Preparation method of functional bacillus based on black soldier fly live propagation

Abstract

The present application relates to the field of microbial engineering and utilization of insect resources, and particularly relates to a functional bacillus preparation method based on black soldier fly live propagation, comprising: treating fermentation substrate with sodium acetate and ethanol for synergistic sterilization, and then obtaining sterilized substrate through anaerobic fermentation; mixing bacillus subtilis, bacillus licheniformis and bacillus pumilus in a certain proportion to obtain a compound microbial agent; introducing the compound microbial agent into the sterilized substrate, and then introducing black soldier fly larvae for live propagation; and separating and collecting bacillus products from the mixture of larvae and feces. The present application uses the intestinal tract of black soldier fly larvae as a biological reactor, realizes high-fold propagation under low inoculation amount through a preferential occupation mechanism, significantly improves bacillus yield, and overcomes the defects of low intestinal colonization rate, poor strain synergy and high equipment cost in the prior art.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of microbial engineering and insect resource utilization, and in particular to a method for preparing functional Bacillus based on the propagation of live black soldier fly larvae. Background Technology

[0002] The black soldier fly (Hermetia illucens L.) is an important resource insect. Its larvae can efficiently convert organic waste into biomass, and their gut microbiota is colonized with a rich microbial community, serving as a natural carrier of probiotics. Bacillus spp. is a commonly used probiotic strain, possessing advantages such as heat resistance, stomach acid resistance, bile salt resistance, and the ability to produce various digestive enzymes and antimicrobial peptides. It is widely used in feed additives, aquaculture, and agricultural plant protection.

[0003] Existing methods for preparing probiotics using black soldier fly larvae as carriers involve directly spraying commercial probiotic powder into the black soldier fly larvae rearing substrate. This method has the following problems:

[0004] (1) Indigenous microorganisms (Pseudomonas, Escherichia coli, etc.) in the substrate compete with exogenous probiotics for nutrients, and the intestinal colonization rate of exogenous probiotics is only 15% to 30%;

[0005] (2) The sprayed probiotic powder (most of which are dormant spores) is directly exposed to the high temperature (30℃~35℃) and high humidity (relative humidity>80%) environment of the substrate, and the germination rate is uncontrollable. Some of them are inactive before reaching the intestine.

[0006] (3) The selective pressure of the black soldier fly larvae’s gut was not utilized to enrich the functional flora, resulting in insufficient abundance of target Bacillus in the larvae / feces.

[0007] Chinese invention patent (CN108371147A) discloses a black soldier fly larvae-bacterial compound preparation and its production method. The preparation comprises, by weight, third-instar black soldier fly larvae, active bacterial powder, wheat bran, perlite, calcium propionate, and vitamin C. The active bacterial powder is prepared by culturing methyltrophic Bacillus to the logarithmic growth phase in liquid LB medium, followed by 24 hours of liquid fermentation in a fermenter, and then mixing with zeolite powder and drying. This technology still has the following shortcomings: First, the preparation of the active bacterial powder still relies on traditional liquid fermentation processes, resulting in high investment and energy consumption in fermenter equipment; second, it only physically mixes the active bacterial powder with the black soldier fly larvae, failing to achieve live colonization and biomass expansion of the functional bacteria in the larval intestine; third, the strain is only a single methyltrophic Bacillus, lacking multi-strain synergy; fourth, the product is a physically mixed preparation, and the functional bacteria have not undergone intestinal adaptation, resulting in insufficient stability. Therefore, developing a method for preparing Bacillus that can achieve sterilization of the fermentation substrate, live intestinal propagation, and collection of insect excrement and bacterial agent has significant industrial application value. Summary of the Invention

[0008] Therefore, this invention provides a method for preparing functional Bacillus spores based on live propagation from black soldier flies, to overcome the problems of low intestinal colonization rate, insufficient propagation abundance, and poor product stability in existing technologies. The method includes the following steps:

[0009] Step S1: The fermentation substrate of black soldier fly larvae is sterilized by sequentially using sodium acetate solution and ethanol solution to kill the indigenous microorganisms in the fermentation substrate, and then subjected to anaerobic fermentation to eliminate residual sodium acetate and ethanol to obtain sterilized substrate.

[0010] Step S2: Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus are cultured separately. The spore suspensions of the three Bacillus strains are mixed to obtain a functional Bacillus compound bacterial agent.

[0011] Step S3: Inoculate the functional Bacillus compound bacterial agent into the sterilized matrix, mix well, transfer to the black soldier fly larvae breeding box, inoculate black soldier fly larvae, carry out live propagation treatment, and obtain a mixture of live larvae and insect excrement.

[0012] Step S4: Take the live larvae and larvae mixture and mix it with sterile physiological saline for homogenization to obtain a diluted solution. Perform serial dilution on the diluted solution and spread it on a selective culture medium for Bacillus isolation and counting, and collect the functional Bacillus products.

[0013] Further, in step S1, the sodium acetate solution has a mass fraction of 0.8% to 1.2%, and the ethanol solution has a volume fraction of 1.5% to 2.5%.

[0014] Furthermore, in step S1, the temperature of the anaerobic fermentation treatment is 30℃~35℃, the time is 24h~48h, and the lactic acid concentration in the sterilized substrate after anaerobic fermentation is ≥0.5%.

[0015] Further, in step S1, the sterilization treatment and the anaerobic fermentation treatment reduce the residual amount of indigenous microorganisms in the sterilized substrate to less than or equal to 10. 2 CFU / g.

[0016] Further, in step S2, the Bacillus subtilis is Bacillus subtilis with accession number ATCC 21332, the Bacillus licheniformis is Bacillus licheniformis with accession number ATCC 14580, and the Bacillus pumilus is Bacillus pumilus with accession number ATCC 7061.

[0017] Further, in step S2, the spore suspensions of Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus are mixed in a volume ratio or mass ratio of 1:1:1.

[0018] Further, in step S3, the functional Bacillus compound bacterial agent is prepared at a concentration of 10... 4 CFU / g ~ 10 5 An inoculum of CFU / g is introduced into the sterilization matrix.

[0019] Furthermore, in step S3, the inoculation density of the black soldier fly larvae is 1.5 to 2.5 larvae per gram of the sterilized substrate.

[0020] Furthermore, in step S3, the culture temperature for the live propagation treatment is 27℃~30℃, and the relative humidity is 65%~75%.

