Culture box and culture method for efficiently culturing beauveria bassiana spore powder

Abstract

The invention discloses a culture box for efficiently culturing beauveria bassiana conidial powder and a culture method.The culture box comprises a transparent box body, a cover body is arranged at the upper end of the box body, a sealing ring matched with the box body for use is arranged at the lower end of the cover body, an air hole is formed in the cover body, and a supporting net is arranged in the air hole; the bottom of the box body is provided with air holes, the air holes are provided with air-permeable sealing films installed on the supporting net, the length and the width of the box body are 20-25 cm, the height of the box body is 5-8 cm, the diameter of the air holes is 2-3 cm, and the bottom of the box body is in a continuous wave shape from left to right. When the culture box is used for culturing beauveria bassiana, pollution can be effectively avoided, the culture box has the advantages of being easy to operate, low in equipment cost and low in technical requirement, and the wavy bottom of the culture box can increase the bottom area, improve the ventilation effect and guarantee balance of the temperature of the bottom in the culture box.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and particularly relates to a culture box for efficiently culturing Beauveria bassiana spore powder and a culturing method thereof. Background Art

[0002] Stiff silkworm is the dried whole worm of the 4th to 5th instar larvae of the silkworm Bombyx mori that have died of natural infection (or artificial inoculation) with Beauveria. As a traditional Chinese medicine, stiff silkworm has significant medicinal value, with effects such as calming endogenous wind and stopping convulsions, expelling wind and relieving pain, resolving phlegm and dissipating nodules, etc. Modern pharmacological research also shows that stiff silkworm contains a variety of pharmacologically active substances, such as more than 10 polypeptides, 12 fatty acids, 4 flavonoids, 6 nucleosides, 8 steroids, as well as coumarin, oligosaccharides, polysaccharides and many other components, with effects such as anti-spasm, anti-epilepsy, anticoagulation, anti-tumor, antibacterial, antioxidant, hypoglycemic and enhancing immune function. Currently, there are more than a hundred Chinese patent prescriptions published that contain the ingredient of stiff silkworm. With the in-depth research on its medicinal value, the clinical application demand for stiff silkworm has also increased accordingly, with a large market prospect. Relevant data reports that the total consumption of stiff silkworm was estimated to be about 400 tons in 1991; the market demand in 2015 was about 750 - 800 tons. After 2020, the market demand has been strong, and the total market demand has remained above 6,000 tons since 2022. However, at present, the stiff silkworm in the Chinese medicine market still mainly comes from the dead silkworms that are naturally infected with Beauveria during the breeding process. With the improvement of silkworm breeding and disease prevention technology, the number of silkworms naturally infected with Beauveria is decreasing, and the stiff silkworm has been in short supply in the international and domestic Chinese herbal medicine markets. Some enterprises and regions directly obtain spores from the silkworm bodies of naturally infected or artificially infected stiff silkworms for re-inoculation. The quality of the Beauveria bassiana spore powder obtained by this method is unstable, with serious contamination by miscellaneous bacteria, and there are risks of the occurrence of bacterial silkworm diseases and incomplete stiffening, which easily causes breeding losses.

[0003] At present, Beauveria bassiana conidia powder is generally produced by a liquid-solid two-phase fermentation process. Liquid-solid two-phase fermentation means that a large amount of mycelia or blastospores are first rapidly produced through liquid fermentation, and then inoculated onto a solid nutrient or inert matrix for solid fermentation to produce aerial conidia that are closest to the natural inoculum form. The production of Beauveria bassiana spore powder has problems such as a relatively long production cycle, easy contamination by miscellaneous bacteria during the solid culture process, and unstable spore quality. Li Yinping, Lei Zhongren, and Wang Haihong used a fresh-keeping bag for fermentation, which could not form a solid fermentation space and did not have ventilation, which was not conducive to spore growth, and the spore powder culture time was long (Chinese Journal of Biological Control, 2013, 29(2)219-226); in the patent "An Industrial Production Method of Beauveria bassiana Bals. var. sinensis Powder" (CN109321470A), the use of open shallow pan fermentation was easily contaminated by environmental microorganisms, and the labor cost for temperature control management such as ventilation was high; in the patent "A Method for Culturing Beauveria bassiana Spore Powder in a Bacterial Bag" (CN110305801 B), using bacterial bag fermentation required placing a mushroom ring and a sterile breathable sealing film on the bacterial bag, and the operation was relatively complex. The artificial cultivation of Beauveria bassiana for inoculating the 5th instar silkworm larvae to produce stiff silkworms has relatively high economic benefits. However, during the artificial production process of stiff silkworms, due to the long production cycle of Beauveria bassiana spore powder, rich medium nutrition, and suitable temperature conditions, it is extremely easy to cause serious contamination by miscellaneous bacteria, resulting in unstable spore powder quality, and there are risks of the occurrence of bacterial silkworm diseases and incomplete stiffening, which are likely to cause breeding losses. Summary of the Invention

