Method for producing hirsutella hepialid mycelium through semi-continuous fed-batch fermentation

Abstract

The invention discloses a method for producing Hirsutella hepialid mycelia through semi-continuous fed-batch fermentation. The method specifically comprises the following steps: (1) strain preparation and shake-flask culture; (2) carrying out amplification culture on the seed solution; (3) semi-continuous feeding fermentation culture; (4) filtering fermentation liquor; and (5) low-temperature vacuum drying. According to the fermentation method disclosed by the invention, under the condition that the equipment investment cost is not increased, the process links can be reduced, the fermentation cost is reduced, the productivity is increased, and the product quality is stabilized. And continuous fermentation production can be realized, and equipment is simple and convenient to maintain and easy to clean.

Description

Technical Field

[0001] The present invention belongs to the technical field of fermentative production of medicinal fungi, and particularly relates to a method for semi-continuous fed-batch fermentation to produce mycelia of Hirsutella hepiali Background Art

[0002] Cordyceps sinensis ( Ophiocordyceps Sinensis ) is a complex of entomogenous fungi parasitizing in the larvae of Hepialus armoricanus and the corpses of its host Hepialus armoricanus larvae. It has the effects of tonifying the kidney and lung, resolving phlegm and relieving cough, etc. It is a precious traditional Chinese medicine with a long medicinal history. Due to its specific requirements for the growth environment, the yield is extremely limited. Coupled with the over-excavation for a long time, the ecological environment has been damaged, making the quantity of this precious medicinal material increasingly scarce. With the continuous increase in the demand for cordyceps, wild cordyceps is becoming increasingly in short supply. Modern pharmacology has confirmed that fermented cordyceps mycelia are similar to the active ingredients of Cordyceps sinensis and have similar pharmacological effects. In view of the relatively low cost and easy control of fermentation methods, the current industrial production of mycelia by liquid deep fermentation has become an important way to solve the current shortage of cordyceps resources.

[0003] Hirsutella hepiali powder, that is, fermented cordyceps sinensis powder. It is the dried powder of the mycelia obtained by liquid fermentation culture of the anamorph of Cordyceps sinensis, a fungus of the family Clavicipitaceae isolated from fresh Cordyceps sinensis in Qinghai. Its main uses are to tonify the lung and kidney, benefit essence and qi, and can be used for the adjuvant treatment of cough, asthma, hemoptysis, low back and back pain caused by deficiency of both the lung and kidney, and chronic bronchitis.

[0004] Microbial fermentation production usually mainly adopts two methods: batch fermentation and continuous fermentation. Batch fermentation has some disadvantages, with relatively large consumption of manpower and material resources. Each batch of fermentation requires operations such as charging, sterilization, inoculation, discharging, and cleaning. The procedures are relatively cumbersome, the fermentation cycle is long, resulting in low production efficiency. Compared with batch fed-batch fermentation, the production intensity of continuous fermentation is greatly improved. However, continuous fermentation also has some problems, such as being easily contaminated by miscellaneous bacteria, the strain is prone to degeneration, and the equipment investment is large. In addition, the product concentration of continuous fermentation is relatively low. The invention patent "A method for rapid deep liquid fermentation to produce Hirsutella sinensis powder (CN201610146343.8)" discloses a process for rapid deep fermentation to produce Hirsutella sinensis. Through this process, the production cycle is extended from 25 days to 55 days, so as to obtain more fermented broth. At the same time, this invention patent also points out that Hirsutella sinensis is severely inhibited by the substrate in the early stage of fermentation and severely inhibited by the product in the later stage of fermentation. Summary of the Invention

[0005] The present invention aims to solve the deficiencies existing in the existing batch fermentation and continuous fermentation. For this purpose, a semi-continuous fed-batch fermentation method for producing Hirsutella hepiali powder is provided. Without increasing the equipment investment cost, the process steps can be reduced, the fermentation cost can be lowered, the production capacity can be increased, and the product quality can be stabilized.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A semi-continuous fed-batch fermentation method for producing Hirsutella hepiali mycelium mainly includes the following steps: (1) Preparation of strains and shake flask culture: Under sterile conditions, transfer the Hirsutella hepiali strain to a slant medium and culture it at 15-20 °C for 40-50 days. Then transfer the well-grown slant strain to a sterilized shake flask medium and culture it at 150-200 r / min and 15-20 °C for 30-45 days to obtain a shake flask seed liquid.

