Beauveria bassiana strain with high toxicity to multiple silkworm varieties and application of Beauveria bassiana strain in production of bombyx batryticatus

Abstract

The invention discloses a Beauveria bassiana strain with high toxicity to a plurality of silkworm varieties and application of the Beauveria bassiana strain in production of bombyx batryticatus, the Beauveria bassiana strain is a Beauveria bassiana KW1 strain, the Beauveria bassiana KW1 strain is preserved in Guangdong Microbial Culture Collection Center on July 30, 2024, and the preservation number of the Beauveria bassiana KW1 strain is GDMCC NO: 64922. According to the method, a spore suspension (the spore content is 1 * 10 < 6 > / mL) of the KW1 strain is used for inoculating a plurality of varieties of healthy five-instar silkworms, and the dead rate is 97.1%-99.5%. When large-scale production of the stiff silkworms is carried out, the dead rate still reaches 96%, the average body length of the obtained stiff silkworms is 4.3 + / -0.495 cm, the average diameter of the obtained stiff silkworms is 6.8 + / -0.703 mm, the maximum weight of a single stiff silkworm can reach 1.14 g, and the average weight of the obtained stiff silkworms is 0.88 + / -0.109 g (equivalent to the weight of 8.8 kg of ten thousands of stiff silkworms), all indexes of the stiff silkworms such as extracts, total ash content and water content conform to and are obviously superior to the standards of Pharmacopoeia of the People's Republic of China (2020 edition), and the stiff silkworms have good market prospects. Therefore, the KW1 strain has a good application prospect in the aspect of large-scale standardized production 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 particularly relates to a Beauveria bassiana strain with high toxicity to multiple silkworm varieties and application thereof in silkworm pupa production. Background Art

[0002] Bombyx batryticatus is the dried body of the 4th to 5th instar larvae of the silkworm (Bombyx mori Linnaeus), an insect of the Bombycidae family, which is killed by natural infection (or artificial inoculation) with Beauveria bassiana (Bals.) Vuillant. It is mostly produced in spring and autumn and has the effects of calming wind and stopping spasms, dispelling wind and relieving pain, and resolving phlegm and dispersing knots. According to statistics, 206 of the 10,169 existing Chinese patent medicines contain Bombyx batryticatus, which are mostly used to treat symptoms such as acute convulsions, epilepsy, and wind-heat headaches in children. The annual demand for Bombyx batryticatus in China is as high as 3,000 tons. With its wide application in medicine, health food, and cosmetics, its demand is expected to increase year by year. However, the production of Bombyx batryticatus is very limited.

[0003] Early silkworms mainly come from dead silkworms infected with Beauveria bassiana from time to time during silkworm breeding. Both the quality and yield of the medicinal materials are very unstable. In recent years, the production method of preparing silkworms by artificially inoculating Beauveria bassiana to infect five-year-old silkworms has attracted more and more attention. For example, CN110352920B provides a method for artificially cultivating white silkworms based on Beauveria bassiana. The average body length and diameter of the produced silkworms are 4.30±0.35cm and 0.5±0.1cm, respectively, and 10,000 silkworms weigh 7-8kg. However, the silkworms currently produced 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 silkworms. The characteristics of Beauveria bassiana strains are a key factor affecting the production efficiency and product quality of silkworms. At present, studies have also shown that different strains of Beauveria bassiana have certain selectivity for silkworm varieties, and the reported strains of Beauveria bassiana infect silkworm varieties are relatively single.

[0004] Therefore, discovering excellent Beauveria bassiana strains that are highly toxic to multiple silkworm species and can be used to mass-produce high-quality silkworm pupae is of great significance to the high-quality development of the silkworm pupae industry. Summary of the invention

