CN109652482A - A kind of antibacterial peptide and the preparation method and application thereof - Google Patents
A kind of antibacterial peptide and the preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to technical field of bioengineering, and in particular to a kind of antibacterial peptide and the preparation method and application thereof.The antibacterial peptide is to carry out fermentation to milk by lactobacillus paracasei and kluyveromyces marxianus hybrid bacterial strain to be made, and mainly comprises the steps that (1) prepares hybrid bacterial strain fermentation mother liquor；(2) inoculation fermentation mother liquor carries out low temperature and long term ferment and prepares whey；(3) crude extract containing antibacterial peptide is made using hyperfiltration technique sepg whey；(4) pass through gel chromatography purpose antibacterial peptide.Antibacterial peptide thermal stability produced by the present invention is good, has no stimulation to human body and has a broad antifungal spectrum, especially significantly inhibits effect to propionibacterium acnes, alternative antibiotic effectively reduces the use of antibiotic, can be used for preventing and treating acne.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of antibacterial peptide and the preparation method and application thereof.
Background technique
Acne is commonly called as whelk, is a kind of chronic inflammatory skin related with gonadal Function imbalance.It is often
Puberty is seen, extrudable flaxen rouge bolt shows as the skin lesions such as black and white acne, papule, warts, tubercle, tumour and scar.
This skin disease will affect beauty, appearance damage be resulted even in when serious, therefore huge psychological burden can be brought to patient
And stress.The cause of disease and pathogenesis of acne are also complex, mainly with propionibacterium acnes infection, androgen, sebum
It is related to secrete the four big reasons such as increase and pilosebaceous follicle opening hyperkeratosis, and propionibacterium acnes have become acne
One of main inducement.
Propionibacterium acnes are subordinate to Actinomycetal, and Propionibacteriaceae, category, propionibacterium acnes kind are a kind of gram sun
Property, anaerobism, no motion of brevibacterium.It is widely present in air, soil, water body and animal body in the especially skin of people.
Propionibacterium acnes can secrete the esterase of decomposable skin keratin and lipid composition, and can enhance the triacylglycerol of inflammatory reaction
Equal metabolites, so as to cause the generation of acne.It often invades the physicochemical properties such as hair follicle and epidermis deep, anaerobism, slow growth
Also make it be not easy to be removed, and become conditioned pathogen.
It the use of antibiotic is at present a kind of important method for treating acne, main antibacterials have Tetracyclines, big ring
Lactone, clindamycin, Compound New Nomin and quinolones etc..But in recent years, because antibiotic usage is lack of standardization, acne third
The drug resistance of acidfast bacilli is also increasingly stronger, therefore the safety of antibiotic also receives more and more queries.If can develop
A kind of to can replace antibiotic to inhibit the substance of propionibacterium acnes, this will become the Gospel of many patients with acne.
Summary of the invention
For overcome the deficiencies in the prior art and disadvantage, the primary purpose of the present invention is that providing a kind of preparation of antibacterial peptide
Method, this method is easy to operate, is easy to large-scale industrial production, and raw material is edible material, green, health, safety
It is high.
Another object of the present invention is to provide the antibacterial peptide that above-mentioned preparation method is prepared, the thermostabilizations of the antibacterial peptide
Property is good, has no stimulation to intact animal, significantly inhibits effect to propionibacterium acnes.
A further object of the present invention is to provide the applications of above-mentioned antibacterial peptide.
