CN110964646B

CN110964646B - Sclerotinia sclerotiorum, application, fermentation medium and preparation method of PF1022A

Info

Publication number: CN110964646B
Application number: CN201911119067.6A
Authority: CN
Inventors: 王昆蓉; 俞岩青; 曾志刚; 田敏; 雷叶明; 马利明
Original assignee: Chengdu University
Current assignee: Chengdu University
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2021-07-27
Anticipated expiration: 2039-11-15
Also published as: CN110964646A

Abstract

The invention discloses a sedum, application, a fermentation medium and a preparation method of PF 1022A; the firmiana firma is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 18132; the sedum basilicum is used for PF1022A production, and has the advantages of fast hypha growth, low hypha concentration, short fermentation time and high fermentation unit.

Description

Sclerotinia sclerotiorum, application, fermentation medium and preparation method of PF1022A

Technical Field

The invention relates to the technical field of biology, and in particular relates to a firmiana, an application, a fermentation medium and a preparation method of PF 1022A.

Background

PF1022A is an anthelmintic with low toxicity to animals, wide prevention and treatment spectrum and good effect, and is the most potential anthelmintic for livestock and pets after macrolide anthelmintics. PF1022A can bind to the amino site of the seven-helix transmembrane receptor like latrophilin isolated from Haemonchus contortus Rudolphi, which acts to induce the influx of external calcium into cells. The mode of action of this new nematicide is quite different from the mechanism of action of known anthelmintics such as benzimidazoles, imidazothiazoles, macrolides. Therefore, the PF1022A insecticide has good effect and also has strong insecticidal effect on drug-resistant parasites.

Currently, PF1022A is synthesized by a solid-phase synthesis method and a biosynthesis method. The solid phase synthesis method has complicated steps and complex process, and is not suitable for large-scale production. The report of producing PF1022A by microbial fermentation is less, and the yield is lower and is limited to laboratory level.

Disclosure of Invention

In view of the above, the present application provides a firmiana, applications thereof, a fermentation medium, and a preparation method of PF 1022A; the sedum basilicum is used for PF1022A production, and has the advantages of fast hypha growth, low hypha concentration, short fermentation time and high fermentation unit.

In order to solve the technical problems, the technical scheme provided by the application is the firmus hypochondriacus, the firmus hypochondriacus (Rosellinia sp.) is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the address is No. 3 of Xilu No.1 of Beijing Korean district, the microorganism research institute of Chinese academy of sciences, the preservation number is CGMCC No.18132, and the preservation date is 2019, 6 months and 19 days.

The invention provides application of the firmiana in production of PF 1022A.

The invention also provides a fermentation medium for culturing the sedum firmum, which comprises, by mass, 1.0-3.5% of maltose, 0.2-1.0% of dextrin, 0.5-2.0% of soybean oil, 0.5-2.0% of malt extract, 0.5-2.5% of soybean meal, 0.5-2.0% of yeast extract, 0.2-0.8% of monopotassium phosphate, 0.2-0.8% of sodium chloride, 0.1-0.3% of magnesium sulfate, 0.1-0.5% of calcium carbonate and the balance of water

Preferably, the fermentation medium comprises, by mass, 1.0-3.5% of maltose, 0.2-1.0% of dextrin, 0.5-2.0% of soybean oil, 0.5-2.0% of malt extract, 0.5-2.5% of soybean meal, 0.5-2.0% of yeast extract, 0.2-0.8% of monopotassium phosphate, 0.2-0.8% of sodium chloride, 0.1-0.3% of magnesium sulfate, 0.1-0.5% of calcium carbonate and the balance of water.

The invention also provides a preparation method of PF1022A, which comprises the following steps: the sedum firmum is fermented to obtain PF 1022A.

Preferably, the method specifically comprises: inoculating the seed liquid of the sclerotium rolfsii into a fermentation culture medium for culture to obtain a fermentation liquid.

Preferably, the method specifically comprises:

the sedum firmum is activated and inoculated in a slant culture medium for culture; obtaining slant strains;

inoculating the slant strains into a seed culture medium for culturing to obtain a seed solution.

