CN109180779B - A kind of method that purifying prepares antibacterial peptide - Google Patents
A kind of method that purifying prepares antibacterial peptide Download PDFInfo
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- CN109180779B CN109180779B CN201811277061.7A CN201811277061A CN109180779B CN 109180779 B CN109180779 B CN 109180779B CN 201811277061 A CN201811277061 A CN 201811277061A CN 109180779 B CN109180779 B CN 109180779B
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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Abstract
The invention discloses the method and apparatus that a kind of purifying prepares antibacterial peptide, when solving antibacterial peptide purification in the prior art, its yield also reaches 50% or more when purity can not be made to be greater than 99%.The present invention includes the method and apparatus of producing synthesis gas, wherein method includes: Step 1: antibacterial peptide crude product solution is made；Step 2: antibacterial peptide crude product solution is added in semi-permeable membrane, pure solution at the beginning of antibacterial peptide is obtained after successively dialysing in buffer and purified water；Step 3: solution pure at the beginning of antibacterial peptide is passed through the consummate rear acquisition consummate solution of antibacterial peptide of RP chromatography；Step 4: the consummate solution of antibacterial peptide is turned salt by HPLC method, antibacterial peptide salting liquid is obtained；It is concentrated under reduced pressure, obtains antibacterial peptide finished product after drying.The present invention can make semi-permeable membrane purifying and reverse-phase chromatography purifying mutually promote, its yield also reaches 50% or more when purity being made to be greater than 99%.
Description
Technical field
The present invention relates to purification fields, and in particular to a kind of method that purifying prepares antibacterial peptide.
Background technique
Antibacterial peptide is a kind of polypeptide with antibacterial activity, by acting on bacterial cell membrane, destroying its integrality and producing
Raw perforated phenomenon destroys its organelle and causes metabolic disorder into intracellular.Bacterium content, which releases, extracellularly keeps bacterium dead
It dies.Antibacterial peptide not only has preferable bactericidal effect to bacterium, fungi, it also plays the role of antiviral activity, promotes wound healing.
Meanwhile antibacterial peptide and lysozyme are combined also with synergetic antibacterial effect.Under the action of both core components, formed to pathogenic
The dual broken wall bactericidal effect of bacterium.
Antibacterial peptide is similar to the MSI-78 of overseas clinical trial, which is completed three phases clinic, is used for diabetic ulcer
Treatment.Currently, few research reports in terms of it is isolated and purified, the especially method of large-scale production.Antibacterial peptide
Common purification process includes reversed-phased high performace liquid chromatographic, gel method, ion-exchange etc., using any single method
Or multiple means, all it is difficult isolated high purity product (> 99%) or yield is lower (< 50%).
A kind of purification process for synthesizing carbetocin is disclosed in CN201511014180.X, it is public in this document
It has opened and technique is combined using semi-permeable membrane purifying and reverse-phase chromatography purifying, it is ensured that finished product meets purity greater than 99%, single miscellaneous
Medicinal crude drug standard of the matter less than 0.1%.
Although it discloses the purity after peptide purification can be made to be greater than 99%, effect of the single impurity less than 0.1%.But
It is to be learnt by detection, when applying this method in antibacterial peptide, its yield also reaches when purity can not still be made to be greater than 99%
50% or more.
Summary of the invention
It is an object of that present invention to provide a kind of method that purifying prepares antibacterial peptide, pass through the reasonable of processing step and parameter
Optimization can make semi-permeable membrane purifying and reverse-phase chromatography purifying mutually promote, its yield also reaches 50% when purity being made to be greater than 99%
More than.
The present invention is achieved through the following technical solutions:
A kind of method that purifying prepares antibacterial peptide, comprising:
Step 1: antibacterial peptide crude product filters after being dissolved by purified water is made antibacterial peptide crude product solution；
Step 2: antibacterial peptide crude product solution is added in semi-permeable membrane, resisted after successively dialysing in buffer and purified water
Pure solution at the beginning of bacterium peptide；
Step 3: solution pure at the beginning of antibacterial peptide is passed through the consummate rear acquisition consummate solution of antibacterial peptide of RP chromatography；The reverse phase
In chromatography, use octadecylsilane chemically bonded silica for stationary phase, the ammonium sulfate solution that concentration is 5mM~50.0mM is A
Phase, acetonitrile are B phase；A phase uses sulphur acid for adjusting pH value to 2.0~3.5；B phase is eluted with 10%~40% gradient, elution
Time is 60min；
Step 4: the consummate solution of antibacterial peptide is turned salt by HPLC method, antibacterial peptide salting liquid is obtained；It is concentrated under reduced pressure, is dry
After obtain antibacterial peptide finished product.
