of North Chen West Lu 1 institute of the Korean district, Beijing.

Kluyveromyces marxianus SYKM16-1 isolated and preserved in this example was activated and cultured to obtain 10% yeast^-7cfu/ml、10^-9cfu/ml and 10^-10A suspension of cfu/ml Kluyveromyces marxianus was used in the following examples.

Example 3

(1) Preparing a mixed strain fermentation mother liquor: weighing 8.00g of milk powder and ionized water, uniformly mixing to obtain 100ml of 8% reduced milk by mass percent, emulsifying and homogenizing the reduced milk, pasteurizing at 65 ℃ for 30min, and cooling to 25 ℃; inoculating 2mL of the mixture under sterile conditions with an initial concentration of 10^-7cfu/mL of Lactobacillus paracasei suspension (prepared in example 1) and 2mL of initial concentration 10^-7cfu/ml Kluyveromyces marxianus suspension (prepared in example 1) is subjected to static culture at a constant temperature of 28 ℃ for 4 days to obtain a fermentation mother liquor;

(2) preparing whey from fermentation mother liquor: uniformly mixing milk powder and ionized water to obtain 1000ml of reduced milk with the mass percentage of 5%, emulsifying and homogenizing the reduced milk, pasteurizing the reduced milk at 65 ℃ for 30min, placing the reduced milk in a fermentation tank, cooling the reduced milk to 32 ℃, inoculating 20ml of fermentation mother liquor prepared in the step (1) in the fermentation tank under an aseptic condition, and performing fermentation culture at 30 ℃ for 6d to obtain fermentation liquor containing whey;

(3) and (3) performing ultrafiltration separation on whey to obtain the antibacterial peptide: filtering larger particles in the whey-containing fermentation liquor prepared in the step (2) by using a sterilized cloth bag, collecting filtrate, and performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 3000 daltons to obtain a crude extract of the antibacterial peptide;

(4) purifying the target antibacterial peptide by gel chromatography: and (2) taking sephadex G-25 as a matrix, taking a phosphate buffer solution with the pH of 7.0 as an eluent, purifying the crude extract of the antibacterial peptide prepared in the step (3) on a column, wherein the loading amount is 1ml, the flow rate is controlled at 1ml/min, detecting the light absorption value of the effluent at 280nm by using a nucleic acid protein detector in real time in the purification process, drawing a curve according to different light absorption values (the abscissa is time and the ordinate is the light absorption value) of the sample at different times, and collecting the effluent of the maximum peak to obtain the antibacterial peptide.

Example 4

(1) Preparing a mixed strain fermentation mother liquor: weighing 12.00g of milk powder and ionized water, uniformly mixing to obtain 100ml of 12% reduced milk, emulsifying and homogenizing the reduced milk, pasteurizing at 75 ℃ for 30min, and cooling to 35 ℃; inoculating 3mL of the mixture under sterile conditions with an initial concentration of 10^-9cfu/mL of Lactobacillus paracasei suspension (prepared in example 1) and 2mL of initial concentration 10^- ⁹A suspension of Kluyveromyces marxianus (prepared in example 1) in cfu/ml is subjected to static culture at a constant temperature of 37 ℃ for 3 days to obtain a fermentation mother liquor;

(2) preparing whey from fermentation mother liquor: uniformly mixing milk powder and ionized water to obtain 1000ml of 8 mass percent reduced milk, emulsifying and homogenizing the reduced milk, carrying out pasteurization at 75 ℃ for 30min, placing the reduced milk in a fermentation tank, cooling the reduced milk to 42 ℃, inoculating 150ml of fermentation mother liquor prepared in the step (1) in the fermentation tank under an aseptic condition, and carrying out fermentation culture at 42 ℃ for 2d to obtain fermentation liquor containing whey;

(3) and (3) performing ultrafiltration separation on whey to obtain the antibacterial peptide: filtering larger particles in the whey-containing fermentation liquor prepared in the step (2) by using a sterilized cloth bag, collecting filtrate, and performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 5000 daltons to obtain a crude extract of the antibacterial peptide;

(4) purifying the target antibacterial peptide by gel chromatography: and (2) taking sephadex G-25 as a matrix, taking a phosphate buffer solution with the pH of 7.0 as an eluent, purifying the crude extract of the antibacterial peptide prepared in the step (3) on a column, wherein the sample loading amount is 2ml, the flow rate is controlled at 1ml/min, detecting the light absorption value of the effluent at 280nm by using a nucleic acid protein detector in real time in the purification process, drawing a curve according to different light absorption values (the abscissa is time and the ordinate is the light absorption value) of the sample at different times, and collecting the effluent of the maximum peak to obtain the antibacterial peptide.

