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

Abstract

The application discloses a strain suitable for large-scale production of high-quality Bombyx Batryticatus and application thereof. The strain is Beauveria bassiana KW2 strain, which was preserved in the Guangdong Microbial Culture Collection Center on November 19, 2024, and the preservation number is GDMCC NO: 65502. The KW2 strain is used for producing Bombyx Batryticatus, and the color of the Bombyx Batryticatus after being stiffened is peach red, and then gradually fades, which is helpful for efficient selection of the Bombyx Batryticatus, can better control the quality of the Bombyx Batryticatus and reduce the labor cost, and is very suitable for large-scale production of the Bombyx Batryticatus. Meanwhile, the mycelium on the surface of the Bombyx Batryticatus is thick and heavy, and the spore powder is less, which is beneficial to production environment management and personnel health. Moreover, the KW2 strain has a 100% silking rate on the silkworm, and the Bombyx Batryticatus is in a cylindrical shape, has high quality, and various indexes such as moisture content all meet the standards; moreover, the content of the white bacillus and ammonium oxalate is 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 Bombyx Batryticatus.

Description

Technical Field

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

[0002] Bombyx batryticatus, also known as the "dead worm," is the dried remains of a silkworm larvae infected with Beauveria bassiana and later died. The characteristics, uses, and therapeutic effects of Bombyx batryticatus were first documented in the medical classic "Shennong's Herbal Classic." Bombyx batryticatus is mild in nature and has the effects of calming, reducing fever, relieving wind and spasms, and resolving phlegm and dispersing nodules. Bombyx batryticatus is rich in medicinal ingredients, such as flavonoids, ammonium oxalate, and beauvericin, which have significant effects in inhibiting cancer cell proliferation, enhancing immunity, killing bacteria, and providing an anticonvulsant effect. With in-depth research on the medicinal value of Bombyx batryticatus, its range of uses has become increasingly broad, and market demand for Bombyx batryticatus is increasing year by year.

[0003] In recent years, the production method of producing Bombyx mori by artificially inoculating fifth-instar silkworms with Beauveria bassiana has garnered increasing attention. The quality of the resulting Bombyx mori is closely linked to the type of Beauveria inoculated. Currently, the overall quality of Bombyx mori produced on the market is low. Bombyx mori often fail to meet the requirements of the Pharmacopoeia of the People's Republic of China, as well as some local and enterprise standards, in one or more parameters, such as dimensions, ash content, extract content, and number of silk gland rings. This significantly impacts the profitability of large-scale Bombyx mori production and its clinical application.

[0004] Furthermore, large-scale production requires a significant workload for sorting dead silkworms. When the commercially available Beauveria bassiana infects silkworms, the silkworms appear white when they become rigid, a color not dissimilar to the silkworms' own bodies. Sorting out the dead silkworms from a large number of silkworms is particularly challenging, requiring experienced personnel and placing high demands on the personnel. Furthermore, if the dead silkworms are not promptly sorted out for subsequent rigidification and drying, the quality and yield of the dead silkworms decrease, impacting their medicinal value. Furthermore, after conventional Beauveria bassiana infects silkworms, the mycelium that grows on the surface of the silkworms resembles lime powder, easily producing large amounts of spore powder. This creates a large amount of dust in the production workshop, which is not only detrimental to the management of the silkworm farming environment but also to the health of the workers involved.

[0005] In addition, studies have shown that beauvericin and ammonium oxalate, as key characteristic ingredients in Bombyx batryticatus for calming wind and relieving spasms, show a significant positive correlation between the levels of ammonium oxalate and beauvericin in Bombyx batryticatus, and that the levels of these two ingredients are also significantly positively correlated with the appearance of Bombyx batryticatus. However, the beauvericin and ammonium oxalate levels in existing Bombyx batryticatus are low and unstable, ranging from 0.0101% to 0.1192%, a difference of approximately 10 times; the ammonium oxalate content ranges from 4.71% to 10.84%. Moreover, beauvericin and ammonium oxalate were not detected in 17.2% of multiple batches. Therefore, the quality of Bombyx batryticatus currently available on the market presents a high risk (Gao Peng et al., 2024, Research on Quality Standards for Bombyx batryticatus Medicinal Materials and Decoction Pieces). Summary of the Invention

[0006] In response to the above-mentioned 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 Microbial 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 conducive to 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 Bombyx batryticatus.