[0005] The present invention aims at the problems of low production efficiency, low quality and lack of excellent Beauveria bassiana strains of silkworm, and provides a Beauveria bassiana strain with high toxicity to multiple silkworm varieties and its application in silkworm production. The strain was deposited in Guangdong Microbial Culture Collection Center on July 30, 2024, and its deposit number is GDMCC NO: 64922. The present invention firstly uses spore suspensions of KW1 strain and other 4 different Beauveria bassiana strains (the spore content is 1×10 6 The fifth-instar silkworms of Yuecan 6 were inoculated with spores of KW1 strain (containing 1×10 6 / mL) inoculated with four different silkworm varieties (Huakang No. 3, Jinxiu No. 2, Jingsong × Haoyue and Liangguang No. 2), the five-instar silkworms had a mortality rate higher than 97%, with the mortality rate (99.5%) and average weight of a single silkworm (0.90 ± 0.099g) of Huakang No. 3 silkworm being the highest. Finally, the present invention selected a healthy five-instar silkworm of Huakang No. 3, and after inoculation with the spore suspension of the KW1 strain, its mortality rate was still as high as 96%, the total weight of the dried silkworms could reach 26kg, the maximum weight of a single silkworm could reach 1.14g, and the average weight was 0.88 ± 0.109g; and the obtained silkworms were hard and brittle, easy to break, and four bright brown or bright black silk gland rings could be seen on the cross section. All indicators of the silkworms (tested by a third-party agency) met the standards of the Pharmacopoeia of the People's Republic of China (2020 edition), and the silkworms had excellent quality, indicating that the Beauveria bassiana KW1 strain has good application prospects in the large-scale standardized production of high-quality silkworms.

[0006] The first object of the present invention is to provide a Beauveria bassiana strain and a bacterial agent.

[0007] The second object of the present invention is to provide the use of the Beauveria bassiana strain and the bacterial agent in producing Bombyx batryticatus.

[0008] The third object of the present invention is to provide a method for producing Bombyx batryticatus based on the Beauveria bassiana strain and the bacterial agent.

[0009] The above-mentioned purpose of the present invention is achieved by the following technical solutions:

[0010] The present invention isolates and purifies the silkworm and identifies a Beauveria bassiana strain, named as Beauveria bassiana KW1 strain. The strain was deposited in Guangdong Microbial Culture Collection Center on July 30, 2024, and its deposit number is GDMCC NO: 64922. The present invention first uses spore suspensions of KW1 strain and other 4 different Beauveria bassiana strains (the spore content is 1×10 6The fifth-instar silkworms of Yuecan 6 were inoculated with spores of KW1 strain (containing 1×10 6 / mL) were inoculated into five-instar silkworms of four different silkworm varieties (Huakang No. 3, Jinxiu No. 2, Jingsong × Haoyue and Liangguang No. 2), and the mortality rates were all higher than 97%, with the mortality rate and average weight of a single Huakang No. 3 silkworm being the highest. Finally, the present invention selected the above-mentioned spore suspension to inoculate a healthy five-instar Huakang No. 3 silkworm, and the mortality rate was still as high as 96%. The total weight of the dried silkworms could reach 26kg, the maximum weight of a single silkworm could reach 1.14g, and the average weight was 0.88±0.109g; and the obtained silkworms were hard and brittle, easy to break, and four bright brown or bright black silk gland rings could be seen on the cross section. All quality indicators (tested by a third-party agency) met the standards of the Pharmacopoeia of the People's Republic of China (2020 edition), and the silkworms were of excellent quality, indicating that the KW1 strain of Beauveria bassiana has good application prospects in the large-scale standardized production of high-quality silkworms.

[0011] The present invention claims protection for a Beauveria bassiana strain.

[0012] Specifically, the strain is Beauveria bassiana KW1 strain, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on July 30, 2024, and its preservation number is GDMCC NO: 64922.

[0013] Specifically, the strain is isolated and purified from a silkworm naturally infected with Beauveria bassiana. The center of the colony of the strain is slightly protruding, the colony is white, grows radially in concentric circles, and has obvious annular grooves; the hyphae are colorless and slender, with a smooth surface, the conidiophores are attached to the nutritional hyphae, and the conidia are spherical or nearly spherical, transparent and smooth.

[0014] The present invention also claims to protect a bacterial agent containing the Beauveria bassiana KW1 strain.

[0015] The present invention also seeks to protect the use of the Beauveria bassiana KW1 strain and the bacterial agent in producing Bombyx batryticatus.

[0016] The present invention also claims a method for producing silkworm pupae, which produces silkworm pupae by inoculating silkworms with the Beauveria bassiana KW1 strain or the bacterial agent.

[0017] The scheme of the present invention is applicable to a variety of silkworm varieties, such as Yuecan No. 6, Huakang No. 3, Jinxiu No. 2, Jingsong×Haoyue, and Liangguang No. 2.

[0018] As a preferred embodiment, when the Beauveria bassiana KW1 strain is used to produce silkworms, the temperature of the production environment (rigidification chamber) is maintained at 25-26°C.

