Factory culture method for hepialus armoricanus larvae

Abstract

The invention discloses a hepialus armoricanus larva factory culture method, and relates to the technical field of biological cultivation, the hepialus armoricanus larva factory culture method comprises the following steps: S1, the hepialus armoricanus larvae are raised under the cycle conditions that the environment temperature is controlled at 10-20 DEG C, the relative humidity is kept between 70% and 90%, and the cycle of 10-14 h light and 10-14 h darkness is alternated; s2, using isaria fumosorosea suspension to spray a first bacterial staining solution in a culture environment of the larvae when the hepialus armoricanus larvae grow to a stage of 3-6 years old; s3, preparing a second bacterial staining solution by using the bacillus suspension, and spraying the second bacterial staining solution in the hepialus armoricanus larva culture environment; and S4, mixing the cordyceps sinensis bacterial suspension and the fecal fungus suspension according to the volume ratio of (1-3): 1 to prepare a third bacterial staining solution, and spraying the third bacterial staining solution in the culture environment of the hepialus armoricanus larvae. According to the scheme, multiple florae are introduced into the culture environment in sequence, infectious microbes in the hepialus armoricanus larva ecological environment are eliminated, the influence of the infectious microbes on cordyceps sinensis bacterial contamination is avoided, and the infection rate of cordyceps sinensis is increased.

Description

Technical Field

[0001] The invention relates to the field of Cordyceps sinensis cultivation, and in particular to a factory cultivation method for bat moth larvae. Background Art

[0002] Cordyceps sinensis is a precious Chinese medicinal material and is believed to have multiple medicinal values ​​in traditional Chinese medicine, such as enhancing immunity, resisting fatigue, and improving kidney function. However, due to its special and limited natural growth environment, it is in short supply in the market and is expensive.

[0003] Under natural conditions, Cordyceps sinensis can only grow in specific high-altitude areas (such as the Qinghai-Tibet Plateau), which limits its production. Artificial cultivation can achieve a stable supply throughout the year, reduce dependence on natural resources, and ensure continuous supply in the market. Collecting Cordyceps sinensis in the wild requires a lot of manpower and material resources, and as resources gradually become depleted, the difficulty of collection continues to increase. In contrast, artificial cultivation can be carried out in a more controlled environment, which helps to reduce costs and improve production efficiency.

[0004] Cordyceps sinensis is a complex formed after Cordyceps sinensis infects bat moth larvae. The formation of Cordyceps sinensis in nature is often in a delicate microbial ecosystem. Various microorganisms in this system interact with each other to maintain a dynamic balance. It is very difficult to rebuild such a balance in an artificial environment. Cordyceps sinensis cultured with bat moth larvae as carriers has a certain impact on the success rate of Cordyceps sinensis infection due to the influence of the bat moth larvae's own state and the influence of various microorganisms in the culture environment. In the prior art, even if the process from culturing bat moth larvae to obtaining Chinese Hirsutosporium species to prepare Cordyceps sinensis suspension and then to infecting bat moth larvae is pollution-free, the success rate of successfully infecting and cultivating complete Cordyceps sinensis is still very low under the influence of the bat moth larvae themselves or the microorganisms in the growth environment or the state of the bat moth larvae themselves. The success rate of Cordyceps sinensis cultivated according to traditional existing technologies is low and cannot meet the increasing demand. Therefore, a new solution is needed to solve these problems. Summary of the invention

[0005] The invention overcomes the shortcomings of the prior art and provides a factory culture method for bat moth larvae.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for cultivating bat moth larvae in a factory, comprising the following steps:

[0007] S1. Raise bat moth larvae under the condition of an ambient temperature of 10-20°C, relative humidity of 70%-90%, and a cycle of 10-14h light and 10-14h dark cycles;

[0008] S2, use 10 6 -10 8 The CFU / mL suspension of Fumosomyces roseus is prepared as a first bacterial staining solution. When the bat moth larvae develop to the 3-6 instar stage, the first bacterial staining solution is sprayed in the culture environment of the larvae, and 100-200 mL of the first bacterial staining solution is sprayed per square meter of the culture environment;

[0009] S3, use 10 6 -10 8 The bacillus suspension with a CFU / mL is configured as a second bacterial staining solution, and the second bacterial staining solution is sprayed in the bat moth larvae culture environment within 12h-48h after step S2, and 100-200mL of the second bacterial staining solution is sprayed per square meter of the culture environment;

[0010] S4, the concentration is 10 6 -10 8 CFU / mL of Cordyceps sinensis suspension was compared with the GenBank accession number MK616520.1 and the concentration of 10 6 -10 8 The suspension of the class Mycetes with CFU / mL is mixed in a volume ratio of 1-3:1 to prepare a third bacterial staining solution; after executing step S3, the bat moth larvae are allowed to resume normal action, and the third bacterial staining solution is sprayed in the culture environment of the bat moth larvae, with 200-500 mL of the third bacterial staining solution sprayed per square meter of the culture environment.