[0007] (2) Subculture of the seed liquid: Prepare a seed medium. After sterilization and cooling, inoculate the shake flask seed liquid obtained in step (1) at an inoculation amount of 1.0-5.0% by volume. Culture it at 15-20 °C, tank pressure of 0.03-0.05 Mpa, aeration ratio of 1:0.4-1:0.6, and stirring speed of 100-200 r / min for 10-25 days to obtain a primary seed liquid. The subculture of the seed liquid can be continued, that is, transfer the primary seed liquid to a secondary seed tank at an inoculation amount of 5-10%. Culture it at 15-20 °C, tank pressure of 0.03-0.05 Mpa, aeration ratio of 1:0.4-1:0.6, and stirring speed of 100-200 r / min for 5-15 days to obtain a secondary seed liquid.

[0008] (3)Semi - continuous fed - batch fermentation culture: Add fermentation medium into fermenter F - 1 with a filling coefficient of 0.6 - 0.8. Sterilize at 121 °C for 30 minutes and then cool. When the temperature of the fermentation medium drops to 15 - 20 °C, under aseptic conditions, inoculate the seed liquid through the transfer pipeline with an inoculation amount of 5 - 10% of the volume of the fermentation medium. After inoculation, the culture conditions are a tank pressure of 0.03 - 0.05 Mpa, an aeration ratio of 1:0.6 - 1:1.5, a stirring speed of 100 - 300 r / min, and a temperature of 15 - 20 °C. When the dissolved oxygen content of the fermentation medium drops to 40 - 50%, under aseptic conditions, connect the bottom pipelines of fermenter F - 1 with the sterilized empty fermenters F - 2 and F - 3, divide the fermented liquid in F - 1 equally into 3 fermenters, then close the bottom valve of the fermenter, and supplement the sterilized and cooled fermentation medium in the feed tank into fermenters F - 1, F - 2, and F - 3. The ratio of the volume of the supplemented fermentation medium to the volume of the fermented liquid in the fermenter is 1:0.5 - 1:0.8. Control the culture conditions of the 3 fermenters as a tank pressure of 0.03 - 0.05 Mpa, aeration of 1:0.6 - 1:1.5, a stirring speed of 100 - 300 r / min, and a temperature of 15 - 20 °C. When the dissolved oxygen content of the fermentation culture drops to 40 - 50%, supplement the sterilized and cooled fermentation medium into fermenters F - 1, F - 2, and F - 3 respectively. Stop feeding when the filling coefficient of fermenters F - 1, F - 2, and F - 3 reaches 0.6 - 0.8, and continue the fermentation culture for 5 - 10 days under the above - mentioned fermentation culture parameters, then discharge the tank to obtain the fermentation liquid of Hirsutella hepiali, and this step can also be repeated according to the specific fermentation scale.

[0009] (4)Filtration of the fermentation liquid: Filter the fermentation liquid of Hirsutella hepiali obtained in step (3) through a ceramic membrane or a plate - and - frame filter press to collect the mycelium.

[0010] (5)Low - temperature vacuum drying: Place the mycelium obtained in step (4) under the conditions of a vacuum degree of - 0.065 - - 0.095 Mpa and a temperature of 60 - 80 °C for drying treatment.

[0011] Furthermore, the slant medium in step (1) is: 200.0 g of potato, 20.0 g of glucose, 10.0 g of peptone, 2.0 g of KH2PO4, 1.0 g of MgSO4·7H2O, 0.1 g of VB1, 20.0 g of agar powder, 1000.0 mL of H2O, and the pH is natural.