[0004] The purpose of the present invention is to provide a culture box and a culture method for efficiently culturing Beauveria bassiana spore powder. The culture box solves the problems that shallow pans and fresh-keeping bags are easily contaminated during the culture process and the operation of culture devices such as bacterial bags is cumbersome. The present invention uses a solid-liquid two-way fermentation method. First, Beauveria bassiana spores are quickly germinated in a liquid medium. In the solid culture stage, the use of the culture box is simple and not easily contaminated, and it can make the most of the advantages of producing aerial conidia on the solid surface, shortening the mycelium growth cycle and increasing the yield of conidia.

[0005] The above technical purpose of the present invention is achieved through the following technical solutions:

[0006] A culture box for efficiently culturing Beauveria bassiana spore powder, including a transparent box body. The upper end of the box body is provided with a cover body. The lower end of the cover body is provided with a sealing ring that cooperates with the box body. The cover body is provided with air holes. A support net is arranged in the air holes. An air-permeable sealing film installed on the support net is arranged on the air holes. The length and width of the box body are 20-25 cm, the height is 5-8 cm, the diameter of the air holes is 2-3 cm, and the bottom of the box body is continuously wavy from left to right.

[0007] By adopting the above technical solution, in the solid culture stage, the rice medium is inoculated with liquid bacteria and then placed in the box body for culture. The breathable sealing film on the cover body can not only ensure good air permeability but also block external miscellaneous bacteria, ensuring that the culture process of Beauveria bassiana is not contaminated and the operation is simple. The wavy bottom increases the bottom area of the box body, has good ventilation, and can make the temperature in the box body more balanced.

[0008] The further setting of the present invention is: a communication hole is opened on the cover body, a support rod is inserted in the communication hole, the lower end of the support rod is inserted into the box body, a temperature sensor and a humidity sensor are installed at the lower end of the support rod, a limiting piece is provided at the lower end of the support rod on the cover body, a threaded sleeve is detachably connected at the upper end of the support rod located on the cover body, a data line is connected to the upper end of the support rod, and the data line is electrically connected to the temperature sensor and the humidity sensor.

[0009] By adopting the above technical solution, the temperature sensor and the humidity sensor are detachably installed in the box body, which is convenient for detecting the humidity and temperature in the box body without affecting the cleaning of the box body.

[0010] The further setting of the present invention is: a vertical plate is provided at the rear end of the box body, a through groove is opened on the vertical plate, two front-to-back placing rods are provided at the front end of the vertical plate at the lower end of each box body, the lower ends of the two placing rods are connected with a pressing rod, a reinforcing rod is provided between the pressing rods, a plurality of first ventilation grooves are opened at the lower end of the pressing rod from front to back, the placing rod is slidably connected to the concave portion at the bottom of the box body, the box body can slide back and forth, the rear end of the placing rod is slidably connected to the vertical plate, the placing rod can slide up and down, a fixing knob is connected to the rear end of the placing rod on the vertical plate, a limiting block is connected to the lower side of the front end of the placing rod, a top plate is provided at the upper end of the vertical plate, and a pressing mechanism is provided on the top plate. The pressing mechanism includes a screw rod, a handle, a connecting plate and a pressing strip. The screw rod is vertically inserted into the middle of the top plate, the screw rod is threadedly connected to the top plate, the handle is connected to the upper end of the screw rod, the connecting plate is rotatably connected to the lower end of the screw rod, the pressing strips are symmetrically arranged on the lower end of the connecting plate, second ventilation grooves are provided on the pressing strips, a handle is provided at the upper end of the top plate, a base is provided at the lower end of the vertical plate, and side rods are provided on both the left and right sides of the vertical plate. The upper and lower ends of the side rods are respectively connected to the top plate and the base.

[0011] By adopting the above technical solution, the culture box is placed on the lower placing rod and pressed by the upper placing rod to fix the culture box. Firstly, it is used in cooperation with the wavy bottom, which is convenient for placement and has good ventilation. Secondly, the placing rod is used to press the culture box from top to bottom, and there is no need to set a locking mechanism between the cover body and the box body. The cover body and the box body can be sealed directly by pressing. Thirdly, it is convenient to fix each culture box together and put them into the culture room for culturing uniformly.