Preferably, the method specifically comprises:

activating and inoculating the hypocrea into a preservation and separation slant culture medium at 25-26 ℃, and culturing for 7-10 days to obtain slant strains;

inoculating slant strains into a seed culture medium at the temperature of 25-26 ℃, and culturing at the rotating speed of 230r/min for 3-5 d to obtain a seed solution;

and inoculating the seed liquid to a fermentation medium at the temperature of 25-26 ℃, rotating speed of 230r/min, and culturing for 3-5 d to obtain a fermentation liquid.

Preferably, the seed solution is inoculated to a fermentation medium at the temperature of 25-26 ℃, the rotating speed of 230r/min, and the fermentation is carried out for 135 hours to obtain the fermentation liquid.

Preferably, the slant culture medium consists of 2 to 4 mass percent of malt extract, 0.4 to 1.0 mass percent of casein peptone, 1.0 to 2.5 mass percent of agar and the balance of water;

the seed culture medium comprises, by mass, 0.5-2.0% of soluble starch, 0.5-1.5% of glucose, 0.2-1.5% of malt extract, 0.2-1.5% of cottonseed meal, 0.2-1.5% of soybean cake meal, 0.2-1.5% of yeast extract, 0.05-0.2% of magnesium sulfate, 0.1-0.3% of sodium chloride, 0.1-0.4% of calcium carbonate and the balance of water;

the fermentation medium comprises, by mass, 1.0-3.5% of maltose, 0.2-1.0% of dextrin, 0.5-2.0% of soybean oil, 0.5-2.0% of malt extract, 0.5-2.5% of soybean flour, 0.5-2.0% of yeast extract, 0.2-0.8% of monopotassium phosphate, 0.2-0.8% of sodium chloride, 0.1-0.3% of magnesium sulfate, 0.1-0.5% of calcium carbonate and the balance of water.

Preferably, the slant culture medium consists of 3% of bud extract, 0.6% of casein peptone, 1.5-2% of agar and the balance of water in percentage by mass;

the seed culture medium comprises, by mass, 1% of soluble starch, 1% of glucose, 0.5% of malt extract, 0.5% of cottonseed meal, 0.5% of soybean cake meal, 0.5% of yeast extract, 0.1% of magnesium sulfate, 0.2% of sodium chloride, 0.2% of calcium carbonate and the balance of water;

the fermentation medium comprises, by mass, 2.5% of maltose, 0.5% of dextrin, 1% of soybean oil, 1% of malt extract, 1.5% of soybean meal, 1% of yeast extract, 0.5% of potassium dihydrogen phosphate, 0.5% of sodium chloride, 0.2% of magnesium sulfate, 0.3% of calcium carbonate and the balance of water.

Preferably, the method further comprises: and separating and extracting PF1022A from the fermentation liquor.

Preferably, the fermentation liquor is extracted by ethyl acetate, and after the fermentation liquor is concentrated under reduced pressure, the fermentation liquor is extracted by ethyl acetate for the second time, and the fermentation liquor is concentrated under reduced pressure to obtain concentrated liquor;

separating the concentrated solution by using a high-pressure preparation liquid chromatography system, and collecting target components;

and concentrating and crystallizing the target component under reduced pressure to obtain a finished PF1022A product.

Compared with the prior art, the detailed description of the application is as follows:

according to the invention, the procymidone basidioides obtained by plasma mutation breeding and a composite mutation method combining ultraviolet mutation is improved in the capability of biosynthesizing PF 1022A; verified by a 10L stainless steel fermentation tank, the fermentation tank has the characteristics of fast hypha growth, low hypha concentration (about 24 percent of bacterial concentration) and short fermentation time (135h) in the 10L fermentation tank, and the maximum fermentation unit on the 10L fermentation tank reaches 1.81 g/L; compared with the fermentation unit 1.481g/L of 192h placed on a 5L fermentation tank reported in the prior patent (patent application No. CN107217007A), the fermentation time is obviously shortened, the yield is improved by 22.2 percent to the maximum extent, the fermentation period is shortened, and the energy consumption is reduced.

