Plant biostimulants, methods of making and using the same

Abstract

The application provides a plant biological stimulant and a preparation method and application thereof, relates to the fields of agricultural biotechnology, organic fertilizer and plant biological stimulant, and the method comprises the following steps: mixing black soldier fly defatted insect powder, pupal shell powder, molasses, specific inorganic salt and water, carrying out pretreatment through super-micro wet crushing, and carrying out heat preservation sterilization; the sterilized slurry is divided into two parts at a ratio of 8:2; 80% of the first fermentation liquor is inoculated with bacillus velezensis for fermentation, pH is adjusted, and then the temperature is increased to 55-60 DEG C for primary autolysis; then 20% of the fermented aspergillus fermentation liquor is introduced for further synergistic secondary enzymolysis at 55-60 DEG C, and then enzyme inactivation is carried out rapidly at 80 DEG C, and the filtrate A and the residue A are collected by filtration; the residue A is resuspended and inoculated with trichoderma harzianum for fermentation and chitin enzymolysis, and the filtrate B is obtained by separation; the filtrate A and the filtrate B are mixed to obtain the plant biological stimulant. The process is scientific and complete in degradation, and realizes the full-amount waste-free high-value utilization of black soldier fly by-products.

Description

Technical Field

[0001] This invention relates to the fields of agricultural biotechnology, organic fertilizers, and plant biostimulants, specifically to a method and its application for preparing plant biostimulants rich in amino acids, small peptides, chitosan, and abundant trace elements through multi-stage fermentation and enzymatic hydrolysis of black soldier fly defatted insect powder and pupal shell powder. Background Technology

[0002] Plant biostimulants are substances and / or microorganisms applied to plants or their rhizosphere. Their function is to stimulate the plant's natural physiological metabolic processes, improve the efficiency of nutrient absorption and utilization, enhance the plant's resistance to abiotic stresses such as drought, waterlogging, salinity, and extreme temperatures, and significantly improve the quality of agricultural products. Compared with traditional chemical fertilizers and pesticides, biostimulants have significant advantages such as lower dosage, safety, environmental friendliness, and no residue, aligning with the development trend of modern green and low-carbon agriculture.

[0003] Black soldier flies (Hermetia illucens L.) are environmental insects that efficiently transform organic waste such as livestock manure and kitchen waste into high-value insect biomass. In the resource utilization of black soldier flies, defatted insect powder can be obtained through a pressing and defatting process. Typical defatted insect powder contains approximately 49% protein, 17% oil, and 8% chitin. Furthermore, the pupal shells left after black soldier flies emerge (mainly composed of pupal shell powder) are also a valuable waste resource rich in chitin (over 30%-40%) and minerals such as calcium.

[0004] Black soldier fly larvae defatting powder contains proteins rich in various essential amino acids in a balanced ratio. Chitin and its deacetylated products, chitosan and chitosan oligosaccharides, are recognized as potent plant immune inducers, capable of inducing systemic resistance (SAR) in plants and promoting root development. However, the presence of residual oil (up to 17%) and high-molecular-weight chitin in the defatting powder presents the following technical bottlenecks when applied directly:

[0005] 1. Difficult to absorb directly: Chitin has a highly crystalline, insoluble, sheet-like structure, which plants cannot absorb directly, and it degrades very slowly in the soil. 2. Fat hinders enzymatic hydrolysis: Up to 17% fat can easily form a hydrophobic barrier, encapsulating proteins and chitin, severely hindering the contact and degradation of proteins and chitin by conventional hydrolytic enzymes; 3. Traditional methods are highly polluting: Traditional methods for extracting chitosan and amino acids usually use strong acid and strong alkali at high temperatures, which not only pollute the environment but also destroy heat-sensitive active peptides and amino acids.

[0006] To address the aforementioned challenges, conventional bio-fermentation typically suffers from limitations such as low efficiency and slow strain adaptation. Defatted insect powder and pupal shell powder have relatively large particles with high lignification and crystallinity; without efficient mechanical crushing and pretreatment, microorganisms and enzymes cannot quickly penetrate them. Furthermore, microorganisms exhibit a strong dependence on trace elements (such as zinc, magnesium, and calcium) during fermentation and proteolysis (for example, many highly efficient proteases and chitinases are metal-dependent enzymes), while ordinary culture media lack these cofactors, limiting the release of extracellular enzyme activity.

[0007] Therefore, how to combine advanced mechanical crushing, refined inorganic salt synergistic formulation, and highly industrially operable temperature-controlled stepwise fermentation and autolytic enzymatic hydrolysis processes to completely convert the proteins in black soldier fly defatted insect powder and pupal shell powder into peptides and amino acids, while efficiently degrading chitin into highly bioactive chitosan and chitosan oligosaccharides, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0008] In view of this, this application provides a plant biostimulant, its preparation method, and its application. This method creatively introduces a specific inorganic salt system into the raw materials, and combines it with a colloid mill ultrafine grinding pretreatment and a temperature-controlled stepwise fermentation enzymatic hydrolysis process (80% of Bacillus belye fermentation broth is neutralized and subjected to primary enzymatic hydrolysis at 55-60℃, then combined with 20% Aspergillus fermentation broth for secondary enzymatic hydrolysis, and finally inactivated at 80℃ in synergistic fermentation with two-stage filter residue Trichoderma harzianum). This effectively solves the problem of residual oil in black soldier fly defatted insect powder hindering enzymatic hydrolysis, achieving efficient protein release and small peptide formation, amino acid enzymatic hydrolysis, and completely overcoming the technical bottleneck of the difficulty in degrading high molecular weight chitin and pupal shell chitin. It yields a plant biostimulant rich in amino acids, highly active small peptides, soluble chitosan / chitosan oligosaccharide, and mineral elements, realizing the full-volume, waste-free, and high-value utilization of black soldier fly processing by-products.

