Novel microbacterium strain capable of producing amylase and cellulase and application of novel microbacterium strain in breeding and growth promotion of hermetia illucens

Abstract

The invention provides a new strain of Microbacterium for promoting growth of larvae of hermetia illucens and application of the new strain. The strain comprises a 16S rDNA sequence shown in SEQ ID NO: 1 or a complementary sequence of the 16S rDNA sequence or a sequence with at least 85% of nucleic acid identity compared with the sequence shown in SEQ ID NO: 1. The Microbacterium sp.Micro. M175 disclosed by the invention is separated from insect sand, and has the characteristic of decomposing starch and cellulose. When the strain is added in the breeding process of the hermetia illucens, growth of larvae can be promoted, and accumulation of protein and fat of the larvae is promoted. Compared with sterile water, after the Micr.M175 is added, the body length and body weight of the hermetia illucens larvae are respectively increased by 2.94% and 21.19%, and the accumulation of protein and fat is respectively increased by 32.41% and 47.74%. Therefore, high-quality and high-efficiency breeding of the hermetia illucens is achieved, and industrial production of hermetia illucens bodies is promoted.

Description

Technical Field

[0001] This application relates to the field of microbial technology, specifically to a novel strain of *Microbacterium* that produces amylase and cellulase and its application in promoting the growth of black soldier fly larvae. Background Technology

[0002] With the rapid development of my country's catering industry and large-scale livestock and poultry farming, the annual production of urban kitchen waste has exceeded 120 million tons, and the annual production of livestock and poultry manure has reached 3.8 billion tons. This type of organic solid waste has a high water content, is easily perishable, and accumulates pathogens, insect eggs, and resistance genes. If not treated in a timely manner, it will emit foul odors, breed mosquitoes and flies, and pollute water bodies through runoff, causing eutrophication and zoonotic diseases, becoming a major source of non-point source pollution in urban and rural areas. Traditional waste treatment methods such as landfill and incineration have problems of secondary pollution and resource waste; composting is time-consuming and easily affected by seasons; and anaerobic fermentation easily acidifies the sludge, requiring further treatment. Therefore, there is an urgent need for a new, efficient, rapid, low-environmentally-risk, and high-value-added biological treatment technology for organic solid waste to overcome the bottlenecks of existing processes.

[0003] Black soldier fly ( Hermetia illucens Black soldier fly larvae (L.) are a resource insect with significant potential for environmental remediation. Their larvae efficiently feed on various organic wastes such as kitchen waste, livestock manure, and agricultural waste, rapidly converting them into their own biomass. This process significantly reduces and neutralizes waste. More importantly, black soldier fly larvae are rich in protein and fat, making them an excellent source of feed protein, thus realizing the resource-based value-added transformation of waste and aligning with the development concept of a circular economy. Some studies have shown that certain microorganisms can decompose macromolecular organic matter, produce beneficial metabolites, and inhibit the growth of pathogens. Theoretically, this could create a better intestinal and feed microenvironment for black soldier flies, thereby promoting their growth and nutrient accumulation, especially in their larvae.

[0004] Microbacteria ( Microbacterium Microbes are Gram-positive, non-spore-forming, irregularly shaped rod-shaped bacteria, formally named in 1919, belonging to the family Microbacteriaceae within the phylum Actinomycetota. Microbes are widely distributed in soil, water, polar regions, deserts, air, and within plants and animals. According to data published in the International Commission on Prokaryotic Systematics (ICSP) database (https: / / lpsn.dsmz.de / ), as of September 2025, the genus *Microbes* has 206 species-level taxonomic units, of which 168 are validly published. Currently, *Microbes*, with its multiple potentials in promoting growth and stress resistance, degrading pollutants, producing enzymes for pharmaceuticals, and forming films for preservation, has become a rising star microbial resource for cross-sectoral applications in agricultural production, environmental governance, pharmaceutical development, and materials protection.

[0005] Among related technologies, Chinese patent CN118006590B proposes a method based on... Microbacterium dextranolyticum A mutant of β-glucosidase can be used for enzymatic conversion production, improving production efficiency; Chinese patent CN108277175B discloses a strain that efficiently degrades 2,4-dinitrotoluenesulfonate. Microbacterium sp. X3. This strain can efficiently degrade TNT red water and red water-contaminated soil, and has good application prospects in the remediation of soils contaminated with nitro compounds; in addition, Chinese patent CN120272385A discloses a Dendrobium microbacterium that has the functions of producing iron carriers, solubilizing potassium, promoting growth, and alleviating plant salt and alkali stress. Microbacterium dendrobii 2T1 is a novel strain of the genus *Microbacterium*, possessing the ability to secrete siderophores and solubilize potassium. Related technologies have demonstrated the efficacy of *Microbacterium* in promoting growth and stress resistance, degrading pollutants, and producing enzymes for pharmaceutical applications; however, research on its application in black soldier fly farming remains lacking.

