CN101525640A - Preparation method of sigma-polylysine - Google Patents
Preparation method of sigma-polylysine Download PDFInfo
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- CN101525640A CN101525640A CN200910071853A CN200910071853A CN101525640A CN 101525640 A CN101525640 A CN 101525640A CN 200910071853 A CN200910071853 A CN 200910071853A CN 200910071853 A CN200910071853 A CN 200910071853A CN 101525640 A CN101525640 A CN 101525640A
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- polylysine
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Abstract
The invention provides a preparation method of sigma-polylysine, comprising the steps of using Virginia streptomycete as an original strain, equivalently mixing spore suspension with diethyl sulphate with a concentration of 5%, respectively oscillating the mixture for different time at 200r/min, sampling each oscillated liquid and coating the liquid on a flat plate, culturing the liquid for 6 days at 26 DEG C to obtain superior strain DES-25, taking 5ml of spore suspension of strain DES-25 and putting the spore suspension in a 9cm dish, opening the cover of the dish at a position 30cm away from a 15w ultraviolet lamp, agitating and radiating the spore suspension for certain time, taking and transferring strain liquid to a sterile test tube, immediately immersing the test tube in ice water for 2h, then coating the strain liquid on the flat plate under red ray, adding 0.5% lithium chloride in the culture medium as an auxiliary mutagenic agent, and culturing by keeping out of the sun for 3 days at 26 DEG C to obtain the Virginia streptomycete Y12, which has yield of epsilon-polylysine of 8.36g/L and serves as the fermentation strain. The epsilon-polylysine can be produced by fermentation through primary seed expansion culture and secondary seed expansion culture, and the yield after extraction is more than 8g/L.
Description
(1) technical field
The present invention relates to a kind of Applied Physics, chemomorphosis means, mutagenesis separates a plant height and produces the epsilon-polylysine streptomycete, relates in particular to a kind of preparation method of epsilon-polylysine.
(2) background technology
Epsilon-polylysine be the eighties by a kind of novel food product fungistat after Nisin that Japan at first finds, have broad-spectrum antibacterial, for the sharp-pointed candidiasis of yeast belong, method rhodotorula bacterium, produce film pichia yeast, rose shadow yeast; Heat-resisting fatty genus bacillus in the gram-positive microorganism, Bacillus coagulans, subtilis; Aerogenesis Arthrobacter in the Gram-negative bacteria, intestinal bacteria etc. cause that food poisoning and putrid bacterium have had strong inhibitory effects, also have certain phage-resistant ability simultaneously.Its chemical constitution is the polypeptide that is made of essential amino acid L-Methionin, and it becomes single Methionin again and forms reinforcer into human nutrition after digestion, have no side effect safe characteristics so polylysine as food preservatives, has.The thermostability of polylysine is very good, and its optimal pH 5-8 that is to say that it has stronger biocidal property in neutral and slightly acidic environment.Epsilon-polylysine is the polypeptide that is made of essential amino acid L-Methionin, and it becomes single Methionin again and forms reinforcer into human nutrition after digestion, have no side effect safe characteristics so polylysine as food preservatives, has.This natural additive for foodstuff of polylysine that on February 24th, 2004, Japanese Chisso company produced has passed through the U.S. FDA authentication as the GRAS material, and the CAS registration number is 28211-04-3, indicates that epsilon-polylysine approved by international standard.The epsilon-polylysine antimicrobial spectrum is wide, and gram-positive microorganism, Gram-negative bacteria, yeast, mould are all had certain bacteriostatic action, and it also has restraining effect to some thermotolerance genus bacillus and some viruses.
Domestic a lot of scientific research institution also carries out deep research to epsilon-polylysine; majority is to separate streptomyces albus from soil; fermentation production of epsilon-polylysine; but the ability of the streptomyces albus of occurring in nature product epsilon-polylysine is lower; general output is no more than 1g/L; but contain a certain amount of α-polylysine in the product; toxic; must separate and in food, to use; bring great difficulty to industrial production; the products production cost is very high simultaneously, thus domestic so far to epsilon-polylysine not large-scale production as yet, only limit to the scientific research stage.This patent is a starting strain with the streptomyces virginiae of Chinese industrial microbial strains preservation center preservation; through physics, chemomorphosis; screen a strain epsilon-polylysine enhanced variant; production capacity can reach 8.36g/L; do not have α-polylysine in the product, greatly reduce production cost, through 5 tons of fermentor tank pilot scales; the ton substratum is produced epsilon-polylysine and can be reached more than 8 kilograms, fully can large-scale production.
