CN112813122A - Fermentation preparation method and culture medium of Aureobasidin A - Google Patents

Fermentation preparation method and culture medium of Aureobasidin A Download PDF

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CN112813122A
CN112813122A CN202110030705.8A CN202110030705A CN112813122A CN 112813122 A CN112813122 A CN 112813122A CN 202110030705 A CN202110030705 A CN 202110030705A CN 112813122 A CN112813122 A CN 112813122A
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culture
fermentation
seed
medium
aureobasidin
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朱进伟
彭湘屏
张敏
王雪峰
孙琼
石磊
汪超
陈世敏
高祥
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Zhejiang Hunda Biotechnology Co ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract

The invention relates to the technical field of microbial pharmacy, in particular to a fermentation preparation method of Aureobasidin A and a culture medium thereof, and particularly relates to a fermentation preparation method and a culture medium thereof suitable for Aureobasidin A high-yield strain CGMCC NO. 20887. According to the method and the culture medium, the process route, the process conditions, the seed culture medium, the fermentation culture medium (especially adding a carbon source regulating factor such as isopropyl magnesium chloride) and the like are optimized, so that the synthesis efficiency of Aureobasidin A can be obviously improved, the yield is stable, the product synthesis period is prolonged, the yield of Aureobasidin A is obviously improved and can reach more than 5.5g/L, and the method and the culture medium are favorable for industrial production of Aureobasidin A.

Description

Fermentation preparation method and culture medium of Aureobasidin A
Technical Field
The invention relates to the technical field of microbial pharmacy, in particular to a fermentation preparation method of Aureobasidin A and a culture medium thereof, and particularly relates to a fermentation preparation method and a culture medium thereof suitable for Aureobasidin A high-yield strain CGMCC NO. 20887.
Background
AureobasidinA is a cyclic lipopeptide antibiotic consisting of 9 amino acid molecules, which was first isolated from the culture broth of the black yeast Aureobasidium pullulans by the Kazutoh Takesako group of Japan in 1991. Subsequent researches prove that AureobasidinA has very strong antifungal capacity and can generate toxicity to yeast at a low concentration of 0.1-0.5 mu g/ml. The fungal species to which they are susceptible include: budding yeast (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Candida glabrata (Candida glabrata), Aspergillus nidulans (Aspergillus nidulans), and Aspergillus niger (Aspergillus niger). The mechanism of action of AureobasidinA is to inhibit the activity of Inositol Phosphamide (IPC) synthase, which is dependent on the growth of fungi, and interfere with sphingolipid synthesis, thereby further killing the strain. However, AureobasidinA does not disrupt DNA, RNA and protein synthesis.
Currently, there are few reports on the AureobasidinA production technology. In 1991, the group of KazutohTakesako first isolated Aureobasidium pullulans from leaves of Islands, and after fermentation culture, the yield of AureobasidinA was about 140. mu.g/ml, and after medium optimization (addition of different amino acids) the yield was up to 312. mu.g/ml (Kazutoh Takesako, Shigetoshi Mizutani, Hitoshi Sakakibara, et al 1996, Precursor Directed Biosynthesis of Aureobasidins). In 2008, Ahmed Abdel-Lateff et al isolated a marine microorganism Aureobasidium sp from the sea area near Posdonia, the fermentation unit of which was no more than 300. mu.g/ml. Because of the strong antifungal activity of AureobasidinA, the application field is gradually expanded from potential human medication to biological pesticide, and therefore, the development of the high-level AureobasidinA production technology has important significance.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a preparation method of Aureobasidin A.
Specifically, the method comprises the following steps:
(1) activating strains;
(2) preparing a bacterial suspension;
(3) expanding and culturing seeds;
(4) and (5) fermenting.
Specifically, the step (1) includes: and inoculating the Aureobasidin A production strain to a solid culture medium, and culturing to obtain the lawn.
In one embodiment of the invention, the Aureobasidin A producing strain is strain CGMCC NO. 20887.
Specifically, the solid medium may be any medium suitable for fungal culture, for example, PDA medium.
Specifically, the temperature of the culture in step (1) may be 20 to 30 ℃ (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ℃), e.g., 22 to 30 ℃, 24 to 26 ℃, 25 ℃.
Specifically, the cultivation humidity in the step (1) may be 40-70% relative humidity (e.g., 40%, 50%, 60%, 70% relative humidity).
Specifically, the culturing time in step (1) may be 1 to 10 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days), e.g., 3 to 6 days, 4 days.
In one embodiment of the present invention, the step (1) includes: taking a strain glycerin tube of an Aureobasidin A production strain, unfreezing in water bath at 30 ℃, uniformly mixing, taking a strain suspension, inoculating the strain suspension to a PDA culture medium plate, and inversely culturing in an incubator at 22-30 ℃ and 40-70% relative humidity for 3-6 days to obtain the lawn.
Specifically, the step (2) includes: diluting the strain obtained after the activation in the step (1) by using a solvent, and dispersing to obtain a strain suspension.
Specifically, the solvent may be sterile physiological saline.
Specifically, for the concentration of the bacterial suspension, each plate of lawn can be diluted with 10-20ml (e.g., 10, 12, 14, 16, 18, 20ml) of solvent.
In an embodiment of the present invention, the step (2) includes: collecting well-grown and qualified flat thallus Porphyrae (such as thick and plump thallus Porphyrae, cream color or olive green), washing with sterile normal saline, transferring into a triangular flask containing glass beads, oscillating, scattering, and mixing to obtain bacterial suspension.
