CN102517336A - Method for improving yield of aureobasidium pullulans melanin by using oxidative stress - Google Patents
Method for improving yield of aureobasidium pullulans melanin by using oxidative stress Download PDFInfo
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- CN102517336A CN102517336A CN2011104265277A CN201110426527A CN102517336A CN 102517336 A CN102517336 A CN 102517336A CN 2011104265277 A CN2011104265277 A CN 2011104265277A CN 201110426527 A CN201110426527 A CN 201110426527A CN 102517336 A CN102517336 A CN 102517336A
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- melanochrome
- aureobasidium pullulans
- fermentation
- oxidative stress
- fermention medium
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Abstract
The invention discloses a method for improving yield of aureobasidium pullulans melanin by using oxidative stress. The method is characterized by comprising the following steps of: activating aureobasidium pullulans for 4 to 8 hours, performing shaking culture for 12 to 36 hours to obtain seed liquid, inoculating the seed liquid in a volume of which is 1 to 10 percent of that of a fermentation medium to the fermentation medium, and performing liquid state fermentation; centrifuging the fermentation solution obtained by fermentation for 5 minutes under the condition of 5,000r/min, performing acidolysis on the supernate by using hydrochloric acid, and then performing precipitation by using methanol to obtain a crude melanin product; dissolving the crude product to obtain a black solution by using an alkali solution; and performing precipitation on the obtained black solution by using hydrochloric acid, and thus obtaining the melanin. Compared with other fermentation medium, the method using the fermentation medium has the advantages that: the yield of the melanin is improved from 2.47g/L to 12.86g/L, the fermentation period is shortened from 7 days to 3 days, the culture substrate is fully utilized, the utilization rate of raw materials is improved, and the production cost is reduced.
Description
Technical field
The invention belongs to by mikrobe liquid state fermentation and produce the melanochrome technical field, specifically utilize environment-stress to improve the method for Aureobasidium pullulans melanochrome output.
Background technology
Melanochrome is a kind of one type of natural pigment that extensively is present in the organism, and it is a heterogeneous type of polyphenol polymer through polyhydroxy phenol (very easily conjugated protein) oxidation formation irregular structure.Melanochrome can be used as the composition, positively charged ion chela and the agent that absorb ultraviolet cosmetic composition, natural hair dye, as the bright protective agent of unbodied semi-conductor and biotic pesticide in industrial or agricultural; Melanochrome medically also has very wide range of application in addition; Have remove radical, the oxidation that stops the Val liposome, radioprotective and anti-oxidant, significant with taxonomic research to pathology, as novel natural drug carrier, be used for treating some nervous system disease relevant, some solubility melanochrome in external effect with inhibition HIV host cells infected with short of melanin, so might become a kind of effective medicine. mostly present melanic production is to extract through chemosynthesis or driven, plant materials.Chemosynthesis melanin reactions process is many, and has certain toxic; Driven, plant materials extracts the problem that there is sample source in melanochrome.Utilize microorganisms producing melanochrome have reaction conditions gentleness, cost low, simple to operate, need not a large amount of advantages such as sample.And the domestic fungi fermentation that utilizes is produced melanic research also seldom, and the present invention utilizes environment-stress to improve the melanic output of Aureobasidium pullulans.
Summary of the invention
The purpose of this invention is to provide a strain and produce melanic aureobasidium pullulans (Aureobasidium Pullulans), and adopt improved culture medium its deep layer liquid state fermentation to be provided the method for melanochrome output.
A kind of method of utilizing oxidative stress to improve Aureobasidium pullulans melanochrome output; It is characterized in that: be shake-flask culture after Aureobasidium pullulans (Aureobasidium Pullulans) the bacterial strain activation of CGMCC3337 earlier deposit number; Be inoculated in fermention medium according to 1～10% inoculum size, carry out liquid state fermentation.Fermented liquid is centrifugal 5min under the condition of 5000r/min, and the gained supernatant is earlier with the 6mol/L hydrochloric acidolysis, and the back is precipitated with 50% methyl alcohol, gets the melanochrome bullion.Above-mentioned bullion obtains dark solution with the dissolving of 1mol/L basic soln.With 3mol/L hydrochloric acid the gained dark solution is precipitated at last, promptly obtain melanochrome.
The present invention adopt implementation step following:
(1) actication of culture: will be deposited in the bacterial classification on the PDA medium slant, and, obtain the activation bacterial strain 22～28 ℃ of following constant temperature culture 4～8 hours;
(2) preparation seed culture medium, its aqueous solution consists of (g/L): glucose 20～40, murphy juice 100～700, Carnis Bovis seu Bubali cream 1～5, zero(ppm) water, pH 4～7,121 ℃ of sterilization 20min; Shake-flask culture the seed culture medium above picking one ring aureobasidium pullulans inserts from the inclined-plane is cultivated 12h～36h, and culture temperature: 27 ± 2 ℃, rotating speed is 150～230rpm.