[0011] Preferably, in the step S1, the ambient temperature is preferably 15°C to 20°C, the relative humidity is preferably 80% to 90%, and the photoperiod is 14 hours of light and 10 hours of darkness.

[0012] Preferably, in the step S2, the preparation of the suspension of Coryna fumosorum comprises: inoculating the purified Coryna fumosorum into a liquid culture medium, culturing on a shaking table at 20° C. to 25° C. for 7-14 days, and then diluting to a desired concentration with sterile water.

[0013] Preferably, in the step S3, the Bacillus is selected from one or more combinations of Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus.

[0014] Preferably, in the step S4, the fungal suspension of Sordella fasciata is prepared by culturing the strain corresponding to GenBank accession number MK616520.1.

[0015] Preferably, in the step S4, the mixing volume ratio of the Cordyceps sinensis suspension to the Sclerotium fungus suspension is preferably 2:1, and the mixture is fully stirred before spraying.

[0016] Preferably, the method further comprises the steps of:

[0017] S5. After each spraying of the bacterial stain, observe the behavioral changes and infection progress of the bat moth larvae in the culture environment every 6h-8h, record any abnormal phenomena, and adjust the environmental conditions or re-spray the first, second and third bacterial stains according to the actual situation.

[0018] Preferably, in step S1, the food source of the bat moth larvae is a mixture of wheat bran, yeast powder and carrot slices.

[0019] Preferably, in steps S2 to S4, each spraying operation is performed using a high-pressure sprayer, and the spray pressure is controlled between 2-20 bar.

[0020] Preferably, in steps S2 to S4, when a high-pressure sprayer is used for spraying, the spray particle size is 50-100 μm.

[0021] The present invention solves the defects existing in the background technology and has the following beneficial effects:

[0022] (1) Spores of fumochrome were used as pioneer bacteria to exclude other bacteria in the culture environment of bat moth larvae, and then Bacillus was used as a competitive bacteria to inhibit the continued growth of spores of fumochrome to ensure the health of bat moth larvae. Finally, Cordyceps sinensis was mixed with a fungus with GenBank accession number MK616520.1 to form an antagonistic zone that prevented other bacteria from spreading and reproducing, thereby reducing the influence of other microorganisms on Cordyceps sinensis infection. Compared with the existing technology, the infection success rate of Cordyceps sinensis and the culture success rate of Cordyceps sinensis were improved.

[0023] (2) Steps S2-S4 ensure that appropriate bacterial flora are introduced at the appropriate time, thereby avoiding excessive bacterial reproduction that affects the introduction of subsequent bacterial flora. Strict control of the time of bacterial flora introduction and the culture environment of the bat moth larvae are used to ensure the health of the bat moth larvae and reduce the impact of the bat moth larvae's own state on the infection rate of Cordyceps sinensis and the Cordyceps sinensis growth rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 It is a flow chart of the implementation steps of the method of the present invention. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0028] Sources

[0029]

[0030]

[0031]

[0032] See also Figure 1 , a bat moth larvae factory culture method, comprising the following steps:

[0033] S1. The bat moth larvae are reared under the condition of the ambient temperature being controlled at 10-20°C, the relative humidity being maintained between 70% and 90%, and the cycle of 10-14h light and 10-14h dark cycle alternating;

[0034] S2, use 10 6 -10 8 A suspension of fumosomyces rosae with a CFU / mL is prepared as the first bacterial staining solution. When the bat moth larvae develop to the 3-6th instar stage, the first bacterial staining solution is sprayed in the culture environment of the larvae, and 100-200 mL of the first bacterial staining solution is sprayed per square meter of the culture environment;

[0035] S3, use 10 6 -10 8 CFU / mL of the Bacillus suspension is configured as a second bacterial staining solution, and the second bacterial staining solution is sprayed in the bat moth larvae culture environment within 12h-48h after step S2, and 100-200mL of the first bacterial staining solution is sprayed per square meter of the culture environment;

[0036] S4, the concentration is 10 6 -10 8 CFU / mL of Cordyceps sinensis suspension was compared with the GenBank accession number MK616520.1 and the concentration of 10 6 -108 CFU / mL of the dung mushroom fungus suspension is mixed in a volume ratio of 1-3:1 to prepare a third bacterial staining solution; within 12h-48h after performing step S3, the third bacterial staining solution is sprayed in the culture environment of the bat moth larvae;

[0037] S5: After each spraying of the bacterial stain, observe the behavioral changes and infection progress of the bat moth larvae every 6h-8h, record any abnormal phenomena, and adjust the environmental conditions or re-spray the second bacterial stain, the third bacterial stain, and the third bacterial stain according to the actual situation.

