CN101486794A - Preparation of epsilon-poly-L-lysine component with high antibacterial activity - Google Patents
Preparation of epsilon-poly-L-lysine component with high antibacterial activity Download PDFInfo
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- CN101486794A CN101486794A CNA200810153709XA CN200810153709A CN101486794A CN 101486794 A CN101486794 A CN 101486794A CN A200810153709X A CNA200810153709X A CN A200810153709XA CN 200810153709 A CN200810153709 A CN 200810153709A CN 101486794 A CN101486794 A CN 101486794A
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Abstract
The invention relates to a method for preparing a high antibacterial activity Epsilon-poly-L-lysine (Epsilon-PL) component; the high antibacterial activity component Epsilon-PL product is obtained by using micro-filtration, ultrafiltration and nanofiltration combination multi-level eparation technology for obtaining Epsilon-PL with different molecular weight distribution, determining the molecular weight range of the optimum antibacterial activity component by bacteriostasis experiment, and further being collected and dried. By using the method, the high-purity Epsilon-PL with different molecular weight ranges can be obtained; and as a membrane separation technology is adopted, any effect on the activity of Epsilon-PL is not caused, thus being favorable to research of the Epsilon-PL with different molecular weight.
Description
Technical field
The invention belongs to technical field of biochemical industry, especially a kind of preparation method of high bacteriostatic activity epsilon-poly-L-lysine component.
Background technology
Epsilon-poly-L-lysine (ε-poly-L-lysine, being called for short ε-PL) is poly-and thing by strain bio synthetic such as streptomyces albus one seed amino acid homotype, is that the amido linkage that the α-carboxyl by the epsilon-amino of monomer L-Methionin and another monomer L-Methionin forms is formed by connecting.ε-PL good water solubility is slightly soluble in ethanol, the thermostability height, and antimicrobial spectrum is wide, can resisting gram-positive bacteria, the activity of Gram-negative bacteria, fungi, heat-resistant bacillus and virus, in human body, be decomposed into L-Methionin.L-Methionin is one of eight necessary seed amino acids of human body, is the agent of a kind of natural biological antiseptic safely and efficiently therefore.In addition, ε-PL also can be used as gene transport agent, medicine capsulating material, electronic material, environment-friendly materials etc., and prospect is widely used.In recent years, along with the transformation of bacterial classification performance and the improvement of fermentation control means, the fermentation production efficiency of ε-PL constantly improves.
At present, the main patent of the relevant ε of China's announcement-PL production has:
1.2000 on July 12,, State Intellectual Property Office has announced that people such as the rock Tian Minzhi of Japan are called the patent of invention of " bacterial strain and the production method that produce epsilon-poly-L-lysine in a large number " in the name of China's application, and the patent No. is 97182253.0.This patent provides a kind of generative capacity of conventional raising bacterial strain ε-PL and the method for scale operation ε-PL.
2.2005 on November 16,, State Intellectual Property Office has announced that application number is 200510037774.2 by " utilizing kitasatosporia PL6-3 to prepare the method for ε-PL and salt thereof " patent of invention of Nanjing University of Technology's application.This patent has proposed a kind of by screening the method for the kitasatosporia PL-3 fermentation production of epsilon-PL that obtains.
3.2007 on November 28,, State Intellectual Property Office has announced that application number is 200610013800.2 by " reflux technique is produced the method for epsilon-poly-L-lysine " patent of invention of applicant oneself application.This patent has proposed the method that a kind of reflux technique is produced ε-PL, promptly in the fermenting process of ε-PL, later stage of leaching process is penetrated liquid as the stream liquid feeding, carries out stream and adds fermentation production of epsilon-PL.Later stage penetrates liquid and uses as the stream liquid feeding, has improved the productive rate of ε-PL, has reduced the discharging with hydrochloride waste of fermented waste fluid and wash-out, and it is towering right to have alleviated environment.On April 9th, 2007, the applicant on the basis of existing patent continuation application the patent of invention of " the mutagenic strain streptomyces albus TUST2 of a kind of a large amount of generation ε-PL and utilize this mutagenic strain TUST2 to adopt the method for fermentative Production ε-PL and salt thereof ", application number 200710057098.4.Production bacterial strain streptomyces albus TUST2 (CGMCC No.1986) fermentation production of epsilon-PL of ε-PL that this patent utilization screens from China's Hainan Province's soil, mutagenesis is also identified, output is at 10～30g/L.
