CN115505605B - Production method of ursolic acid - Google Patents

Production method of ursolic acid Download PDF

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CN115505605B
CN115505605B CN202211366063.XA CN202211366063A CN115505605B CN 115505605 B CN115505605 B CN 115505605B CN 202211366063 A CN202211366063 A CN 202211366063A CN 115505605 B CN115505605 B CN 115505605B
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glucose
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王颖
贾男
臧国伟
那雪梅
李敬知
李春
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Beijing Institute of Technology BIT
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Abstract

The invention relates to the technical field of fermentation engineering, and particularly provides a production method of ursolic acid, which comprises two-stage fermentation culture of saccharomyces cerevisiae (yeast)Saccharomyces cerevisiae) The production method provided by the application has the advantages that the yield of the ursolic acid is 2331.44 +/-180.87 mg/L or more, and a demonstration effect is provided for accelerating the production process for synthesizing the ursolic acid by the biological method and realizing industrial application as soon as possible.

Description

Production method of ursolic acid
Technical Field
The invention relates to the technical field of fermentation engineering, in particular to a production method of ursolic acid, and particularly relates to a fermentation method for producing the ursolic acid by utilizing saccharomyces cerevisiae high-density fermentation and application thereof.
Background
Ursolic acid is a ursolic alkane type pentacyclic triterpene compound naturally existing in plants, and a plurality of researches show that the ursolic acid has physiological activities of resisting inflammation, oxidation, tumor and the like. At present, the ursolic acid is obtained from plants mainly, but the growth cycle of the plants is long, and serious ecological problems can be caused by excessive felling. The chemical synthesis method has the technical problems of complex process, low extraction efficiency and the like. The microbial fermentation method is well-paid attention by people because of the advantages of mild conditions, no occupation of cultivated land, green and controllable process and the like. However, the current production of ursolic acid in saccharomyces cerevisiae is a certain distance away from the lowest standard of industrial production. In order to further release the capability of the engineering saccharomyces cerevisiae for producing the ursolic acid, the amplification culture of the fermentation process of the engineering strains is necessary. Therefore, the optimal conditions of the strain fermentation are determined by optimizing the fermentation conditions of the system and developing a large-scale fermentation process, and the method has important significance for promoting the synthesis of ursolic acid by a microbial fermentation method to realize industrial production as soon as possible.
Disclosure of Invention
In order to solve the defects of the prior art, the application provides a method for producing ursolic acid by saccharomyces cerevisiae high-density fermentation, and the result shows that the yield of the ursolic acid is greatly improved by at least 20 times by adopting the method for 8 days of fermentation through the amplification and optimization of the fermentation production process, and the specific yield of the ursolic acid reaches 2331.44 +/-180.87 mg/L and above. Specifically, the method comprises the following steps:
in the first aspect of the invention, the production method of ursolic acid is provided, and the production method comprises two-stage fermentation culture of saccharomyces cerevisiae (Saccharomyces cerevisiae) The two-stage fermentation comprises:
the first stage is as follows: glucose is used as a carbon source, and a yeast nitrogen source without amino acid is used as a nitrogen source.
Preferably, the initial concentration of the carbon source in the first stage is 50-70g/L. Further preferably 55 to 65g/L. For example 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70g/L.
Preferably, the initial concentration of the nitrogen source in the first stage is 15-25g/L. Further preferably 18 to 22g/L. E.g., 15, 16, 17, 18, 19, 20, 20.1, 21, 22, 23, 24, 25g/L.
And a second stage: ethanol is used as a carbon source, and a yeast nitrogen source without amino acid is used as a nitrogen source.
Preferably, the initial concentration of the nitrogen source in the second stage is 15-25g/L. More preferably 18 to 22g/L. E.g., 15, 16, 17, 18, 19, 20, 20.1, 21, 22, 23, 24, 25g/L.
Preferably, the time for feeding ethanol in the second stage is the logarithmic growth phase of the cell growth, and more preferably, the early stage of the logarithmic growth phase.