A kind of purification process be combineding with each other by semi-permeable membrane purifying and reverse-phase chromatography purifying is disclosed in the prior art,
The purpose is to improve the purity of finished product, purity is made to reach 99.5% or more.
Based on the above issues, inventor is learnt by experimental verification: using semi-permeable membrane purifying and reverse chromatograms in this document
After the method that method purifying be combined with each other is to antibacterial peptide purification, although the purity of antibacterial peptide can be improved well, reach purity
99.5% or more, but the raising of the yield of actually antibacterial peptide is not significant, or even is not improved, can pass through reference examples
Result control learn.
And in order to improve the yield of antibacterial peptide, industry technical staff usually passes through the Parameter Conditions during optimized purification
To improve yield and purity after purification.But in the actual process, since the substance of purifying is different, the purification process of use and
Condition is different, and purification effect is also not quite similar.For polypeptide, when the method purified using reverse phase liquid chromatography method
When, when purity after purification reaches 99.5% or more, yield is often below 50%, even lower than 40%, and yield is low, cost
Height, and it is unsatisfactory for the requirement of industrialization.
After the present invention be combined with each other by using semi-permeable membrane purifying and reverse-phase chromatography purifying, reversed liquid has been advanced optimized
Reverse-phase chromatography condition in phase chromatography when the purity of antibacterial peptide finished product reaches 99.5% or more, while making antibacterial peptide finished product
Yield be higher than 50%, effect is very significant.
In addition, the semi-permeable membrane dialysis purification in the present invention is easy to operate, it is not necessarily to special installation, semi-permeable membrane can repeat to make
With greatly reducing cost；Good protective effect is provided to consummate reverse-phase chromatography filler simultaneously, extends chromatograph packing material
Replacement cycle and service life, reduce the cost in reverse-phase chromatography consummate stage accordingly.
The present invention carries out preliminary purification using Immunohistochemistry, can carry out chromatogram purification without concentration, it is dense to reduce high temperature
Contracting step reduces antibacterial peptide high temperature degradation probability.
Further, the process of the step 2 are as follows:
Firstly, antibacterial peptide crude product solution is encased in semi-permeable membrane, it is placed in buffer, stirring is percolated to slow at room temperature
Fliud flushing pH=2.0~3.0；Then the semi-permeable membrane of the peptide solution containing antibacterial is transferred in purified water, at room temperature stirring diafiltration to half
PH value of solution=3.5~4.5 in permeable membrane.
Further, the volume ratio of the buffer and antibacterial peptide crude product solution is (10~40): 1；The purified water and anti-
The volume ratio of bacterium peptide solution is (50~60): 1.
Further, the buffer is acetate buffer, and the pH value of the acetate buffer is 3.5.
Further, the semi-permeable membrane is cellulose ester membrane or regenerated cellulose film, and molecular cut off is 500 dalton.
Further, described that salt is turned to turn salt by ammonium acetate and acetic acid condition ion exchange by HPLC method.
Further, it is no more than 35 DEG C that condition is concentrated during the reduced pressure, and vacuum degree is 0.08Mpa or more.
Further, the drying is realized by the way of decompression freeze-drying, operating process are as follows: pre-freeze is to -50~-10 DEG C
It is kept for 2~4 hours, keeps vacuum degree constant in 0.01~1.00mbar, heating is 12~36 hours dry.
Further, the antibacterial peptide crude product is synthesized by liquid phase synthesizing method or solid-phase synthesis.
