Beauveria bassiana fungicide capable of greatly improving silkworm death time concentration ratio and high-quality stiff silkworm production method

Abstract

The invention belongs to the technical field of stiff silkworm preparation. More specifically, the invention relates to a beauveria bassiana microbial inoculum capable of greatly improving silkworm death time concentration and a high-quality stiff silkworm production method. The invention provides a beauveria bassiana microbial inoculum which contains a strain of beauveria bassiana and a fermentation product of another strain of beauveria bassiana, wherein the fermentation product is obtained by removing thalli. When the bombyx mori is infected by the microbial inoculum, the time that all bombyx mori is dead can be shortened, the bombyx mori dead time is concentrated on the sixth day after inoculation, the time span is only 24 h, the production efficiency of bombyx mori can be improved, the labor for feeding and picking silkworms in the dead stage is effectively reduced, and the production cost is reduced; in addition, according to the method for producing the bombyx batryticatus, a very good infection effect can be achieved by spraying the fungicide once for inoculation, and the working procedures for producing the bombyx batryticatus are reduced. And the bombyx batryticatus prepared by the method for producing the bombyx batryticatus is uniform in size, good in consistency of product quality parameters and stable in quality, and the clinical application effect can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparing silkworm corpuscles, and more specifically relates to a Beauveria bassiana fungus agent that significantly increases the concentration of silkworm death time and a method for producing high-quality silkworm corpuscles. Background Art

[0002] White silkworm, also known as "dead silkworm" or "dead sky worm," refers to the dried bodies of the fourth and fifth instar larvae of the silkworm, Bombyx mori Linnaeus, infected (or artificially inoculated) with the fungus Beauveria bassiana (Bals.) Vuillant. It is typically produced in spring and autumn. It has the properties of calming wind and relieving spasms, dispelling wind and relieving pain, and resolving phlegm and dispersing nodules. It is commonly used for treating liver wind with phlegm, epilepsy, acute convulsions in children, tetanus, stroke, wind-heat headaches, red eyes and sore throat, itchy urticaria, and mumps. According to statistics, 206 of the 10,169 existing Chinese patent medicines contain dead silkworm. Annual domestic demand for dead silkworm is as high as 3,000 tons, and with its widespread application in medicine, health foods, and cosmetics, demand is expected to increase annually.

[0003] Currently, large-scale production of Bombyx batryticatus typically involves infecting silkworms with a suspension of Beauveria bassiana spores. This method is simple to operate and can improve rigor mortis by regulating the spore concentration. However, many of the currently produced Bombyx batryticatus suffer from quality issues such as uneven size. Existing solutions to this problem primarily involve screening for dominant Beauveria bassiana strains and improving silkworm breeding techniques. However, little attention has been paid to the long time span over which silkworms die. This long time span necessitates multiple feeding and sorting operations during the rigor mortis phase. Furthermore, the resulting Bombyx batryticatus exhibits significant individual variability due to varying growth times, resulting in a high degree of dispersion in product quality parameters, resulting in poor and unstable quality, which in turn affects clinical efficacy.

[0004] Therefore, it is of great significance to develop a method for producing silkworm pupae that increases the concentration of silkworm death time, thereby reducing individual differences in silkworm pupae products, improving the consistency and quality of silkworm pupae products, and reducing production costs. Summary of the Invention

[0005] The present invention aims to overcome the above-mentioned problems in the existing production of Bombyx batryticatus and provide a bacterial agent and an infection method thereof that can greatly increase the concentration of silkworm death time during the production process of Bombyx batryticatus.

[0006] The first object of the present invention is to provide a Beauveria bassiana agent and its application in producing Bombyx batryticatus.

[0007] The second object of the present invention is to provide a method for producing high-quality Bombyx batryticatus and the resulting Bombyx batryticatus.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The invasion of Beauveria bassiana by silkworms requires the combined effects of mechanical pressure and enzymolysis. The enzymolysis occurs after spore attachment and germination, when Beauveria bassiana degrades the silkworm's epidermis by secreting proteases, chitinases, and lipases, thereby increasing the number of invasions and the lethality of the fungus. Different strains of Beauveria bassiana often have certain differences in their genomes, with differences in the types and amounts of secreted proteases. Extensive research has found that relying on a single strain of Beauveria bassiana, even one with a high lethality rate, is difficult to effectively alleviate the problem of a long time span for silkworm morbidity. However, by combining two strains of Beauveria bassiana, mixing a spore suspension from one strain with a fermentation product from another strain that has been detoxified, and then inoculating the silkworm with it, the time span for morbidity can be significantly shortened.