The purpose of the invention is achieved by the following technical solution:
A kind of preparation method of antibacterial peptide, comprises the following steps:
(1) it prepares hybrid bacterial strain fermentation mother liquor: milk powder and water is mixed, obtain the reduction that mass percent is 8~20%
Cream is emulsified homogeneous, then sterilizes, cooling；By lactobacillus paracasei bacteria suspension and this Kluyveromyces sp under aseptic condition
Suspension accesses in recombined milk after cooling, 25~42 DEG C constant temperature stationary culture 2~4 days, obtain fermentation mother liquor；
(2) whey is prepared with fermentation mother liquor: milk powder and water is mixed, obtain the recombined milk that mass percent is 5~20%,
It is emulsified homogeneous, is then sterilized, it is cooling；Fermentation mother liquor made from step (1) is accessed into reduction after cooling under aseptic condition
In cream, then 25~42 DEG C fermented and cultured 2~30 days, obtain the fermentation liquid containing whey；
(3) antibacterial peptide is made in ultra-filtration and separation whey: by the filtering fermentation liquor containing whey made from step (2), collecting filter
Liquid, then through molecular cut off be 3000~150000 dalton ultrafiltration membrane ultrafiltration, obtain the crude extract of antibacterial peptide；
(4) gel chromatography purpose antibacterial peptide: using sephadex SephadexG-25 as matrix, with phosphate-buffered
Column purification on the crude extract of antibacterial peptide made from step (3) is obtained antibacterial peptide as eluent by solution；
The mass percent of recombined milk described in step (1) is preferably 8~15%；
Lactobacillus paracasei described in step (1) (Lactobacillus paracasei) (number SLBC16-4)
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 26th, 2016, deposit number is
CGMCC NO.13498；
(number is kluyveromyces marxianus described in step (1) (Kluyveromyces marxianus)
SYKM16-1 it) is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 26th, 2016, is protected
Hiding number is CGMCC NO.13499；
The initial concentration of lactobacillus paracasei bacteria suspension described in step (1) is preferably 10-7~10-10cfu/ml；
The initial concentration of lactobacillus paracasei bacteria suspension described in step (1) is more preferably 10-9~10-10cfu/
ml；
The inoculum concentration of lactobacillus paracasei bacteria suspension described in step (1) is preferably percent by volume 2~10%；
The inoculum concentration of lactobacillus paracasei bacteria suspension described in step (1) be more preferably percent by volume 5~
10%；
The initial concentration of kluyveromyces marxianus bacteria suspension described in step (1) is preferably 10-7~10-10cfu/ml；
The initial concentration of kluyveromyces marxianus bacteria suspension described in step (1) is more preferably 10-9~10- 10cfu/ml；
The inoculum concentration of kluyveromyces marxianus bacteria suspension described in step (1) is preferably percent by volume 2~10%；
The inoculum concentration of kluyveromyces marxianus bacteria suspension described in step (1) is more preferably percent by volume 5
~10%；
Sterilizing described in step (1) is preferably 60~82 DEG C of 20~30min of pasteurization；
Cooling described in step (1) is preferably cooled to 25~42 DEG C；
The temperature of stationary culture described in step (1) is preferably 25~37 DEG C；
The time of stationary culture described in step (1) is preferably 2~3 days；
The mass percent of recombined milk described in step (2) is preferably 8~15%；
The inoculum concentration of fermentation mother liquor described in step (2) is preferably percent by volume 1~15%；
The inoculum concentration of fermentation mother liquor described in step (2) is more preferably percent by volume 5~10%；
Sterilizing described in step (2) is preferably 60~82 DEG C of 20~30min of pasteurization；
Cooling described in step (2) is preferably cooled to 25~42 DEG C；
The temperature of fermented and cultured described in step (2) is preferably 25~37 DEG C；
The time of fermented and cultured described in step (2) is preferably 7~28 days；
Filtering described in step (3) preferably uses larger particles in sterilized Bag filter fermentation liquid；
The molecular cut off of ultrafiltration membrane described in step (3) is preferably 3000~10000 dalton；
The pH of phosphate buffer solution described in step (3) is preferably 7.0；
The flow velocity of upper column purification described in step (4) is preferably 0.5~10ml/min；
The flow velocity of upper column purification described in step (4) is more preferably 1~5ml/min；
Nucleic acid-protein detector test is preferably used in purification process described in step (4), collects the outflow of maximum peak
Liquid；
The wavelength of the detection is 254~280nm；
The wavelength of the detection is preferably 260~280nm；
A kind of antibacterial peptide is prepared by above-mentioned preparation method；
The antibacterial peptide prevents and treats the application in acne products in preparation；
Compared with prior art, the invention has the following advantages:
(1) purer antibacterial peptide can be obtained by ultrafiltration and step purifying in the present invention, and method is easy to operate, is easy to industry
Metaplasia produces.
(2) present invention prepares antibacterial using probiotics such as lactobacillus paracasei and kluyveromyces marxianus come fermented milk
Peptide, bacterial strain uses therefor are edible bacterial strain, and raw materials used is edible material, effect harmless to the human body, and safety is high.
(3) antibacterial peptide produced by the present invention can tolerate 121 DEG C of high temperature, and thermal stability is good, has no stimulation and resists to human body
Bacterium spectrum is wide, especially significantly inhibits effect to propionibacterium acnes, alternative antibiotic effectively reduces the use of antibiotic, can
For preventing and treating acne.
(4) antibacterial peptide produced by the present invention is made of 12~100 amino acid residues, contains hydrophobic amino acid, Duo Shuoshi
Amphipathic cation there is antibacterium, fungi, virus, helminth and selective induction tumour cell apoptosis etc. occurs a variety of
Bioactivity.Because it is with has a broad antifungal spectrum, stability is good, almost without toxic action and is not likely to produce anti-to normal animal cells
The characteristics such as pharmacological property, antibacterial peptide probably become the substitute of conventional antibiotic.