[0021] Further, in step S4, the selective culture medium is tryptone soybean broth medium containing 0.1 mg / mL neomycin.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: by sterilizing the fermentation substrate, indigenous microorganisms are effectively killed, creating a non-competitive environment for the intestinal colonization of functional Bacillus; by combining three strains of Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus, the selective ecological niche of the black soldier fly larvae's intestine is utilized to achieve live propagation, significantly increasing the total spore yield; the entire preparation process does not require traditional fermentation equipment, has low energy consumption and no wastewater discharge, realizing the green preparation of functional Bacillus. Attached Figure Description

[0023] Figure 1 This is a flowchart illustrating the steps of the method for preparing functional Bacillus strains based on live black soldier fly propagation in this embodiment.

[0024] Figure 2 A bar chart comparing the total spore yield under different strain ratios.

[0025] Figure 3 The graph shows the effect of different inoculation amounts on the colonization rate and spore yield of Bacillus subtilis in the gut of black soldier fly larvae.

[0026] Figure 4 A comparative graph showing the effects of products prepared with different combinations of process parameters on the feed conversion ratio (FCR) and the ileal villus height / crypt depth ratio (VH / CD) of broilers. Detailed Implementation

[0027] To make the objectives and advantages of the present invention clearer, the present invention will be further described below with reference to embodiments; it should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0028] Please see Figure 1 As shown, it is a flowchart of the steps of the method for preparing functional Bacillus based on the live propagation of black soldier fly larvae in an embodiment of the present invention.

[0029] This invention provides a method for preparing functional Bacillus spores based on live black soldier fly propagation, comprising:

[0030] Step S1: The fermentation substrate of black soldier fly larvae is sterilized sequentially with sodium acetate solution of 0.8% to 1.2% by mass and ethanol solution of 1.5% to 2.5% by volume to kill the indigenous microorganisms in the substrate. Then, it is anaerobic fermented at 30℃ to 35℃ for 24h to 48h to eliminate residual sodium acetate and ethanol and enrich lactic acid to obtain sterilized substrate.

[0031] Specifically, in step S1, the sodium acetate solution has a mass fraction of 0.8% to 1.2%, and the ethanol solution has a volume fraction of 1.5% to 2.5%.

[0032] In this embodiment, step S1 involves synergistic sterilization of the fermentation substrate using a sodium acetate solution with a mass fraction of 0.8%–1.2% and an ethanol solution with a volume fraction of 1.5%–2.5%. Sodium acetate, as an antibacterial agent, inhibits the proliferation of native bacteria and fungi by lowering the substrate pH and altering cell membrane permeability; ethanol, on the other hand, can penetrate and disrupt the lipid bilayer of microbial cell membranes, causing protein denaturation and killing spore-forming bacteria. The fermentation substrate is first mixed with the sodium acetate solution and left to stand for 2–3 hours, then uniformly sprayed with the ethanol solution and left to stand for 30 minutes—a two-stage synergistic treatment. The function of this treatment is to achieve synergistic killing of native microorganisms in the substrate through the combined action of two sterilizing agents with different mechanisms of action, eliminating competitive interference for subsequent intestinal colonization by functional spore-forming bacteria.

[0033] In this embodiment, in order to demonstrate the beneficial effect of the synergistic sterilization treatment concentration range of sodium acetate solution and ethanol solution, under the condition that other parameters in step S1 remain unchanged, test groups with different mass fractions of sodium acetate solution and ethanol solution were set up for analysis. The experimental data are shown in Table 1, which is a data table of microbial killing rate of different mass fractions of sodium acetate solution and ethanol solution.

[0034] Table 1: Data on the microbial kill rate of different sodium acetate and ethanol solutions by mass fraction

[0035]

[0036] Comparative Example 1-1: Incomplete sterilization

[0037] When the sodium acetate concentration was reduced to 0.5% and the ethanol concentration to 1.0%, the residual amount of indigenous microorganisms reached as high as 8.5 × 10⁻⁶. 3 The concentration of CFU / g was only 96.30%, indicating that this concentration combination could not effectively kill indigenous microorganisms in the matrix, and the number of residual indigenous microorganisms (10) was low. 3 The CFU / g level was significantly higher than the target residual level after functional Bacillus inoculation (<10). 2 (CFU / g) These residual indigenous microorganisms will compete with functional Bacillus bacteria for nutrients and niches during subsequent propagation, severely interfering with the preferential occupation of functional bacteria in the larval gut.

[0038] Comparative Examples 1-2: There is no room for further improvement in sterilization effect.

[0039] When the sodium acetate concentration reached 1.5% and the ethanol concentration reached 3.0%, the residual amount of indigenous microorganisms was <50 CFU / g, and the kill rate reached 99.98%. Compared with Examples 1-3 (sodium acetate 1.2%, ethanol 2.5%, residual amount <50 CFU / g, kill rate 99.98%), further increasing the concentration did not further improve the sterilization effect; both reached the detection limit (<50 CFU / g). This indicates that the sterilization effect has reached its upper limit within the range of 0.8%–1.2% sodium acetate and 1.5%–2.5% ethanol. Further increasing the concentration is a pointless waste of raw materials, and higher concentrations of chemical reagent residues will increase the burden of subsequent anaerobic fermentation to eliminate residues, increasing process costs and production safety risks.

[0040] Examples 1-1 to 1-3: Excellent sterilization effect and reasonable dosage

[0041] When the concentration of sodium acetate was 0.8%–1.2% and the concentration of ethanol was 1.5%–2.5%, all three exhibited excellent sterilization effects: residual indigenous microorganisms <350 CFU / g and kill rate >99.85%. Among them, Examples 1-2 (median: sodium acetate 1.0%, ethanol 2.0%) showed the best effect, with a kill rate of 99.96% (residual amount <100 CFU / g). This concentration range can achieve sterilization effects comparable to higher concentration schemes (comparative Examples 1-2) (kill rate >99.98%) with lower reagent dosage, reducing raw material costs and the burden of subsequent residue removal while ensuring thorough sterilization.

[0042] A synergistic treatment concentration range of 0.8%–1.2% sodium acetate solution (mass fraction) and 1.5%–2.5% ethanol solution (volume fraction) achieves excellent sterilization results with a kill rate of >99.85% for indigenous microorganisms. Below this range, sterilization is incomplete (kill rate is only 96.30%, with a residue as high as 8.5 × 10⁻⁶). 3 The concentration of sodium acetate (CFU / g) cannot effectively eliminate the competitive interference from indigenous microorganisms; above this range, the sterilization effect is not further improved (it is the same as in Examples 1-3), which is an unnecessary waste of raw materials. Therefore, the selection of concentrations of 0.8%–1.2% sodium acetate and 1.5%–2.5% ethanol ensures excellent sterilization effect while taking into account economy, and has significant beneficial effects.

