Strain suitable for large-scale production of high-quality stiff silkworm and application thereof

Abstract

The invention discloses a bacterial strain suitable for large-scale production of high-quality stiff silkworm and application of the bacterial strain. The strain is a beauveria bassiana KW2 strain, and is preserved in Guangdong Microbial Culture Collection Center on November 19, 2024, and the preservation number is GDMCC NO: 65502. When the KW2 strain is used for producing stiff silkworms, the stiff silkworms are pink in color and then gradually fade, so that efficient sorting of the stiff silkworms is facilitated, the quality of the stiff silkworms can be better controlled, the labor cost can be better reduced, and the method is very suitable for large-scale production of the stiff silkworms. Meanwhile, hyphae on the surface of the stiff silkworm are oily and thick, less spore powder is produced, and production environment management and personnel health are facilitated. The KW2 strain has a silkworm death rate of 100%, the bombyx batryticatus is cylindrical and high in quality, and various indexes such as moisture and the like meet standards. Moreover, the content of beauveria bassiana and ammonium oxalate is relatively high, and the medicinal value is high. The KW2 strain has a very good application prospect in improving the large-scale safe production efficiency of high-quality stiff silkworms.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of traditional Chinese medicine silkworm pupa, and specifically relates to a strain suitable for efficient picking and obtaining high-quality silkworm pupa in large-scale production and application thereof. Background Art

[0002] Bombyx Batryticatus, also known as dead worm, is a complex of silkworm larvae that have been infected and died by Beauveria bassiana and then dried. The medical classic Shennong's Herbal Classic first recorded the characteristics, uses and efficacy of Bombyx batryticatus. Bombyx batryticatus is mild in nature and has the effects of calming fever, dispelling wind and relieving spasms, and resolving phlegm and dispersing knots. Bombyx batryticatus is rich in medicinal ingredients, such as flavonoids, ammonium oxalate, and beauvericin, which have significant effects on inhibiting cancer cell proliferation, enhancing immunity, sterilization, and anticonvulsant. With the in-depth study of the medicinal value of Bombyx batryticatus, its medicinal range is very wide, and the current market demand for Bombyx batryticatus is also increasing year by year.

[0003] In recent years, the production method of preparing silkworm pupae by artificially inoculating Beauveria bassiana to infect the fifth-instar silkworm has attracted more and more attention. The quality of the prepared silkworm pupae is closely related to the inoculated Beauveria bassiana. The overall quality of silkworm pupae produced on the market is not high. The silkworm pupae usually cannot meet the requirements of the "Pharmacopoeia of the People's Republic of China" and some local standards and enterprise standards in one or more indicators such as appearance size, ash content, extract content, and number of silk gland rings, which greatly affects the large-scale production efficiency and clinical application effect of silkworm pupae.

[0004] In addition, in large-scale production, the workload of picking dead silkworms is heavy. During the process of silkworm infection by Beauveria bassiana on the market, silkworms are white when they are rigid, which is not much different from the color of the silkworm itself. Especially in large-scale production, it is very difficult to pick the dead silkworms from a large number of silkworms. Experienced staff are required to carry out the picking work, which has high requirements for employees. Moreover, if the dead silkworms are not picked out in time for subsequent rigidification and drying, the quality and yield of the dead silkworms will decrease, affecting their medicinal value. In addition, after the common Beauveria bassiana infects the silkworm, the mycelium growing on the surface of the dead silkworm is in the shape of lime powder, which is easy to produce a large amount of spore powder. In the production workshop, a large amount of dust is not only not conducive to the management of the production environment of the silkworm breeding, but also not conducive to the health of the production workers.

[0005] In addition, studies have also shown that beauvericin and ammonium oxalate are important characteristic ingredients for calming wind and stopping spasms in Bombyx batryticatus. The contents of ammonium oxalate and beauvericin in Bombyx batryticatus are significantly positively correlated, and the contents of these two ingredients are also significantly positively correlated with the appearance of Bombyx batryticatus. However, the content of beauvericin and ammonium oxalate in existing Bombyx batryticatus is low and unstable. The content of beauvericin is between 0.0101% and 0.1192%, which is about 10 times different; the content of ammonium oxalate is between 4.71% and 10.84%; and among multiple batches, 17.2% of the batches did not detect beauvericin and ammonium oxalate. The existing Bombyx batryticatus on the market has a high quality risk (Gao Peng et al., 2024, Research on Quality Standards of Bombyx batryticatus Medicinal Materials and Decoction Pieces). Summary of the invention

