CN110105437A - Method, the purposes of Antimicrobial Peptides From Plants and Antimicrobial Peptides From Plants of separating plant antibacterial peptide - Google Patents
Method, the purposes of Antimicrobial Peptides From Plants and Antimicrobial Peptides From Plants of separating plant antibacterial peptide Download PDFInfo
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- CN110105437A CN110105437A CN201910381420.1A CN201910381420A CN110105437A CN 110105437 A CN110105437 A CN 110105437A CN 201910381420 A CN201910381420 A CN 201910381420A CN 110105437 A CN110105437 A CN 110105437A
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- sesami nigrum
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Botany (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides method, the purposes of Antimicrobial Peptides From Plants and Antimicrobial Peptides From Plants of separating plant antibacterial peptide, and the method for separating plant antibacterial peptide adds acetum in Semen sesami nigrum the following steps are included: take the Semen sesami nigrum of certain mass；By the Semen sesami nigrum and acetum, ultrasound is carried out in ultrasonic generator, filters to obtain extracting solution；By the extracting solution heating water bath, it is then down to room temperature；The extracting solution is centrifuged, precipitating is abandoned, harvests supernatant；The supernatant is successively dialysed and is concentrated, reverse-phase chromatography sample is obtained；The reverse-phase chromatography sample is subjected to reversed-phase high performance liquid chromatography separation, obtains the isolated Antimicrobial Peptides From Plants.1. directly being extracted using Semen sesami nigrum as raw material, the cumbersome process of peeling degreasing is eliminated, reduces the application of organic reagent, reduces operation difficulty, improve production efficiency, reduce production cost, meet industrialized production.2. Semen sesami nigrum antibacterial peptide prepared by has broad-spectrum antibacterial effect, all has very strong bacteriostasis to Candida albicans, copper aluminium Pseudomonas alba, staphylococcus aureus, Escherichia coli, haemophilus influenzae.It can be widely applied to the fields such as external sterilizing product and poultry and livestock feed additive.
Description
Technical field
The present invention relates to field of biological pharmacy, and in particular, to method, Antimicrobial Peptides From Plants and the plant of separating plant antibacterial peptide
The purposes of object antibacterial peptide.
Background technique
Antibacterial peptide is that living organism generated one kind in the defense reaction for resisting pathogenic microorganism is antimicrobial with one
The small peptide of a little malignant cells.It is the important component of body innate immune system, to bacterium, fungi, helminth, disease
Poison, tumour cell etc. have extensive inhibiting effect.Natural antibacterial peptide is usually the small molecule being made of 12-60 amino acid
Cationic polypeptide, due to usually having 2-7 positive electricity rich in basic amino acids, antibacterial peptides such as lysine, arginine and histidines
Lotus, isoelectric point are greater than 7, show stronger cationic character.
The study found that the immunocyte of animals and plants can inhibit micro- with a variety of such as phagocyte and mucomembranous epithelial cell
The antibacterial peptide of biological growth exists, the mechanism of action be first antibacterial peptide can well be made of amphiphatic molecule, particularly
It is combined in the cell membrane of electronegativity, this is the structure basis that antibacterial peptide and cell membrane interact, and prevents to destroy cell wall
Synthesis and film effect, hole is formed on adipose membrane, so that important content is leaked, with intracellular mitochondrial, nucleic acid molecule
Etc. important organelles interaction, eventually lead to thallus death.Currently, the antibacterial peptide wide variety isolated from animals and plants,
It is widely distributed, it is found in the biology such as insect, fish, mammal, amphibian and plant.For the anti-of separate sources
There has been no systematic researches for the application of bacterium peptide.Especially Antimicrobial Peptides From Plants, it is especially unstable, easily by proteolytic enzymes hydrolize,
To substantially reduce its antibacterial activity.Thus, the research about Antimicrobial Peptides From Plants need to be goed deep into.
Summary of the invention
The present invention is directed to solve at least some of the technical problems in related technologies.For this purpose, of the invention
One purpose is to propose a kind of means that can effectively inhibit bacterium and fungi to be proliferated.
The first aspect of the present invention, the present invention provide a kind of isolated Antimicrobial Peptides From Plants.According to an embodiment of the invention, this
The isolated Antimicrobial Peptides From Plants of invention can effectively inhibit the growth of bacterium and fungi, and then can prevent or treat microorganism sense
Infectious diseases.