Specifically, the step (3) includes: inoculating the bacterial suspension obtained in the step (2) into a primary seed culture medium, performing primary culture, and then inoculating a primary culture solution into a secondary seed culture medium, and performing secondary culture.
Specifically, in the above-mentioned first-stage culture, the amount of the inoculum may be 0.05 to 1% (by volume, for example, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%), for example, 0.1 to 1%, 0.2 to 0.8%, 0.4 to 0.6%, 0.5%.
Specifically, in the primary culture, the culture temperature may be 20 to 30 ℃ (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ℃), e.g., 22 to 30 ℃, 24 to 26 ℃, 25 ℃.
Specifically, in the primary culture, the culture time may be 15 to 72 hours (e.g., 16, 18, 20, 22, 24, 26, 28, 30, 32, 36, 42, 48, 54, 60, 66, 72 hours), for example, 15 to 30 hours.
Specifically, in the primary culture, the culture pH may be 1.5 to 8.0 (e.g., 1.5, 2, 3, 4, 5, 5.5, 6, 6.5, 7, 7.5, 8), particularly 5.5 to 6.5.
Specifically, the above-mentioned first-stage culture may be carried out in a triangular flask.
Specifically, the primary culture is shaking culture, and the shaking frequency can be 100-.
In one embodiment of the invention, the primary culturing comprises: inoculating the bacterial suspension obtained in the step (2) into a first-level seed culture medium, and performing shake culture, wherein the inoculation amount is 0.1-1%, the rotating speed of a shaking table is 180-250rpm, and the seed age is 15-30 h.
Specifically, in the above secondary culture, the amount of the inoculum may be 0.05 to 1% (by volume, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%), for example, 0.05 to 0.5%, 0.06 to 0.4%, 0.08 to 0.2%, 0.1%.
Specifically, in the secondary culture, the culture temperature may be 20 to 30 ℃ (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ℃), e.g., 22 to 30 ℃, 24 to 26 ℃, 25 ℃.
Specifically, in the secondary culture, the culture time may be 8 to 60 hours (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36, 42, 48, 54, 60 hours), for example, 15 to 30 hours.
Specifically, in the secondary culture, the culture pH may be 1.5 to 8.0 (e.g., 1.5, 2, 3, 4, 5, 5.5, 6, 6.5, 7, 7.5, 8), particularly 5.5 to 6.5.
Specifically, the secondary culture can be carried out in a seed tank.
Specifically, the above-mentioned secondary culture is a stirring culture, and the stirring speed may be 50-400rpm (e.g., 50, 100, 150, 200, 250, 300, 350, 400rpm), such as 150-300 rpm.
Specifically, in the secondary culture, the dissolved oxygen value is 5 to 60% (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%), particularly 30 to 60%.
In one embodiment of the invention, the secondary culturing comprises: inoculating the first-stage seed culture solution into a second-stage seed culture medium, performing expanded culture in a seed tank, wherein the inoculation amount is 0.05-0.5%, the stirring speed is 150-300rpm, the dissolved oxygen value is not less than 30%, and the seed age is 15-30 h.
Specifically, the primary medium and the secondary medium may be the same or different; in one embodiment of the present invention, the primary medium is the same as the secondary medium.
Specifically, the step (3) includes: inoculating the seed culture solution expanded and cultured in the step (2) into a fermentation culture medium, and performing fermentation culture.
Specifically, the amount of the inoculum in the fermentation culture may be 0.5 to 20% (by volume, for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%), for example, 0.5 to 10%, 0.5 to 5%, 1%.
Specifically, in the above fermentation culture, the culture temperature may be 20 to 30 ℃ (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ℃), e.g., 22 to 30 ℃, 24 to 26 ℃, 25 ℃.
Specifically, in the above fermentation culture, the culture time may be 4 days or more (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12 days), for example, 8 to 10 days.
Specifically, in the above fermentation culture, the culture pH may be 1.5 to 8.0 (e.g., 1.5, 2, 3, 4, 5, 5.5, 6, 6.5, 7, 7.5, 8), particularly 5.5 to 6.5; the culture pH can be adjusted and controlled by ammonia water.
Specifically, in the above fermentation culture, the dissolved oxygen value is 5 to 50% (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%), for example, 10 to 40%, particularly 10 to 15%.
Specifically, the fermentation culture is a stirring culture, and the stirring speed may be 100-.
In one embodiment of the present invention, the fermentation culture comprises: inoculating the seed culture solution expanded in the step (2) into a fermentation culture medium, performing fermentation culture for more than 4 days, wherein the seed transfer amount is 0.5-10%, the stirring rotation speed is 200-.
In one embodiment of the present invention, the above method further comprises step (5): separating and purifying Aureobasidin A.
Specifically, the step (5) includes: and (4) carrying out solvent extraction on the fermentation liquor obtained in the step (4).
Specifically, in the step (5), the extraction solvent may be ethanol.
Specifically, in the step (5), the volume ratio of the fermentation liquid to the solvent may be 1:2 to 10 (e.g., 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10), e.g., 1:4 to 8.
Specifically, in the step (5), the leaching time may be 10 to 60min (e.g., 10, 20, 30, 40, 50, 60min), for example, 20 to 40 min.
The present invention also provides a seed culture medium which can be used for the culture (e.g., the primary culture and/or the secondary culture described above) of an Aureobasidin A-producing strain (e.g., strain CGMCC NO. 20887).
Specifically, the above seed culture medium contains a carbon source and an organic nitrogen source.
Specifically, the carbon source is contained in the seed culture medium in an amount of 1 to 10% (by weight, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%), for example, 1 to 5%.