The normal pressure room temperature plasma (ARTP) mutagenesis system adopted by the invention has the characteristics of low temperature, high active particle concentration and the like, damages genetic materials of strains, induces biological cells to start an SOS repair mechanism, and finally stably inherits to form mutant strains. Therefore, the atmospheric pressure room temperature plasma (ARTP) mutagenesis system can be applied to the culture of the firmicutes as a rapid and simple method.

The invention completely meets the requirements of mutagenesis and screening of industrial PF1022A producing strains, can be applied to industrial fermentation production, and has great economic value.

Drawings

FIG. 1 is a graph showing the results of fermentation titer, cell growth and pH of a mutagenized strain obtained by the composite mutagenesis screening of the present invention.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.

Example 1

Isolation and characterization of Strain SIIA-16-1#

1. Isolation of Strain SIIA-16-1 #:

separating and screening soil sample of Yaan mountain of Sichuan to obtain original strain SIIA-16-1# producing PF 1022A.

Test methods and procedures:

1. preparation of the culture Medium

(1) The formula is as follows: 10.0g of glucose, 5.0g of peptone, 20g of agar, 20g of K2HPO41.0 g, 0.5g of MgSO4 & 7H2O 0.5, 100mL of 1/3000 Bengal aqueous solution, 100mL of 0.03 streptomycin diluent and 800mL of tap water.

(2) The method comprises the following operation steps:

1) mixing the raw materials according to the formula, and boiling until the agar is dissolved.

2) The hot tubes were sub-packaged in 18mm x 180mm tubes, 15mL each, tampons were stoppered, labelled, loaded into small wire baskets and partially wrapped with old newspapers.

3) Sterilizing with high pressure steam at 121 deg.C for 30 min.

Wherein, the preparation process of the 0.03 percent streptomycin solution comprises the following steps: taking a domestic streptomycin 1 g/bottle, sucking 10mL of sterile water by using a sterile straw to dissolve to obtain a 10% streptomycin solution, and taking 0.3mL of the solution to be dissolved in a 100mL sterile volumetric flask to obtain a 0.03% streptomycin solution.

2. Preparation of soil dilution

By the method of "dilution plate countingDiluting the soil sample to 10^-5。

Weighing 10g of soil sample, placing the soil sample into a triangular flask containing 90mL of sterile water, shaking for about 20 minutes to fully mix the soil and the water, and dispersing the bacteria. lmL10 were pipetted with a 1mL sterile pipette under sterile operating conditions^-1The bacterial liquid with the concentration is placed in a tube of 9mL sterile water, the pipette is flushed for three times, and the pipette is shaken up to obtain 10^-2And (4) concentration bacterial liquid. Same method, sequentially diluting to 10^-5。

3. Plate separation

(1) Inverted plate

The Martin's medium to which the streptomycin solution was added was poured to make the medium contain 30. mu.g of streptomycin per ml.

(2) Coating of

The three plates of each medium described above are labeled 10 each^-1、10^-2、10^-3Three dilutions were made, then separately transferred from 10 using three 1mL sterile pipettes^-1、10^-2、10^-3Three tubes of soil dilution were each pipetted 0.1mL in number into plates of the dilution labeled and gently spread evenly over the surface of the medium with a sterile glass spreader bar.

4. Constant temperature culture

And (3) inverting the plate inoculated with the soil diluent to culture at 28 ℃ for 5-7 d.

5. Bacteria selection and purification

Selecting typical fungus colony transfer slant from the plate with single colony, making plate for purity check, and if not, selecting the colony to prepare bacterial suspension for further dilution and separation until pure culture is obtained.

6. Counting

And selecting a plate with 10-100 colonies appearing on each dish, and calculating the content of soil fungi (including yeast).

After the counted dilution is selected, the number of colonies growing on the plate can be counted, and the counting result is calculated according to the following formula:

bacterial count per g sample-average number of colonies repeated several times at the same dilution X10 Xdilution

The viable count of each gram of original soil sample is calculated, and if the viable count is converted into the viable count of each gram of dry soil, the viable count is divided by the mass fraction of the dry soil in the soil sample (the mass of the dried soil/the mass of the original soil sample).