The second aspect of the present invention, the present invention provides the methods of separating plant antibacterial peptide, comprising the following steps:
(1) Semen sesami nigrum for taking certain mass, adds acetum in Semen sesami nigrum；
(2) by the Semen sesami nigrum and acetum, ultrasound is carried out in ultrasonic generator, filters to obtain extracting solution；
(3) by the extracting solution heating water bath, then it is down to room temperature；
(4) extracting solution is centrifuged, abandons precipitating, harvest supernatant；
(5) supernatant is successively dialysed and is concentrated, obtain reverse-phase chromatography sample；
(6) the reverse-phase chromatography sample is subjected to reversed-phase high performance liquid chromatography separation, obtains the isolated plants antimicrobial
Peptide.
The volume for 5-20 times of the acetum Semen sesami nigrum being added in the step (1), the acetum
Concentration be 1-10%.
The ultrasonic time is 10-35min.
The water bath heating temperature in the step (3) is 60-100 DEG C, time 5-30min.
Centrifugal rotational speed described in the step (4) is 8000-14000rpm, centrifugation time 5-20min.
The time of dialysis in the step (5) is 24-72 hours.
The step (6) further includes steps of
A, the reverse-phase chromatography sample is subjected to reversed-phase high performance liquid chromatography separation, wherein the reverse-phase chromatographic column is reverse phase
C4 silicagel column；
B, by the sample loading to the reverse phase C4 silicagel column, and successively utilize the distilled water containing trifluoroacetic acid and
Acetonitrile containing trifluoroacetic acid carries out elution processing to the C4 silicagel column, obtains elution samples；
C, the elution samples are concentrated, obtain the isolated Antimicrobial Peptides From Plants.
The content containing trifluoroacetic acid in trifluoroacetic acid distilled water in the step (B) is 0.1%-0.5%, institute
The content for stating trifluoroacetic acid in the acetonitrile containing trifluoroacetic acid is 0.1%-0.5%, ethane nitrile content 75%-90%.
The elution processing time in the step (B) is 30-60 minutes.
In the step (1) is 100g by the Semen sesami nigrum；Add 7% acetum of 5 times of Semen sesami nigrum volumes
500mL is in Semen sesami nigrum；Ultrasonic generator power is 200W-300W in the step (2), and frequency is to be surpassed in 40kHz
Sound, extraction time 20min；The water bath heating temperature in the step (3) is 80 DEG C；Centrifugation in the step (4)
Condition is 4 DEG C of centrifugation 5min under the conditions of 10000rpm；The time of dialysis in the step (5) is 48 hours.The step
(B) content containing trifluoroacetic acid in trifluoroacetic acid distilled water in is 0.1%, in the acetonitrile containing trifluoroacetic acid
The content of trifluoroacetic acid is 0.1%, ethane nitrile content 90%, and the elution processing time in the step (B) is 30 minutes.
Since vegetable seeds is Semen sesami nigrum, the yield of isolated Antimicrobial Peptides From Plants is higher.
The third aspect of the present invention, the Antimicrobial Peptides From Plants obtained using the above-mentioned separating plant antibacterial peptide method of the present invention, tool
It has the following characteristics that
PH value: the pH value using acidometer measurement antibacterial peptide is 2.82；
Isoelectric point: the basic protein that the PI value using Nephelometric Determination antibacterial peptide is 9.4-10.2；
Molecular weight: utilizing SDS-PAGE electrophoresis and laser desorption ionization time of flight mass spectrometry method, and measuring molecular weight is 4-
6kd。
The antibacterial peptide, which has the feature that, weighs sample 1mL in hydrolysis bottle, and after performic acid processing, 1mL hydrochloric acid is added
110 DEG C of hydrolysis 22~for 24 hours, hydrolysate measures amino acid classes using UPLC method are as follows: glycine (Glycine), cysteine
(Cysteine), arginine (Argnine), lysine (Lysine), histidine (Histidine), alanine (Alanine),
Threonine (Threonine), aspartic acid (Asparagine), leucine (Leucine), tyrosine (Tyrosine), phenylpropyl alcohol
Propylhomoserin (Phenylephrine), serine (Serine), glutamic acid (Glutamic acid), valine (Valine).