[0038] Isaria fumosorosea, whose scientific name is Isaria fumosorosea, can be observed under a microscope, and its hyphae are smooth and transparent, with uniform diameter and partitions, without obvious lock-like union phenomenon, and its spore-producing structure presents a bottleneck feature. On PDA culture medium, this strain grows rapidly, forming white flocculent colonies with neat edges, and its aerial hyphae are well developed, which can secrete yellow pigments and produce a large number of oval chain spores. Introducing Isaria fumosorosea into the culture environment of bat moth larvae can make Isaria fumosorosea grow rapidly and form bacterial infection on bat moth larvae. The purpose of its introduction is to use Isaria fumosorosea as a pioneer bacterial community to exclude other miscellaneous bacteria in the culture environment of bat moth larvae, so that it can effectively occupy the ecological niche of bat moth larvae, reduce other microorganisms, and avoid the influence of potential other microorganisms on the success rate of Cordyceps sinensis bacterial infection.

[0039] In step S3, Bacillus is introduced, which is a probiotic that can secrete antimicrobial substances such as antimicrobial peptides. After the introduction of Spores fumoides to eliminate most of the miscellaneous bacteria in the culture environment of the bat moth larvae, if the Spores fumoides multiply in large numbers, the bat moth larvae can be killed within 2 days at the fastest, and the nutrients of the bat moth larvae can be absorbed, which greatly affects the infection of Cordyceps sinensis. Therefore, the purpose of introducing Bacillus is to compete with Spores fumoides, so that a more balanced ecological environment that is conducive to the infection of Cordyceps sinensis is formed in the body of the bat moth larvae and in the culture environment, thereby improving the health of the bat moth larvae and the success rate of Cordyceps sinensis culture.

[0040] In step S4, a third bacterial dye solution obtained by mixing the spores of the class Hypocrea with GenBank accession number MK616520.1 and Cordyceps sinensis is added to the culture environment into which the spores of the class Hypocrea and Bacillus are introduced successively. The spores of the class Hypocrea grow slowly on the milk culture medium, and the aerial hyphae are short and evenly distributed. The colony is soft in texture, and the outer layer is covered with short white hyphae, while the inner layer is dark green. The hyphae structure of the spores of the class Hypocrea is relatively dense, and when mixed with Cordyceps sinensis, it can form an antagonistic zone as a physical barrier, thereby preventing the growth of the spores of the class Hypocrea and Bacillus. The continued infection of Cordyceps sinensis can be greatly improved. In addition, the above-mentioned dung scale fungi will secrete some secondary metabolites, which can interfere with the growth signal pathways of fumosomal spores and Bacillus and weaken their competitiveness. This chemical signal interference helps to further reduce the impact of fumosomal spores and Bacillus and promote the successful infection of Cordyceps sinensis. By mixing dung scale fungi and Cordyceps sinensis, on the one hand, Cordyceps sinensis and dung scale fungi are mixed to form an antagonistic zone, and on the other hand, Cordyceps sinensis can quickly infect the bat moth larvae and cultivate Cordyceps sinensis, thereby greatly improving the success rate of Cordyceps sinensis cultivation.

[0041] Specifically, in step S1, the ambient temperature is preferably 15°C-20°C, the relative humidity is preferably 80%-90%, and the photoperiod is 14 hours of light and 10 hours of darkness. The precise control of environmental conditions simulates the optimal environment for the natural growth of bat moth larvae, helps to enhance their health, promote their normal development, and improve the success rate of subsequent bacterial infection steps.

[0042] Specifically, in step S2, the preparation of the suspension of Fumosoraceous Corynaeus includes: inoculating the purified Fumosoraceous Corynaeus into a liquid culture medium, culturing on a shaker at 20°C-25°C for 7-14 days, and then diluting to the required concentration with sterile water. This method ensures the quality and activity of the Fumosoraceous Corynaeus suspension, enables it to effectively exclude foreign bacteria, and creates favorable conditions for the introduction of subsequent strains.

