CN117467725A - Fermentation production process of paecilomyces hepiali mycelium powder with high nucleoside content - Google Patents
Fermentation production process of paecilomyces hepiali mycelium powder with high nucleoside content Download PDFInfo
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- 239000002777 nucleoside Substances 0.000 title claims abstract description 24
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- OFEZSBMBBKLLBJ-UHFFFAOYSA-N cordycepine Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)CC1O OFEZSBMBBKLLBJ-UHFFFAOYSA-N 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12P19/38—Nucleosides
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- C12N1/14—Fungi; Culture media therefor
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/79—Paecilomyces
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Abstract
The invention discloses a fermentation production process of paecilomyces hepialid mycelium powder with high nucleoside content, belonging to the technical field of microorganisms. Aiming at the characteristics of relatively low nucleoside content, long fermentation period and complex fermentation process of the paecilomyces hepiali fermentation product, the invention develops a fermentation production process of paecilomyces hepiali mycelium powder with high nucleoside content, and the process steps of the invention specifically comprise: bacterial strain slant inoculation culture, primary shake flask seed culture, secondary shake flask seed culture, seed tank culture, fermentation tank fermentation and hypha post-treatment. The product obtained by the invention has the adenosine content of 0.4 percent, the total amount of adenosine, guanosine and uridine of 1.72 percent, and the nucleoside content is effectively improved and is far higher than that of a commercial product.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a fermentation production process of paecilomyces hepialid mycelium powder with high nucleoside content.
Background
Cordyceps sinensis is only one of 190 kinds of Cordyceps sinensis in China, and is mainly produced on Qinghai-Tibet plateau, and is also called "Cordyceps sinensis" for short. Cordyceps sinensis is a compound of stroma and larva corpse of Cordyceps sinensis of Clavipitaceae on larva of Hepialidae, and has main active ingredients of cordycepin, cordycepic acid, cordyceps polysaccharide, etc. beneficial to human body absorption, and Cordyceps sinensis is a traditional rare medicated diet tonic in China, has good taste, and has effects of nourishing lung and kidney, relieving cough, benefiting deficiency, and nourishing essence and qi. Cordyceps sinensis has the effects of nourishing lung yin, tonifying kidney yang, relieving cough, resolving phlegm, resisting cancer and preventing aging, and is a product for balancing and tonifying yin and yang, and has no contraindication. Cordyceps sinensis can be used for treating tuberculosis, hemoptysis, impotence, spermatorrhea, etc.; the pharmacological effects of the cordyceps sinensis are irreplaceable, the ecological environment is bad, and the like, so that the cordyceps sinensis is precious and rare, along with the continuous improvement of the human living standard, the demand of people for cordyceps sinensis is increased, the resource amount is reduced, and the contradiction between supply and demand is more and more prominent. In order to solve the problem, in recent years, researchers in the biomedical field of China successively separate a plurality of cordyceps fungus strains, and attempt to produce cordyceps mycelia by using an artificial fermentation mode so as to replace natural cordyceps sinensis. Through continuous efforts, several products similar to natural cordyceps in pharmacological, toxicological and efficacy components and the like are produced, and mass production under modern conditions can be realized.
For example, paecilomyces hepiali (Paecilomyces hepiali) is a fungus isolated from fresh Cordyceps sinensis belonging to Penicillium (Paecilomyces Bain) genus Penicillium (Moniliaceae). In 1982, researchers at the institute of medicine and science of China separated from fresh Cordyceps sinensis collected by Qinghai Hualong to obtain Cs-4 strain, and the Cs-4 strain was identified as Paecilomyces hepiali (Hemsl. Li Xiaoming, shao Aijuan, etc. Paecilomyces hepiali name, J. Fungus journal, 2008 (5): 641-644), which was obtained by culturing Paecilomyces hepiali by artificial submerged fermentation, and has been widely used as a substitute for Cordyceps sinensis in Chinese patent medicines and health foods.
The fermentation culture of Cordyceps strain is a precondition for preparing Cordyceps products, and the traditional Cordyceps strain fermentation process has the problems of low fermentation yield and long fermentation time. Thus, processes such as stationary fermentation, batch fermentation, and fed-batch fermentation have appeared, but these fermentation processes still have various problems such as high bacterial contamination rate, long fermentation period, complicated operation process, and low content of active ingredient in the fermentation product. In particular, the nucleoside content of the typical active ingredient of the product obtained by the traditional fermentation process is still at a low level, so how to optimize the fermentation process to obtain paecilomyces hepialid mycelium with high nucleoside content is a technical problem to be solved at present.
Disclosure of Invention
Aiming at the characteristics of relatively low nucleoside content, long fermentation period and complex fermentation process of the paecilomyces hepiali fermentation product in the prior art, the invention develops a fermentation production process of paecilomyces hepiali mycelium powder with high nucleoside content, and the nucleoside content of the obtained product is greatly improved.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a fermentation production process of paecilomyces hepialid mycelium powder with high nucleoside content comprises the following steps: bacterial strain slant inoculation culture, primary shake flask seed culture, secondary shake flask seed culture, seed tank culture, fermentation tank fermentation and hypha post-treatment.