[0006] In view of the above technical problems, the present invention provides a strain suitable for efficient picking and obtaining high-quality silkworm pupae in large-scale production and its application. The strain is the Beauveria bassiana KW2 strain, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 19, 2024. Its deposit number is GDMCC NO: 65502, and its deposit address is: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou. The KW2 strain is used to produce silkworm pupae. After rigidification, the color of the silkworm is red (pink), and then gradually fades, and the silkworm pupae is cylindrical, which is helpful for the selection of silkworm pupae, better control of the quality of silkworm pupae and reduction of labor costs, and is very suitable for large-scale production of silkworm pupae. At the same time, the mycelium on the surface of the silkworm pupae is oily and thick, and there is less spore powder, which is beneficial to production environment management and personnel health. Moreover, the KW2 strain has a 100% mortality rate for silkworms, and its quality is high. Various indicators such as moisture content meet the standards. It also has high content of beauvericin and ammonium oxalate and high medicinal value, indicating that the KW2 strain has good application prospects in large-scale, standardized and safe production of high-quality silkworms.

[0007] The first object of the present invention is to provide a Beauveria bassiana KW2 strain.

[0008] The second object of the present invention is to provide a bacterial agent containing the Beauveria bassiana KW2 strain.

[0009] The third object of the present invention is to provide the use of the Beauveria bassiana KW2 strain and the bacterial agent in producing Bombyx batryticatus.

[0010] The fourth object of the present invention is to provide a method for producing silkworm pupae.

[0011] The above-mentioned purpose of the present invention is achieved by the following technical solutions:

[0012] The present invention studied and isolated a new strain of Beauveria bassiana, named Beauveria bassiana KW2 strain, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 19, 2024, and its deposit number is GDMCC NO: 65502. When the KW2 strain is inoculated into silkworms, the color of the silkworm surface changes from white to pink after the silkworm dies, and then gradually fades, which is conducive to the picking of silkworms during the production process, better control of the quality of silkworms and reduction of labor costs, and is very suitable for the large-scale production of silkworms. At the same time, the mycelium grown on the surface of the silkworm is oily and thick, and it is not easy to produce a large amount of spore powder, which is beneficial to the production environment management and personnel health. Moreover, the KW2 strain has a 100% mortality rate for silkworms, and the silkworms are cylindrical. The total weight of the dried silkworms can reach 26kg, and the maximum weight of a single silkworm can reach 1.12g; the obtained silkworms are hard and brittle, easy to break, and several bright brown or bright black silk gland rings can be seen on the cross-section. All indicators (tested by third-party agencies) have met the standards of the "Pharmacopoeia of the People's Republic of China" (2020 edition). The content of characteristic ingredients beauvericin and ammonium oxalate is high, and the silkworms are of excellent quality.

[0013] Therefore, the present invention claims protection for a Beauveria bassiana KW2 strain.

[0014] Specifically, the Beauveria bassiana KW2 strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 19, 2024, and its preservation number is GDMCC NO: 65502.

[0015] The present invention also claims to protect a bacterial agent containing the Beauveria bassiana KW2 strain.

[0016] The present invention also claims to protect the use of the Beauveria bassiana KW2 strain or the bacterial agent in producing Bombyx batryticatus.

[0017] The present invention also claims a method for producing silkworm pupae.

[0018] Specifically, the Beauveria bassiana KW2 strain or the bacterial agent is used to inoculate silkworms to produce silkworm pupae.

[0019] Since the silkworms appear pink in their initial stages after they die, it helps to efficiently pick the dead silkworms.

[0020] Therefore, preferably, in the method for producing silkworm pupae of the present invention, the picking operation is performed when the silkworms change color after death.

[0021] Preferably, the silkworm is sorted when its color turns red after it dies. More preferably, the silkworm is sorted when its color turns pink after it dies.

[0022] As a preferred embodiment, when the Beauveria bassiana KW2 strain is used to produce silkworms, the temperature of the production environment (rigidification chamber) is maintained at 25-26°C.