[0006] Developing new strains of Microbacterium that promote the growth and nutrient accumulation of black soldier fly larvae is of great significance for advancing black soldier fly farming and the efficient conversion of organic solid waste. Summary of the Invention

[0007] This application addresses at least one of the problems of the related technology in the following aspects.

[0008] Therefore, embodiments of this application provide a novel microbacterial strain and its related applications.

[0009] The first aspect of this application provides a microbacterium containing the 16S rDNA sequence shown in SEQ ID NO: 1 or its complementary sequence or a sequence with at least 85% identity to SEQ ID NO: 1.

[0010] In some embodiments, the microbacteria have a 16S rDNA sequence as shown in SEQ ID NO: 1, and are those with accession number CGMCC No. 35903. Microbacterium sp. Micr.M175 was deposited at the China General Microbiological Culture Collection Center on September 12, 2025.

[0011] The second aspect of this application provides a method for culturing microbes as described in any embodiment of the first aspect of this application, the method comprising inoculating the microbes into a culture medium and culturing them at a temperature of 25 to 37°C, preferably 28 to 30°C, and most preferably 30°C.

[0012] In some embodiments, the pH of the culture medium is 7.0 to 8.0, preferably 7.4 to 7.8, and more preferably 7.6.

[0013] In some embodiments, the culture medium is 2216E, and the 2216E culture medium comprises: 5.0 g peptone, 1.0 g yeast extract, 0.1 g ferric citrate, 19.45 g sodium chloride, 5.98 g magnesium chloride, 3.24 g sodium sulfate, 1.8 g calcium chloride, 0.55 g potassium chloride, 0.16 g sodium carbonate, 0.08 g potassium bromide, 0.034 g strontium chloride, 0.022 g boric acid, 0.004 g sodium silicate, 0.0024 g sodium fluoride, 0.0016 g ammonium nitrate, and 0.008 g disodium hydrogen phosphate.

[0014] A third aspect of this application provides a microbial agent comprising microbes as described in any embodiment of the first aspect of this application and microbiologically acceptable excipients.

[0015] In some embodiments, the microbial agent is a liquid microbial agent or a solid microbial agent.

[0016] In some embodiments, the dosage form of the microbial agent is selected from: wettable powder, water-dispersible granules, suspension concentrate, emulsion, granules, or combinations thereof.

[0017] The fourth aspect of this application proposes the use of microbacteria as proposed in any embodiment of the first aspect or microbial agents as proposed in any embodiment of the second aspect in promoting the growth of black soldier fly larvae and / or increasing the accumulation of protein and fat in black soldier fly bodies.

[0018] The fifth aspect of this application proposes the application of microbacteria as proposed in any embodiment of the first aspect of this application or microbial agents as proposed in any embodiment of the second aspect of this application in the conversion of organic solid waste by black soldier flies.

[0019] In some embodiments, the organic waste is selected from one or more of the following: wheat bran, kitchen waste, livestock and poultry manure, distiller's grains, and food processing waste.

[0020] In some embodiments, the application includes the following steps: (1) The microbacterium or the microbial agent is prepared at 10 8 The organic solid waste was inoculated at a ratio of cfu / g to obtain a mixed matrix; (2) After the mixed substrate is cultured at 28 ℃ and 70% relative humidity for 1 day, 1200 6-day-old black soldier fly larvae are inoculated and cultured for a longer period of time to obtain biomass.

[0021] The embodiments of this application achieve the following beneficial effects: This application is the first to isolate and identify Microbacterium genus ( ). MicrobacteriumA new strain of fungus under sp.) Microbacterium sp. Micr.M175 possesses amylase and cellulase production capabilities, enabling it to decompose starch and cellulose in the environment and convert them into bioavailable nutrients. Micr.M175 exhibits a significant growth-promoting effect on black soldier fly larvae and facilitates nutrient accumulation in the larvae. Therefore, the application of Micr.M175 enables high-quality and efficient rearing of black soldier flies, which is conducive to promoting the industrial production of black soldier fly feed. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 According to Embodiment 2 of this application Microbacterium Phylogenetic tree of sp. Micr.M175 based on 16S rDNA; Figure 2 According to Embodiment 2 of this application Microbacterium Phylogenetic tree of sp. Micr.M175 based on genome-wide ANI; Figure 3 This is a plate growth diagram of Micr.M175 according to Example 3 of this application; Figure 4 The microstructure of Micr.M175 according to Embodiment 3 of this application is shown; Figure 5 The growth of Micr.M175 according to Example 4 of this application on soluble starch medium is shown; Figure 6 The growth of Micr.M175 according to Example 4 of this application on sodium carboxymethyl cellulose medium is shown; Figure 7 The results of salt gradient culture of Micr.M175 according to Example 5 of this application are shown; Figure 8 The growth of the insect body length according to Embodiment 6 of this application is shown; Figure 9 The growth of the insect body weight according to Example 6 of this application is shown; Figure 10 The growth of insects treated with Micr.M175 according to Example 6 of this application is shown in comparison with the control group. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0025] The large-scale generation and improper disposal of food waste, livestock and poultry manure, and agricultural waste (such as crop straw and fruit and vegetable residues) have become a serious environmental challenge facing society. Meanwhile, the global protein supply has continued to tighten, and the "protein shortage" in my country's feed industry is becoming increasingly prominent. In 2024, the domestic feed protein gap exceeded 42 million tons, with an import dependency rate still above 80%, mainly relying on imported soybeans.