Specifically, in the seed culture medium, the carbon source may be selected from: one or more of glucose, sucrose, glycerol, lactose, maltose, dextrin, starch, and soybean oil; more specifically, the carbon source may include glucose, and further include one or more carbon sources selected from the group consisting of: one or more of sucrose, glycerol, lactose, maltose, dextrin, starch, and soybean oil; in one embodiment of the invention, the carbon source comprises or consists of: glucose; in another embodiment of the invention, the carbon source comprises or consists of: glucose and starch.
Specifically, the content of the organic nitrogen source in the seed culture medium is 0.1 to 5% (by weight, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%), for example, 0.5 to 2%.
Specifically, in the seed culture medium, the organic nitrogen source is selected from: one or more of yeast extract, peptone, casein, corn steep liquor, soybean cake powder, corn flour, corn protein powder, cottonseed cake powder, and peanut cake powder; more specifically, the organic nitrogen source comprises a yeast extract, further comprising a source selected from: one or more of peptone, casein, corn steep liquor, soybean cake powder, corn flour, corn protein powder, cottonseed cake powder and peanut cake powder; in one embodiment of the invention, the organic nitrogen source comprises or consists of: a yeast extract; in another embodiment of the invention, the organic nitrogen source comprises or consists of: yeast extract and corn steep liquor.
Specifically, the seed culture medium may further contain an inorganic salt.
Specifically, the content of the inorganic salt in the seed culture medium is 0.01 to 1% (by weight, for example, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%), for example, 0.05 to 0.5%.
Specifically, in the seed culture medium, the inorganic salt is selected from: one or more of magnesium salt, potassium salt, sodium salt, calcium salt, ferric salt, ferrous salt, zinc salt, manganese salt, copper salt and cobalt salt; more specifically, the inorganic salt comprises or consists of: magnesium, potassium, sodium salts; in one embodiment of the invention, the inorganic salt comprises or consists of: dipotassium phosphate, magnesium sulfate and sodium chloride.
In one embodiment of the present invention, the above seed culture medium comprises or consists of: 1-10% glucose (e.g., 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 8%, 10%), 0.1-5% yeast extract (e.g., 0.1%, 0.2%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 1%, 2%, 3%, 4%).
In one embodiment of the present invention, the above seed culture medium comprises or consists of: glucose 2%, yeast extract 0.67%.
In another embodiment of the present invention, the above seed culture medium comprises or consists of: 1-5% (e.g., 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%) glucose, 0.5-5% (e.g., 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.5%, 2%, 3%, 4%, 5%) starch, 0.1-4% (e.g., 0.1%, 0.2%, 0.4%, 0.5%, 0.6%, 0.8%, 1%, 2%, 3%, 4%) corn steep liquor, 0.1-1% (e.g., 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1%), dipotassium hydrogen phosphate 0.05-0.5% (e.g., 0.05%, 0.1%, 0.15%, 0.2%, 0.3%, 0.4%, 0.5%) magnesium sulfate, 0.01-0.1% (e.01%, 0.02%, 0.04%, 0.05%, 0.08%, 0.05%, 0.5% sodium chloride, 0.05%, 0.5%, 0.1, 0.3%, 0.4%, 0.5%).
In one embodiment of the present invention, the above seed culture medium comprises or consists of: 2% of glucose, 1% of starch, 0.5% of yeast extract, 0.2% of corn steep liquor, 0.1% of dipotassium phosphate, 0.05% of magnesium sulfate and 0.1% of sodium chloride.
The invention also provides a fermentation medium which can be used for the fermentation culture of the Aureobasidin A production strain (such as the strain CGMCC NO. 20887).
Specifically, the fermentation medium comprises: carbon source, organic nitrogen source, inorganic nitrogen source and inorganic salt.
Specifically, the carbon source is contained in the fermentation medium in an amount of 1 to 20% (by weight, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%), for example, 5 to 15%.
Specifically, in the above fermentation medium, the carbon source may be selected from: one or more of glucose, sucrose, glycerol, lactose, maltose, dextrin, starch, and soybean oil; more specifically, the carbon source may include glucose, and further include one or more carbon sources selected from the group consisting of: one or more of sucrose, glycerol, lactose, maltose, dextrin, starch, and soybean oil; in one embodiment of the invention, the carbon source is glucose; in another embodiment of the invention, the carbon source is glucose, starch and dextrin.
Specifically, the content of the organic nitrogen source in the fermentation medium is 1 to 20% (by weight, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%), for example, 5 to 10%.
Specifically, in the fermentation medium, the organic nitrogen source is selected from: one or more of yeast extract, peptone, casein, corn steep liquor, soybean cake powder, corn flour, corn protein powder, cottonseed cake powder, and peanut cake powder; in one embodiment of the invention, the organic nitrogen source is peptone; in another embodiment of the invention, the organic nitrogen source is yeast extract and soybean meal.
Specifically, the content of the inorganic nitrogen source in the fermentation medium is 0.1 to 5% (by weight, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%), for example, 0.1 to 1%.
Specifically, in the fermentation medium, the inorganic nitrogen source is selected from: ammonium sulfate, ammonium chloride, ammonium nitrate, sodium nitrate, urea; more specifically, the inorganic nitrogen source comprises or consists of: ammonium sulfate, sodium nitrate.
Specifically, the content of the inorganic salt in the fermentation medium is 0.01 to 1% (by weight, for example, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%), for example, 0.05 to 0.5%.
Specifically, in the fermentation medium, the inorganic salt is selected from: one or more of magnesium salt, potassium salt, sodium salt, calcium salt, ferric salt, ferrous salt, zinc salt, manganese salt, copper salt and cobalt salt; more specifically, the inorganic salt comprises or consists of: potassium salt, sodium salt; in one embodiment of the invention, the inorganic salt comprises or consists of: dipotassium phosphate and sodium nitrate.