Preferably, the ethanol is fed in the second stage at the beginning of 40-50h (e.g. 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 h) of the fermentation.
Preferably, the feeding rate of the ethanol in the second stage is 28-36mL/h. For example 28, 28.5, 28.8, 29, 30, 31, 32, 33, 34, 35, 36mL/h.
Preferably, after the addition of ethanol, the concentration of ethanol is controlled to be kept below 5 g.
Preferably, the feeding of the glucose feed medium is started at 10-12h (e.g., 10h, 10.5h, 11h, 11.5h, 12 h) of the fermentation, and the feeding is stopped at 35-38h (e.g., 35, 36, 37, 38 h).
Glucose feed medium included per liter: 400-600g glucose, 10-15g yeast nitrogen source without amino acid, 1-2g abscisic acid mixture, 8-10g KH 2 PO 4 ，2-3g MgSO 4 ，3-4g K 2 SO 4 ，0.2-0.35g Na 2 SO 4 8-12mL of trace metal solution and 10-15mL of vitamin solution.
Further preferably, the feeding rate of the glucose feed medium can be adjusted according to actual conditions, for example, glucose is added under the condition that the concentration of ethanol produced by the saccharomyces cerevisiae is not more than 10g/L, and the feeding rate of the glucose feed medium is preferably 30mL/h to 80mL/h, such as 30, 40, 50, 60, 70, 80mL/h.
Preferably, the addition of galactose is started after the glucose is depleted. For example, the glucose-depleted state is estimated to be 1 to 3 hours (e.g., 1, 2, 3 hours) after the supply of the glucose feed medium is stopped, and the addition of galactose is started.
Further preferably, galactose is added as an inducer at 35-45h (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 h) of the fermentation, and the final concentration of galactose is 55-65g/L (e.g., 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 g/L) after addition.
The addition can be fed-batch or intermittent.
In one embodiment of the present invention, the galactose is added intermittently.
Preferably, a vitamin solution and/or a trace metal solution are/is added in the fermentation process.
In one embodiment of the invention, the galactose feed medium, the vitamin solution and the trace metal solution are added every 10-12h (e.g. 10h, 10.5h, 11h, 11.5h, 12 h) after 40-50h (e.g. 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 h) of the fermentation. Specifically, if in a 5L fermentor, 2 to 3 liters of fermentation system, approximately 5 to 10mL (e.g., 5, 6, 7, 8, 9, 10 mL) of vitamin solution is added, approximately 5 to 10mL (e.g., 5, 6, 7, 8, 9, 10 mL) of trace metal solution is added, and 40 to 60mL (e.g., 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 mL) of galactose feed medium is added. If the fermentation system is other fermentation systems, the fermentation system is added according to the proportion by referring to the addition amount of 2-3 liters.
In one embodiment of the invention, the galactose feed medium comprises per liter: 400-600g galactose, 10-15g yeast nitrogen source without amino acid, 1-2g abscisic acid mixture, 8-10g KH 2 PO 4 ，2-3g MgSO 4 ，3-4g K 2 SO 4 ，0.2-0.35g Na 2 SO 4 8-12mL of trace metal solution and 10-15mL of vitamin solution;
in one embodiment of the invention, the vitamin solution comprises per liter: 0.01-0.1g of biotin, 0.5-2g of calcium pantothenate, 0.5-2g of nicotinic acid, 20-30g of inositol, 0.5-2g of thiamine hydrochloride, 0.5-2g of pyridoxal hydrochloride and 0.1-0.3g of p-aminobenzoic acid;
in one embodiment of the invention, the trace metal solution comprises per liter: 10-20g of EDTA, znSO 4 •7H 2 O 5-6g，MnCl 2 •4H 2 0.2-0.4g of O and anhydrous CuSO 4 0.1-1g，CoCl 2 •6H 2 O 0.2-0.6g，Na 2 MoO 4 •2H 2 O 0.3-0.8g，CaCl 2 •2H 2 O 2-3.5g，FeSO 4 •7H 2 O 2-3.5g。
Preferably, the pH is maintained between 5 and 6, e.g. 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6 during the fermentation.
