CN117187076A - Paecilomyces hepialid mutagenesis strain with high adenosine yield and preparation method and application thereof - Google Patents
Paecilomyces hepialid mutagenesis strain with high adenosine yield and preparation method and application thereof Download PDFInfo
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Abstract
The application discloses a paecilomyces hepialid mutagenesis strain with high adenosine yield, a preparation method and application thereof, wherein the strain is preserved in China general microbiological culture collection center (CGMCC) No.20758 of China national institute of sciences microbiological culture Collection center (China Committee for culture Collection). The strain provided by the application can greatly enhance the expression capability under the condition of little increase of dead weight, and realizes the utilization of raw materials with larger proportion.
Description
Technical Field
The application belongs to the technical field of microbial engineering, and particularly relates to a paecilomyces hepialid mutagenesis strain with high adenosine yield, a preparation method and application thereof.
Background
Cordyceps sinensis is a complex of various parasitic fungi growing on the insect body, is mainly distributed in the Tibetan, yunnan and other areas of China, and is a precious health care product. The body contains rich nutrients such as nucleosides, sterols, essential amino acids, etc., and the nutrients have the effects of preventing and treating various diseases of human body. However, due to the influence of growth conditions, etc., the Cordyceps sinensis has extremely low yield and is not in demand in the market.
Paecilomyces hepiali is fungus separated from Cordyceps sinensis, and the chemical components of mycelium obtained by fermenting and culturing the fungus are basically similar to the main components and pharmacological actions of natural Cordyceps sinensis. Adenosine is a main functional component in Cordyceps sinensis, and has remarkable preventing and treating effects on cardiovascular diseases, chronic bronchitis, liver cirrhosis, hyperlipidemia, etc. The adenosine content in the paecilomyces hepialid mycelia is also one of the main indexes for investigating the quality level of paecilomyces hepialid mycelia powder (the adenosine content is more than or equal to 0.2% specified by Chinese pharmacopoeia is qualified paecilomyces hepialid powder).
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.
The present application has been made in view of the above and/or problems occurring in the prior art.
Therefore, the application aims to overcome the defects in the prior art and provide the paecilomyces hepialid mutagenesis strain with high adenosine yield.
In order to solve the technical problems, the application provides the following technical scheme: a paecilomyces hepialid mutagenesis strain with high adenosine yield is characterized in that: comprises that the paecilomyces hepialid mutant strain is preserved in China general microbiological culture Collection center (CGMCC No. 20758), the preservation time is 10 months and 29 days in 2020, and the preservation address is North Chen West Lu No. 1 of the Korean region of Beijing city.
Another object of the present application is to provide a method for preparing a highly adenosine-producing paecilomyces hepiali mutant strain.
In order to solve the technical problems, the application provides the following technical scheme: a preparation method of a paecilomyces hepialid mutagenesis strain with high adenosine yield comprises the following steps: protoplast preparation: culturing Paecilomyces hepialid in shake flask liquid culture medium, cleaning, adding lysozyme, performing enzymolysis in water bath shaking table, filtering, and collecting filtrate;
atmospheric room temperature plasma mutagenesis: diluting the filtrate, cleaning, re-suspending in physiological saline, performing plasma treatment, eluting and coating on a culture medium;
activation screening: and (3) singly picking strains, performing activation culture on a culture medium, and picking dominant strains after the culture is finished by using a shaking table.
As a preferable scheme of the preparation method of the paecilomyces hepiali mutagenesis strain with high adenosine yield, the application comprises the following steps,
protoplast preparation: culturing Paecilomyces hepialid in shake flask liquid culture medium, cleaning, adding lysozyme, performing enzymolysis in water bath shaking table, filtering, and collecting filtrate;
atmospheric room temperature plasma mutagenesis: diluting the filtrate, cleaning, re-suspending in physiological saline, performing plasma treatment, eluting and coating on a culture medium;
activation screening: and (3) singly picking strains, performing activation culture on a culture medium, and picking dominant strains after the culture is finished by using a shaking table.
As a preferred scheme of the preparation method of the paecilomyces hepiali mutagenesis strain with high adenosine yield, the application comprises the following steps: the paecilomyces hepiali mutant strain is fermented at the end to prepare the adenosine.
As a preferred scheme of the preparation method of the paecilomyces hepiali mutagenesis strain with high adenosine yield, the preparation method comprises the following steps:
inoculating the strain into shake flask seed culture medium for culturing, wherein the culture medium comprises, by weight, 5-10% of bran, 2-4% of glucose, 1-3% of peptone, 1-2% of soybean peptide and KH 2 PO 4 0.1-0.3％、MgSO 4 0.05-0.15％。
As a preferred scheme of the preparation method of the paecilomyces hepiali mutagenesis strain with high adenosine yield, the application comprises the following steps: the expression level of the paecilomyces hepialid mutant strain after the culture is finished is improved by 50% or more compared with that of the wild type, and the dry weight of the thalli is increased by 5% or less.