[0026] Faced with the aforementioned challenges, bioconversion using black soldier fly larvae is considered a promising solution. This technology can efficiently and cost-effectively reduce and recycle organic waste. Simultaneously, its products—the larvae themselves—can serve as high-quality protein feed, and their excrement as organic fertilizer, aligning with the concept of a circular economy. In March 2025, the Ministry of Agriculture and Rural Affairs included dried black soldier fly larvae (powder) and defatted black soldier fly larvae powder in the "Feed Raw Material Catalogue" (draft for comments). Dried black soldier fly larvae have a protein content exceeding 40%, effectively replacing soybeans and providing the feed industry with an innovative and high-quality protein source.

[0027] However, the black soldier fly conversion technology still faces industrial technological bottlenecks in practical applications, resulting in low production efficiency. Pretreatment by inoculating the substrate with microbial agents can decompose macromolecules in the substrate, enhance nutrient absorption by the insects, and thus promote insect growth and the accumulation of protein and fat in the insects.

[0028] This application provides an embodiment of a microbacterium genus ( ). Microbacterium A new strain of microbacterium Microbacterium sp. Micr.M175 (hereinafter referred to as "Micr.M175") possesses excellent stress resistance, including salt tolerance, enabling it to specifically adapt to organic waste environments. It also exhibits amylase and cellulase production, allowing it to decompose starch and cellulose in the environment and convert them into bioavailable nutrients. Furthermore, Micr.M175 demonstrates a significant growth-promoting effect on black soldier fly larvae and promotes the accumulation of protein and fat in the larvae, thus providing a better theoretical basis and solution for black soldier fly farming and the industrialization of insect protein production.

[0029] The first aspect of this application provides a microbacterium containing the 16S rDNA sequence shown in SEQ ID NO: 1 or its complementary sequence or a sequence with at least 85% identity to SEQ ID NO: 1.

[0030] In this application embodiment, the percentage of identity typically describes the degree to which two sequences are identical; that is, it typically describes the percentage of nucleotides that correspond to the same nucleotides in the reference sequence at their sequence positions. In this application embodiment, the "sequence with at least 85% identity" refers to a sequence having an identity of any value (including endpoint values) between 85% and 100% compared to the sequence shown in SEQ ID NO: 1, for example, it can have sequence identity of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, as well as sequence identity represented by an infinite number of decimals between two adjacent integers, for example, having at least 98.57%, 99.64%, 99.7%, 99.8%, or 99.9% sequence identity compared to the sequence shown in SEQ ID NO: 1.

[0031] In the embodiments of this application, the proposed "Micr.M175" was identified as a member of the genus Microbacterium (Microbacterium). Microbacterium A new species under sp.) is named " Microbacterium The entry for sp. Micr.M175 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 35903. The classification name is... Microbacterium sp. Micr.M175, deposited on September 12, 2025.

[0032] In this embodiment, the strain Micr.M175 having the sequence shown in SEQ ID NO: 1 can be understood by those skilled in the art as the original strain. A strain with a genome sequence having at least 85% identity with the sequence shown in SEQ ID NO: 1 can be understood by those skilled in the art as a variant strain of the strain Micr.M175 having the sequence shown in SEQ ID NO: 1. It is understood that strains (such as strain Micr.M175) can undergo spontaneous mutations or be artificially cultured to form variant strains, for example, by nucleotide deletions, additions, or substitutions. The "variant strain" and "strain Micr.M175" have highly identical gene sequences and extremely similar biological functions. The mutated genes do not substantially affect the conserved sequences of strain Micr.M175, and therefore do not affect the genetic stability of strain Micr.M175. More specifically, this "variant strain" is also a strain of the species Micr.M175, exhibiting the physiological activity characteristics of species Micr.M175. The specific species corresponding to Micr.M175 and all its bacterial strains also fall within the protection scope of this application.

[0033] The novel strain Micr.M175 isolated in this application embodiment can effectively promote the growth of black soldier fly larvae and significantly increase the accumulation of protein and fat in the larvae, thereby enriching the available microbial resources and providing a better theoretical basis and solution for black soldier fly farming. This is of vital significance for promoting the industrialization of black soldier fly biological treatment technology.

[0034] The second aspect of this application provides a method for culturing microbes as described in any of the embodiments of the first aspect, comprising inoculating the microbes into a culture medium for culture, wherein the culture temperature is 25 to 37°C, preferably 28 to 30°C, and most preferably 30°C.