[0023] As a preferred embodiment, the relative humidity of the production environment (rigidity chamber) is not less than 85%.

[0024] As an optional embodiment, the inoculation method is spraying a spore suspension, and the spore content of the spore suspension is not less than 1×10 6 Pieces / mL.

[0025] As a preferred experimental scheme, the sterilization and drying of the silkworm should start on the 3rd to 4th day after the silkworm dies.

[0026] Specifically, the sterilization and drying is a process in which the silkworms are first sterilized to kill the Beauveria bassiana and its spores and then dried.

[0027] Specifically, the sterilization condition is heat treatment at 85-95° C. for 40-60 min.

[0028] Preferably, the sterilization condition is heat treatment at 90°C for 50 minutes.

[0029] Specifically, the drying method of the silkworm is staged drying: the temperature of the first stage is 38-45°C, lasting 8-12 hours; the temperature of the second stage is 50-70°C, lasting 8-12 hours.

[0030] More specifically and preferably, the temperature of the first stage is 40° C. and lasts for 10 h; the temperature of the second stage is 60° C. and lasts for 10 h.

[0031] Specifically, the drying is performed until the moisture content of the silkworm is less than 13%.

[0032] The silkworm produced by the method of the present invention has high quality, and all indicators meet the standards of the Pharmacopoeia of the People's Republic of China (2020 edition), and the characteristic components beauvericin and ammonium oxalate have high contents, and the silkworm has excellent quality. The silkworm produced by the method of the present invention should also be within the scope of protection of the present invention.

[0033] The present invention has the following beneficial effects:

[0034] The invention provides a Beauveria bassiana KW2 strain, which has excellent performance in the process of infecting silkworms. The silkworms are pink on the second day after they die, and then gradually fade. The silkworms are cylindrical, which is helpful for picking and sorting the silkworms, can better control the quality of the silkworms and reduce labor costs, and is very suitable for large-scale production of silkworms.

[0035] At the same time, the mycelium on the surface of Bombyx mori is oily and thick, and there are fewer spores on the body surface, which is more beneficial to the production environment, and is conducive to the management of the Bombyx mori breeding production environment and the health of production workers.

[0036] The KW2 strain is also highly toxic to silkworms. 6 The healthy fifth-instar silkworms of Huakang 3 were inoculated with the KW2 strain spore suspension at a concentration of 100 / mL, and the mortality rate reached 100%, with the average weight of each silkworm reaching 0.90±0.096 g.

[0037] In addition, in the large-scale production of silkworms, the spore suspension of the KW2 strain is used to inoculate a healthy five-year-old Huakang No. 3 silkworm. The total weight of the dried silkworms can reach 26kg, and the maximum weight of a single silkworm can reach 1.12g; the resulting silkworms are hard and brittle, easy to break, and several bright brown or bright black silk gland rings can be seen on the cross section. Other indicators such as impurities, moisture, acid-insoluble ash content (tested by a third-party agency) have reached the standards of the Pharmacopoeia of the People's Republic of China (2020 edition). Moreover, its characteristic ingredients, beauvericin and ammonium oxalate, have high contents, and the silkworms are of excellent quality.

[0038] The Beauveria bassiana KW2 strain provided by the present invention has good application prospects in the large-scale, standardized and safe production of high-quality silkworm pupa. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 These are the front and back colony morphology images and conidia micrographs of the isolated strains; the scale of the front and back colony images is 1 cm, and the scale of the conidia image is 100 μm.

[0040] Figure 2 This is a phylogenetic tree constructed based on the 18S rDNA+ITS sequence of Beauveria bassiana KW2 strain.

[0041] Figure 3 The results of analysis on the mortality rate of silkworms infected with different strains of Beauveria bassiana; KW2 represents the KW2 strain of Beauveria bassiana, KW1 represents the KW1 strain of Beauveria bassiana, and HN6 represents the HN6 strain of Beauveria bassiana.

[0042] Figure 4 This is a comparative analysis of the phenotypic characteristics of different Beauveria bassiana strains at different time periods during the rigidification process of silkworms; A in the figure is the Beauveria bassiana KW2 strain, and B in the figure is the Beauveria bassiana KW1 strain.

[0043] Figure 5 This is the manifestation of the rigid state of silkworms after being infected by the KW2 strain of Beauveria bassiana in production.