7. Strain preservation

Transferring the separated fungus SIIA-16-1# to a PDA slant for culturing, and storing in ice book after growing.

2. Characterization of Strain SIIA-16-1#

2.1. The shape of the thallus: the thallus is fungus, and does not produce sexual spore or asexual spore.

2.2. Colony morphology: on the plate, the mycelium of the colony is in the form of white villi, and the reverse side of the colony is white to yellowish. Dark brown spots with a diameter of 2-3 mm were observed on the reverse side of the colonies after about 3 weeks of growth.

2.3. The culture characteristics are as follows:

the strain SIIA-16-1# was inoculated on various synthetic media and organic media, cultured at 25-26 ℃ for 7 days, and the corresponding growth characteristics were recorded (see Table 1).

TABLE 1

Kind of culture Medium	Growth conditions	Color of colony back	Soluble pigment
				Potato glucose culture medium	Good, white colony	White to light yellow	Is free of
Potato carrot culture medium	Good, white colony	White to light yellow brown	Is free of
				Malt extract culture medium	Good, white colony	White to light yellow	Is free of
Oat porridge culture medium	Good, white colony	White to light yellow	Is free of

2.4. Physiological and biochemical characteristics of the strain SIIA-16-1 #:

culturing on solid nutrient medium (CzA agar, LcA agar and CMA agar), finding that the optimum growth temperature is 25-26 deg.C, the growth is slow below 25 deg.C, and the strain does not grow above 37 deg.C. The strain is fungus and grows aerobically; glucose, starch slurry, sucrose, dextrin, glycerol, molasses, animal and vegetable oils can be utilized.

CzA agar Medium composition: 3% of glucose, 0.3% of sodium nitrate, 0.1% of dipotassium phosphate, 0.05% of potassium chloride, 0.05% of magnesium sulfate, 0.001% of ferrous sulfate and 1.5% of agar, and the pH is natural;

LcA agar Medium composition: 0.1% of glucose, 0.1% of monopotassium phosphate, 0.02% of magnesium sulfate, 0.02% of potassium chloride, 0.2% of sodium nitrate, 0.02% of yeast extract and 1.5% of agar, wherein the pH is natural;

CMA agar medium composition: corn flour 4%, agar 1.5%, and natural pH.

Example 2

1. Isolation, mutagenesis and characterization of mutagenized Strain SIIA-18-56#

1. Isolation and mutagenesis of mutagenized Strain SIIA-18-56#, and the like

1.1 preparation of bacterial suspension:

separating and screening soil sample of Yaan mountain of Sichuan to obtain original strain SIIA-16-1# producing PF 1022A. Adding 10mL of sterile normal saline into the slant culture of the strain SIIA-16-1# for washing, pouring into a shake flask with cullet, and shaking to disperse to prepare a bacterial suspension for later use.

1.2 composite mutagenesis treatment:

(1) 10 μ L of 1.1 medium bacterial suspension was aspirated by a pipette onto a 1cm diameter slide glass, and placed in an atmospheric pressure room temperature plasma mutagenesis system (ARTP mutagenesis system) with a treatment distance of 2mm using helium as a working gas, a power of 120W, and a ventilation amount of 10 SLM. Respectively treating for 0s, 15s, 30s, 45s, 60s and 80s, diluting the bacterial suspension after the mutagenesis treatment, coating the bacterial suspension on a separation culture medium, and culturing for 7d at 25-26 ℃. According to colony counting and shake flask screening results, the lethality and the positive rate under each irradiation dose are counted.

The experimental results show that: when the ARTP treatment time is 80s, the lethality is close to 100 percent; the positive mutation rate of the strain is 38.2 percent at most and the lethality rate is 92 percent when the mutagenesis time is 45 s. In consideration of obtaining a sufficient number of strains after the complex mutagenesis, the treatment time of ARTP for the complex mutagenesis in this experiment was selected to be 30 seconds.