[0035] In some embodiments, the pH of the culture medium is 7.0 to 8.0, preferably 7.4 to 7.8, and more preferably 7.6.

[0036] In some embodiments, the culture medium is 2216E medium. In some specific embodiments, the formulation of 2216E medium is as follows (g / L): peptone 5.0 g, yeast extract 1.0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 5.98 g, sodium sulfate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium carbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 0.034 g, boric acid 0.022 g, sodium silicate 0.004 g, sodium fluoride 0.0024 g, ammonium nitrate 0.0016 g, and disodium hydrogen phosphate 0.008 g.

[0037] In some specific embodiments, the method may include one or more of the following steps, or the culture may be performed using any of the following culture media: S1. Strain activation: Micr.M175 was streaked onto solid agar plates and incubated at 30 ℃ for 48 h. The Micr.M175 used for streaking can be taken from bacterial suspension or single colony. S2. Liquid seed preparation: The activated Micr.M175 from step S1 was inoculated into liquid seed culture medium and cultured with shaking at 30 ℃ and 150 r / min for 24 h to obtain liquid seeds. S3. Fermentation: The liquid seed prepared in step S2 was inoculated into the fermentation medium at a volume ratio of 1%, and cultured under shaking and aeration conditions of 150 r / min and 30 ℃ for 24 h to obtain the microbial agent Micr.M175.

[0038] In some embodiments, the viable count of Micr.M175 in the bacterial agent may be 10.8 Up to 10 10 cfu / mL.

[0039] In some embodiments, the formulation of the solid plate culture medium may be: 5.0 g / L peptone, 1.0 g / L yeast extract, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L ammonium nitrate, 0.008 g / L disodium hydrogen phosphate, 15-20 g / L agar, dissolved in distilled water and brought to a final volume of 1 L, with a pH of 7.6 ± 0.2.

[0040] In some embodiments, the liquid seed culture medium may be formulated as follows: 5.0 g / L peptone, 1.0 g / L yeast extract, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L ammonium nitrate, 0.008 g / L disodium hydrogen phosphate, dissolved in distilled water and brought to a final volume of 1 L, with a pH of 7.6 ± 0.2.

[0041] In some embodiments, the fermentation medium may be formulated as follows: 5.0 g / L peptone, 1.0 g / L yeast extract, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L ammonium nitrate, 0.008 g / L disodium hydrogen phosphate, dissolved in distilled water and brought to a final volume of 1 L, with a pH of 7.6 ± 0.2.

[0042] In some embodiments, the culture medium is subjected to autoclaving for a time of 15 to 20 minutes.

[0043] In some embodiments, the method further includes concentrating and / or freeze-drying the prepared microbial agent.

[0044] In some embodiments, the microbial agent is in the form of a liquid agent or a solid agent.

[0045] It is understood that this application does not intend to limit the types of culture media and specific parameters of the culture media (such as pH, content of each component, etc.) involved in the microbial culture method proposed in the embodiments. Other culture media or culture conditions can be selected according to actual needs, as long as the normal growth of microbial bacteria can be guaranteed.

[0046] In some embodiments, the microbacteria exhibit salt tolerance. In some embodiments, the microbacteria tolerate NaCl concentrations ranging from 0-5%. The microbacteria proposed in this application have strong salt tolerance and can specifically adapt to organic waste environments.

[0047] The third aspect of this application provides a microbial agent comprising microbes as described in any embodiment of the first aspect of this application and microbiologically acceptable excipients.

[0048] In this embodiment of the application, the microbial agent may contain any of the above-mentioned microbes, including: microbes containing the 16S rDNA sequence shown in SEQ ID NO: 1 or its complementary sequence or a sequence with at least 85% identity with SEQ ID NO: 1; microbes having the 16S rDNA sequence shown in SEQ ID NO: 1; and / or microbes having the accession number CGMCC No. 35903. Microbacterium sp. Micr.M175; at the same time, since the above strains also include mutant strains of the same species as Micr.M175, the specific species of Micr.M175 and all its bacterial strains also fall within the protection scope of the microbial agents of this application.

[0049] In this application, "microbiologically acceptable excipients" refers to all components other than live microbial strains used in the preparation of microbial agents, provided they do not adversely affect the survival, stability, or functional activity of the microbes. These excipients do not significantly inhibit microbial growth and are safe for the environment, plants, animals, and humans. In some specific embodiments, such excipients may include: carriers / fillers, protectants / lyophilization stabilizers, nutrients / metabolite activators, suspending agents / thickeners, surfactants / wetting and dispersing agents, binders, disintegrants, pH adjusters / buffers, preservatives, etc. This application does not limit the specific types of excipients.

[0050] In some embodiments, the dosage form of the microbial agent is selected from: wettable powder, water-dispersible granules, suspension concentrate, emulsion, granules, or combinations thereof.