[0012] A further setting of the present invention is that a heating layer is provided at the lower end of the box body, and the heating layer is arranged in a continuous wavy shape along the lower end of the box body.

[0013] By adopting the above technical solution, the temperature of the box body can be controlled and heated separately, avoiding temperature imbalance in the culture room and resulting in temperature deviation in some box bodies. Of course, it is also suitable for culturing in a sterile room.

[0014] A culture method for efficiently culturing Beauveria bassiana spore powder includes the following steps:

[0015] S1. Subculture of Beauveria bassiana mother strain: Pick and inoculate the purified and preserved Beauveria bassiana mother strain on the SDY liquid medium, and culture it at 26±1°C with shaking at 130-180 rpm for 2-3 days. Then spread it on a culture dish and culture it in an incubator at 26±1°C for 3-4 days until the surface of the medium is covered with white spore powder.

[0016] S2. Inoculate the liquid medium: Under sterile conditions, scrape the spore powder on the surface of the Beauveria bassiana mother strain from the surface of the medium and inoculate it into the SDY liquid medium. Culture it at 26±1°C with shaking at 130-180 rpm for about 1-2 days until the concentration of germinated spores in the suspension reaches 10 8 cells / mL to 10 9 cells / mL of liquid seeds.

[0017] S3. Inoculate the solid medium: Inoculate the above liquid seeds into the rice solid medium in the culture box. The inoculation amount of the liquid seed solution is based on the volume ratio of the liquid strain to the mass of the medium, which is 10-15 mL / 100 g. Shake the culture box or stir with a spoon to evenly distribute the bacterial liquid on the rice medium, and then transfer it to an incubator or culture room and culture it at 26-30°C for 5-6 days.

[0018] S4. Screen the spore powder: Pour out the rice medium with good growth process and dry it at 30-40°C, separate the medium to obtain the spore powder.

[0019] Furthermore, step S1 can be prepared in advance. The obtained spores can be stored in a 4°C refrigerator. Each time when culturing spore powder, scrape the Beauveria bassiana spore powder from the surface of the culture medium for liquid culture. This step can increase the spore concentration during liquid culture, greatly save the liquid culture time, and also avoid the contamination and reduced vitality caused by the frequent exposure of the mother culture.

[0020] Furthermore, the liquid medium SDY medium described in step S1: 40 g of white sugar, 10 g of yeast powder, 10 g of peptone, 1000 mL of water. After mixing, perform high-pressure steam sterilization at 121°C for 30 min.

[0021] The culture medium in the petri dish described in step S1 is: 64 g of rice flour, 13 g of wheat flour, 0.5 g of KNO3, 0.5 g of K2HPO4·3H2O, 1.0 g of CaCO3, 20 g of agar, 1000 mL of water. After mixing this solid medium, it needs to be boiled over low heat and then sealed in a glass conical flask, and perform high-pressure steam sterilization at 121°C for 30 min.

[0022] Furthermore, the rice solid medium described in step S3: 1000 g of rice, 0 - 1 g of KNO3, 0 - 1 g of K2HPO4·3H2O, 0 - 1.5 g of CaCO3, 0 - 6 g of silkworm pupa powder, 200 mL of water. After mixing, perform high-pressure steam sterilization at 121°C for 30 min.

[0023] Furthermore, the rice solid medium described in step S3: 1000 g of rice, 0.5 g of KNO3, 0.5 g of K2HPO4·3H2O, 1 g of CaCO3, 5 g of silkworm pupa powder, 200 mL of water. After mixing, perform high-pressure steam sterilization at 121°C for 30 min.

[0024] Furthermore, the rice solid medium accounts for 15 - 20% of the volume of the culture box, and the inoculation liquid inoculum volume is in accordance with the volume-to-medium mass ratio of 10 - 15 mL / 100 g.

[0025] In summary, the present invention has the following beneficial effects:

[0026] Firstly, when culturing Beauveria bassiana in the culture box of the present invention, it can effectively avoid contamination, and has the advantages of simple operation, low equipment cost and low technical requirements. Moreover, the wavy bottom of the culture box can increase the bottom area, improve the ventilation effect, and ensure the temperature balance at the bottom of the culture box.

[0027] Secondly, the culture box of the present invention is placed on the lower placement rod and pressed and fixed by the upper placement rod, and has the advantages of good ventilation, convenient sealing and convenient unified movement.