Compared with prior art, the present invention having the following advantages and benefits:
1, the present invention has given full play to the advantages of respective purification technique, significantly improves aimed purity, can prepare pure
Degree is greater than 99.5%, and Medicinal crude drug of the single impurity less than 0.1% breaches the bottleneck of existing high-purity；
2, the present invention reduces consummate number and step because of the effect of dialysis, shortens purifying process route, saved at
This；
3, the present invention greatly improves purification efficiency and yield, and purifying yield is greater than when purity is higher than 99.5%
50%, it is more suitable for industrialization production, effect is more significant.
Detailed description of the invention
Attached drawing described herein is used to provide to further understand the embodiment of the present invention, constitutes one of the application
Point, do not constitute the restriction to the embodiment of the present invention.In the accompanying drawings:
Fig. 1 is sample detection map after purification in embodiment 1.
Fig. 2 is sample high resolution mass spectrum figure after purification in embodiment 1.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below with reference to embodiment and attached drawing, to this
Invention is described in further detail, and exemplary embodiment of the invention and its explanation for explaining only the invention, are not made
For limitation of the invention.
Embodiment 1
A kind of method that purifying prepares antibacterial peptide, comprising:
(1) sample treatment: chromatographic purity is obtained up to 50% or more the thick peptide of antibacterial peptide using solid-phase synthesis, uses purified water
It is dissolved according to the concentration of 50g/L, stirring uses the water system membrane filtration of 0.45um, filters out insoluble matter, filtered after being completely dissolved
Liquid is antibacterial peptide crude product after filtrate is dry.
(2) just pure: 3.2L purified water is added in 160g antibacterial peptide crude product, is configured to 50mg/ml antibacterial peptide crude product solution,
Then filtering with microporous membrane is used, it is spare to obtain antibacterial peptide crude product solution.
Antibacterial peptide crude product solution is fitted into the RC semi-permeable membrane that molecular cut off is 500 dalton, about total capacity volume
60%, as in 32L acetate buffer, stirring is permeated at room temperature.
The semi-permeable membrane of peptide solution containing antibacterial is transferred in the purified water of 160L, is taken out after stirring infiltration at 20~30 DEG C,
Pure solution for standby at the beginning of obtaining antibacterial peptide.
(3) consummate
Reverse-phase chromatography condition: using octadecylsilane chemically bonded silica as the high-performance liquid chromatogram preparation column of stationary phase, pillar
Partial size 10um, aperture 100A.Pillar diameter and length are as follows: 80x250mm.A phase in mobile phase are as follows: 10mM ammonium sulfate passes through sulphur
Acid adjustment pH to 2.0；B phase in mobile phase are as follows: acetonitrile；Flow velocity: 320ml/min；Gradient: 10%~40%, Detection wavelength:
220nm；Single applied sample amount about 16g.
Chromatographic column balances after being rinsed well with 50% or more acetonitrile, by solution loading pure at the beginning of gained antibacterial peptide, on single
Sample amount about 16g.Linear gradient elution 60min collects target peak, quantitative according to HPLC analysis detection, by the antibacterial peptide of collection in
35 DEG C of water temperature it is below under the conditions of vacuum rotary steam it is dense be reduced to acetonitrile largely screwed out, obtain the consummate solution for standby of antibacterial peptide.
(4) turn salt: by the consummate solution of gained antibacterial peptide, after reduced pressure, salt method, i.e. ammonium acetate and acetic acid being changed using HPLC
Turn salt by ion exchange；It will turn the qualified fraction after salt to merge, carry out being concentrated under reduced pressure into 0.5g/50ml, thickening temperature does not surpass
35 DEG C are crossed, then freeze-drying obtains the antibacterial peptide solution that purity is higher than 99.5%.
By calculating: the purity of the present embodiment is 99.7%, and purifying yield is 56%.
Embodiment 2
The present embodiment is the comparative examples of embodiment 1, uses documents CN201511014180.X in the present embodiment
In purification process and condition antibacterial peptide is handled, treatment process and result are as follows:
Step 1 takes the thick peptide of 5g antibacterial peptide, and 1% acetum 50ml is added, is configured to the thick peptide solution of 10% antibacterial peptide.