Further, the process steps of the invention are specifically as follows:
(1) Bacterial strain slant inoculation culture: inoculating the purchased strain into a slant culture medium MM, culturing at 23-25deg.C for 7-8 days until mycelia grow over the slant, marking the name and date of preparation of strain on the slant, wrapping with kraft paper, and storing at 4deg.C;
(2) First-stage shake flask seed culture: filling 250mL of primary shake flask seed culture medium into a 500mL triangular flask, sealing with gauze, sterilizing at 121 ℃ for 20 minutes, cooling for later use, picking 3 pieces of the bacteria-carrying agar blocks obtained in the step (1) by using a burning sterilized inoculation hook, inoculating, sealing with gauze, and culturing at 23-25 ℃ for 4-7d at 130-180r/min to obtain primary shake flask seed liquid;
(3) Culturing secondary shake flask seeds: filling 250mL of secondary shake flask seed culture medium into a 500mL triangular flask, sealing with gauze, sterilizing at 121 ℃ for 20 minutes, cooling for standby, sucking 10mL of primary shake flask seed liquid with a sterilizing gun head, inoculating the primary shake flask seed liquid into the secondary shake flask culture medium, and culturing at 23-25 ℃ for 4-7d at 130-180r/min to obtain secondary shake flask seed liquid;
(4) Seed pot culture: inoculating the secondary shake flask seed liquid into a seed tank culture medium according to a mass ratio of 1:80-160, and introducing air volume of 1.0-1.3vvm, tank pressure of 0.02-0.05MPa, temperature of 20-25 ℃, pH and dissolved oxygen nature, and fermenting for 2-3 days to obtain seed tank fermentation liquid;
(5) Fermenting in a fermentation tank: transferring the fermentation liquor of the seed tank into a fermentation tank culture medium for fermentation, wherein the volume ratio of the transferred seed is 1: (7-15), the ventilation rate is 1.0-1.3vvm, the tank pressure is 0.02-0.05MPa, the temperature is 20-25 ℃, the pH is natural, the dissolved oxygen is natural, the fermentation period is 3-4 days, the pH value is stable or slightly rises, the fermentation time is 65-72h, the residual sugar is about 0.9-1.2%, and the fermentation tank is arranged;
(6) Mycelium post-treatment: filtering the material obtained in the step (5) by using 400-500 meshes of gauze, drying for 40-48 hours at 65-75 ℃, controlling the water content of the material to be lower than 7%, grinding, and sieving by using a 80-mesh sieve to obtain the final product.
Further, the strain used in the step (1) is Paecilomyces hepiali (Paecilomyces hepiali) which is purchased from China general microbiological culture collection center, and the strain number is CGMCC No.3.14870. The strain used in the invention can be purchased through China general microbiological culture Collection center without repeated preservation.
Further, the composition of the slant culture medium MM in the step (1) is as follows: KH (KH) 2 PO 4 1.0 g，（NH 4 ） 2 HPO 4 1.5 g，MgSO 4 ·7H 2 O0.6g,Thiamine HCl500 mug, agar powder 15g, distilled water is added to 1L of sterilization pouring plate.
Further, the first-stage shake flask seed culture medium in the step (2) comprises the following components: glucose 2-3%, peptone 1-1.5%, KH 2 PO 4 0.1%、MgSO 4 ·7H 2 O0.03%, the balance being water, the pH is natural, and the mass percentage is calculated.
Further, the step (3) is a secondary shake flask seed cultureThe composition of the base is: 30-60% of wheat bran extract, 2-3% of glucose, 0.5% of peptone and KH 2 PO 4 0.1%，MgSO 4 0.03 percent of pH value, and the mass percent of the pH value is natural.
Further, the composition of the seed tank culture medium in the step (4) is as follows: 30-60% of wheat bran extract, 2-3% of glucose, 2-3.5% of nitrogen source and KH 2 PO 4 0.06%、MgSO 4 ·7H 2 0.04% of O, 0.2-0.3% of simethicone, and the balance of water, wherein the pH is natural, and the weight percentage is calculated; the nitrogen source is peptone and low-temperature soybean cake powder, and the mass ratio of the peptone to the low-temperature soybean cake powder is 2:5.
further, the preparation method of the wheat bran extract comprises the following steps: wheat bran and water are mixed according to the mass ratio of 1: (30-50), mixing, extracting with ultrasonic wave with ultrasonic frequency of 40-60kHz, ultrasonic time of 40-60min, ultrasonic temperature of 70-80deg.C, and filtering with 400-500 mesh gauze.
Further, the composition of the fermentation tank culture medium in the step (5) is as follows: glucose 2-3%, nitrogen source 2-3.5%, KH 2 PO 4 0.06%、MgSO 4 ·7H 2 0.04% of O, 0.2-0.3% of simethicone, and the balance of water, wherein the pH is natural, and the weight percentage is calculated; the nitrogen source is peptone and low-temperature soybean cake powder, and the mass ratio of the peptone to the low-temperature soybean cake powder is 2:5.
further, in the step (6), a small steel mill is used in grinding, and the rotating speed is 24000r/min.