Example 5

(1) Preparing a mixed strain fermentation mother liquor: weighing 14.00g of milk powder and ionized water, mixing uniformly to obtain 100ml of 14% reduced milk, emulsifying, homogenizing, pasteurizing at 80 deg.C for 25min, and cooling to 40 deg.C; inoculating 4mL of the mixture under sterile conditions with an initial concentration of 10^-10cfu/mL of Lactobacillus paracasei suspension (prepared in example 1) and 2mL of initial concentration 10^- ¹⁰A suspension of Kluyveromyces marxianus (prepared in example 1) in cfu/ml is subjected to static culture at a constant temperature of 25 ℃ for 2 days to obtain a fermentation mother liquor;

(2) preparing whey from fermentation mother liquor: uniformly mixing milk powder and ionized water to obtain 1000ml of reduced milk with the mass percentage of 14%, emulsifying and homogenizing the reduced milk, carrying out pasteurization at 80 ℃ for 25min, placing the reduced milk in a fermentation tank, cooling the reduced milk to 25 ℃, inoculating 40ml of fermentation mother liquor prepared in the step (1) into the fermentation tank under an aseptic condition, and carrying out fermentation culture at 25 ℃ for 8d to obtain fermentation liquor containing whey;

(3) and (3) performing ultrafiltration separation on whey to obtain the antibacterial peptide: filtering larger particles in the whey-containing fermentation liquor prepared in the step (2) by using a sterilized cloth bag, collecting filtrate, and performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 8000 daltons to obtain a crude extract of the antibacterial peptide;

(4) purifying the target antibacterial peptide by gel chromatography: and (2) taking sephadex G-25 as a matrix, taking a phosphate buffer solution with the pH of 7.0 as an eluent, purifying the crude extract of the antibacterial peptide prepared in the step (3) on a column, wherein the sample loading amount is 3ml, the flow rate is controlled at 3ml/min, detecting the light absorption value at 268nm of the effluent by using a nucleic acid protein detector in real time in the purification process, drawing a curve according to different light absorption values (the abscissa is time and the ordinate is the light absorption value) of the sample at different times, and collecting the effluent of the maximum peak to obtain the antibacterial peptide.

Example 6

(1) Preparing a mixed strain fermentation mother liquor: weighing 15.00g of milk powder, uniformly mixing with ionized water to obtain 100ml of reduced milk with the mass percentage of 15%, emulsifying and homogenizing the reduced milk, pasteurizing at 82 ℃ for 20min, and cooling to 42 ℃; inoculating 4mL of the mixture under sterile conditions with an initial concentration of 10^-10cfu/mL of Lactobacillus paracasei suspension (prepared in example 1) and 6mL of initialAt a concentration of 10^- ¹⁰A suspension of Kluyveromyces marxianus (prepared in example 1) in cfu/ml is subjected to static culture at a constant temperature of 25 ℃ for 2 days to obtain a fermentation mother liquor;

(2) preparing whey from fermentation mother liquor: uniformly mixing milk powder and ionized water to obtain 1000ml of reduced milk with the mass percentage of 15%, emulsifying and homogenizing the reduced milk, carrying out pasteurization at 82 ℃ for 20min, placing the reduced milk in a fermentation tank, cooling the reduced milk to 42 ℃, inoculating 60ml of fermentation mother liquor prepared in the step (1) into the fermentation tank under an aseptic condition, and carrying out fermentation culture at 25 ℃ for 12d to obtain fermentation liquor containing whey;

(3) and (3) performing ultrafiltration separation on whey to obtain the antibacterial peptide: filtering larger particles in the whey-containing fermentation liquor prepared in the step (2) by using a sterilized cloth bag, collecting filtrate, and performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 10000 Dalton to obtain a crude extract of the antibacterial peptide;