Preferably, the temperature is maintained between 25-35 ℃ during the fermentation, such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 ℃.
Preferably, the dissolved oxygen during the fermentation is 30-40%, such as 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40%. Further preferably, the dissolved oxygen of the fermentation liquor is controlled to be 30-40% by the cascade rotating speed of the dissolved oxygen.
Preferably, the stirring speed during the fermentation is controlled between 300-800rpm, such as 300, 400, 500, 600, 700, 800rpm. In one embodiment of the invention, the stirring speed is controlled at 300-800rpm after initial control of the stirring speed for fermentation of 300rpm for 1-5h (e.g., 1, 2, 3, 4, 5 h).
In one embodiment of the invention, the stirring speed is coupled with dissolved oxygen, and the stirring speed fluctuates within the range of 300-800rpm on the premise of ensuring that the dissolved oxygen is 30-40%.
Preferably, the air flow during the fermentation is controlled to be 2-5vvm, such as 2, 3, 4, 5vvm.
In one embodiment of the present invention, the production method comprises:
a) Activating strains: taking out the frozen glycerol strain from an ultralow temperature refrigerator at minus 80 ℃, and culturing the glycerol strain on an SD defective solid culture medium until a single colony appears;
b) Seed liquid culture: selecting single colony, inoculating to seed culture medium, and culturing in a shaker at 25-35 deg.C and 150-250rpm for 30-40h;
c) The first stage fermentation culture: inoculating 8-12% of the inoculum size into a fermentation initial culture medium which takes glucose as a carbon source and takes a yeast nitrogen source without amino acid as a nitrogen source, culturing under the conditions that the pH value is maintained at 5-6, the temperature is maintained at 25-35 ℃, the dissolved oxygen is 30-40%, feeding glucose feed supplement culture medium after 10-12h of culture, wherein the feeding rate is 30-80mL/h, and stopping feeding in 35-38 h; preferably, the stirring speed is controlled at 300-800rpm, and the air flow is controlled at 2-5vvm.
D) And (3) second-stage fermentation culture: ethanol is used as a carbon source, and a yeast nitrogen source without amino acid is used as a nitrogen source, wherein the time of adding ethanol in a flow manner is 40-50h, and the flow rate is 28-36mL/h. After the ethanol is added, the concentration of the ethanol is controlled to be kept below 5 g;
wherein, galactose is added as an inducer at the 35 th-45 th hour of fermentation, and the final concentration of the galactose is 55-65g/L after the addition; and adding a galactose supplementary culture medium, a vitamin solution and a trace metal solution every 10-12h after the fermentation for 40-50 h;
in one embodiment of the invention, the SD-deficient solid medium comprises per liter: 5-8g of yeast nitrogen source without amino acid, 15-25g of glucose, 0.5-1g of abscisic acid mixture and 15-25g of agar powder, and adjusting the pH value of the culture medium to 6-6.5;
in one embodiment of the invention, the seed medium comprises per liter: 15-25g/L yeast nitrogen source without amino acid, 50-70g/L glucose and 2-3g/L abscisic acid mixture;
in one embodiment of the invention, the fermentation starting medium comprises per liter: 50-70g glucose, 15-25g yeast nitrogen source without amino acid, 2-3g abscisic acid mixture, 7-10g KH 2 PO 4 ，2-4g MgSO 4 ，0.5-1g ZnSO 4 ·7H 2 O,8-12mL of trace metal solution, 10-15mL of vitamin solution, and the pH of the culture medium is 5-6 (preferably 5.5).
Preferably, the fermentation period is 5-10 days, such as 5, 6, 7, 8, 9, 10 days.