The beneficial effects are that: (1) According to the invention, the purchased strain is subjected to activation culture by using the slant culture medium MM, and the strain is better ensured not to be mutated by using the MM culture medium with relatively poor nutrient content;
(2) In the primary shake flask seed culture process, the culture medium components are all water-soluble components, so that the method is convenient for intuitively observing the bacteria-infection condition in the process of transferring the solid culture medium to the liquid culture medium, and can make corresponding treatment in time;
(3) The invention adds a proper amount of wheat bran extracting solution into the secondary shake flask culture medium and the seed tank culture medium, and the wheat bran extracting solution is beneficial to the rapid proliferation of the bacterial amount in the seed solution, shortens the fermentation period and is beneficial to the accumulation of active substances;
(4) The higher ventilation is adopted in the fermentation culture, so that the metabolic capacity of strains can be enhanced, the accumulation of active substances is increased, and meanwhile, relatively more simethicone defoamer is added, so that the foaming phenomenon generated in the fermentation production process is effectively inhibited;
(5) The product obtained by the invention has the adenosine content of 0.4 percent, the total amount of adenosine, guanosine and uridine of 1.72 percent, and the nucleoside content is effectively improved and is far higher than that of a commercial product.
Detailed Description
The technical scheme of the present invention is further described below with reference to specific examples, but is not limited thereto.
Example 1: a fermentation production process of paecilomyces hepialid mycelium powder with high nucleoside content comprises the following steps: bacterial strain slant inoculation culture, primary shake flask seed culture, secondary shake flask seed culture, seed tank culture, fermentation tank fermentation and hypha post-treatment.
The process steps of the embodiment specifically include:
(1) Bacterial strain slant inoculation culture: inoculating the purchased strain into a slant culture medium MM, culturing at 23deg.C for 7-8 days until mycelium grows to form a slant, marking the name and preparation date of the strain on the grown slant, wrapping with kraft paper, and storing at 4deg.C;
(2) First-stage shake flask seed culture: filling 250mL of primary shake flask seed culture medium into a 500mL triangular flask, sealing with gauze, sterilizing at 121 ℃ for 20 minutes, cooling for later use, picking 3 pieces of the bacteria-carrying agar blocks obtained in the step (1) by using a burning sterilized inoculation hook, inoculating, sealing with gauze, and culturing at 23 ℃ for 7d at 130r/min to obtain primary shake flask seed liquid;
(3) Culturing secondary shake flask seeds: 250mL of secondary shake flask seed culture medium is filled into a 500mL Erlenmeyer flask, a gauze is sealed, sterilization is carried out for 20 minutes at 121 ℃, cooling is carried out for standby, 10mL of primary shake flask seed liquid is sucked by a sterilization gun head, and is connected into the secondary shake flask culture medium, and the culture is carried out for 7d at 23 ℃ and 130 r/min; obtaining a second-stage shake flask seed liquid;
(4) Seed pot culture: inoculating the secondary shake flask seed liquid into a seed tank culture medium according to a mass ratio of 1:160, and obtaining seed tank fermentation liquid, wherein the aeration rate is 1.04vvm, the tank pressure is 0.02-0.05MPa, the temperature is 20 ℃, the pH is natural, the dissolved oxygen is natural, and the fermentation time is 3 days;
(5) Fermenting in a fermentation tank: transferring the fermentation liquor of the seed tank into a fermentation tank culture medium for fermentation, wherein the volume ratio of the transfer is 1:15, the ventilation quantity is 1.04vvm, the tank pressure is 0.02-0.05MPa, the temperature is 20 ℃, the pH is natural, the dissolved oxygen is natural, the fermentation is carried out until the 3 rd day, the pH value is stable or slightly rises, the fermentation time is 72 hours, and the residual sugar is 0.9 percent, and then, the fermentation tank is put down;
(6) Mycelium post-treatment: filtering the material obtained in the step (5) by using 400-500 mesh gauze, drying for 48 hours at 65 ℃, controlling the water content of the material to be lower than 7%, grinding and sieving by using an 80 mesh sieve to obtain a final product.
The strain used in the step (1) is Paecilomyces hepiali (Paecilomyces hepiali) which is purchased from China general microbiological culture collection center, and the strain number is CGMCC No.3.14870. The strain used in the invention can be purchased through China general microbiological culture Collection center without repeated preservation.
The composition of the slant culture medium MM in the step (1) is as follows: KH (KH) 2 PO 4 1.0 g，（NH 4 ） 2 HPO 4 1.5 g，MgSO 4 ·7H 2 O0.6g,Thiamine HCl500 mug, agar powder 15g, distilled water is added to 1L of sterilization pouring plate.
The first-level shake flask seed culture medium in the step (2) comprises the following components: glucose 2%, peptone 1%, KH 2 PO 4 0.1%、MgSO 4 ·7H 2 O0.03%, the balance being water, the pH is natural, and the mass percentage is calculated.