Detailed description of the invention
Fig. 1 is the Gram's staining and colonial morphology figure of lactobacillus paracasei, wherein A: Gram's staining (10 × 100),
B: colonial morphology.
Fig. 2 is the methylene blue and colonial morphology figure of kluyveromyces marxianus, wherein A: methylene blue (10 × 40),
B: colonial morphology.
Fig. 3 is real-time monitoring sample OD in the SephadexG-25 gel-purification of embodiment 8280Variation diagram, wherein
Abscissa is time, ordinate OD280Value.
Fig. 4 is inhibition zone result figure of the antibacterial peptide made from embodiment 8 to propionibacterium acnes ATCC6919.
Fig. 5 is that patients with acne tries out the effect assessment figure after antibacterial peptide made from embodiment 8.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
The separation and identification of 1 lactobacillus paracasei bacterial strain of embodiment
(1) lactobacillus paracasei is to separate to obtain from Kefir granule.Specific separation method is as follows:
1) fermentation liquid of 100 μ l Kefir granules is taken to be connected in liquid MRS culture medium, 37 DEG C of constant temperature stationary culture 48h.2) will
Bacterium solution carries out gradient dilution, takes 100 μ l dilution bacterium solution to be coated on MRS plate, 37 DEG C of constant temperature stationary culture 48h.3) according to bacterium
The form fallen chooses the biggish single colonie of bacterium colony, lines on new MPS plate, and after 37 DEG C of constant temperature stationary culture 48h, it is blue to carry out leather
Albert'stain Albert and microscopy observe cellular morphology.4) after microscopy is judged as pure culture, pure culture is connected in MRS fluid nutrient medium, 37
After DEG C constant temperature stationary culture 48h, the sterile glycerol that percent by volume is 30% is added, mixing is placed on -80 DEG C and saves backup.
(2) bacterial strain is identified
1. separated obtained lactobacillus paracasei Gram's staining is purple, be presented elongated rod shape, arrangement in single or
In pairs, it is seen that Figure 1A.
2. circle is presented in separated obtained lactobacillus paracasei colonial morphology, surface is smooth, swells, opaque, edge
Neatly, color is creamy white, it is seen that Figure 1B.
3. the Physiology and biochemistry qualification result of separated obtained lactobacillus paracasei is as follows:
The Physiology and biochemistry qualification result of 1 lactobacillus paracasei of table
Note: "+" indicates that result is the positive；" one " indicates that result is feminine gender.
4. the rDNA-ITS full-length nucleotide sequence of separated obtained lactobacillus paracasei are as follows:
GTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTT
GTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTA
ACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCT
CTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTT
GTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCT
GCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCT
GTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATA
TATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCGCT
TCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGAT
AACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGCC
GACAACAGTTACTCTGCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAAGCCTTCTTCACTCACGCGGC
GTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAG
TCCCAATGTGGCC
In conjunction with Physiology and biochemistry identification and its rDNA-ITS full-length nucleotide sequence, the results show that the bacterial strain is secondary cheese cream bar
The Strain Designation is lactobacillus paracasei (Lactobacillus paracasei) SLBC16-4 by a new strains for bacterium, in
On December 26th, 2016 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is
CGMCC NO.13498, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica.
Lactobacillus paracasei (Lactobacillus paracasei) the SLBC16-4 activation that the present embodiment separation is saved
Culture, it is 10 that concentration, which is made,-7cfu/ml、10-9Cfu/ml and 10-10The lactobacillus paracasei bacteria suspension of cfu/ml, for following
Embodiment.
The separation and identification of 2 kluyveromyces marxianus of embodiment
(1) kluyveromyces marxianus is to separate to obtain from Kefir granule.Specific separation method is as follows:
1) fermentation liquid of 100 μ l Kefir granules is taken to be connected in YPD fluid nutrient medium, 28 DEG C of constant temperature stationary culture 72h.2) will
Bacterium solution carries out gradient dilution, takes 100 μ l dilution bacterium solution to be coated on YPD plate, 28 DEG C of culture 72h.3) according to the form of bacterium colony
Choose biggish single colonie, line on new YPD plate, after 28 DEG C of culture 72h, carries out methylene blue and microscopy, observe cell
Form.4) after microscopy is judged as pure culture, pure culture is connected in YPD fluid nutrient medium, after 28 DEG C of culture 72h, body is added
The sterile glycerol that product percentage is 30%, mixing are placed on -80 DEG C and save backup.