[0010] Therefore, the present invention provides a Beauveria bassiana agent capable of significantly increasing the concentration of silkworm death time, comprising a Beauveria bassiana strain and a fermentation product of another Beauveria bassiana strain.

[0011] As an optional specific embodiment, the Beauveria bassiana inoculum contains a spore suspension of one strain of Beauveria bassiana and a fermentation product of another strain of Beauveria bassiana.

[0012] The fermentation product is a fermentation liquid with the bacterial cells removed.

[0013] In this solution, a mixture of a spore suspension of one Beauveria bassiana strain and a fermentation product (containing multiple enzymes) of another Beauveria bassiana strain is used to infect silkworms. The enzymes in the fermentation product immediately enzymatically degrade the silkworm's epidermis, and the enzymatic degradation time is longer than using the spore suspension alone. In addition, the hydrolytic enzyme systems of the two Beauveria bassiana strains vary to a certain extent, and using the mixture can bring into play a wider variety of hydrolytic enzymes, resulting in a stronger ability to depolymerize the epidermis. Therefore, the present invention utilizes a spore suspension of one Beauveria bassiana strain and a fermentation product of another Beauveria bassiana strain to act on silkworms, effectively promoting the degradation of the silkworm's epidermis, improving the efficiency of Beauveria bassiana infection in silkworms, thereby increasing the concentration of silkworm deaths during the production process, and thus improving the efficiency of large-scale production.

[0014] As a preferred specific embodiment, one strain of Beauveria bassiana is Beauveria bassiana KW2, and the other strain of Beauveria bassiana is Beauveria bassiana KW1; the Beauveria bassiana KW1 was deposited in the Guangdong Provincial Center for Microbiological Culture Collection on July 30, 2024, and its preservation number is GDMCC NO: 64922; the Beauveria bassiana KW2 was deposited in the Guangdong Provincial Center for Microbiological Culture Collection on November 19, 2024, and its preservation number is GDMCC NO: 65502.

[0015] More specifically and preferably, the Beauveria bassiana agent contains a spore suspension of Beauveria bassiana KW2 strain and a fermentation product of Beauveria bassiana KW1 strain.

[0016] Preferably, the volume ratio of the spore suspension to the fermentation product is 1:(0.5-4).

[0017] More preferably, the volume ratio of the spore suspension to the fermentation product is 1:1.

[0018] As an optional embodiment, the preparation method of the spore suspension of Beauveria bassiana KW2 strain is as follows: spore powder of KW2 strain is added to sterile water containing Tween-80, mixed evenly, and filtered to obtain the spore suspension of KW2 strain.

[0019] As an optional embodiment, the concentration of the spore suspension of the KW2 strain is 2×10 6 pieces / mL.

[0020] Optionally, the filtration is performed using filter paper.

[0021] Optionally, the concentration of Tween-80 is 0.05-0.15% w / v.

[0022] As an optional embodiment, the preparation method of the fermentation product of the Beauveria bassiana KW1 strain is as follows: the KW1 strain is inoculated into a silkworm powder induction medium for fermentation to obtain a fermentation culture liquid, and the fermentation culture liquid is filtered and sterilized to obtain the fermentation product of the Beauveria bassiana KW1 strain; the formula of the silkworm powder induction medium is 40 g / L glucose, 10 g / L peptone, 10 g / L yeast powder and 35-45 g / L silkworm powder.

[0023] As an optional embodiment, the filtration sterilization method is: the fermentation culture broth is first filtered using filter paper to obtain a coarse filtrate, and the coarse filtrate is then filtered using a 0.22 μm filter membrane.

[0024] As an optional embodiment, the preparation method of the fermentation product of Beauveria bassiana KW1 strain is as follows: spore powder of KW1 strain is added to sterile water containing Tween-80 and mixed evenly, filtered with filter paper, and then prepared into 1×10 6 The method comprises the following steps: preparing a spore suspension of a KW1 strain at a volume ratio of 0.5-1.5% by volume, adding the spore suspension of the KW1 strain to a silkworm powder induction culture medium, culturing the culture medium on a shaker at 23-27° C. and 130-170 r / min for 4-6 days, filtering the culture medium with filter paper to obtain a coarse filtrate, and then filtering the culture medium with a 0.22 μm filter membrane to obtain a fermentation product of the KW1 strain.