In one embodiment of the present invention, the fermentation medium comprises or consists of: 1-5% (e.g., 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 5%) of glucose, 1-10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%) of dextrin, 1-5% (e.g., 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 5%) of starch, 1-5% (e.g., 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 5%) of yeast extract, 1-10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%) of soybean cake powder, 0.1-1% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%) of dipotassium hydrogen phosphate, 0.05-0.5% (e.g., 0.05%, (e.1%, 0.05%), 0.1%, 0.15%, 2%, 3%, 4%, 5%), sodium nitrate 0.05-0.5% (e.g., 0.05%, 0.1%, 0.15%, 2%, 3%, 4%, 5%).
In one embodiment of the present invention, the fermentation medium comprises or consists of: 3% of glucose, 5% of dextrin, 3% of starch, 3% of yeast extract, 4% of soybean cake powder, 0.5% of ammonium sulfate, 0.1% of dipotassium hydrogen phosphate and 0.1% of sodium nitrate.
In one embodiment of the present invention, the fermentation medium further comprises a carbon source regulator.
Specifically, the carbon source regulator is contained in the fermentation medium in an amount of 0.01 to 1% (by weight, for example, 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%), for example, 0.05 to 0.5%.
Specifically, the carbon source regulator is a compound having an isopropyl group, for example, isopropyl magnesium chloride.
In one embodiment of the present invention, the fermentation medium comprises or consists of: 3% of glucose, 5% of dextrin, 3% of starch, 3% of yeast extract, 4% of soybean cake powder, 0.5% of ammonium sulfate, 0.1% of dipotassium hydrogen phosphate, 0.1% of sodium nitrate and 0.1% of isopropyl magnesium chloride.
The invention also provides application of the seed culture medium in culture (particularly seed culture) of Aureobasidin A production strains (such as the strain CGMCC NO. 20887).
The invention also provides application of the fermentation medium in culture (particularly fermentation culture) of the Aureobasidin A production strain (such as the strain CGMCC NO. 20887).
The invention also provides application of the method, the seed culture medium and the fermentation culture medium in preparation of Aureobasidin A.
The invention also provides a combination of materials for preparing Aureobasidin A by fermentation, which comprises the seed culture medium and the fermentation culture medium.
In particular, the above combination may also include a species activation medium, which may be any medium suitable for fungal culture, for example, PDA medium.
In particular, the above combination may also include a solvent for the preparation of the bacterial suspension, such as sterile physiological saline.
The Aureobasidin A production strain CGMCC NO.20887 related in the invention is an Aureobasidin A production strain obtained by screening and mutation of an inventor, is classified and named as Aureobasidin A, is preserved in China general microbiological culture Collection center (address: No. 1 Siro-1 of the sunward area, Beijing, institute of microbiology, China academy of sciences) within 10 months and 14 days of 2020, and has a preservation number of: CGMCC NO.20887, sometimes referred to herein simply as strain CGMCC NO. 20887.
The Aureobasidin A production strain (particularly strain CGMCC NO.20887) is used as a production strain, and the yield of Aureobasidin A can reach more than 5.5g/L by optimizing a process route, process conditions, a seed culture medium, a fermentation culture medium (particularly adding a carbon source regulating factor such as isopropyl magnesium chloride) and the like. Wherein, the seed culture medium is optimized to ensure that the seed culture medium has vigorous activity, quickly removes the substrate effect of fermentation nutrition and accelerates the start of the auxin; through the optimization of a fermentation formula, especially the addition of isopropyl magnesium chloride, the carbon source metabolism of the strain can be effectively regulated, so that the synthesis efficiency of Aureobasidin A is remarkably improved. In addition, the key process condition of dissolved oxygen in the Aureobasidin A fermentation process is controlled, so that the stable yield can be maintained, and the product synthesis period can be prolonged. Finally, the yield of Aureobasidin A is obviously improved, and the industrial production of Aureobasidin A is facilitated.
Drawings
FIG. 1 shows the results of AureobasidinA production in the fermentation broths of examples 1 to 6.
FIG. 2 shows the effect of seed vigor on the growth rate (a), substrate consumption rate (b), and AureobasidinA synthesis rate (c) of a fermentation strain.
FIG. 3 shows a comparison of AureobasidinA synthesis curves in examples 3, 4 and 5.
FIG. 4 shows the effect of adding isopropyl magnesium chloride to the fermentation medium on Aureobasidin A synthesis and carbon source conversion.
Detailed Description
Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
The "lawn" refers to a dense colony with certain morphological structure characteristics, which is formed by the propagation of mother cells on a solid culture medium, and is generally formed by the aggregation of a large number of colonies.
The "inoculum amount" refers to the ratio of the volume of the seed solution added to the volume of the culture solution after inoculation.
"fermentation broth" refers to a mixture obtained by inoculating a liquid culture medium with a microorganism strain and culturing the mixture for a certain period of time, wherein the mixture contains microorganism cells and metabolites thereof, unused medium components (if possible), and the like.
The disclosures of the various publications, patents, and published patent specifications cited herein are hereby incorporated by reference in their entirety.