[0012] Furthermore, the shake - flask medium in step (1) is 30.0 g of glucose, 5.0 g of yeast extract powder, 10.0 g of peptone, 2.0 g of KH2PO4, 1.0 g of MgSO4·7H2O, 0.1 g of VB1. After dissolving each component in water and combining them, make up the volume to 1000 mL and adjust the pH to 6.0 - 6.5.

[0013] Furthermore, the seed culture medium in step (2) is composed of 1.0 - 3.0% glucose, 0.5 - 1.0% yeast extract powder, 0.5 - 1.0% peptone, 0.5 - 1.0% milk powder, 0.1 - 0.3% KH2PO4, 0.1 - 0.3% MgSO4·7H2O, 0.01 - 0.02% VB1, 0.02 - 0.05% antifoaming agent, and the pH is adjusted to 6.0 - 6.5.

[0014] Furthermore, the fermentation culture medium in step (3) is composed of 1.0 - 3.0% glucose, 0.5 - 1.0% yeast extract powder, 0.5 - 1.0% peptone, 1.0 - 2.0% corn flour, 0.1 - 0.3% KH2PO4, 0.1 - 0.3% MgSO4·7H2O, 0.01 - 0.02% VB1, 0.02 - 0.05% antifoaming agent, and the pH is adjusted to 6.0 - 6.5.

[0015] Furthermore, the filtration by ceramic membrane or plate - frame filter press in step (4) is a conventional solid - liquid separation method, and corresponding ceramic membrane or filter cloth can be selected for operation.

[0016] The present invention provides a new method for liquid - state fermentation to produce Hirsutella hepiali powder. The advantages of this method are as follows: (1) The fermentation process adopted in the method of the present invention can reduce the technological links such as strain preparation, shake - flask culture in step 1 and seed - liquid scale - up culture in step 2 without increasing the investment in fixed assets. In the process of expanding culture from the original strain to the first - stage seed, 70 - 95 days can be saved; in the process of expanding culture from the first - stage seed to the second - stage seed, 5 - 15 days can be saved. The fermentation cycle is shortened, the utilization rate of equipment is improved, thereby increasing the production capacity and reducing the production cost.

[0017] (2) The product quality is stable: The fermentation broth of batch fermentation is uniform, the fed - batch fermentation culture medium is the same, the properties of the final fermented broth are stable, and the quality of the mycelium separated by filtration is stable.

[0018] (3) When the production scale of the method of the present invention is expanded, there is no need to add equipment such as supporting first - stage and second - stage seed tanks, reducing the investment in fixed assets and the input cost of production equipment.

[0019] (4) The method of the present invention can achieve continuous fermentation production, and the equipment maintenance is simple, convenient and easy to clean. Specific Embodiments

[0020] To make the content of the present invention easier to understand, the following further describes the technical solutions of the present invention in combination with specific embodiments, but does not limit the scope of the present invention.

[0021] The Hirsutella hepiali strain AF-Os2103 in the following embodiments was provided by Alpha (Fujian) Biotechnology Co., Ltd. and was identified by the China Center for Industrial Culture Collection, with the strain number AF-Os2103.

[0022] The following traditional process refers to CN1478886A, a method for fermenting Hirsutella sinensis liquid strain.

[0023] Example 1 1) Under aseptic conditions, transfer the Hirsutella hepiali strain to a slant medium and culture it at 18°C for 45 days. Then, inoculate the well-grown slant strain into a sterilized shake flask medium and culture it at 180 r / min and 18°C for 35 days to obtain a shake flask seed liquid.

[0024] 2) Inoculate the cultured Hirsutella hepiali shake flask seed liquid into a seed tank with an inoculation amount of 2% and culture it for 16 days to obtain a seed liquid. The seed medium composition is 3.0% glucose, 0.8% yeast extract powder, 1.0% peptone, 0.5% milk powder, 0.02% KH2PO4, 0.02% MgSO4·7H2O, 0.01% VB1, 0.02% antifoaming agent, and the pH is adjusted to 6.0 - 6.5. The seed tank culture conditions are a tank pressure of 0.04 Mpa, an aeration ratio of 1:0.4, a stirring speed of 150 r / min, and a culture temperature of 18°C.