(3) summary of the invention
The object of the invention is to provide a kind of preparation method of epsilon-polylysine.
The object of the present invention is achieved like this: related per-cent is mass ratio except that other has indicating among the present invention, and product of the present invention adopts such method to prepare:
1. starting strain is originated
Starting strain is bought in Chinese industrial microbial strains preservation center, title and being numbered: streptomyces virginiae CICC11013.
2. substratum configuration
Zulkovsky starch (g/L) 50, (NH
4)
2SO
4(g/L) 10, yeast extract (g/L) 7.5, K
2HPO
4(g/L) 0.8, KH
2PO
4(g/L) 1.36, MgSO
47H
2O (g/L) 0.75, ZnSO
47H
2O (g/L) 0.06, FeSO
47H
2O 0.045 (the solid inclined-plane adds 2% agar), all the other are deionized water.
3. mutagenic processes
3.1 monospore suspension preparation
Take out and send out bacterial strain streptomyces virginiae one articulating on slant medium, cultivate 7d for 26 ℃ and treat that the spore maturation is paved with the inclined-plane, wash with stroke-physiological saline solution, the vibration activation disperses spore in the stroke-physiological saline solution of granulated glass sphere is housed, obtain monospore suspension with the filter paper filtering mycelia, adjusting spore concentration is 10
8Individual/mL.
3.2 ethyl sulfate (DES) mutagenesis
With the ethyl sulfate balanced mix of spore suspension and 5% concentration, vibrate 30,60,90,120,150 respectively at 200r/min, 180min, add 25% Sulfothiorine 1ml termination reaction after each oscillation treatment immediately.The sampling dilution is coated with flat board, with same operation, use cell diluent spread plate to compare without DES mutagenesis, place under 26 ℃ the condition and cultivate 6d, choose 30 strains, obtained DES-25 through primary dcreening operation, multiple sieve, its epsilon-polylysine output reaches 5.45g/L, produces the epsilon-polylysine ability than former bacterial strain and has improved 5 times.
3.3 the compound lithium chloride mutagenesis of ultraviolet
The spore suspension 5ml that gets the bacterial strain DES-25 for preparing is in the aseptic plate of Φ 9cm.Ultraviolet lamp is opened preheating 20min to stablize light wave, the plate that fills spore suspension is placed on the magnetic stirring apparatus, apart from the 15w ultraviolet lamp apart from the 30cm place, open the ware lid, stir the irradiation certain hour and get a certain amount of the commentaries on classics in sterile test tube, and immerse immediately in the frozen water behind the 2h, dilution is coated with flat board under ruddiness, and the lithium chloride of interpolation 0.5% is as comutagen in the substratum, and it is parallel that every sample is made three wares, 26 ℃ of lucifuges are cultivated, statistics total number of bacterial colony and picking 30 strains after 3 days are through primary dcreening operation, multiple sieve has obtained DES-25-12, and its epsilon-polylysine output reaches 8.36g/L, with this bacterial strain called after streptomyces virginiae Y12, as fermentation strain.
4. fermentor tank pilot scale
4.1 first order seed preparation
Y12 makes spore suspension with streptomyces virginiae, and adjusting spore concentration is 10
8Individual/mL, be inoculated in some 500ml triangular flask liquid nutrient mediums by 5% inoculum size, triangular flask liquid amount 200ml, 26 ℃ of shaking tables were cultivated 180 rev/mins of shaking speed 72 hours.
4.2 secondary seed preparation
Substratum is identical with first order seed, presses 5% inoculum size seeding tank enlarged culturing 72 hours, prepares fermentation.
4.3 epsilon-polylysine fermentation
Fermention medium is identical with seed culture medium, is inoculated in 5 tons of fermentor tanks by 5% inoculum size, and 26 ℃, ventilate and cultivated 108 hours, finish fermentation.
4.4 the extraction of epsilon-polylysine
Behind the fermentation liquid filtration sterilization body, filtrate is transferred pH to 8.5 with NaOH, refilter and remove out precipitation, filtrate is used cationic exchange resin adsorption, washes and wash-out with 0.2mol/L acetic acid and 0.1mol/L hydrochloric acid respectively, elutriant is neutralized to pH 6.5 with NaOH, carry out reduction vaporization again and concentrate, concentrated solution with decolorizing with activated carbon after, add ethanol and ether (2: 1) mixed solution, the throw out that obtains after the filtration is epsilon-polylysine, does not contain α-polylysine after testing.