(2) bacterial strain is identified
1. separated obtained kluyveromyces marxianus is found through methylene blue, present bluish violet, form be circle or
Ellipse, one end budding, it is seen that Fig. 2A.
2. separated obtained kluyveromyces marxianus colonial morphology is rounded, surface is smooth wet, and color is in milky white
Color is surface bulge, neat in edge, opaque, it is seen that Fig. 2 B.
3. the Physiology and biochemistry qualification result of separated obtained kluyveromyces marxianus is as follows:
Note: "+" indicates that producing depressed fruit is the positive；" one " indicates that producing depressed fruit is feminine gender.
4. the rDNA-ITS full-length nucleotide sequence of separated obtained kluyveromyces marxianus are as follows:
AAGGAGACAAACACCAGCGAGTCTTTATAACACCTATGAGTCTCTATGACCCAAGCTTACCACGAATTG
GCGCAAACCTAAGACGTAGATGTGCAAGAGTCGAGTCCATAGACTTGACACGCAGCCCTGCTCACGCAGATGGCAAC
GGCTAGCCACTTTCAAGTTAACCCGAGACGAGTATCACTCACTACCAAACCCAAAGGTTTGAGAGAGAAATGACGCT
CAAACAGGCATGCCCCCTGGAATACCAGAGGGCGCAATGTGCGTTCAAAGATTTGATGATTCACGAAAATCTGCAAT
TCACAATACATATCGCAATTCGCTGCGTTCTTCATCGATGCGAGAACCAAGAGATCCGTTGTTGAAAGTTTTGAATA
TTAAATTTTATAGTATAATAATAGTTTTTCATAATACAAAATATTGTTTGTGTTTATGTCCACTGGAGAGACGAGCT
CTCCAGGGAAGTAGTTCATAGAGAAAAAACTCCATTGTGTTTAGGATGAGAAATAGAAAACTGATAGCAGAGAATCA
AGAACTGGCCGCGCAATTAAGCGCAGGCCTTGTTCAGACGATTCCCCCAGCAATCTATTCATTCATAATCTTTAATG
ATCCTTGCGCAGGTCA
In conjunction with Physiology and biochemistry identification and its rDNA-ITS full-length nucleotide sequence, the results show that the bacterial strain is Marx's Crewe
The Strain Designation is kluyveromyces marxianus (Kluyveromyces marxianus) SYKM16- by one new strains of dimension
1, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 26th, 2016, deposit number is
CGMCC NO.13499, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica.
Kluyveromyces marxianus (Kluyveromyces marxianus) SYKM16-1 that the present embodiment separation is saved
Activation culture, it is 10 that concentration, which is made,-7cfu/ml、10-9Cfu/ml and 10-10The kluyveromyces marxianus bacteria suspension of cfu/ml,
For following embodiments.
Embodiment 3
(1) it prepares hybrid bacterial strain fermentation mother liquor: weighing milk powder 8.00g and ionized water is uniformly mixed, obtain 100ml mass hundred
Divide the recombined milk than being 8%, is emulsified homogeneous, 65 DEG C of pasteurization 30min are cooled to 25 DEG C；2mL is accessed under aseptic condition
Initial concentration is 10-7The lactobacillus paracasei bacteria suspension (embodiment 1 is made) and 2mL initial concentration of cfu/ml is 10-7cfu/ml
Kluyveromyces marxianus bacteria suspension (embodiment 1 be made), 28 DEG C constant temperature stationary culture 4 days, obtain fermentation mother liquor；
(2) whey is prepared with fermentation mother liquor: milk powder and ionized water is uniformly mixed, obtaining 1000ml mass percent is
5% recombined milk is emulsified homogeneous, and 65 DEG C of pasteurize 30min are placed in fermentor, sterile after it is cooled to 32 DEG C
Under the conditions of access 20ml step (1) made from fermentation mother liquor in fermentor, 30 DEG C of fermented and cultured 6d obtain the hair containing whey
Zymotic fluid；
(3) antibacterial peptide is made in ultra-filtration and separation whey: the hair containing whey made from sterilized Bag filter step (2)
Biggish particle in zymotic fluid, collect filtrate, then with molecular cut off be 3000 dalton ultrafiltration membrane ultrafiltration, obtain antibacterial peptide
Crude extract；
(4) gel chromatography purpose antibacterial peptide: being 7.0 with pH using sephadex SephadexG-25 as matrix
Phosphate buffer is as eluent, by column purification on the crude extract of antibacterial peptide made from step (3), applied sample amount 1ml, stream
Speed control is in 1ml/min, purification process in real time using the light absorption value at nucleic acid-protein detector test efflux 280nm, root
According to sample different time difference light absorption value (abscissa is the time, and ordinate is light absorption value), curve is drawn, the stream of maximum peak is collected
Liquid out obtains antibacterial peptide.
