CN111849793A - Yeast JHpharm5-1 for high-yield ergosterol and mutagenesis method thereof - Google Patents

Yeast JHpharm5-1 for high-yield ergosterol and mutagenesis method thereof Download PDF

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CN111849793A
CN111849793A CN202010795931.0A CN202010795931A CN111849793A CN 111849793 A CN111849793 A CN 111849793A CN 202010795931 A CN202010795931 A CN 202010795931A CN 111849793 A CN111849793 A CN 111849793A
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CN111849793B (en
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吴建中
丁叶
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Changzhou Yuepeng Technology Co ltd
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Nanjing Jinhao Medical Technology Co ltd
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Abstract

The invention discloses a yeast JHpharm5-1 for high yield of ergosterol, which is named as Saccharomyces cerevisiae with the preservation number of CGMCC No.20137 and is preserved in China general microbiological culture Collection Center (CCM) 24.06.2020. The yeast JHpharm5-1 with high yield of ergosterol can be applied to ergosterol production, the yield of the yeast JHpharm5-1 is high to 7.2g/100g of dry bacteria, the yield is improved by 53% compared with the highest yield of the existing strains, and the production cost can be reduced while the yield is improved.

Description

Yeast JHpharm5-1 for high-yield ergosterol and mutagenesis method thereof
Technical Field
The invention relates to a yeast JHpharm5-1 for high yield of ergosterol and a mutagenesis method thereof.
Background
Ergosterol, also known as ergosterol, is a colorless needle-like or plate-like crystal, which is a precursor for the production of vitamin D2, is present in the cell membranes of fungi and some protists such as trypanosomes and plays an important role in ensuring the integrity of membrane structure, the activity of membrane-bound enzymes, membrane fluidity, cell viability and substance transport. Ergosterol can be used for the function research of antifungal drugs such as amphotericin B and analogues thereof and the research of ergosterol biosynthesis pathways in various fungi.
Ergosterol is extracted from ergot at the earliest, the main method for producing ergosterol at home and abroad at present is a microbial fermentation method, and the strains used for fermentation mainly comprise saccharomycetes, aspergillus, penicillium and the like. The yeast has high growth rate and relatively high ergosterol yield, so that the ergosterol is the most commonly used strain in the current industrial production, and the ergosterol produced by yeast fermentation at home and abroad at the level of 0.1-4.7 g/100g of dry bacteria still has very large available space.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defect that the yield of ergosterol produced by a microbial fermentation method in the prior art is only 0.1-4.7 g/100g, and provides a yeast JHpharm5-1 for high yield of ergosterol and a mutagenesis method thereof.
In order to solve the technical problems, the invention provides the following technical scheme:
the yeast JHpharm5-1 for high yield of ergosterol is Saccharomyces cerevisiae with the preservation number of CGMCC No.20137, is preserved in China general microbiological culture Collection center (CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC.
The strain adopts an aerobic culture mode, carbon sources for culturing the strain can be glucose, fructose, sucrose, lactose, soluble starch, corn steep liquor and the like, and nitrogen sources for the strain can be yeast extract, peptone, soybean cake powder and the like. The optimal temperature range for the growth of the strain is 28-32 ℃, the pH range is 5-8, and the optimal pH range is 5.5-6.0. Inorganic salts such as magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and sodium nitrate can be added in the process of culturing the strain.
The strain has the advantages of large and thick colony, smooth surface of the colony, wetness, viscosity, uniform texture of the colony, uniform color and milk white color.
The mutation breeding method of the yeast JHpharm5-1 with high ergosterol yield comprises the following steps:
s1, culturing the yeast starting strain on an LB (lysogeny broth) culture medium for 2-4 days, then measuring biomass and ergosterol yield, and screening out a dominant strain;
s2, uniformly dispersing the fermentation liquor of the dominant strain, and diluting the fermentation liquor to 10 cell numbers according to a 10-fold dilution method7Taking 0.1ml of bacterial liquid, uniformly coating the bacterial liquid in a sterile empty plate, air-drying, and carrying out N+Performing ion implantation mutagenesis, washing cells with sterile water, diluting by 10-fold dilution method, coating the diluted cells into a culture medium, culturing at 30 ℃, selecting a single colony, performing shake flask culture for three days, detecting biomass and ergosterol yield, and screening the yeast strain with high ergosterol yield.
Preferably, N+The ion implantation dose is 100 x 2.6 x 1013N+/cm2、145*2.6*1013N+/cm2、190*2.6*1013N+/cm2、235*2.6*1013N+/cm2And 280 x 2.6 x 1013N+/cm2,N+The ion beam implantation energy was 15 KeV.
The culture process comprises the following steps:
(1) seed culture: inoculating a loopful bacteria from the activated plate to the liquid seed culture medium, and culturing for 24h at 30 ℃.
(2) The seed culture medium comprises the following components: carbon source: 20-35 g/L, nitrogen source: 15-45 g/L, the balance of water, pH: 5 to 7,. Wherein the carbon source is at least one selected from glucose, fructose, sucrose, lactose, soluble starch, corn steep liquor and the like, the nitrogen source is at least one selected from yeast extract, peptone, bean cake powder and the like, and the inorganic salt is at least one selected from magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium nitrate and the like.