(4) embodiment
For a more detailed description below in conjunction with specific embodiment to the present invention:
1. starting strain is originated
Starting strain is bought in Chinese industrial microbial strains preservation center, title and being numbered: streptomyces virginiae CICC11013.
2. substratum configuration
Zulkovsky starch (g/L) 50, (NH
4)
2SO
4(g/L) 10, yeast extract (g/L) 7.5, K
2HPO
4(g/L) 0.8, KH
2PO
4(g/L) 1.36, MgSO
47H
2O (g/L) 0.75, ZnSO
47H
2O (g/L) 0.06, FeSO
47H
2O 0.045 (the solid inclined-plane adds 2% agar), all the other are deionized water.
3. mutagenic processes
3.1 monospore suspension preparation
Take out and send out bacterial strain streptomyces virginiae one articulating on slant medium, cultivate 7d for 26 ℃ and treat that the spore maturation is paved with the inclined-plane, wash with stroke-physiological saline solution, the vibration activation disperses spore in the stroke-physiological saline solution of granulated glass sphere is housed, obtain monospore suspension with the filter paper filtering mycelia, adjusting spore concentration is 10
8Individual/mL.
3.2 ethyl sulfate (DES) mutagenesis
With the ethyl sulfate balanced mix of spore suspension and 5% concentration, vibrate 30,60,90,120,150 respectively at 200r/min, 180min, add 25% Sulfothiorine 1ml termination reaction after each oscillation treatment immediately.The sampling dilution is coated with flat board, with same operation, use cell diluent spread plate to compare without DES mutagenesis, place under 26 ℃ the condition and cultivate 6d, choose 30 strains, obtained DES-25 through primary dcreening operation, multiple sieve, tool epsilon-polylysine output reaches 5.45g/L, produces the epsilon-polylysine ability than former bacterial strain and has improved 5 times.
3.3 the compound lithium chloride mutagenesis of ultraviolet
The spore suspension 5ml that gets the bacterial strain DES-25 for preparing is in the aseptic plate of Φ 9cm.Ultraviolet lamp is opened preheating 20min to stablize light wave, the plate that fills spore suspension is placed on the magnetic stirring apparatus, apart from the 15w ultraviolet lamp apart from the 30cm place, open the ware lid, stir the irradiation certain hour and get a certain amount of the commentaries on classics in sterile test tube, and immerse immediately in the frozen water behind the 2h, dilution is coated with flat board under ruddiness, and the lithium chloride of interpolation 0.5% is as comutagen in the substratum, and it is parallel that every sample is made three wares, 26 ℃ of lucifuges are cultivated, statistics total number of bacterial colony and picking 30 strains after 3 days are through primary dcreening operation, multiple sieve has obtained DES-25-12, and its epsilon-polylysine output reaches 8.36g/L, with this bacterial strain called after streptomyces virginiae Y12, as fermentation strain.
4. batch process epsilon-polylysine
4.1 first order seed preparation
Y12 makes spore suspension with streptomyces virginiae, and adjusting spore concentration is 10
8Individual/mL, be inoculated in 45 500ml triangular flask liquid nutrient mediums by 5% inoculum size, triangular flask liquid amount 200ml, 26 ℃ of shaking tables were cultivated 180 rev/mins of shaking speed 72 hours.
4.2 secondary seed preparation
Substratum is identical with first order seed, is inoculated in 250 liters of seeding tanks in 175 liters of substratum by 5% inoculum size, and enlarged culturing 72 hours is prepared fermentation.
4.3 epsilon-polylysine fermentation
Fermention medium is identical with seed culture medium, in 5 tons of fermentor tanks, is inoculated in 3.5 tons of substratum by 5% inoculum size, and 26 ℃, ventilate and cultivated 108 hours, finish fermentation.
4.4 the extraction of epsilon-polylysine
Behind the fermentation liquid filtration sterilization body, filtrate is transferred pH to 8.5 with NaOH, refilter and remove out precipitation, filtrate is used cationic exchange resin adsorption, wash and wash-out with 0.2mol/L acetic acid and 0.1mol/L hydrochloric acid respectively, elutriant is neutralized to pH 6.5 with NaOH, carrying out reduction vaporization again concentrates, concentrated solution with decolorizing with activated carbon after, add ethanol and ether (2: 1) mixed solution, the throw out that obtains after the filtration was epsilon-polylysine, with 80 ℃ of air seasonings of throw out 10 hours, survey moisture less than 5%, claim that its weight is 28.5 kilograms.