[0043] Specifically, the Bacillus is selected from one or more combinations of Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus. These Bacillus can secrete substances such as antimicrobial peptides to inhibit the growth of other harmful microorganisms, while maintaining the ecological balance in the culture environment, reducing the risk of excessive growth of Spore spores, and protecting the bat moth larvae from harm.

[0044] Specifically, the fungal suspension of the class Sordellae is cultured and prepared according to the strain corresponding to the GenBank accession number MK616520.1, ensuring the consistency of its characteristics and functions, facilitating the formation of antagonistic zones, and preventing continued infection by other microorganisms.

[0045] Specifically, the mixing volume ratio of the Cordyceps sinensis suspension and the Scutellaria fungus suspension is preferably 2:1, and they are fully stirred before spraying to ensure uniform distribution of the two bacterial suspensions. This mixing ratio and uniform distribution can better exert the synergistic effect of the two strains, form a more effective physical barrier and chemical defense mechanism, and significantly improve the successful infection rate of Cordyceps sinensis.

[0046] Specifically, the dynamic monitoring of the bat moth larvae in step S5 can timely discover and solve possible problems, ensure that each step can be carried out as expected, and maximize the quality and yield of the final product.

[0047] Specifically, in step S1, the food source of the bat moth larvae is a mixture of wheat bran, yeast powder and carrot slices, which provides the bat moth larvae with rich nutrients such as carbohydrates, cellulose and protein required for their growth, so as to support the healthy growth of the bat moth larvae. Adequate nutritional supply helps to enhance the immunity and tolerance of the larvae, and is beneficial to improve the success rate of Cordyceps sinensis infection.

[0048] Specifically, in steps S2 to S4, each spraying operation is performed using a high-pressure sprayer, the spray pressure is controlled between 2-20 bar, the spray particle size is 50-100 μm, and the spraying method is to start from one side of the culture environment and gradually advance to the other side to ensure that there are no omissions, so as to ensure that the bacterial staining liquid evenly covers the entire culture environment, improve the bacterial staining effect, and reduce the risk of failure caused by local unevenness. The high-pressure sprayer should be calibrated before each spraying to ensure that the amount of bacterial staining liquid sprayed per square meter of the culture environment is accurate. In addition, the high-pressure sprayer needs to be thoroughly cleaned before and after each spraying to prevent cross-contamination between different strains.

[0049] Example 1

[0050] This embodiment provides a method for cultivating bat moth larvae in a factory, comprising the following steps:

[0051] S1. The ambient temperature was controlled at 17±2℃, the relative humidity was maintained between 80%±10%, and the bat moth larvae were raised under the conditions of a 14-hour light cycle and a 10-hour dark cycle, and the larvae were fed with a mixture of wheat bran, yeast powder and carrot slices in a mass ratio of 3:1:1 as feed every day;

[0052] S2, use (1.0±0.1)*10 7A suspension of fumosomyces rosae with a CFU / mL was prepared as the first bacterial staining solution. When the bat moth larvae developed to the 4th instar stage, the first bacterial staining solution was sprayed in the culture environment of the larvae. Based on the floor area of ​​the culture environment, 150±10mL of the first bacterial staining solution was sprayed per square meter of the culture environment using a high-pressure sprayer at a working pressure of 10 bar, and the spray particle size was controlled to be 50-100μm.

[0053] S3, use (1.0±0.1)*10 7 CFU / mL of Bacillus subtilis suspension is configured as a second bacterial staining solution, and the second bacterial staining solution is sprayed in the bat moth larvae culture environment 12 hours after step S2. Based on the floor area of ​​the culture environment, 150±10mL of the second bacterial staining solution is sprayed per square meter of the culture environment using a high-pressure sprayer at a working pressure of 10 bar, and the spray particle size is controlled to be 50-100μm;

[0054] S4, the concentration is (1.0±0.1)*10 7 CFU / mL of Cordyceps sinensis suspension and GenBank accession number MK616520.1, concentration (1.0±0.1)*10 7 The fungal suspension of the class of dung beetles with a CFU / mL is mixed in a volume ratio of 1:1 to prepare a third bacterial staining solution; after executing step S3, the bat moth larvae are allowed to resume normal action state, and the third bacterial staining solution is sprayed in the culture environment of the bat moth larvae. Based on the floor area of ​​the culture environment, 300±10mL of the third bacterial staining solution is sprayed per square meter of the culture environment using a high-pressure sprayer at a working pressure of 10 bar, and the spray particle size is controlled to be 50-100μm.

