A special device for producing morchella esculenta strain and a high-efficiency production method

Abstract

This invention relates to the field of medicinal plant propagation technology, and discloses a special device and efficient production method for *Agaricus buergerianus* spawn production. The aseptic operation device includes a sterilization chamber and an inoculation chamber that are isolated from each other. The sterilization chamber is equipped with a stepped sterilization rack and matching tray and drain trough adapted for the step-by-step sterilization of *Agaricus buergerianus* explants. The inoculation chamber is equipped with an operating plate and a shelf. The two chambers are connected by a transfer door with a transfer block. Each chamber has a circular operating hole on the front for wearing rubber gloves, which enables closed-loop aseptic operation throughout the entire *Agaricus buergerianus* spawn production process. The production method of this invention is based on the above device and achieves stable production of *Agaricus buergerianus* spawn through steps such as explant sterilization, isolation and purification of germinating bacteria, primary mother culture propagation, secondary original culture preparation, and tertiary cultivation spawn production, combined with a special culture medium system.

Description

Technical Field

[0001] This invention relates to the field of medicinal plant propagation technology, specifically to a special device and efficient production method for producing *Agaricus fasciatus* spawn. Background Technology

[0002] Under natural conditions, the seeds of *Cistanche deserticola* lack endosperm and must rely on symbiotic fungi for nutrition to germinate, resulting in an extremely low natural reproduction rate. Wild resources are nearing depletion due to over-harvesting, making artificial cultivation the only path to ensure the sustainable development of the *Cistanche deserticola* industry. The spawn is the core foundation of *Cistanche deserticola* artificial cultivation. Traditional spawn production mainly relies on asexual reproduction from wild tubers, which not only severely damages wild resources but also presents problems such as difficulty in spawn isolation, low purity of the obtained spawn, susceptibility to contamination by other microorganisms during production, weak mycelial viability, long propagation cycles, and high production costs. Furthermore, existing production models lack specialized and simple operating devices suitable for the step-by-step disinfection and aseptic inoculation process of *Cistanche deserticola* spawn. The production process is mostly open or semi-open, making it difficult to effectively isolate the disinfection and inoculation processes, leading to cross-contamination and inability to accurately control environmental parameters. This results in extremely poor batch stability of the spawn, failing to meet the stable spawn supply requirements for large-scale artificial cultivation of *Cistanche deserticola*, and severely restricting the standardized and regulated development of the *Cistanche deserticola* artificial cultivation industry. To address the numerous shortcomings of the existing technologies, this invention provides a specialized device with a simple structure, convenient operation, and the ability to achieve a closed-loop aseptic operation throughout the entire disinfection and inoculation process. Simultaneously, it is equipped with a standardized and efficient production method based on this device, which can effectively reduce the contamination rate of the strain, shorten the propagation cycle, and improve the viability and batch stability of the strain. This device is suitable for the large-scale production needs of individuals and small-scale production units, promoting the healthy development of the artificial cultivation industry of *Cistanche deserticola*. Summary of the Invention

[0003] The present invention aims to provide a special device and efficient production method for the production of Maoci mushroom spawn, so as to solve the defects of existing Maoci mushroom spawn production processes, such as difficulty in separation, high contamination rate, high risk of cross-contamination, poor batch stability, and lack of special and suitable aseptic operation device.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: a special device for the production of *Gnaphalium affine* spawn, comprising a sterile operating chamber, the interior of which is divided into an independent disinfection chamber and an inoculation chamber by a partition. A sealable transfer door is installed on the partition, and a transfer block for opening and closing is provided on the transfer door. An ultraviolet lamp is fixed to the top of the disinfection chamber, and a stepped disinfection rack is fixed to the bottom of the inner cavity of the disinfection chamber. A tray is placed on the stepped disinfection rack, and a drain trough is provided below the stepped disinfection rack. An ultraviolet lamp is fixed to the top of the inoculation chamber, and an operating plate is provided to the bottom of the inner cavity of the inoculation chamber. A shelf is fixed to the inner wall of the inoculation chamber. Operating holes are provided on the front of both the disinfection chamber and the inoculation chamber, and rubber gloves are sealed and fixed to the operating holes.

[0005] Preferably, as an improvement, the stepped disinfection rack is provided with three layers of bearing steps with height differences, and each bearing step has an independent tray placed on it.

[0006] Preferably, as an improvement, the transfer door is a push-pull sealing door, and the transfer block is fixed to the front of the transfer door, which can drive the transfer door to slide horizontally to open and close. In the closed state, the disinfection chamber and the inoculation chamber are completely isolated.