(2) Sucking 5mL of the bacterial suspension in 1.1 into a sterile culture dish by using a pipette gun, and irradiating the sterile culture dish with ultraviolet (the power is 30W, and the lamp distance is 35cm) for 0s, 20s, 30s, 40s, 60s, 90s and 120s respectively. Diluting the bacterial suspension subjected to the mutagenesis treatment, coating the bacterial suspension on a separation culture medium, and culturing for 7d at 25-26 ℃. According to colony counting and shake flask screening results, the lethality and the positive rate under each irradiation dose are counted.

The experimental results show that: the firmicutes is sensitive to ultraviolet rays, and the lethality rate is close to 100% when the irradiation time is 120 s. The positive mutation rate of the strain is 32.3 percent at most when the irradiation time is 40s, and the lethality rate is 88.6 percent. In consideration of obtaining sufficient strain number after the complex mutagenesis, the UV treatment time adopted by the complex mutagenesis in the experiment is selected to be 30 s.

(3) After the bacterial suspension in 1.1 was treated with the irradiation dose selected in (1) (ARTP treatment time 30s), the cells on the slide glass were washed with sterile physiological saline and immediately (within 30s) subjected to complex mutagenesis treatment with the ultraviolet irradiation time (power 30W, lamp distance 35cm, UV treatment time 30s) selected in (2).

1.3 primary screening by a flat plate:

separating a culture medium: the nutrient medium consists of 3 percent of bud extract, 0.6 percent of casein peptone, 1.5 to 2 percent of agar and the balance of water in percentage by mass, the pH value is 5.6, and the high pressure (1.05 kg/cm) is 121 DEG C²) Steam sterilizing for 30 min.

Diluting with physiological saline in a gradient manner, taking 0.1mL of diluted physiological saline, coating the diluted physiological saline on a separation culture medium, and culturing for 7-10 days at 25-26 ℃.

1.4 shaking flask re-screening:

slant culture medium: the nutrient medium consists of 3 percent of bud extract, 0.6 percent of casein peptone, 1.5 to 2 percent of agar and the balance of water in percentage by mass, the pH value is 5.6, and the high pressure (1.05 kg/cm) is 121 DEG C²) Steam sterilizing for 30 min.

Selecting single colonies of the sedum cruzi grown by the primary screening of the flat plate to inoculate on a slant culture medium, and culturing for 7-10 days at 25-26 ℃;

selecting mutant strain with fermentation unit higher than 30% of original strain, i.e. mutant strain SIIA-18-56 #.

2. Characterization and identification of mutant strain SIIA-18-56#

2.1 cell morphology: the thallus is fungus, and does not produce sexual spore or asexual spore.

2.2 colony morphology: on the plate, the mycelium of the colony is in the form of grey white villi, and the reverse side of the colony is grey white to light yellow.

2.3 culture characteristics:

the strain SIIA-18-56# was inoculated on various synthetic media and organic media, cultured at 25-26 ℃ for 7 days, and the corresponding growth characteristics were recorded (see Table 2).

TABLE 2

Kind of culture Medium	Growth conditions	Color of colony back	Soluble pigment
				Potato glucose culture medium	Good, gray-white bacterial colony	Off-white to light yellow	Is free of
Potato carrot culture medium	Good, gray-white bacterial colony	Off-white to light yellow brown	Is free of
				Malt extract culture medium	Good, gray-white bacterial colony	Off-white to light yellow	Is free of
Oat porridge culture medium	Good, gray-white bacterial colony	Off-white to light yellow	Is free of

2.4 physiological and biochemical characteristics of mutant strain SIIA-18-56 #:

culturing on a solid nutrient medium with rich nutrition (culturing on CzA agar, LcA agar and CMA agar respectively) to find that the optimal growth temperature is 25-26 deg.C, the growth is slow below 25 deg.C, and the strain does not grow above 37 deg.C. The strain is fungus and grows aerobically; glucose, starch, sucrose, dextrin, glycerol, molasses, fructose, maltose, animal and vegetable oils can be utilized; trehalose and xylose were not utilized.