[0055] Example 2

[0056] The difference between this embodiment and embodiment 1 is that in step S4, the volume ratio of the Cordyceps sinensis suspension to the Sphaerocephala fungus suspension is changed to 1.5:1;

[0057] Example 3

[0058] The difference between this embodiment and embodiment 1 is that in step S4, the volume ratio of the Cordyceps sinensis suspension to the Sphaerocephala fungus suspension is changed to 2:1;

[0059] Example 4

[0060] The difference between this embodiment and embodiment 1 is that in step S4, the volume ratio of the Cordyceps sinensis suspension to the Sphaerocephala fungus suspension is changed to 2.5:1;

[0061] Example 5

[0062] The difference between this embodiment and embodiment 1 is that in step S4, the volume ratio of the Cordyceps sinensis suspension to the Sphaerocephala fungus suspension is changed to 3:1;

[0063] Example 6

[0064] Follow these steps to culture and infect bat moth larvae:

[0065] S1. The ambient temperature was controlled at 17±2℃, the relative humidity was maintained between 80%±10%, and the bat moth larvae were raised under the conditions of a 14-hour light cycle and a 10-hour dark cycle, and the larvae were fed with a mixture of wheat bran, yeast powder and carrot slices in a mass ratio of 3:1:1 as feed every day;

[0066] S2, use (1.0±0.1)*10 7 A suspension of fumosomyces rosae with a CFU / mL was prepared as the first bacterial staining solution. When the bat moth larvae developed to the 4th instar stage, the first bacterial staining solution was sprayed in the culture environment of the larvae. Based on the floor area of ​​the culture environment, 150±10mL of the first bacterial staining solution was sprayed per square meter of the culture environment using a high-pressure sprayer at a working pressure of 10 bar, and the spray particle size was controlled to be 50-100μm.

[0067] S3, use (1.0±0.1)*10 7 CFU / mL of Bacillus subtilis suspension is configured as a second bacterial staining solution, and the second bacterial staining solution is sprayed in the bat moth larvae culture environment 12 hours after step S2. Based on the floor area of ​​the culture environment, 150±10mL of the second bacterial staining solution is sprayed per square meter of the culture environment using a high-pressure sprayer at a working pressure of 10 bar, and the spray particle size is controlled to be 50-100μm;

[0068] S4, the concentration is (1.0±0.1)*10 7 CFU / mL of Cordyceps sinensis suspension and GenBank accession number MK616520.1, concentration (1.0±0.1)*10 7 The fungal suspension of the class of dung beetles with a CFU / mL is mixed in a volume ratio of 2:1 to prepare a third bacterial staining solution; after executing step S3, the bat moth larvae are allowed to resume normal action state, and the third bacterial staining solution is sprayed in the culture environment of the bat moth larvae. Based on the floor area of ​​the culture environment, 300±10mL of the third bacterial staining solution is sprayed per square meter of the culture environment using a high-pressure sprayer at a working pressure of 10 bar, and the spray particle size is controlled to be 50-100μm.

[0069] Example 7

[0070] Different from Example 6, the time for executing step S3 is changed to 24 hours after executing step S2.

[0071] Example 8

[0072] Different from Example 6, the time for executing step S3 is changed to 36 hours after executing step S2.

[0073] Example 9

[0074] Different from Example 6, the time for executing step S3 is changed to 48 hours after executing step S2.

[0075] Comparative Example 1

[0076] When the bat moth larvae obtained by step S1 in Example 1 reached the 4th instar, they were directly treated with (1.0±0.1)*10 7 The Cordyceps sinensis suspension with CFU / mL was sprayed on the culture environment with a spray volume of 300±10mL using a high-pressure sprayer to directly carry out bacterial infection.

[0077] Comparative Example 2

[0078] S1. The ambient temperature was controlled at 17±2℃, the relative humidity was maintained between 80%±10%, and the bat moth larvae were raised under the conditions of a 14-hour light cycle and a 10-hour dark cycle, and the larvae were fed with a mixture of wheat bran, yeast powder and carrot slices in a mass ratio of 3:1:1 as feed every day;

[0079] S2, use (1.0±0.1)*10 7 A suspension of fumosomyces rosae with a CFU / mL was used as the first spraying liquid. When the bat moth larvae developed to the 4th instar stage, the first spraying liquid was sprayed in the culture environment of the larvae. Based on the floor area of ​​the culture environment, 150±10mL of the first spraying liquid was sprayed per square meter of the culture environment using a high-pressure sprayer at a working pressure of 10 bar, and the spray particle size was controlled to be 50-100μm;

[0080] S3, the concentration is (1.0±0.1)*10 7 CFU / mL of Cordyceps sinensis suspension is used as the second spray liquid; 24 hours after executing step S2, the second spray liquid is sprayed in the culture environment of the bat moth larvae. Based on the floor area of ​​the culture environment, 300±10mL of the second spray liquid is sprayed per square meter of the culture environment using a high-pressure sprayer at a working pressure of 10 bar, and the spray particle size is controlled to be 50-100μm.