The fourth aspect of the present invention, the Antimicrobial Peptides From Plants obtained the present invention provides above-mentioned separating plant antibacterial peptide method
Purposes, the antibacterial peptide can be used as preventing and treating microbial infectious diseases.
The invention has the advantages that 1. using Semen sesami nigrum as raw material, directly extract, eliminates the cumbersome process of peeling degreasing, reduce
The application of organic reagent, reduces operation difficulty, improves production efficiency, reduces production cost, meets industrialized production.2.
Prepared Semen sesami nigrum antibacterial peptide has broad-spectrum antibacterial effect, to Candida albicans, copper aluminium Pseudomonas alba, Staphylococcus aureus
Bacterium, Escherichia coli, haemophilus influenzae all have very strong bacteriostasis.It can be widely applied to external sterilizing product and poultry man
The fields such as animal fodder additive.
Detailed description of the invention
Fig. 1 is the result and negative control figure antibacterial to Candida albicans of Antimicrobial Peptides From Plants obtained in embodiment 1-3.
Fig. 2 is the result and negative control figure antibacterial to Escherichia coli of Antimicrobial Peptides From Plants obtained in 1-3 embodiment.
Fig. 3 is the result and negative control antibacterial to staphylococcus aureus of Antimicrobial Peptides From Plants obtained in embodiment 1-3
Figure.
Fig. 4 is the result and negative control antibacterial to pseudomonas aeruginosa of Antimicrobial Peptides From Plants obtained in embodiment 1-3
Figure.
Fig. 5 is bacteriostasis rate result figure of the antibacterial peptide extracting solution to Candida albicans and haemophilus influenzae.
Specific embodiment
A kind of method of separating plant antibacterial peptide, comprising the following steps:
(1) Semen sesami nigrum for taking certain mass, adds acetum in Semen sesami nigrum；
(2) by Semen sesami nigrum and acetum, ultrasound is carried out in ultrasonic generator, filters to obtain extracting solution；
(3) by extracting solution heating water bath, then it is down to room temperature；
(4) extracting solution is centrifuged, abandons precipitating, harvest supernatant；
(5) supernatant is successively dialysed and is concentrated, obtain reverse-phase chromatography sample；
(6) reverse-phase chromatography sample is subjected to reversed-phase high performance liquid chromatography separation, obtains the Antimicrobial Peptides From Plants of separation.
The volume for the 5-20 times of Semen sesami nigrum of acetum being added in step (1), the concentration of acetum are 1-10%.
Ultrasonic time is 10-35min.
Water bath heating temperature in step (3) is 60-100 DEG C, time 5-30min.
Centrifugal rotational speed is 8000-14000rpm, centrifugation time 5-20min in step (4).
The time of dialysis in step (5) is 24-72 hours.
Step (6) further includes steps of
A, reverse-phase chromatography sample is subjected to reversed-phase high performance liquid chromatography separation, wherein reverse-phase chromatographic column is reverse phase C4 silica gel
Column；
B, it by sample loading to reverse phase C4 silicagel column, and successively utilizes the distilled water containing trifluoroacetic acid and contains trifluoro
The acetonitrile of acetic acid carries out elution processing to C4 silicagel column, obtains elution samples；
C, elution samples are concentrated, obtain the Antimicrobial Peptides From Plants of separation.
The content containing trifluoroacetic acid in trifluoroacetic acid distilled water in step (B) is 0.1%-0.5%, contains trifluoro second
The content of trifluoroacetic acid is 0.1%-0.5%, ethane nitrile content 75%-90% in the acetonitrile of acid.
The elution processing time in step (B) is 30-60 minutes.
In step (1) is 100g by Semen sesami nigrum；Add the 7% acetum 500mL of 5 times of Semen sesami nigrum volumes in black sesame
In fiber crops；Ultrasonic generator power is 200W-300W in step (2), and frequency is for progress ultrasound, extraction time in 40kHz
20min；Water bath heating temperature in step (3) is 80 DEG C；Centrifugal condition in step (4) is 4 DEG C under the conditions of 10000rpm
It is centrifuged 5min；The time of dialysis in step (5) is 48 hours.Contain trifluoro second in trifluoroacetic acid distilled water in step (B)
The content of acid is 0.1%, and the content of trifluoroacetic acid is 0.1% in the acetonitrile containing trifluoroacetic acid, ethane nitrile content 90%, step
(B) the elution processing time in is 30 minutes.