[0028] Thirdly, the present invention uses a solid-liquid two-way fermentation method. First, Beauveria bassiana spores are cultured in a liquid medium to germinate quickly. In the solid culture stage, a culture box is used, which is simple to operate and not easily contaminated, and can maximize the utilization of the advantages of producing aerial conidia on the solid surface, shortening the mycelium growth cycle and increasing the yield of conidia. The culture box in this method can be reused, which not only saves costs and reduces pollution in production, but also has a simple usage method. The raw materials and equipment used in the culture are easily available and low in cost. The obtained spore powder has a high spore content and strong activity and can be used for inoculation in the production of the traditional Chinese medicine Bombyx batryticatus. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a schematic diagram of the overall structure in the present invention;

[0030] Figure 2 is a sectional view in the present invention;

[0031] Figure 3 is a rear view of the present invention.

[0032] In the figure: 1. box body; 11. heating layer; 2. cover body; 21. sealing ring; 22. communication hole; 3. ventilation hole; 31. support net; 32. ventilation sealing film; 4. support rod; 41. temperature sensor; 42. humidity sensor; 43. limit piece; 44. threaded sleeve; 45. data line; 5. vertical plate; 51. through groove; 6. placing rod; 61. pressing rod; 62. fixing knob; 63. limit block; 64. first ventilation groove; 65. strengthening rod; 7. top plate; 71. handle; 8. pressing mechanism; 81. screw rod; 82. handle; 83. connecting plate; 84. pressing strip; 85. second ventilation groove; 9. base; 10. side rod. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The present invention will be further described in detail below with reference to the accompanying drawings.

[0034] In the description of the present invention, it should be understood that the orientation or positional relationship indicated by the terms "upper", "lower", "left", "right", "front", "rear", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention.

[0035] In addition, terms such as "horizontal" and "vertical" do not mean that the components are required to be absolutely horizontal or hanging, but can be slightly inclined. For example, "horizontal" only means that its direction is more horizontal relative to "vertical", and does not mean that the structure must be completely horizontal, but can be slightly inclined.

[0036] In the description of the present invention, it should also be noted that unless otherwise clearly specified and defined, the terms "set", "connected", and "coupled" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, a direct connection, or an indirect connection through an intermediate medium, and it can be the communication inside two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0037] Example 1. A culture box for efficiently culturing Beauveria bassiana spore powder, as Figures 1 to 3 shown, includes a transparent box body 1 for facilitating the observation of the culture state. A cover body 2 is provided at the upper end of the box body 1. A sealing ring 21 for cooperating with the box body 1 is provided at the lower end of the cover body 2, which facilitates the cover body 2 to seal the box body 1 to avoid contamination. A ventilation hole 3 is provided on the cover body 2. A support net 31 is provided inside the ventilation hole 3. A ventilation sealing film 32 installed on the support net 31 is provided on the ventilation hole 3, which can not only ensure good air permeability but also block external miscellaneous bacteria. The bottom of the box body 1 is in a continuous wavy shape from left to right, which increases the bottom area of the box body 1 and is also convenient for heat dissipation, ensuring the temperature balance inside the box body 1. The length and width of the box body 1 are 20 - 25 cm, and the height is 5 - 8 cm, while the diameter of the ventilation hole 3 is 2 - 3 cm. Of course, the length and width of the box body 1 can be any value between 20 cm, 25 cm or 20 - 25 cm, the height can be any value between 5 cm, 8 cm or 5 - 8 cm, and the diameter of the ventilation hole 3 can be any value between 2 cm, 3 cm or 2 - 3 cm. Compared with the existing culture dishes and shallow trays, it has a larger capacity space.

[0038] A communication hole 22 is opened on the cover body 2. A support rod 4 is inserted into the communication hole 22. The lower end of the support rod 4 is inserted into the box body 1. A temperature sensor 41 and a humidity sensor 42 are installed at the lower end of the support rod 4. A limiting piece 43 is provided at the lower end of the support rod 4 on the cover body 2. The support rod 4 is located at the upper end of the cover body 2 and is detachably connected with a threaded sleeve 44. The upper end of the support rod 4 is connected with a data line 45, and the data line 45 is electrically connected to the temperature sensor 41 and the humidity sensor 42. It is convenient to use the temperature sensor 41 and the humidity sensor 42 to detect the temperature and humidity inside the box body 1, so as to regulate the culture room. Of course, such installation is convenient for cleaning the box body 1 for repeated use.

