CN102363797A - Method for producing epsilon-poly-L-lysine - Google Patents
Method for producing epsilon-poly-L-lysine Download PDFInfo
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Abstract
The invention relates to a method for producing epsilon-poly-L-lysine. The method is characterized in that a medium containing 1-10g/L of glycine is added at an early or middle stage of culture. By adding glycine in a fermentation process in the invention, glycine which enters a folic acid metabolism approach allows sufficient one-carbon groups to be supplied to a biosynthesis process, the anabolism activity of bacterial strains to be improved, the synthesis of a precursor L-lysine and epsilon-poly-L-lysine to be increased, and the accumulation amount of epsilon-poly-L-lysine to be 20-50% higher than the accumulation amount obtained through original technologies.
Description
Technical field
The invention belongs to field of fermentation engineering, especially a kind of working method of epsilon-poly-L-lysine.
Background technology
(ε-PL) is the outer linear polymers of a kind of microbial source born of the same parents to epsilon-poly-L-lysine, only is formed by connecting through α-carboxyl and epsilon-amino 25-35 L-lysine residue.1977, Shima and Sakai found this seed amino acid polymer first in the fermentation clear liquid of Streptomyces albulus NBRC 14147 bacterial strains.Epsilon-poly-L-lysine is a kind of polycation polypeptide to the human body safety non-toxic, can suppress gram negative bacterium, gram-positive microorganism, fungi even some virus, and antimicrobial spectrum is extensive.After through the ε-PL security of mouse experiment proof, Japan, Korea S, the U.S. etc. national with it as antiseptics for natural food and widespread use.ε-PL and verivate range of application thereof are wider, except that can be used for food antiseptic, can also be as antiobesity agent, medicine and genophore, retentiveness material, and the Coating Materials in biochip and the biological electronics etc.
According to retrieval; Find following patent documentation about epsilon-poly-L-lysine; Wherein CN1260004 discloses a kind of bacterial strain and working method of mass production epsilon-poly-L-lysine, and this method is through good air culture bacteria strain B21021 (FERM BP-5926) and separation and purification gained epsilon-poly-L-lysine.Bacterial strain B21021 obtains through little streptomyces albus lysinopolymerus subspecies 11011A-1 bacterial strain (FERM BP-1109) is carried out mutagenic treatment, and it is that 10mg/mL or higher S-(2-amino-ethyl)-L-halfcystine (AEC) have resistance to concentration.CN101285046 discloses a kind of mutagenic strain TUST2 and has utilized this mutagenic strain to produce the method for epsilon-polylysine and salt thereof; Be that the TUST1 that obtains with separation in Chinese Hainan Province soil is a starting strain; The bacterial strain TUST2 (CGMCC No.1986) that utilizes physics and chemomorphosis method to select; This bacterial strain has resistance to 10mg/mL or greater concn AEC and 2mg/mL glycocoll, produces sour amount under the optimal conditions and can reach 10～30g/L.Fermented liquid obtains the epsilon-polylysine product that MWD is 4000～6500Da after processing such as centrifugal, filtration and weakly acidic cation-exchange resin then.CN101525640 discloses a kind of preparation method of epsilon-polylysine, and this method is to utilize streptomyces virginiae Y12, through one-level, secondary seed enlarged culturing, and fermentation production of epsilon-polylysine, extracting the back yield can reach more than the 8g/L.Bacterial strain Y12 is that starting strain passes through mutafacient system acquisitions such as ethyl sulfate, ultraviolet.CN102086441A discloses the brown streptomycete bacterial strain of a kind of ash and has utilized this bacterial strain to prepare the method for epsilon-polylysine and salt thereof; This method is utilized beige streptomycete LS-H1 fermentation production of epsilon-polylysine; Accumulation volume is 0.7～20g/L under optimal conditions, and fermented liquid obtains epsilon-polylysine and salt thereof through centrifugal, IX then.CN101671703 discloses a kind of novel method that improves epsilon-poly-L-lysine output; Be to serve as to produce bacterial strain with Streptomyces diastatochromogenes or Streptomyces albulus; In the process of fermentative prodn epsilon-poly-L-lysine; Product begins after the accumulation, and stream adds L-Methionin improving output in substratum, obtains the epsilon-poly-L-lysine hydrochloride after then that fermented liquid is centrifugal, IX absorption, decolouring, the vacuum-drying.