[0044] Figure 6These are some samples of silkworm products obtained by infecting silkworms with the KW2 strain of Beauveria bassiana. Figure A shows part of the dried silkworm products, with a scale of 1 cm; Figure B shows the cross-section of the silkworm products, with a scale of 1 mm. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0046] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0047] In this article, "mortality rate" refers to the ratio of the number of healthy fifth-instar silkworms that died of white muscardine infection caused by Beauveria bassiana before spinning silk and cocooning to the total number of inoculated silkworms. It is an important indicator of the success rate of silkworm breeding. When different strains infect the same silkworm, if the same concentration of spore suspension is used for infection, the strain with a higher mortality rate has a higher toxicity to the silkworm.

[0048] Sabouraud medium (SDAY medium): weigh 40g of glucose, 10g of peptone, 10g of yeast extract powder, and 15g of agar powder, add distilled water to completely dissolve and dilute to 1000mL. After high temperature and high pressure sterilization for 30min, spread on a plate for later use.

[0049] Example 1 Isolation and Identification of Beauveria bassiana

[0050] 1. Isolation and purification of strains

[0051] In the clean bench, the silkworm samples obtained from the natural infection of Beauveria bassiana during silkworm breeding (provided by a silkworm farmer in Guangzhou, the silkworm species is Liangguang No. 2) were soaked in 75% alcohol for 30 seconds, rinsed with sterile water 2-3 times, and the surface moisture was absorbed by absorbent paper, and then placed on the SDAY medium plate, with the front side of the SDAY medium plate facing up, and placed in a constant temperature and humidity incubator at 25°C and a relative humidity of 90%. When pure white colonies grow on the silkworm body, the colonies are picked up in time with an inoculation needle, and the colonies are purified and cultured on a new SDAY medium plate by multiple rounds of plate streaking method, and the colony morphology is observed. The morphological characteristics of hyphae and conidia are observed under an optical microscope.

[0052] Single colonies without other foreign bacteria under microscopic examination were identified, including morphological identification and 18S rDNA+ITS sequencing molecular identification, and a pure colony identified as Beauveria bassiana was selected as the target strain and recorded as Beauveria bassiana KW2 strain.

[0053] The specific morphological identification methods and 18S rDNA+ITS sequencing molecular identification methods are as follows "2. Morphological identification of strains" and "3. Molecular biological identification of strains":

[0054] 2. Morphological identification of strains

[0055] The isolated and purified strain was inoculated onto a new SDAY medium plate and cultured in a constant temperature and humidity incubator at 25°C and 90% relative humidity for 7 days. The color and shape of the colonies were observed and recorded. The type, size and shape of the conidia of the strain were observed and recorded under a microscope.

[0056] The colony morphology and conidia morphology of the isolated and purified strains on SDAY medium plates were analyzed. The results are as follows Figure 1 As shown. Figure 1 It can be seen that the center of the colony is slightly protruding, the colony is white, and it grows outward in a circular shape, with annular grooves on the surface; the hyphae are colorless and slender, with a smooth surface, and its conidiophores are attached to the vegetative hyphae. The conidia are spherical or nearly spherical, transparent and smooth. Based on the above characteristics, the strain was preliminarily determined to be Beauveria bassiana.

[0057] 3. Molecular biological identification of strains

[0058] The isolated and purified strain was inoculated on a new SDAY medium plate, placed in a constant temperature and humidity incubator at 25°C and 90% relative humidity for 7 days, and then the spores and hyphae on the surface of the medium were scraped off, and the genomic DNA of the strain was extracted using a fungal genomic DNA extraction kit (purchased from Beijing Solebow Technology Co., Ltd.). Primers 18S-F and ITS4-R were designed to amplify the gene fragment containing the 18S rDNA+ITS sequence of the strain. The nucleotide sequences of the primers are shown below, and Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the primers.

[0059] Primer 18S-F: 5′-TATCTGGTTGATTCTGCCAG-3′

[0060] Primer ITS4-R: 5′-TCCTCCGCTTATTGATATGC-3′

[0061] Reaction system: 25 μL PrimeSTAR Max Premix (2X) (purchased from Shanghai Biosai Biotechnology Co., Ltd.), 1 μL each of primers 18S-F and ITS4-R (concentration 10 μM), 1 μL genomic DNA, and add ultrapure water to 50 μL.