Embodiment 4
(1) it prepares hybrid bacterial strain fermentation mother liquor: weighing milk powder 12.00g and ionized water is uniformly mixed, obtain 100ml mass
The recombined milk that percentage is 12%, is emulsified homogeneous, 75 DEG C of pasteurization 30min are cooled to 35 DEG C；It is accessed under aseptic condition
3mL initial concentration is 10-9The lactobacillus paracasei bacteria suspension (embodiment 1 is made) and 2mL initial concentration of cfu/ml is 10- 9The kluyveromyces marxianus bacteria suspension (embodiment 1 be made) of cfu/ml, 37 DEG C constant temperature stationary culture 3 days, it is female to obtain fermentation
Liquid；
(2) whey is prepared with fermentation mother liquor: milk powder and ionized water is uniformly mixed, obtaining 1000ml mass percent is
8% recombined milk is emulsified homogeneous, and pasteurize 30min, is placed in fermentor at 75 DEG C, after it is cooled to 42 DEG C, nothing
Fermentation mother liquor made from 150ml step (1) is accessed under the conditions of bacterium in fermentor, 42 DEG C of fermented and cultured 2d are obtained containing whey
Fermentation liquid；
(3) antibacterial peptide is made in ultra-filtration and separation whey: the hair containing whey made from sterilized Bag filter step (2)
Biggish particle in zymotic fluid, collect filtrate, then with molecular cut off be 5000 dalton ultrafiltration membrane ultrafiltration, obtain antibacterial peptide
Crude extract；
(4) gel chromatography purpose antibacterial peptide: being 7.0 with pH using sephadex SephadexG-25 as matrix
Phosphate buffer is as eluent, by column purification on the crude extract of antibacterial peptide made from step (3), applied sample amount 2ml, stream
Speed control is in 1ml/min, purification process in real time using the light absorption value at nucleic acid-protein detector test efflux 280nm, root
According to sample different time difference light absorption value (abscissa is the time, and ordinate is light absorption value), curve is drawn, the stream of maximum peak is collected
Liquid out obtains antibacterial peptide.
Embodiment 5
(1) it prepares hybrid bacterial strain fermentation mother liquor: weighing milk powder 14.00g and ionized water is uniformly mixed, obtain 100ml mass
The recombined milk that percentage is 14%, is emulsified homogeneous, 80 DEG C of pasteurization 25min are cooled to 40 DEG C；It is accessed under aseptic condition
4mL initial concentration is 10-10The lactobacillus paracasei bacteria suspension (embodiment 1 is made) and 2mL initial concentration of cfu/ml is 10- 10The kluyveromyces marxianus bacteria suspension (embodiment 1 be made) of cfu/ml, 25 DEG C constant temperature stationary culture 2 days, it is female to obtain fermentation
Liquid；
(2) whey is prepared with fermentation mother liquor: milk powder and ionized water is uniformly mixed, obtaining 1000ml mass percent is
14% recombined milk is emulsified homogeneous, and pasteurize 25min, is placed in fermentor at 80 DEG C, after it is cooled to 25 DEG C,
Fermentation mother liquor made from 40ml step (1) is accessed under aseptic condition in fermentor, 25 DEG C of fermented and cultured 8d are obtained containing whey
Fermentation liquid；
(3) antibacterial peptide is made in ultra-filtration and separation whey: the hair containing whey made from sterilized Bag filter step (2)
Biggish particle in zymotic fluid, collect filtrate, then with molecular cut off be 8000 dalton ultrafiltration membrane ultrafiltration, obtain antibacterial peptide
Crude extract；
(4) gel chromatography purpose antibacterial peptide: being 7.0 with pH using sephadex SephadexG-25 as matrix
Phosphate buffer is as eluent, by column purification on the crude extract of antibacterial peptide made from step (3), applied sample amount 3ml, stream
Speed control is in 3ml/min, purification process in real time using the light absorption value at nucleic acid-protein detector test efflux 268nm, root
According to sample different time difference light absorption value (abscissa is the time, and ordinate is light absorption value), curve is drawn, the stream of maximum peak is collected
Liquid out obtains antibacterial peptide.