The Antimicrobial Peptides From Plants that above-mentioned separating plant antibacterial peptide method obtains, have the feature that
PH value: the pH value using acidometer measurement antibacterial peptide is 2.82；
Isoelectric point: the basic protein that the PI value using Nephelometric Determination antibacterial peptide is 9.4-10.2；
Molecular weight: utilizing SDS-PAGE electrophoresis and laser desorption ionization time of flight mass spectrometry method, and measuring molecular weight is 4-
6kd。
The antibacterial peptide, which has the feature that, weighs sample 1mL in hydrolysis bottle, and after performic acid processing, 1mL hydrochloric acid is added
110 DEG C of hydrolysis 22~for 24 hours, hydrolysate measures amino acid classes using UPLC method are as follows: glycine (Glycine), cysteine
(Cysteine), arginine (Argnine), lysine (Lysine), histidine (Histidine), alanine (Alanine),
Threonine (Threonine), aspartic acid (Asparagine), leucine (Leucine), tyrosine (Tyrosine), phenylpropyl alcohol
Propylhomoserin (Phenylephrine), serine (Serine), glutamic acid (Glutamic acid), valine (Valine).
The purposes for the Antimicrobial Peptides From Plants that above-mentioned separating plant antibacterial peptide method obtains, the antibacterial peptide can be used as preventing and control
Treat microbial infectious diseases.
Embodiment 1
A kind of preparation method directly extracting antibacterial peptide from Semen sesami nigrum using ultrasonic wave:
1. weighing 100g Semen sesami nigrum；
2. adding 7% acetum 500mL of 5 times of Semen sesami nigrum volumes in Semen sesami nigrum；
3. Semen sesami nigrum and acetum to be 200W-300W in power, be surpassed in frequency 40kHz ultrasonic generator
Sound, extraction time 20min filter to obtain extracting solution；
4. by the extracting solution under the conditions of 10000rpm 4 DEG C of centrifugation 5min, abandon precipitating, to harvest supernatant；
5. then 80 DEG C of heating water bath 10min of the extracting solution are down to room temperature；
6. the supernatant is dialysed 48 hours, concentration obtains reverse-phase chromatography sample；
7. the reverse-phase chromatography sample is carried out reversed-phase high performance liquid chromatography separation, wherein the reverse-phase chromatographic column is reverse phase
C4 silicagel column；
8. by the sample loading to the reverse phase C4 silicagel column, and successively utilizing double containing 0.1% trifluoroacetic acid
It steams water and 90% acetonitrile containing 0.1% trifluoroacetic acid carries out elution processing 30 minutes to the C4 silicagel column, to be washed
De- sample；
9. the elution samples are concentrated, to obtain the isolated Antimicrobial Peptides From Plants.
Embodiment 2
A kind of preparation method directly extracting antibacterial peptide from Semen sesami nigrum using ultrasonic wave:
1. weighing 100g Semen sesami nigrum；
2. adding 5% acetum 1000mL of 10 times of Semen sesami nigrum volumes in Semen sesami nigrum；
3. Semen sesami nigrum and acetum to be 200W-300W in power, be surpassed in frequency 40kHz ultrasonic generator
Sound, extraction time 35min filter to obtain extracting solution；
4. by the extracting solution under the conditions of 8000rpm 4 DEG C of centrifugation 15min, abandon precipitating, to harvest supernatant；
5. then 60 DEG C of heating water bath 30min of the extracting solution are down to room temperature；
6. the supernatant is dialysed 72 hours, concentration obtains reverse-phase chromatography sample；
7. the reverse-phase chromatography sample is carried out reversed-phase high performance liquid chromatography separation, wherein the reverse-phase chromatographic column is reverse phase
C4 silicagel column；
8. by the sample loading to the reverse phase C4 silicagel column, and successively utilizing double containing 0.3% trifluoroacetic acid
It steams water and 80% acetonitrile containing 0.3% trifluoroacetic acid carries out elution processing 60 minutes to the C4 silicagel column, to be washed
De- sample；
9. the elution samples are concentrated, to obtain the isolated Antimicrobial Peptides From Plants.