The acetic acid solution of antibacterial peptide is encased in molecular cut off as in 500 dalton RC semi-permeable membranes, loading amount is by step 2
The 70% of semi-permeable membrane loading amount volume is placed in 2L acetate buffer (pH3.5) buffer, and stirring is percolated extremely at 20~30 DEG C
PH of buffer=2.0~3.0.
Step 3 goes to the semi-permeable membrane of the acetic acid solution containing antibacterial peptide in 3L purified water, and diafiltration is stirred at 20~30 DEG C
To pH value of solution=3.5~4.5 in semi-permeable membrane bag.
Step 4 takes out the antibacterial peptide solution in semi-permeable membrane, is purified using RP chromatography, collects and contain antibacterial peptide
Eluting fraction, specific chromatographic condition is as follows；
Chromatographic column: using octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are 50mm × 250mm
Mobile phase: A:1% glacial acetic acid aqueous solution B: acetonitrile
Detection wavelength: 230nm
Flow velocity: 80ml/min.
Gradient is as shown in table 2:
Table 2
Eluent containing antibacterial peptide is concentrated out 75~85% by step 5, controls temperature less than 60 DEG C, vacuum degree is
After concentration, residue is poured into lyophilized plate, is put into freeze dryer by 0.09Mpa, is kept the temperature after extremely -45~-40 DEG C of pre-freeze
2h opens vacuum pump and vacuumizes and keep vacuum degree constant in 100KPa or less.Period heats up naturally, is dried in vacuo 12h, freeze-drying
After, obtain antibacterial peptide sterling.
By detection obtain its purity be 98.5%, yield 44%.
Embodiment 3
The present embodiment is the comparative example of embodiment 1, in the present embodiment only with the reverse-phase chromatography in embodiment 1 into
Row purifying, detailed process and experimental result are as follows:
(1) sample treatment: antibacterial peptide purified water is dissolved according to the concentration of 50mg/mL, stirring is used after being completely dissolved
The water system membrane filtration of 0.45um, filters out insoluble matter, obtains filtrate, is antibacterial peptide crude product after filtrate is dry.
(2) just pure: purified water is added in 2g antibacterial peptide crude product, 35% antibacterial peptide crude product solution is configured to, then with micro-
It is spare to obtain antibacterial peptide crude product solution for hole membrane filtration.
(3) consummate
Reverse-phase chromatography condition: using octadecylsilane chemically bonded silica as the high-performance liquid chromatogram preparation column of stationary phase, pillar
Partial size 10um, aperture 100A.Pillar diameter and length are as follows: 20x250mm.A phase in mobile phase are as follows: 30mM ammonium sulfate passes through sulphur
Acid adjustment pH to 2.5；B phase in mobile phase are as follows: acetonitrile；Flow velocity: 20ml/min；Gradient: 10%~40%, Detection wavelength:
220nm；Single applied sample amount about 50mg.
Chromatographic column balances after being rinsed well with 50% or more acetonitrile, by gained antibacterial peptide crude product solution loading, on single
Sample amount about 50mg.Linear gradient elution 60min collects target peak, quantitative according to HPLC analysis detection, by the antibacterial peptide of collection in
35 DEG C of water temperature it is below under the conditions of vacuum rotary steam it is dense be reduced to acetonitrile largely screwed out, obtain the consummate solution for standby of antibacterial peptide.
(4) turn salt: by the consummate solution of gained antibacterial peptide, after reduced pressure, salt method, i.e. ammonium acetate and acetic acid being changed using HPLC
Turn salt by ion exchange；It will turn the qualified fraction after salt to merge, carry out being concentrated under reduced pressure into 0.5g/50ml, thickening temperature does not surpass
35 DEG C are crossed, the purity being then freeze-dried is higher than 98% antibacterial peptide solution.
Obtaining its purity by detection is 98.8%, calculated yield 41.2%.
Embodiment 4
A kind of method that purifying prepares antibacterial peptide, detailed process and experimental result are as follows:
(1) sample treatment: 2g antibacterial peptide crude product is dissolved with the concentration of 50mg/mL with 40ml purified water, stirring is completely dissolved
The water system membrane filtration for using 0.45um afterwards, obtains antibacterial peptide crude product solution.