[0007] Preferably, as an improvement, both the disinfection chamber and the inoculation chamber have two parallel operating holes on their front sides, and each operating hole is sealed with a long-arm rubber glove that extends into the interior of the corresponding chamber.

[0008] A method for efficient production of *Gnaphalium affine* spawn based on any one of the above-described devices includes the following steps: (1) Selection and disinfection of explants: Select healthy mycorrhizae, tubers or mature capsules of Sagittaria trifolia as explants, and use them after surface disinfection and rinsing with sterile water; (2) Isolation and purification of germinating fungi: The treated explants were inoculated into the isolation medium and cultured in the dark at 22~26℃. After the mycelium grew, the fungi were purified by 2~3 tube transfers to obtain high-purity mother culture of symbiotic germinating fungi of *Cymbidium goeringii*. (3) Propagation of primary mother culture: The purified germinating bacteria were inoculated into modified PDA slant medium and cultured at 23-25℃ for 10-15 days to obtain primary mother culture; (4) Preparation of secondary primary culture: The primary mother culture is inoculated into the primary culture medium, bottled or bagged and sterilized by high temperature and high pressure. After cooling, it is cultured in the dark at 23~26℃ for 25~35 days. When the mycelium is fully grown, it is the secondary primary culture. (5) Production of third-level spawn: The second-level original spawn is transferred to the spawn culture medium, and after the same sterilization, inoculation and culture conditions, it is cultured for 30 to 40 days to obtain the third-level spawn that can be directly used for the production of Maocigu.

[0009] Preferably, as an improvement, the specific process of surface disinfection in step (1) is as follows: after the explant is rinsed with running water, it is disinfected with 75% ethanol for 10-30 seconds, disinfected with 0.1% mercuric chloride or sodium hypochlorite solution for 5-10 minutes, rinsed with sterile water 3-5 times, and dried with sterile filter paper.

[0010] Preferably, as an improvement, the isolation culture medium comprises: 200g potato, 20g glucose, 15-18g agar, 1-2g yeast extract, 1000mL water, pH 5.5-6.5.

[0011] Preferably, as an improvement, the modified PDA slant culture medium comprises: 200g potato, 20g sucrose, 16g agar, 1-1.5g peptone, 0.5g magnesium sulfate, 0.5g potassium dihydrogen phosphate, and 1000mL water.

[0012] Preferably, as an improvement, the original culture medium and the cultivated culture medium contain, by weight percentage: 35-45% broadleaf sawdust, 20-25% cottonseed hulls, 15-20% wheat bran, 5-10% corn flour, 5-8% humus, 1-1.5% gypsum, 1-1.5% sucrose, and a moisture content of 60-65%.

[0013] Preferably, as an improvement, the high-temperature and high-pressure sterilization conditions in step (4) are 121°C for 1.5 to 2 hours, and the resulting strains can be directly used for aseptic sowing of Maocigu fruit pods, induction of dragon eggs and artificial cultivation.

[0014] The beneficial effects of this solution are as follows: 1. The aseptic operation device of the present invention has a simple structure, low manufacturing cost, and convenient operation. The stepped disinfection carrier is specifically adapted to the core process of step-by-step disinfection of Maocigu explants, which can effectively reduce the risk of contamination by miscellaneous bacteria during the disinfection process, achieve complete isolation between the disinfection and inoculation processes, and avoid cross-contamination from the source.

[0015] 2. The device of the present invention, through the combination of a closed cavity, an operating hole with rubber gloves, and a sealable transfer door, can achieve completely aseptic operation of the entire process of disinfection and inoculation of *Gnaphalium affine* spawn production, without exposing materials to contact with the external environment, controlling the contamination rate of the entire production process to within 5%, and ensuring the stability of spawn quality.

[0016] 3. The production method of the present invention adopts a standardized process of mycorrhizal tissue separation combined with multi-stage purification and propagation, and with the aseptic environment control of special equipment, it can effectively improve the purity of the symbiotic strain of *Cistanche deserticola*. At the same time, a special culture medium system is configured for the growth characteristics of the symbiotic germination fungus of *Cistanche deserticola*, which can significantly accelerate the mycelial growth rate, shorten the overall propagation cycle of the strain by more than 30%, and greatly improve production efficiency and reduce production costs.

[0017] 4. This invention, through the combination of specialized equipment and standardized production methods, enables standardized control of the entire production process of Maocigu mushroom spawn, effectively improving the batch stability of the spawn. The resulting spawn exhibits strong vitality and stable genetic traits, and can be directly adapted to the entire process of aseptic sowing, egg induction, and artificial cultivation of Maocigu mushroom pods.