(4) purifying the target antibacterial peptide by gel chromatography: and (2) taking sephadex G-25 as a matrix, taking a phosphate buffer solution with the pH of 7.0 as an eluent, purifying the crude extract of the antibacterial peptide prepared in the step (3) on a column, wherein the loading amount is 3.5ml, the flow rate is controlled at 6ml/min, detecting the light absorption value of 269nm of the effluent by using a nucleic acid protein detector in real time in the purification process, drawing a curve according to different light absorption values (the abscissa is time and the ordinate is the light absorption value) of the sample at different times, and collecting the effluent of the maximum peak to obtain the antibacterial peptide.

Example 7

(1) Preparing a mixed strain fermentation mother liquor: weighing 20.00g of milk powder, mixing with ionized water uniformly to obtain 100ml of 20% reduced milk, emulsifying, homogenizing, pasteurizing at 82 deg.C for 30min, and cooling to 37 deg.C; inoculating 10mL of the mixture under the aseptic condition with the initial concentration of 10^-10cfu/mL of Lactobacillus paracasei suspension (prepared in example 1) and 10mL of initial concentration 10^- ¹⁰cfu/ml Kluyveromyces marxianus suspension (prepared in example 1) is subjected to static culture at a constant temperature of 30 ℃ for 2 days to obtain a fermentation mother liquor;

(2) preparing whey from fermentation mother liquor: uniformly mixing milk powder and ionized water to obtain 1000ml of reduced milk with the mass percentage of 20%, emulsifying and homogenizing the reduced milk, carrying out pasteurization at 82 ℃ for 30min, placing the reduced milk in a fermentation tank, cooling the reduced milk to 37 ℃, inoculating 80ml of fermentation mother liquor prepared in the step (1) into the fermentation tank under an aseptic condition, and carrying out fermentation culture at 25 ℃ for 30d to obtain fermentation liquor containing whey;

(3) and (3) performing ultrafiltration separation on whey to obtain the antibacterial peptide: filtering larger particles in the whey-containing fermentation liquor prepared in the step (2) by using a sterilized cloth bag, collecting filtrate, and performing ultrafiltration by using an ultrafiltration membrane with the molecular weight cutoff of 150000 daltons to obtain a crude extract of the antibacterial peptide;

(4) purifying the target antibacterial peptide by gel chromatography: and (2) taking sephadex G-25 as a matrix, taking a phosphate buffer solution with the pH of 7.0 as an eluent, purifying the crude extract of the antibacterial peptide prepared in the step (3) on a column, wherein the sample loading amount is 4ml, the flow rate is controlled at 5ml/min, detecting the light absorption value of the effluent at 280nm by using a nucleic acid protein detector in real time in the purification process, drawing a curve according to different light absorption values (the abscissa is time and the ordinate is the light absorption value) of the sample at different times, and collecting the effluent of the maximum peak to obtain the antibacterial peptide.

Example 8

(1) Preparing a mixed strain fermentation mother liquor: weighing 10.00g of milk powder and ionized water, uniformly mixing to obtain 100ml of 10% reduced milk, emulsifying and homogenizing the reduced milk, pasteurizing at 82 ℃ for 30min, and cooling to 35 ℃; inoculating 5mL of the mixture under sterile conditions with the initial concentration of 10^-10cfu/mL of Lactobacillus paracasei suspension (prepared in example 1) and 5mL of initial concentration 10^- ¹⁰A suspension of Kluyveromyces marxianus (prepared in example 1) in cfu/ml is subjected to static culture at a constant temperature of 25 ℃ for 2 days to obtain a fermentation mother liquor;

(2) preparing whey from fermentation mother liquor, uniformly mixing milk powder and ionized water to obtain 1000ml of reduced milk with the mass percentage of 10%, emulsifying and homogenizing the reduced milk, carrying out pasteurization at 82 ℃ for 30min, placing the reduced milk in a fermentation tank, cooling the reduced milk to 37 ℃, inoculating 80ml of fermentation mother liquor prepared in the step (1) into the fermentation tank under an aseptic condition, and carrying out fermentation culture at 25 ℃ for 28 days to obtain fermentation liquor containing whey;