The composition of the secondary shake flask seed culture medium in the step (3) is as follows: 30% of wheat bran extract, 2% of glucose, 0.5% of peptone and KH 2 PO 4 0.1%，MgSO 4 0.03 percent of pH value, and the mass percent of the pH value is natural.
The composition of the seed tank culture medium in the step (4) is as follows: 30% of wheat bran extract, 2% of glucose, 2% of nitrogen source and KH 2 PO 4 0.06%、MgSO 4 ·7H 2 0.04% of O, 0.2% of simethicone, the balance of water, and natural pH, and the weight percentage is calculated; the nitrogen source is peptone and low-temperature soybean cake powder, and the mass ratio of the peptone to the low-temperature soybean cake powder is 2:5.
the preparation method of the wheat bran extract comprises the following steps: wheat bran and water are mixed according to the mass ratio of 1:50, mixing, extracting with ultrasonic wave with ultrasonic frequency of 40kHz and ultrasonic time of 40min at 70deg.C, and filtering with 400-500 mesh gauze.
The composition of the fermentation tank culture medium in the step (5) is as follows: glucose 2%, nitrogen source 2%, KH 2 PO 4 0.06%、MgSO 4 ·7H 2 0.04% of O, 0.2% of simethicone, the balance of water, and natural pH, and the weight percentage is calculated; the nitrogen source is peptone and low-temperature soybean cake powder, and the mass ratio of the peptone to the low-temperature soybean cake powder is 2:5.
and (3) grinding in the step (6) by using a small steel mill, wherein the rotating speed is 24000r/min.
Example 2: a fermentation production process of paecilomyces hepialid mycelium powder with high nucleoside content comprises the following steps: bacterial strain slant inoculation culture, primary shake flask seed culture, secondary shake flask seed culture, seed tank culture, fermentation tank fermentation and hypha post-treatment.
The process steps of the embodiment specifically include:
(1) Bacterial strain slant inoculation culture: inoculating the purchased strain into a slant culture medium MM, culturing at 25deg.C for 7-8 days until mycelia grow to form a slant, marking the name and preparation date of the strain on the slant, wrapping with kraft paper, and storing at 4deg.C;
(2) First-stage shake flask seed culture: filling 250mL of primary shake flask seed culture medium into a 500mL triangular flask, sealing with gauze, sterilizing at 121 ℃ for 20 minutes, cooling for later use, picking 3 pieces of the bacteria-carrying agar blocks obtained in the step (1) by using a burning sterilized inoculation hook, inoculating, sealing with gauze, and culturing at 25 ℃ for 4 days at 180r/min to obtain primary shake flask seed liquid;
(3) Culturing secondary shake flask seeds: 250mL of secondary shake flask seed culture medium is filled into a 500mL triangular flask, a gauze is sealed, the temperature is 121 ℃, sterilization is carried out for 20 minutes, cooling is carried out for standby, 10mL of primary shake flask seed liquid is sucked by a sterilization gun head, and is connected into the secondary shake flask culture medium, and the culture is carried out for 4d at the temperature of 25 ℃ and at the speed of 180 r/min; obtaining a second-stage shake flask seed liquid;
(4) Seed pot culture: inoculating the secondary shake flask seed liquid into a seed tank culture medium according to a mass ratio of 1:160, and obtaining seed tank fermentation liquid, wherein the aeration rate is 1.3vvm, the tank pressure is 0.02-0.05MPa, the temperature is 25 ℃, the pH is natural, the dissolved oxygen is natural, and the fermentation time is 3 days;
(5) Fermenting in a fermentation tank: transferring the fermentation liquor of the seed tank into a fermentation tank culture medium for fermentation, wherein the volume ratio of the transfer is 1:10, the ventilation quantity is 1.3vvm, the tank pressure is 0.02-0.05MPa, the temperature is 25 ℃, the pH is natural, the dissolved oxygen is natural, the fermentation is carried out until the 3 rd day, the pH value is stable or slightly rises, the fermentation time is 70h, the residual sugar is 1.0%, and the fermentation tank is put down;
(6) Mycelium post-treatment: filtering the material obtained in the step (5) by using 400-500 mesh gauze, drying at 75 ℃ for 48 hours, controlling the water content of the material to be lower than 7%, grinding, and sieving by using an 80-mesh sieve to obtain the final product.
The strain used in the step (1) is Paecilomyces hepiali (Paecilomyces hepiali) which is purchased from China general microbiological culture collection center, and the strain number is CGMCC No.3.14870. The strain used in the invention can be purchased through China general microbiological culture Collection center without repeated preservation.
The composition of the slant culture medium MM in the step (1) is as follows: KH (KH) 2 PO 4 1.0 g，（NH 4 ） 2 HPO 4 1.5 g，MgSO 4 ·7H 2 O0.6g,Thiamine HCl500 mug, agar powder 15g, distilled water is added to 1L of sterilization pouring plate.