As a preferred scheme of the preparation method of the paecilomyces hepiali mutagenesis strain with high adenosine yield, the application comprises the following steps: under the condition of subculturing the paecilomyces hepialid mutagenesis strain, the adenosine expression level of the 10 th generation strain is attenuated by 5.5%.
As a preferred scheme of the preparation method of the paecilomyces hepiali mutagenesis strain with high adenosine yield, the application comprises the following steps: the expression level of the paecilomyces hepialid mutant strain is improved by 65% or more compared with that of the wild type adenosine at the level of a fermentation tank, and the dry weight of the bacterial body is increased by 10% or less.
As a preferred scheme of the preparation method of the paecilomyces hepiali mutagenesis strain with high adenosine yield, the application comprises the following steps: under the condition that the paecilomyces hepialid mutagenesis strain is cultured in a shake flask, the culture medium comprises, by weight, 1-2% of glucose, 1-2% of soybean cake powder, 0.2-0.6% of soybean peptide, 1-2% of sucrose, 0.1-0.3% of potassium dihydrogen phosphate, 0.05-0.15% of magnesium sulfate and 0.05-0.1% of defoaming agent.
As a preferred scheme of the preparation method of the paecilomyces hepiali mutagenesis strain with high adenosine yield, the application comprises the following steps: the culture medium used by the paecilomyces hepialid mutant strain at the level of a fermentation tank is 2-4% of soybean cake powder, 0.5-1% of peptone, 2-4% of glucose, 2-4% of molasses and 0.1-0.2% of defoamer
The application has the beneficial effects that:
the application provides a paecilomyces hepialid mutagenesis strain with high adenosine yield and a preparation method and application thereof, and the paecilomyces hepialid mutagenesis strain is obtained by mutagenesis and selection through a normal-pressure room-temperature plasma mutagenesis method. The strain provided by the application can realize larger improvement of the adenosine yield under the condition of no or little increase of dead weight.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a physical diagram of the strain of example 1 of the present application after cultivation and visual observation;
FIG. 2 is a schematic diagram of a fermenter for performing three-stage fermentation according to an embodiment of the present application;
in the figure, figure a is a physical diagram of the fermenter in a higher level inclined top view; FIG. b is a physical view of the fermentation tank at a lower level with inclination.
Detailed Description
In order that the above-recited objects, features and advantages of the present application will become more apparent, a more particular description of the application will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, but the present application may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present application is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the application. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The paecilomyces hepialid mutagenesis strain used in the application is preserved in China general microbiological culture Collection center of China Committee of microbiological culture Collection center of China academy of sciences of China, which is No. 1, 3 in the Korean region of Beijing, 10 months in 2020, and the preservation number is CGMCC No.20758, and the preservation address is No. 3 in the North west, 1, 3 in the Korean region of Beijing;
the paecilomyces hepialid mutagenesis strain used in the application is obtained by using paecilomyces hepialid (purchased from China general microbiological culture collection center (CGMCC) 3.15540) as a starting strain, treating an activated bacterial liquid by using muramidase to obtain paecilomyces hepialid protoplast, and then adopting a normal-pressure room-temperature plasma mutagenesis treatment technology to breed the paecilomyces hepialid mutagenesis strain with high adenosine yield.
Example 1
The application is used for preparing paecilomyces hepialid mutant strains:
(1) Protoplast preparation: paecilomyces hepialid strain is planted in shake flask liquid culture medium, cultured for 5 days at 26 ℃ and 150rpm, centrifuged to obtain wet mycelium, then dissolved and washed for 3-5 times by 0.6MKCl, 3mg/ml lywallzyme solution is added, enzymatic hydrolysis is carried out for 1 hour at 20 ℃ by using a water bath shaker and 100rpm, and then double-layer mirror wiping paper is used for filtration, thus obtaining filtrate.
(2) Atmospheric room temperature plasma mutagenesis: diluting the filtrate to 108cfu/ml, washing with physiological saline for 2-3 times, re-suspending in physiological saline containing 5% glycerol, controlling ARTP power to 120W, air amount to 10SLM, treating for 120s, eluting, and spreading onto slant plate for culturing for 10 days.
(3) Activation screening: the strain is selected one by using a sterile inoculating loop, activated and cultured in a shake flask culture medium, cultured in a shaking table at a constant temperature of 26 ℃ and at 150rpm for 7 days, and the dry weight of mycelium and the adenosine content are measured to screen dominant strains.
The adenosine content detection is determined by referring to the Chinese pharmacopoeia rule 0512.