[0019] As a preferred embodiment, the relative humidity of the production environment (rigidity chamber) is not less than 85%.

[0020] 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.

[0021] The present invention has the following beneficial effects:

[0022] The present invention provides a strain of Beauveria bassiana KW1 which is highly toxic to a variety of silkworm varieties. The strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on July 30, 2024, and its deposit number is GDMCC NO: 64922. When using the Beauveria bassiana KW1 strain to produce silkworms, a strain with a spore content of 1×10 6 The healthy fifth-instar silkworms of Yuecan 6 were inoculated with a spore suspension of 1×10 / mL, and the mortality rate reached 99.5%, which was much higher than that of the other four strains (the mortality rates were all less than 18.5%). 6 The fifth-instar silkworms of four different silkworm varieties (Huakang 3, Jinxiu 2, Jingsong×Haoyue and Liangguang 2) inoculated with 1% parasite (mL) had a mortality rate higher than 97%, with Huakang 3 having the highest mortality rate (99.5%) and average single silkworm weight (0.90±0.099g).

[0023] In addition, the present invention uses the above-mentioned spore suspension to inoculate a healthy five-year-old silkworm of Huakang No. 3, and the dead rate is still as high as 96%, and the weight of all silkworms can reach 26kg; the average body length of a single silkworm is 4.3±0.495cm, the average diameter is 6.8±0.703mm, the maximum weight of a single silkworm can reach 1.14g, and the average weight is 0.88±0.109g; and the obtained silkworm is hard and brittle, easy to break, and four bright brown or bright black silk gland rings can be seen in the cross section. Other indicators such as extract, impurities, moisture, total ash, acid-insoluble ash and other contents (third-party agency detection) have reached the "Pharmacopoeia of the People's Republic of China" (2020 edition) standards, and the silkworm is of excellent quality. The Beauveria bassiana KW1 strain provided by the present invention has good application prospects in the large-scale standardized production of high-quality silkworms. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 2This is a phylogenetic tree constructed based on the 18S rDNA+ITS sequence of Beauveria bassiana KW1 strain.

[0026] Figure 3 The results are to analyze the mortality rate of the fifth instar silkworm of Yuechan 6 infected with Beauveria bassiana KW1 strain and four other different strains of Beauveria bassiana (all with spores containing 1×10 6 / mL of spore suspension).

[0027] Figure 4 Comparative analysis of the mortality rate of different silkworm varieties inoculated with Beauveria bassiana KW1 (spore suspension containing 1×10 6 / mL).

[0028] Figure 5 The silkworm products obtained by infecting silkworms with the KW1 strain of Beauveria bassiana. A in the figure is the dried silkworm product (with aerial hyphae and conidia in the form of white powdery frost on the surface); B in the figure is the cross section of the silkworm product (with bright black silk gland rings), and the scale is 1 mm. DETAILED DESCRIPTION

[0029] 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.

[0030] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0031] 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 reflecting 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; if the concentration of spore suspension required to achieve the same mortality rate is different, then the strain with a lower concentration has a higher toxicity to the silkworm. In addition, when the same spore suspension (same strain and spore content) is used to infect different varieties of silkworms, the variety with a higher mortality rate is more susceptible to Beauveria bassiana, which also means that Beauveria bassiana is more toxic to the silkworm variety.

[0032] 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.

[0033] Example 1 Isolation and Identification of Beauveria bassiana

[0034] 1. Isolation and purification of strains

[0035] In the clean bench, the silkworm samples obtained by natural infection with Beauveria bassiana during silkworm breeding (provided by a silkworm farmer in Guangzhou, the silkworm species was Yuecan No. 6) were soaked in 75% alcohol for 30 seconds, rinsed with sterile water for 3 times, and placed on sterile absorbent paper. When the moisture on the surface of the silkworm was completely removed, it was placed on a Sabouraud medium (SDAY medium) plate, with the front side of the SDAY medium plate facing upward, and cultured in a constant temperature and humidity incubator at 25°C and a relative humidity (RH) of 90%. When pure white colonies grow, pick them up with an inoculation needle in time, purify and culture them on new SDAY medium plates through multiple rounds of plate streaking method, and then pick single colonies for microscopic examination. Select colonies without other bacteria under microscopic examination for identification, including morphological identification and 18S rDNA+ITS sequencing analysis and identification. Select a pure colony identified as Beauveria bassiana as the target strain and record it as Beauveria bassiana KW1 strain.