[0025] 3) In a 500L fermenter F-1, add the fermentation medium with a filling coefficient of 0.75. After sterilization and cooling, inoculate the seed liquid into the fermenter at an inoculation amount of 10%. The fermentation medium composition is 2.5% glucose, 1.5% corn flour, 1% peptone, 0.5% yeast powder, 0.2% KH2PO4, 0.2% MgSO4·7H2O, 0.01% VB1, 0.03% antifoaming agent, and the pH value is adjusted to 6.0 - 6.5. After 13 days of fermentation culture, when the dissolved oxygen in the fermentation drops to 48%, under aseptic conditions, the fermentation broth is evenly distributed into fermenters F-1, F-2, and F-3, and 125L of sterilized and cooled fermentation medium is supplemented according to a volume ratio of 1:1 respectively, and fermentation continues for 7 days. When the dissolved oxygen in the fermentation culture drops to 43%, supplement the sterilized and cooled fermentation medium again. When the filling coefficients of the fermentation tanks of F-1, F-2, and F-3 reach 0.75, stop feeding and continue fermentation culture for 5 days, then discharge the tank to obtain the Hirsutella hepiali fermentation broth. The fermentation culture conditions are a tank pressure of 0.04 Mpa, an aeration ratio of 1:0.6, a stirring speed of 200 r / min, and a temperature of 20°C.

[0026] 4) Filtration of the fermentation broth: Select a plate and frame filter press for filtration, with a 200-mesh filter cloth, and a filtration pressure of 0.04 - 0.12 Mpa. Filter the Hirsutella hepiali fermentation broth obtained in step 3) through the plate and frame filter to obtain 25.81 kg, 27.05 kg, and 25.26 kg of wet Hirsutella hepiali mycelia respectively. The water content of the wet mycelia is 65 - 70%.

[0027] 5) Vacuum low-temperature drying: Put the 25.81 kg, 27.05 kg, and 25.26 kg of wet Hirsutella hepiali mycelia obtained in step 4) into a vacuum drying unit for drying to obtain 8.775 kg, 8.925 kg, and 8.588 kg of mycelia respectively. The water content of the mycelia is 3 - 4%. The drying conditions are a temperature of 60°C and a vacuum degree of -0.085 Mpa. Conduct quality inspection according to the method in the drug standard WS3-181(Z-60)-2006(Z)-2010. The test results show that the adenosine content is not less than 0.15%, the mannitol content is not less than 10%, and the amino acid content is not less than 33%.

[0028] 6) Table 1 shows the culture results of the 500L fermenter for the process of the present invention and the traditional batch process. The final filling coefficient of the fermenter is 0.75 for both.

[0029] Table 1 Culture Results of the 500L Fermenter for the Process of the Present Invention and the Traditional Batch Process It can be clearly seen from the above table that 7.665 kg of mycelia can be obtained by fermentation using the traditional process, while using the process of the present invention, an average of 8.763 kg of mycelia can be obtained from three fermenters. After calculation, the mycelia yield has increased by 14.3%. In addition, the quality of the mycelia product obtained by using the process of the present invention is better than that of the product produced by the traditional process. In summary, using the process of the present invention can improve the yield and quality while reducing the production cost.

[0030] Example 2 1) Under aseptic conditions, transfer the Hirsutella hepiali strain to a slant medium and culture it at 18°C for 45 days. Then inoculate the well-grown slant strain into the sterilized shake flask medium and culture it at 180 r / min and 18°C for 35 days to obtain the shake flask seed liquid.

[0031] 2) Inoculate the cultured Hirsutella hepiali shake flask seed liquid into the seed tank at an inoculation amount of 3%, and culture for 14 days to obtain the primary seed liquid. Then, inoculate the primary seed liquid into the secondary seed tank at an inoculation amount of 10% and culture for 8 days to obtain the secondary seed liquid. The seed culture medium consists of 3.0% glucose, 0.8% yeast extract powder, 1.0% peptone, 0.5% milk powder, 0.02% KH2PO4, 0.02% MgSO4·7H2O, 0.01% VB1, 0.02% antifoaming agent, and the pH is adjusted to 6.0 - 6.5. The culture conditions in the seed tank are a tank pressure of 0.04 Mpa, an aeration ratio of 1:0.4, a stirring speed of 150 r / min, and a culture temperature of 18°C.