[0011] The above-mentioned purpose of the present invention is achieved through 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 Microbial 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's body surface changes from white to pink after the silkworm dies, and then gradually fades, which is conducive to the selection 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 body is oily and thick, and it is not easy to produce a large amount of spore powder, which is beneficial to the management of the production environment and the health of personnel. 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 26 kg, and the maximum weight of a single silkworm can reach 1.12 g; 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), and 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 Microbial Culture Collection Center on November 19, 2024, and its preservation number is GDMCC NO: 65502.

[0015] The present invention also claims protection for 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 protection for a method for producing Bombyx batryticatus.

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

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

[0020] Therefore, preferably, in the method for producing dead silkworms 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 using the Beauveria bassiana KW2 strain to produce silkworms, the temperature of the production environment (rigidification room) 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 be started 3 to 4 days 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 minutes.

[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, preferably, the temperature of the first stage is 40° C. and lasts for 10 hours; and the temperature of the second stage is 60° C. and lasts for 10 hours.

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

[0032] The silkworm produced by the above method of the present invention is of high quality, and all indicators meet the standards of the Pharmacopoeia of the People's Republic of China (2020 edition), and the content of the characteristic components beauvericin and ammonium oxalate is high, and the silkworm is of 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 present invention provides a Beauveria bassiana KW2 strain. The KW2 strain performs excellently in the process of infecting silkworms. The silkworms appear pink on the second day after death, and then gradually fade. The silkworms become cylindrical, which is conducive to the picking and sorting of the silkworms, can better control the quality of the silkworms and reduce labor costs. The KW2 strain is very suitable for the large-scale production of silkworms.

[0035] At the same time, the mycelium on the surface of the silkworm 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 silkworm breeding production environment and the health of production workers.

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

[0037] In addition, in the large-scale production of silkworms, the spore suspension of the KW2 strain was 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 and easy to break. Several bright brown or bright black silk gland rings can be seen on the cross section. Other indicators such as impurities, moisture, and acid-insoluble ash content (tested by a third-party agency) all meet the standards of the "Pharmacopoeia of the People's Republic of China" (2020 edition). In addition, its characteristic ingredients, beauvericin and ammonium oxalate, are high in content, 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 show the analysis of 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; Figure A is the Beauveria bassiana KW2 strain, and Figure B 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 a part of the dried silkworm product, with a scale of 1 cm; Figure B shows a cross-section of the silkworm product, with a scale of 1 mm. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. 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 were commercially available.

[0047] In this article, the "mortality rate" refers to the ratio of healthy fifth-instar silkworms that die from Beauveria bassiana infection before spinning cocoons to the total number of inoculated silkworms. It is a key indicator of the success rate of silkworm farming. When different strains infect the same silkworm species, using the same concentration of spore suspension, the strain with a higher mortality rate is more virulent to that silkworm.

[0048] Sabouraud's medium (SDAY medium): Weigh 40 g of glucose, 10 g of peptone, 10 g of yeast extract powder, and 15 g of agar powder. Dissolve completely in distilled water and adjust the volume to 1000 mL. Sterilize the mixture under high temperature and high pressure for 30 minutes, then spread onto a plate for later use.

[0049] Example 1 Isolation and Identification of Beauveria bassiana

[0050] 1. Isolation and purification of strains

[0051] In a clean bench, samples of silkworms obtained from natural infection with Beauveria bassiana during silkworm breeding (provided by a silkworm farmer in Guangzhou, with the silkworm spawn being Liangguang No. 2) were soaked in 75% alcohol for 30 seconds, rinsed two to three times with sterile water, and surface moisture absorbed with absorbent paper. The samples were then placed on SDAY medium plates, facing upward, and incubated in a constant temperature and humidity incubator at 25°C and 90% relative humidity. When pure white colonies appeared on the silkworms, they were promptly picked with an inoculating needle and purified by multiple rounds of plate streaking on new SDAY medium plates. Colony morphology was observed, and the morphological characteristics of hyphae and conidia were observed under an optical microscope.