Above-mentioned patent document mainly concentrates on the production bacterial classification and zymotechnique of epsilon-poly-L-lysine, but does not all study the influence of glycocoll to its leavening property, has essential distinction with this patent.
Summary of the invention
The working method that the purpose of this invention is to provide a kind of epsilon-poly-L-lysine; The present invention produces the method for epsilon-poly-L-lysine through increasing the external source medium component; The glycocoll that in substratum, contains 1～10g/L makes the epsilon-poly-L-lysine accumulation volume improve 20～50% than original technology.
The present invention realizes that the technical scheme of purpose is:
A kind of working method of epsilon-poly-L-lysine contains glycocoll in the substratum.
And the concentration of said glycocoll is 1～10g/L.
And said glycocoll is being cultivated the adding of early stage or mid-term.
Advantage of the present invention and positively effect are:
1, the present invention is through add glycocoll during the fermentation; Glycocoll can get into the folic acid metabolism approach; For biosynthetic process provides a competent carbon-based group; Improve bacterial strain anabolism vigor, increase the synthetic of precursor L-Methionin and epsilon-poly-L-lysine, make the epsilon-poly-L-lysine accumulation volume improve 20～50% than original technology.
2, the present invention has changed original zymotechnique, has significantly increased production bacterial strain metabolic activity, has improved the productive rate of epsilon-poly-L-lysine, has increased its accumulation volume to a certain extent, has reduced cost, can be used for plant-scale fermentative prodn.
Embodiment
Through specific embodiment the present invention is made further detailed description below, following examples are descriptive, are not determinate, can not limit protection scope of the present invention with this.
At first need to prove:
1. by the preservation of Chinese common micro-organisms culture presevation administrative center, deposit number is CGMCC No.1986 to the used streptomyces albus (Streptomyces albulus) of following examples, and preservation date is on March 23rd, 2007.
2. by the preservation of Chinese common micro-organisms culture presevation administrative center, deposit number is CGMCC No.3145 to the used streptomyces diastatochromogenes (Streptomyces diastatochromogenes) of following examples, and preservation date is on June 29th, 2009.
The present invention is that example is explained the present invention with above-mentioned two bacterial classifications; But the scope of used bacterial classification is not limited only to above-mentioned two kinds; Bacterial classification as mentioning in the background technology of the present invention all can be applied among the present invention; Because principle is identical, the concrete operations step is easy to the technician and knows that rationally the present invention gives an example no longer one by one.
During the fermentation stream add L-Methionin be adopt " a kind of novel method that improves epsilon-poly-L-lysine output " (number of patent application is: 200910069517.5, publication number is: the stream process CN101671703).
4. example 1 and 4 narrations is existing manufacturing technique, embodiment 2 and embodiment 3 innovative approach for comparing with embodiment 1, and embodiment 5 and embodiment 6 are the innovative approach that compares with embodiment 4.
Embodiment 1:
A kind of working method of epsilon-poly-L-lysine, step is following:
In the 100mL substratum, insert the spore of 1～2 ring streptomyces diastatochromogenes or streptomyces albus, 30 ℃ of shaking culture 30h; Inoculum size with 6% (V/V) inserts in the 100mL fermention medium (initial pH 6.8) then, cultivates 72h for 30 ℃.In the fermenting process, the epsilon-poly-L-lysine production peak is 0.4g/L.
Embodiment 2:
A kind of working method of epsilon-poly-L-lysine, step is following:
In the 100mL substratum, insert the spore of 1～2 ring streptomyces diastatochromogenes or streptomyces albus, contain glycocoll 3g/L in the substratum, 30 ℃ of shaking culture 30h; Inoculum size with 6% (V/V) inserts in the 100mL fermention medium (initial pH 6.8) then, cultivates 72h for 30 ℃.In the fermenting process, the epsilon-poly-L-lysine production peak is 0.5g/L.
Embodiment 3:
A kind of working method of epsilon-poly-L-lysine, step is following:
In the 100mL substratum, insert the spore of 1～2 ring streptomyces diastatochromogenes or streptomyces albus, 30 ℃ of shaking culture 48h; Inoculum size access with 10% (V/V) contains in the 100mL fermention medium of glycocoll 2g/L then, and initial pH cultivates 72h for 4.5,30 ℃.In the fermenting process, the epsilon-poly-L-lysine production peak is 0.7g/L.