Preferably, said Saccharomyces cerevisiae (A), (B) isSaccharomyces cerevisiae) Expresses CYP450 enzyme, CPR enzyme, glyceraldehyde-3-phosphate dehydrogenase and NADH dependent hydroxymethylglutarate reductase. Wherein,
the CYP450 enzyme is derived from loquat;
the CPR enzyme is from Lotus corniculatus;
the glyceraldehyde-3-phosphate dehydrogenase is derived from Kluyveromyces lactis;
the NADH-dependent hydroxymethylglutarate reductase is derived from Bacillus silicate.
Preferably, the product obtained by the production method also comprises alpha-balsam alcohol.
In a second aspect of the present invention, there is provided ursolic acid obtained by the production method of the first aspect (see fig. 1).
In a third aspect of the present invention, there is provided a method for simultaneously producing ursolic acid and α -balsamic alcohol, said method comprising the production method of the first aspect.
In a fourth aspect of the invention, there is provided ursolic acid and α -balsamic alcohol obtained by the process of the third aspect.
In a fifth aspect of the present invention, there is provided an application of ursolic acid obtained by the above-mentioned production method or method in preparing anti-inflammatory, anti-oxidant or anti-tumor products.
The production method of the invention has the following advantages:
1. according to the invention, through the optimization of fermentation conditions, including galactose induction conditions and addition rules, culture medium conditions, feeding and addition time, fermentation conditions (temperature, pH, rotating speed, dissolved oxygen and the like), and the optimization of fermentation period, the yield of ursolic acid under the condition of a fermentation tank can reach 2331.44 +/-180.87 mg/L, compared with the horizontal yield of a shake flask, the yield is improved by about 25 times, the production capacity of a strain is greatly released, and meanwhile, the yield of alpha-balsamic alcohol can reach 1.21 g/L.
2. The carbon source feeding mode has the obvious characteristic that galactose is intermittently added in the fermentation process of the saccharomyces cerevisiae, so that the galactose can be used as an inducer in the fermentation process, and the fermentation carbon source can be more effectively supplemented.
The "batch" as described herein represents a single charge, but may comprise several single charges throughout the fermentation cycle. Of course, the specific feeding time can be determined according to actual conditions, such as consumption of the fed materials or a certain concentration of the fed materials to be guaranteed, or a certain interval time can be determined for feeding. The amounts charged may be the same or different.
The early stage of the logarithmic growth phase is to averagely divide the logarithmic growth phase into 3 sections according to time, wherein the first section is the early stage of the logarithmic growth phase.
The feeding of the invention refers to the continuous feeding of materials into a fermentation system, and can be started at the beginning of fermentation, can be started at a certain specific moment in the fermentation process, can be stopped at the end of fermentation, and can be stopped at a certain specific moment in the fermentation process. But there may be multiple feedthroughs of the same material, simultaneous feedthroughs of multiple materials, or separate feedthroughs, throughout the fermentation cycle. For example, multiple feeding of the same material may start feeding at a specific time in the whole fermentation cycle, end feeding after a certain time, stop feeding after a certain time, end feeding after a certain time, and the like. The start time, the end time and the fed-batch time of the fed-batch are adjusted according to actual requirements, such as the fed-batch speed, the concentration of fed-batch materials, the required concentration in a fermentation system and the like. The speed of the feeding can also be adjusted according to specific requirements.
The "material" of the present invention may be a medium or its components, water, a reagent for adjusting pH, etc., which are required for conventional fermentation, and may be gas, liquid, solid, semi-solid, etc.
The "inflammation" of the present invention may be inflammation occurring in any tissue including, but not limited to, adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph nodes, lymphoblasts, maxilla, mediastinum, mesenterium, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsil, trachea, uterus, vulva, leukocytes. Further preferably, the inflammation is selected from systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, organ specific inflammatory diseases, allergy (e.g. allergic rhinitis), folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, autoimmune diseases, chronic prostatitis, glomerulonephritis, hypersensitivity, colitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, transplant rejection, vasculitis or interstitial cystitis.