[0062] Reaction conditions: 98°C for 5 min; 98°C for 10 sec, 55°C for 30 sec, 72°C for 1 min, 30 cycles; 72°C for 5 min.

[0063] The PCR product obtained by the above reaction was detected by agarose gel electrophoresis, and the target fragment was purified by gel excision using a common agarose gel DNA recovery kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The target fragment was ligated to pMD TM The ligation product was obtained on the 19-T vector; the ligation product was transformed into E. coli TOP10 competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd.), and the specific operation steps are shown in the instructions. Then, the E. coli transformants were amplified by colony PCR using primers 18S-F and ITS4-R for preliminary testing, and the positive clones were selected and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing analysis.

[0064] The obtained 18S rDNA+ITS gene sequence was subjected to homology analysis by NCBI / BLASTn, and it was found that the strains with the most similar gene sequences to the strain were all Beauveria bassiana, indicating that the strain belonged to Beauveria bassiana. Among them, the ITS sequence of Beauveria bassiana HN6 was completely consistent with that of the strain. Further comparative analysis of the genomes of the strain and Beauveria bassiana HN6 showed that the genome sizes of the strain and Beauveria bassiana HN6 were 36.6Mb and 37.1Mb, respectively, and the total number of genes encoded by the two genomes were 9880 and 10,000, respectively. This shows that there are obvious differences in the genomes of the strain and the HN6 strain, and they are two different strains.

[0065] The phylogenetic tree was constructed using MEGA11.0 software. Figure 2 As shown, this strain clusters with other Beauveria bassiana strains.

[0066] In summary, the strain isolated and purified from the silkworm was identified as the genus Beauveria and the species Beauveria bassiana, named Beauveria bassiana KW2 strain, and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 19, 2024, and its preservation number is GDMCC NO: 65502.

[0067] Example 2 Determination of the mortality rate and phenotypic characteristics of silkworms infected with different strains of Beauveria bassiana

[0068] 1. Experimental methods

[0069] The experiment was divided into 3 experimental groups, each with 210 healthy Huakang 3 silkworms (fourth-instar sleeping silkworms), and 3 replicates were set up in each group. The healthy five-instar silkworms of Huakang 3 were inoculated with Beauveria bassiana KW2, Beauveria bassiana KW1, and Beauveriabassiana HN6, respectively, to study the mortality rate of silkworms infected with different Beauveria bassiana strains, so as to evaluate the virulence of the KW2 strain. Among them, Beauveria bassiana HN6 was donated by Professor Guo Xijie of Jiangsu University of Science and Technology; Beauveria bassiana KW1 was isolated and purified from Bombyx mori by our research group and was deposited in Guangdong Provincial Microbiological Culture Collection Center on July 30, 2024, and its deposit number is GDMCC NO: 64922.

[0070] The specific method is as follows:

[0071] (1) Selection of fourth-instar dormant silkworms: Select fourth-instar dormant silkworms that eat normally and are in good health from the silkworm beds, transfer them to a rigidification room, and raise them. After the silkworms wake up, inoculate them with Beauveria bassiana;

[0072] (2) Preparation of the inoculation environment: Use constant temperature and humidity equipment to maintain the temperature of the rigid room at 25-26°C and the humidity not less than 85%; use a mechanical ventilation system to supply and exhaust air regularly every day, and connect a spray sterilization device at the exhaust port to prevent the leakage of Beauveria bassiana and its spores;

[0073] (3) Preparation of spore suspension: The KW2 strain of Beauveria bassiana was inoculated on a SDAY medium plate and cultured in a constant temperature and humidity incubator at 25°C and 90% relative humidity for 15 to 20 days. Then, a spore suspension containing 1×10 6 / mL of spore suspension;

[0074] (4) Spraying and inoculation of fifth-instar silkworms: The selected fourth-instar sleeping silkworms are transferred to a rigidification room. 2 to 3 days after waking up, the fifth-instar silkworms are sprayed and inoculated with the spore suspension using an agricultural sprayer. The spore suspension is sprayed on the surface of the silkworms for the first time until the silkworms are wet. The second spraying is performed 15 minutes later. Mulberry is given 15 minutes later, and then the spore suspension is sprayed on the surface of mulberry leaves for the third inoculation. After 15 minutes, the spore suspension is sprayed on the surface of mulberry leaves again for the fourth inoculation.