[0009] This application provides the following technical solution: a method for preparing plant biostimulants using black soldier fly defatted insect powder and pupal shell powder, comprising the following steps: S1. Preparation and pretreatment of multi-component fermentation substrate: By weight, mix 80-120 parts of black soldier fly defatted insect powder, 10-30 parts of pupal shell powder, 20-50 parts of molasses, 1-3 parts of ammonium sulfate, 0.5-1.5 parts of magnesium sulfate, 1-4 parts of potassium dihydrogen phosphate, 0.1-0.5 parts of zinc sulfate, and 300-600 parts of water evenly, and adjust the pH of the mixture to 6.0-7.5; the evenly mixed material is first subjected to wet ultra-fine grinding, then heated to 90-95℃ and kept at that temperature for 30-60 minutes for sterilization, and after cooling, a sterile fermentation substrate is obtained; The black soldier fly defatted insect powder contains, by weight percentage, 45%-52% protein, 12%-20% oil and 6%-10% chitin; S2. First-stage split fermentation and two-stage enzymatic hydrolysis: S21. Split inoculation: The sterile fermentation substrate is divided into a first fermentation broth and a second fermentation broth at a mass ratio or volume ratio of 8:2; Bacillus velezensis is inoculated into 80% of the total mass of the first fermentation broth, and aerobic fermentation is carried out at a temperature of 30-38℃ for 18-72 hours, preferably 24 hours; Aspergillus is inoculated into 20% of the total mass of the second fermentation broth, and aerobic fermentation is carried out at a temperature of 28-32℃; S22. Primary autolysis and enzymatic hydrolysis: Add saturated lime milk to the first fermentation broth after fermentation, adjust the pH value to 6.5-7.0, and then heat to 55-60℃ for incubation autolysis and primary enzymatic hydrolysis. The enzymatic hydrolysis time is 6-12 hours, preferably 8 hours. S23. Secondary synergistic enzymatic hydrolysis: After the primary autolytic enzymatic hydrolysis is completed, the fermented second fermentation broth is added to the first fermentation broth, and secondary enzymatic hydrolysis is continued at a temperature of 55-60℃ for 6-12 hours, preferably 8 hours; S24. Inactivation and separation: After the secondary enzymatic hydrolysis is completed, the mixed fermentation broth is heated to 80℃ for enzyme inactivation and sterilization treatment for 30-45 minutes, followed by solid-liquid separation, and the first-stage fermentation filtrate and the first-stage fermentation residue are collected separately. S3. Second stage fermentation and enzymatic hydrolysis: The first-stage fermentation residue was mixed with water at a weight ratio of 1:(3-6) and resuspended. The pH of the mixture was adjusted to 4.5-6.0. Trichoderma harzianum was inoculated and fermented and enzymatically hydrolyzed at a temperature of 25-30℃ for 96-168 hours. After fermentation, solid-liquid separation was performed, and the second-stage fermentation filtrate was collected. S4. Compound preparation: The first stage fermentation filtrate and the second stage fermentation filtrate are mixed at a volume ratio of (1.5-3):1, and homogenized, concentrated or dried to obtain a plant biostimulant rich in amino acids, small peptides and chitosan, and containing abundant trace elements.

[0010] As a preferred embodiment of the present invention, in step S1, the black soldier fly defatted insect powder contains 49% protein, 17% oil and 8% chitin by weight percentage.

[0011] As a preferred embodiment of the present invention, the scientific basis and function of adding inorganic salts in step S1 are as follows: Ammonium sulfate: provides an inorganic ammonium source for rapid absorption by Bacillus belye and Aspergillus in the early stage of fermentation, shortens the fermentation lag phase, and promotes their rapid entry into the logarithmic growth phase and secretion of extracellular hydrolases; Magnesium sulfate: Magnesium ions (Mg) 2+ It is an essential cofactor for a variety of kinases and nucleic acid hydrolases in microorganisms, which helps stabilize the ribosome structure of the cells and significantly improves fermentation efficiency; Potassium dihydrogen phosphate: provides plants with essential phosphorus and potassium nutrients, and at the same time plays an excellent pH buffering role during fermentation, stabilizing the microbial microenvironment; Zinc sulfate: Zinc ions (Zn) 2+ Zinc is an essential component of the active sites of many metalloneutral / alkaline proteases secreted by Bacillus and Aspergillus. Exogenous zinc supplementation can greatly activate the catalytic efficiency of proteolytic enzymes, and zinc is also an important trace element in plants.

[0012] As a preferred embodiment of the present invention, in step S1, the uniformly mixed material is first subjected to wet ultrafine pulverization using a colloid mill. The gap between the grinding teeth of the colloid mill is controlled at 10-30 μm. Through high-speed shearing and grinding, the fibrin in the insect powder and the hard chitinous skeleton in the pupal shell are subjected to strong physical destruction, resulting in a median particle size D50 ≤ 40 μm. The ultrafine particle size greatly increases the contact area between the subsequent enzymes and the matrix, increasing the autolytic hydrolysis rate by more than double.

[0013] As a preferred embodiment of the present invention, in step S21, the inoculation amount of Bacillus velezensis is 2%-5% of the total mass of the first fermentation broth, and the effective viable count of the bacterial solution is ≥1.0×10⁻⁶. 9 CFU / mL; the inoculated Aspergillus is either Aspergillus oryzae or Aspergillus niger, and the inoculation amount is 2%-5% of the total mass of the second fermentation broth, with a spore suspension concentration ≥1.0×10⁻⁶. 7 The number of cells / mL and the aerobic fermentation time of the second fermentation broth is 24-48 hours.