[0032] 3) In the 1000L fermenter F-1, add the fermentation medium with a filling coefficient of 0.75. After sterilization and cooling, inoculate the cultured seed liquid into the fermenter at an inoculation amount of 10%. The fermentation medium consists of 2.5% glucose, 1.5% corn flour, 1% peptone, 0.5% yeast powder, 0.2% KH2PO4, 0.2% MgSO4·7H2O, 0.01% VB1, 0.03% antifoaming agent, and the pH value is adjusted to 6.0 - 6.5. After 12 days of fermentation culture, when the dissolved oxygen in the fermentation drops to 50%, under aseptic conditions, the fermentation broth is evenly distributed into fermenters F-1, F-2, and F-3, and 250L of the sterilized and cooled fermentation medium is supplemented at a volume ratio of 1:1 respectively, and fermentation continues for 7 days. When the dissolved oxygen in the fermentation culture drops to 47%, the sterilized and cooled fermentation medium is supplemented again. When the filling coefficients of the fermentation tanks of F-1, F-2, and F-3 reach 0.75, stop feeding, continue fermentation culture for 5 days, and then discharge the tank to obtain the Hirsutella hepiali fermentation broth. The fermentation culture conditions are a tank pressure of 0.04 Mpa, an aeration ratio of 1:0.8, a stirring speed of 180 r / min, and a temperature of 20°C.

[0033] 4) Filtration of the fermentation broth: Select a plate and frame filter press for filtration, with a 200-mesh filter cloth and a filtration pressure of 0.04 - 0.12 Mpa. Filter the fermentation broth from the 3 fermenters to obtain 2.2 kg, 2.15 kg, and 2.10 kg of wet mycelia of Hirsutella hepiali respectively. The water content of the wet mycelia is 65 - 70%.

[0034] 5) Vacuum low-temperature drying: Put the 56.48 kg, 53.01 kg, and 53.61 kg of wet mycelia of Hirsutella hepiali obtained in step 4) into a vacuum drying unit for drying to obtain 18.075 kg, 18.023 kg, and 18.225 kg of mycelia respectively. The water content of the mycelia is 3 - 4%. The drying conditions are a temperature of 60°C and a vacuum degree of -0.085 Mpa. Quality inspection is carried out according to the method in the drug standard WS3-181(Z-60)-2006(Z)-2010. The test results show that the adenosine content is not less than 0.14%, the mannitol content is not less than 10%, and the amino acid content is not less than 34%.

[0035] 6) The following table shows the culture results of the 1000L fermenter for the process of the present invention and the traditional batch process, and the final filling coefficient of the fermenter is 0.75 for both.

[0036] Table 2 Culture results of the 1000L fermenter for the process of the present invention and the traditional batch process It can be clearly seen from the above table that 16.245 kg of mycelia can be obtained by fermentation using the traditional process, while using the process of the present invention, an average of 18.229 kg of mycelia can be obtained from three fermenters. After calculation, the mycelia yield has increased by 12.2%. In addition, the quality of the mycelia product obtained by using the process of the present invention is better than that of the product produced by the traditional process. In summary, using the process of the present invention can improve the yield and quality while reducing the production cost.