(2) solution of antibacterial peptide is encased in molecular cut off is in 500 dalton RC semi-permeable membranes, and loading amount is semi-permeable membrane dress
The 70% of volume is measured, is placed in 800mL acetate buffer (pH3.5) buffer, stirring is percolated to pH of buffer at 25 DEG C
=3.0.
The semi-permeable membrane of the solution of antibacterial peptide is gone in 2L purified water, stirring is percolated to solution in semi-permeable membrane bag at 25 DEG C
PH=3.5.
(3) consummate: high-performance liquid chromatogram preparation column is using octadecylsilane chemically bonded silica as stationary phase, the partial size of pillar
10um, aperture 100A.Pillar diameter and length are as follows: 30x250mm.A phase in mobile phase are as follows: 50mM ammonium sulfate is adjusted by sulfuric acid
PH to 2.5；B phase in mobile phase are as follows: acetonitrile；Flow velocity: 20ml/min；Gradient: 10%~40%, Detection wavelength: 220nm；
Chromatographic column balances after being rinsed well with 50% or more acetonitrile, by gained antibacterial peptide crude product solution loading, on single
Sample amount about 100mg.Linear gradient elution 60min collects target peak, quantitative according to HPLC analysis detection, by the antibacterial peptide of collection
In 35 DEG C of water temperature it is below under the conditions of vacuum rotary steam it is dense be reduced to acetonitrile largely screwed out, it is standby to obtain the consummate solution of antibacterial peptide
With.
(4) turn salt: by the consummate solution of gained antibacterial peptide, after reduced pressure, salt method, i.e. ammonium acetate and acetic acid being changed using HPLC
Turn salt by ion exchange；It will turn the qualified fraction after salt to merge, carry out being concentrated under reduced pressure into 0.5g/50ml, thickening temperature does not surpass
35 DEG C are crossed, the purity being then freeze-dried is higher than 99.5% antibacterial peptide solution.
Obtaining its purity by detection is 99.9%, calculated yield 63%.
Embodiment 5
A kind of method that purifying prepares antibacterial peptide, detailed process and experimental result are as follows:
(1) sample treatment: 10g antibacterial peptide crude product is dissolved with the concentration of 50mg/mL with 200ml purified water, stirring is completely molten
The water system membrane filtration of Xie Houyong 0.45um obtains antibacterial peptide crude product solution.
(2) just pure: it is in 500 dalton RC semi-permeable membranes that the solution of antibacterial peptide, which is encased in molecular cut off, and loading amount is half
The 65% of permeable membrane loading amount volume is placed in 8000mL acetate buffer (pH3.5) buffer, and stirring is percolated to slow at 35 DEG C
Fliud flushing pH=3.0.
The semi-permeable membrane of the solution of antibacterial peptide is gone in 10L purified water, stirring is percolated to molten in semi-permeable membrane bag at 35 DEG C
Liquid pH=3.5.
(4) consummate: using octadecylsilane chemically bonded silica as the high-performance liquid chromatogram preparation column of stationary phase, the partial size of pillar
10um, aperture 100A.Pillar diameter and length are as follows: 30x250mm.A phase in mobile phase are as follows: 50mM ammonium sulfate is adjusted by sulfuric acid
PH to 2.5；B phase in mobile phase are as follows: acetonitrile；Flow velocity: 20ml/min；Gradient: 10%~40%, Detection wavelength: 220nm；
Chromatographic column balances after being rinsed well with 50% or more acetonitrile, by gained antibacterial peptide crude product solution loading, on single
Sample amount about 5g.Linear gradient elution 60min collects target peak, quantitative according to HPLC analysis detection, by the antibacterial peptide of collection in water
The dense acetonitrile that is reduced to of vacuum rotary steam is largely screwed out under the conditions of 35 DEG C of temperature is below, obtains the consummate solution for standby of antibacterial peptide.
(4) turn salt: by the consummate solution of gained antibacterial peptide, after reduced pressure, salt method, i.e. ammonium acetate and acetic acid being changed using HPLC
Turn salt by ion exchange；It will turn the qualified fraction after salt to merge, carry out being concentrated under reduced pressure into 0.5g/50ml, thickening temperature does not surpass
35 DEG C are crossed, the purity being then freeze-dried is higher than 99.5% antibacterial peptide solution.
Obtaining its purity by detection is 99.7%, calculated yield 57%.