(4) purifying the target antibacterial peptide by gel chromatography: and (2) taking sephadex G-25 as a matrix, taking a phosphate buffer solution with the pH of 7.0 as an eluent, purifying the crude extract of the antibacterial peptide prepared in the step (3) on a column, wherein the sample loading amount is 5ml, the flow rate is controlled at 5ml/min, detecting the light absorption value of the effluent at 280nm by using a nucleic acid protein detector in real time in the purification process, drawing a curve according to different light absorption values (the abscissa is time and the ordinate is the light absorption value) of the sample at different times, and collecting the effluent of the maximum peak (figure 3, peak 2) to obtain the antibacterial peptide, wherein the concentration of the antibacterial peptide is 148.23 mu g/ml.

Example 9 analysis of bacteriostatic Effect

(1) Adopting agar diffusion method as bacteriostatic loop

Diluting the antibacterial peptide prepared in the embodiment 8 by one time with deionized water to obtain an antibacterial peptide diluent; under the aseptic condition, 15mL of BHI solid culture medium is poured into the aseptic plate, and 4 Oxford cups with the outer diameter of 7.8 +/-0.1 mm are placed after the solid culture medium is solidified for 1 hour. To BHI solid Medium at about 50 ℃ was added 1mL of 1X 10⁸cfu/mL propionibacterium acnes suspension is evenly mixed and poured into the solidified BHI flat plate, and the oxford cup is taken out after solidification. After labeling, 150. mu.l of differently pretreated antimicrobial peptide dilutions or sterile water were added, A: and (3) treating the antibacterial peptide diluent at 115 ℃ for 20min, wherein B: antimicrobial peptide dilutions without any pretreatment, C: and (3) treating the antibacterial peptide diluent at 121 ℃ for 20min, and D: sterile water. After the plate is placed at 37 ℃ for anaerobic culture for 72h, the diameter of the inhibition zone is measured by a vernier caliper.

The results are shown in FIG. 4, wherein the diameter of the inhibition zone A is 17.20 + -0.1 mm; the diameter of the inhibition zone B is 21.30 +/-0.1 mm; the diameter of the bacteriostatic circle C is 13.50 +/-0.1 mm; d diameter is 7.8 +/-0.1 mm.

(2) Trial effect of acne patients

After washing the face of a patient with acne for 2 times by using clear water, a small amount of antibacterial peptide diluent (the antibacterial peptide prepared in example 8 is diluted by deionized water by one time) is dipped in a cotton swab and smeared on the affected part of acne for 1 time every 4 hours and 3 times every day, and the change condition of acne is recorded by photographing every day.

As a result, as shown in FIG. 5, after applying the antimicrobial peptide 5d prepared in example 8, inflammatory pustules were remarkably eliminated.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> southern China university of agriculture