[0014] Bacillus berreatus rapidly proliferates in the first fermentation broth (80% proportion) for 24 hours, using molasses and defatted insect powder protein to rapidly produce a large amount of alkaline / neutral protease and natural lipopeptide surfactants that can efficiently emulsify 17% of residual oil. Aspergillus ferments in the second fermentation broth (20% of the total volume) for 24-48 hours, specifically accumulating highly active saccharifying enzymes, acidic proteases, peptidases, cellulases, and preliminary chitin-degrading enzymes.

[0015] As a preferred embodiment of the present invention, in step S22, saturated lime milk (i.e., calcium hydroxide suspension) is used to adjust the pH to 6.5-7.0.

[0016] Compared to conventional strong alkalis (such as NaOH), the introduction of saturated lime slurry has extremely high technical rationality: First, calcium ions (Ca... 2+ It is an excellent heat stabilizer and activator for neutral / alkaline proteases secreted by Bacillus belyssus and other organisms. The introduction of calcium ions can prevent the rapid thermal inactivation of proteases during the subsequent high-temperature enzymatic hydrolysis process at 55-60℃, and significantly prolong the half-life of the enzyme. Secondly, it avoids the introduction of excess sodium ions into the biostimulant (high sodium can easily lead to soil compaction and salinization, which can damage plants), while calcium ions are an essential medium-level nutrient element for plants, which can enhance the cell wall strength and disease resistance of crops.

[0017] In a preferred embodiment of the present invention, in step S22, the temperature of the primary autolytic hydrolysis is controlled at 55-60°C (preferably 58°C), and the hydrolysis time is 8 hours. At this temperature, the vegetative cells of *Bacillus belyssus* are inactivated by thermal shock and undergo autolysis, releasing their intracellular coenzymes and beneficial metabolites. Simultaneously, the extracellular thermostable proteases secreted by the *Bacillus belyssus* are released into the atmosphere. 2+ With the aid of hydrolysis, insect powder protein is subjected to extremely vigorous hydrolysis, which releases a large number of active peptides in a short period of time.

[0018] In a preferred embodiment of the present invention, in step S23, 20% of the Aspergillus fermentation broth is combined and introduced into the first fermentation broth at 55-60°C, and secondary synergistic enzymatic hydrolysis is continued at 55-60°C for 8 hours. The complementary enzyme system provided by Aspergillus (such as peptidases that further cleave polypeptides into short peptides and free amino acids, and chitin-degrading enzymes that initially open chitin rings) synergistically enhances the Bacillus enzyme system at 55-60°C, achieving an ultra-high protein and small peptide conversion rate that cannot be achieved by single fermentation.

[0019] As a preferred embodiment of the present invention, in step S24, the temperature is raised to 80°C and maintained for 30-45 minutes to achieve complete enzyme inactivation and completely terminate the reaction. At the same time, the fermentation broth is pasteurized, which greatly improves the shelf-life stability of the product.

[0020] As a preferred embodiment of the present invention, in step S3, the inoculated *Trichoderma harzianum* is a suspension of *Trichoderma harzianum* spores, and the inoculation amount is 2%-6% of the total mass of the filter residue resuspension, with a spore suspension concentration ≥1.0×10⁻⁶. 7 Chitinase was added exogenously to the filter residue resuspension at the start of fermentation or within 24 hours of fermentation, at a rate of 50-200 U / g filter residue (based on dry weight).

[0021] The filter residue from the first-stage solid-liquid separation contains the majority of the recalcitrant chitin from the insect powder and pupal shells. Since the proteins have been largely removed by hydrolysis in the first stage (the filter residue mainly consists of chitin and a very small amount of resistant fibrin), the inoculation of *Trichoderma harzianum* in the second stage is not inhibited by high concentrations of soluble polypeptides / amino acids, thus allowing *Trichoderma harzianum* to use chitin as its sole / primary carbon source and secrete highly active chitinase and chitosanase in excess. This increases the chitin degradation rate in the pupal shells and insect powder to over 78%, completely solving the industrial problem of the extremely difficult degradation of lignified pupal shells.

[0022] As a preferred embodiment of the present invention, in step S4, after mixing the filtrates from the two stages, a refining process is further included: ultrasonic homogenization at 60-70°C for 20-30 minutes to promote the physical shearing of macromolecular peptides and the emulsification stability of the mixture.

[0023] This invention also provides a plant biostimulant prepared according to the above method, wherein the biostimulant contains ≥110 g / L of free amino acids, ≥55 g / L of active small peptides with a molecular weight less than 1000 Da, and ≥18 g / L of total water-soluble chitosan and chitosan oligosaccharides (molecular weight ≤3000 Da). Furthermore, the biostimulant prepared by this invention is rich in naturally chelated calcium, magnesium, zinc, potassium, and other nutrients, wherein the calcium ion content is ≥8 g / L, the magnesium ion content is ≥2 g / L, and the zinc ion content is ≥0.5 g / L.

[0024] As a preferred embodiment of the present invention, the plant biostimulant is in liquid form or solid powder form; wherein, when the plant biostimulant is in liquid form, it further contains 0.1%-0.5% by mass of preservative and 1%-5% by mass of plant growth regulator; when the plant biostimulant is in solid powder form, it is prepared by low-temperature vacuum concentration of a mixture of the first-stage fermentation filtrate and the second-stage fermentation filtrate, followed by spray drying.

[0025] This invention also provides the application of plant biostimulants in promoting crop growth, enhancing crop resistance to drought and salinity stress, and preventing soil-borne fungal diseases in crops.