Embodiment 4:
A kind of working method of epsilon-poly-L-lysine, step is following:
(1) in the 5L fermentor tank of 2.7L substratum is housed, the seed culture fluid of inoculation 300mL streptomyces diastatochromogenes or streptomyces albus, 30 ℃ of cultivations treat that DO reduces to be controlled to be 30% automatically at 30% o'clock that 120h is supported in good air culture, air velocity is 4.0～6.0L/min;
(2) when pH reduces to 6.0 left and right sides, stream adds ammoniacal liquor, and (it is 6.0 that the concentration 10～30%g/mL) of ammoniacal liquor is kept pH, when the remaining sugar concentration in the fermented liquid is reduced to 10g/L, adds glucose (400g/L) and (NH through stream
4)
2SO
4(80g/L) make remaining sugar concentration reach 12g/L; Simultaneously, no longer control pH, let it reduce to 4.0 and maintain about 4.0 naturally;
(3) cultivate 120h, finish fermentation, the highest accumulation epsilon-poly-L-lysine is 11.9g/L in the fermented liquid;
(4) centrifugal removal thalline and part solid substance; Utilize D152 macropore weakly acidic cation-exchange resin that supernatant is exchanged absorption and wash-out; Elutriant is after the decolouring of D392 macroporous weakly basic anion exchange resin, again through filtering vacuum concentration; Cyclone dryer is dry, obtains the epsilon-poly-L-lysine hydrochloride.
Embodiment 5:
A kind of working method of epsilon-poly-L-lysine, step is following:
(1) in the 5L fermentor tank of 2.7L substratum is housed; Inoculation contains the seed culture fluid of glycocoll (3g/L) 300mL streptomyces diastatochromogenes or streptomyces albus, and 30 ℃ of cultivations are treated that DO reduces to be controlled to be 30% automatically at 30% o'clock; Aerobic cultivation 120h, air velocity is 4.0～6.0L/min;
(2) when pH reduces to 6.0 left and right sides, stream adds ammoniacal liquor, and (it is 6.0 that the concentration 10～30%g/mL) of ammoniacal liquor is kept pH, when the remaining sugar concentration in the fermented liquid is reduced to 10g/L, adds glucose (400g/L) and (NH through stream
4)
2SO
4(80g/L) make remaining sugar concentration reach 12g/L; Simultaneously, no longer control pH, let it reduce to 4.0 and maintain about 4.0 naturally;
(3) cultivate 120h, finish fermentation, the highest accumulation epsilon-poly-L-lysine 12.9g/L in the fermented liquid;
(4) centrifugal removal thalline and part solid substance; Utilize D152 macropore weakly acidic cation-exchange resin that supernatant is exchanged absorption and wash-out; Elutriant is after the decolouring of D392 macroporous weakly basic anion exchange resin, again through filtering vacuum concentration; Cyclone dryer is dry, obtains the epsilon-poly-L-lysine hydrochloride.
Embodiment 6:
A kind of working method of epsilon-poly-L-lysine, step is following:
(1) in the 5L fermentor tank of 2.7L substratum is housed; Inoculation contains the seed culture fluid of glycocoll (3g/L) 300mL streptomyces diastatochromogenes or streptomyces albus, and 30 ℃ of cultivations are treated that DO reduces to be controlled to be 30% automatically at 30% o'clock; Aerobic cultivation 120h, air velocity is 4.0～6.0L/min;
(2) when pH reduces to 6.0 left and right sides, stream adds ammoniacal liquor, and (it is 6.0 that the concentration 10～30%g/mL) of ammoniacal liquor is kept pH, when the remaining sugar concentration in the fermented liquid is reduced to 10g/L, adds glucose (400g/L) and (NH through stream
4)
2SO
4(80g/L), make remaining sugar concentration reach 12g/L, and stream add L-Methionin (10g/L) in fermented liquid; Simultaneously, no longer control pH, let it reduce to 4.0 and maintain about 4.0 naturally;
(3) cultivate 120h, finish fermentation, the highest accumulation epsilon-poly-L-lysine 15.2g/L in the fermented liquid;
(4) centrifugal removal thalline and part solid substance; Utilize D152 macropore weakly acidic cation-exchange resin that supernatant is exchanged absorption and wash-out; Elutriant is after the decolouring of D392 macroporous weakly basic anion exchange resin, again through filtering vacuum concentration; Cyclone dryer is dry, obtains the epsilon-poly-L-lysine hydrochloride.