(3) fermention medium: existing fermention medium, its aqueous solution consists of (g/L): glucose 40, murphy juice: 400, Carnis Bovis seu Bubali cream: 1, FeSO
40.02, zero(ppm) water, 121 ℃ of sterilization 20min; Or the fermention medium of the present invention's proposition, its aqueous solution consists of (g/L): glucose 40, murphy juice 400, Carnis Bovis seu Bubali cream 1, FeSO
40.02, H
2O
25mmoL/L～25mmoL/L, zero(ppm) water, 4,121 ℃ of sterilizations of pH 20min.
(4) seed liquor is inoculated in 10L and controls automatically in the fermentor tank, fermention medium is respectively the fermention medium of existing fermention medium and the present invention's proposition, and liquid amount is 7L; Inoculum size is 1%～10% (V/V), and the fermentation stirring velocity is 200～500r/min, and leavening temperature is 25 ℃; Air flow is 0.5～1 (V/V); Tank pressure is 0.01～0.02Mpa, and pH 4.0～7.0, ferments respectively 3～5 days.Fermented liquid centrifugal 5min under the condition of 5000r/min that fermentation obtains, gained supernatant are earlier with the 6mol/L hydrochloric acidolysis, and the back is precipitated with 50% methyl alcohol, get the melanochrome bullion.Above-mentioned bullion obtains dark solution with the dissolving of 1mol/L basic soln.With 3mol/L hydrochloric acid the gained dark solution is precipitated at last, promptly obtain melanochrome.
Said basic soln is selected from sodium hydroxide, Pottasium Hydroxide or ammonia soln.
Added H in the said substratum
2O
2, H
2O
2Can decompose generation oxygen, oxygen is the high activity material, when oxidation is incomplete, then can generate active oxygen.Active oxygen is the principal mode of radical in the body, can in the homergy of cell, produce, and external environment such as radiation, redox active medicine, metals ion also can stimulate the generation of ROS.Under the normal circumstances, the generation of ROS and anti-ROS level are equilibrated, when ROS produces greater than anti-ROS level, promptly can attack body, cause the damage of lipid, protein, DNA, or even the death of cell, Here it is so-called oxidative stress.Organism is in order to deal with this response to oxidative stress some protection mechanisms of evolving out, the variation on the producer adjusting level, thus induce synthesis of melanin, protect and self do not receive the injury of ROS.Be in the H of sublethal dose when Aureobasidium pullulans
2O
2When middle, thereby it will adapt to the stimulation generation melanochrome of this stressed condition to lethal dose.Therefore in the fermention medium of Aureobasidium pullulans, add the H of sublethal dose
2O
2The time, will improve the mould melanic output of short stalk widely.
Bacterial classification of the present invention uses bacterial classification in University Of Science and Technology Of Tianjin's application, and application number is CN200910071018, and denomination of invention is a kind of mutagenic strain Aureobasidium pullulans TKPM00006 and cultural method thereof of a large amount of generation Beta-polymalic acids, and the open date is 2011.02.23.Disclose Aureobasidium pullulans (Aureobasidium pullulans) TKPM00006 in this patent, culture presevation number is CGMCC3337, preservation on the 10.14th in 2009.
Beneficial effect
The fermentative medium formula that the present invention proposes is compared with existing fermention medium, and melanochrome output is brought up to 12.86g/L by 2.47g/L; Fermentation period had 7 days to shorten to 3 days; The substratum substrate is fully used, has improved utilization ratio of raw materials, reduced production cost.
Embodiment
Embodiment 1:
(1) actication of culture: will be deposited in bacterium on the PDA medium slant, and, obtain the activation bacterial strain 22 constant temperature culture 4 hours;
(2) getting a ring Aureobasidium pullulans bacterial classification access is equipped with in 500 milliliters of baffle plate bottles of 50 milliliters of substratum.The aqueous solution of seed culture medium consists of (g/L): glucose 20, murphy juice 100, Carnis Bovis seu Bubali cream 1, zero(ppm) water, 4,121 ℃ of sterilizations of pH 20min; Shake bottle in 25 ℃, rotating speed is to cultivate 24h on the shaking table of 150r/min, as seed liquor.
(3) fermention medium: existing fermention medium, its aqueous solution consists of (g/L): glucose 40, murphy juice 400, Carnis Bovis seu Bubali cream 1, FeSO
40.02,4,121 ℃ of sterilizations of pH 20min.121 ℃ of high pressure steam sterilization 20min; Or the fermention medium of the present invention's proposition, its aqueous solution consists of (g/L): glucose 40, murphy juice 400, Carnis Bovis seu Bubali cream 1, FeSO
40.02, H
2O
25mmoL/L, zero(ppm) water, 4,121 ℃ of sterilizations of pH 20min.