Claims (5)
1. the preparation method of an epsilon-polylysine is characterized in that:
The invention provides a kind of preparation method of epsilon-polylysine.With the streptomyces virginiae is starting strain, and with the ethyl sulfate balanced mix of spore suspension and 5% concentration, at the 200r/min different time that vibrates respectively, each vibration sampling is coated with flat board, places under 26 ℃ the condition to cultivate 6 days, has obtained superior strain DES-25.The spore suspension 5ml that gets bacterial strain DES-25 is in the 9cm plate, apart from 15w ultraviolet lamp 30cm place, open the ware lid, stir the irradiation certain hour, get bacterium liquid and change in sterile test tube, and immerse immediately in the frozen water behind the 2h, be coated with flat board under ruddiness, the lithium chloride of interpolation 0.5% is as comutagen in the substratum, and 26 ℃ of lucifuges are cultivated and obtained streptomyces virginiae Y12 after 3 days, its epsilon-polylysine output reaches 8.36g/L, with it as fermentation strain.Through one-level, secondary seed enlarged culturing, the fermentation production of epsilon-polylysine product extracts back yield＞8g/L.
2. the preparation method of a kind of epsilon-polylysine according to claim 1 is characterized in that:
Described seed culture medium and fermention medium are: Zulkovsky starch (g/L) 50, (NH
4)
2SO
4(g/L) 10, yeast extract (g/L) 7.5, K
2HPO
4(g/L) 0.8, KH
2PO
4(g/L) 1.36, MgSO
47H
2O (g/L) 0.75, ZnSO
47H
2O (g/L) 0.06, FeSO
47H
2O 0.045 (the solid inclined-plane adds 2% agar), all the other are deionized water.
3. the preparation method of a kind of epsilon-polylysine according to claim 1 is characterized in that:
Described spore suspension concentration 10
8Individual/mL.
4. the preparation method of a kind of epsilon-polylysine according to claim 1 is characterized in that:
Described one-level, secondary seed enlarged culturing, culture temperature are 26 ℃, 180 rev/mins of first order seed shaking speed.
5. the preparation method of a kind of epsilon-polylysine according to claim 1 is characterized in that:
Described epsilon-polylysine leavened prod moisture＜5%.
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Family
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827889A (en) * | 2012-09-07 | 2012-12-19 | 吉林中粮生化科技有限公司 | Method for fermentation production of epsilon-polylysine by corn soaking water |
| CN103834594A (en) * | 2014-03-06 | 2014-06-04 | 成都大学 | Epsilon-polylysine bacterium and preparation and fermentation method thereof |
| CN104789550A (en) * | 2015-05-11 | 2015-07-22 | 江南大学 | Method for breeding epsilon-poly-L-lysine tolerant high-producing strain |
| CN104962592A (en) * | 2015-07-29 | 2015-10-07 | 苏州科技学院 | Continuous fed-batch feeding batch fermentation process for producing epsilon-polylysine by adopting cassava starch as raw material |
| CN106434421A (en) * | 2016-08-15 | 2017-02-22 | 山东省药学科学院 | Epsilon-polylysine high-yielding strain and epsilon-polylysine production method |
-
2009
- 2009-04-22 CN CN200910071853A patent/CN101525640A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827889A (en) * | 2012-09-07 | 2012-12-19 | 吉林中粮生化科技有限公司 | Method for fermentation production of epsilon-polylysine by corn soaking water |
| CN103834594A (en) * | 2014-03-06 | 2014-06-04 | 成都大学 | Epsilon-polylysine bacterium and preparation and fermentation method thereof |
| CN104789550A (en) * | 2015-05-11 | 2015-07-22 | 江南大学 | Method for breeding epsilon-poly-L-lysine tolerant high-producing strain |
| CN104962592A (en) * | 2015-07-29 | 2015-10-07 | 苏州科技学院 | Continuous fed-batch feeding batch fermentation process for producing epsilon-polylysine by adopting cassava starch as raw material |
| CN106434421A (en) * | 2016-08-15 | 2017-02-22 | 山东省药学科学院 | Epsilon-polylysine high-yielding strain and epsilon-polylysine production method |
| CN106434421B (en) * | 2016-08-15 | 2019-06-11 | 山东省药学科学院 | One plant of epsilon-polylysine superior strain and production epsilon-polylysine method |
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Open date: 20090909 |