[0039] A vertical plate 5 is provided at the rear end of the box body 1. A through groove 51 is formed in the vertical plate 5. At the front end of the vertical plate 5, two front-to-back placing rods 6 are provided at the lower end of each box body 1. A pressing rod 61 is connected to the lower ends of the two placing rods 6. A reinforcing rod 65 is provided between the pressing rods 61. A plurality of first ventilation grooves 64 are formed in the lower end of the pressing rod 61 from front to back. The placing rod 6 is slidably connected to the recess at the bottom of the box body 1. The box body 1 can slide back and forth. The rear end of the placing rod 6 is slidably connected to the vertical plate 5. The placing rod 6 can slide up and down. A fixing knob 62 is connected to the rear end of the placing rod 6 on the vertical plate 5. The fixing knob 62 can fix the placing rod 6, which is convenient for taking out the culture box and avoiding large-range sliding of the placing rod 6. A limiting block 63 is connected to the lower side of the front end of the placing rod 6. A top plate 7 is provided at the upper end of the vertical plate 5. The top plate 7 is provided with a pressing mechanism 8. The pressing mechanism 8 includes a screw rod 81, a handle 82, a connecting plate 83 and a pressing strip 84. The screw rod 81 is vertically inserted into the middle of the top plate 7. The screw rod 81 is threadedly connected to the top plate 7. The handle 82 is connected to the upper end of the screw rod 81. The connecting plate 83 is rotatably connected to the lower end of the screw rod 81. The pressing strips 84 are symmetrically arranged on the lower end of the connecting plate 83. Second ventilation grooves 85 are provided on the pressing strips 84. A handle 71 is provided at the upper end of the top plate 7. A base 9 is provided at the lower end of the vertical plate 5. Side rods 10 are provided on both the left and right sides of the vertical plate 5. The upper and lower ends of the side rods 10 are respectively connected to the top plate 7 and the base 9. The vertical plate 5, the bottom plate, the side rods 10 and the base 9 form a support frame, which is convenient for supporting the placing rod 6. After placing the culture box on the lower placing rod 6, the upper placing rod 6 is pressed down, so that the culture box can be fixed, and the cover body 2 can seal the box body 1, which is also convenient for uniformly moving the culture box.

[0040] A heating layer 11 is provided at the lower end of the box body 1. The heating layer 11 is arranged in a continuous wavy shape along the lower end of the box body 1. Heating the bottom of the box body 1 through the heating layer 11 can, firstly, heat in the culture room to improve the problem of uneven temperature in the culture room, and secondly, facilitate temperature control culture in the sterile room.

[0041] Example 2: A culture method for efficiently culturing Beauveria bassiana spore powder, comprising the following steps:

[0042] 1. Preparation and sterilization of the culture medium

[0043] Liquid culture medium SDY medium: 40 g of white sugar, 10 g of yeast powder, 10 g of peptone, 1000 mL of water. After mixing, it is treated with high-pressure steam sterilization at 121 °C for 30 min.

[0044] Weigh various raw materials according to the formula, stir evenly, and load them into a glass conical flask. The liquid filling amount is between 1 / 3 and 2 / 3 of the volume of the conical flask. The bottle mouth is sealed with a sterile breathable sealing film and a rubber band, and sterilized with high-pressure steam at 121 °C for 30 min.

[0045] The culture medium in the petri dish is as follows: 64 g of rice flour, 13 g of wheat flour, 0.5 g of KNO3, 0.5 g of K2HPO4·3H2O, 1.0 g of CaCO3, 20 g of agar, and 1000 mL of water. After mixing this solid medium, it needs to be boiled over a gentle flame and then sealed in a glass conical flask, followed by autoclaving at 121 °C for 30 min.

[0046] Weigh various raw materials according to the formula, stir evenly, boil over a gentle flame for 5 minutes while keeping stirring during the boiling process, seal in a glass conical flask, with the filling volume between 1 / 3 and 2 / 3 of the conical flask volume, and perform autoclaving at 121 °C for 30 min. Wait until the temperature drops to about 60 - 50 °C and then pour it into the petri dish for standby.

[0047] Rice solid medium: 1000 g of rice, 0.5 g of KNO3, 0.5 g of K2HPO4·3H2O, 1 g of CaCO3, 5 g of silkworm chrysalis powder, 200 mL of water. After mixing, perform autoclaving at 121 °C for 30 min.

[0048] Stir evenly, fill into the culture box with a filling amount of 350 g, and perform autoclaving at 121 °C for 30 min.