[0018] 5. The overall solution of this invention has a high degree of standardization, does not require complex large-scale equipment, and can be well adapted to the needs of individuals and small production units for large-scale production of Maoci mushroom spawn. It has extremely high agricultural promotion value and can effectively promote the standardized development of the artificial cultivation industry of Maoci mushroom. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of an embodiment of a special device for producing Maocigu mushroom spawn according to the present invention; Figure 2 This is a schematic diagram of an embodiment of a special device for producing Maocigu mushroom spawn according to the present invention; Figure 3 for Figure 2 Top view of the middle tray; Figure 4 The image shows a real mushroom cultivated using the special device and efficient production method for producing *Cymbidium goeringii* spawn according to the present invention. Figure 5 The image shows a real mushroom cultivated using the special device and efficient production method for producing *Cymbidium goeringii* spawn according to the present invention. Figure 6 The image shows a real mushroom cultivated using the special device and efficient production method for producing *Cymbidium goeringii* spawn according to the present invention.

[0020] The reference numerals in the accompanying drawings include: 1. Sterilization chamber; 2. Ultraviolet lamp; 3. Transfer door; 301. Transfer block; 4. Inoculation chamber; 5. Shelf; 6. Operating plate; 7. Drain trough; 8. Tray; 801. Hole plug; 9. Stepped sterilization rack; 10. Rubber glove; 11. Operating hole. Detailed Implementation

[0021] The following detailed description illustrates the specific implementation method: The basic implementation examples are as follows: Figure 1 - Appendix Figure 3 As shown: The aseptic operating device for producing *Cymbidium goeringii* spawn used in this embodiment includes an aseptic operating chamber. The interior of the aseptic operating chamber is divided into an independent disinfection chamber 1 and an inoculation chamber 4 by a partition. A closable transfer door 3 is installed on the partition, and a transfer block 301 for opening and closing is provided on the transfer door 3. An ultraviolet lamp 2 is fixed to the top of the disinfection chamber 1. A stepped disinfection rack 9 is fixed to the bottom of the inner cavity of the disinfection chamber 1. A tray 8 is placed on the stepped disinfection rack 9. A drain hole is opened at the bottom of the tray 8, and a plug 801 is installed on the drain hole. A drain trough 7 is provided below the stepped disinfection rack 9. An ultraviolet lamp 2 is fixed to the top of the inoculation chamber 4. An operating plate 6 is provided at the bottom of the inner cavity of the inoculation chamber 4. A shelf 5 is fixed to the inner wall of the inoculation chamber 4. Operating round holes 11 are opened on the front of the chamber of both the disinfection chamber 1 and the inoculation chamber 4. A rubber glove 10 is sealed and fixed on the operating round hole 11.

[0022] The stepped sterilization rack 9 has three layers of support steps with different heights. Each support step has an independent tray 8, and the drainage holes at the bottom of the tray 8 correspond vertically to the drainage groove 7 below. The transfer door 3 is a sliding sealed door. The transfer block 301 is fixed to the front of the transfer door 3, which can drive the transfer door 3 to slide horizontally to open and close. In the closed state, the sterilization chamber 1 and the inoculation chamber 4 are completely isolated. Both the sterilization chamber 1 and the inoculation chamber 4 have two parallel operating holes 11 on their front sides. Each operating hole 11 is sealed with a long-arm rubber glove 10, which extends into the interior of the corresponding chamber.

[0023] The specific implementation process is as follows: Step 1: Pre-treatment of the device. Close the transfer door 3, turn on the ultraviolet lamps 2 on the top of the sterilization chamber 1 and the inoculation chamber 4 respectively, and irradiate continuously for 30 minutes to sterilize the internal space of the sterilization chamber 1 and the inoculation chamber 4. After sterilization, turn off the ultraviolet lamps 2. Open the sealed feeding port of the sterilization chamber 1, and place 75% ethanol solution, 0.1% mercuric chloride solution, sterile water, sterile filter paper, and sterile forceps into the corresponding trays 8 respectively. The tray 8 on the leftmost first step of the stepped sterilization rack 9 contains 75% ethanol solution, the tray 8 on the middle second step contains 0.1% mercuric chloride solution, and the tray 8 on the rightmost third step contains sterile water, sterile filter paper, and sterile forceps. The drainage holes at the bottom of all trays 8 are sealed with plugs 801. After closing the feeding port, turn on the ultraviolet lamps 2 again to sterilize the materials in the sterilization chamber 1 continuously for 20 minutes.