Above-described specific embodiment has carried out further the purpose of the present invention, technical scheme and beneficial effects
It is described in detail, it should be understood that being not intended to limit the present invention the foregoing is merely a specific embodiment of the invention
Protection scope, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should all include
Within protection scope of the present invention.
Claims (6)
1. a kind of method that purifying prepares antibacterial peptide characterized by comprising
Step 1: antibacterial peptide crude product filters after being dissolved by purified water is made antibacterial peptide crude product solution；
Step 2: antibacterial peptide crude product solution is added in semi-permeable membrane, antibacterial peptide is obtained after successively dialysing in buffer and purified water
First pure solution；
Step 3: solution pure at the beginning of antibacterial peptide is passed through the consummate rear acquisition consummate solution of antibacterial peptide of RP chromatography；The reverse-phase chromatography
In method, use octadecylsilane chemically bonded silica for stationary phase, the ammonium sulfate solution that concentration is 5mM~50.0mM is A phase, second
Nitrile is B phase；A phase uses sulphur acid for adjusting pH value to 2.0~3.5；B phase is eluted with 10%~40% gradient, elution time
For 60min；
Step 4: the consummate solution of antibacterial peptide is turned salt by HPLC method, antibacterial peptide salting liquid is obtained；After being concentrated under reduced pressure, being dry
To antibacterial peptide finished product；
The process of the step 2 are as follows:
Firstly, antibacterial peptide crude product solution is encased in semi-permeable membrane, it is placed in buffer, at room temperature stirring diafiltration to buffer
PH=2.0~3.0；Then the semi-permeable membrane of the peptide solution containing antibacterial is transferred in purified water, at room temperature stirring diafiltration to semi-permeable membrane
Interior pH value of solution=3.5~4.5；
The volume ratio of the buffer and antibacterial peptide crude product solution is (10~40): 1；The body of the purified water and antibacterial peptide solution
Product is than being (50~60): 1；
The antibacterial peptide crude product is synthesized by liquid phase synthesizing method or solid-phase synthesis.
2. the method that a kind of purifying according to claim 1 prepares antibacterial peptide, which is characterized in that the buffer is acetic acid
Salt buffer, the pH value of the acetate buffer are 3.5.
3. the method that a kind of purifying according to claim 1 prepares antibacterial peptide, which is characterized in that the semi-permeable membrane is fiber
Cellulose ester film or regenerated cellulose film, molecular cut off are 500 dalton.
4. the method that a kind of purifying according to claim 1 prepares antibacterial peptide, which is characterized in that described to be turned by HPLC method
Salt is to turn salt by ion exchange by ammonium acetate and acetic acid.
5. the method that a kind of purifying according to claim 1 prepares antibacterial peptide, which is characterized in that the reduced pressure process
Middle concentration condition is no more than 35 DEG C, and vacuum degree is 0.08 or more.
6. the method that a kind of purifying according to claim 1 prepares antibacterial peptide, which is characterized in that described dry using decompression
The mode of freeze-drying realizes, operating process are as follows: pre-freeze is kept for 2~4 hours to -50~-10 DEG C, keeps vacuum degree constant 0.01
~1.00mbar, heating are 12~36 hours dry.
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| CN111690035A (en) * | 2020-06-05 | 2020-09-22 | 广州颜如玉生物科技有限公司 | Method for improving yield of antibacterial peptide |
| CN111662362B (en) * | 2020-07-31 | 2021-05-28 | 成都诺和晟泰生物科技有限公司 | Method for purifying carbetocin |
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| CN103936832A (en) * | 2014-04-23 | 2014-07-23 | 江南大学 | Antibacterial peptide derived from anchovy cooking waste liquid and antibacterial peptide separation method |
| CN103923166A (en) * | 2014-05-05 | 2014-07-16 | 西华大学 | Separation and purification method of bamboo leaf antioxidative peptide |
| CN105399799A (en) * | 2015-12-31 | 2016-03-16 | 郑州大明药物科技有限公司 | Purifying method for carbetocin |
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