A "tumor" as described herein may be any undesirable cell proliferation (or any disease that manifests itself as undesirable cell proliferation), neoplasm, or increased predisposition or risk of undesirable cell proliferation, neoplasm, or tumor. It may be benign or malignant, or primary or secondary (metastatic). A neoplasm can be any abnormal growth or proliferation of a cell and can be located in any tissue. Examples of tissues include adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph nodes, lymphoblasts, maxilla, mediastinum, mesenterium, myometrium, nasopharynx, omentum, mouth, ovary, pancreas, parotid, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsil, trachea, uterus, vulva, leukocytes. Further preferably, the tumor is selected from the group consisting of prostate cancer, breast cancer, liver cancer, glioma (e.g., glioma), intestinal cancer, cervical cancer, non-small cell lung cancer, pancreatic cancer, gastric cancer, bladder cancer, skin cancer, striated muscle cancer, squamous cell cancer, nasopharyngeal cancer, ovarian cancer, placental villus cancer, lymphoma (e.g., non-hodgkin's lymphoma, cutaneous T-cell lymphoma), leukemia, rectal adenocarcinoma, medulloblastoma, meningioma, neurofibroma (e.g., neurofibrosarcoma), ependymoma, schwannoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myelogenous leukemia, acute myelogenous leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermoid carcinoma, colon cancer, thymus cancer, blood cancer, head and neck cancer, and oropharyngeal cancer.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: is the structural formula of the fermentation product ursolic acid.
FIG. 2: results of medium concentration optimization.
FIG. 3: pH optimization result chart.
FIG. 4: and (4) a galactose induction time optimization result graph.
FIG. 5: ethanol feeding is started at 42h, and the fermentation curve of the saccharomyces cerevisiae WN85 strain is in the condition of a 5L fermentation tank.
FIG. 6: ethanol feeding is started at 48h, and the fermentation curve of the saccharomyces cerevisiae WN85 strain is in a 5L fermentation tank.
Detailed Description
The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The strain used in the experiment is saccharomyces cerevisiae (Saccharomyces cerevisiae) An engineering strain WN85 (WN 85 for short) expressing CYP450 enzyme, CPR enzyme, glyceraldehyde-3-phosphate dehydrogenase and NADH dependent hydroxymethyl glutarate reductase, see patent CN202210432868.3。
The yeast nitrogen source without amino acids was purchased from beijing solilebao scientific ltd, cat #: y8040.
The abscisic acid mixture is prepared by purchasing various amino acid components from Beijing Soilebao science and technology Limited, and the formula is shown in Table 1.
TABLE 1
Figure 224495DEST_PATH_IMAGE001
Example 1: shake flask fermentation condition culture medium optimization
The invention firstly optimizes the culture medium condition at the shake flask level, and in order to balance the growth of the strain and the biosynthesis of ursolic acid, the whole fermentation process is divided into two stages: the first stage is a strain growth stage using glucose as a carbon source, and the second stage is a stage of adding galactose after glucose is exhausted to induce the accumulation of ursolic acid. The fermentation medium comprises the following components: the 1 XSD glucose medium contains 6.7 g/L yeast nitrogen source without amino acid, 20 g/L glucose, 0.8 g/L abscisic acid mixture. The corresponding 1 XSD galactose medium also contained 6.7 g/L yeast nitrogen source without amino acids, 20 g/L galactose, 0.8 g/L abscisic acid mixture. The 2X, 3X, 4X and 5X culture media are multiplied by 2, 3, 4 and 5 in turn according to the mixture ratio.
Taking the activated plate, selecting single colony, inoculating in 1X SD glucose culture medium to prepare fermentation seed liquid according to initial OD 600 The WN85 strain is respectively transferred to 2X, 3X, 4X and 5X glucose culture media in the inoculation amount of 0.1, the culture media are replaced to 2X, 3X, 4X and 5X galactose culture media in the same conditions after being cultured for 48 hours with shaking at 30 ℃, the culture media are continuously cultured for 3 days to extract products, wherein the rotation speed is 200rpm, the pH value is maintained at 6.2 in the fermentation process, and the result is shown in figure 2.