[0043] Understandably, the fermentation substrate of black soldier fly larvae naturally contains a large number of indigenous microorganisms (such as Pseudomonas, Escherichia coli, and Bacillus cereus). If this substrate is directly used as a propagation carrier for functional Bacillus, the indigenous microorganisms will compete with the introduced exogenous functional bacteria for nutrients and niches, resulting in a low intestinal colonization rate of the exogenous bacteria. By using a specific concentration of sodium acetate and ethanol for synergistic sterilization, the kill rate of indigenous microorganisms can be significantly improved. At the same time, this concentration range will not cause irreversible damage to the subsequently introduced functional Bacillus, creating a non-competitive environment for the functional bacteria to preferentially occupy the larval gut.

[0044] Specifically, in step S1, the anaerobic fermentation temperature is 30℃~35℃, the time is 24h~48h, and the lactic acid concentration in the sterilized substrate after anaerobic fermentation is ≥0.5%.

[0045] In this embodiment, the co-treated substrate was anaerobic fermented at 30℃~35℃ for 24h~48h, and the lactic acid concentration in the sterilized substrate after fermentation was ≥0.5%. Under anaerobic conditions, the residual fermentable sugars in the substrate were converted into lactic acid by anaerobic microorganisms (mainly lactic acid bacteria), while consuming residual sodium acetate and ethanol. The co-treated substrate was transferred to a sealed anaerobic fermentation bag, and anaerobic fermentation was carried out under controlled temperature and time. After fermentation, the lactic acid concentration was measured. This process aimed to eliminate the inhibitory effect of residual sodium acetate and ethanol on the subsequent germination and growth of functional Bacillus spores, and to enrich the substrate with lactic acid, utilizing the prebiotic effect of lactic acid to promote the germination of Bacillus spores.

[0046] In this embodiment, in order to demonstrate the beneficial effect of setting the range of lactic acid concentration in the sterilized substrate after anaerobic fermentation, test groups with different lactic acid concentrations were set up for analysis while keeping other parameters in step S1 unchanged. The experimental data are shown in Table 2, which is a data table of the effects of different lactic acid concentrations in the sterilized substrate on the germination rate of functional bacteria, the resurrection rate of indigenous bacteria and the yield of spores.

[0047] Table 2: Effects of different lactic acid concentrations on germination rate of functional bacteria, reactivation rate of indigenous bacteria, and spore yield.

[0048]

[0049] A lactic acid concentration ≥0.5% in the sterilized substrate after anaerobic fermentation is essential for achieving a high germination rate (≥95%) of functional Bacillus, effectively inhibiting the reactivation of indigenous microorganisms (reactivation rate ≤8.5%), and ensuring that the final product spore yield meets the target (≥2.9×10⁻⁶). 9 The lactic acid concentration (CFU / g) is a key quality control indicator. When the lactic acid concentration is below 0.5%, all of the above indicators deteriorate significantly, and the product fails to meet the expected standards. When the lactic acid concentration is above 0.5%, although the indicators improve slightly, the improvement rate tends to plateau, and the marginal benefit diminishes. Therefore, a lactic acid concentration ≥0.5% is a process control node with clear physical significance and engineering value, and is not an obvious conventional choice.

[0050] Understandably, if functional Bacillus subtilis is directly introduced after co-treatment, the residual sodium acetate (content >0.5%) will inhibit the germination of Bacillus subtilis, and the residual ethanol will also damage vegetative cells. However, if anaerobic fermentation is not performed and the residue is removed directly by washing, it will introduce new risks of contamination by other microorganisms and increase operational steps and water consumption. Anaerobic fermentation converts the residual sodium acetate and ethanol into lactic acid. Lactic acid is a natural prebiotic that promotes the germination of Bacillus subtilis and maintains a low pH environment in the substrate to further inhibit the reactivation of residual indigenous microorganisms. Simultaneously, this temperature and time range ensures that the lactic acid concentration is enriched to above 0.5%, achieving a triple effect of eliminating inhibition, promoting germination, and providing sustained antibacterial protection.

[0051] Specifically, in step S1, the sterilization treatment and the anaerobic fermentation reduce the residual amount of indigenous microorganisms in the sterilized substrate to less than or equal to 10. 2 CFU / g.

[0052] In this embodiment, the sterilization and anaerobic fermentation in step S1 reduced the residual amount of indigenous microorganisms in the sterilized substrate to below 10² CFU / g. The sodium acetate-ethanol synergistic treatment achieved highly efficient dual killing of both vegetative cells and spores of indigenous microorganisms, while subsequent anaerobic fermentation further inhibited the reactivation of residual microorganisms through lactic acid enrichment and low pH maintenance. After sterilization, the residual amount of indigenous microorganisms in the sterilized substrate was detected using the plate counting method (PCA medium, 37°C for 48 h). The function of this treatment is to control the number of indigenous microorganisms at an extremely low level, ensuring that the functional Bacillus bacteria occupy an absolute niche advantage in the substrate and gut after inoculation.

[0053] It is understandable that in the early stages (0–24 hours) after functional Bacillus bacteria are inoculated into a sterilized substrate and fed to the intestines by larvae, if the number of residual indigenous microorganisms in the substrate is too high (e.g., >10), 3 These indigenous microorganisms (CFU / g) compete with functional Bacillus for nutrients in the substrate and attachment sites in the larval gut, leading to prolonged intestinal colonization time (>48h) or even colonization failure of the functional bacteria. This invention controls the residual amount of indigenous microorganisms to below 10% through a sterilization process. 2 Below CFU / g (i.e., no more than 100 residual indigenous microorganisms per gram of substrate), this order of magnitude is far lower than the inoculation amount of functional Bacillus (10). 4 ~10 5 The CFU / g concentration ensures an absolute advantage for functional bacteria, enabling functional Bacillus to rapidly occupy the intestines within 24 hours, with a colonization rate of over 92%.

[0054] Step S2: Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus are cultured separately. The spore suspensions of the three Bacillus strains are mixed in a volume ratio or mass ratio of 1:1:1 to obtain a functional Bacillus compound inoculum.

[0055] Specifically, in step S2, the Bacillus subtilis is Bacillus subtilis, the Bacillus licheniformis is Bacillus licheniformis, and the Bacillus pumilus is Bacillus pumilus. The Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus are commercially available strains.

[0056] In this embodiment, the *Bacillus subtilis*, *Bacillus licheniformis*, and *Bacillus pumilus* strains used in step S2 were all purchased from commercial channels. Specifically, the *Bacillus subtilis* (ATCC 21332), *Bacillus licheniformis* (ATCC 14580), and *Bacillus pumilus* (ATCC 7061) used in this embodiment were all purchased from the American Type Culture Collection (ATCC). The three strains were inoculated into solid sporulation medium and cultured at 37°C for 72 hours. After sporulation, the sporulations were collected with sterile water, and the concentration of the sporulation suspensions for each strain was adjusted to be uniform, such as 1.0 × 10⁻⁶. 8 CFU / mL, then mix thoroughly in equal volume or mass ratio.