(3) The most preferred mode of production is: activating the strain, and carrying out aerobic fermentation at 28-32 ℃ for 24-72 h, wherein the liquid loading amount is 100ml of fermentation liquid/500 ml of triangular flask, and the rotating speed is 200 rpm.
The culture medium for aerobic fermentation is as follows: 80/L glucose, 20g/L peptone, 10g/L yeast powder, 3g/L anhydrous magnesium sulfate, 1.5g/L dipotassium phosphate, 1.5g/L potassium dihydrogen phosphate, 2g/L sodium nitrate and the balance of water, wherein the pH value is 6. The yield reaches the highest after fermentation for 48 hours.
The invention has the following beneficial effects:
the yeast JHpharm5-1 with high yield of ergosterol can be applied to ergosterol production, the yield of the yeast JHpharm5-1 is high to 7.2g/100g of dry bacteria, the yield is improved by 53% compared with the highest yield of the existing strains, and the production cost can be reduced while the yield is improved.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.
Example 1 mutagenic screening of Saccharomyces cerevisiae JHpharm5-1
More than 300 yeasts respectively purchased from platforms such as a Beinai organism, an vast Ling organism and the like are cultured for 3 days at 30 ℃ by utilizing an LB culture medium through a high-throughput screening technology, the biomass and the ergosterol yield are measured, and a strain with the most vigorous activity is screened to be used as a dominant strain.
After the fermentation liquor of the strain is uniformly dispersed, diluting the strain to 10 cell numbers according to a 10-fold dilution method7Taking 0.1ml of bacterial liquid, uniformly coating the bacterial liquid in a sterile empty plate, air-drying, and carrying out N+Ion beam implantation with an implant dose of (100, 145, 190, 235, 280) × 2.6 × 1013N+/cm2,N+Injecting energy of an ion beam is 15KeV, washing cells with 1ml of sterile water after radiation is finished, diluting the cells by a 10-fold dilution method, coating the cells into a culture medium, culturing the cells for one day at 30 ℃, selecting a single colony, culturing the single colony for three days in a shaking flask, detecting biomass and ergosterol yield, and screening out a strain which grows fastest and has the highest ergosterol yield. And named as Saccharomyces cerevisiae JHpharm 5-1.
The strain has a plate culture form: the colony is large and thick, the surface of the colony is smooth, moist and sticky, the texture of the colony is uniform, the color is uniform, and the colony is milky white.
Example 2: fermentation production of ergosterol by Saccharomyces cerevisiae JHpharm5-1 seed culture: inoculating a loopful bacteria from the activated plate to the liquid seed culture medium, and culturing for 24h at 30 ℃.
The seed culture medium comprises the following components: 20g/L of glucose, 10g/L of yeast powder, 20g/L of peptone, 1.5g/L of dipotassium hydrogen phosphate and the balance of water, wherein the pH value is 6.0. Culturing for 24h, inoculating 5ml into the fermentation liquid, and aerobically fermenting at 30 deg.C for 48h, wherein the liquid loading is 50ml fermentation liquid/250 ml triangular flask, and the rotation speed is 200 rpm.
The culture medium for aerobic fermentation is as follows: 80/L glucose, 20g/L peptone, 10g/L yeast powder, 3g/L anhydrous magnesium sulfate, 1.5g/L dipotassium phosphate, 1.5g/L potassium dihydrogen phosphate, 2g/L sodium nitrate and the balance of water, wherein the pH value is 6. The yield reaches the highest after fermentation for 48 hours. The yield of the strain reaches 7.2g/100g of dry thalli, and is improved by 53 percent compared with the initial strain.
Ergosterol determination method:
the standard curve for ergosterol was determined first: accurately weighing 0.1g of ergosterol standard substance, placing the ergosterol standard substance in a 100ml volumetric flask, dissolving the ergosterol standard substance by using absolute ethyl alcohol, fixing the volume to a scale mark to obtain the concentration of the standard substance solution of 1g/L, diluting the ergosterol standard substance solution into a series of concentrations (0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0, unit g/L) by using absolute ethyl alcohol, measuring peak areas corresponding to different concentrations by using a high performance liquid chromatograph of a series of standard substance solutions with different concentrations, performing regression calculation by using the concentration of ergosterol as a horizontal coordinate and the peak area as a vertical coordinate to obtain the standard regression equation of ergosterol, wherein y is 1.418x-1.501, and R is2＝0.9998
Determination of ergosterol content: centrifuging 5ml of fermentation liquor at 3500r/min for 10 minutes, discarding the supernatant, centrifuging and washing the precipitate for 3 times by using 20ml of 50% potassium hydroxide and 15ml of absolute ethyl alcohol, transferring the precipitate into a ground triangular flask, heating the mixture in a water bath to 85-90 ℃, saponifying the mixture for 3 hours, cooling the mixture to room temperature after saponification is finished, adding 8ml of deionized water and 20ml of petroleum ether, violently shaking the mixture for 15 minutes, standing the mixture for 30 minutes, and taking the upper layer extract after layering to measure the ergosterol content.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (7)