[0026] Compared with the prior art, the beneficial effects that at least one technical solution adopted in the embodiments of this specification can achieve include at least: 1. An extremely rigorous synergistic technology system of "mechanical crushing - inorganic salt activation - cascade temperature control": a. For the first time, hard lignified pupal shells and defatted insect powder were pulverized to the micron level (D50≤40μm) using a colloid mill wet pulverization method, exposing the internal crystal structure; b. Scientifically added zinc, magnesium, and calcium inorganic salt cofactors. Zinc ions directly activate the protease catalytic center, and calcium ions introduced by saturated lime milk are highly stable and activate Bacillus protease at high temperatures of 55-60℃, maximizing the efficiency of primary and secondary enzymatic hydrolysis in the first stage. The release rate of peptides and free amino acids is increased by more than 180% compared to traditional room temperature mixed fermentation.

[0027] 2. Innovative 80%:20% split fermentation and segmented sequential enzymatic hydrolysis process: a. It avoids the drawback of excessive inhibition of Aspergillus by Bacillus due to differences in growth rates when multiple microorganisms are mixed. 80% of the slurry is used for the rapid proliferation of Bacillus to secrete large amounts of proteases and lipopeptides; 20% of the slurry is used for separate fermentation of Aspergillus to accumulate complementary enzyme systems.

[0028] b. By designing a sequence of "autolysis at 55-60℃ for 8 hours of primary enzymatic hydrolysis + introduction of Aspergillus solution for synergistic secondary enzymatic hydrolysis for 8 hours", the reaction not only utilizes the high-temperature autolysis to decompose the bacterial protein, but also fully leverages the synergistic degradation effect of the bacterial and fungal complex enzyme system at the optimal temperature. Finally, the reaction is rapidly inactivated at 80℃, ensuring thoroughness and ease of industrial control.

[0029] 3. The two-stage "division of labor" greatly improves the conversion rate of chitin: a. After the protein was completely removed in the first stage, Trichoderma harzianum was inoculated in the second stage. This completely eliminated the feedback inhibition of the chitin-degrading enzyme system of Trichoderma harzianum by the high concentration of soluble carbon / nitrogen sources (i.e., the carbon metabolite repression was relieved), which enabled Trichoderma harzianum to "target" and specifically degrade the crystalline chitin in the pupal shell and insect powder residue, with a degradation rate of over 80%, and obtained a high concentration of natural chitosan and chitosan oligosaccharides.

[0030] 4. The product has significant multifunctionality and is rich in trace elements: a. The addition of saturated lime milk with zinc sulfate and magnesium sulfate makes the final product not only contain highly active small peptides, amino acids and chitosan, but also naturally contain chelated calcium, magnesium, zinc, potassium and other trace elements that are easily absorbed by plants. It has multiple biostimulation functions such as promoting root growth, drought resistance, salt and alkali resistance and rhizosphere disease prevention. Detailed Implementation

[0031] The embodiments of this application are described in detail below.

[0032] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. This application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0033] The sources of the raw materials and strains used in the embodiments of this invention are as follows: 1. Black soldier fly defatted insect powder: Analysis of main nutritional components (mass fraction): protein 49.2%, oil 17.1%, chitin 8.3%, moisture 6.2%, ash 8%.

[0034] 2. Black soldier fly pupa shell powder: Collect empty pupa shells after black soldier flies emerge from their larvae, rinse them with water to remove attached mud and impurities, dry them at 60℃ to a moisture content of 8%, and pre-crush them using an ultra-fine pulverizer.

[0035] 3. Sugarcane molasses: Total sugar content 52.4%, moisture content 22.1%.

[0036] 4. Inorganic salts: industrial grade ammonium sulfate, magnesium sulfate, potassium dihydrogen phosphate, zinc sulfate.

[0037] 5. Strains: a. Bacillus velezensis, colony count 2.0 × 10⁻⁶ 10 CFU / g can be purchased through commercial channels, such as from Henan Jiubang Biotechnology Co., Ltd. b. *Aspergillus oryzae*, spore count 1.5 × 10⁻⁶ 10 Spores / g can be purchased through commercial channels, such as from Shandong Hezhong Kangyuan Biotechnology Co., Ltd. c. *Aspergillus niger*, spore count 1.2 × 10⁻⁶ 10 Spores / g can be purchased through commercial channels, such as from Shandong Hezhong Kangyuan Biotechnology Co., Ltd. d. *Trichoderma harzianum*, spore count 2.5 × 10⁻⁶ 10Spores / g can be purchased through commercial channels, such as from Shandong Hezhong Kangyuan Biotechnology Co., Ltd.

[0038] This invention discloses a method for preparing plant biostimulants using black soldier fly defatted insect powder and pupal shell powder, and its application. The method of this invention includes: 1. Black soldier fly defatted insect powder, pupal shell powder, molasses, and specific inorganic salts (ammonium sulfate, magnesium sulfate, potassium dihydrogen phosphate, zinc sulfate) and water are mixed and pretreated by ultra-fine wet grinding in a colloid mill, and then sterilized at 90-95℃. 2. First-stage splitting and two-stage enzymatic hydrolysis: The sterilized slurry was split at an 8:2 ratio. 80% of the first fermentation broth was inoculated with Bacillus belye and fermented for 24 hours. The pH was adjusted to 6.5-7.0 using saturated lime milk, and the temperature was raised to 55-60℃ for 8 hours for primary autolytic enzymatic hydrolysis (using calcium ions for heat-stable protection of proteases). Subsequently, the broth was combined with 20% of the fermented Aspergillus oryzae and continued to undergo secondary enzymatic hydrolysis at 55-60℃ for 8 hours. Then, the enzymes were rapidly inactivated at 80℃, and the filtrate A and filter residue A were collected by filtration. 3. Second stage fermentation: After resuspending the filter residue A, inoculate it with Trichoderma harzianum for enzymatic hydrolysis of chitin, and separate to obtain filtrate B; 4. Compound preparation: Filtrate A and filtrate B are compounded and mixed to obtain plant biostimulants rich in amino acids, highly active small peptides, water-soluble chitosan / chitosan oligosaccharides, and chelated calcium, magnesium, zinc, potassium and other elements.