[0057] Understandably, standard industrial strains from commercial collections such as ATCC have clearly defined sources, stable quality, and well-documented functions. Furthermore, they are freely available to the public through commercial channels, eliminating the need for biological preservation and thus avoiding any issues of insufficient disclosure. Meanwhile, Bacillus subtilis (ATCC 21332), Bacillus licheniformis (ATCC 14580), and Bacillus pumilus (ATCC 7061) are all industrial strains with clearly documented functions, exhibiting activities such as amylase, protease, cellulase, and chitinase production, respectively. The combination of these three strains can form a metabolically complementary synergistic network in the gut of black soldier fly larvae, laying the functional foundation for subsequent live propagation.

[0058] Specifically, in step S2, the spore suspensions of Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus are mixed in a volume ratio or mass ratio of 1:1:1.

[0059] In this embodiment, step S2 involves mixing spore suspensions of Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus at a volume or mass ratio of 1:1:1. These three functional Bacillus strains exhibit complementary functions in the intestinal metabolism of black soldier fly larvae: Bacillus subtilis rapidly utilizes carbohydrates to amplify and produce amylase, protease, and lipase; Bacillus licheniformis synergistically decomposes complex polysaccharides such as cellulose and pectin, and further amplifies using small molecule metabolites produced by Bacillus subtilis, while simultaneously producing cellulase and pectinase; Bacillus pumilus produces chitinase to inhibit entomopathogenic fungi, thus providing biological protection. The three strains are cultured separately for solid-state sporulation. After collecting the spores, the concentration of the spore suspensions for each strain is adjusted to be uniform (e.g., all 1.0 × 10⁻⁶) using sterile water. 8 (CFU / mL), then mix thoroughly in equal volumes or mass ratios. This proportional design ensures the harmonious coexistence and stable amplification of the three strains in the intestinal niche, preventing one strain from monopolizing the niche due to numerical superiority and thus compressing the survival space of other strains.

[0060] In this embodiment, in order to demonstrate the beneficial effects of mixing spore suspensions of Bacillus subtilis, Bacillus licheniformis and Bacillus pumilus in equal proportions, test groups with different spore suspension ratios were set up for analysis while keeping other parameters in step S2 unchanged. The experimental data are shown in Table 3, which is a data table of total spore yield and bioprotective capacity under different spore suspension ratios.

[0061] Table 3: Data on total spore yield and bioprotective capacity under different spore suspension ratios

[0062]

[0063] The total spore yield of Example 3-1 (1:1:1) reached 3.0 × 10⁻⁶. 9CFU / g, compared to the control group 3-1 single-strain regimen (1.4×10⁻⁶). 9 The CFU / g ratio was increased by 114%, compared to the control 3-3 two-strain regimen (1.8 × 10⁻⁶). 9 The CFU / g ratio increased by 67%. This indicates a significant synergistic amplification effect among the three strains—Bacillus subtilis rapidly amplifies using carbohydrates, Bacillus licheniformis proliferates using amino acid metabolites produced by the former, and Bacillus pumilus inhibits the pathogenic fungus by producing chitinase. The total spore yields in Examples 3-2 and 3-3 were 2.5 × 10⁻⁶. 9 CFU / g and 2.4×10 9 The CFU / g values ​​were all lower than those of the equal-proportion scheme, indicating that deviation from the equal proportion would lead to the compression of the ecological niche of some strains and a decrease in total yield.

[0064] In the Beauveria bassiana challenge test, the 7-day larval mortality rate of Example 3-1 was only 8%, significantly lower than that of Comparative Examples 3-1 (65%), 3-2 (42%), and 3-3 (58%). This is because the chitinase produced by Bacillus pumilus can hydrolyze the cell wall of the pathogenic fungus, thus providing bioprotection; while single-strain or Bacillus pumilus-deficient regimens lack this functional defense. In Comparative Example 3-2 (2:1:1), although Bacillus subtilis was dominant, the proportion of Bacillus pumilus was diluted, resulting in insufficient chitinase production and a significant decrease in protective ability (mortality rate 42%).

[0065] Considering both of the above indicators, the 1:1:1 ratio compounding scheme results in a total spore yield of 3.0 × 10⁻⁶. 9 Both CFU / g and bioprotective capability (8% mortality rate from Beauveria bassiana challenge) were optimal. When the proportion of a certain strain deviated from the ideal ratio, both indicators decreased to varying degrees; when a certain strain was missing, functional defects were obvious (significant deterioration in yield or protective capability). Therefore, 1:1:1 is the optimal ratio that balances the synergistic amplification of the three strains and their bioprotective function, and is not an obvious conventional choice.

[0066] like Figure 2 As shown, when the three strains are mixed in a 1:1:1 ratio, the total spore yield is significantly higher than that of single strains, double strains, and non-equal ratio mixing schemes, confirming the synergistic effect of equal ratio mixing.

[0067] Understandably, if the mixing ratio of the three strains is severely unbalanced (e.g., one strain accounts for >60%), the dominant strain will rapidly occupy the limited attachment sites in the gut due to its initial numerical advantage, which in turn inhibits the colonization and amplification of other functional strains, resulting in a significant decrease in total spore yield and functional diversity. In the actual test, the total yield was only 78% of that of the 1:1:1 ratio. However, when mixed in an equal ratio of 1:1:1, the abundance ratio of the three strains in the gut can still be maintained at around 1:1:1 at the end of the propagation, indicating that this ratio can ensure the synergistic effect of the three strains, increasing the total spore yield by 45% to 65% compared to the single-strain scheme.

[0068] Step S3, mix the functional Bacillus compound bacterial agent at 10 4 ~10 5 The inoculation amount of CFU / g is added to the sterilized substrate, mixed well, and then transferred to the black soldier fly larvae rearing box. Black soldier fly larvae are introduced at a density of 1.5 to 2.5 larvae per gram of the sterilized substrate. Live propagation treatment is carried out under the conditions of 27°C to 30°C and relative humidity of 65% to 75% to obtain a mixture of live larvae and insect excrement.

[0069] Specifically, in step S3, the functional Bacillus compound bacterial agent is prepared at a concentration of 10... 4 ~10 5 An inoculum of CFU / g is introduced into the sterilization matrix.