<120> antibacterial peptide, preparation method and application thereof

<130> 1

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 929

<212> DNA

<213> Artificial Sequence

<220>

<223> rDNA-ITS full-length base sequence of Lactobacillus paracasei

<400> 1

gtagcacgtg tgtagcccag gtcataaggg gcatgatgat ttgacgtcat ccccaccttc 60

ctccggtttg tcaccggcag tcttactaga gtgcccaact aaatgctggc aactagtcat 120

aagggttgcg ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacaacc 180

atgcaccacc tgtcattttg cccccgaagg ggaaacctga tctctcaggt gatcaaaaga 240

tgtcaagacc tggtaaggtt cttcgcgttg cttcgaatta aaccacatgc tccaccgctt 300

gtgcgggccc ccgtcaattc ctttgagttt caaccttgcg gtcgtactcc ccaggcggaa 360

tgcttaatgc gttagctgcg gcactgaagg gcggaaaccc tccaacacct agcattcatc 420

gtttacggca tggactacca gggtatctaa tcctgttcgc tacccatgct ttcgagcctc 480

agcgtcagtt acagaccaga cagccgcctt cgccactggt gttcttccat atatctacgc 540

atttcaccgc tacacatgga gttccactgt cctcttctgc actcaagttt cccagtttcc 600

gatgcgcttc ctcggttaag ccgagggctt tcacatcaga cttaaaaaac cgcctgcgct 660

cgctttacgc ccaataaatc cggataacgc ttgccaccta cgtattaccg cggctgctgg 720

cacgtagtta gccgtggctt tctggttgga taccgtcacg ccgacaacag ttactctgcc 780

gaccattctt ctccaacaac agagttttac gacccgaaag ccttcttcac tcacgcggcg 840

ttgctccatc agacttgcgt ccattgtgga agattcccta ctgctgcctc ccgtaggagt 900

ttgggccgtg tctcagtccc aatgtggcc 929

<210> 2

<211> 624

<212> DNA

<213> Artificial Sequence

<220>

<223> rDNA-ITS full-length base sequence of Kluyveromyces marxianus

<400> 2

aaggagacaa acaccagcga gtctttataa cacctatgag tctctatgac ccaagcttac 60

cacgaattgg cgcaaaccta agacgtagat gtgcaagagt cgagtccata gacttgacac 120

gcagccctgc tcacgcagat ggcaacggct agccactttc aagttaaccc gagacgagta 180

tcactcacta ccaaacccaa aggtttgaga gagaaatgac gctcaaacag gcatgccccc 240

tggaatacca gagggcgcaa tgtgcgttca aagatttgat gattcacgaa aatctgcaat 300

tcacaataca tatcgcaatt cgctgcgttc ttcatcgatg cgagaaccaa gagatccgtt 360

gttgaaagtt ttgaatatta aattttatag tataataata gtttttcata atacaaaata 420

ttgtttgtgt ttatgtccac tggagagacg agctctccag ggaagtagtt catagagaaa 480

aaactccatt gtgtttagga tgagaaatag aaaactgata gcagagaatc aagaactggc 540

cgcgcaatta agcgcaggcc ttgttcagac gattccccca gcaatctatt cattcataat 600

ctttaatgat ccttgcgcag gtca 624

Claims

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

(1) preparing a mixed strain fermentation mother liquor: mixing milk powder and water to obtain 8-20% of reduced milk by mass percent, emulsifying and homogenizing the reduced milk, sterilizing and cooling; inoculating lactobacillus paracasei suspension and kluyveromyces marxianus suspension into the cooled reduced milk under aseptic condition, and performing static culture at constant temperature of 25-42 ℃ for 2-4 days to obtain fermentation mother liquor;

lactobacillus paracasei described in step (1) (ii)Lactobacillus paracasei) Is preserved in the China general microbiological culture collection management center in 2016, 12 months and 26 days, and the preservation number is CGMCC NO. 13498;

kluyveromyces marxianus yeast described in step (1) (ii)Kluyveromyces marxianus) Is preserved in the China general microbiological culture collection management center in 2016, 12 months and 26 days, and the preservation number is CGMCC NO. 13499.

2. The method for producing an antimicrobial peptide according to claim 1, wherein:

the initial concentration of the lactobacillus paracasei suspension in the step (1) is 10^-7～10^-10cfu/ml；

The inoculation amount of the lactobacillus paracasei suspension in the step (1) is 2-10% by volume.

3. The method for producing an antimicrobial peptide according to claim 1, wherein:

the initial concentration of the Kluyveromyces marxianus suspension in the step (1) is 10^-7～10^-10cfu/ml；

The inoculation amount of the kluyveromyces marxianus suspension in the step (1) is 2-10% by volume.

4. The method for producing an antimicrobial peptide according to claim 1, wherein:

the temperature of the static culture in the step (1) is 25-37 ℃;

the standing culture time in the step (1) is 2-3 days.

5. The method for producing an antimicrobial peptide according to claim 1, wherein:

the temperature of the fermentation culture in the step (2) is 25-37 ℃;

the fermentation culture time in the step (2) is 7-28 days.

6. The method for producing an antimicrobial peptide according to claim 1, wherein:

the molecular weight cutoff of the ultrafiltration membrane in the step (3) is 3000-10000 Dalton.

7. The method for producing an antimicrobial peptide according to claim 1, wherein:

and (4) purifying by using the upper column at a flow rate of 0.5-10 ml/min.

8. An antibacterial peptide, characterized by being prepared by the preparation method of any one of claims 1 to 7.

9. Use of the antimicrobial peptide of claim 8 for the preparation of a product for the prevention and treatment of acne.