The first-level shake flask seed culture medium in the step (2) comprises the following components: glucose 3%, peptone 1.5%, KH 2 PO 4 0.1%、MgSO 4 ·7H 2 O0.03%, the balance being water, the pH is natural, and the mass percentage is calculated.
The composition of the secondary shake flask seed culture medium in the step (3) is as follows: 60% of wheat bran extract, 3% of glucose, 0.5% of peptone and KH 2 PO 4 0.1%，MgSO 4 0.03 percent of pH value, and the mass percent of the pH value is natural.
The composition of the seed tank culture medium in the step (4) is as follows: 60% of wheat bran extract, 3% of glucose, 3.5% of nitrogen source and KH 2 PO 4 0.06%、MgSO 4 ·7H 2 0.04% of O, 0.3% of simethicone, the balance of water, and natural pH, and the weight percentage is calculated; the nitrogen source is peptone and low-temperature soybean cake powderThe mass ratio of the two is 2:5.
the preparation method of the wheat bran extract comprises the following steps: wheat bran and water are mixed according to the mass ratio of 1:30, extracting with ultrasonic wave with ultrasonic frequency of 60kHz and ultrasonic time of 60min at 80deg.C, and filtering with 400-500 mesh gauze.
The composition of the fermentation tank culture medium in the step (5) is as follows: glucose 3%, nitrogen source 3.5%, KH 2 PO 4 0.06%、MgSO 4 ·7H 2 0.04% of O, 0.3% of simethicone, the balance of water, and natural pH, and the weight percentage is calculated; the nitrogen source is peptone and low-temperature soybean cake powder, and the mass ratio of the peptone to the low-temperature soybean cake powder is 2:5.
and (3) grinding in the step (6) by using a small steel mill, wherein the rotating speed is 24000r/min.
Example 3: a fermentation production process of paecilomyces hepialid mycelium powder with high nucleoside content comprises the following steps: bacterial strain slant inoculation culture, primary shake flask seed culture, secondary shake flask seed culture, seed tank culture, fermentation tank fermentation and hypha post-treatment.
The process steps of the embodiment specifically include:
(1) Bacterial strain slant inoculation culture: inoculating the purchased strain into a slant culture medium MM, culturing at 25deg.C for 7-8 days until mycelia grow to form a slant, marking the name and preparation date of the strain on the slant, wrapping with kraft paper, and storing at 4deg.C;
(2) First-stage shake flask seed culture: filling 250mL of primary shake flask seed culture medium into a 500mL triangular flask, sealing with gauze, sterilizing at 121 ℃ for 20 minutes, cooling for later use, picking 3 pieces of the bacteria-carrying agar blocks obtained in the step (1) by using a burning sterilized inoculation hook, inoculating, sealing with gauze, and culturing at 25 ℃ for 5 days at 150r/min to obtain primary shake flask seed liquid;
(3) Culturing secondary shake flask seeds: 250mL of secondary shake flask seed culture medium is filled into a 500mL triangular flask, a gauze is sealed, the temperature is 121 ℃, sterilization is carried out for 20 minutes, cooling is carried out for standby, 10mL of primary shake flask seed liquid is sucked by a sterilization gun head, and is connected into the secondary shake flask culture medium, and the culture is carried out for 5 days at 25 ℃ and 150 r/min; obtaining a second-stage shake flask seed liquid;
(4) Seed pot culture: inoculating the secondary shake flask seed liquid into a seed tank culture medium according to a mass ratio of 1:80, and carrying out tank pressure of 0.02-0.05MPa, temperature of 23 ℃, pH nature, dissolved oxygen nature and fermentation time of 3 days to obtain a seed tank fermentation liquid;
(5) Fermenting in a fermentation tank: transferring the fermentation liquor of the seed tank into a fermentation tank culture medium for fermentation, wherein the volume ratio of the transferred seed is 1:7, the aeration rate is 1.2vvm, the tank pressure is 0.02-0.05MPa, the temperature is 23 ℃, the pH is natural, the dissolved oxygen is natural, the fermentation period is 4 days, the pH value is stable or slightly rises, the fermentation time is 65h, the residual sugar is 1.2%, and the fermentation tank is put down;
(6) Mycelium post-treatment: filtering the material obtained in the step (5) by using 400-500 mesh gauze, drying at 70 ℃ for 48 hours, controlling the water content of the material to be lower than 7%, grinding, and sieving by using an 80-mesh sieve to obtain the final product.
The strain used in the step (1) is Paecilomyces hepiali (Paecilomyces hepiali) which is purchased from China general microbiological culture collection center, and the strain number is CGMCC No.3.14870. The strain used in the invention can be purchased through China general microbiological culture Collection center without repeated preservation.
The composition of the slant culture medium MM in the step (1) is as follows: KH (KH) 2 PO 4 1.0 g，（NH 4 ） 2 HPO 4 1.5 g，MgSO 4 ·7H 2 O0.6g,Thiamine HCl500 mug, agar powder 15g, distilled water is added to 1L of sterilization pouring plate.