[0039] Example 1: Preparation of plant biostimulants using Aspergillus oryzae and Bacillus belye The preparation process steps in this embodiment are as follows: S1. Preparation of multi-component fermentation substrate and ultrafine grinding and sterilization using colloid mill. 1. Weigh out the following ingredients according to the specified weight proportions: 100 kg of black soldier fly defatted insect powder, 20 kg of black soldier fly pupa shell powder, 30 kg of sugarcane molasses, 2 kg of ammonium sulfate, 1 kg of magnesium sulfate, 2.5 kg of potassium dihydrogen phosphate, and 0.3 kg of zinc sulfate.

[0040] 2. Add to 450 kg of water, stir thoroughly in a premixing tank, and adjust the pH of the mixed slurry to 7.0 using 1 mol / L KOH.

[0041] 3. Turn on the material pump and force the uniformly mixed slurry through a pipeline colloid mill for wet ultrafine grinding. Set the mill tooth gap to 15μm and perform two cycles of grinding. Take a sample and measure the median particle size D of the material. 50 It is 28μm.

[0042] 4. Pump the slurry treated by the colloid mill into the primary fermenter, heat it to 92°C and keep it at that temperature for 45 minutes for sterilization, and then cool it to 35°C to obtain a sterile fermentation substrate.

[0043] S2. First-stage split fermentation and two-stage enzymatic hydrolysis 1. Split inoculation: The sterile slurry after sterilization and cooling is divided into two parts at a mass ratio of 8:2: the first fermentation liquid (accounting for 80% of the total mass, i.e., about 484 kg) is pumped into fermenter A; the second fermentation liquid (accounting for 20% of the total mass, i.e., about 121 kg) is pumped into fermenter B.

[0044] a. Inoculate Bacillus belye seed culture (5.0 × 10⁻⁶ viable cells) into fermenter A (first fermentation broth). 9 The inoculum was 3% of the mass of the first fermentation broth (i.e., 14.5 kg). Aerobic fermentation was carried out for 24 hours at 36°C, 180 rpm stirring speed, and 0.8 vvm aeration rate.

[0045] b. Inoculate fermenter B (second fermentation broth) with a suspension of Aspergillus oryzae spores (spore count 2.0 × 10⁻⁶). 8 The inoculum size was 3% of the second fermentation broth mass (i.e., 3.6 kg). Aerobic fermentation was carried out at 30°C, 120 rpm, and 0.5 vvm for 36 hours to allow for the accumulation of abundant extracellular chitinase and acidic protease.

[0046] 2. Primary autolytic enzymatic hydrolysis: After fermentation in fermenter A (Bacillus belye) for 24 hours, saturated lime milk (calcium hydroxide suspension with a mass percentage concentration of 10%) is slowly pumped in to adjust the pH of the first fermentation broth to 6.8.

[0047] a. Then, quickly shut off the aeration, reduce the stirring speed to 100 rpm, and turn on the steam to raise the temperature of fermenter A to 58°C. Maintain this temperature for 8 hours for autolytic enzymatic hydrolysis. At this point, the Bacillus bacteria become inactive and rupture due to heat, and the extracellular, highly thermostable proteases it secretes undergo powerful hydrolysis of the insect meal proteins under the thermal stability protection of calcium ions.

[0048] 3. Secondary synergistic enzymatic hydrolysis: After 8 hours of primary autolytic enzymatic hydrolysis in fermenter A, the fermented Aspergillus oryzae broth (20% ratio, totaling approximately 124.6 kg) from fermenter B is pumped into fermenter A.

[0049] a. Maintain the temperature of fermenter A at 58℃ and continue the secondary synergistic enzymatic hydrolysis for 8 hours.

[0050] 4. Inactivation and separation: After the two-stage co-enzymatic hydrolysis is completed, the temperature of fermenter A is raised to 80°C and kept at this temperature for 40 minutes to inactivate the enzyme and terminate the hydrolysis reaction.

[0051] a. The inactivated liquid is separated into solid and liquid components by a plate and frame filter press to collect the first-stage fermentation filtrate (filtrate A, about 480 L) and wet filter residue (filter residue A, wet weight about 130 kg, with a measured moisture content of 55%).

[0052] S3. Second-stage fermentation and enzymatic hydrolysis 1. Put 130 kg of wet filter residue A (containing about 58.5 kg of dry matter) into another fermenter, add 300 kg of water and stir evenly (equivalent to a dry residue to water weight ratio of about 1:5.1), add 3 kg of sugarcane molasses as a carbon source for Trichoderma initiation, and use citric acid to adjust the pH of the mixture to 5.0.

[0053] 2. Inoculate the mixture with a suspension of Trichoderma harzianum spores (spore concentration 5.0 × 10⁻⁶). 8 The inoculum was 3% of the total mass of the suspension (i.e., 12.9 kg). Simultaneously, commercial chitinase was added exogenously at a rate of 100 U / g filter residue (dry weight, totaling 5,850,000 U).

[0054] 3. Control the fermentation temperature at 28℃, the stirring speed at 130 rpm, the aeration rate at 0.6 vvm, and continue fermentation and enzymatic hydrolysis for 120 hours. After fermentation, perform solid-liquid separation again by centrifugation and filtration, and collect the second-stage fermentation filtrate (filtrate B, approximately 310 L).

[0055] S4. Compounding and Refining The collected filtrate A and filtrate B were mixed at a volume ratio of 2:1 (360 L of filtrate A and 180 L of filtrate B), and homogenized using high-speed ultrasonic treatment in a mixing tank (temperature 65℃, ultrasonic power 1200 W, treatment time 25 minutes). After homogenization, 0.2% potassium sorbate was added as a preservative, and the mixture was concentrated to 1 / 3 of its original volume by vacuum evaporation at 55℃, yielding a dark brown liquid plant biostimulant product with a slightly acidic aroma (denoted as Stimulant-E1).