[0051] In this embodiment, the microbial agent may also be a fermentation broth containing any of the aforementioned microbes, the fermentation broth containing the microbe and its metabolites, which can be further used as biological nutrients. In some embodiments, the fermentation medium for the fermentation broth may be 2216E medium. It is understood that the fermentation medium for the microbe fermentation broth in this embodiment only needs to provide for the normal growth and metabolic fermentation of the microbes, and this application does not limit the specific type of medium.

[0052] The microbial agent containing the novel strain Micr.M175 proposed in this application can effectively promote the growth of black soldier fly larvae and significantly increase the accumulation of protein and fat in the larvae. This enriches the available microbial resources and provides a better solution and foundation for black soldier fly farming, which is of vital importance to promoting the industrialization of black soldier fly biological treatment technology.

[0053] The fourth aspect of this application proposes the use of microbacteria as described in any embodiment of the first aspect of this application and / or microbial agents as described in any embodiment of the third aspect in promoting the growth of black soldier fly larvae and / or promoting the accumulation of protein and fat in the body of black soldier fly larvae.

[0054] The fifth aspect of this application proposes the use of microbacteria as described in any embodiment of the first aspect of this application and / or microbial preparations as described in any embodiment of the third aspect of this application in the conversion of organic solid waste by black soldier flies.

[0055] In some embodiments, the organic solid waste includes: wheat bran, kitchen waste, livestock and poultry manure, and food processing waste.

[0056] In some embodiments, the application includes the following steps: (1) dispensing the microbacterium or the microbial agent at 10 8 (2) The mixed matrix was inoculated into organic solid waste at a ratio of cfu / g to obtain a mixed matrix; (3) The mixed matrix was cultured at 28°C and 70% relative humidity for 1 day, and then inoculated with 1200 6-day-old black soldier fly larvae to continue the culture to obtain biomass.

[0057] In this embodiment, Micr.M175 or its fermentation broth (e.g., fermentation broth based on 2216E medium), or microbial agents or compound nutrients containing it, can be introduced into the rearing substrate before or during the rearing of black soldier fly larvae, thereby promoting the growth of black soldier fly larvae and / or promoting the accumulation of protein and fat in the larvae's bodies. In some embodiments, the rearing substrate may contain any of the components or combinations thereof described in the fourth aspect of this application.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0059] Unless otherwise specified, the quantitative analysis experiments in the following examples are all repeated three times, and the results are averaged.

[0060] Example 1: Isolation of a new strain, Micr.M175 One g of insect residue obtained from the conversion of kitchen waste by black soldier flies was added to 100 mL of sterile water and mixed at 150 rpm for 15 min to obtain a mixture. The mixture was then diluted with sterile water to a concentration of 10. -1 Up to 10 -8 A gradient concentration was established. The gradient dilutions were spread onto 2216E solid medium (g / L: peptone 5.0 g, yeast extract 1.0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 5.98 g, sodium sulfate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium carbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 0.034 g, boric acid 0.022 g, sodium silicate 0.004 g, sodium fluoride 0.0024 g, ammonium nitrate 0.0016 g, disodium hydrogen phosphate 0.008 g, agar 15 g). Sterile water without bacterial culture was also spread as a blank control. After spreading, the medium was incubated upside down at 30 ℃ for 48–72 hours. Single colonies were picked and amplified in liquid 2216E medium, and then repeatedly streaked to amplify and purify the strain, which was numbered Micr.M175.

[0061] The 16S rDNA gene of the Micr.M175 monoclonal strain was sequenced, and the specific sequence is shown in SEQ ID NO: 1.

[0062] Micr.M175 16S rDNA gene sequence (SEQ ID NO: 1): Example 2: Identification and evolutionary status determination of the new strain Micr.M175 The nearly full-length 16S rDNA sequence (SEQ ID NO: 1) of Micr.M175, approximately 1.4 kb, obtained through sequencing, was compared with the 16S rDNA gene database of EzBioCloud (https: / / www.ezbiocloud.net / ). The comparison results showed that the two strains in the database with the highest homology to the 16S rDNA gene of Micr.M175 were... Microbacterium abyssi A18JL241 T (Serial number: OR603944) and Microbacterium shaanxiense CCNWSP60 T (Sequence number: KJ735510), with similarities of 98.10% and 98.03% respectively (both less than the new strain determination standard of 98.65%). Based on this 16S rDNA information, strain Micr.M175 can be identified as belonging to... Microbacterium A new species of the genus.

[0063] Furthermore, sequences of closely related strains of Micr.M175 were selected, and a phylogenetic tree based on 16S rDNA was constructed using MAGA with neighbor-joining. The results are as follows: Figure 1 As shown. It can be seen that Micro.M175 and Microbacterium The strains are in a stable cluster, indicating that the strains are Microbacterium Member; meanwhile, Micr.M175 and Microbacterium Other species within the genus branched out, which also proves that the Micr.M175 isolated in Example 1 is... Microbacterium A new species within the genus.