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 fermentative preparation of AureobasidinA
Taking a working strain glycerin pipe of a strain CGMCC NO.20887, thawing, taking a suspension of two-ring bacteria, inoculating a solid culture medium, culturing for 72 hours at 25 ℃, taking a ring lawn, inoculating a seed culture medium, culturing for 48 hours at 25 ℃, then inoculating into a fermentation culture medium according to 1% of inoculation amount, fermenting at 25 ℃, air flow of 1vvm, stirring at 100rpm, supplementing a fresh supplemented culture medium with an initial feeding volume of 1/5 after culturing for 56 hours, continuing to culture at 25 ℃, air flow of 1vvm, stirring at 100rpm, and finishing culturing for 8 days to obtain a fermentation liquid containing AureobasidinA. Taking 1ml of the fermentation liquor, soaking the fermentation liquor in 5 times volume of absolute ethyl alcohol for more than half an hour, centrifuging the fermentation liquor to obtain supernatant, filtering the supernatant by using a 0.45-micrometer membrane, and analyzing and detecting the fermentation liquor by using HPLC (high performance liquid chromatography), wherein the content of AureobasidinA in the fermentation liquor is 0.98 g/L.
Seed culture medium: 2% of glucose and 0.67% of yeast basic nitrogen source. Sterilizing at pH6.5 at 121 deg.C for 30min before sterilization.
Fermentation medium: glucose 2%, (NH)4)2SO40.5％，KH2PO40.15％，MgSO4*7H2O0.05％，CaCl2*2H2O 0.01％，NaCl 0.01％，FeCl3*6H2O 0.5μg/ml，ZnSO4*7H2O0.5. mu.g/ml. Sterilizing at pH6.5 at 121 deg.C for 30min before sterilization.
A supplemented medium: glucose 10%, (NH4)2SO42.5%, polypeptone 5%, KH2PO40.75％，MgSO4*7H2O 0.25％，CaCl2*2H2O 0.05％，NaCl 0.05％，FeCl3*6H2O 2.5μg/ml，ZnSO4*7H2O2.5. mu.g/ml. Sterilizing at pH6.5 at 121 deg.C for 30min before sterilization.
Example 2 AureobasidinA fermentation preparation method optimization
Activating strains: taking a working strain glycerin tube, thawing in a water bath at 30 ℃, uniformly mixing, then inoculating 0.1ml of strain suspension to a PDA culture medium plate, uniformly coating and bundling by using a coating rod, and inversely covering in an incubator with the relative humidity of 40-70% and the temperature of 22-30 ℃ for activation culture for 4 days, wherein the lawn is rich and full and is cream or olive green.
Preparing a bacterial suspension: washing each flat plate with 15ml sterile normal saline, transferring into a triangular flask containing two spoons of glass beads, oscillating for 30min, and scattering to obtain bacterial suspension.
Seed expanding culture: inoculating the prepared bacterial suspension into a shake flask seed culture medium with the inoculation amount of 0.5%, and performing shake culture on a shaking table at 25 ℃ and 220rpm for 24 h. Then inoculating the shake flask seed culture solution into a seed tank by 0.1% of inoculum size for secondary expansion culture, controlling the tank temperature at 25 ℃, stirring at 150-300rpm, and culturing for 18h, wherein the dissolved oxygen is more than or equal to 30%.
Fermentation control culture: transferring the expanded seed culture solution into a fermentation culture medium according to the seed transfer amount of 1%, controlling the fermentation temperature to be 25 ℃, stirring at 150-500 rpm, dissolving oxygen at 30-40% and pH at 5.5-6.5, and finishing the culture for 8 days to obtain the fermentation liquor containing AureobasidinA.
And (3) yield detection: taking 1ml of fermentation liquor, soaking the fermentation liquor in 5 times volume of absolute ethyl alcohol for more than half an hour, centrifuging the fermentation liquor to obtain supernatant, filtering the supernatant by using a 0.45-micrometer membrane, and analyzing and detecting the fermentation liquor by using HPLC (high performance liquid chromatography), wherein the content of Aureobasidin A in the fermentation liquor is 1.73 g/L.
Seed culture medium: 2% of glucose and 0.67% of yeast basic nitrogen source. Sterilizing at pH6.5 at 121 deg.C for 30min before sterilization.
Fermentation medium: glucose 3.67%, (NH)4)2SO40.92 percent, 0.83 percent of polypeptone and KH2PO40.275％，MgSO4*7H2O 0.092％，CaCl2*2H2O 0.018％，NaCl 0.018％，FeCl3*6H2O0.92μg/ml，ZnSO4*7H2O0.92. mu.g/ml. Sterilizing at pH6.5 at 121 deg.C for 30min before sterilization.
Example 3 AureobasidinA seed formulation optimization
Activating strains: taking a working strain glycerin tube, thawing in a water bath at 30 ℃, uniformly mixing, then inoculating 0.1ml of strain suspension to a PDA culture medium plate, uniformly coating and bundling by using a coating rod, and inversely covering in an incubator with the relative humidity of 40-70% and the temperature of 22-30 ℃ for activation culture for 4 days, wherein the lawn is rich and full and is cream or olive green.
Preparing a bacterial suspension: washing each flat plate with 15ml sterile normal saline, transferring into a triangular flask containing two spoons of glass beads, oscillating for 30min, and scattering to obtain bacterial suspension.
Seed expanding culture: inoculating the prepared bacterial suspension into an optimized shake flask seed culture medium with the inoculation amount of 0.5%, and carrying out shake culture on a shaking table at 25 ℃ and 220rpm for 24 h. Then inoculating the shake flask seed culture solution into a seed tank by 0.1% of inoculum size for secondary expansion culture, controlling the tank temperature at 25 ℃, stirring at 150-300rpm, and culturing for 18h, wherein the dissolved oxygen is more than or equal to 30%.
Fermentation control culture: transferring the expanded seed culture solution into a fermentation culture medium according to the seed transfer amount of 1%, controlling the fermentation temperature to be 25 ℃, stirring at 150-500 rpm, dissolving oxygen at 30-40% and pH at 5.5-6.5, and finishing the culture for 8 days to obtain the fermentation liquor containing AureobasidinA.