Embodiment 3
A kind of preparation method directly extracting antibacterial peptide from Semen sesami nigrum using ultrasonic wave:
1. weighing 100g Semen sesami nigrum；
2. adding 1% acetum 500mL of 20 times of Semen sesami nigrum volumes in Semen sesami nigrum；
3. Semen sesami nigrum and acetum to be 200W-300W in power, be surpassed in frequency 40kHz ultrasonic generator
Sound, extraction time 10min filter to obtain extracting solution；
4. by the extracting solution under the conditions of 12000rpm 4 DEG C of centrifugation 10min, abandon precipitating, to harvest supernatant；
5. then 100 DEG C of heating water bath 15min of the extracting solution are down to room temperature；
6. the supernatant is dialysed 24 hours, concentration obtains reverse-phase chromatography sample；
7. the reverse-phase chromatography sample is carried out reversed-phase high performance liquid chromatography separation, wherein the reverse-phase chromatographic column is reverse phase
C4 silicagel column；
8. by the sample loading to the reverse phase C4 silicagel column, and successively utilizing double containing 0.5% trifluoroacetic acid
It steams water and 75% acetonitrile containing 0.5% trifluoroacetic acid carries out elution processing 45 minutes to the C4 silicagel column, to be washed
De- sample；
9. the elution samples are concentrated, to obtain the isolated Antimicrobial Peptides From Plants.
Experimental example 1
Application of the present invention in antibacterial
1. materials and methods
1.1 tested material Semen sesami nigrum antibacterial peptide extracting solutions
1.2 experiment thallus Candida albicans (BNCC341327), staphylococcus aureus (BNCC186335), Escherichia coli
(BNCC336902), pseudomonas aeruginosa (BNCC337940), haemophilus influenzae (BNCC337544)
1.3 experimental materials: diameter is that 5mm circle Xinhua No.1 qualitative filter paper piece is placed in after pressuresteam sterilization is handled
120 DEG C dry it is spare.
1.4 experimental method
(a) preparation of bacteria inhibition tablet: sterile and dry filter paper is taken.Every 20 μ L of dropwise addition antibacterial peptide solution, then will filter
The scraps of paper are lain against in clean sterilized petri dishes, are set spare after spontaneously drying at room temperature.
(b) preparation of negative control print: taking sterile dry filter paper piece, and every is added dropwise the 20 μ L of acetum of identical PH,
It is spare after drying.
(c) inoculation of test organisms: dipping concentration with sterile cotton swab is that 5 × 105cfu/ml-5 × 106cfu/ml is tested
Bacteria suspension is uniformly smeared 3 times in nutrient agar planar surface.Every to smear 1 time, plate should rotate 60 °, finally by cotton
Swab is smeared one week around plate edge.Plate is covered, drying at room temperature 5min is set.
(d) bacteriostatic agent print is placed with: test is placed with 1 microbiological contamination plate every time, and each plate is placed with 3 test prints, and 1
Negative control print, totally 4.It is placed with aseptic nipper coupongs in planar surface.At a distance of 25mm or more between each print center,
Periphery with plate is at a distance of 15mm or more.After being placed with, with the light pressure-like piece of aseptic nipper, it is made to be tightly attached to planar surface.It covers
Plate, sets 37 DEG C of incubators, and culture 16h~18h observes result.Simultaneously with the diameter (including patch) of vernier caliper measurement antibacterial ring size
Record.Test repeats 3 times.