[0049] 2. Cultivation of Beauveria bassiana mother culture

[0050] Using the tissue isolation method or spore isolation method, the pure culture of Beauveria bassiana that is isolated, purified from the stiff silkworm, has consistency and stability, and is stored in a glass test tube or petri dish is the mother culture. Under aseptic operation conditions, scrape the spore powder from the surface of the mother culture thallus with an inoculation loop, put it into a liquid seed culture flask cooled to room temperature, and quickly seal the bottle mouth with a sterile breathable sealing film and a rubber band. The liquid seed is cultured on a rotary shaker at a temperature of 26 ± 1 °C and a rotation speed of 130 - 180 rpm for about 2 - 3 days, and then spread on a petri dish and cultured in an incubator at 26 ± 1 °C for 3 - 4 days until the surface of the medium is covered with white spore powder.

[0051] 3. Inoculation of Beauveria bassiana into liquid medium for cultivation

[0052] Under aseptic conditions, scrape the Beauveria bassiana spore powder from the surface of the medium, inoculate it into the SDY liquid medium, and culture it on a shaker at 26 ± 1 °C and 130 - 180 rpm for about 1 - 2 days until the concentration of budding spores in the fermentation liquid reaches 10 8 cells / mL to 10 9 cells / mL, and then transfer the suspension to the rice solid medium. The inoculation volume of the liquid bacterial solution is in accordance with the volume - to - medium mass ratio of 10 - 15 mL / 100 g.

[0053] 4. Inoculation of liquid strain into rice solid medium

[0054] After the rice solid medium is cooled to room temperature, 50 mL of liquid inoculum is added to each culture box, and the rice solid medium accounts for 15-20% of the volume of the culture box. Shake the culture box or stir with a spoon to evenly distribute the liquid inoculum on the rice medium, and transfer it to an incubator or culture room for cultivation at 26-30 °C for 5-6 days. As Beauveria bassiana metabolizes water, the water content of the solid medium decreases, and under the stress of reduced water content, Beauveria bassiana rapidly produces a large amount of spore powder around the 4th day of cultivation.

[0055] 5. Sieving the spore powder after drying

[0056] Pour the rice medium with well-grown spore powder into an oven tray lined with filter paper, dry it in a forced-air drying oven at 30-40 °C, and pass it through a 200-mesh sieve with an electric shaker to separate the rice matrix and obtain the spore powder.

[0057] 6. Determination of sporulation amount and spore content

[0058] Determination of sporulation amount: Weigh the sieved spore powder, retain two decimal places in the result, and set 3 replicates. Determination of spore content: Take 0.1 g of spore powder and suspend it in 10 mL of 0.1% Tween-80 solution, stir well to completely dissolve it, and count the spores with a hemocytometer and calculate.

[0059] 7. Determination of viable spore rate

[0060] Take an appropriate amount of spore powder and add it to 50 mL of SDY medium to prepare a spore suspension with a concentration of 1.0×10 6 spores / mL. Under the condition of 25 °C, culture it at 130 r / min for 24 h, and microscopically examine the germination rate, which is the viable spore rate (%) of the spore powder. The standard for spore germination is that the germ tube length is equal to or greater than the spore diameter.

[0061] 8. Virulence determination

[0062] Take spore powder and prepare spore suspensions with concentrations of 1×10 7 , 5×10 7 spores / mL with 0.1% Tween-80 sterile water. Use the spraying method to inoculate the 5th instar silkworms 24 h old respectively, so that the body surface of the silkworms is just moist. Feed the silkworms with mulberry leaves 30 min after inoculation. The culture conditions are 26-28 °C and relative humidity 80%. Observe and record the mortality of the silkworms within 1-7 d. Each treatment group has 100 silkworms and 3 replicates.

[0063] Example 3: The difference between this example and Example 1 is that the tested rice medium is 1000 g of rice, 0.5 g of KNO3, 0.5 g of K2HPO4·3H2O, 1 g of CaCO3, and 200 mL of water, and other steps are the same as those in Example 1.

[0064] Example 4: The difference between this example and Example 1 is that the tested rice culture medium consists of 1000 g of rice, 5 g of silkworm pupa powder, and 200 mL of water. Other steps are the same as in Example 1.

[0065] Example 5: The difference between this example and Example 1 is that the tested rice culture medium consists of 1000 g of rice and 200 mL of water. Other steps are the same as in Example 1.

[0066] Example 6: The difference between this example and Example 1 is that the tested rice culture medium is a rice solid medium: 1000 g of rice, 1 g of KNO3, 1 g of K2HPO4·3H2O, 1.5 g of CaCO3, 6 g of silkworm pupa powder, and 200 mL of water. Other steps are the same as in Example 1.