There are some researches show that ε-PL range of molecular weight distributions that microbial fermentation is produced is wider, the ε-PL of different polymerization degree has different physico-chemical properties and purposes; In addition, ε-PL molecular weight difference, its fungistatic effect has very big difference.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of preparation method of high bacteriostatic activity epsilon-poly-L-lysine component is provided, ε-PL that this method will have explained hereafter now carries out stage treatment according to different molecular weight (different poly-and degree), and collection has the ε-PL component of high bacteriostatic activity.
Technical scheme of the present invention is:
A kind of preparation method of high bacteriostatic activity epsilon-poly-L-lysine component, the step of its preparation method is:
(1). ε-PL fermented liquid behind centrifugal removal thalline and albumen, is obtained ε-PL filtrate by decolouring;
(2). ε-PL filtrate is joined in micro-filtration, ultrafiltration and the nanofiltration membrane combination tripping device of the various molecular weight cut-offs that assemble in advance, separate and obtain respectively the ε-PL component filtrate of various molecular weight distribution;
(3). checking ε-PL filtrate molecular weight ranges is in 2KDa～5KDa, and by the ε-PL product that concentrates, frozen drying or spraying drying obtain high bacteriostatic activity.
And, the material of described filter membrane is polysulfones, polymeric amide, bacteria cellulose, regenerated cellulose or other appropriate pH values at 2～14 filter membrane, the aperture of its microfiltration membrane is 0.2 μ m, the molecular weight cut-off of ultrafiltration and nanofiltration membrane is respectively 25KDa, 10kDa, 5kDa, 2kDa, 1kDa, adopts micro-filtration, ultrafiltration and nanofiltration membrane combination tripping device to carry out separation and purification.
And the operating pressure of described filtration stage treatment can be at 0.05～1.0MPa, and the pH of filtering fermentation liquor is neutral, and temperature is 20～40 ℃.
Advantage of the present invention and positively effect are:
The present invention carries out film combination technique stage treatment to epsilon-poly-L-lysine by the molecular weight size, determine to have the molecular weight part of high bacteriostatic activity by bacteriostatic experiment, thereby preparation has the epsilon-poly-L-lysine product that the suitable molecular weight of high bacteriostatic activity distributes.Use method of the present invention and can obtain the highly purified ε-PL filtrate of different molecular weight ranges,, can not produce any influence, and help the research of the ε-PL of various different molecular weights the activity of ε-PL because what adopt is membrane separation technique.
Embodiment
The present invention is further described below in conjunction with embodiment; Following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Need to prove, fermented liquid of the present invention is according to the preparation of the method in the method for epsilon-poly-L-lysine " reflux technique produce " patent, and used bacterial classification is the bacterial classification in " the mutagenic strain streptomyces albus TUST2 of a kind of a large amount of generation ε-PL and utilize this mutagenic strain TUST2 to adopt the method for fermentative Production ε-PL and salt thereof " patent.
The material of the used filter membrane of the present invention can for polysulfones, polymeric amide, bacteria cellulose, regenerated cellulose or other appropriate pH values at 2～14 filter membrane, the aperture of microfiltration membrane is 0.2 μ m, the molecular weight cut-off of ultrafiltration and nanofiltration membrane is respectively 25KDa, 10kDa, 5kDa, 2kDa, 1kDa, adopts micro-filtration, ultrafiltration and nanofiltration membrane combination tripping device to carry out separation and purification.