[0037] The above are only the preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims

1. A semi-continuous fed-batch fermentation method for producing Hirsutella hepiali mycelium, characterized in that: Specifically, it includes the following steps: (1) Preparation of strains and shake flask culture: Under sterile conditions, transfer the Hirsutella hepiali strain to a slant medium and culture it at 15 - 20°C for 40 - 50 days. Then transfer the well-grown slant strain into a shake flask medium and culture it at 150 - 200 r / min and 15 - 20°C for 30 - 45 days to obtain a shake flask seed solution; (2) Subculture of the seed solution for expansion: Inoculate the seed solution obtained in step (1) into a seed medium to obtain a primary seed solution; then continue to inoculate the primary seed solution into a secondary seed tank with an inoculation amount of 5 - 10%, and culture it at 15 - 20°C, a tank pressure of 0.03 - 0.05 Mpa, an aeration ratio of 1:0.4 - 1:0.6, and a stirring speed of 100 - 200 r / min for 5 - 15 days to obtain a secondary seed solution; (3) Semi-continuous fed-batch fermentation culture: Inoculate the seed solution obtained in step (2) into the fermentation medium of fermenter F-1 with a filling coefficient of 0.6 - 0.

8. The culture conditions are a tank pressure of 0.03 - 0.05 Mpa, an aeration ratio of 1:0.6 - 1:1.5, a stirring speed of 100 - 300 r / min, and a temperature of 15 - 20°C. When the dissolved oxygen content in the fermentation medium drops to 40 - 50%, connect the bottom pipelines of fermenter F-1 with fermenters F-2 and F-3 so that the fermentation broth in F-1 is evenly divided into 3 fermenters, then supplement the fermentation medium and continue the culture. When the dissolved oxygen content in the fermentation culture drops to 40 - 50%, supplement the fermentation medium again and continue the fermentation culture for 5 - 10 days and then discharge the tank to obtain the Hirsutella hepiali fermentation broth. Repeat this fed-batch step according to the specific fermentation scale; (4) Filtration of the fermentation broth: Filter the Hirsutella hepiali fermentation broth obtained in step (3) through a ceramic membrane or a plate and frame filter press to collect the mycelium; (5) Low-temperature vacuum drying: Place the mycelium obtained in step (4) under the conditions of a vacuum degree of -0.065 - -0.095 Mpa and a temperature of 60 - 80°C for drying treatment.

2. The method according to claim 1, wherein: The components of the slant medium in step (1) include 200.0 g of potato, 20.0 g of glucose, 10.0 g of peptone, 2.0 g of KH2PO4, 1.0 g of MgSO4·7H2O, 0.1 g of VB1, 20.0 g of agar powder, 1000.0 mL of H2O, and the pH is natural.

3. The method according to claim 1, wherein: The components of the shake flask medium in step (1) include 30.0 g of glucose, 5.0 g of yeast extract powder, 10.0 g of peptone, 2.0 g of KH2PO4, 1.0 g of MgSO4·7H2O, and 0.1 g of VB1.

4. The method according to claim 1, characterized in that: The components of the seed medium in step (2) include 1.0 - 3.0% of glucose, 0.5 - 1.0% of yeast extract powder, 0.5 - 1.0% of peptone, 0.5 - 1.0% of milk powder, 0.1 - 0.3% of KH2PO4, 0.1 - 0.3% of MgSO4·7H2O, 0.01 - 0.02% of VB1, 0.02 - 0.05% of antifoaming agent, and the inoculation amount is 1.0 - 5.0% of the seed medium.

5. The method according to claim 1, wherein: The components of the fermentation medium described in step (3) include 1.0 - 3.0% glucose, 0.5 - 1.0% yeast extract powder, 0.5 - 1.0% peptone, 1.0 - 2.0% corn flour, 0.1 - 0.3% KH2PO4, 0.1 - 0.3% MgSO4·7H2O, 0.01 - 0.02% VB1, 0.02 - 0.05% antifoaming agent, and the inoculation amount of the seed liquid is 5 - 10%.

6. The method according to claim 1, wherein: In step (3), the volume ratio of the supplemented fermentation medium to the fermentation broth volume in the fermenter is 1:0.5 - 1:0.

8.

7. The method according to claim 1, characterized in that: In step (3), the culture conditions for the three fermenters are a tank pressure of 0.03 - 0.05 Mpa, an aeration ratio of 1:0.6 - 1:1.5, a stirring speed of 100 - 300 r / min, and a temperature of 15 - 20 °C.

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