CMA agar medium composition: corn flour 4%, agar 1.5%, and natural pH.

2.5 identification

Combining the above classification data analysis, the mutagenized strain SIIA-18-56# belongs to Sechium Rosellinia, which is different from the reported related strains in Sechium in morphological, physiological and biochemical characteristics, so the mutagenized strain SIIA-18-56# is named as Rosellinia sp.

The mutagenic strain SIIA-18-56# is a sedum firma (Rosellinia sp.), is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and is No. 3 of West Luo No.1 of North Chen of the Korean district, Beijing, and the microorganism research institute of Chinese academy of sciences, with the preservation number of CGMCC No.18132 and the preservation date of 2019, 6 months and 19 days.

Example 3

Culture of mutagenized Strain SIIA-18-56#

1. Preparation of a culture medium:

slant culture medium: the slant culture medium comprises, by mass, 3% of bud extract, 0.6% of casein peptone, 1.5-2% of agar and the balance of water; pH5.6, high pressure at 121 ℃ (1.05 kg/cm)²) Steam sterilizing for 30 min;

seed culture medium: the seed culture medium comprises, by mass, 1% of soluble starch, 1% of glucose, 0.5% of malt extract, 0.5% of cottonseed meal, 0.5% of soybean cake meal, 0.5% of yeast extract, 0.1% of magnesium sulfate, 0.2% of sodium chloride, 0.2% of calcium carbonate and the balance of water; pH7.0, high pressure at 121 ℃ (1.05 kg/cm)²) Steam sterilizing for 30 min;

fermentation medium: the fermentation medium comprises, by mass, 2.5% of maltose, 0.5% of dextrin, 1% of soybean oil, 1% of malt extract, 1.5% of soybean meal, 1% of yeast extract, 0.5% of potassium dihydrogen phosphate, 0.5% of sodium chloride, 0.2% of magnesium sulfate, 0.3% of calcium carbonate and the balance of water; pH7.0, high pressure at 121 ℃ (1.05 kg/cm)²) Steam sterilizing for 30 min.

2. Activating strains:

the strain SIIA-18-56# in example 2 preserved in glycerol is transferred to a slant culture medium and cultured for 7-10 days at 25-26 ℃ to obtain slant strains.

3. And (3) seed culture in a shaking flask:

selecting slant strains with good growth, inoculating into the seed culture medium (25 mL in a 250mL triangular flask) prepared in the step 1, and culturing at 25-26 ℃ and 230r/min for 3-5 d to obtain seed liquid.

4. And (3) shake flask fermentation culture:

the seed solution prepared in 3 was inoculated into the fermentation medium prepared in 1 (25 mL in a 250mL Erlenmeyer flask) at an inoculum size of 10% (v/v), and cultured at a rotation speed of 230r/min for 132 hours to obtain a fermentation broth.

5. Separating and extracting PF1022A from the fermentation liquor:

taking 1mL of fermentation liquor, adding 2mL of acetone, shaking and extracting for 1h, taking supernatant, and carrying out HPLC detection, wherein the yield of PF1022A reaches 1.68 g/L.

Example 4

Production of PF1022A by mutant strain SIIA-18-56#

1. Preparation of a culture medium:

slant culture medium: the slant culture medium comprises, by mass, 3% of bud extract, 0.6% of casein peptone and 1.5-2% of agarAnd the balance of water; pH5.6, high pressure at 121 ℃ (1.05 kg/cm)²) Steam sterilizing for 30 min;

2. Activating strains:

3. And (3) seed culture in a shaking flask:

4. Fermentation culture:

transferring the seed solution prepared in the step 3 into a sterilized 10L stainless steel fermentation tank filled with 6L fermentation medium under the protection of flame according to the inoculation amount of 10% (v/v), wherein the culture conditions are that the rotation speed of a stirring shaft is 420r/min, the temperature is 25-26 ℃, the ventilation amount is 1:1(VVM), the tank pressure is 0.05Mpa, and the fermentation is stopped after 135h of culture to obtain the fermentation liquid.