[0064] In addition, download from GenBank Microbacterium The complete genome sequences of *Micr. M175* and other closely related genera were obtained. FastANI analysis was used to calculate the average nucleotide identity (ANI) between *Micr. M175* and other strains, revealing that *Micr. M175* is similar to... Microbacterium ihumii and Microbacterium aerolatum The highest ANI values ​​were 81.70% and 81.60% respectively (both less than the 95% threshold for new strains). Phylogenetic tree construction was performed using the default parameters of the OrthoFinder software, and the results are as follows... Figure 2 As shown. Based on this, it can be further determined that strain Micr.M175 belongs to... Microbacterium A new species of the genus.

[0065] Therefore, combining the 16S rDNA sequence alignment and genome-wide ANI calculation results, Micr.M175 can be identified as... Microbacterium A new species of fungus in the genus was named Microbacterium sp. Micr.M175 is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 35903.

[0066] Example 3: Morphological observation of the new strain Micr.M175 After cultivation, it was observed that the new bacterial strain Micr.M175 provided in this application appeared pale yellow and opaque on 2216E solid medium plates, with round and glossy colonies. Figure 3 ).

[0067] Figure 4 Microscopic imaging of the microbacterium Micr.M175, an embodiment of this application. For example... Figure 4 As shown, Micr.M175 is a Gram-positive bacterium with rod-shaped cells.

[0068] Example 4: The new strain Micr.M175 has the ability to decompose starch and cellulose. 4.1 Verification of Starch Decomposition Characteristics A small amount of strain Micr.M175 was picked up with an inoculation loop and placed in the center of a soluble starch medium plate. The plate was then incubated at 30 °C for 48 hours. Lugol's iodine solution was added, and the growth was observed. The results showed that Micr.M175 grew well on soluble starch medium and exhibited a clear transparent zone. Figure 5 The ratio of transparent zone diameter D to colony diameter d is 1.7, indicating that the strain has a certain starch-decomposing ability. This suggests that Micr.M175 in this embodiment can be used to decompose starch in the substrate and the digestive tract of black soldier fly larvae, thereby providing more absorbable nutrients for larval growth and promoting the growth of black soldier fly larvae.

[0069] The formula for soluble starch culture medium is as follows: g / L: 5 g yeast extract, 10 g peptone, 10 g sodium chloride, 2 g soluble starch, 15 g agar; pH value is 7.0±0.2, autoclave at 121 ℃ for 20 min.

[0070] 4.2 Verification of Cellulose Decomposition Characteristics Using an inoculation loop, strain Micr.M175 was picked and spotted in the center of a sodium carboxymethyl cellulose (CCMC) agar plate. The plate was then incubated at 30 °C for 48 hours. After incubation, 3 mL of a 1 mg / mL Congo red solution was evenly distributed onto the plate and stained for 30 min. The plate was then rinsed with 1 mol / L sodium chloride solution for 30-60 min before observing the results. The results showed a clear zone appearing on the CCMC agar plate. Figure 6 The ratio of transparent zone diameter D to colony diameter d was 2.0, confirming that the strain has cellulose decomposition ability. This suggests that Micr.M175 in this embodiment can be used to decompose cellulose in the substrate and the digestive tract of black soldier fly larvae, thereby providing more absorbable nutrients for larval growth and promoting the growth of black soldier fly larvae.

[0071] The formula for sodium carboxymethyl cellulose medium is as follows (g / L): 5 g sodium carboxymethyl cellulose, 2.5 g yeast extract, 5 g peptone, 0.5 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, 15 g agar, pH 6.0 ± 0.2, autoclaved at 121 ℃ for 20 min.

[0072] Example 5: The new strain Micr.M175 exhibits salt tolerance. Use 1-12% ( w / v Salt tolerance tests were conducted on *Micr. M175* in 2216E liquid medium with a gradient of 1% NaCl, with a salt-free 2216E medium (0% NaCl) as a control. *Micr. M175* seed culture was inoculated at a 1% inoculum, placed on a shaker, and cultured for 24 hours. The absorbance (OD) of each culture was then measured at 600 nm. 600 Each group has 3 parallels. The results are as follows: Figure 7 As shown.

[0073] Figure 7 The results of salt gradient culture according to this embodiment are shown, where the horizontal axis represents different salt concentrations (0-12%) and the vertical axis represents OD. 600 Values, where OD 600 The higher the value, the higher the bacterial concentration in the cultured bacterial solution. Figure 7 It is evident that Micr.M175 exhibits a certain degree of adaptability to saline environments, functioning even in concentrations below 5% ( w / v Micr.M175 exhibits rapid growth within a NaCl salinity range. Its salt tolerance provides a favorable foundation for its subsequent cultivation and utilization, such as the rearing of black soldier fly larvae using waste as a substrate.