The first-level shake flask seed culture medium in the step (2) comprises the following components: glucose 3%, peptone 1%, KH 2 PO 4 0.1%、MgSO 4 ·7H 2 O0.03%, the balance being water, the pH is natural, and the mass percentage is calculated.
The composition of the secondary shake flask seed culture medium in the step (3) is as follows: 60% of wheat bran extract, 3% of glucose, 0.5% of peptone and KH 2 PO 4 0.1%，MgSO 4 0.03 percent of pH value, and the mass percent of the pH value is natural.
The composition of the seed tank culture medium in the step (4) is as follows: wheat bran extract 50%, glucose 3%, nitrogen source 3%, KH 2 PO 4 0.06%、MgSO 4 ·7H 2 O0.04%, simethicone 0.2%, water in balance, natural pH, according to mass percentA percentage meter; the nitrogen source is peptone and low-temperature soybean cake powder, and the mass ratio of the peptone to the low-temperature soybean cake powder is 2:5.
the preparation method of the wheat bran extract comprises the following steps: wheat bran and water are mixed according to the mass ratio of 1:30, extracting with ultrasonic wave with ultrasonic frequency of 50kHz and ultrasonic time of 50min at 75deg.C, and filtering with 400-500 mesh gauze.
The composition of the fermentation tank culture medium in the step (5) is as follows: glucose 3%, nitrogen source 2%, KH 2 PO 4 0.06%、MgSO 4 ·7H 2 0.04% of O, 0.3% of simethicone, the balance of water, and natural pH, and the weight percentage is calculated; the nitrogen source is peptone and low-temperature soybean cake powder, and the mass ratio of the peptone to the low-temperature soybean cake powder is 2:5.
and (3) grinding in the step (6) by using a small steel mill, wherein the rotating speed is 24000r/min.
Example 4: a fermentation production process of paecilomyces hepialid mycelium powder with high nucleoside content comprises the following steps: bacterial strain slant inoculation culture, primary shake flask seed culture, secondary shake flask seed culture, seed tank culture, fermentation tank fermentation and hypha post-treatment.
The process steps of the embodiment specifically include:
(1) Bacterial strain slant inoculation culture: inoculating the purchased strain into a slant culture medium MM, culturing at 25deg.C for 7-8 days until mycelia grow to form a slant, marking the name and preparation date of the strain on the slant, wrapping with kraft paper, and storing at 4deg.C;
(2) First-stage shake flask seed culture: filling 250mL of primary shake flask seed culture medium into a 500mL triangular flask, sealing with gauze, sterilizing at 121 ℃ for 20 minutes, cooling for later use, picking 3 pieces of the bacteria-carrying agar blocks obtained in the step (1) by using a burning sterilized inoculation hook, inoculating, sealing with gauze, and culturing at 25 ℃ for 7 days at 130r/min to obtain primary shake flask seed liquid;
(3) Culturing secondary shake flask seeds: 250mL of secondary shake flask seed culture medium is filled into a 500mL triangular flask, a gauze is sealed, the temperature is 121 ℃, sterilization is carried out for 20 minutes, cooling is carried out for standby, 10mL of primary shake flask seed liquid is sucked by a sterilization gun head, and is connected into the secondary shake flask culture medium, and the culture is carried out for 6d at 25 ℃ and 150 r/min; obtaining a second-stage shake flask seed liquid;
(4) Seed pot culture: inoculating the secondary shake flask seed liquid into a seed tank culture medium according to a mass ratio of 1:100, and obtaining seed tank fermentation liquid, wherein the aeration rate is 1.3vvm, the tank pressure is 0.02-0.05MPa, the temperature is 23 ℃, the pH is natural, the dissolved oxygen is natural, and the fermentation time is 2 days;
(5) Fermenting in a fermentation tank: transferring the fermentation liquor of the seed tank into a fermentation tank culture medium for fermentation, wherein the volume ratio of the transfer is 1:8, the ventilation quantity is 1.3vvm, the tank pressure is 0.02-0.05MPa, the temperature is 23 ℃, the pH is natural, the dissolved oxygen is natural, the fermentation is carried out until the 3 rd day, the pH value is stable or slightly rises, the fermentation time is 71h, and the residual sugar is 1.0%, and then the fermentation tank is put down;
(6) Mycelium post-treatment: filtering the material obtained in the step (5) by using 400-500 mesh gauze, drying for 40 hours at 65 ℃, controlling the water content of the material to be lower than 7%, grinding and sieving by using an 80 mesh sieve to obtain a final product.
The strain used in the step (1) is Paecilomyces hepiali (Paecilomyces hepiali) which is purchased from China general microbiological culture collection center, and the strain number is CGMCC No.3.14870. The strain used in the invention can be purchased through China general microbiological culture Collection center without repeated preservation.
The composition of the slant culture medium MM in the step (1) is as follows: KH (KH) 2 PO 4 1.0 g，（NH 4 ） 2 HPO 4 1.5 g，MgSO 4 ·7H 2 O0.6g,Thiamine HCl500 mug, agar powder 15g, distilled water is added to 1L of sterilization pouring plate.