[0052] Single colonies without other foreign bacteria were identified under microscopic examination, including morphological identification and 18S rDNA+ITS sequencing molecular identification. 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 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 The colony is white, with a slightly raised center and a circular, outward-spreading growth pattern. Its surface is characterized by annular grooves. The hyphae are colorless, elongated, and smooth. Conidiophores are attached to the vegetative hyphae. The conidia are spherical or nearly spherical, transparent, and smooth. Based on these characteristics, the strain was preliminarily identified as Beauveria bassiana.

[0057] 3. Molecular biological identification of strains

[0058] The isolated and purified strain was inoculated onto new SDAY medium plates and cultured in a constant temperature and humidity incubator at 25°C and 90% relative humidity for 7 days. Spores and hyphae were then scraped from the surface of the culture medium, and genomic DNA was extracted using a fungal genomic DNA extraction kit (purchased from Beijing Solaibao Technology Co., Ltd.). Primers 18S-F and ITS4-R were designed to amplify a gene fragment containing the strain's 18S rDNA and ITS sequences. The nucleotide sequences of these primers are shown below. These primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[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 Baisai 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 recovered and purified using a common agarose gel DNA recovery kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). The target fragment was ligated into pMD TM The ligation product was obtained on the 19-T vector and transformed into competent E. coli TOP10 (purchased from Beijing Qingke Biotechnology Co., Ltd.) according to the instructions. The E. coli transformants were then amplified by colony PCR using primers 18S-F and ITS4-R for preliminary testing. Positive clones were selected and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing analysis.

[0064] Homology analysis of the resulting 18S rDNA + ITS gene sequence using NCBI / BLASTn revealed that the strains most similar to this strain's gene sequence were all Beauveria bassiana, indicating that this strain belongs to the same species. The ITS sequence of Beauveria bassiana HN6 was completely identical to that of this strain. Further comparative analysis of the genomes of this strain and Beauveria bassiana HN6 revealed that the genome sizes of this strain and Beauveria bassiana HN6 were 36.6 Mb and 37.1 Mb, respectively, and that the total number of genes encoded by these two genomes was 9,880 and 10,000, respectively. This indicates that this strain and HN6 have significant genomic differences, indicating that they are distinct 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 deposited in the Guangdong Provincial Microbial Culture Collection on November 19, 2024, with the deposit number GDMCC NO: 65502.

[0067] Example 2 Determination of Mortality Rate and Phenotypic Characteristics of Silkworms Infected by Different Beauveria bassiana Strains

[0068] 1. Experimental methods

[0069] The experiment was divided into three experimental groups, each with 210 healthy Huakang 3 silkworms (fourth-instar sleeping silkworms), and three replicates per group. Healthy fifth-instar Huakang 3 silkworms were inoculated with Beauveria bassiana KW2, Beauveria bassiana KW1, and Beauveriabassiana HN6, respectively. The mortality rates of silkworms infected with different Beauveria bassiana strains were studied, thereby evaluating the virulence of the KW2 strain. 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 deposited in the Guangdong Provincial Center for Microbiological Culture Collection on July 30, 2024, with the deposit number GDMCC NO: 64922.

[0070] The specific method is as follows:

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

[0072] (2) Preparation of the inoculation environment: Use constant temperature and humidity equipment to maintain the indoor temperature of the inoculation chamber at 25-26°C and the humidity not less than 85%; use a mechanical ventilation system to provide regular air supply and exhaust 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 Spore suspension of 1000 / mL;

[0074] (4) Spraying and inoculating the fifth-instar silkworms: Transfer the selected fourth-instar dormant silkworms to a rigidification room. 2-3 days after waking up, spray the fifth-instar silkworms with the spore suspension using an agricultural sprayer. Spray the spore suspension onto the surface of the silkworms for the first time until the silkworms are wet. Spray the second time 15 minutes later. Start feeding mulberry leaves 15 minutes later. Then spray the spore suspension onto the surface of mulberry leaves for the third inoculation. 15 minutes later, spray the spore suspension onto 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 on 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 and feed a small amount of mulberry leaves, 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, put away the silkworm net;

[0076] (6) Drying and storage of silkworm pupae: When the silkworms are completely rigidified (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 rigidified. 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 finally less than 13%. Finally, the dried silkworms 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.