Further optimizing the pH value according to OD 600 Transferring the WN85 strain to a 3X glucose medium, culturing at 30 deg.C for 48 hr under shaking, changing to the 3X galactose medium under the same conditions, and culturing for 3 days to obtain the final product, wherein the rotation speed is 200rpmThe result is shown in FIG. 3.
Further optimization of galactose addition time, as OD 600 The inoculation amount of =0.1 was inoculated in a 3 Xglucose medium from WN85 strain, the culture was continued by changing to the 3 Xgalactose medium under the same conditions after shaking culture at 30 ℃ for 10-60 hours, the total days of culture was 5 days, and the product was extracted, wherein the rotation speed was 200rpm, and the pH was maintained at 5.5 during the fermentation, and the results are shown in FIG. 4.
Example 2: amplification and optimization of saccharomyces cerevisiae ursolic acid fermentation process
As the yield difference of the ursolic acid synthesized by the strain in the 3X culture medium and the 4X culture medium is not large, the 3X fermentation culture medium is finally selected for fed-batch fermentation in consideration of economic cost.
Strain activation: taking out the frozen glycerol strain from an ultralow temperature refrigerator at minus 80 ℃, streaking the glycerol strain on a plate, and culturing the glycerol strain in an incubator at 30 ℃ for 3 days until a single colony appears.
SD-deficient solid medium (1 ×): 6.7 g/L yeast nitrogen source without amino acid, 20 g/L glucose, 0.8 g/L abscisic acid mixture, agar powder 20 g/L, adjusting the pH of the culture medium to 6.2, and sterilizing at 115 ℃ for 15 minutes.
First-order seed liquid is cultured in a test tube: the single colony on the activated plate is selected and inoculated in a test tube containing a culture medium (3X SD glucose medium), and is placed in a shaking table at 200rpm and at 30 ℃ for culturing for 16 hours to obtain mature primary seed liquid.
Shake flask culture of secondary seed liquid: the obtained first-stage seed liquid is transferred to a 100 mL shaking flask containing a culture medium (3 XSD glucose medium) according to the inoculation proportion of 10 percent, and is placed in a shaking table for culturing for 36 hours at the temperature of 30 ℃ and at the rpm of 200, so as to obtain mature second-stage seed liquid.
Shake flask culture of the third seed liquid: the obtained secondary seed liquid is transferred to a 500 mL shaking flask containing a culture medium (3 XSD glucose medium) according to the inoculation proportion of 10 percent, and is placed in a shaking table at 200rpm and 30 ℃ for culturing for 36 hours to obtain mature tertiary seed liquid.
Culturing in a fermentation tank: the liquid loading capacity of 5L fermentation tank is 2.1L, and mature tertiary seed liquid is inoculated according to the inoculation ratio of 10% (after the seed liquid is inoculated into the fermentation tank, the seed liquid is initially inoculatedStarting OD 600 = 10.35) is inoculated in a fermentation tank, a proper amount of antifoaming agent is added, the temperature of the tank is controlled to be 30 ℃ by using condensed water, the air flow is controlled to be 3 vvm, the initial stirring rotation speed is controlled to be 300rpm, the dissolved oxygen is controlled to be about 30%, the pH of the fermentation tank is automatically controlled to be about 5.5 by using ammonia water, the dissolved oxygen cascade rotation speed is set to be 300-800rpm after fermenting for two hours, glucose feeding fermentation medium is started to flow into the fermentation tank after 12 hours of fermentation are consumed (the feeding speed is 30-80mL/h, the concentration of ethanol is guaranteed to be less than 10 g/L), the glucose feeding is stopped at 36 hours of fermentation, galactose is supplemented once at 38 hours of the fermentation process until the final concentration of the system is 60g/L, then galactose is supplemented once at 12 hours from the 3 th day of fermentation, the feeding ethanol is started at 42 hours of the fermentation process, the concentration of the ethanol feeding is controlled to be less than 5g/L all the time in the fermentation process, specifically 12mL of the feeding is started every 3 th day of the strain, the feeding period is 8 days of the strain, the feeding is started every 3 th day of the fermentation process, the medical use of an injector, the micro-fermentation process, the concentration of the vitamin solution is always controlled to be less than 5g/L, the concentration of the vitamin extraction process, and the vitamin extraction result is always maintained in the whole fermentation process after the fermentation process is controlled to be less than 5mL of the vitamin extraction process. Specifically, the output of the ursolic acid can reach 2331.44 +/-180.87 mg/L, and the output of the alpha-balsamic alcohol can reach 1.21 g/L.