[0081] Comparative Example 3

[0082] S1. The ambient temperature was controlled at 17±2℃, the relative humidity was maintained between 80%±10%, and the bat moth larvae were raised under the conditions of a 14-hour light cycle and a 10-hour dark cycle, and the larvae were fed with a mixture of wheat bran, yeast powder and carrot slices in a mass ratio of 3:1:1 as feed every day;

[0083] S2, use (1.0±0.1)*10 7 CFU / mL of Bacillus subtilis suspension is used as the first spraying liquid. When the bat moth larvae develop to the 4th instar stage, the first spraying liquid is sprayed in the bat moth larvae culture environment. Based on the floor area of ​​the culture environment, 150±10mL of the first spraying liquid is sprayed per square meter of the culture environment using a high-pressure sprayer at a working pressure of 10 bar, and the spray particle size is controlled to be 50-100μm;

[0084] S3, the concentration is (1.0±0.1)*10 7 CFU / mL of Cordyceps sinensis suspension is used as the second spray liquid. 24 hours after executing step S2, the second spray liquid is sprayed in the culture environment of the bat moth larvae. Based on the floor area of ​​the culture environment, 300±10mL of the second spray liquid is sprayed per square meter of the culture environment using a high-pressure sprayer at a working pressure of 10 bar, and the spray particle size is controlled to be 50-100μm.

[0085] Comparative Example 4

[0086] S1. The ambient temperature was controlled at 17±2℃, the relative humidity was maintained between 80%±10%, and the bat moth larvae were raised under the conditions of a 14-hour light cycle and a 10-hour dark cycle, and the larvae were fed with a mixture of wheat bran, yeast powder and carrot slices in a mass ratio of 3:1:1 as feed every day;

[0087] S2, the concentration is (1.0±0.1)*10 7 CFU / mL of Cordyceps sinensis suspension and GenBank accession number MK616520.1, concentration (1.0±0.1)*10 7 The CFU / mL of the Scutellaria fungal suspension is mixed in a volume ratio of 2:1 to prepare a first spray liquid; the first spray liquid is sprayed in the culture environment of the bat moth larvae. Based on the floor area of ​​the culture environment, 300±10mL of the first spray liquid is sprayed per square meter of the culture environment using a high-pressure sprayer at a working pressure of 10 bar, and the spray particle size is controlled to be 50-100μm.

[0088] Comparative Example 5

[0089] S1. The ambient temperature was controlled at 17±2℃, the relative humidity was maintained between 80%±10%, and the bat moth larvae were raised under the conditions of a 14-hour light cycle and a 10-hour dark cycle, and the larvae were fed with a mixture of wheat bran, yeast powder and carrot slices in a mass ratio of 3:1:1 as feed every day;

[0090] S2, use (1.0±0.1)*10 7 A suspension of fumosomyces rosae with a CFU / mL was used as the first spraying liquid. When the bat moth larvae developed to the 4th instar stage, the first spraying liquid was sprayed in the culture environment of the larvae. Based on the floor area of ​​the culture environment, 150±10mL of the first spraying liquid was sprayed per square meter of the culture environment using a high-pressure sprayer at a working pressure of 10 bar, and the spray particle size was controlled to be 50-100μm;

[0091] S3, the concentration is (1.0±0.1)*10 7 CFU / mL of Cordyceps sinensis suspension and GenBank accession number MK616520.1, concentration (1.0±0.1)*10 7 The suspension of the class Sclerotium with a CFU / mL is mixed in a volume ratio of 2:1 to prepare a second spray liquid; 24 hours after executing step S2, the second spray liquid is sprayed in the culture environment of the bat moth larvae. Based on the floor area of ​​the culture environment, 300±10mL of the second spray liquid is sprayed per square meter of the culture environment using a high-pressure sprayer at a working pressure of 10 bar, and the spray particle size is controlled to be 50-100μm.