[0075] (5) Collection of dead silkworms: Regularly observe the fifth-instar silkworms after inoculation. When the silkworms become dead, they should be picked out in time and spread out in a clean silkworm plaque. During this period, the ambient temperature should be maintained at 25-26°C and the relative humidity should not be lower than 85%. When the dead silkworm mortality rate reaches 20%, lay out the fifth-instar silkworm net, feed a small amount of mulberry leaves, and then stop feeding mulberry leaves (a small amount of mulberry leaves is to cover the silkworm net with a layer of mulberry leaves, so as to completely cover the silkworm net). After 48 hours, collect the silkworm net;

[0076] (6) Drying and storage of silkworm pupae: When the silkworms are completely rigid (the surface of the silkworms is covered with Beauveria bassiana hyphae and the color of the silkworm body cannot be distinguished), sterilization and drying are generally carried out 3 to 4 days after the silkworms are rigid. The wet silkworms are placed in an oven and first heat-treated at 90°C for 50 minutes to kill Beauveria bassiana and its spores; then the silkworms are dried in stages, with the first stage at 40°C for 10 hours and the second stage at 60°C for 10 hours. The moisture content of the silkworms is less than 13%; finally, the dried silkworms are collected and sorted. They are packed into airtight packaging bags according to the specification of 1kg / bag, the air is extracted and stored at 20°C.

[0077] At the same time, control experiments were carried out using Beauveria bassiana KW1 and Beauveria bassiana HN6.

[0078] 2. Experimental results

[0079] The results of the analysis of the mortality rate of silkworms infected with different strains of Beauveria bassiana are shown in the figure. Figure 3 As shown: the rigor mortis rate of silkworm infected by Beauveriabassiana KW2 strain reached 100%, the rigor mortis rate of Beauveria bassiana KW1 was 99.02%, and the rigor mortis rate of Beauveria bassiana HN6 was only 3.9%.

[0080] In the process of using different strains of Beauveria bassiana to infect silkworms, it was found that Beauveria bassiana KW2 strain had unique performance characteristics during the rigidification process of silkworms. Taking Beauveria bassiana KW2 strain and Beauveriabassiana KW1 as examples, the phenotypic characteristics of different strains during the rigidification process of silkworms were analyzed.

[0081] Comparative analysis of the phenotypic characteristics of different strains of Beauveria bassiana at different time periods during the rigidification process of silkworms Figure 4 As shown. Figure 4It can be seen that the silkworms inoculated with KW2 strains showed a pink color on the second day after they died, while the silkworms inoculated with KW1 strains basically maintained their original body color. This obvious change in body color helps to more intuitively judge whether the silkworms have died in the mass production process, which is convenient for manual picking. Secondly, from the comparison of the body shape of the silkworms after they died after being infected by KW2 strains and KW1 strains, the silkworms made by KW2 strains showed cylindrical shapes, while the silkworms made by KW1 strains clearly showed that the connection between the head and chest and the abdomen was very narrow and thin, and it was very easy to "break heads" during the picking process, which ultimately affected the appearance of the silkworms. Finally, the mycelium on the surface of the silkworms made by KW2 strains was oily and thick, while the mycelium on the surface of the silkworms made by KW1 strains produced a large amount of spore powder, which was as delicate as lime powder. In the production process, less spore powder is conducive to the hygienic maintenance of the silkworm production environment and can better ensure the health of production workers.

[0082] Example 3 Method for large-scale production of silkworm using Beauveria bassiana KW2 strain

[0083] 1. Experimental Materials

[0084] Beauveria bassiana KW2 strain and a healthy silkworm of Huakang 3

[0085] 2. Experimental methods

[0086] Healthy fifth-instar silkworms were inoculated with Beauveria bassiana KW2 strain, and the effect of Beauveria bassiana KW2 strain on the mortality rate of silkworms and the quality of silkworms during large-scale production was analyzed.

[0087] The specific method of inoculating the Beauveria bassiana KW2 strain into silkworms to prepare silkworm pupae is as described in Example 2.