Claims (3)
1. the working method of an epsilon-poly-L-lysine is characterized in that: contain glycocoll in the substratum.
2. the working method of epsilon-poly-L-lysine according to claim 1, it is characterized in that: the concentration of said glycocoll is 1～10g/L.
3. the working method of epsilon-poly-L-lysine according to claim 1 is characterized in that: said glycocoll adds in early stage or mid-term cultivating.
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Cited By (6)
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| CN104004796A (en) * | 2014-04-18 | 2014-08-27 | 天津科技大学 | Epsilon-polylysine fermentation method by homoserine accumulation |
| CN104193988A (en) * | 2014-09-01 | 2014-12-10 | 江南大学 | Method for flocculating and sterilizing fermentation solution of epsilon-polylysine |
| CN105368887A (en) * | 2015-11-05 | 2016-03-02 | 天津科技大学 | Fermentation production process of Epsilon-poly-L-lysine |
| CN108498401A (en) * | 2018-05-25 | 2018-09-07 | 钱兴 | A kind of preparation method of mouthwash |
| CN109112169A (en) * | 2017-06-26 | 2019-01-01 | 武汉臻智生物科技有限公司 | Polylysine glyceride and preparation method thereof, purposes and the method for preparing polylysine |
| CN111471633A (en) * | 2020-03-13 | 2020-07-31 | 天津科技大学 | Gene engineering high-yield strain streptomyces diastatochromogenes and method for improving yield of polylysine |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004796A (en) * | 2014-04-18 | 2014-08-27 | 天津科技大学 | Epsilon-polylysine fermentation method by homoserine accumulation |
| CN104004796B (en) * | 2014-04-18 | 2017-06-13 | 天津科技大学 | A kind of fermentation process of the ε polylysines for accumulating homoserine |
| CN104193988A (en) * | 2014-09-01 | 2014-12-10 | 江南大学 | Method for flocculating and sterilizing fermentation solution of epsilon-polylysine |
| CN105368887A (en) * | 2015-11-05 | 2016-03-02 | 天津科技大学 | Fermentation production process of Epsilon-poly-L-lysine |
| CN105368887B (en) * | 2015-11-05 | 2019-01-22 | 天津科技大学 | A kind of fermentation manufacturing technique of epsilon-poly-L-lysine |
| CN109112169A (en) * | 2017-06-26 | 2019-01-01 | 武汉臻智生物科技有限公司 | Polylysine glyceride and preparation method thereof, purposes and the method for preparing polylysine |
| CN109112169B (en) * | 2017-06-26 | 2021-12-14 | 安泰生物工程股份有限公司 | Polylysine glyceride, preparation method and application thereof, and method for preparing polylysine |
| CN108498401A (en) * | 2018-05-25 | 2018-09-07 | 钱兴 | A kind of preparation method of mouthwash |
| CN111471633A (en) * | 2020-03-13 | 2020-07-31 | 天津科技大学 | Gene engineering high-yield strain streptomyces diastatochromogenes and method for improving yield of polylysine |
| CN111471633B (en) * | 2020-03-13 | 2022-12-06 | 天津科技大学 | Gene engineering high-yield strain streptomyces diastatochromogenes and method for improving yield of epsilon-polylysine |
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Effective date of registration: 20160330 Address after: 317200 No. 18 Feng Dong Road, Fuk Creek Street, Tiantai County, Zhejiang Patentee after: ZHEJIANG SILVER-ELEPHANT BIO-ENGINEERING CO., LTD. Address before: 300457 No. thirteen, 29 Avenue, Tanggu economic and Technological Development Zone, Tianjin Patentee before: Tianjin University of Science & Technology |
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Application publication date: 20120229 Assignee: ZHEJIANG SHENGDA BIO-PHARM Co.,Ltd. Assignor: ZHEJIANG SILVER-ELEPHANT BIO-ENGINEERING Co.,Ltd. Contract record no.: X2021330000154 Denomination of invention: one kind e- Production method of poly-L-lysine Granted publication date: 20130501 License type: Common License Record date: 20210828 |
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