[0077] At the same time, Beauveria bassiana KW1 and Beauveria bassiana HN6 were used as control experiments.

[0078] 2. Experimental results

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

[0080] During the infection of silkworms with different strains of Beauveria bassiana, it was discovered that Beauveria bassiana KW2 exhibited unique characteristics during the rigidification process. Using Beauveria bassiana KW2 and Beauveriabassiana KW1 as examples, the phenotypic characteristics of the rigidification process after infection with different strains of silkworms were analyzed.

[0081] Comparative analysis of phenotypic characteristics of silkworms infected with different Beauveria bassiana strains at different time periods during the rigidification process. Figure 4 As shown. Figure 4As can be seen, silkworms inoculated with the KW2 strain developed a pinkish color the day after death, while those inoculated with the KW1 strain maintained their original color. This distinct color change facilitates intuitive identification of dead silkworms during mass production, facilitating manual sorting. Secondly, a comparison of the body shapes of dead silkworms infected with the KW2 and KW1 strains revealed that KW2-inoculated silkworms were cylindrical, while KW1-inoculated silkworms exhibited a significantly narrow and thin junction between the cephalothorax and abdomen, making them prone to breaking off during sorting and ultimately affecting their appearance. Finally, the mycelium of KW2-inoculated silkworms appeared oily and thick, while that of KW1-inoculated silkworms produced copious amounts of spore powder, as fine as lime powder. Less spore powder improves the hygiene of silkworm production and the health of 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, Bombyx mori, from Zhanghuakang 3

[0085] 2. Experimental methods

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

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

[0088] Random inspection of the quality of silkworm samples: Following the sampling principles for traditional Chinese medicines specified in the Pharmacopoeia of the People's Republic of China (2020 edition), silkworm samples were selected for random inspection. The diameter, length, and weight of each silkworm in the sample were measured and statistically analyzed. Subsequently, the samples were sent to a third-party agency for testing. The test indicators included impurities, moisture, total ash, acid-insoluble ash, extractables, 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 efficient sorting during silkworm production.

[0091] The use of Beauveria bassiana KW2 strain inoculated a Huakang 3 healthy silkworm, the dead 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. According to the measurement results of the silkworm, the average body 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 prepared 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 inspected 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 indicators 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 Bombyx batryticatus be above 0.05%. The uracil content of the Bombyx batryticatus prepared in this embodiment is 0.11%, which meets and significantly exceeds the minimum requirement.

[0097] The beauvericin and ammonium oxalate contents were significantly positively correlated with the appearance of the Bombyx batryticatus. The Bombyx batryticatus prepared in this example had a beauvericin content of 0.21% and an ammonium oxalate content of 15.3%, both significantly higher than commercially available Bombyx batryticatus. Comparisons of multiple batches revealed no significant differences in beauvericin and ammonium oxalate content between batches.

[0098] The embodiments above are preferred implementations of the present invention, but the implementations 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 considered equivalent replacement methods and are included in the scope of protection 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 Microbial Culture Collection Center on November 19, 2024, with the deposit 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 microbial agent according to claim 2 in producing Bombyx batryticatus.

4. A method for producing Bombyx batryticatus, characterized in that: The silkworm is inoculated with the Beauveria bassiana KW2 strain according to claim 1 or the microbial agent according to claim 2 to produce a dead silkworm.

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

6. The method according to claim 5, characterized in that The silkworms are picked 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℃, which lasts for 8-12 hours; the temperature of the second stage is 50-70℃, which lasts for 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 3-4 days after the silkworm pupae die.

10. The Bombyx batryticatus produced according to the method of any one of claims 4 to 9.

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