[0092] Comparative Example 6

[0093] S1. The ambient temperature was controlled at 17±2℃, the relative humidity was maintained between 80%±10%, and the bat moth larvae were raised under the conditions of a 14-hour light cycle and a 10-hour dark cycle, and the larvae were fed with a mixture of wheat bran, yeast powder and carrot slices in a mass ratio of 3:1:1 as feed every day;

[0094] S2, use (1.0±0.1)*10 7 CFU / mL of Bacillus subtilis suspension is prepared as the first spraying liquid. When the bat moth larvae develop to the 4th instar stage, the first spraying liquid is sprayed in the bat moth larvae culture environment. Based on the floor area of ​​the culture environment, 150±10mL of the second bacterial staining liquid is sprayed per square meter of the culture environment using a high-pressure sprayer at a working pressure of 10 bar, and the spray particle size is controlled to be 50-100μm;

[0095] S3, the concentration is (1.0±0.1)*10 7CFU / mL of Cordyceps sinensis suspension and GenBank accession number MK616520.1, concentration (1.0±0.1)*10 7 The suspension of the class Sclerotium with a CFU / mL is mixed in a volume ratio of 2:1 to prepare a second spray liquid; 24 hours after executing step S2, the second spray liquid is sprayed in the culture environment of the bat moth larvae. Based on the floor area of ​​the culture environment, 300±10mL of the second spray liquid is sprayed per square meter of the culture environment using a high-pressure sprayer at a working pressure of 10 bar, and the spray particle size is controlled to be 50-100μm.

[0096] Test Case

[0097] Taking 100 bat moth larvae as the sample base, culture and fungus infection were carried out under the conditions of Examples 1-9 and Comparative Examples 1-4, respectively, and the average lethal time of the bat moth larvae, the average length of the stroma formed, and the number of stroma successfully formed by the successful fungus infection of Cordyceps sinensis were recorded. The formula:

[0098] Infection rate = the number of larvae infected by Cordyceps sinensis to form stroma / sample base number, the infection rate is calculated;

[0099] The test results are shown in Table 1.

[0100] Table 1

[0101]

[0102]

[0103] From the analysis of the data in Table 1, we can see that:

[0104] (1) In comparative examples 6-9, the number of successful infection with Cordyceps sinensis in Example 9 is lower than that in other examples or comparative examples. The reason is that after 48 hours, the fumosomal spores have occupied most of the ecological environment of the bat moth larvae, resulting in the death of most of the bat moth larvae. The subsequent introduction of Bacillus subtilis can only help the remaining larvae with stronger resistance to recover their vitality. The subsequent examples 6-8 are compared with comparative example 1. The infection rate of Cordyceps sinensis is improved. It can be concluded that the present invention has a higher Cordyceps sinensis grass rate than the prior art.

[0105] (2) Comparative Examples 1-5, when the volume ratio of Cordyceps sinensis to the dung fungus with GenBank accession number MK616520.1 was changed from 1 to 3:1, different numbers of mature grass were obtained. The reason for this change is that when the volume ratio was 1:1 and 1.5:1, most of the Cordyceps sinensis and the dung fungus were mixed. Although an antagonistic zone was formed, there was a certain mutual inhibition between the two. Only a small part of the Cordyceps sinensis occupied and absorbed the nutrients in the bat moth larvae, which had a certain impact on the bacterial infection of the Cordyceps sinensis. When the volume ratio exceeded 2:1, there was a sufficient number of Cordyceps sinensis. Even if a part of the Cordyceps sinensis was mixed to form an antagonistic zone, the other part of the Cordyceps sinensis began to reproduce in the bat moth larvae, and finally formed a fruiting body that grew into Cordyceps sinensis. However, when the ratio of Cordyceps sinensis to the dung fungus was subsequently increased to more than 2:1, the continued increase of Cordyceps sinensis would reduce the length of the stroma formed, affecting the medicinal value of Cordyceps sinensis.

[0106] (3) The average length of the stroma formed in Examples 1-9 and the length of the stroma formed in Comparative Example 1 are both about 40 mm, indicating that the Cordyceps sinensis cultured according to the scheme of the present invention has a higher Cordyceps sinensis fungus infection success rate and Cordyceps sinensis grass yield rate than the prior art, and the medicinal value of Cordyceps sinensis is almost the same as that of the prior art;

[0107] (4) From the data listed in Comparative Examples 1-6, it can be concluded that the lack of the addition of one or two of the bacterial suspensions does not improve the success rate of bacterial infection, and even reduces the infection rate of Cordyceps sinensis. In addition, in the comparative example where Bacillus subtilis is not introduced, the lethal time is relatively fast. The reasons are as follows: on the one hand, the miscellaneous bacteria in the bat moth larvae will affect the success rate of bacterial infection with Cordyceps sinensis. On the other hand, without the addition of any bacterial species, the ecological balance of the colony in the bat moth larvae will be broken, resulting in a decrease in the nutrients absorbed by the Cordyceps sinensis, thereby reducing the infection rate, which is not conducive to controlling the bacterial infection of Cordyceps sinensis. The introduction of Bacillus subtilis can improve the disease resistance of the bat moth larvae to a certain extent, but cannot significantly increase the infection rate of Cordyceps sinensis.