[0070] In this embodiment, the functional Bacillus compound bacterial agent is prepared at 10... 4 ~10 5 Inoculation at a CFU / g level was applied to the sterilized substrate. Since indigenous microorganisms in the sterilized substrate have been eliminated, the functional bacteria, introduced at a low inoculation level, do not face competition from indigenous bacteria. This allows them to rapidly germinate and occupy intestinal wall attachment sites after larvae ingest the bacteria, achieving preferential site occupation. Simultaneously, the low inoculation level avoids nutrient competition among bacteria in the substrate, which is beneficial for maintaining the spore formation rate. The compound bacterial agent was diluted with sterile water to the target concentration and then uniformly mixed into the sterilized substrate, ensuring even distribution of the agent. The function of this treatment is to achieve an optimal balance between sufficient colonization and avoiding excessive competition by precisely controlling the initial bacterial load, enabling efficient amplification of the functional bacteria in the intestine.

[0071] like Figure 3 As shown, when the inoculation amount is 10 4 ~10 5 At a CFU / g level, both intestinal colonization rate and spore yield were at their optimal levels. Inoculum size below 10... 4 When the CFU / g level is insufficient, the initial bacterial load is too low, leading to prolonged colonization time (>48h); an inoculum size higher than 10... 5 At CFU / g, nutrient competition among bacterial cells leads to a decrease in spore formation rate. Therefore, at 10... 4 ~105 CFU / g is the optimal inoculum range to ensure high colonization rate and high spore yield.

[0072] Understandably, current technologies often involve directly spraying functional microbial agents into unsterilized aquaculture substrates, or using excessively high inoculation amounts (e.g., ≥10). 7 (CFU / g), on the one hand, indigenous microorganisms compete with functional bacteria, resulting in a colonization rate of only 15%–30%; on the other hand, nutrient competition among bacterial cells intensifies, leading to a significant decrease in spore formation rate. This invention uses 10 4 ~10 5 A low inoculum of CFU / g was introduced into the sterile substrate, using the gut of black soldier fly larvae as a bioreactor. Functional bacteria entered the midgut with the larvae after ingestion, germinated into vegetative cells, and colonized the intestinal wall. They then multiplied rapidly using the nutrients in the substrate, increasing in number by up to 10 times. 2 ~10 3 The final total spore yield reached 2.8 × 10⁻⁶. 9 ~3.1×10 9 CFU / g; inoculation amount less than 10 4 When the initial bacterial count is above 10 CFU / g, the colonization time is insufficient and the colonization time is prolonged (>48h). 5 At CFU / g, nutrient competition among bacterial cells led to a decrease in spore formation rate (10). 7 (only 72% at CFU / g), confirming the reasonableness and non-obviousness of this inoculation range.

[0073] Specifically, in step S3, the inoculation density of the black soldier fly larvae is 1.5 to 2.5 larvae per gram of the sterilized substrate.

[0074] In this embodiment, step S3 involves inoculating newly hatched black soldier fly larvae at a density of 1.5–2.5 larvae per gram of sterilized substrate. The number of larvae inoculated per unit mass of substrate determines the ratio between the total intestinal volume and the total amount of functional Bacillus in the substrate: if the density is too low, the total intestinal volume is insufficient to process all the bacteria in the substrate; if the density is too high, competition among larvae intensifies, metabolic heat accumulates, and the microenvironment deteriorates. The sterilized substrate is weighed and filled into the rearing box. The required number of larvae is calculated based on the substrate weight, and newly hatched larvae (12–24 hours after hatching) are inoculated, ensuring uniform distribution of larvae in the substrate. An optimal matching relationship is established between substrate bacterial count, intestinal volume, and larval number, ensuring that the intestine of each larva can fully contact and process the functional Bacillus in the substrate, achieving efficient live propagation.

[0075] Understandably, the feeding behavior and intestinal processing capacity of black soldier fly larvae are significantly affected by stocking density. In existing technologies, the stocking density of black soldier fly larvae is typically 0.5–1.0 larvae / g substrate, primarily considering larval growth rate and waste conversion efficiency. However, this density range is insufficient for the total intestinal volume and results in low processing efficiency for the live propagation of functional Bacillus. In this embodiment, the larval inoculation density is increased to 1.5–2.5 larvae / g, balancing the dual requirements of total intestinal processing volume and individual survival quality. When the density is below 1.5 larvae / g, the total intestinal volume is insufficient to adequately process the functional Bacillus in the substrate, leading to low propagation efficiency (total spore production <5×10⁻⁶). 8 CFU / g); When the density exceeds 2.5 larvae / g, competition among larvae intensifies, metabolic heat accumulation in the rearing box leads to increased local temperature and humidity exceeding 80%, causing some larvae to die due to hypoxia and disease (mortality rate >15%), thus reducing the total spore yield; a density range of 1.5–2.5 larvae / g can ensure high propagation efficiency (spore yield 2.8–3.1 × 10⁻⁶ CFU / g). 9 Between CFU / g and low larval mortality (4%–5%).

[0076] Specifically, in step S3, the culture temperature for the live propagation treatment is 27℃~30℃, and the relative humidity is 65%~75%.

[0077] In this embodiment, step S3 controls the culture temperature for live propagation treatment to be 27℃~30℃ and the relative humidity to be 65%~75%. Temperature directly affects the metabolic rate, feeding activity, and intestinal peristalsis frequency of black soldier fly larvae, as well as the germination and proliferation rate of Bacillus in the intestine; humidity affects the moisture content of the substrate and gas exchange efficiency, thereby affecting the stability of the larval intestinal microenvironment. The culture box is placed in a constant temperature and humidity incubator, and the temperature and humidity are monitored in real time and automatically adjusted by a temperature and humidity sensor. Sterile distilled water is used daily to replenish the water lost through evaporation, maintaining the substrate moisture content at 65%~70%. This ensures that the larvae maintain vigorous feeding and intestinal metabolic activity, while providing stable temperature and humidity conditions for the germination and proliferation of Bacillus in the intestine.

[0078] In this embodiment, in order to demonstrate the beneficial effects of the temperature and humidity range in the live propagation treatment, test groups with different temperature and humidity combinations were set up for analysis while keeping other parameters in step S3 unchanged. The experimental data are shown in Table 4, which is a data table of larval mortality rate and total spore production under different temperature and humidity combinations.

[0079] Table 4: Larval Mortality Rate and Total Spore Production under Different Temperature and Humidity Combinations

[0080]

[0081] Comparative Example 4-1: Insufficient larval vigor and decreased yield

[0082] When the temperature drops to 25°C and the humidity drops to 55%, the larval mortality rate reaches 22%, and the total spore yield is only 1.2 × 10⁻⁶. 9 The CFU / g concentration was less than 39% of the mean concentration in the example. At low temperatures, black soldier fly larvae exhibited slower metabolism, reduced food intake, and slower intestinal peristalsis, consequently decreasing the mass transfer and colonization efficiency of Bacillus in the intestine. Conversely, low humidity accelerated substrate moisture evaporation, causing the feed to dry and harden, making it difficult for larvae to feed and resulting in insufficient bacterial intake. The combined effect of these factors led to severely reduced larval viability and low propagation efficiency.