5. Separating and extracting PF1022A from the fermentation liquor:

pouring 6L of fermentation liquor into a storage tank, adding 6L of ethyl acetate into the storage tank, stirring for 2h at a stirring shaft rotation speed of 50r/min, adding a demulsifier, standing overnight, and removing the fermentation liquor on the lower layer after delamination; transferring the ethyl acetate extract into a concentration device for vacuum concentration, and recovering the vacuum concentrated organic solvent;

performing secondary extraction on the fermentation liquor by using the recovered organic solvent (ethyl acetate), and performing reduced pressure concentration to obtain a concentrated solution; and (2) separating the concentrated solution by using a high-pressure preparation liquid chromatography system, wherein the preparation column is a C18 column, the detection wavelength is 220nm, the mobile phase is acetonitrile-water (volume ratio is 80:20), collecting the target component, concentrating under reduced pressure to dry to obtain a PF1022A component, and crystallizing to obtain 7.6g of a PF1022A finished product.

Example 5

This example differs from example 4 in that:

slant culture medium: the slant culture medium comprises, by mass, 4% of malt extract, 0.4% of casein peptone, 1.0-2.5% of agar and the balance of water;

seed culture medium: the seed culture medium comprises, by mass, 2.0% of soluble starch, 1.5% of glucose, 1.5% of malt extract, 0.2% of cottonseed meal, 1.5% of soybean cake meal, 0.2% of yeast extract, 0.05% of magnesium sulfate, 0.1% of sodium chloride, 0.4% of calcium carbonate and the balance of water, and the pH value is 7.0;

fermentation medium: the fermentation medium comprises, by mass, 1.0% of maltose, 1.0% of dextrin, 2.0% of soybean oil, 0.5% of malt extract, 2.5% of soybean meal, 0.5% of yeast extract, 0.8% of monopotassium phosphate, 0.8% of sodium chloride, 0.3% of magnesium sulfate, 0.5% of calcium carbonate and the balance of water, and the pH value is 7.0.

All other conditions were in full agreement with example 4.

Pouring the obtained 5.6L of fermentation liquor into a storage tank, adding 5.6L of ethyl acetate into the storage tank, stirring for 2h at the rotation speed of a stirring shaft of 50r/min, adding a demulsifier, standing overnight, and removing the fermentation liquor on the lower layer after delamination; transferring the ethyl acetate extract into a concentration device for vacuum concentration, and recovering the vacuum concentrated organic solvent;

performing secondary extraction on the fermentation liquor by using the recovered organic solvent (ethyl acetate), and performing reduced pressure concentration to obtain a concentrated solution; and (2) separating the concentrated solution by using a high-pressure preparation liquid chromatography system, wherein the preparation column is a C18 column, the detection wavelength is 220nm, the mobile phase is acetonitrile-water (volume ratio is 80:20), collecting the target component, concentrating under reduced pressure to dry to obtain a PF1022A component, and crystallizing to obtain 6.6g of a PF1022A finished product.

Example 6

This example differs from example 4 in that:

slant culture medium: the slant culture medium comprises, by mass, 2% of malt extract, 1.0% of casein peptone, 1.0-2.5% of agar and the balance of water;

seed culture medium: the seed culture medium comprises, by mass, 0.5% of soluble starch, 0.5% of glucose, 0.2% of malt extract, 1.5% of cottonseed meal, 0.2% of soybean cake meal, 1.5% of yeast extract, 0.2% of magnesium sulfate, 0.3% of sodium chloride, 0.1% of calcium carbonate and the balance of water, and the pH value is 7.0;

fermentation medium: the fermentation medium comprises, by mass, 3.5% of maltose, 0.2% of dextrin, 0.5% of soybean oil, 2.0% of malt extract, 0.5% of soybean flour, 2.0% of yeast extract, 0.2% of monopotassium phosphate, 0.2% of sodium chloride, 0.1% of magnesium sulfate, 0.1% of calcium carbonate and the balance of water, and the pH value is 7.0.

All other conditions were in full agreement with example 4.