Embodiment 6
(1) it prepares hybrid bacterial strain fermentation mother liquor: weighing milk powder 15.00g and ionized water is uniformly mixed, obtain 100ml mass
The recombined milk that percentage is 15%, is emulsified homogeneous, 82 DEG C of pasteurization 20min are cooled to 42 DEG C；It is accessed under aseptic condition
4mL initial concentration is 10-10The lactobacillus paracasei bacteria suspension (embodiment 1 is made) and 6mL initial concentration of cfu/ml is 10- 10The kluyveromyces marxianus bacteria suspension (embodiment 1 be made) of cfu/ml, 25 DEG C constant temperature stationary culture 2 days, it is female to obtain fermentation
Liquid；
(2) whey is prepared with fermentation mother liquor: milk powder and ionized water is uniformly mixed, obtaining 1000ml mass percent is
15% recombined milk is emulsified homogeneous, and pasteurize 20min, is placed in fermentor at 82 DEG C, after it is cooled to 42 DEG C,
Fermentation mother liquor made from 60ml step (1) is accessed under aseptic condition in fermentor, 25 DEG C of fermented and cultured 12d are obtained containing cream
Clear fermentation liquid；
(3) antibacterial peptide is made in ultra-filtration and separation whey: the hair containing whey made from sterilized Bag filter step (2)
Biggish particle in zymotic fluid, collect filtrate, then with molecular cut off be 10000 dalton ultrafiltration membrane ultrafiltration, obtain antibacterial peptide
Crude extract；
(4) gel chromatography purpose antibacterial peptide: being 7.0 with pH using sephadex SephadexG-25 as matrix
Phosphate buffer is as eluent, by column purification on the crude extract of antibacterial peptide made from step (3), applied sample amount 3.5ml,
Flow control uses the light absorption value at nucleic acid-protein detector test efflux 269nm in real time in 6ml/min, purification process,
According to sample different time difference light absorption value (abscissa is the time, and ordinate is light absorption value), curve is drawn, maximum peak is collected
Efflux obtains antibacterial peptide.
Embodiment 7
(1) it prepares hybrid bacterial strain fermentation mother liquor: weighing milk powder 20.00g and ionized water is uniformly mixed, obtain 100ml mass
The recombined milk that percentage is 20%, is emulsified homogeneous, 82 DEG C of pasteurization 30min are cooled to 37 DEG C；It is accessed under aseptic condition
10mL initial concentration is 10-10The lactobacillus paracasei bacteria suspension (embodiment 1 is made) and 10mL initial concentration of cfu/ml is 10- 10The kluyveromyces marxianus bacteria suspension (embodiment 1 be made) of cfu/ml, 30 DEG C constant temperature stationary culture 2 days, it is female to obtain fermentation
Liquid；
(2) whey is prepared with fermentation mother liquor: milk powder and ionized water is uniformly mixed, obtaining 1000ml mass percent is
20% recombined milk is emulsified homogeneous, and pasteurize 30min, is placed in fermentor at 82 DEG C, after it is cooled to 37 DEG C,
Fermentation mother liquor made from 80ml step (1) is accessed under aseptic condition in fermentor, 25 DEG C of fermented and cultured 30d are obtained containing cream
Clear fermentation liquid；
(3) antibacterial peptide is made in ultra-filtration and separation whey: the hair containing whey made from sterilized Bag filter step (2)
Biggish particle in zymotic fluid, collect filtrate, then with molecular cut off be 150000 dalton ultrafiltration membrane ultrafiltration, obtain antibacterial peptide
Crude extract；
(4) gel chromatography purpose antibacterial peptide: being 7.0 with pH using sephadex SephadexG-25 as matrix
Phosphate buffer is as eluent, by column purification on the crude extract of antibacterial peptide made from step (3), applied sample amount 4ml, stream
Speed control is in 5ml/min, purification process in real time using the light absorption value at nucleic acid-protein detector test efflux 280nm, root
According to sample different time difference light absorption value (abscissa is the time, and ordinate is light absorption value), curve is drawn, the stream of maximum peak is collected
Liquid out obtains antibacterial peptide.