And (3) yield detection: taking 1ml of fermentation liquor, soaking the fermentation liquor in 5 times volume of absolute ethyl alcohol for more than half an hour, centrifuging the fermentation liquor to obtain supernatant, filtering the supernatant by using a 0.45-micrometer membrane, and analyzing and detecting the fermentation liquor by using HPLC (high performance liquid chromatography), wherein the content of Aureobasidin A in the fermentation liquor is 1.96 g/L.
Optimizing a seed culture medium: 2% of glucose, 1% of starch, 0.5% of yeast extract powder, 0.2% of corn steep liquor dry powder, 0.1% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate and 0.1% of sodium chloride. Sterilizing at pH6.5 at 121 deg.C for 30min before sterilization.
Fermentation medium: glucose 3.67%, (NH)4)2SO40.92 percent, 0.83 percent of polypeptone and KH2PO40.275％，MgSO4*7H2O 0.092％，CaCl2*2H2O 0.018％，NaCl 0.018％，FeCl3*6H2O0.92μg/ml，ZnSO4*7H2O0.92. mu.g/ml. Sterilizing at pH6.5 at 121 deg.C for 30min before sterilization.
Example 4 AureobasidinA fermentation Medium optimization
Activating strains: taking a working strain glycerin tube, thawing in a water bath at 30 ℃, uniformly mixing, then inoculating 0.1ml of strain suspension to a PDA culture medium plate, uniformly coating and bundling by using a coating rod, and inversely covering in an incubator with the relative humidity of 40-70% and the temperature of 22-30 ℃ for activation culture for 4 days, wherein the lawn is rich and full and is cream or olive green.
Preparing a bacterial suspension: washing each flat plate with 15ml sterile normal saline, transferring into a triangular flask containing two spoons of glass beads, oscillating for 30min, and scattering to obtain bacterial suspension.
Seed expanding culture: inoculating the prepared bacterial suspension into an optimized shake flask seed culture medium with the inoculation amount of 0.5%, and carrying out shake culture on a shaking table at 25 ℃ and 220rpm for 24 h. Then inoculating the shake flask seed culture solution into a seed tank by 0.1% of inoculum size for secondary expansion culture, controlling the tank temperature at 25 ℃, stirring at 150-300rpm, and culturing for 18h, wherein the dissolved oxygen is more than or equal to 30%.
Fermentation control culture: transferring the expanded seed culture solution into a fermentation culture medium according to the seed transfer amount of 1%, controlling the fermentation temperature to be 25 ℃, stirring at 150-500 rpm, dissolving oxygen at 30-40% and pH at 5.5-6.5, and finishing the culture for 8 days to obtain the fermentation liquor containing AureobasidinA.
And (3) yield detection: soaking 1ml of fermentation liquor in 5 times volume of absolute ethyl alcohol for more than half an hour, centrifuging to obtain supernatant, filtering with a 0.45-micron membrane, and analyzing and detecting by HPLC, wherein the content of Aureobasidin A in the fermentation liquor is 3.01 g/L.
Optimizing a seed culture medium: 2% of glucose, 1% of starch, 0.5% of yeast extract powder, 0.2% of corn steep liquor dry powder, 0.1% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate and 0.1% of sodium chloride. Sterilizing at pH6.5 at 121 deg.C for 30min before sterilization.
Optimizing a fermentation medium: 3% of glucose, 5% of dextrin, 3% of starch, 3% of yeast extract powder, 4% of soybean cake powder, 0.5% of ammonium sulfate, 0.1% of dipotassium hydrogen phosphate and 0.1% of sodium nitrate. Sterilizing at pH6.5 at 121 deg.C for 30min before sterilization.
Example 5 AureobasidinA fermentation Key Process parameter optimization
Activating strains: taking a working strain glycerin tube, thawing in a water bath at 30 ℃, uniformly mixing, then inoculating 0.1ml of strain suspension to a PDA culture medium plate, uniformly coating and bundling by using a coating rod, and inversely covering in an incubator with the relative humidity of 40-70% and the temperature of 22-30 ℃ for activation culture for 4 days, wherein the lawn is rich and full and is cream or olive green.
Preparing a bacterial suspension: washing each flat plate with 15ml sterile normal saline, transferring into a triangular flask containing two spoons of glass beads, oscillating for 30min, and scattering to obtain bacterial suspension.
Seed expanding culture: inoculating the prepared bacterial suspension into an optimized shake flask seed culture medium with the inoculation amount of 0.5%, and carrying out shake culture on a shaking table at 25 ℃ and 220rpm for 24 h. Then inoculating the shake flask seed culture solution into a seed tank by 0.1% of inoculum size for secondary expansion culture, controlling the tank temperature at 25 ℃, stirring at 150-300rpm, and culturing for 18h, wherein the dissolved oxygen is more than or equal to 30%.
Fermentation control culture: transferring the expanded seed culture solution into a fermentation culture medium according to the seed transfer amount of 1%, controlling the fermentation temperature to be 25 ℃, stirring at 150-500 rpm, dissolving oxygen at 10-15% and pH at 5.5-6.5, and finishing the culture for 10 days to obtain the fermentation liquor containing AureobasidinA.
And (3) yield detection: taking 1ml of fermentation liquor, soaking the fermentation liquor in 5 times volume of absolute ethyl alcohol for more than half an hour, centrifuging the fermentation liquor to obtain supernatant, filtering the supernatant by using a 0.45-micrometer membrane, and analyzing and detecting the fermentation liquor by using HPLC (high performance liquid chromatography), wherein the content of Aureobasidin A in the fermentation liquor is 3.67 g/L.