(4) seed liquor is inoculated in 10L and controls automatically in the fermentor tank, fermention medium is respectively the fermention medium of existing fermention medium and the present invention's proposition, and liquid amount is 7L; Inoculum size is 1% (V/V), and the fermentation stirring velocity is 200r/min, and leavening temperature is 25 ℃; Air flow is 0.5 (V/V); Tank pressure is 0.01Mpa, and pH 4.0, ferments respectively 3 days.Fermented liquid centrifugal 5min under the condition of 5000r/min that fermentation obtains, gained supernatant are earlier with the 6mol/L hydrochloric acidolysis, and the back is precipitated with 50% methyl alcohol, get the melanochrome bullion.Above-mentioned bullion obtains dark solution with the dissolving of 1mol/L basic soln.With 3mol/L hydrochloric acid the gained dark solution is precipitated at last, promptly obtain melanochrome.
(5) fermented liquid obtains the melanochrome of 7.49g/L and 11.24g/L respectively after above-mentioned processing, adopts fermention medium of the present invention, and melanic output has improved 50.1%.
Embodiment 2:
(1) actication of culture: will be deposited in the bacterial classification on the PDA medium slant, and, obtain the activation bacterial strain 28 ℃ of following constant temperature culture 6 hours.
(2) getting a ring Aureobasidium pullulans bacterial classification access is equipped with in 500 milliliters of baffle plate bottles of 50 milliliters of substratum.The aqueous solution of seed culture medium consists of (g/L): glucose 40, murphy juice 700, Carnis Bovis seu Bubali cream 5, zero(ppm) water, 4,121 ℃ of sterilizations of pH 20min; Shake bottle in 29 ℃, rotating speed is to cultivate 36h on the shaking table of 230r/min, as seed liquor.
(3) fermention medium: existing fermention medium, its aqueous solution consists of (g/L): glucose 40, murphy juice 400, Carnis Bovis seu Bubali cream 1, FeSO
40.02, zero(ppm) water, 4,121 ℃ of sterilizations of pH 20min; Or the fermention medium of the present invention's proposition, its aqueous solution consists of (g/L): glucose 40, murphy juice 400, Carnis Bovis seu Bubali cream 1, FeSO
40.02, H
2O
225mmoL/L, zero(ppm) water, 4,121 ℃ of sterilizations of pH 20min.
(4) seed liquor is inoculated in 10L and controls automatically in the fermentor tank, fermention medium is respectively the fermention medium of existing fermention medium and the present invention's proposition, and liquid amount is 7L; Inoculum size is 10% (V/V), and the fermentation stirring velocity is 500r/min, and leavening temperature is 29 ℃; Air flow is 1 (V/V); Tank pressure is 0.01Mpa, and pH 4.0, ferments respectively 4 days.Fermented liquid centrifugal 5min under the condition of 5000r/min that fermentation obtains, gained supernatant are earlier with the 6mol/L hydrochloric acidolysis, and the back is precipitated with 50% methyl alcohol, get the melanochrome bullion.Above-mentioned bullion obtains dark solution with the dissolving of 1mol/L basic soln.With 3mol/L hydrochloric acid the gained dark solution is precipitated at last, promptly obtain melanochrome.
(5) fermented liquid obtains the melanochrome of 6.95g/L and 11.74g/L respectively after above-mentioned processing, adopts fermention medium of the present invention, and melanic output has improved 68.9%.
Embodiment 3:
(1) actication of culture: will be deposited in the bacterial classification on the PDA medium slant, and, obtain the activation bacterial strain 25 ℃ of following constant temperature culture 6 hours;
(2) seed culture: get a ring Aureobasidium pullulans bacterial classification access and be equipped with in 500 milliliters of baffle plate bottles of 50 milliliters of substratum.The aqueous solution of seed culture medium consists of (g/L): glucose 20, murphy juice 100, Carnis Bovis seu Bubali cream 1, zero(ppm) water, 4,121 ℃ of sterilizations of pH 20min; Shaking bottle is 25 ℃ in temperature, and rotating speed is to cultivate 24h on the shaking table of 200r/min, as seed liquor;
(3) fermention medium: existing fermention medium, its aqueous solution consists of (g/L): glucose 40, murphy juice 400, Carnis Bovis seu Bubali cream 1, FeSO
40.02, zero(ppm) water, 4,121 ℃ of sterilizations of pH 20min; Or the fermention medium of the present invention's proposition, its aqueous solution consists of (g/L): glucose 40, and murphy juice 400, Carnis Bovis seu Bubali cream: 1, FeSO
40.02, H
2O
215mmoL/L, zero(ppm) water, 4,121 ℃ of sterilizations of pH 20min.