In addition, the ethanol feeding time was replaced by starting the feeding at 48h of the fermentation process under different conditions, and the results are shown in FIG. 6. Specifically, the output of the ursolic acid can reach 1567.4 +/-329.45 mg/L, and the output of the alpha-balsamic alcohol can reach 1.75g/L.
The initial fermentation medium used in the fermentor consisted of: 60g/L glucose, 20.1g/L yeast nitrogen source without amino acid, 2.4g/L abscisic acid mixture, 8g/L KH 2 PO 4 ，3g/L MgSO 4 ，0.72g/L ZnSO 4 ·7H 2 O,10mL/L trace metal solution, 12mL/L vitamin solution, and the pH of the culture medium is 5.5.
The glucose feed medium used in the first stage of fermentation consisted of: 500g/L glucose, 13.4g/L yeast nitrogen source without amino acid, 1.6g/L abscisic acid mixture, 9g/L KH 2 PO 4 ，2.5g/L MgSO 4 ，3.5g/L K 2 SO 4 ，0.28g/L Na 2 SO 4 10mL/L trace metal solution and 12mL/L vitamin solution.
The composition of the induced galactose feeding medium used in the fermentation process is as follows: 500g/L galactose, 13.4g/L yeast nitrogen source without amino acid, 1.6g/L abscisic acid mixture, 9g/L KH 2 PO 4 ，2.5g/L MgSO 4 ，3.5g/L K 2 SO 4 ，0.28g/L Na 2 SO 4 10mL/L trace metal solution and 12mL/L vitamin solution.
Vitamin solution composition (1L): 0.05g of biotin, 1g of calcium pantothenate, 1g of nicotinic acid, 25g of inositol, 1g of thiamine hydrochloride, 1g of pyridoxal hydrochloride and 0.2g of p-aminobenzoic acid.
Composition of trace metal solution (1L): EDTA 15g, znSO 4 •7H 2 O 5.75g，MnCl 2 •4H 2 0.32g of O, anhydrous CuSO 4 0.5g，CoCl 2 •6H 2 O 0.47g，Na 2 MoO 4 •2H 2 O 0.48g，CaCl 2 •2H 2 O 2.9g，FeSO 4 •7H 2 O 2.8g。
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are all within the protection scope of the present invention.
It should be noted that, in the above embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present invention does not separately describe various possible combinations.