[0067] Example 7: The difference between this example and Example 1 is that the tested rice culture medium is a rice solid medium: 1000 g of rice, 0.2 g of KNO3, 0.2 g of K2HPO4·3H2O, 0.5 g of CaCO3, 2 g of silkworm pupa powder, and 200 mL of water. Other steps are the same as in Example 1.

[0068] The sporulation amount, virulence, and the weight of the finished Bombyx batryticatus of Beauveria bassiana cultured in the above-mentioned different culture media in Examples 2 - 7 were analyzed, and the results are shown in Table 1.

[0069]

[0070] Table 1 Effects of different rice culture media on the sporulation amount, virulence, and the weight of the finished Bombyx batryticatus of Beauveria bassiana

[0071] Summary: As can be seen from Table 1, when using the culture box to culture Beauveria bassiana, the sporulation amount is all 1.85 g / 100 g or more, the spore content is more than 4×10 10 spores / g, the viable spore rate is more than 95%. When spraying the 5th instar silkworms with a spore concentration of 1×10 7 spores / mL for 7 days, the mortality rate is all above 80%. When spraying with a spore concentration of 5×10 7 spores / mL for 7 days, the mortality rate is all above 92%. Among them, the rice culture medium formula used in combination with the culture box is: 1000 g of rice, 0.5 g of KNO3, 0.5 g of K2HPO4·3H2O, 1 g of CaCO3, 5 g of silkworm pupa powder, and 200 mL of water, and all the results are the best. The sporulation amount reaches 2.20 ± 0.36 g / 100 g, the spore content is 1.24×10 11 spores / g, the viable spore rate is 98%, and the virulence to silkworms is the highest. The present invention can quickly obtain spores by using the culture box in combination with the culture method and the rice solid culture medium, and the results such as the spore yield, spore content, viable spore rate, and the virulence to silkworms are all relatively ideal.

[0072] This specific embodiment is only an interpretation of the present invention and does not limit the present invention. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as it is within the scope of the claims of the present invention, it is protected by the patent law.

Claims

1. A culture box for efficiently culturing Beauveria bassiana spore powder, comprising a transparent box body (1), characterized in that: The upper end of the box body (1) is provided with a cover body (2). The lower end of the cover body (2) is provided with a sealing ring (21) for cooperating with the box body (1). The cover body (2) is provided with a ventilation hole (3). A support net (31) is arranged in the ventilation hole (3). A ventilation sealing film (32) installed on the support net (31) is arranged on the ventilation hole (3). The length and width of the box body (1) are 20 - 25 cm, and the height is 5 - 8 cm. The diameter of the ventilation hole (3) is 2 - 3 cm. The bottom of the box body (1) is in a continuous wavy shape from left to right.

2. The culture box for efficiently culturing Beauveria bassiana spore powder according to claim 1, wherein: A communication hole (22) is opened on the cover body (2). A support rod (4) is inserted into the communication hole (22). The lower end of the support rod (4) is inserted into the box body (1). A temperature sensor (41) and a humidity sensor (42) are installed at the lower end of the support rod (4). A limiting piece (43) is arranged at the lower end of the support rod (4) on the lower end of the cover body (2). The support rod (4) is located at the upper end of the cover body (2) and is detachably connected with a threaded sleeve (44). The upper end of the support rod (4) is connected with a data line (45). The data line (45) is electrically connected to the temperature sensor (41) and the humidity sensor (42).

3. The culture box for efficiently culturing Beauveria bassiana spore powder according to claim 2, wherein: A vertical plate (5) is arranged at the rear end of the box body (1). A through groove (51) is opened on the vertical plate (5). At the front end of the vertical plate (5), two front - and - rear - facing placing rods (6) are arranged at the lower end of each box body (1). The lower ends of the two placing rods (6) are connected with a pressing rod (61). A reinforcing rod (65) is arranged between the pressing rods (61). A plurality of first ventilation grooves (64) are opened at the lower end of the pressing rod (61) from front to back. The placing rod (6) is slidably connected to the recess at the bottom of the box body (1). The box body (1) can slide back and forth. The rear end of the placing rod (6) is slidably connected to the vertical plate (5). The placing rod (6) can slide up and down. The placing rod (6) is connected with a fixing knob (62) at the rear end of the vertical plate (5). A limiting block (63) is connected to the lower side of the front end of the placing rod (6). A top plate (7) is arranged at the upper end of the vertical plate (5). The top plate (7) is provided with a pressing mechanism (8). The pressing mechanism (8) includes a screw rod (81), a handle (82), a connecting plate (83) and a pressing strip (84). The screw rod (81) is vertically inserted into the middle of the top plate (7). The screw rod (81) is threadedly connected to the top plate (7). The handle (82) is connected to the upper end of the screw rod (81). The connecting plate (83) is rotatably connected to the lower end of the screw rod (81). The pressing strips (84) are symmetrically arranged on the left and right sides at the lower end of the connecting plate (83). Second ventilation grooves (85) are arranged on the pressing strips (84). A handle (71) is arranged at the upper end of the top plate (7). A base (9) is arranged at the lower end of the vertical plate (5). Side rods (10) are arranged on both the left and right sides of the vertical plate (5). The upper and lower ends of the side rods (10) are respectively connected to the top plate (7) and the base (9).