Optimizing a seed culture medium: 2% of glucose, 1% of starch, 0.5% of yeast extract powder, 0.2% of corn steep liquor dry powder, 0.1% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate and 0.1% of sodium chloride. Sterilizing at pH6.5 at 121 deg.C for 30min before sterilization.
Optimizing a fermentation medium: 3% of glucose, 5% of dextrin, 3% of starch, 3% of yeast extract powder, 4% of soybean cake powder, 0.5% of ammonium sulfate, 0.1% of dipotassium hydrogen phosphate and 0.1% of sodium nitrate. Sterilizing at pH6.5 at 121 deg.C for 30min before sterilization.
Example 6 Effect of addition of isopropyl magnesium chloride to AureobasidinA fermentation Medium
Activating strains: taking a working strain glycerin tube, thawing in a water bath at 30 ℃, uniformly mixing, then inoculating 0.1ml of strain suspension to a PDA culture medium plate, uniformly coating and bundling by using a coating rod, and inversely covering in an incubator with the relative humidity of 40-70% and the temperature of 22-30 ℃ for activation culture for 4 days, wherein the lawn is rich and full and is cream or olive green.
Preparing a bacterial suspension: washing each flat plate with 15ml sterile normal saline, transferring into a triangular flask containing two spoons of glass beads, oscillating for 30min, and scattering to obtain bacterial suspension.
Seed expanding culture: inoculating the prepared bacterial suspension into an optimized shake flask seed culture medium with the inoculation amount of 0.5%, and carrying out shake culture on a shaking table at 25 ℃ and 220rpm for 24 h. Then inoculating the shake flask seed culture solution into a seed tank by 0.1% of inoculum size for secondary expansion culture, controlling the tank temperature at 25 ℃, stirring at 150-300rpm, and culturing for 18h, wherein the dissolved oxygen is more than or equal to 30%.
Fermentation control culture: transferring the expanded seed culture solution into a fermentation culture medium according to the seed transfer amount of 1%, controlling the fermentation temperature to be 25 ℃, stirring at 150-500 rpm, dissolving oxygen at 10-15% and pH at 5.5-6.5, and finishing the culture for 10 days to obtain the fermentation liquor containing AureobasidinA.
And (3) yield detection: taking 1ml of fermentation liquor, soaking the fermentation liquor in 5 times volume of absolute ethyl alcohol for more than half an hour, centrifuging the fermentation liquor to obtain supernatant, filtering the supernatant by using a 0.45-micrometer membrane, and analyzing and detecting the fermentation liquor by using HPLC (high performance liquid chromatography), wherein the content of Aureobasidin A in the fermentation liquor is 5.52 g/L.
Optimizing a seed culture medium: 2% of glucose, 1% of starch, 0.5% of yeast extract powder, 0.2% of corn steep liquor dry powder, 0.1% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate and 0.1% of sodium chloride. Sterilizing at pH6.5 at 121 deg.C for 30min before sterilization.
Optimized and added with 0.1% isopropyl magnesium chloride fermentation medium: 3% of glucose, 5% of dextrin, 3% of starch, 3% of yeast extract powder, 4% of soybean cake powder, 0.5% of ammonium sulfate, 0.1% of dipotassium hydrogen phosphate, 0.1% of isopropyl magnesium chloride and 0.1% of sodium nitrate. Sterilizing at pH6.5 at 121 deg.C for 30min before sterilization.
The results of AureobasidinA production in the fermentation broth of each example are shown in FIG. 1.
The effects of seed vigor on the growth rate (a), substrate consumption rate (b), and AureobasidinA synthesis rate (c) of the fermentation strain are shown in fig. 2.
The ratio of aureobasidinA synthesis curves in examples 3, 4 and 5 is shown in FIG. 3.
The effect of adding isopropyl magnesium chloride to the fermentation medium on AureobasidinA synthesis and carbon source conversion is shown in fig. 4.
It can be seen from the examples 1 and 2 that, in the AureobasidinA fermentation preparation process, the AureobasidinA yield is obviously improved after the bacterial suspension preparation is increased, the secondary seed expanding culture is increased, and the fermentation pH is controlled, and the improvement amplitude is 76.5%.
It can be seen from the examples 2 and 3 that, after the seed culture medium is optimized, the growth rate of the strain, the substrate consumption rate and the synthesis rate of AureobasidinA are obviously increased in the early stage of the fermentation stage, and finally the yield of AureobasidinA is increased by 13.3%.
It can be seen from examples 3, 4 and 5 that the yield of AureobasidinA is respectively improved by 53.6% and 22% by fermentation formula optimization and dissolved oxygen control, which indicates that the fermentation medium and dissolved oxygen have significant influence on the yield of AureobasidinA.
It can be seen from examples 5 and 6 that after isopropyl magnesium chloride is added to the fermentation medium, the conversion rate from the carbon source to the product is obviously increased, and finally, the yield of AureobasidinA is obviously increased, and the increase rate reaches 50.4%.
In conclusion, the Aureobasidin A high-yield strain CGMCC NO.20887 is used as a production strain, and the strain has vigorous activity by further optimizing a seed culture medium, and quickly removes the substrate effect of fermentation nutrition to accelerate the start of production. Through the optimization of a fermentation formula, especially the addition of isopropyl magnesium chloride, the carbon source metabolism of the strain can be effectively regulated, so that the synthesis efficiency of Aureobasidin A is remarkably improved. In addition, the key process condition of dissolved oxygen in the Aureobasidin A fermentation process is controlled, so that the stable yield can be maintained, and the product synthesis period can be prolonged. Finally, the yield of Aureobasidin A is obviously improved, and the production cost is reduced, so that the method is favorable for realizing commercial production.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.