The strain is identified by the strain, and belongs to Paecilomyces hepiali, and the strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 20758 in the national academy of sciences of China microbiological culture Collection center (China) No. 1, no. 3 of the national academy of sciences of microorganisms in the Korean of Beijing in the 10 th month of the year 2020.
Example 2
The present example was used to perform laboratory level expression comparison of paecilomyces hepiali mutant strains:
inoculating the mutant strain and original strain into shake flask seed culture medium (testa Tritici 10%, glucose 3%, peptone 1%, soybean peptide 1%, KH) 2 PO 4 0.15％、MgSO 4 0.075 In the culture at 26 ℃ and 150rpm for 5 days, the adenosine content of the paecilomyces hepiali mutant strain is obviously improved, the dry weight of mycelium is slightly improved, wherein the adenosine content can reach 0.413g/l, and the adenosine content is improved by 54.1% compared with that of the parent strain; the dry weight of the thalli reaches 15.63g/l, which is improved by 3.78 percent compared with the original strain.
TABLE 1 comparison of active ingredient contents of original strain and mutant strain
| Dry weight (g/l) | Adenosine (g/l) | |
| Original strain | 15.06 | 0.268 |
| Mutant strains | 15.63 | 0.413 |
| Increase ratio (%) | 3.78 | 54.1 |
As can be seen from Table 1, the expression level of the mutant strain provided by the application is greatly improved compared with that of the original strain, and the dry weight of the strain is extremely reduced, namely, the strain provided by the application can realize the improvement of the expression product without self weight increment, and the upper limit of the expression level is improved while a larger proportion of raw materials are used for expressing the product.
Example 3
The present example was used to examine genetic stability of paecilomyces hepiali mutant strains:
the mutagenized strain was continuously passaged for ten generations, each of which was inoculated into the shake flask liquid medium of example 3, cultured at 26℃and 150rpm for 4 days, and the content of the active ingredient was measured after the fermentation was completed. Table 2 investigation of genetic stability of mutagenized strains
As shown in Table 2, the decrease in the dry weight and the expression level of the cells after 10 passages was very small, and the decrease in the performance of the cells after the passages was very small.
Example 4
This example was used to perform fermenter level expression capacity comparison:
three-stage fermentation comparison of Paecilomyces hepiali mutant strain and original strain
(1) Seed tank seed culture
The seed culture medium is: glucose 2%, soybean cake powder 2%, soybean peptide 0.4%, sucrose 2%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.1% and defoaming agent 0.05%. The seed tank culture method comprises the following steps: inoculating the shake flask seed liquid into a 10L primary seed tank (7L liquid loading amount) according to 1-5% of inoculation amount, fermenting at 26 ℃ under 0.05MPa, ventilating at 0.2vvm, and naturally culturing for 70h; then, a 30L secondary seed tank (21L of liquid filling amount) is inoculated according to the inoculation amount of 5 percent, the fermentation temperature is 24 ℃, the tank pressure is 0.05MPa, the ventilation amount is 0.2vvm, the initial PH is natural, and the culture is carried out for 64 hours.
(2) Fermentation tank culture
The fermentation medium is: 3% of soybean cake powder, 0.5% of peptone, 2% of glucose, 4% of molasses and 0.1% of defoaming agent. The fermentation tank culture method comprises the following steps: inoculating the second-stage seed liquid into a 100L fermentation tank (70L of liquid filling amount) according to 10% of inoculation amount, naturally making the initial PH, the tank pressure of 0.05MPa, and culturing for 48h at 24 ℃ and ventilation of 0.2vvm when the fermentation period is 0-20 h; the fermentation period is 20-36 hours, the temperature is 22 ℃, and the ventilation rate is 0.4vvm; the fermentation period is 36-48h, the temperature is 20 ℃, and the ventilation rate is 0.6vvm. The content of adenosine and mycelium of the paecilomyces hepialid mutant strain is obviously improved after fermentation, wherein the content of the adenosine can reach 0.522g/l, and is improved by 66.2 percent compared with the initial strain; the dry weight of the thalli reaches 22.06g/l, which is 9.48 percent higher than that of the original strain.
The adenosine content detection is determined by referring to the Chinese pharmacopoeia rule 0512.
TABLE 3 comparison of active ingredient contents of three-stage fermentation original strains and mutant strains
| Dry weight (g/l) | Adenosine (g/l) | |
| Original strain | 20.15 | 0.314 |
| Mutant strains | 22.06 | 0.522 |
| Increase ratio (%) | 9.48 | 66.2 |
As can be seen from table 3, at the fermenter level significantly more expression levels can be achieved than at the laboratory level, while at the same time less dry weight increase is achieved on the dry weight of the cells than the original strain.