(4) seed liquor is inoculated in 10L and controls automatically in the fermentor tank, fermention medium is respectively the fermention medium of existing fermention medium and the present invention's proposition, and liquid amount is 7L; Inoculum size is 6% (V/V), and the fermentation stirring velocity is 300r/min, and leavening temperature is 25 ℃; Air flow is 0.8 (V/V); Tank pressure is 0.01Mpa, and pH 4, ferments respectively 7 days and 5 days.Fermented liquid centrifugal 5min under the condition of 5000r/min that fermentation obtains, gained supernatant are earlier with the 6mol/L hydrochloric acidolysis, and the back is precipitated with 50% methyl alcohol, get the melanochrome bullion.Above-mentioned bullion obtains dark solution with the dissolving of 1mol/L basic soln.With 3mol/L hydrochloric acid the gained dark solution is precipitated at last, promptly obtain melanochrome.
(5) fermented liquid obtains the melanochrome of 8.46g/L and 12.86g/L respectively after above-mentioned processing, adopts fermention medium of the present invention, and melanic output has improved 52%.
Claims (5)
1. method of utilizing oxidative stress to improve aureobasidium pullulans melanochrome output; It is characterized in that: earlier with shake-flask culture after Aureobasidium pullulans (Aureobasidium Pullulans) the bacterial strain activation; Shake-flask seed liquid is inoculated in fermention medium according to 1～10% of fermention medium volume, ferments, fermented liquid is centrifugal 5min under the condition of 5000r/min; Use methanol extraction behind the gained supernatant hydrochloric acidolysis; Get the melanochrome bullion, above-mentioned bullion obtains with hydrochloric acid the gained dark solution being precipitated after the dark solution with the basic soln dissolving, promptly obtains melanochrome.
2. as weighing 1 said a kind of method of utilizing oxidative stress to improve aureobasidium pullulans melanochrome output, step is following:
(1) bacterial strain activation: will be deposited in bacterium on the PDA medium slant, and, obtain the activation bacterial strain 22～28 ℃ of constant temperature culture 4～8 hours;
(2) shake-flask culture: picking one ring aureobasidium pullulans inserts shake-flask culture the seed culture medium from the inclined-plane;
(3) liquid state fermentation: seed liquor is inoculated in 10L controls automatically in the fermentor tank, fermention medium liquid amount 7L, inoculum size is 1%～10%; The fermentation stirring velocity is 200～500r/min; Leavening temperature is 25 ℃, and air flow is 0.5～1 (V/V), and tank pressure is 0.01～0.02Mpa; PH 4.0～7.0, ferment 3～5 days;
(4) melanochrome extracts: fermented liquid is centrifugal 5min under the condition of 5000r/min; Use methanol extraction behind the gained supernatant hydrochloric acidolysis, get the melanochrome bullion, above-mentioned bullion obtains dark solution with the basic soln dissolving; The back is precipitated the gained dark solution with hydrochloric acid, promptly obtains melanochrome.
3. as weighing 1 said a kind of method of utilizing oxidative stress to improve aureobasidium pullulans melanochrome output, be characterised in that said basic soln is sodium hydroxide, Pottasium Hydroxide or ammonia soln.
4. as weighing 1 said a kind of method of utilizing oxidative stress to improve aureobasidium pullulans melanochrome output,
Be characterised in that said fermention medium consists of (g/L): glucose 40, murphy juice 400, Carnis Bovis seu Bubali cream 1, FeSO
40.02, H
2O
25mmoL/L～25mmoL/L, zero(ppm) water, pH 4.
5. as weighing 1 said a kind of method of utilizing oxidative stress to improve aureobasidium pullulans melanochrome output,
Be characterised in that said fermention medium consists of (g/L): glucose 40, murphy juice 400, Carnis Bovis seu Bubali cream 1, FeSO
40.02, H
2O
215mmoL/L, zero(ppm) water, pH 4.6. the application of an Aureobasidium pullulans bacterial strain (Aureobasidium Pullulans) CGMCC3337 in the melanochrome fermentation.
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Cited By (4)
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| CN105586369A (en) * | 2015-12-15 | 2016-05-18 | 天津北洋百川生物技术有限公司 | Method for preparing polymalic acid by adding oxygen carriers |
| CN106906244A (en) * | 2017-04-28 | 2017-06-30 | 福建农林大学 | A kind of incense ashes bacterium melanin fermentation preparation |
| CN108936313A (en) * | 2018-09-12 | 2018-12-07 | 恩施硒谷科技股份有限公司 | A kind of method of high effect culture product containing organic selenium |
| CN114134047A (en) * | 2021-11-17 | 2022-03-04 | 广西大学 | Method for producing melanin by using nitrogen-free culture medium of aureobasidium pullulans |
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Cited By (5)
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Application publication date: 20120627 |