The first-level shake flask seed culture medium in the step (2) comprises the following components: glucose 3%, peptone 1.5%, KH 2 PO 4 0.1%、MgSO 4 ·7H 2 O0.03%, the balance being water, the pH is natural, and the mass percentage is calculated.
The composition of the secondary shake flask seed culture medium in the step (3) is as follows: 40% of wheat bran extract, 3% of glucose, 0.5% of peptone and KH 2 PO 4 0.1%，MgSO 4 0.03 percent of pH value, and the mass percent of the pH value is natural.
The composition of the seed tank culture medium in the step (4) is as follows: wheat bran extract 50%, glucose 2.5%, nitrogen source 3%, KH 2 PO 4 0.06%、MgSO 4 ·7H 2 O 0.04%0.2% of simethicone, the balance being water, and the pH is natural, and the weight percentage is calculated; the nitrogen source is peptone and low-temperature soybean cake powder, and the mass ratio of the peptone to the low-temperature soybean cake powder is 2:5.
the preparation method of the wheat bran extract comprises the following steps: wheat bran and water are mixed according to the mass ratio of 1:40, mixing, extracting with ultrasonic wave with ultrasonic frequency of 45kHz, ultrasonic time of 50min, ultrasonic temperature of 80deg.C, and filtering with 400-500 mesh gauze.
The composition of the fermentation tank culture medium in the step (5) is as follows: glucose 2.5%, nitrogen source 2.5%, KH 2 PO 4 0.06%、MgSO 4 ·7H 2 0.04% of O, 0.3% of simethicone, the balance of water, and natural pH, and the weight percentage is calculated; the nitrogen source is peptone and low-temperature soybean cake powder, and the mass ratio of the peptone to the low-temperature soybean cake powder is 2:5.
and (3) grinding in the step (6) by using a small steel mill, wherein the rotating speed is 24000r/min.
Comparative example 1: fermented Cordyceps powder (CS-4) is used as reference material, and is used in Chinese food and drug inspection institute.
Comparative example 2: paecilomyces hepiali mycelium powder, jiangsu Shenhua pharmaceutical Co., ltd.
Comparative example 3: the commercial fermented cordyceps sinensis powder (CS-4) is a crude drug, and is a country of Jiangxi medicine Limited liability company.
Comparative example 4: commercially available capsule (Jinshuibao capsule).
Comparative example 5: this comparative example was conducted in the same manner as in example 4, except that the wheat bran extract was not added to the secondary shake flask medium and the seed pot medium in step (3) and step (4). Namely:
the composition of the secondary shake flask seed culture medium in the step (3) is as follows: glucose 3%, peptone 0.5%, KH 2 PO 4 0.1%，MgSO 4 0.03 percent of water and the balance of natural pH value, and the mass percent of the water is calculated.
The composition of the seed tank culture medium in the step (4) is as follows: glucose 2.5%, nitrogen source 3%, KH 2 PO 4 0.06%、MgSO 4 ·7H 2 0.04% of O, 0.2% of simethicone, the balance of water, and natural pH, and the weight percentage is calculated; the nitrogen source is peptone and low temperatureSoybean cake powder, wherein the mass ratio of the soybean cake powder to the soybean cake powder is 2:5.
comparative example 6: this comparative example was the same as example 4 except that Paecilomyces hepialid with the strain number of CGMCC No.3.15540 was selected, namely:
the strain used in the step (1) is Paecilomyces hepiali, and is purchased from China general microbiological culture collection center, and the strain number is CGMCC No.3.15540. The strain can be purchased through China general microbiological culture Collection center without repeated preservation.
And (3) detecting relevant active ingredients:
the detection of the content of adenosine, guanosine and uridine according to the related method of the first edition of the Chinese pharmacopoeia 2020 edition shows that the content of adenosine, guanosine and uridine is as follows:
table 1 test results (mass percent)
It should be noted that the above-mentioned embodiments are merely some, but not all embodiments of the preferred mode of carrying out the invention. It is evident that all other embodiments obtained by a person skilled in the art without making any inventive effort, based on the above-described embodiments of the invention, shall fall within the scope of protection of the invention.
Claims (9)
1. A fermentation production process of paecilomyces hepialid mycelium powder with high nucleoside content is characterized by comprising the following steps: bacterial strain slant inoculation culture, primary shake flask seed culture, secondary shake flask seed culture, seed tank culture, fermentation tank fermentation and hypha post-treatment.