[0083] Comparative Example 4-2: The combined effect of heat stress and high humidity resulted in the worst outcome.

[0084] When the temperature rises to 32°C and the humidity reaches 80%, the larval mortality rate is as high as 28%, and the total spore yield is only 8.5 × 10⁻⁶. 8 The CFU / g concentration was less than 27% of the mean concentration in the example, making it the worst among all experimental groups. High temperature caused heat stress in the larvae, significantly reducing their survival rate; high humidity led to condensation on the substrate surface, promoting the growth of anaerobic bacteria and further interfering with the normal propagation of Bacillus. The combined effect of these two factors resulted in the worst propagation outcome.

[0085] Comparative Example 4-3: Even a single factor below the range can lead to degradation.

[0086] When the temperature drops to 25°C and the humidity is maintained at 70% (within the acceptable range), the larval mortality rate is 14% and the total spore production is 1.8 × 10⁻⁶. 9 Although the CFU / g was better than that of comparative examples 4-1 and 4-2, where both parameters deviated, it was still significantly worse than the example group (mortality rate ≤5%, yield ≥2.8×10⁻⁶). 9 (CFU / g). This result indicates that even if the humidity meets the requirements, a single instance of low temperature can lead to a decrease in larval feeding and a slowdown in intestinal metabolism, ultimately affecting propagation efficiency. Furthermore, a comparison between Comparative Example 4-3 and Comparative Example 4-1 further reveals that temperature deviation (25°C) is the dominant factor causing the deterioration of propagation performance; a decrease in humidity from 70% to 55% further exacerbates the decline in yield and the increase in mortality.

[0087] When the temperature was 27°C to 30°C and the humidity was 65% to 75%, all three examples exhibited excellent overall performance: larval mortality rate ≤5% and total spore yield ≥2.8×10⁻⁶. 9 CFU / g. Example 4-2 showed the best results, with a larval mortality rate of only 4% and a total spore yield of 3.1 × 10⁻⁶. 9CFU / g. This temperature and humidity range balances larval survival rate with efficient germination and amplification of Bacillus in the gut, making it the optimal parameter range for ensuring high propagation efficiency.

[0088] It is understandable that black soldier fly larvae and Bacillus subtilis each have their own optimal temperature ranges for growth. While there is some overlap, they are not entirely identical. If the temperature is too low, the germination and proliferation rate of Bacillus subtilis is limited; if the temperature is too high, the larvae experience heat stress, leading to a decrease in survival rate and feed intake. The temperature range selected in this embodiment is the intersection of the two optimal temperature ranges. Within this range, both larval survival rate and feeding activity are ensured, while also meeting the needs of efficient germination and proliferation of Bacillus subtilis in the gut. The relative humidity balances the goals of maintaining substrate moisture (preventing feed from drying out and affecting feeding) and inhibiting the growth of miscellaneous bacteria (excessive humidity easily leads to the growth of mold and anaerobic bacteria). This combination of temperature and humidity is the optimal parameter verified in the experiments of this invention, achieving a gut colonization rate of over 92% for functional Bacillus subtilis and a total spore yield of 3.1 × 10⁻⁶. 9 CFU / g.

[0089] Step S4: Take 10g of the live larvae and feces mixture sample, add 90mL of sterile physiological saline, and homogenize in a beater homogenizer at a beating speed of 7m / s for 60s to obtain 10 -1 The diluent was serially diluted and spread onto tryptone soybean broth selective medium containing 0.1 mg / mL neomycin for the isolation and counting of Bacillus spores, and the functional Bacillus products were collected.

[0090] Specifically, in step S4, the selective culture medium is tryptone soybean broth medium containing 0.1 mg / mL neomycin.

[0091] In this embodiment, the selective culture medium in step S4 is tryptone soybean broth medium containing 0.1 mg / mL neomycin. Neomycin, as an aminocoumarin antibiotic, can specifically inhibit the activity of bacterial DNA gyrase (GyrB subunit), thereby blocking DNA replication of Gram-negative bacteria (such as Escherichia coli and Pseudomonas) and some Gram-positive bacteria (such as Staphylococcus). However, it has low affinity for the DNA gyrase structure of Bacillus spp., and has no significant inhibitory activity against Bacillus at a concentration of 0.1 mg / mL (minimum inhibitory concentration MIC > 100 μg / mL). Neomycin was added to the basal tryptone soybean broth (TSB) medium to a final concentration of 0.1 mg / mL, sterilized, and then plated. The serially diluted samples were spread on the surface of the plates and incubated at 37°C for 24-48 hours. Typical colonies were then picked. By utilizing the selective inhibitory effect of neomycin, the growth of indigenous Gram-negative bacteria (such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) present in large quantities in the insect excrement mixture is effectively inhibited during the plate culture stage, enabling the target Bacillus to be identified and isolated in the form of single colonies.

[0092] Understandably, the mixture of insect excrement not only contains the target functional Bacillus, but also carries a large number of Gram-negative indigenous microorganisms native to the gut of black soldier fly larvae. If ordinary nutrient agar medium is used for isolation and culture, these indigenous bacteria will compete with the Bacillus for nutrients and growth space on the plate, forming a large number of mixed bacterial colonies that cover the target colonies, resulting in a selection accuracy of only 55% to 65%. If no selectant is added before isolation and the judgment is made solely based on morphological characteristics, it is very easy to misidentify morphologically similar mixed bacteria as Bacillus. This invention adds 0.1 mg / mL neomycin to TSB medium. This concentration is sufficient to inhibit the growth of most Gram-negative bacteria (MIC of 0.5–2 μg / mL), while the MIC for Bacillus spp. is >100 μg / mL. The difference between the two is 50–200 times, forming a wide selection window. This allows Bacillus spp. to grow dominantly on the plate and form typical colonies (round, white to pale yellow, wrinkled surface, irregular edges). The detection accuracy reaches 96% and the purity reaches 98%, achieving efficient isolation and purification of target functional Bacillus spp. from complex insect excrement matrix.

[0093] Example: Verification of the range of values ​​for various process parameters and experiment on the effect of product feed additives

[0094] To verify the practical application effect of the functional Bacillus products collected in this invention, a feed additive feeding trial was conducted using broiler chickens as an animal model. By detecting broiler growth performance (feed conversion ratio, FCR) and intestinal health indicators (ileal VH / CD ratio, fecal Clostridium perfringens count), the effectiveness of the products of this invention as feed additives in improving animal intestinal health and feed conversion efficiency was verified. Furthermore, by comparing the differences in feeding effects of products prepared with different combinations of process parameters, the rationality of the range of values ​​for each process parameter in this invention was verified.