Claims (7)

1. The production method of ursolic acid is characterized in that the production method comprises two-stage fermentation culture of saccharomyces cerevisiae (C. Cerevisiae)Saccharomyces cerevisiae) The production method comprises the following steps:
a) Activating strains: taking out the frozen glycerol strain from an ultralow temperature refrigerator at minus 80 ℃, and culturing the glycerol strain on an SD defective solid culture medium until a single colony appears;
b) Seed liquid culture: selecting single colony, inoculating in seed culture medium, and culturing in shaker at 25-35 deg.C and 150-250rpm for 30-40h;
c) The first stage fermentation culture: inoculating 8-12% of the inoculum size into a fermentation initial culture medium which takes glucose as a carbon source and takes a yeast nitrogen source without amino acid as a nitrogen source, culturing under the conditions that the pH value is maintained to be 5-6, the temperature is maintained to be 25-35 ℃, and the dissolved oxygen is 30-40%, feeding the glucose feed medium when culturing is started for 10-12h, and stopping feeding in 35-38 h;
d) And (3) second-stage fermentation culture: taking ethanol as a carbon source and a yeast nitrogen source without amino acid as a nitrogen source, wherein the ethanol feeding time is that ethanol is fed from 40 th to 50 th of fermentation, the feeding rate is 28 mL/h to 36mL/h, and after the ethanol is added, the concentration of the ethanol is controlled to be kept below 5 g;
wherein, galactose is added as an inducer at the 35 th-45 th hour of fermentation, and the final concentration of the galactose is 55-65g/L after the addition; and adding a galactose feeding medium, a vitamin solution and a trace metal solution every 10-12h after the fermentation for 40-50 h;
SD-deficient solid media include per liter: 5-8g of yeast nitrogen source without amino acid, 15-25g of glucose, 0.5-1g of abscisic acid mixture and 15-25g of agar powder, and adjusting the pH value of the culture medium to 6-6.5;
the seed culture medium comprises per liter: 15-25g/L yeast nitrogen source without amino acid, 50-70g/L glucose and 2-3g/L abscisic acid mixture;
fermentation initiation medium includes per liter: 50-70g glucose, 15-25g yeast nitrogen source without amino acid, 2-3g abscisic acid mixture, 7-10g KH 2 PO 4 ，2-4g MgSO 4 ，0.5-1g ZnSO 4 ·7H 2 O,8-12mL of trace metal solution, 10-15mL of vitamin solution and the pH value of the culture medium is 5-6.
2. The production process according to claim 1, characterized in thatCharacterized in that the glucose feed medium comprises per liter: 400-600g glucose, 10-15g yeast nitrogen source without amino acid, 1-2g abscisic acid mixture, 8-10g KH 2 PO 4 ，2-3g MgSO 4 ，3-4g K 2 SO 4 ，0.2-0.35g Na 2 SO 4 8-12mL of trace metal solution and 10-15mL of vitamin solution.
3. The production method according to claim 1, wherein,
galactose feed medium included per liter: 400-600g galactose, 10-15g yeast nitrogen source without amino acid, 1-2g abscisic acid mixture, 8-10g KH 2 PO 4 ，2-3g MgSO 4 ，3-4g K 2 SO 4 ，0.2-0.35g Na 2 SO 4 8-12mL of trace metal solution and 10-15mL of vitamin solution;
the vitamin solution comprises per liter: 0.01-0.1g of biotin, 0.5-2g of calcium pantothenate, 0.5-2g of nicotinic acid, 20-30g of inositol, 0.5-2g of thiamine hydrochloride, 0.5-2g of pyridoxal hydrochloride and 0.1-0.3g of p-aminobenzoic acid;
the trace metal solution comprises per liter: 10-20g of EDTA, znSO 4 •7H 2 O 5-6g，MnCl 2 •4H 2 0.2-0.4g of O and anhydrous CuSO 4 0.1-1g，CoCl 2 •6H 2 O 0.2-0.6g，Na 2 MoO 4 •2H 2 O 0.3-0.8g，CaCl 2 •2H 2 O 2-3.5g，FeSO 4 •7H 2 O 2-3.5g。
4. The method according to any one of claims 1 to 3, wherein Saccharomyces cerevisiae (Saccharomyces cerevisiae)Saccharomyces cerevisiae) Expresses CYP450 enzyme, CPR enzyme, glyceraldehyde-3-phosphate dehydrogenase and NADH dependent hydroxymethylglutarate reductase.
5. The production method according to claim 4, wherein,
the CYP450 enzyme is derived from loquat;
the CPR enzyme is from Lotus corniculatus;
the glyceraldehyde-3-phosphate dehydrogenase is derived from Kluyveromyces lactis;
the NADH-dependent hydroxymethylglutarate reductase is derived from a Bacillus silicate.
6. A method for simultaneously producing ursolic acid and α -amyrin, comprising the production method of any one of claims 1 to 5.
7. Use of ursolic acid obtained by the production method or method according to any one of claims 1-6 in the preparation of anti-inflammatory, antioxidant or anti-tumor products.
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