1. The yeast JHpharm5-1 for high yield of ergosterol is Saccharomyces cerevisiae with the preservation number of CGMCC No.20137, and is preserved in China general microbiological culture Collection Center (CCM) 24.06.2020.
2. The ergosterol-producing yeast JHpharm5-1 as claimed in claim 1, wherein the strain has a plate culture form of large and thick colonies, smooth surface, moistness, viscosity, uniform colony texture, uniform color, and milky white color.
3. The method for mutagenizing and breeding the yeast JHpharm5-1 with high ergosterol yield according to claim 1, comprising the following steps:
s1, culturing the yeast starting strain on an LB (lysogeny broth) culture medium for 2-4 days, then measuring biomass and ergosterol yield, and screening out a dominant strain;
s2, uniformly dispersing the fermentation liquor of the dominant strain, and diluting the fermentation liquor to 10 cell numbers according to a 10-fold dilution method7Taking 0.1ml of bacterial liquid, uniformly coating the bacterial liquid in a sterile empty plate, air-drying, and carrying out N+Performing ion implantation mutagenesis, washing cells with sterile water, diluting by 10-fold dilution method, coating the diluted cells into a culture medium, culturing at 30 ℃, selecting a single colony, performing shake flask culture for three days, detecting biomass and ergosterol yield, and screening the yeast strain with high ergosterol yield.
4. The method for mutagenizing and breeding the ergosterol-highly-producing yeast JHpharm5-1 as claimed in claim 3, wherein N is N+The ion implantation dose is 100 x 2.6 x 1013N+/cm2、145*2.6*1013N+/cm2、190*2.6*1013N+/cm2、235*2.6*1013N+/cm2And 280 x 2.6 x 1013N+/cm2,N+Ion beam implantation energy of 15KeV。
5. The use of the yeast JHpharm5-1 for high yield of ergosterol according to claim 1 in the production of ergosterol.
6. Use according to claim 5, wherein the seed medium used for the fermentative ergosterol production consists of: carbon source: 20-35 g/L, nitrogen source: 15-45 g/L, the balance of water, pH: 5-7; wherein the carbon source is selected from at least one of glucose, fructose, sucrose, lactose, soluble starch and corn steep liquor, the nitrogen source is selected from at least one of yeast extract, peptone and soybean cake powder, and the inorganic salt is selected from at least one of magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and sodium nitrate.
7. The use according to claim 5, wherein the fermentation medium employed for the fermentative production of ergosterol consists of: 80/L of glucose, 20g/L of peptone, 10g/L of yeast powder, 3g/L of anhydrous magnesium sulfate, 1.5g/L of dipotassium phosphate, 1.5g/L of monopotassium phosphate, 2g/L of sodium nitrate and the balance of water, wherein the pH value is 6, and the yield reaches the highest after fermentation for 48 hours;
the fermentation mode is that after the strain is activated, aerobic fermentation is carried out for 24-72 hours at 28-32 ℃, the liquid loading amount is 100ml fermentation liquid/500 ml triangular flask, and the rotating speed is 200 rpm.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1388243A (en) * 2001-05-30 2003-01-01 中国科学院微生物研究所 One brewer's yeast engineering saccharomycete strain and the production process of alcohol and ergosterin with the strain
CN102911883A (en) * 2012-09-27 2013-02-06 华南理工大学 Saccharomyces ergosterol high-producing strain, and breeding method and application for same
CN106434401A (en) * 2016-10-21 2017-02-22 广东五洲药业有限公司 Yeast strain enriched in ergosterol and method for preparing ergot yeast powder

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1388243A (en) * 2001-05-30 2003-01-01 中国科学院微生物研究所 One brewer's yeast engineering saccharomycete strain and the production process of alcohol and ergosterin with the strain
CN102911883A (en) * 2012-09-27 2013-02-06 华南理工大学 Saccharomyces ergosterol high-producing strain, and breeding method and application for same
CN106434401A (en) * 2016-10-21 2017-02-22 广东五洲药业有限公司 Yeast strain enriched in ergosterol and method for preparing ergot yeast powder

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HE XP等: "Overexpression of asterol C-24（28）reductasse increase ergosterol production in Saccharomyces cerevisiae.", 《BIOTECHNOLOGY LETTERS》 *
曹龙辉等: "麦角甾醇的研究进展", 《中国酿造》 *
林晓珊等: "酵母麦角固醇生物合成及其基因调控的研究", 《生物学杂志》 *
莫湘筠等: "产麦角固醇酵母的选育及其发酵条件的研究", 《食品与发酵工业》 *

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