Pouring the obtained 5.8L of fermentation liquor into a storage tank, adding 5.8L of ethyl acetate into the storage tank, stirring for 2h at the rotation speed of a stirring shaft of 50r/min, adding a demulsifier, standing overnight, and removing the fermentation liquor on the lower layer after delamination; transferring the ethyl acetate extract into a concentration device for vacuum concentration, and recovering the vacuum concentrated organic solvent;

performing secondary extraction on the fermentation liquor by using the recovered organic solvent (ethyl acetate), and performing reduced pressure concentration to obtain a concentrated solution; and (2) separating the concentrated solution by using a high-pressure preparation liquid chromatography system, wherein the preparation column is a C18 column, the detection wavelength is 220nm, the mobile phase is acetonitrile-water (volume ratio is 80:20), collecting the target component, concentrating under reduced pressure to dry to obtain a PF1022A component, and crystallizing to obtain 6.8g of a PF1022A finished product.

Example 7

Production of PF1022A by mutant strain SIIA-18-56#

This example differs from example 4 in that:

4. fermentation culture:

transferring the seed solution prepared in the example 4 into a sterilized 10L stainless steel fermentation tank filled with 6L fermentation medium under the protection of flame according to the inoculation amount of 10% (v/v), wherein the culture conditions are that the rotation speed of a stirring shaft is 420r/min, the temperature is 25-26 ℃, the ventilation quantity is 1:1(VVM), the tank pressure is 0.05Mpa, and the fermentation is stopped after culturing for 66h, 72h, 90h, 94h, 114h, 118h, 135h, 144h, 159h and 166h, so as to obtain fermentation liquid.

The fermentation unit of PF1022A and the concentration of mycelia in the fermentation broth were measured by HPLC, and the titer, the amount of bacterial growth and the pH were shown in Table 3, respectively.

TABLE 3

Table 3 and FIG. 1 show that the mutant strain SIIA-18-56# obtained by composite mutagenesis screening has the characteristics of fast hypha growth, low hypha concentration (about 24% of the hypha concentration) and short fermentation time (135h) in a 10L fermentation tank, and the maximum fermentation unit reaches 1.81g/L on a 10L stainless steel fermentation tank. Not only greatly shortens the fermentation period (the fermentation time reported in the patent is 192h, and the tank placing unit is 1.481g/L), reduces the energy consumption, but also improves the fermentation unit to a certain extent.

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

Claims

1. The firmus hypochondriacus (Roselliiasp.) is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the preservation number of CGMCCNo.18132.

2. Use of the firmiana sp of claim 1 for the production of PF 1022A.

3. A preparation method of PF1022A, comprising: fermenting the hypocrea according to claim 1 to obtain PF 1022A.

4. The method according to claim 3, comprising in particular: inoculating the seed liquid of the sclerotium rolfsii into a fermentation culture medium for culture to obtain a fermentation liquid.

5. The method according to claim 4, wherein the method comprises:

6. The method according to claim 4, wherein the method comprises:

and inoculating the seed liquid to a fermentation medium at the temperature of 25-26 ℃, rotating speed of 230-420 r/min, and culturing for 3-5 d to obtain fermentation liquid.

7. The production method according to claim 6,

the slant culture medium comprises, by mass, 2-4% of malt extract, 0.4-1.0% of casein peptone, 1.0-2.5% of agar and the balance of water;

the seed culture medium comprises, by mass, 0.5-2.0% of soluble starch, 0.5-1.5% of glucose, 0.2-1.5% of malt extract, 0.2-1.5% of cottonseed meal, 0.2-1.5% of soybean cake meal, 0.2-1.5% of yeast extract powder, 0.05-0.2% of magnesium sulfate, 0.1-0.3% of sodium chloride, 0.1-0.4% of calcium carbonate and the balance of water;

8. The method of manufacturing according to claim 4, further comprising: and separating and extracting PF1022A from the fermentation liquor.

9. The method according to claim 8, wherein the fermentation broth is extracted with ethyl acetate, concentrated under reduced pressure, extracted with ethyl acetate twice, and concentrated under reduced pressure to obtain a concentrated solution;