[0036] The specific morphological identification and 18S rDNA+ITS sequencing analysis and identification methods are as follows "2. Morphological identification of strains" and "3. Molecular biological identification of strains":

[0037] 2. Morphological identification of strains

[0038] The isolated and purified strain was inoculated onto SDAY medium plates and cultured at 25°C for 7 days. The shape and color of the colonies were observed and recorded. The type, shape and size of the conidia were observed and recorded under a microscope.

[0039] The colony and conidia morphology of the isolated and purified strains on SDAY medium plates were analyzed. Figure 1 As shown. Figure 1 It can be seen that the colony has a slight protrusion in the center, is white, grows radially in concentric circles, and has obvious annular grooves; the hyphae are colorless and slender, with a smooth surface, and the 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.

[0040] 3. Molecular biological identification of strains

[0041] The isolated and purified strain was inoculated on a SDAY medium plate and cultured at 25°C for 7 days. The spores and hyphae 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.). The gene fragment containing the 18SrDNA+ITS sequence of the strain was amplified using primers 18S-F and ITS4-R. The nucleotide sequences of the primers are as follows:

[0042] Primer 18S-F: 5′-TATCTGGTTGATTCTGCCAG-3′

[0043] Primer ITS4-R: 5′-TCCTCCGCTTATTGATATGC-3′

[0044] Reaction system: 25 μL PrimeSTAR Max Premix (2X), 1 μL each of primers 18S-F and ITS4-R (concentration 10 μM), 1 μL genomic DNA of the strain, and add ultrapure water to 50 μL.

[0045] Reaction conditions: pre-denaturation at 98°C for 5 min; 98°C for 10 sec, 55°C for 30 sec, 72°C for 1 min, 30 cycles; extension at 72°C for 5 min.

[0046] After the PCR product was detected by agarose gel electrophoresis, the target fragment was recovered and purified using a common agarose gel DNA recovery kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) and ligated to pMD TM 19-T vector and transformed into E. coli TOP10 competent cells. Then, colony PCR was used to perform preliminary detection of E. coli transformants, and positive clones were selected and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing analysis.

[0047] The homology analysis of the 18S rDNA+ITS sequence of the strain by NCBI / BLASTn showed that the strains most similar to the strain were all Beauveria bassiana, indicating that the strain belonged to Beauveria bassiana. Among them, the 18S rDNA+ITS sequence of Beauveria bassiana HN6 was completely consistent with that of the strain. At the same time, the genomes of the strain and Beauveria bassiana HN6 were compared and analyzed. The results showed that the genome sizes of the strain and Beauveria bassiana HN6 were 38.3Mb and 37.1Mb, respectively, and the total number of genes encoded by the two genomes were 10488 and 10000, respectively. It can be seen that there are obvious differences in the genomes of the two strains, and they are two different strains.

[0048] The phylogenetic tree was constructed using MEGA11.0 software. Figure 2 As shown, this strain clusters with other Beauveria bassiana strains.

[0049] In summary, the strain isolated and purified from the silkworm was identified as the genus Beauveria and the species Beauveria bassiana, named Beauveria bassiana KW1 strain, and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 30, 2024, with the preservation number GDMCC NO: 64922.

[0050] Example 2 Determination of the Mortality Rate of Silkworms Infected by Different Strains of Beauveria bassiana

[0051] 1. Experimental methods

[0052] The experiment was divided into 5 experimental groups, each with 210 healthy silkworms of Yuecan No. 6 (fourth-instar sleeping silkworms), and 3 replicates were set in each group. Beauveria bassiana KW1, Beauveria bassiana ARSEF 2860, Beauveria bassiana HN6, Beauveria bassiana ACCC 30812 and Beauveria bassiana ACCC30886 were used to inoculate healthy five-instar silkworms of Yuecan No. 6, respectively, to study the mortality rate of silkworms infected with different Beauveria bassiana, so as to evaluate the virulence of KW1 strain. Among them, Beauveria bassiana ACCC 30812 and Beauveria bassiana ACCC 30886 were from the China Agricultural Microbial Culture Collection Management Center, Beauveria bassiana HN6 was donated by Professor Guo Xijie of Jiangsu University of Science and Technology, and Beauveria bassiana ARSEF 2860 was donated by Professor Wang Chengshu of Shanghai Institute of Plant Physiology and Ecology. The specific method is as follows:

[0053] (1) Selection of fourth-instar dormant silkworms: Select fourth-instar dormant silkworms with healthy bodies and normal feeding from the silkworm beds, transfer them to a rigidification room for feeding, and inoculate them with Beauveria bassiana after they wake up from dormancy;

[0054] (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 the exhaust port to a spray sterilization device to prevent the leakage of Beauveria bassiana and its spores;

[0055] (3) Preparation of spore suspension: Select Beauveria bassiana grown on SDAY medium plates for 15 to 20 days and use sterile water containing 0.1% Tween-80 to prepare a spore suspension with a spore content of 1×10 6 / mL of spore suspension;

[0056] (4) Spraying and inoculation of fifth-instar silkworms: Selected fourth-instar sleeping silkworms are transferred to a rigidification room. 2-3 days after waking up, the spore suspension is added to an agricultural sprayer and sprayed and inoculated on the fifth-instar silkworms. The spore suspension is sprayed on the surface of the silkworms for the first time until the surface of the silkworm body is wet. After 15 minutes, the second spraying is performed. After 15 minutes, mulberry is started, and then the spore liquid is sprayed on the surface of mulberry leaves for the third inoculation. After 15 minutes, the spore liquid is sprayed on the surface of mulberry leaves again for the fourth inoculation.

[0057] (5) Collection of dead silkworms: Regularly observe the fifth-instar silkworms after inoculation. When the silkworms become dead, pick them out in time and spread them out on a clean silkworm plaque. When the dead silkworm mortality rate reaches 20%, lay out the fifth-instar silkworm net and feed them a small amount of mulberry leaves. (A small amount of mulberry leaves means covering the silkworm net with a layer of mulberry leaves, which should be sufficient to completely cover the silkworm net.) After 48 hours, collect the silkworm net and maintain the ambient temperature at 25-26°C and the relative humidity at not less than 85%.

[0058] (6) Drying and storage of silkworm pupae: After the ossification is completed (when the surface of the silkworm pupae is covered with Beauveria bassiana hyphae and the color of the silkworm body cannot be distinguished), the wet silkworm pupae are placed in an oven and first heat treated at 90°C for 50 minutes to kill Beauveria bassiana and its spores; then continue to dry at 60°C until the moisture content of the silkworm pupae is less than 13%; finally, the dried silkworm pupae are collected and sorted. They are packed into airtight packaging bags according to the specification of 1 kg / bag, the air is extracted and stored at 20°C.

[0059] 2. Experimental results

[0060] Analysis of the results of the mortality rate of silkworms infected with different strains of Beauveria bassiana. Figure 3 As shown. Figure 3 It can be seen that the rigor mortis rate of Beauveria bassiana KW1 strain infecting silkworms is as high as 99.5%, while the rigor mortis rates of the other four strains of Beauveria bassiana infecting silkworms are all lower than 20%, specifically: the rigor mortis rates of Beauveria bassiana ARSEF 2860, Beauveriabassiana HN6, Beauveria bassiana ACCC 30812 and Beauveria bassiana ACCC 30886 are 18.5%, 3.9%, 1.95% and 17.07% respectively.

[0061] Example 3 Analysis of the mortality rate of different varieties of silkworms inoculated with Beauveria bassiana KW1 strain and the weight of the obtained silkworms

[0062] 1. Experimental Materials

[0063] Beauveria bassiana KW1 strain, healthy fifth-instar silkworms from four different silkworm species (Huakang 3, Jinxiu 2, Jingsong×Haoyue and Liangguang 2)

[0064] 2. Experimental methods

[0065] The experiment was divided into 4 experimental groups (each silkworm species as one group), each group had 210 healthy silkworms (fourth instar sleeping silkworms), and each group was set up with 3 replicates. The four silkworms were inoculated with the Beauveria bassiana KW1 strain, and the mortality rate of Beauveria bassiana to silkworms and the average weight of a single silkworm were counted. The specific method of infecting silkworms with the Beauveria bassiana KW1 strain is referred to Example 2.

[0066] 3. Experimental results

[0067] Comparative analysis of the mortality rate of different varieties of silkworms inoculated with Beauveria bassiana KW1 strain. Figure 4 As shown. Figure 4 It can be seen that the silkworm rigidification process was completed on the 9th day after inoculation with the Beauveria bassiana KW1 strain, and the rigidification rates of different silkworm species were all over 97%, specifically: Huakang No. 3, 99.5%; Jinxiu No. 2, 97.1%; Jingsong×Haoyue, 97.7%; Liangguang No. 2, 99.1%.