[0074] Example 6: Effects of the new strain Micr.M175 on the growth and nutrient accumulation of black soldier fly larvae. 6.1 Growth-promoting effect of the new strain Micr.M175 on black soldier fly larvae After activation, the Milr. M175 strain was inoculated into 2216E liquid medium and fermented at 30 °C with shaking at 150 r / min for 1 day. Then, the OD of the bacterial culture was measured. 600 Adjust to 1.0, take the bacterial culture, centrifuge at 5000 rpm for 10 min, remove the supernatant, resuspend the bacterial cells in sterile water, and set aside for use.

[0075] Furthermore, the Micr.M175 strain was used at 10 8 cfu·g -1 The strain was inoculated into 1.2 kg of wheat bran (humidity 58% after inoculation) at a ratio of 1:1, and a control group was set up, in which an equal volume of sterile water was used instead of the Micr.M175 strain. Three replicates were set up for each group.

[0076] Further, one day after inoculation, 1200 six-day-old black soldier fly larvae were inoculated into each group and cultured for approximately eight days in a climate chamber at 28 ℃ and 70% relative humidity. Larval samples were collected on days 0, 4, 6, and 8, rinsed clean, dried with absorbent paper, and their length and weight were measured. Specific measurement data are as follows: Figure 8 and Figure 9 As shown in the figure, the growth of black soldier fly larvae in the treatment group and the control group is as follows: Figure 10 As shown in the figure. The experimental results were analyzed using GraphPad Prism statistical analysis software.

[0077] Figure 8 and Figure 9 The growth trends of larval body length and weight in this embodiment are shown separately. After inoculation with Micr.M175, the body length and weight of black soldier fly larvae were significantly higher than those of the control group on days 4, 6, and 8. Compared with the control group, on day 4 after inoculation, the body length and weight of black soldier fly larvae in the Micr.M175 group increased by 37.76% and 88.41%, respectively; on day 8 after inoculation, the body length and weight of black soldier fly larvae in the Micr.M175 group increased by 2.94% and 21.19%, respectively. This indicates that Micr.M175 can significantly promote the growth of body length and weight of black soldier fly larvae.

[0078] Figure 10 The data shows comparisons between selected worms grown for 4, 6, and 8 days, respectively, and the control group. Figure 10 The effect of the Micr.M175 strain on the growth of black soldier fly larvae in terms of body length and weight can also be clearly seen.

[0079] 6.2 The new strain Micr.M175 improves the insect production rate and substrate reduction rate of the material. On the 8th day of rearing, weigh and record the weight of the larvae and the substrate, and calculate the larval production rate and substrate reduction rate. Larval production rate = 100 × (fresh weight of larvae on day 8 / fresh weight of initial material); Substrate reduction rate = 100 × (dry weight of substrate on day 8 - dry weight of initial material) / dry weight of initial material.

[0080] Table 1. Insect production rate and substrate reduction rate in the insect-bacterial experiment.

[0081] As shown in Table 1, after applying the Micr.M175 strain, the insect production rate at harvest reached 8.94%, an increase of 36.74% compared to the control group. Furthermore, the substrate reduction rate significantly increased to 35.20%, significantly higher than the control group. This demonstrates that the Micr.M175 strain proposed in this application can achieve efficient black soldier fly rearing and simultaneously achieve highly efficient reduction of organic solid waste.

[0082] The Micr.M175 proposed in this application has broad application prospects in shortening the black soldier fly breeding cycle, increasing the commercial value of the insects, and efficiently treating organic solid waste.

[0083] 6.3 The new bacterial strain Micr.M175 promotes protein accumulation in black soldier fly larvae. The fresh insects obtained in step 6.1 were dried, ground into powder, and approximately 2 mg of the powder was weighed and packaged in a tin boat. The powder was then placed in an elemental analyzer (Elementar, UNICUBE, Germany). The combustion tube temperature was set to 1100 ℃, the reduction tube temperature to 850 ℃, acetanilide was used as the standard, the method was selected as 2 mg Standard-80s, and the remaining parameters were set to default. The nitrogen (N) content in the sample was then determined. The protein yield of the insects was further calculated based on the weight and moisture content of the black soldier fly larvae. The specific calculation formula is: Crude protein yield (g / 1000 larvae) = (Percentage of nitrogen in the sample / 100) × 6.25 × Average dry weight per larva × 1000. The calculation results are shown in Table 2.

[0084] Table 2 Protein production of black soldier fly larvae

[0085] As shown in Table 1, compared with the control group, the protein production of the experimental group treated with Micr.M175 strain showed a highly significant increase throughout the entire growth cycle. On day 4 and day 8 of rearing, the protein production of the insect body increased by 103.57% and 32.41%, respectively. Moreover, the protein production of the experimental group on day 4 was comparable to that of the control group on day 8, indicating that Micr.M175 strain played a highly significant role in promoting protein accumulation in the larvae. These results prove that the Micr.M175 strain proposed in this application can effectively promote protein accumulation in black soldier fly larvae and greatly improve the protein production of black soldier fly larvae during rearing.