The present invention filters the operating pressure of stage treatment can be at 0.05～1.0MPa, and the pH of filtering fermentation liquor is neutral, and temperature is 25 ℃.
The present invention collects ε-PL solution that the highest molecular weight section of bacteriostatic activity is 2KDa～5KDa, and ε-PL product is made in spray-dried or lyophilize, is used as food preservatives.
Provide embodiments of the invention below:
Embodiment 1:
Content 20g/L ε-PL fermented liquid centrifugation thalline, gained solution can carry out the separation of film combination tripping device after decolouring.
Get 2000ml ε-PL filtrate, step by step through 0.2 μ m microfiltration membrane, 25KDa, 10KDa, 5KDa, 2KDa, 1KDa ultra-filtration membrane and nanofiltration membrane.Wherein the working pressure of micro-filtration is 0.05MPa, molecular weight cut-off is that the working pressure of the ultra-filtration membrane of 25KDa, 10KDa, 5KDa is 0.15MPa, molecular weight cut-off is that the working pressure of the ultra-filtration membrane of 2KDa is 0.2MPa, and the working pressure of the nanofiltration membrane of 1KDa is 0.7MPa.
Microfiltration membrane is 0 to the rejection of ε-PL filtrate, sees through the long-pending 2000ml that is about of liquid; After the 25KDa ultra-filtration membrane, the volume ratio that sees through liquid and concentrated solution is about 10:1; After the 10KDa ultra-filtration membrane, the volume ratio that sees through liquid and trapped fluid is about 7:1; After the 5KDa ultra-filtration membrane, the volume ratio that sees through liquid and trapped fluid is about 7:1; After the 2KDa ultra-filtration membrane, the volume ratio that sees through liquid and trapped fluid is about 2:1; By after the 1KDa nanofiltration membrane, the volume ratio that sees through liquid and concentrated solution is about 1:1 at last.
Do bacteriostatic experiment at each different components then.
By result's (seeing attached list 1) of bacteriostatic experiment checking, drawing the best molecular weight section of fungistatic effect is 2KDa～5KDa, collects this a part of solution, concentrates through lyophilize and makes high bacteriostatic activity ε-PL product.
Embodiment 2:
ε-PL product powder is configured to 20g/L solution 2L (pH is for neutral, and temperature is 25 ℃), carries out membrane filtration by micro-filtration, ultrafiltration with nanofiltration combined films tripping device successively again and separates.Wherein the working pressure of micro-filtration is 0.1MPa, and molecular weight cut-off is that the working pressure of the ultra-filtration membrane of 25KDa, 10KDa, 5KDa is 0.2MPa, and molecular weight cut-off is that the working pressure of the ultra-filtration membrane of 2KDa is 0.25MPa, and the working pressure of the nanofiltration membrane of 1KDa is 1MPa.
The ε of above-mentioned 2000ml-PL solution sees through microfiltration membrane fully; Afterwards, by the 25KDa ultra-filtration membrane, the volume ratio that sees through liquid and trapped fluid is about 10:1; By the 10KDa ultra-filtration membrane, the volume ratio that sees through liquid and trapped fluid is about 8:1 again; By after the 5KDa ultra-filtration membrane, the volume ratio that sees through liquid and trapped fluid is about 7:1 then; Then by the 2KDa ultra-filtration membrane, the volume ratio that sees through liquid and trapped fluid is about 2:1; By after the 1KDa nanofiltration membrane, the volume ratio that sees through liquid and trapped fluid is about 1:1 at last.
Do bacteriostatic experiment at each different components then.
By the bacteriostatic experiment result, can draw the best molecular weight section of fungistatic effect is 2KDa～5KDa, collects this a part of solution, concentrates after high bacteriostatic activity ε-PL product is made in lyophilize.