[0068] In addition, the average weight of each silkworm of the four types of silkworms is slightly different, Huakang No. 3 is 0.90±0.099g, Jinxiu No. 2 is 0.79±0.102g, Jingsong×Haoyue is 0.88±0.105g, and Liangguang No. 2 is 0.73±0.102g. Among them, 80% of the silkworms produced by Huakang No. 3 and Jingsong×Haoyue weigh more than 0.8g, while only 50% of the silkworms produced by Liangguang No. 2 and Jinxiu No. 2 weigh 0.8g. Therefore, judging by the mortality rate and the average weight of each silkworm, Huakang No. 3 is the most suitable silkworm species for preparing silkworms, so this silkworm species is selected for large-scale silkworm production.

[0069] Example 4 Method for large-scale production of silkworm using Beauveria bassiana KW1 strain

[0070] 1. Experimental Materials

[0071] Beauveria bassiana KW1 strain and a healthy silkworm of Huakang 3

[0072] 2. Experimental methods

[0073] A healthy five-instar silkworm of the Huakang 3 variety was inoculated with the Beauveria bassiana KW1 strain, and the mortality rate of the silkworm and the quality of the silkworm were analyzed.

[0074] The specific method of infecting silkworms with the Beauveria bassiana KW1 strain to produce silkworm pupae is shown in Example 2.

[0075] Random inspection of the quality of silkworm samples: According to 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 length, diameter and weight of each silkworm in the sample were measured, and statistical analysis was performed. Then, they 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 and uracil.

[0076] 3. Experimental results

[0077] The KW1 strain of Beauveria bassiana was used to inoculate a healthy silkworm, Huakang No. 3, and the mortality rate reached 96%. The total weight of the silkworms obtained was up to 26 kg. Figure 5 As shown in A, the measurement results show that the average body length of a single silkworm is 4.3±0.495cm, the average diameter is 6.8±0.703mm, and the average weight is 0.88±0.109g (the maximum weight is 1.14g). The obtained silkworm is hard and brittle, easy to break, and the cross section is as follows Figure 5 As shown in B, four bright brown or bright black silk gland rings can be seen in the cross section.

[0078] 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).

[0079] Table 1 Comparative analysis of the detection indexes of homemade silkworm pupa and the Pharmacopoeia of the People's Republic of China

[0080]

[0081]

[0082] In addition, according to the requirement of Guangdong Province's "Bombyx Batryticatus Formula Granules", the total content of adenine and adenosine in Bombyx Batryticatus is 0.04% to 0.24%. The total content of adenine and adenosine in Bombyx Batryticatus prepared in this embodiment is 0.10% (including 0.01% adenine and 0.09% adenosine), which meets the requirement.

[0083] Some pharmaceutical companies in the industry require that the uracil content of Bombyx mori is above 0.05%. The uracil content of the Bombyx mori prepared in this embodiment is 0.11%, which meets and significantly exceeds the minimum requirement.

[0084] 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 strain KW1, characterized in that: The Beauveria bassiana strain KW1 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 30, 2024, and its preservation number is GDMCC NO: 64922.

2. A bacterial agent, characterized in that Contains the Beauveria bassiana strain KW1 as claimed in claim 1.

3. Use of the Beauveria bassiana strain KW1 according to claim 1 in producing Bombyx batryticatus.

4. Use of the microbial agent according to claim 2 in producing Bombyx batryticatus.

5. A method for producing silkworm pupae, characterized in that: The silkworm is produced by inoculating silkworms with the Beauveria bassiana strain KW1 described in claim 1 or the bacterial agent described in claim 2.

6. The method according to claim 5, characterized in that The variety of the silkworm is selected from any one, any two or any several of Yuecan No. 6, Huakang No. 3, Jinxiu No. 2, Jingsong×Haoyue, and Liangguang No.

2.

7. The method according to claim 5, characterized in that The temperature of the production environment is maintained at 25-26℃.

8. The method according to claim 5, characterized in that The relative humidity of the production environment shall not be less than 85%.

9. The method according to claim 5, characterized in that The inoculation method is spore liquid spraying.

10. The method according to claim 9, characterized in that The spore content of the spore liquid is not less than 1×10 6 Pieces / mL.

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