1.5 test results: antibacterial peptide extracting solution is false single to Candida albicans, Escherichia coli, staphylococcus aureus, verdigris
The antibacterial result of born of the same parents bacillus referring to Fig.1-4, Fig. 1 be above-mentioned three kinds of embodiments obtained in Antimicrobial Peptides From Plants to Candida albicans press down
The result and negative control figure of bacterium, wherein the upper left Fig. 1 is the antibacterial of embodiment 2 as a result, the lower-left Fig. 1 is the antibacterial knot of embodiment 3
Fruit, the bottom right Fig. 1 are the antibacterial of embodiment 1 as a result, Fig. 1 upper right is negative control.Fig. 2 is obtained in above-mentioned three kinds of embodiments
The Antimicrobial Peptides From Plants result antibacterial to Escherichia coli and negative control figure, wherein the upper left Fig. 2 is the antibacterial of embodiment 2 as a result, figure
2 lower-lefts are the antibacterial of embodiment 3 as a result, the bottom right Fig. 2 is the antibacterial of embodiment 1 as a result, Fig. 2 upper right is negative control.Fig. 3 is
Antimicrobial Peptides From Plants obtained in above-mentioned three kinds of embodiments result and negative control figure antibacterial to staphylococcus aureus, wherein
The upper left Fig. 3 is the antibacterial of embodiment 2 as a result, the lower-left Fig. 3 is the antibacterial of embodiment 3 as a result, the bottom right Fig. 3 is the antibacterial of embodiment 1
As a result, Fig. 3 upper right is negative control.Fig. 4 is Antimicrobial Peptides From Plants obtained in above-mentioned three kinds of embodiments to pseudomonas aeruginosa
Antibacterial result and negative control figure, wherein the upper left Fig. 4 is the antibacterial of embodiment 2 as a result, the lower-left Fig. 4 is the suppression of embodiment 3
Bacterium is as a result, the bottom right Fig. 4 is the antibacterial of embodiment 1 as a result, Fig. 4 upper right is negative control.By the comparison in Fig. 1-4 it is found that implementing
The fungistatic effect of the resulting antibacterial peptide extracting solution of example 1 is best.
Experimental example 2
Application of the present invention in antibacterial
1. materials and methods
1.1 tested material Semen sesami nigrum antibacterial peptide extracting solutions
1.2 experiment thallus Candida albicans (BNCC341327), haemophilus influenzae (BNCC337544)
1.3 experimental method
(a) bacteriostatic solution prepare: by antibacterial peptide extracting solution with physiological saline do to be serially diluted into again various concentration by
It is spare to set 45 DEG C~50 DEG C constant temperature water baths for test solution.
(b) double concentration cultures are prepared: 2 times of dehydrated mediums are dissolved in 1000ml water.It is heated to boiling dissolution.So
It is spare to set 45 DEG C~50 DEG C water-baths by 121 DEG C of pressuresteam sterilization 15min afterwards.
(c) preparation containing antibacterial liquid culture medium: the antimicrobial fluid for taking 10ml to be serially diluted respectively is added in plate.It will be at 45 DEG C
Double culture medium 10ml in~50 DEG C of water-baths, is added in plate, and side edged rocks plate, makes antibacterial peptide extracting solution and culture medium
It mixes well.
(d) 1 μ L-2 μ L is taken with sample injector (bacteria containing amount is about 107Cfu/ml) bacteria suspension dibbling is trained in extracting solution containing antibacterial peptide
Support the plate of base.
(e) it is in kind inoculated with the plate for being free of antipathogenic composition, as positive control.
(f) plate after inoculation is placed in 35 DEG C of incubators, inversion culture 18h~for 24 hours, observe result.
1.4 test results: antibacterial peptide extracting solution is to the bacteriostasis rate result of Candida albicans and haemophilus influenzae referring to figure
5.Wherein, the cylinder with Fill Color indicates the inhibiting rate to Candida albicans, and the cylinder of Fill Color does not indicate convection current
The inhibiting rate of haemophilus influenza, according to result it is found that antibacterial peptide extracting solution has suppression to Candida albicans and haemophilus influenzae
Effect processed is greater than 80% to the inhibiting rate of bacterium when concentration reaches 16mg/mL.
Claims (13)
1. the method for separating plant antibacterial peptide, which comprises the following steps:
(1) Semen sesami nigrum for taking certain mass, adds acetum in Semen sesami nigrum；
(2) by the Semen sesami nigrum and acetum, ultrasound is carried out in ultrasonic generator, filters to obtain extracting solution；
(3) by the extracting solution heating water bath, then it is down to room temperature；
(4) extracting solution is centrifuged, abandons precipitating, harvest supernatant；
(5) supernatant is successively dialysed and is concentrated, obtain reverse-phase chromatography sample；
(6) the reverse-phase chromatography sample is subjected to reversed-phase high performance liquid chromatography separation, obtains the isolated Antimicrobial Peptides From Plants.
2. the method for separating plant antibacterial peptide as described in claim 1, which is characterized in that
The volume for 5-20 times of the acetum Semen sesami nigrum being added in the step (1), the acetum it is dense
Degree is 1-10%.