Table 1
Claims (3)
1. the preparation method of a high bacteriostatic activity epsilon-poly-L-lysine component, it is characterized in that: preparation method's step is:
(1). ε-PL fermented liquid behind centrifugal removal thalline and albumen, is obtained ε-PL filtrate by decolouring;
(2). ε-PL filtrate is joined in micro-filtration, ultrafiltration and the nanofiltration membrane combination tripping device of the various molecular weight cut-offs that assemble in advance, separate and obtain respectively the ε-PL component filtrate of various molecular weight distribution;
(3). checking ε-PL filtrate molecular weight ranges is in 2KDa～5KDa, and by the ε-PL product that concentrates, frozen drying or spraying drying obtain high bacteriostatic activity.
2. the preparation method of high bacteriostatic activity epsilon-poly-L-lysine component according to claim 1, it is characterized in that: the material of described filter membrane is polysulfones, polymeric amide, bacteria cellulose, regenerated cellulose or other appropriate pH values at 2～14 filter membrane, the aperture of its microfiltration membrane is 0.2 μ m, the molecular weight cut-off of ultrafiltration and nanofiltration membrane is respectively 25KDa, 10kDa, 5kDa, 2kDa, 1kDa, adopts micro-filtration, ultrafiltration and nanofiltration membrane combination tripping device to carry out separation and purification.
3. the preparation method of high bacteriostatic activity epsilon-poly-L-lysine component according to claim 1 is characterized in that: the operating pressure of described filtration stage treatment can be at 0.05～1.0Mpa, and the pH of filtering fermentation liquor is neutral, and temperature is 20～40 ℃.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105778118A (en) * | 2016-03-30 | 2016-07-20 | 浙江新银象生物工程有限公司 | Preparation method of epsilon-polylysine |
| CN106380592A (en) * | 2016-11-04 | 2017-02-08 | 江南大学 | Method for extracting epsilon-polylysine and hydrochloride thereof from fermentation liquid |
| CN111777760A (en) * | 2020-07-03 | 2020-10-16 | 中国科学院过程工程研究所 | Polylysine separation and purification process and application |
| CN112237844A (en) * | 2020-10-28 | 2021-01-19 | 江南大学 | Method for improving high polymerization degree epsilon-polylysine in product |
-
2008
- 2008-12-03 CN CN200810153709XA patent/CN101486794B/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105778118A (en) * | 2016-03-30 | 2016-07-20 | 浙江新银象生物工程有限公司 | Preparation method of epsilon-polylysine |
| CN106380592A (en) * | 2016-11-04 | 2017-02-08 | 江南大学 | Method for extracting epsilon-polylysine and hydrochloride thereof from fermentation liquid |
| CN111777760A (en) * | 2020-07-03 | 2020-10-16 | 中国科学院过程工程研究所 | Polylysine separation and purification process and application |
| CN112237844A (en) * | 2020-10-28 | 2021-01-19 | 江南大学 | Method for improving high polymerization degree epsilon-polylysine in product |
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| CN101486794B (en) | 2010-11-17 |
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Owner name: ZHEJIANG SILVER ELEPHANT BIOENGINEERING CO., LTD. Free format text: FORMER OWNER: TIANJIN UNIVERSITY OF SCIENCE + TECHNOLOGY Effective date: 20120327 Free format text: FORMER OWNER: YINXIANG BIOLOGICAL ENGINEERING CO., LTD., ZHEJIANG PROV. Effective date: 20120327 |
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Effective date of registration: 20120327 Address after: 317200, Tiantai County, Zhejiang province hi tech Industrial Park, Feng Lu / South Ring Road Patentee after: ZHEJIANG SILVER-ELEPHANT BIO-ENGINEERING CO., LTD. Address before: 300457 Tianjin economic and Technological Development Zone, No. thirteenth Main Street, No. 29 Co-patentee before: Yinxiang Biological Engineering Co., Ltd., Zhejiang Prov. Patentee before: Tianjin University of Science & Technology |