[0056] Example 2: Preparation of plant biostimulants using Aspergillus niger and Bacillus belye The steps in this embodiment are basically the same as in embodiment 1, with the only difference being: 1. In step S21, Aspergillus oryzae is replaced with the same inoculum of Aspergillus niger spore suspension; 2. No exogenous chitinase is added during the second stage of fermentation. The fermentation time is extended to 144 hours, relying entirely on the chitin-degrading enzyme system secreted by Trichoderma harzianum itself. 3. The remaining material ratios, inoculation amounts, and compounding and refining steps are completely consistent with those in Example 1. The resulting liquid plant biostimulant product is designated as Stimulant-E2.

[0057] Comparative Example 1: Conventional single-mixed aerobic fermentation (without colloid milling and autolytic enzymatic hydrolysis processes) This comparative example is used to compare the effects of the special pretreatment and segmented fermentation processes of the present invention.

[0058] 1. Weigh out 100 kg of black soldier fly defatted insect powder, 20 kg of black soldier fly pupa shell powder, 30 kg of sugarcane molasses, 2 kg of ammonium sulfate, 1 kg of magnesium sulfate, 2.5 kg of potassium dihydrogen phosphate, and 0.3 kg of zinc sulfate.

[0059] 2. Add 450 kg of water and stir well to adjust the pH to 7.0. Sterilize directly at 85°C for 40 minutes without grinding in a colloid mill.

[0060] 3. Cool to 30°C and simultaneously inoculate with Bacillus belye (3%), Aspergillus oryzae (3%) and Trichoderma harzianum (3%). Do not perform split fermentation, do not use saturated lime milk for neutralization, and do not perform high-temperature cascade enzymatic hydrolysis at 58°C.

[0061] 4. Ferment continuously at 30℃ for 144 hours. After fermentation, the filtrate was collected by single-stage pressure filtration, homogenized by ultrasound, and concentrated 3 times to obtain the biostimulant product (referred to as Comparative Example-D1).

[0062] Comparative Example 2: Conventional Acid-Base Chemical Hydrolysis Method This comparative example is used to compare the degree of damage to active ingredients caused by conventional chemical treatment methods.

[0063] 1. Weigh 100 kg of black soldier fly defatted insect powder and 20 kg of black soldier fly pupa shell powder, add 450 kg of 10% hydrochloric acid solution, and acid hydrolyze at 95℃ for 12 hours.

[0064] 2. After filtration, the filter residue is deacetylated with a 40% sodium hydroxide solution at 100°C for 6 hours to hydrolyze chitin.

[0065] 3. Mix the two liquids, neutralize the pH to 6.5 with alkali, concentrate under reduced pressure by 3 times to produce the stimulant product (referred to as comparative example-D2).

[0066] Effect Example Example 1: Detection and Comparative Analysis of Main Components and Trace Elements of Plant Biostimulants The main active ingredients of the plant biostimulants prepared in Examples 1-2 and Comparative Examples 1-2 were quantitatively analyzed. The detection indicators included: total free amino acids (determined using an amino acid analyzer), small peptide (molecular weight <1000 Da) content, total water-soluble chitosan and chitosan oligosaccharides (molecular weight <3000 Da), comprehensive chitin degradation rate, and the content of chelated calcium, magnesium, zinc, and potassium ions (determined using inductively coupled plasma mass spectrometry, ICP-MS). The detection results are shown in Table 1 below.

[0067] Table 1: Comparison of core active ingredients of biostimulants, chitin degradation rate and mineral elements in each group

[0068] Technical Principle Analysis: 1. A significant leap in chitin degradation rate: The chitin degradation rates in Examples 1 and 2 reached 84.5% and 78.2%, respectively, far exceeding those of Comparative Example 1. This is attributed to the unique colloid milling wet ultrafine grinding method of this invention, which produces extremely fine particles while avoiding the mutual inhibition of multiple bacterial co-occurrences. In the second stage, Trichoderma harzianum is used to overexpress chitinase under carbon source limitation.

[0069] 2. A dramatic increase in the content of small peptides and amino acids: The content of small peptides and amino acids in Example 1 was significantly higher than that in the comparative example. This is because: First, the addition of zinc sulfate activated the neutral protease activity of Bacillus; second, the high concentration of calcium ions introduced by saturated lime milk provided excellent thermal stability protection for the protease structure at 58°C, enabling it to maintain extremely high enzyme activity during 8+8 hours of high-temperature enzymatic hydrolysis; simultaneously, the split fermentation and temperature-controlled enzymatic hydrolysis process maximized the synergistic effect of the extracellular enzymes of the two strains.

[0070] 3. Natural chelation of micronutrients: Because magnesium sulfate and zinc sulfate are added to the formula, and saturated lime milk (calcium hydroxide) is used instead of NaOH for neutralization, the organic acids, small peptides and amino acids in the fermentation process form natural organic chelates with these calcium, magnesium and zinc ions that are easily absorbed by plants, eliminating the tedious secondary compounding process in the later stage and making the nutrients more balanced.

[0071] Example 2: Effects on the growth and root promotion of greenhouse tomatoes The experiment was conducted in a modern multi-span greenhouse, using tomatoes (variety "Fenbeibei") as the test crop. After transplanting, the tomato seedlings were randomly divided into 5 groups of 30 plants each, and the following treatments were administered: 1. Blank control group (CK): Irrigated with clean water; 2. Example 1 group: Administer a 500-fold diluted solution of stimulant-E1; 3. Example 2 group: Administered a 500-fold diluted solution of stimulant-E2; 4. Comparative Example 1: Apply a 500-fold diluted Comparative Example-D1 solution; 5. Comparative Example 2: Apply Comparative Example-D2 diluted 500 times.