[0024] The second step is the selection and disinfection of explants. Healthy, disease-free, and mechanically undamaged mycorrhizae of *Sagittaria sagittifolia* are selected as explants. After rinsing with running water for 30 minutes to remove surface soil and impurities, they are placed in the disinfection chamber 1. The operator places both hands inside the rubber gloves 10 on the operating hole 11 on the front of the disinfection chamber 1, and completes the entire disinfection process within the closed disinfection chamber 1: using sterile forceps to pick up the explant, placing it in the 75% ethanol solution in the tray 8 of the first step, immersing it for 20 seconds, then removing it and draining off any residual solution; then placing the explant in the 0.1% mercuric chloride solution in the tray 8 of the second step, immersing it for 8 minutes, then removing it and draining off any residual solution; subsequently, transferring the explant to the tray 8 of the third step, rinsing it repeatedly with sterile water 4 times, leaving the waste liquid in the tray 8; after rinsing, removing the plug 801 on the drain hole of the tray 8, allowing the waste liquid to flow into the drain trough 7 below through the drain hole; finally, using sterile filter paper to absorb the moisture from the surface of the explant, completing the explant disinfection process.

[0025] The third step is aseptic inoculation and material transfer. The operator opens the transfer door 3 via transfer block 301, transferring the sterilized explants and sterile forceps from the sealed cavity into the inoculation chamber 4. The transfer door 3 is then closed to maintain isolation between the sterilization chamber 1 and the inoculation chamber 4. The operator places both hands inside rubber gloves 10 through the operating holes 11 on the front of the inoculation chamber 4 and performs the inoculation operation on the operating plate 6 of the inoculation chamber 4. The explants are cut into 5mm segments and inoculated into pre-prepared and sterilized isolation culture medium containing 200g potato, 20g glucose, 16g agar, 1.5g yeast extract, and 1000mL water, with the pH adjusted to 6.0.

[0026] The fourth step is the isolation and purification of the germinating bacteria. The inoculated isolation medium is placed in the dark at a constant temperature of 24℃. After mycelia grow from the cut of the explant, it is placed back into the inoculation chamber 4 to complete two tube transfers for purification. Samples contaminated with other bacteria are removed, and the healthy mycelia are retained to obtain a high-purity mother culture of *Cistanche deserticola* symbiotic germination bacteria.

[0027] Step 5: Propagation of the primary mother culture. The purified germinating mother culture is inoculated into modified PDA slant medium in inoculation chamber 4. The modified PDA slant medium contains 200g potato, 20g sucrose, 16g agar, 1.2g peptone, 0.5g magnesium sulfate, 0.5g potassium dihydrogen phosphate, and 1000mL water. After inoculation, it is incubated at a constant temperature of 24℃ for 14 days. After the mycelium has fully grown on the slant, the primary mother culture is obtained.

[0028] Step 6: Preparation of secondary primary culture. The primary mother culture is inoculated into the primary culture medium in inoculation chamber 4. The primary culture medium contains, by weight percentage, 40% broadleaf sawdust, 23% cottonseed hulls, 18% wheat bran, 8% corn flour, 7% humus, 1.5% gypsum, and 1.5% sucrose, with the water content adjusted to 63%. After the culture medium is bottled, it is sterilized at 121℃ under high temperature and high pressure for 2 hours. After cooling, the inoculation is completed. After inoculation, it is placed in the dark at a constant temperature of 24℃ for 28 days. After the mycelium has fully grown in the culture bottle, the secondary primary culture is obtained.

[0029] Step 7: Production of tertiary spawn. The secondary spawn is transferred to the spawn culture medium in inoculation chamber 4. The spawn culture medium has the same formula as the spawn culture medium. After the same sterilization and inoculation operations, it is placed in the same constant temperature dark culture conditions as the spawn culture for 35 days. After the mycelium has fully grown in the culture bottle and is growing evenly and vigorously, the tertiary spawn that can be directly used for the production of Maocigu is obtained.

[0030] The contamination rate of the strain obtained in this embodiment during the entire production process is 3%, and the propagation cycle is shortened by more than 30% compared with the traditional process. The obtained strain can be directly used for aseptic sowing of *Sagittaria sagittifolia* pods, induction of egg formation, and artificial cultivation. After sowing, germination is uniform, egg formation is normal, and the survival rate of cultivated specimens is high. Actual product images are shown below. Figure 4 , 5 As shown in Figure 6.