[0025] The application of the above-mentioned microbial agent in the production of Bombyx batryticatus should also be within the scope of protection of the present invention.

[0026] The present invention also provides a method for producing batryticatus, which comprises inoculating silkworms with the above-mentioned microbial agent to produce batryticatus.

[0027] As an optional embodiment, the method for producing silkworm pupae is: spraying the above-mentioned microbial agent on the surface of the silkworm body (until the silkworm body is wet but not dripping), and feeding mulberry leaves after 30 minutes.

[0028] As an optional implementation scheme, after feeding the silkworms until the mortality rate reaches 5%, a layer of mulberry leaves is covered after laying the silkworm net (to completely cover the silkworm net), and the net is collected after 18-30 hours (preferably 24 hours). After the rigidification is completed, the silkworms are sterilized and dried to a moisture content of less than 13% to obtain the silkworm corpse product.

[0029] As an optional implementation scheme, after feeding until the mortality rate of silkworms reaches 5%, a layer of mulberry leaves is covered after laying a silkworm net (to completely cover the silkworm net), and the net is collected after 18-30 hours (preferably 24 hours), and the silkworms are allowed to rigidify for 48-72 hours. After rigidification, the silkworms are moved into an oven for drying to obtain finished silkworms.

[0030] As an optional embodiment, the inoculation method is spraying inoculation.

[0031] As an optional embodiment, the silkworm is a fourth-instar silkworm or a fifth-instar silkworm.

[0032] As an optional implementation scheme, the temperature of the production environment is maintained at 24-28°C and the relative humidity is not less than 85%.

[0033] Preferably, the temperature of the production environment is maintained at 25-26°C and the relative humidity is not less than 85%.

[0034] As an optional embodiment, the spore content of Beauveria bassiana in the bacterial agent in the method for producing silkworm is not less than 1×10 6 pieces / mL.

[0035] The present invention also claims protection for the silkworm batryticatus produced by the above method.

[0036] The present invention has the following beneficial effects:

[0037] (1) The present invention provides a microbial agent, which is prepared by mixing a spore suspension of Beauveria bassiana strain KW2 with a fermentation broth of Beauveria bassiana strain KW1. Infecting silkworms with the microbial agent of the present invention can shorten the time it takes for all silkworms to die, and concentrate the time of death on the sixth day after inoculation, with a time span of only 24 hours, thereby helping to improve the production efficiency of silkworms, reduce production costs, and increase economic benefits.

[0038] (2) The method for producing silkworm pupae of the present invention facilitates the subsequent regulation of the death time of silkworms, so that silkworms die in batches when their fresh weight is the largest at the fifth instar, thereby increasing the yield of silkworm pupae, effectively reducing the labor required for feeding and sorting silkworms during the dead stage, and reducing the production cost of silkworm pupae. In addition, the method for producing silkworm pupae of the present invention can achieve a good infection effect by spraying a bacterial agent once for inoculation, thereby reducing the number of steps in silkworm pupae production.

[0039] (3) The silkworm pupae produced by the method for producing silkworm pupae of the present invention are uniform in size, and the consistency of product quality parameters is very good. In addition, various indicators such as extract, total ash content, moisture content, etc. all meet and significantly exceed the standards of the "Pharmacopoeia of the People's Republic of China" (2020 edition). The quality is stable, which helps to improve the clinical application effect. DETAILED DESCRIPTION

[0040] The present invention is further described below with reference to specific examples, which, however, are not intended to 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.

[0041] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0042] 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.

[0043] Silkworm powder-induced enzyme production medium: Dissolve 40g of glucose, 10g of peptone, 10g of yeast powder, and 40g of silkworm powder in distilled water until completely dissolved and dilute to 1000mL. Sterilize the mixture under high temperature and high pressure for 30min, then allow to cool and set aside.

[0044] The following examples demonstrate the present invention by combining a spore suspension of Beauveria bassiana strain KW2 with a fermentation broth from which the thallus bodies of Beauveria bassiana strain KW1 have been removed, and inoculating silkworms with the mixed solution.

[0045] The Beauveria bassiana KW1 strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on July 30, 2024, and its deposit number is GDMCC NO: 64922.

[0046] 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.