[0072] Each plant was irrigated with 200 mL of the solution every 10 days, for a total of 4 applications. Fifteen days after the last application, the increases in plant height, stem diameter, chlorophyll SPAD value, total root length, root volume, and fresh root weight were measured. The results are shown in Table 2.

[0073] Table 2: Effects of different biostimulant treatments on tomato plant growth and root development

[0074] Conclusions on root and growth promotion: After applying the biostimulant of Example 1 of this invention, the total root length of tomatoes increased by 117.2% and the fresh root weight increased by 188.2% compared with the blank control (CK). This is not only due to the synergistic root-promoting effect of active small peptides and chitosan / chitosan oligosaccharides, but also due to the high content of chelated calcium, magnesium, and zinc ions in the product. Magnesium ions significantly promoted chlorophyll synthesis (SPAD value increased to 52.4), zinc ions promoted the synthesis of endogenous auxin (IAA), and calcium ions enhanced root tip cell division. The synergistic effect of these multiple stimuli demonstrated an extremely powerful "root-promoting, seedling-strengthening, and leaf-greening" effect.

[0075] Example 3: Tests on resistance to abiotic stresses such as salt and alkali and drought To verify the abiotic stress resistance effect of the prepared plant biostimulant, a greenhouse simulation experiment was conducted on the salt and drought resistance of tomato seedlings.

[0076] 1. Salt resistance test When tomato seedlings reached the stage of having four true leaves, they were subjected to salt stress treatment by irrigating with a high-salt solution containing 150 mmol / L NaCl. Two days before and three days after salt stress treatment, foliar sprays were applied to the leaves of three different plants: control group (CK), Example 1 (E1, diluted 400 times), and Comparative Example 1 (D1, diluted 400 times). Seven days after high salt stress, the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in the tomato leaves were measured.

[0077] 2. Drought resistance test When tomato seedlings reached the stage of 5 true leaves, watering was stopped to induce natural drought stress. On the day watering was stopped and on the 5th day, the root zone was irrigated with clean water (CK), Example 1 (E1, diluted 400 times), and Comparative Example 1 (D1, diluted 400 times), respectively. After 12 days of continuous drought, the average wilting index of the plants was observed, and the proline (Pro) content in the leaves was measured. The test data are shown in Table 3.

[0078] Table 3: Effects of biostimulants on physiological indicators of salt and drought resistance in tomato seedlings

[0079] Analysis of the physiological mechanisms of stress resistance: In tomatoes treated with Example 1, under severe salt stress, the level of MDA (dimethylaminoalkanoate), a lipid peroxidation product, was only 38.7% of that in the control group, while SOD activity increased significantly. Under severe drought, there was virtually no wilting, and the proline content reached 465.8 μg / g. This indicates that the biostimulant of this invention can extremely effectively stimulate the plant's protective mechanisms against abiotic stresses, which is closely related to chitosan oligosaccharides and bioactive peptides as environmental elicitors, and the activation of drought signal transduction by the trace element zinc.

[0080] Example 4: Effects of improving rhizosphere soil microecology and inhibiting soil-borne pathogenic fungi Using *Fusarium oxysporum* (the pathogen of tomato wilt) as the target bacterium, plate confrontation experiments and rhizosphere soil drenching experiments were conducted (the detection indicators and methods are the same as described above). The data are shown in Table 4.

[0081] Table 4: Antibacterial activity of plant biostimulants and their effects on rhizosphere soil microbial communities

[0082] Conclusions on antibacterial and microecological improvement: Example 1 showed an extremely strong direct antibacterial rate of up to 82.4%, and in the rhizosphere soil, the number of pathogenic Fusarium oxysporum decreased by three orders of magnitude after application of this product, while the number of beneficial bacteria (Trichoderma and Bacillus) increased by nearly four thousand times.

[0083] This is mainly due to two factors: First, the micronization of chitin via colloid mill significantly reduces its molecular weight, resulting in even lower molecular weight chitosan oligosaccharides (mostly less than 2000 Da) produced during degradation. This strongly stimulates the proliferation of antagonistic microorganisms such as soil actinomycetes, thus forming a powerful natural defense barrier. Second, the antimicrobial lipopeptides and trichomoniasis-like natural disease-preventing factors produced during fermentation are perfectly preserved during temperature-controlled enzymatic hydrolysis. In summary, this invention, through unique micronization pretreatment via colloid mill, inorganic salt-assisted enhancement, and the innovative "8:2 split fermentation and autolysis + cascade enzymatic hydrolysis process," successfully transforms black soldier fly defatted insect powder and pupal shell powder into high-activity, easily absorbed, and rich in trace elements high-quality plant biostimulants. All performance indicators and application effects far exceed those of conventional single-mixed fermentation or chemical degradation methods, demonstrating extremely high promotion and application value.

[0084] The invention employs a scientific process and achieves thorough degradation, enabling the complete and waste-free high-value utilization of black soldier fly byproducts.