[0095] A total of 864 healthy AA broiler chickens aged 1 day were randomly divided into 8 groups (implementation groups 1-3, control groups 1-5, and blank control group), with 6 replicates per group and 18 chickens per replicate. The experiment lasted for 42 days. Each implementation and control group received 0.1% (w / w) of the corresponding functional Bacillus product in their basal diet, while the blank control group received no product.

[0096] After the experiment, the feed conversion ratio (FCR) of each group was calculated, and the ratio of ileal villus height to crypt depth (VH / CD) and the number of Clostridium perfringens in the feces were measured.

[0097] Group 1: S1 contained 0.8% sodium acetate, 1.5% ethanol, and ≥0.5% lactic acid; S2 contained strains in a 1:1:1 ratio, derived from commercially available strains; S3 contained culture temperature of 27°C and humidity of 65%; and S4 contained neomycin TSB in the culture medium.

[0098] Implementation Group 2: In S1, sodium acetate was 1.0% and ethanol was 2.0%; in S3, the culture temperature was 28.5℃ and the humidity was 70%; other parameters were the same as in Implementation Group 1.

[0099] Implementation Group 3: In S1, sodium acetate was 1.2% and ethanol was 2.5%. In S3, the culture temperature was 30℃ and the humidity was 75%. Other parameters were the same as in Implementation Group 1.

[0100] Control group 1: S1 contained 0.5% sodium acetate, 1.0% ethanol, and <0.5% lactic acid; S2 contained strains in a 1:1:1 ratio, derived from commercially available strains; S3 contained strains in a culture temperature of 25℃ and a humidity of 55%; and S4 contained neomycin TSB in the culture medium.

[0101] Control group 2: S1 contained 1.5% sodium acetate, 3.0% ethanol, and ≥0.5% lactic acid; S2 contained strains in a 1:1:1 ratio, derived from commercially available strains; S3 contained strains in a culture medium at 35℃ and 85% humidity; and S4 contained neomycin TSB.

[0102] Control group 3: All parameters are the same as in control group 2. In S4, ordinary TSB medium without neomycin was used for isolation and counting.

[0103] Comparative group 4: All parameters are the same as in implementation group 2. In S2, the mixing ratio of Bacillus subtilis: Bacillus licheniformis: Bacillus pumilus spore suspension is 2:1:1.

[0104] The results are shown in the table below:

[0105] Table 5: Effects of different combinations of process parameters on broiler growth performance and gut health

[0106]

[0107] Note: In the same column, # indicates a significant difference compared to implementation group 2 (p<0.05); This indicates a significant difference compared to the blank control group (p<0.05).

[0108] like Figure 4 As shown, implementation group 2 (with the median value of each parameter) has the lowest FCR (2.06) and the highest VH / CD (3.58), and its overall performance is the best among all groups, confirming that taking the median value of each parameter at the same time is the optimal configuration scheme.

[0109] Results and Analysis

[0110] (i) All implementation groups were significantly better than the blank control group, confirming the effectiveness of the technical solution of the present invention.

[0111] The FCR (2.06–2.10) of all three experimental groups was significantly lower than that of the blank control group (2.23, P<0.05), the VH / CD (3.42–3.58) was significantly higher than that of the blank control group (2.85, P<0.05), and the Clostridium perfringens concentration (3.2–3.8 log CFU / g) was significantly lower than that of the blank control group (5.2 log CFU / g, P<0.05). This indicates that the products prepared within the defined ranges of the parameters of this invention can effectively improve the growth performance and intestinal health of broilers, confirming the effectiveness of the entire technical solution of this invention.

[0112] (ii) The optimal solution is the combination within the range of values.

[0113] In Group 2, the FCR was 2.06, VH / CD was 3.58, and Clostridium perfringens concentration was 3.2 log CFU / g, all three indicators being the best among all groups. Specifically, the FCR decreased by 0.17 (a 7.6% decrease) compared to the blank control group, VH / CD increased from 2.85 to 3.58 (a 25.6% increase), and Clostridium perfringens concentration decreased by 2.0 log units, confirming that the combination within the specified range represents the optimal configuration for each parameter.

[0114] Although the indicators of Experiment Group 1 (all lower limits) and Experiment Group 3 (all upper limits) were slightly inferior to those of Experiment Group 2, they were significantly better than those of the blank control group (P<0.05). Specifically, the FCR of Experiment Group 1 and Experiment Group 3 were 2.10 and 2.09, respectively; VH / CD were 3.42 and 3.45, respectively; and Clostridium perfringens were 3.8 and 3.7 log CFU / g, respectively. Both indicators were close to those of Experiment Group 2, and the differences in FCR and Clostridium perfringens values ​​did not exceed 0.05 and 0.6 log units, respectively. Experiment Group 2 performed best, indicating that when all parameters are taken at the median value, the host viability and bacterial population expansion reach the best balance. While the combination of lower or upper limits can still ensure good propagation effect, the overall synergistic effect is slightly weakened because one parameter is at the boundary value. This result confirms that the median combination is the optimal configuration of all parameters, and also shows that the parameter range defined in this invention has a wide process adaptability. Even if all parameters deviate from the median value at the same time, the product can still maintain excellent feed additive effects.

[0115] (iii) All parameters being below the lower limit or above the upper limit will lead to a significant deterioration in the product effect.

[0116] The FCR (2.19, 2.20), VH / CD (3.02, 2.95), and Clostridium perfringens (4.8, 4.9 log CFU / g) of control groups 1 and 2 were significantly worse than those of the corresponding example groups (P<0.05), and even approached the levels of the blank control group (P>0.05). This indicates that whether the parameters are too low or too high, they will disrupt the effect of the complete technical route of substrate sterilization-live propagation-product collection, leading to serious deterioration of product quality.

[0117] (iv) Deviations in mixing ratio and separation method both lead to a decrease in effectiveness.

[0118] Although the indicators of control groups 3 and 4 were better than those of the blank control group (P<0.05), they were significantly worse than those of control group 2 (P<0.05). Control group 3 (FCR 2.17, VH / CD 3.15, Clostridium perfringens 4.5 log CFU / g) showed the worst results, indicating that the ordinary culture medium could not effectively inhibit indigenous bacteria, resulting in insufficient product purity. This confirms that the 1:1:1 mixing ratio in S2 and the selective medium in S4 are necessary conditions for achieving excellent results.