3. the method for separating plant antibacterial peptide as described in claim 1, which is characterized in that the ultrasonic time is 10-
35min。
4. the method for separating plant antibacterial peptide as described in claim 1, which is characterized in that the water in the step (3)
Bathing heating temperature is 60-100 DEG C, time 5-30min.
5. the method for separating plant antibacterial peptide as described in claim 1, which is characterized in that be centrifuged described in the step (4)
Revolving speed is 8000-14000rpm, centrifugation time 5-20min.
6. the method for separating plant antibacterial peptide as described in claim 1, which is characterized in that dialysis in the step (5)
Time is 24-72 hours.
7. the method for separating plant antibacterial peptide as described in claim 1, which is characterized in that the step (6) further comprises
Following steps:
A, the reverse-phase chromatography sample is subjected to reversed-phase high performance liquid chromatography separation, wherein the reverse-phase chromatographic column is reverse phase C4 silicon
Rubber column gel column；
B, it by the sample loading to the reverse phase C4 silicagel column, and successively utilizes the distilled water containing trifluoroacetic acid and contains
The acetonitrile of trifluoroacetic acid carries out elution processing to the C4 silicagel column, obtains elution samples；
C, the elution samples are concentrated, obtain the isolated Antimicrobial Peptides From Plants.
8. the method for separating plant antibacterial peptide as claimed in claim 6, which is characterized in that described in the step (B) contains
The content for having trifluoroacetic acid in trifluoroacetic acid distilled water is 0.1%-0.5%, trifluoro second in the acetonitrile containing trifluoroacetic acid
The content of acid is 0.1%-0.5%, ethane nitrile content 75%-90%.
9. the method for separating plant antibacterial peptide as claimed in claim 7, which is characterized in that at the elution in the step (B)
Managing the time is 30-60 minutes.
10. the method for separating plant antibacterial peptide as claimed in claim 6, which is characterized in that in the step (1) will be described
Semen sesami nigrum is 100g；Add the 7% acetum 500mL of 5 times of Semen sesami nigrum volumes in Semen sesami nigrum；It is ultrasonic in the step (2)
Wave generator power is 200W-300W, and frequency is that ultrasound, extraction time 20min are carried out in 40kHz；In the step (3)
The water bath heating temperature is 80 DEG C；Centrifugal condition in the step (4) is 4 DEG C of centrifugation 5min under the conditions of 10000rpm；
The time of dialysis in the step (5) is 48 hours.Described in the step (B) contains trifluoro in trifluoroacetic acid distilled water
The content of acetic acid is 0.1%, and the content of trifluoroacetic acid is 0.1% in the acetonitrile containing trifluoroacetic acid, and ethane nitrile content is
90%, the elution processing time in the step (B) is 30 minutes.
11. a kind of plants antimicrobial that the method using any separating plant antibacterial peptide as described in claim 1-10 is isolated
Peptide, which is characterized in that the antibacterial peptide has the feature that
PH value: the pH value using acidometer measurement antibacterial peptide is 2.82；
Isoelectric point: the basic protein that the PI value using Nephelometric Determination antibacterial peptide is 9.4-10.2；
Molecular weight: utilizing SDS-PAGE electrophoresis and laser desorption ionization time of flight mass spectrometry method, and measuring molecular weight is 4-6kd.
12. Antimicrobial Peptides From Plants as claimed in claim 11, which is characterized in that the antibacterial peptide, which has the feature that, weighs sample
Product 1mL is in hydrolysis bottle, after performic acid processing, 110 DEG C of 1mL hydrochloric acid hydrolysis 22 of addition~for 24 hours, hydrolysate is surveyed using UPLC method
Determine amino acid classes are as follows: glycine (Glycine), cysteine (Cysteine), arginine (Argnine), lysine
(Lysine), histidine (Histidine), alanine (Alanine), threonine (Threonine), aspartic acid
(Asparagine), leucine (Leucine), tyrosine (Tyrosine), phenylalanine (Phenylephrine), serine
(Serine), glutamic acid (Glutamic acid), valine (Valine).
13. the purposes of the Antimicrobial Peptides From Plants as described in claim 11 or 12, which is characterized in that be used as and prevent and treat microorganism
Infectious diseases.
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