[0047] Example 1 An infection method for increasing the concentration of silkworm death time during the production of silkworm blast

[0048] 1. Rejuvenation and propagation of strains

[0049] Pure glycerol stocks of Beauveria bassiana KW1 and KW2 were streaked onto SDAY solid culture medium, and then cultured in a constant temperature and humidity incubator at 25°C and 90% relative humidity for 5 days. After single colonies grew on the culture medium, single colonies were picked and spot-inoculated onto SDAY solid culture medium, and cultured in the same environment for 15-20 days to complete the rejuvenation and propagation of the strains.

[0050] 2. Preparation of KW2 spore suspension

[0051] The spore powder of Beauveria bassiana KW2 strain after propagation was collected, sterile water containing 0.1% Tween-80 was added, vortexed and mixed, filtered through three layers of filter paper, and the concentration of the spore suspension was counted by a hemocytometer to prepare 2×10 6 Spore suspension with a concentration of spores / mL.

[0052] 3. Preparation of KW1 Induced Fermentation Broth

[0053] The spore powder of Beauveria bassiana KW1 strain after propagation was collected, sterile water containing 0.1% Tween-80 was added, vortexed and mixed, filtered through three layers of filter paper, and the concentration of the spore suspension was counted by a hemocytometer to prepare 1×10 6 Spore suspension with a concentration of spores / mL.

[0054] The KW1 spore suspension was added to the silkworm powder-induced enzyme production medium at a volume ratio of 1%, and cultured on a shaker at 25°C and 150 r / min for 5 days. The crude filtrate was filtered through three layers of filter paper, and then filtered through a 0.22 μm filter membrane to obtain the KW1 fermentation broth.

[0055] IV. Experimental Methods

[0056] The silkworm variety used in the experiment was the Huakang No. 3 reciprocal hybrid. The experiment was divided into two groups (KW2 spore liquid group and mixed liquid group), with 432 healthy silkworms in each group, and 3 parallels were set up in each group. When the selected silkworms grew to the second day of the fifth instar, an agricultural sprayer was used to spray and inoculate Beauveria bassiana onto silkworms with similar individual length and activity. The observation was continued for 9 days, and the infection status of the silkworms was observed every day, and the death of the silkworms was recorded.

[0057] 1. Mixed liquid group: KW2 spore suspension and KW1 fermentation liquid were mixed at a volume ratio of 1:1 to obtain a spore suspension with fermentation liquid added, which was labeled as mixed liquid. The mixed liquid was evenly sprayed on the silkworms using a sprayer until the silkworms were wet. Before spraying, ensure that there were no residual leaves on the silkworm bed. Feed the silkworms with mulberry leaves 30 minutes after spraying.

[0058] 2. KW2 spore liquid group: prepare the same volume of KW2 spore suspension separately. For the first inoculation, spray the spore suspension onto the surface of the silkworm until the silkworm's body surface is wet. Spray it for the second time 15 minutes later. Start feeding mulberry leaves 15 minutes later, and then spray the spore suspension onto the surface of mulberry leaves for the third inoculation. After 15 minutes, spray the spore suspension onto the surface of mulberry leaves again for the fourth inoculation.

[0059] The temperature in the production environment (rigidity chamber) is maintained at 25-26°C, with a relative humidity of no less than 85%. The silkworms are fed 3-4 times a day, ensuring they are well fed. When the mortality rate of the silkworms reaches approximately 5%, a silkworm net is added and covered with a layer of mulberry leaves, enough to completely cover the net. After 24 hours, the net is collected and cleaned of impurities. Rigidity is continued for 48-72 hours. Once rigidity is complete, the silkworms are placed in an oven for drying to obtain the finished product.

[0060] The time course analysis of the rigor mortis of silkworms infected by the two methods is shown in Table 1 below. The results show that rigor mortis began to appear in the KW2 spore liquid group on the 6th day after inoculation, with a rigor mortis rate of only 28%, and reached 100% on the 9th day after inoculation; while the rigor mortis rate in the mixed liquid group was 0% on the 5th day and reached 100% on the 6th day. The rigor mortis of silkworms was concentrated on the 6th day after inoculation, with a time span of only 24 hours.

[0061] Table 1 Time course analysis of the mortality rate of infected silkworms treated with mixed solution group and spore solution group

[0062] Sterilization time (Day) D5 D6 D7 D8 D9 KW2 spore liquid group 0％ 28.0％ 53.2％ 87.2％ 100％ Mixed liquid group 0％ 100％ / / /

[0063] Example 2

[0064] The quality parameters of the silkworms prepared by the two infection methods were measured, and the specific data are shown in Table 2. As shown in Table 2, there are slight differences in the quality of the silkworms prepared by the two different methods. Although the average diameter of the silkworms treated with the mixed solution group is slightly lower than that of the spore solution group, their average length is higher than that of the spore solution group, and the variance of the quality parameters of the silkworms treated with the mixed solution group is smaller.