[0086] 6.4 The new strain Micr.M175 promotes amino acid accumulation in black soldier fly larvae. This embodiment further determined the amino acid content in black soldier fly larvae. The specific method was as follows: approximately 50 mg of larval meal was weighed (preparation of larval meal was the same as described in section 6.3 above), and the protein was hydrolyzed using hydrochloric acid. The amino acid content was then determined using a German Manmerborough A300 fully automated amino acid analyzer, with a sample loading volume of 20 μL. The amino acid yield was further calculated based on the larval weight and moisture content. The specific calculation formula is: Amino acid yield (g / 1000 larvae) = (Amino acid content percentage / 100) × Average dry weight per larva × 1000. The calculation results are shown in Table 3.

[0087] Table 3. Amino acid yield of black soldier fly larvae (g / 1000 larvae)

[0088] As shown in Table 3, compared with the control group, the application of the new strain Micr.M175 provided in this application significantly increased the content of five essential amino acids—threonine, isoleucine, leucine, histidine, and lysine—in black soldier fly larvae. p The concentration of amino acids in larvae was <0.05, and the improvement rate was 18.97%-41.43%, indicating that this strain greatly optimized and promoted the amino acid synthesis and metabolism of larvae and significantly improved the nutritional value of larvae.

[0089] 6.5 The new strain Micr.M175 promotes fat accumulation in black soldier fly larvae. Approximately 0.3 g of D8 larval meal was weighed (meal preparation is the same as in section 6.2 above). The fat content of the meal was determined using a fat analyzer (Haineng, SOX406). The protein yield of the larvae was further calculated by combining this with the body weight and moisture content of the black soldier fly larvae. Fat yield (g / 1000 larvae) = (fat content percentage / 100) × dry weight of a single larva × 1000 larvae. The calculation results are shown in Table 4.

[0090] Table 4. Fat production of black soldier fly larvae

[0091] As shown in Table 4, the body fat yield of larvae in the control group and the experimental group treated with Micr.M175 strain was 3.34 g and 4.93 g, respectively. Compared with the control group, the body fat yield of larvae in the experimental group treated with Micr.M175 strain increased by 47.74%, which was significantly higher than that of the control group. This indicates that the Micr.M175 strain proposed in this application can effectively promote fat accumulation in black soldier fly larvae.

[0092] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0093] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A microbacterium, characterized in that, Contains the 16S rDNA sequence shown in SEQ ID NO: 1 or its complementary sequence or a sequence with at least 85% identity to SEQ ID NO:

1.

2. The microbacteria according to claim 1, characterized in that, The microbacterium has a 16S rDNA sequence as shown in SEQ ID NO: 1, and is protected under accession number CGMCC No. 35903. Microbacterium sp. Micr.M175 was deposited at the China General Microbiological Culture Collection Center on September 12, 2025.

3. A method for culturing microbes as described in claim 1 or 2, characterized in that, The method includes inoculating the microbes into a culture medium and culturing them at a temperature of 25 to 37°C, preferably 28 to 30°C, and most preferably 30°C. The pH of the culture medium is 7.0 to 8.0, preferably 7.4 to 7.8, and more preferably 7.

6. Optionally, the culture medium is 2216E medium, which, in g / L, comprises: 5.0 g peptone, 1.0 g yeast extract, 0.1 g ferric citrate, 19.45 g sodium chloride, 5.98 g magnesium chloride, 3.24 g sodium sulfate, 1.8 g calcium chloride, 0.55 g potassium chloride, 0.16 g sodium carbonate, 0.08 g potassium bromide, 0.034 g strontium chloride, 0.022 g boric acid, 0.004 g sodium silicate, 0.0024 g sodium fluoride, 0.0016 g ammonium nitrate, and 0.008 g disodium hydrogen phosphate.

4. A microbial inoculant, characterized in that, Includes microbes as described in claim 1 or 2 and microbiologically acceptable excipients.

5. The microbial agent according to claim 5, characterized in that, The dosage form of the microbial agent is selected from: wettable powder, water-dispersible granules, suspension concentrate, emulsion, granules, or combinations thereof.

6. The use of the microbacteria as described in claim 1 or 2 or the microbial agent as described in claim 4 or 5 in promoting the growth of black soldier fly larvae and / or increasing the accumulation of protein and fat in black soldier fly bodies.

7. The application of the microbacteria as described in claim 1 or 2 or the microbial agent as described in claim 4 or 5 in the conversion of organic solid waste by black soldier flies.

8. The application according to claim 7, characterized in that, The organic solid waste includes: wheat bran, kitchen waste, livestock and poultry manure, distiller's grains, and food processing waste.

9. The application according to claim 7 or 8, characterized in that, Includes the following steps: (1) The microbacterium or the microbial agent is prepared at 10 8 The organic solid waste was inoculated at a ratio of cfu / g to obtain a mixed matrix; (2) After the mixed substrate is cultured at 28 ℃ and 70% relative humidity for 1 day, 1200 6-day-old black soldier fly larvae are inoculated and cultured for a longer period of time to obtain biomass.

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