[0088] Random inspection of the quality of silkworm samples: In accordance with the sampling principles of traditional Chinese medicine stipulated in the Pharmacopoeia of the People's Republic of China (2020 edition), silkworm samples were selected for random inspection, and the diameter, body length and weight of each silkworm in the sample were measured, and statistical analysis was performed. Subsequently, the samples were sent to a third-party agency for testing. The test indicators included impurities, moisture, total ash, acid-insoluble ash, extracts, aflatoxins, heavy metals and harmful elements, 33 banned pesticides, and the content of adenine, adenosine, uracil, beauvericin and ammonium oxalate.

[0089] 3. Experimental results

[0090] When Beauveria bassiana KW2 is used in the large-scale production of silkworms, it can be seen from the rigidification process that the dead silkworms appear pink, while the surviving silkworms still show their original color. There are obvious differences in the apparent morphology, such as Figure 5 As shown, this color difference is conducive to the efficient picking during silkworm production.

[0091] The use of Beauveria bassiana KW2 strain inoculated a healthy silkworm Huakang 3, the mortality rate can reach 97%, the total weight of the silkworm can reach 26kg. Figure 6 shown. Figure 6 A in the figure is a picture of the finished product of the dried silkworm. The measurement results of the silkworm show that the average length of a single silkworm is 4.1±0.455cm, the average diameter is 7.0±0.517mm, and the average weight is 0.88±0.115g (the maximum weight is 1.12g). The silkworm is hard and brittle, easy to break, and the cross section is as follows: Figure 5 As shown in B, several bright brown or bright black silk gland rings can be seen in the cross section.

[0092] Some samples were randomly selected and sent to a third-party agency for testing, and the test results are shown in Table 1. As can be seen from Table 1, all indicators of Bombyx batryticatus meet the standards of the Pharmacopoeia of the People's Republic of China (2020 edition).

[0093] Table 1 Comparative analysis of the detection indexes of homemade silkworm pupa and the Pharmacopoeia of the People's Republic of China

[0094]

[0095] In addition, according to the requirements of Guangdong Province's "Bombyx Batryticatus Formula Granules", the total content of adenosine and adenine in Bombyx Batryticatus is 0.04% to 0.24%. The total content of adenosine and adenine in Bombyx Batryticatus prepared in this embodiment is 0.09% (including 0.08% adenosine and 0.01% adenine), which meets the requirements.

[0096] Some pharmaceutical companies in the industry require that the uracil content in the silkworm must be above 0.05%. The uracil content of the silkworm prepared in this embodiment is 0.11%, which meets and is significantly higher than the minimum requirement.

[0097] The content of beauvericin and ammonium oxalate is significantly positively correlated with the appearance of the white silkworm. The beauvericin content of the silkworm prepared in this example is 0.21%, and the ammonium oxalate content is 15.3%, which are significantly higher than the silkworm on the market. After comparing multiple batches, the difference in the content of beauvericin and ammonium oxalate in the silkworm is not significant between batches.

[0098] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A Beauveria bassiana KW2 strain, characterized in that: The Beauveria bassiana KW2 strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 19, 2024, with the collection number GDMCC NO: 65502.

2. A bacterial agent containing the Beauveria bassiana KW2 strain according to claim 1.

3. Use of the Beauveria bassiana KW2 strain according to claim 1 or the bacterial agent according to claim 2 in producing Bombyx batryticatus.

4. A method for producing silkworm pupae, characterized in that: The silkworm is inoculated with the Beauveria bassiana KW2 strain described in claim 1 or the bacterial agent described in claim 2 to produce a silkworm.

5. The method according to claim 4, characterized in that The silkworms are picked out when they become dead and change color.

6. The method according to claim 5, characterized in that The silkworms are picked out when their color turns red after they die.

7. The method according to claim 4, characterized in that The drying method of silkworm pupa is staged drying: the temperature of the first stage is 38-45°C, lasting 8-12 hours; the temperature of the second stage is 50-70°C, lasting 8-12 hours.

8. The method according to claim 7, characterized in that Dry until the moisture content of the silkworm is less than 13%.

9. The method according to claim 7, characterized in that: Drying time of silkworm pupae: Drying is carried out on the 3rd to 4th day after the silkworm pupae die.

10. The silkworm pupae produced according to the method of any one of claims 4 to 9.

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