It should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the same, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted without departing from the spirit and scope of the technical solution of the present application, which is intended to be covered in the scope of the claims of the present application.
Claims (9)
1. A paecilomyces hepialid mutagenesis strain with high adenosine yield is characterized in that: comprises that the paecilomyces hepialid mutant strain is preserved in China general microbiological culture Collection center (CGMCC No. 20758), the preservation time is 10 months and 29 days in 2020, and the preservation address is North Chen West Lu No. 1 of the Korean region of Beijing city.
2. A preparation method of a paecilomyces hepialid mutagenesis strain with high adenosine yield is characterized by comprising the following steps: comprises the following steps of the method,
protoplast preparation: culturing Paecilomyces hepialid in shake flask liquid culture medium, cleaning, adding lysozyme, performing enzymolysis in water bath shaking table, filtering, and collecting filtrate;
atmospheric room temperature plasma mutagenesis: diluting the filtrate, cleaning, re-suspending in physiological saline, performing plasma treatment, eluting and coating on a culture medium;
activation screening: and (3) singly picking strains, performing activation culture on a culture medium, and picking dominant strains after the culture is finished by using a shaking table.
3. An application of paecilomyces hepiali mutagenesis strain for high adenosine yield is characterized in that: the paecilomyces hepiali mutant strain is fermented at the end to prepare the adenosine.
4. Use of a highly adenosine producing paecilomyces hepiali mutant strain according to claim 3, characterized in that: the method comprises the following steps:
inoculating the strain into shake flask seed culture medium for culturing, wherein the culture medium comprises, by weight, 5-10% of bran, 2-4% of glucose, 1-3% of peptone, 1-2% of soybean peptide and KH 2 PO 4 0.1-0.3％、MgSO 4 0.05-0.15％。
5. Use of a highly productive adenosine paecilomyces hepiali mutant strain according to claim 3 or 4, characterized in that: the expression level of the paecilomyces hepialid mutant strain after the culture is finished is improved by 50% or more compared with that of the wild type, and the dry weight of the thallus is increased by 5% or less.
6. Use of a highly productive adenosine paecilomyces hepiali mutant strain according to claim 3 or 4, characterized in that: under the condition of subculturing the paecilomyces hepialid mutagenesis strain, the adenosine expression level of the 10 th generation strain is attenuated by 5.5%.
7. Use of a highly productive adenosine paecilomyces hepiali mutant strain according to claim 3 or 4, characterized in that: the paecilomyces hepiali mutant strain has 65% or more improvement compared with the wild adenosine expression level, and the dry weight of the thallus is 10% or less.
8. Use of a highly adenosine producing paecilomyces hepiali mutant strain according to claim 3, characterized in that: under the condition that the paecilomyces hepialid mutagenesis strain is cultured in a shake flask, the culture medium comprises, by weight, 1-2% of glucose, 1-2% of soybean cake powder, 0.2-0.6% of soybean peptide, 1-2% of sucrose, 0.1-0.3% of potassium dihydrogen phosphate, 0.05-0.15% of magnesium sulfate and 0.05-0.1% of defoaming agent.
9. Use of a highly adenosine producing paecilomyces hepiali mutant strain according to claim 3, characterized in that: the paecilomyces hepiali mutant strain uses 2-4% of soybean cake powder, 0.5-1% of peptone, 2-4% of glucose, 2-4% of molasses and 0.1-0.2% of defoamer at the fermentation tank level.
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| CN104911109A (en) * | 2015-05-28 | 2015-09-16 | 吉林大学 | High-yield paecilomyces hepiali mutant strain and cultivation method |
| CN105779299A (en) * | 2016-01-07 | 2016-07-20 | 江苏苏中药业集团股份有限公司 | Paecilomyces hepialid strain capable of realizing high yield of adenosine and mannite type substances and application |
| CN112322504A (en) * | 2020-11-30 | 2021-02-05 | 江苏神华药业有限公司 | Method for increasing adenosine content in paecilomyces hepiali fermentation mycelium |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911109A (en) * | 2015-05-28 | 2015-09-16 | 吉林大学 | High-yield paecilomyces hepiali mutant strain and cultivation method |
| CN105779299A (en) * | 2016-01-07 | 2016-07-20 | 江苏苏中药业集团股份有限公司 | Paecilomyces hepialid strain capable of realizing high yield of adenosine and mannite type substances and application |
| CN112322504A (en) * | 2020-11-30 | 2021-02-05 | 江苏神华药业有限公司 | Method for increasing adenosine content in paecilomyces hepiali fermentation mycelium |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115873718A (en) * | 2022-10-25 | 2023-03-31 | 江苏神华药业有限公司 | Liquid fermentation medium of armillaria mellea mycelium, and preparation method and application thereof |
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