2. The fermentation production process of paecilomyces hepialid mycelium powder with high nucleoside content according to claim 1, which is characterized by comprising the following steps:
(1) Bacterial strain slant inoculation culture: inoculating the purchased strain into a slant culture medium MM, culturing at 23-25deg.C for 7-8 days until mycelium grows over the slant, and storing at 4deg.C;
(2) First-stage shake flask seed culture: filling 250mL of primary shake flask seed culture medium into a 500mL triangular flask, sealing with gauze, sterilizing at 121 ℃ for 20 minutes, cooling for later use, picking 3 pieces of the bacteria-carrying agar blocks obtained in the step (1) by using a burning sterilized inoculation hook, inoculating, sealing with gauze, and culturing at 23-25 ℃ for 4-7d at 130-180r/min to obtain primary shake flask seed liquid;
(3) Culturing secondary shake flask seeds: filling 250mL of secondary shake flask seed culture medium into a 500mL triangular flask, sealing with gauze, sterilizing at 121 ℃ for 20 minutes, cooling for standby, sucking 10mL of primary shake flask seed liquid with a sterilizing gun head, inoculating the primary shake flask seed liquid into the secondary shake flask culture medium, and culturing at 23-25 ℃ for 4-7d at 130-180r/min to obtain secondary shake flask seed liquid;
(4) Seed pot culture: inoculating the secondary shake flask seed liquid into a seed tank culture medium according to a mass ratio of 1:80-160, and introducing air volume of 1.0-1.3vvm, tank pressure of 0.02-0.05MPa, temperature of 20-25 ℃, pH and dissolved oxygen nature, and fermenting for 2-3 days to obtain seed tank fermentation liquid;
(5) Fermenting in a fermentation tank: transferring the fermentation liquor of the seed tank into a fermentation tank culture medium for fermentation, wherein the volume ratio of the transferred seed is 1: (7-15), the ventilation rate is 1.0-1.3vvm, the tank pressure is 0.02-0.05MPa, the temperature is 20-25 ℃, the pH is natural, the dissolved oxygen is natural, the fermentation period is 3-4 days, the pH value is stable or slightly rises, the fermentation lasts for 65-72h, the residual sugar is 0.9-1.2%, and the fermentation tank is arranged;
(6) Mycelium post-treatment: filtering the material obtained in the step (5) by using 400-500 meshes of gauze, drying for 40-48 hours at 65-75 ℃, controlling the water content of the material to be lower than 7%, grinding, and sieving by using a 80-mesh sieve to obtain the final product.
3. The process for producing the high-nucleoside-content paecilomyces hepiali mycelium powder by fermentation according to claim 2, wherein the strain used in the step (1) is paecilomyces hepialiPaecilomyces hepiali) Purchased from China general microbiological culture collection center, and the strain number is CGMCC No.3.14870.
4. A fermentation production process of high-nucleoside-content paecilomyces hepiali mycelium powder according to claim 2, which comprisesCharacterized in that the composition of the slant culture medium MM in the step (1) is as follows: KH (KH) 2 PO 4 1.0g，（NH 4 ） 2 HPO 4 1.5g，MgSO 4 ·7H 2 O0.6g,Thiamine HCl500 mug, agar powder 15g, distilled water is added to 1L of sterilization pouring plate.
5. The process for producing the paecilomyces hepialid mycelium powder with high nucleoside content by fermentation according to claim 2, wherein the primary shake flask seed culture medium in the step (2) comprises the following components: glucose 2-3%, peptone 1-1.5%, KH 2 PO 4 0.1%、MgSO 4 ·7H 2 O0.03%, the balance being water, the pH is natural, and the mass percentage is calculated.
6. The process for producing the paecilomyces hepialid mycelium powder with high nucleoside content by fermentation according to claim 2, wherein the composition of the secondary shake flask seed culture medium in the step (3) is as follows: 30-60% of wheat bran extract, 2-3% of glucose, 0.5% of peptone and KH 2 PO 4 0.1%，MgSO 4 0.03 percent of pH value, and the mass percent of the pH value is natural.
7. The process for producing the paecilomyces hepialid mycelium powder with high nucleoside content by fermentation according to claim 2, wherein the composition of the seed tank culture medium in the step (4) is as follows: 30-60% of wheat bran extract, 2-3% of glucose, 2-3.5% of nitrogen source and KH 2 PO 4 0.06%、MgSO 4 ·7H 2 0.04% of O, 0.2-0.3% of simethicone, and the balance of water, wherein the pH is natural, and the weight percentage is calculated; the nitrogen source is peptone and low-temperature soybean cake powder, and the mass ratio of the peptone to the low-temperature soybean cake powder is 2:5.
8. the process for producing the high-nucleoside-content paecilomyces hepialid mycelium powder by fermentation according to claim 6 or 7, wherein the preparation method of the wheat bran extract is as follows: wheat bran and water are mixed according to the mass ratio of 1: (30-50), mixing, extracting with ultrasonic wave with ultrasonic frequency of 40-60kHz, ultrasonic time of 40-60min, ultrasonic temperature of 70-80deg.C, and filtering with 400-500 mesh gauze.
9. The process for producing the paecilomyces hepialid mycelium powder with high nucleoside content by fermentation according to claim 2, wherein the composition of the fermentation tank culture medium in the step (5) is as follows: glucose 2-3%, nitrogen source 2-3.5%, KH 2 PO 4 0.06%、MgSO 4 ·7H 2 0.04% of O, 0.2-0.3% of simethicone, and the balance of water, wherein the pH is natural, and the weight percentage is calculated; the nitrogen source is peptone and low-temperature soybean cake powder, and the mass ratio of the peptone to the low-temperature soybean cake powder is 2:5.
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