[0085] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing plant biostimulants using black soldier fly defatted insect powder and pupal shell powder, characterized in that, Includes the following steps: S1. Preparation and pretreatment of multi-component fermentation substrate: By weight, mix 80-120 parts of black soldier fly defatted insect powder, 10-30 parts of pupal shell powder, 20-50 parts of molasses, 1-3 parts of ammonium sulfate, 0.5-1.5 parts of magnesium sulfate, 1-4 parts of potassium dihydrogen phosphate, 0.1-0.5 parts of zinc sulfate, and 300-600 parts of water evenly, and adjust the pH of the mixture to 6.0-7.5; the evenly mixed material is first subjected to wet ultra-fine grinding, then heated to 90-95℃ and kept at that temperature for 30-60 minutes for sterilization, and after cooling, a sterile fermentation substrate is obtained; The black soldier fly defatted insect powder contains, by weight percentage, 45%-52% protein, 12%-20% oil and 6%-10% chitin; S2. First-stage split fermentation and two-stage enzymatic hydrolysis: S21. Split inoculation: The sterile fermentation substrate is divided into a first fermentation broth and a second fermentation broth at a mass ratio or volume ratio of 8:2; Bacillus berberis is inoculated into the first fermentation broth, which accounts for 80% of the total mass, and aerobic fermentation is carried out at a temperature of 30-38℃ for 18-72 hours; Aspergillus is inoculated into the second fermentation broth, which accounts for 20% of the total mass, and aerobic fermentation is carried out at a temperature of 28-32℃; S22. Primary autolysis and enzymatic hydrolysis: Add saturated lime milk to the first fermentation broth after fermentation, adjust the pH value to 6.5-7.0, and then heat to 55-60℃ for incubation autolysis and primary enzymatic hydrolysis. The enzymatic hydrolysis time is 6-12 hours. S23. Secondary synergistic enzymatic hydrolysis: After the primary autolytic enzymatic hydrolysis is completed, the fermented second fermentation broth is added to the first fermentation broth, and secondary enzymatic hydrolysis is continued at a temperature of 55-60℃ for 6-12 hours. S24. Inactivation and separation: After the secondary enzymatic hydrolysis is completed, the mixed fermentation broth is heated to 80℃ for enzyme inactivation and sterilization treatment for 30-45 minutes, followed by solid-liquid separation, and the first-stage fermentation filtrate and the first-stage fermentation residue are collected separately. S3. Second stage fermentation and enzymatic hydrolysis: The first stage fermentation residue was mixed with water at a weight ratio of 1:(3-6) and resuspended. The pH of the mixture was adjusted to 4.5-6.

0. Trichoderma harzianum was inoculated and fermented and enzymatically hydrolyzed at a temperature of 25-30℃ for 96-168 hours. After fermentation, solid-liquid separation was performed, and the second stage fermentation filtrate was collected. S4. Compound preparation: The first stage fermentation filtrate and the second stage fermentation filtrate are mixed at a volume ratio of (1.5-3):1, and homogenized, concentrated or dried to obtain a plant biostimulant rich in amino acids, small peptides and chitosan, and containing abundant trace elements.

2. The method according to claim 1, characterized in that, In step S1, the black soldier fly defatted insect powder contains 49% protein, 17% oil and 8% chitin by mass percentage; and the uniformly mixed material is first subjected to wet ultrafine grinding treatment by a colloid mill, wherein the gap between the grinding teeth of the colloid mill is controlled at 10-30μm, and the median particle size D50 of the treated slurry is ≤40μm.

3. The method according to claim 1, characterized in that, In step S21, the inoculum amount of *Bacillus belye* is 2%-5% of the mass of the first fermentation broth, and the effective viable count of the bacterial solution is ≥1.0 × 10⁻⁶. 9 CFU / mL; the inoculated Aspergillus is either Aspergillus oryzae or Aspergillus niger, the inoculation amount is 2%-5% of the mass of the second fermentation broth, and the spore suspension concentration is ≥1.0×10⁻⁶. 7 The fermentation rate is 100 cells / mL, and the fermentation time of the second fermentation broth is 24-48 hours.

4. The method according to claim 1, characterized in that, In step S21, during the fermentation process of the first fermentation broth 18-72 hours after inoculation with Bacillus belye, the fermentation temperature is controlled at 35-37℃, the stirring speed is 150-200rpm, and the aeration rate is 0.5-1.2 vvm.

5. The method according to claim 1, characterized in that, In step S22, the saturated lime milk is a calcium hydroxide aqueous suspension with a mass percentage concentration of 8%-12%; in steps S22 and S23, the temperature of the primary autolytic enzymatic hydrolysis and the secondary synergistic enzymatic hydrolysis is maintained at 55-60℃, the stirring speed is controlled at 100-120 rpm, and no aeration is performed.

6. The method according to claim 1, characterized in that, In step S3, the inoculated Trichoderma harzianum is a suspension of Trichoderma harzianum spores, and the inoculation amount is 2%-6% of the total mass of the filter residue resuspension. The concentration of the Trichoderma harzianum spore suspension is ≥1.0×10⁻⁶. 7 Chitinase was added exogenously to the filter residue resuspension at the start of fermentation or within 24 hours of fermentation, at a rate of 50-200 U / g filter residue.

7. The method according to claim 1, characterized in that, In step S4, the obtained plant biostimulant contains ≥110 g / L of free amino acids, ≥55 g / L of small peptides with a molecular weight less than 1000 Da, ≥18 g / L of water-soluble chitosan and chitosan oligosaccharides, and contains chelated calcium, magnesium and zinc ions, wherein the calcium ion content is ≥8 g / L, the magnesium ion content is ≥2 g / L, and the zinc ion content is ≥0.5 g / L.

8. The plant biostimulant prepared by the method according to any one of claims 1-7.

9. The plant biostimulant according to claim 8, characterized in that, The plant biostimulant is in liquid or solid powder form; wherein... When the plant biostimulant is in liquid form, it also contains 0.1%-0.5% preservative and 1%-5% plant growth regulator by mass fraction. When the plant biostimulant is in solid powder form, it is prepared by low-temperature vacuum concentration of a mixture of the first-stage fermentation filtrate and the second-stage fermentation filtrate, followed by spray drying.

10. The application of the plant biostimulant of claim 8 in promoting crop growth, enhancing crop resistance to drought and salinity stress, and preventing soil-borne fungal diseases in crops.

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