The foregoing embodiments and methods described in this disclosure may vary based on the abilities, experience, and preferences of those skilled in the art.
The mere order in which the steps of a method are listed in the present invention does not constitute any limitation on the order of the steps of the method.

Claims (14)

1.A preparation method of Aureobasidin A comprises the following steps:
(1) activating strains of an Aureobasidin A production strain;
(2) preparing a bacterial suspension;
(3) expanding and culturing seeds;
(4) fermenting;
wherein the step (3) comprises: inoculating the bacterial suspension obtained in the step (2) into a primary seed culture medium, performing primary culture, and then inoculating a primary culture solution into a secondary seed culture medium, and performing secondary culture.
2. The method of claim 1, wherein in the first stage culture, the amount of inoculum is 0.05-1%, and the culture time is 15-72 h;
preferably, the primary culturing comprises: inoculating the bacterial suspension obtained in the step (2) into a first-level seed culture medium, and performing shake culture, wherein the inoculation amount is 0.1-1%, the rotating speed of a shaking table is 180-250rpm, and the seed age is 15-30 h.
3. The method of claim 2, wherein in the secondary culture, the amount of inoculation is 0.05-1%, and the culture time is 8-60 h;
preferably, the secondary culturing comprises: inoculating the primary seed culture solution into a secondary seed culture medium, performing expanded culture in a seed tank, wherein the inoculation amount is 0.05-0.5%, the stirring speed is 150-300rpm, the dissolved oxygen is more than or equal to 30%, and the seed age is 15-30 h.
4. The method of claim 1, wherein the amount of inoculum in said fermentation culture is 0.5-20% and the dissolved oxygen value is 5-50%;
preferably, the fermentation culture comprises: inoculating the seed culture solution expanded in the step (2) into a fermentation culture medium, performing fermentation culture for more than 4 days, wherein the seed transfer amount is 0.5-10%, the stirring rotation speed is 200-.
5. The method according to any one of claims 1 to 4, wherein the Aureobasidin A producing strain is strain CGMCC NO. 20887.
6. The method according to claim 5, wherein the Aureobasidin A-producing strain is cultured at a temperature of 20 to 30 ℃ and a pH of 1.5 to 8.0;
preferably, the Aureobasidin A producing strain is cultured at a temperature of 24 to 26 ℃ and a pH of 5.5 to 6.5.
7. The method of claim 5, wherein the seed medium used in step (3) comprises: 1-10% of glucose and 0.1-5% of yeast extract; or the like, or, alternatively,
the seed culture medium comprises: 1-5% of glucose, 0.5-5% of starch, 0.1-4% of yeast extract, 0.1-1% of corn steep liquor, 0.05-0.5% of dipotassium phosphate, 0.01-0.1% of magnesium sulfate and 0.05-0.5% of sodium chloride.
8. The method of claim 5, wherein the fermentation medium used in step (4) comprises: 1-5% of glucose, 1-10% of dextrin, 1-5% of starch, 1-5% of yeast extract, 1-10% of soybean cake powder, 0.1-1% of ammonium sulfate, 0.05-0.5% of dipotassium phosphate and 0.05-0.5% of sodium nitrate.
9. The method of claim 8, wherein the fermentation medium further comprises a carbon source regulator that is an isopropyl group-containing compound present in the fermentation medium in an amount of 0.01 to 1%;
preferably, the carbon source regulating factor is isopropyl magnesium chloride, and the content of the isopropyl magnesium chloride in the fermentation medium is 0.05-0.5%.
10. A seed culture medium comprising: 1-5% of glucose, 0.5-5% of starch, 0.1-4% of yeast extract, 0.1-1% of corn steep liquor, 0.05-0.5% of dipotassium phosphate, 0.01-0.1% of magnesium sulfate and 0.05-0.5% of sodium chloride;
preferably, the seed medium comprises: 2% of glucose, 1% of starch, 0.5% of yeast extract, 0.2% of corn steep liquor, 0.1% of dipotassium phosphate, 0.05% of magnesium sulfate and 0.1% of sodium chloride.
11. A fermentation medium comprising: 1-5% of glucose, 1-10% of dextrin, 1-5% of starch, 1-5% of yeast extract powder, 1-10% of soybean cake powder, 0.1-1% of ammonium sulfate, 0.05-0.5% of dipotassium phosphate and 0.05-0.5% of sodium nitrate;
preferably, the fermentation medium comprises: 3% of glucose, 5% of dextrin, 3% of starch, 3% of yeast extract powder, 4% of soybean cake powder, 0.5% of ammonium sulfate, 0.1% of dipotassium hydrogen phosphate and 0.1% of sodium nitrate.
12. The fermentation medium of claim 1, further comprising a carbon source regulating factor, wherein the carbon source regulating factor is a compound containing an isopropyl group, and the content of the compound in the fermentation medium is 0.01-1%;
preferably, the carbon source regulating factor is isopropyl magnesium chloride, and the content of the isopropyl magnesium chloride in the fermentation medium is 0.05-0.5%.
13. Use of the seed culture medium of claim 10 or the fermentation medium of claim 11 or 12 in the culture of an Aureobasidin a producing strain;
preferably, the Aureobasidin A producing strain is strain CGMCC NO. 20887.
14. Use of the method of any one of claims 1 to 9, the seed medium of claim 10 or the fermentation medium of claim 11 or 12 for the preparation of Aureobasidin a.
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