Embodiment 8
(1) it prepares hybrid bacterial strain fermentation mother liquor: weighing milk powder 10.00g and ionized water is uniformly mixed, obtain 100ml mass
The recombined milk that percentage is 10%, is emulsified homogeneous, 82 DEG C of pasteurization 30min are cooled to 35 DEG C；It is accessed under aseptic condition
5mL initial concentration is 10-10The lactobacillus paracasei bacteria suspension (embodiment 1 is made) and 5mL initial concentration of cfu/ml is 10- 10The kluyveromyces marxianus bacteria suspension (embodiment 1 be made) of cfu/ml, in 25 DEG C constant temperature stationary culture 2 days, it is female to obtain fermentation
Liquid；
(2) milk powder and ionized water are uniformly mixed with fermentation mother liquor preparation whey, obtaining 1000ml mass percent is
10% recombined milk is emulsified homogeneous, and pasteurize 30min, is placed in fermentor at 82 DEG C, after it is cooled to 37 DEG C,
Fermentation mother liquor made from 80ml step (1) is accessed under aseptic condition in fermentor, 25 DEG C of fermented and cultured 28d are obtained containing cream
Clear fermentation liquid；
(3) antibacterial peptide is made in ultra-filtration and separation whey: the hair containing whey made from sterilized Bag filter step (2)
Biggish particle in zymotic fluid, collect filtrate, then with molecular cut off be 150000 dalton ultrafiltration membrane ultrafiltration, obtain antibacterial peptide
Crude extract；
(4) gel chromatography purpose antibacterial peptide: being 7.0 with pH using sephadex SephadexG-25 as matrix
Phosphate buffer is as eluent, by column purification on the crude extract of antibacterial peptide made from step (3), applied sample amount 5ml, stream
Speed control is in 5ml/min, purification process in real time using the light absorption value at nucleic acid-protein detector test efflux 280nm, root
According to sample different time difference light absorption value (abscissa is the time, and ordinate is light absorption value), curve is drawn, the stream of maximum peak is collected
Liquid (Fig. 3, peak 2) out, obtains antibacterial peptide, and concentration is 148.23 μ g/ml.
The analysis of 9 fungistatic effect of embodiment
(1) inhibition zone is done using agar diffusion method
The deionized water of antibacterial peptide made from embodiment 8 is diluted one times, obtains antibacterial peptide dilution；Under aseptic condition,
15mL BHI solid medium is poured into sterilized petri dishes, it is 7.8 ± 0.1mm Oxford cup that 4 outer diameters are put after it solidifies 1h.
1mL 1 × 10 is added into about 50 DEG C of BHI solid mediums8The propionibacterium acnes bacteria suspension of cfu/mL, is poured into after mixing
In BHI plate through solidifying, Oxford cup is taken out after solidification.150 μ l are added after label to dilute by the antibacterial peptide of different pretreatments
Liquid or sterile water, A: antibacterial peptide dilution is in 115 DEG C of processing 20min, B: antibacterial peptide dilution is without any pretreatment, C: antibacterial peptide
Dilution 121 DEG C of processing 20min, D: sterile water.After plate is placed in 37 DEG C of Anaerobic culturel 72h, inhibition zone is measured with vernier caliper
Diameter.
As a result see Fig. 4, wherein A antibacterial circle diameter is 17.20 ± 0.1mm；B antibacterial circle diameter is 21.30 ± 0.1mm；C
Antibacterial circle diameter is 13.50 ± 0.1mm；D diameter is 7.8 ± 0.1mm.
(2) patients with acne trial effect
After patients with acne is first washed one's face 2 times with clear water, a small amount of antibacterial peptide dilution is dipped with cotton swab and (is resisted made from embodiment 8
Bacterium peptide dilutes one times with deionized water) it is applied to acne affected part, it is smeared 1 time every 4h, smears 3 times, photograph to record daily daily
The situation of change of acne.
As a result see Fig. 5, after smearing antibacterial peptide 5d made from embodiment 8, inflammatory warts is obviously eliminated.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>Agricultural University Of South China
<120>a kind of antibacterial peptide and the 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>the rDNA-ITS full-length nucleotide 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>the rDNA-ITS full-length nucleotide 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 (10)
1. a kind of preparation method of antibacterial peptide, characterized by comprising the steps of:
(1) it prepares hybrid bacterial strain fermentation mother liquor: milk powder and water is mixed, obtain the recombined milk that mass percent is 8~20%, it will
Its emulsifying homogeneous, then sterilizes, cooling；Lactobacillus paracasei bacteria suspension and this kluyveromyces bacteria suspension are connect under aseptic condition
Enter in recombined milk after cooling, 25~42 DEG C constant temperature stationary culture 2~4 days, obtain fermentation mother liquor；
(2) whey is prepared with fermentation mother liquor: milk powder and water is mixed, the recombined milk that mass percent is 5~20% is obtained, by it
Then emulsifying homogeneous sterilizes, cooling；Fermentation mother liquor made from step (1) is accessed in recombined milk after cooling under aseptic condition,
Then 25~42 DEG C fermented and cultured 2~30 days, obtain the fermentation liquid containing whey；
(3) antibacterial peptide is made in ultra-filtration and separation whey: by the filtering fermentation liquor containing whey made from step (2), filtrate is collected, then
It is the ultrafiltration membrane ultrafiltration of 3000~150000 dalton through molecular cut off, obtains the crude extract of antibacterial peptide；
(4) gel chromatography purpose antibacterial peptide: using sephadex SephadexG-25 as matrix, with phosphate buffer solution
Column purification on the crude extract of antibacterial peptide made from step (3) is obtained into antibacterial peptide as eluent.