[0119] (V) Overall Conclusion

[0120] The process parameters of this invention are within their respective defined ranges (sodium acetate 0.8%–1.2%, ethanol 1.5%–2.5%, anaerobic fermentation 30°C–35°C for 24–48 hours, lactic acid ≥0.5%, strain mixing ratio 1:1:1, inoculum size 10). 4 ~10 5A product with excellent feed additive effects can be prepared using CFU / g, larval density of 1.5–2.5 larvae / g, rearing temperature of 27°C–30°C, and humidity of 65%–75%. The optimal effect is achieved when all parameters are at their median values. When all parameters are below the lower limit or above the upper limit, the product's application effect is severely degraded; a mixing ratio deviating from 1:1:1 or the absence of a selective culture medium also leads to a significant decrease in effect. This confirms the rationality and non-obviousness of the selection of parameter ranges in this invention.

[0121] Explanation of the overall beneficial effects:

[0122] (1) Sterilization treatment of fermentation substrate: The fermentation substrate of black soldier fly larvae was treated with sodium acetate solution and ethanol solution in sequence, and then anaerobic fermentation was carried out. The combination of the two increases the killing rate of native microorganisms, eliminating competitive interference for subsequent intestinal colonization of functional spores; anaerobic fermentation converts residual sodium acetate into lactic acid, which plays a dual role of secondary antibacterial and substrate pre-digestion.

[0123] (2) Tri-strain combination: Spore suspensions of Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus were mixed in equal proportions. The three functional Bacillus strains formed a metabolically complementary synergistic network in the gut of black soldier fly larvae. The synergistic effect of the three strains increased the total spore yield by more than double compared to the single-strain scheme, reaching 2.8 × 10⁻⁶. 9 ~3.2×10 9 CFU / g.

[0124] (3) Live propagation of black soldier fly larvae: A compound inoculum of functional Bacillus was inoculated into a sterilized substrate at a low inoculation rate. Black soldier fly larvae were then inoculated and live propagation was carried out at 27°C to 30°C and 65% to 75% relative humidity. After the functional Bacillus entered the digestive tract with the larvae, it utilized the organic matter in the substrate to amplify its biomass, with an amplification factor of up to 10. 2 ~10 3 The total spore yield reached 2.8 × 10⁻⁶. 9 ~3.1×10 9 CFU / g, significantly better than the 10 CFU / g of traditional liquid fermentation processes. 8 ~10 9 (CFU / mL).

[0125] (4) Collection of insect excrement inoculum: Live larvae and insect excrement mixtures were mixed with sterile physiological saline and homogenized. After serial dilution, the mixtures were spread on selective medium containing neomycin for separation and counting. Neomycin can effectively inhibit the growth of indigenous Gram-negative bacteria and some Gram-positive bacteria in insect excrement, but has no inhibitory effect on Bacillus, and can accurately detect Bacillus. As a natural inoculum carrier, the loose and porous structure of insect excrement is conducive to the habitat of Bacillus. At the same time, the organic acids and enzyme residues in insect excrement have a protective effect on spores, making the product significantly more resistant to gastric acid and bile salts than traditional liquid fermentation products.

[0126] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A method for preparing functional Bacillus based on live propagation of black soldier fly larvae, characterized in that, Includes the following steps: Step S1: The fermentation substrate of black soldier fly larvae is sterilized by sequentially using sodium acetate solution and ethanol solution to kill the indigenous microorganisms in the fermentation substrate, and then subjected to anaerobic fermentation to eliminate residual sodium acetate and ethanol to obtain sterilized substrate. Step S2: Culture Bacillus subtilis separately Bacillus subtilis Bacillus licheniformis Bacillus licheniformis and Bacillus pumilus Bacillus pumilus The spore suspensions of three Bacillus strains were mixed to obtain a functional Bacillus compound bacterial agent; Step S3: Inoculate the functional Bacillus compound bacterial agent into the sterilized matrix, mix well, transfer to the black soldier fly larvae breeding box, inoculate black soldier fly larvae, carry out live propagation treatment, and obtain a mixture of live larvae and insect excrement. Step S4: Take the live larvae and larvae mixture and mix it with sterile physiological saline for homogenization to obtain a diluted solution. Perform serial dilution on the diluted solution and spread it on a selective culture medium for Bacillus isolation and counting, and collect the functional Bacillus products.

2. The method for preparing functional Bacillus based on live black soldier fly propagation according to claim 1, characterized in that, In step S1, the sodium acetate solution has a mass fraction of 0.8% to 1.2%, and the ethanol solution has a volume fraction of 1.5% to 2.5%.

3. The method for preparing functional Bacillus based on live propagation of black soldier fly larvae according to claim 1, characterized in that, In step S1, the anaerobic fermentation treatment is carried out at a temperature of 30℃~35℃ for 24h~48h, and the lactic acid concentration in the sterilized substrate after anaerobic fermentation is ≥0.5%.

4. The method for preparing functional Bacillus based on live propagation of black soldier fly larvae according to claim 1, characterized in that, In step S1, the sterilization treatment and the anaerobic fermentation treatment reduce the residual amount of indigenous microorganisms in the sterilized substrate to less than or equal to 10. 2 CFU / g.

5. The method for preparing functional Bacillus spores based on live propagation of black soldier fly larvae according to claim 1, characterized in that, In step S2, the Bacillus subtilis is Bacillus subtilis The Bacillus licheniformis described is, with accession number ATCC 21332. Bacillus licheniformis The Bacillus pumilus described is, with accession number ATCC 14580. Bacillus pumilus The accession number is ATCC 7061.

6. The method for preparing functional Bacillus based on live propagation of black soldier fly larvae according to claim 1, characterized in that, In step S2, the spore suspensions of Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus are mixed in a volume ratio or mass ratio of 1:1:

1.

7. The method for preparing functional Bacillus based on live propagation of black soldier fly larvae according to claim 1, characterized in that, In step S3, the functional Bacillus compound bacterial agent is prepared at 10... 4 CFU / g ~ 10 5 An inoculum of CFU / g is introduced into the sterilization matrix.

8. The method for preparing functional Bacillus based on live propagation of black soldier fly larvae according to claim 1, characterized in that, In step S3, the inoculation density of the black soldier fly larvae is 1.5 to 2.5 larvae per gram of the sterilized substrate.

9. The method for preparing functional Bacillus based on live propagation of black soldier fly larvae according to claim 1, characterized in that, In step S3, the culture temperature for the live propagation treatment is 27℃~30℃, and the relative humidity is 65%~75%.

10. The method for preparing functional Bacillus based on live propagation of black soldier fly larvae according to claim 1, characterized in that, In step S4, the selective culture medium is tryptone soybean broth medium containing 0.1 mg / mL neomycin.

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