[0031] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A special device for producing *Agaricus blazei* spawn, characterized in that: The device includes a sterile operating chamber, the interior of which is divided into an independent disinfection chamber and an inoculation chamber by a partition. A sealable transfer door is installed on the partition, and a transfer block for opening and closing is provided on the transfer door. An ultraviolet lamp is fixed to the top of the disinfection chamber, and a stepped disinfection rack is fixed to the bottom of the inner cavity of the disinfection chamber. A tray is placed on the stepped disinfection rack, and a drain trough is provided below the stepped disinfection rack. An ultraviolet lamp is fixed to the top of the inoculation chamber, and an operating plate is provided to the bottom of the inner cavity of the inoculation chamber. A shelf is fixed to the inner wall of the inoculation chamber. Operating holes are provided on the front of both the disinfection chamber and the inoculation chamber, and rubber gloves are sealed and fixed to the operating holes.

2. The special device for producing *Agaricus blazei* spawn according to claim 1, characterized in that: The stepped disinfection rack is equipped with three layers of support steps with different heights, and each support step has an independent tray.

3. The special device for producing *Agaricus blazei* spawn according to claim 2, characterized in that: The transfer door is a push-pull sealed door. The transfer block is fixed to the front of the transfer door and can drive the transfer door to slide horizontally to open and close. In the closed state, the disinfection chamber and the inoculation chamber are completely isolated.

4. The special device for producing *Agaricus blazei* spawn according to claim 3, characterized in that: Both the disinfection chamber and the inoculation chamber have two parallel operating holes on their front sides. Each operating hole is sealed with a long-arm rubber glove that extends into the corresponding chamber.

5. A method for efficient production of *Gnaphalium affine* spawn based on the apparatus described in any one of claims 1 to 4, characterized in that: Includes the following steps: (1) Selection and disinfection of explants: Select healthy mycorrhizae, tubers or mature capsules of Sagittaria trifolia as explants, and use them after surface disinfection and rinsing with sterile water; (2) Isolation and purification of germinating fungi: The treated explants were inoculated into the isolation medium and cultured in the dark at 22~26℃. After the mycelium grew, the fungi were purified by 2~3 tube transfers to obtain high-purity mother culture of symbiotic germinating fungi of *Cymbidium goeringii*. (3) Propagation of primary mother culture: The purified germinating bacteria were inoculated into modified PDA slant medium and cultured at 23-25℃ for 10-15 days to obtain primary mother culture; (4) Preparation of secondary primary culture: The primary mother culture is inoculated into the primary culture medium, bottled or bagged and sterilized by high temperature and high pressure. After cooling, it is cultured in the dark at 23~26℃ for 25~35 days. When the mycelium is fully grown, it is the secondary primary culture. (5) Production of third-level spawn: The second-level original spawn is transferred to the spawn culture medium, and after the same sterilization, inoculation and culture conditions, it is cultured for 30 to 40 days to obtain the third-level spawn that can be directly used for the production of Maocigu.

6. The method for efficient production of *Agaricus blazei* spawn according to claim 5, characterized in that: The specific process of surface disinfection in step (1) is as follows: after rinsing the explant with running water, it is disinfected with 75% ethanol for 10-30 seconds, disinfected with 0.1% mercuric chloride or sodium hypochlorite solution for 5-10 minutes, rinsed with sterile water 3-5 times, and dried with sterile filter paper.

7. The efficient production method of *Agaricus blazei* strain according to claim 6, characterized in that: The isolation culture medium contains: 200g potato, 20g glucose, 15-18g agar, 1-2g yeast extract, 1000mL water, pH 5.5-6.

5.

8. The method for efficient production of *Agaricus blazei* strain according to claim 7, characterized in that: The modified PDA slant culture medium contains: 200g potato, 20g sucrose, 16g agar, 1-1.5g peptone, 0.5g magnesium sulfate, 0.5g potassium dihydrogen phosphate, and 1000mL water.

9. The efficient production method of *Agaricus blazei* spawn according to claim 8, characterized in that: The original culture medium and the cultivated culture medium contain, by weight percentage: 35-45% broadleaf sawdust, 20-25% cottonseed hulls, 15-20% wheat bran, 5-10% corn flour, 5-8% humus, 1-1.5% gypsum, 1-1.5% sucrose, and a moisture content of 60-65%.

10. The method for efficient production of *Agaricus blazei* strain according to claim 9, characterized in that: The high-temperature and high-pressure sterilization conditions in step (4) are 121°C for 1.5 to 2 hours. The resulting strains can be directly used for aseptic sowing of Maocigu fruit pods, induction of dragon eggs, and artificial cultivation.

Images