[0065] Table 2 Quality parameters of Bombyx batryticatus prepared by mixed liquid group and spore liquid group

[0066] Average diameter (mm) Average length (cm) Average weight (g) KW2 spore liquid group 7.0±0.517 4.1±0.455 0.88±0.115 Mixed liquid group 6.0±0.459 4.2±0.246 0.87±0.093

[0067] Example 3

[0068] Some samples of the mixed liquid group and KW2 spore liquid group were sent to a third-party agency for testing, and the test results are shown in Table 3. As can be seen from Table 3, all indicators of the silkworm meet the standards of the "Pharmacopoeia of the People's Republic of China" (2020 edition). In addition, according to the Guangdong Province "Bombyx Batryticatus Formula Granules", the total content of adenine and adenosine in the silkworm is required to be 0.04% to 0.24%. The total content of adenine and adenosine in the silkworm treated with the mixed liquid group is 0.07% (including 0.01% adenine and 0.06% adenosine), which meets the requirements.

[0069] In summary, the quality of the silkworms in the mixed liquid group and the KW2 spore liquid group met the requirements, and the death time span of the silkworms infected with the mixed liquid was significantly shortened, and the size was more uniform, which is more suitable for large-scale production of silkworms in enterprises.

[0070] Table 3 Comparative analysis of the detection indexes of Bombyx batryticatus in the mixed solution group and the Pharmacopoeia of the People's Republic of China

[0071]

[0072] Traditional spore suspensions for silkworm infection generally require multiple sprayings to ensure a good infection effect (e.g., Chinese patents CN110352920B and CN106614423B). However, the present invention achieves a good infection effect with a spore suspension of the same concentration added to the fermentation liquid, requiring only a single spraying, thereby reducing the number of steps required for silkworm production. Furthermore, the mortality rate of infected silkworms can reach 100% within 24 hours, allowing silkworm nets to be used for silkworm collection, reducing the workload of manual silkworm sorting.

[0073] It can be seen from this that in the production of silkworm pupae, using spore suspension with added fermentation liquid to infect silkworms can increase the concentration of silkworm death time, thereby improving the production efficiency of silkworm pupae, reducing production costs, increasing economic benefits, and being more in line with the large-scale production of silkworm pupae by enterprises.

[0074] 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A Beauveria bassiana inoculum agent, characterized in that: Contains the fermentation product of one strain of Beauveria bassiana and another strain of Beauveria bassiana.

2. The microbial agent according to claim 1, characterized in that The invention contains a spore suspension of one strain of Beauveria bassiana and a fermentation product of another strain of Beauveria bassiana, wherein the fermentation product is a fermentation liquid with the bacterial bodies removed.

3. The bacterial agent according to claim 1 or 2, characterized in that One strain of Beauveria bassiana is Beauveria bassiana KW2, and the other strain of Beauveria bassiana is Beauveria bassiana KW1; the Beauveria bassiana KW1 was deposited in the Guangdong Provincial Center for Microbiological Culture Collection on July 30, 2024, and its preservation number is GDMCC NO: 64922; the Beauveria bassiana KW2 was deposited in the Guangdong Provincial Center for Microbiological Culture Collection on November 19, 2024, and its preservation number is GDMCC NO: 65502.

4. The bacterial agent according to claim 3, characterized in that Contains a spore suspension of Beauveria bassiana KW2 and a fermentation product of Beauveria bassiana KW1.

5. The bacterial agent according to claim 4, characterized in that The volume ratio of the spore suspension to the fermentation product is 1:(0.5-4).

6. Use of the microbial agent according to any one of claims 1 to 5 in producing Bombyx batryticatus.

7. A method for producing Bombyx batryticatus, characterized in that: The silkworm is inoculated with the microbial agent according to any one of claims 1 to 5 to produce a dead silkworm.

8. The method according to claim 7, characterized in that The inoculation method is spraying inoculation.

9. The method according to claim 7, characterized in that The temperature of the production environment is maintained at 24-28°C and the relative humidity is not less than 85%.

10. The Bombyx batryticatus produced according to the method of any one of claims 7 to 9.

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