4. The culture box for efficiently culturing Beauveria bassiana spore powder according to claim 3, characterized in that: A heating layer (11) is provided at the lower end of the box body (1), and the heating layer (11) is arranged in a continuous wavy shape along the lower end of the box body (1).

5. The culturing method for efficiently culturing Beauveria bassiana spore powder according to claim 1, wherein It includes the following steps: S1. Large-scale cultivation of Beauveria bassiana mother culture: Pick and inoculate the purified and preserved Beauveria bassiana mother culture into the SDY liquid medium, and cultivate it with shaking at 26±1°C and 130-180 rpm for 2-3 days. Then spread it on the culture dish and cultivate it in an incubator at 26±1°C for 3-4 days until the surface of the medium is covered with white spore powder; S2. Inoculate the liquid medium: Under aseptic conditions, scrape the spore powder on the surface of the mother culture of Beauveria bassiana from the surface of the medium, inoculate it into the SDY liquid medium, and culture it with shaking at 26 ± 1 °C and 130 - 180 rpm for about 1 - 2 days until the concentration of germinated spores in the suspension reaches 10 8 cells / mL to 10 9 cells / mL of the liquid seed; S3. Inoculate the solid medium: Inoculate the above liquid culture into the rice solid medium in the culture box. The inoculation amount of the liquid culture is based on the volume ratio of the liquid strain to the medium mass of 10-15 mL / 100 g. Shake the culture box or stir with a spoon to evenly distribute the bacterial liquid on the rice medium, and then transfer it to an incubator or culture room at 26-30°C and cultivate for 5-6 days; S4. Screen the spore powder: Pour out the well-growing rice medium and dry it at 30-40°C, separate the medium to obtain the spore powder.

6. The culture method for efficiently culturing Beauveria bassiana spore powder according to claim 5, characterized in that: Step S1 can be prepared in advance. The obtained spores can be stored in a 4°C refrigerator. Each time spore powder is cultured, scrape the spore powder of Beauveria bassiana from the surface of the medium for liquid culture. This step can increase the spore concentration during liquid culture, greatly save the liquid culture time, and also avoid the pollution and reduced vitality caused by the frequent exposure of the mother culture.

7. The culture method for efficiently culturing Beauveria bassiana spore powder according to claim 6, characterized in that: The liquid medium SDY medium described in step S1: 40 g of white sugar, 10 g of yeast powder, 10 g of peptone, 1000 mL of water. After mixing, perform high-pressure steam sterilization at 121°C for 30 min; The medium in the culture dish described in step S1 is: 64 g of rice flour, 13 g of wheat flour, 0.5 g of KNO3, 0.5 g of K2HPO4·3H2O, 1.0 g of CaCO3, 20 g of agar, 1000 mL of water. After mixing this solid medium, it needs to be boiled over low heat and then sealed in a glass conical flask, and perform high-pressure steam sterilization at 121°C for 30 min.

8. The culture method for efficiently culturing Beauveria bassiana spore powder according to claim 7, characterized in that: The rice solid medium described in step S3: 1000 g of rice, 0-1 g of KNO3, 0-1 g of K2HPO4·3H2O, 0-1.5 g of CaCO3, 0-6 g of silkworm pupa powder, 200 mL of water. After mixing, perform high-pressure steam sterilization at 121°C for 30 min.

9. The culture method for efficiently culturing Beauveria bassiana spore powder according to claim 8, characterized in that: The rice solid medium described in step S3: 1000 g of rice, 0.5 g of KNO3, 0.5 g of K2HPO4·3H2O, 1 g of CaCO3, 5 g of silkworm pupa powder, 200 mL of water. After mixing, perform high-pressure steam sterilization at 121°C for 30 min.

10. The culture method for efficiently culturing Beauveria bassiana spore powder according to claim 9, characterized in that: The said rice solid medium accounts for 15-20% of the volume of the culture box, and the inoculation amount of the liquid strain is based on the volume ratio of 10-15 mL / 100 g of the medium mass.

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