2. the preparation method of antibacterial peptide according to claim 1, it is characterised in that:
Lactobacillus paracasei described in step (1) (Lactobacillus paracasei) was in preservation on December 26 in 2016
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO.13498；
Kluyveromyces marxianus described in step (1) (Kluyveromyces marxianus) was on December 26th, 2016
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO.13499.
3. the preparation method of antibacterial peptide according to claim 1, it is characterised in that:
The initial concentration of lactobacillus paracasei bacteria suspension described in step (1) is 10-7~10-10cfu/ml；
The inoculum concentration of lactobacillus paracasei bacteria suspension described in step (1) is percent by volume 2~10%.
4. the preparation method of antibacterial peptide according to claim 1, it is characterised in that:
The initial concentration of kluyveromyces marxianus bacteria suspension described in step (1) is 10-7~10-10cfu/ml；
The inoculum concentration of kluyveromyces marxianus bacteria suspension described in step (1) is percent by volume 2~10%.
5. the preparation method of antibacterial peptide according to claim 1, it is characterised in that:
The temperature of stationary culture described in step (1) is 25~37 DEG C；
The time of stationary culture described in step (1) is 2~3 days.
6. the preparation method of antibacterial peptide according to claim 1, it is characterised in that:
The temperature of fermented and cultured described in step (2) is 25~37 DEG C；
The time of fermented and cultured described in step (2) is 7~28 days.
7. the preparation method of antibacterial peptide according to claim 1, it is characterised in that:
The molecular cut off of ultrafiltration membrane described in step (3) is 3000~10000 dalton.
8. the preparation method of antibacterial peptide according to claim 1, it is characterised in that:
The flow velocity of upper column purification described in step (4) is 0.5~10ml/min.
9. a kind of antibacterial peptide, it is characterised in that be prepared by preparation method according to any one of claims 1 to 8.
10. antibacterial peptide as claimed in claim 9 prevents and treats the application in acne products in preparation.
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| CN110499287A (en) * | 2019-08-30 | 2019-11-26 | 博雅干细胞科技有限公司 | The method for simply preparing placenta mesenchyma stem cell excretion body |
| CN114681588A (en) * | 2022-02-17 | 2022-07-01 | 中山大学 | Application of polypeptide EN-9 in preparation of product for treating acne |
| CN116063380A (en) * | 2022-09-30 | 2023-05-05 | 内蒙古农业大学 | Antifungal peptide separated from lactobacillus paracasei and preparation method thereof |
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| CN103834583A (en) * | 2014-02-28 | 2014-06-04 | 武运 | Hypolipidemic mare yogurt prepared by mixed fermentation of mixed bacteria, and preparation method thereof |
| CN104212857A (en) * | 2014-04-02 | 2014-12-17 | 曹庸 | Method for separation purification of antibacterial peptide from lactobacillus paracasei fermented milk as raw material |
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| WO2005115341A2 (en) * | 2004-05-27 | 2005-12-08 | Advanced Bionutrition Corporation | Microparticles for oral delivery |
| CN101451158A (en) * | 2007-12-04 | 2009-06-10 | 黑龙江大学 | Separation and purification method of antibiotic peptides produced by Lactobacillus paracasei |
| CN103540553A (en) * | 2013-11-01 | 2014-01-29 | 新疆农业大学 | Method for mixing and fermenting compound strain to prepare mare milk vinegar and prepared mare milk vinegar |
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| CN114681588A (en) * | 2022-02-17 | 2022-07-01 | 中山大学 | Application of polypeptide EN-9 in preparation of product for treating acne |
| CN114681588B (en) * | 2022-02-17 | 2023-06-06 | 中山大学 | Application of polypeptide EN-9 in preparation of product for treating acne |
| CN116063380A (en) * | 2022-09-30 | 2023-05-05 | 内蒙古农业大学 | Antifungal peptide separated from lactobacillus paracasei and preparation method thereof |
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