[0108] The above is based on the ideal embodiment of the present invention. Through the above description, relevant personnel can make various changes and modifications without departing from the technical concept of the present invention. The technical scope of the present invention is not limited to the content in the specification, and the technical scope must be determined according to the scope of the claims.

Claims

1. A method for cultivating bat moth larvae in a factory, characterized in that: The following steps are involved: S1. Raise bat moth larvae under the condition of an ambient temperature of 10-20°C, relative humidity of 70%-90%, and a cycle of 10-14h light and 10-14h dark cycles; S2, use 10 6 -10 8 A suspension of fumosomyces rosae with a CFU / mL is prepared as a first bacterial staining solution, and when the bat moth larvae develop to the 3-6 instar stage, the first bacterial staining solution is sprayed in the culture environment of the larvae, and 100-200 mL of the first bacterial staining solution is sprayed per square meter of the culture environment; S3, use 10 6 -10 8 The bacillus suspension with a CFU / mL is configured as a second bacterial staining solution, and the second bacterial staining solution is sprayed in the bat moth larvae culture environment within 12h-48h after step S2, and 100-200mL of the second bacterial staining solution is sprayed per square meter of the culture environment; S4, the concentration is 10 6 -10 8 CFU / mL of Cordyceps sinensis suspension and concentration of 10 6 -10 8 The suspension of the class Mycetes with CFU / mL is mixed in a volume ratio of 1-3:1 to prepare a third bacterial staining solution; after executing step S3, the bat moth larvae are allowed to resume normal action, and the third bacterial staining solution is sprayed in the culture environment of the bat moth larvae, with 200-500 mL of the third bacterial staining solution sprayed per square meter of the culture environment.

2. A method for cultivating bat moth larvae in a factory according to claim 1, characterized in that: In the step S1, the ambient temperature is preferably 15°C to 20°C, the relative humidity is preferably 80% to 90%, and the photoperiod is 14 hours of light and 10 hours of darkness.

3. The method for cultivating bat moth larvae in a factory according to claim 1, characterized in that: In the step S2, the preparation of the suspension of Corysporum fumosorum comprises: inoculating the purified Corysporum fumosorum into a liquid culture medium, culturing in a shaking incubator at 20° C. to 25° C. for 7-14 days, and then diluting to a desired concentration with sterile water.

4. The method for cultivating bat moth larvae in a factory according to claim 1, characterized in that: In the step S3, the Bacillus is selected from one or more combinations of Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus.

5. The method for cultivating bat moth larvae in a factory according to claim 1, characterized in that: In the step S4, the fungal suspension of the class Sordella is prepared by culturing the strain corresponding to the GenBank accession number MK616520.

1.

6. The method for cultivating bat moth larvae in a factory according to claim 1, characterized in that: In the step S4, the mixing volume ratio of the Cordyceps sinensis suspension to the Sclerotium fungus suspension is preferably 2:1, and the mixture is fully stirred before spraying.

7. The factory-scale cultivation method of bat moth larvae according to any one of claims 1 to 6, characterized in that: Also includes the steps: S5. After each spraying of the bacterial stain, observe the behavioral changes and infection progress of the bat moth larvae in the culture environment every 6h-8h, record any abnormal phenomena, and adjust the environmental conditions or re-spray the second, third and fourth bacterial stains according to the actual situation.

8. The method for cultivating bat moth larvae in a factory according to claim 1, characterized in that: In the step S1, the food source of the bat moth larvae is a mixture of wheat bran, yeast powder and carrot slices.

9. The method for cultivating bat moth larvae in a factory according to claim 1, characterized in that: In the steps S2 to S4, each spraying operation is performed using a high-pressure sprayer, and the spray pressure is controlled between 2-20 bar.

10. A method for cultivating bat moth larvae in a factory according to claim 9, characterized in that: In the steps S2 to S4, when a high pressure sprayer is used for spraying, the spray particle size is 50-100 μm.

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