Hermetia illucens fermentation liquor as well as preparation method and application thereof

Abstract

The invention discloses hermetia illucens fermentation liquor as well as a preparation method and application thereof. The hermetia illucens fermentation liquor is obtained by uniformly mixing hermetia illucens powder, wheat bran, enzymolysis miscanthus sinensis powder, sterile water, an aerobic leavening agent and papain, performing aerobic fermentation to obtain a hermetia illucens primary fermentation material, and then adding an anaerobic leavening agent for anaerobic fermentation. According to the invention, bacillus subtilis and aspergillus oryzae are introduced as aerobic leavening agents and cooperate with papain to ferment hermetia illucens, and enzymatic miscanthus powder is introduced to cooperate with an anaerobic leavening agent prepared by compounding bacillus amyloliquefaciens, lactobacillus plantarum and a plant composite extract; and the hermetia illucens fermentation liquor with high nutritional value and high quality is prepared, so that the hermetia illucens fermentation liquor has higher value and better effect in feed application.

Description

Technical Field

[0001] This invention relates to the field of feed preparation technology, and in particular to a black soldier fly fermentation broth, its preparation method, and its application. Background Technology

[0002] With the rapid development of animal husbandry, the shortage of major feed resources, mainly corn, soybeans, and fishmeal, is becoming increasingly serious. Insects, as a sustainable high-protein feed ingredient, have received widespread attention in recent years, with houseflies, mealworms, and black soldier flies showing significant potential for use as feed insects. The black soldier fly, scientifically known as *Bufo lucidae*, belongs to the genus *Bufo* in the family Bandidae of the order Diptera. As a saprophytic insect, it is characterized by its wide diet, ease of rearing, rapid reproduction, and high bioconversion rate. It can harmlessly treat organic waste such as livestock and poultry manure, kitchen waste, and food industry byproducts. Furthermore, its larvae are rich in nutrients, containing approximately 40% protein and 30% lipids, as well as active substances such as antimicrobial peptides, chitin, and lauric acid, and minerals such as calcium and phosphorus, making it highly valuable for applications.

[0003] Currently, extensive research has been conducted both domestically and internationally on black soldier fly larvae as feed for aquatic products, livestock, and pets. Studies have revealed that black soldier fly larvae suffer from unstable nutritional composition, and their high chitin content leads to reduced feed intake and decreased digestibility. To address these issues, current research has found that fermenting black soldier fly larvae can positively impact their nutritional value, reduce adverse effects from feeding, and improve the digestibility of their nutrients.

[0004] Chinese patent CN117256731B discloses a black soldier fly larvae fermentation broth, its preparation method, and its application in shrimp feed. This invention's black soldier fly larvae fermentation broth is obtained from a mixture of Bacillus subtilis and a compound microbial fermentation feed. The feed includes matured black soldier fly larvae, flour, wheat bran, brown sugar, water, and a mixed alcohol extract. The raw materials for the mixed alcohol extract include galangal leaves, olive leaves, and areca nut pulp. The compound microorganisms include fermentative yeast, Rhodopseudomonas palustris, and Lactobacillus plantarum. Using the black soldier fly larvae fermentation broth provided by this invention as shrimp feed not only improves the shrimp's antioxidant activity and resistance to Aeromonas hydrophila, but also increases shrimp weight and survival rate. However, the nutrient content of this black soldier fly larvae fermentation broth is insufficient, resulting in low nutritional value. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the present invention provides a method for preparing and applying black soldier fly fermentation broth. The black soldier fly fermentation broth provided by the present invention not only has high nutritional value, but also effectively reduces the adverse effects of feeding and solves the problem of low nutrient digestibility.

[0006] To achieve the above objectives, the present invention provides a method for preparing black soldier fly fermentation broth, comprising the following steps, in parts by weight: (1) Mix 20-30 parts of black soldier fly powder, 3-6 parts of wheat bran, 3-5 parts of enzymatic hydrolyzed Miscanthus powder, and 45-60 parts of sterile water evenly to obtain a mixture. Then add 1-2% of the total weight of the mixture of aerobic fermentation agent and 0.1-0.3% of the total weight of the mixture of papain and mix evenly. Then ferment aerobically at 35-37℃ for 1-3 days to obtain the primary fermentation material of black soldier fly. (2) Mix the primary fermentation material of black soldier fly with the anaerobic fermentation agent at a weight ratio of 100:1-2, and then seal and anaerobic ferment at 35-37℃ for 3-5 days to obtain the black soldier fly fermentation liquid. Preferably, the enzymatically hydrolyzed Miscanthus powder is Miscanthus powder that has been treated with a compound enzyme or Miscanthus powder that has been pretreated with a water-γ-valerol mixed solvent, a p-toluenesulfonic acid-γ-valerol mixed solvent, or an ethyl lactate-γ-valerol mixed solvent as pretreatment solvents before being treated with a compound enzyme.

[0007] Preferably, the aerobic fermentation agent is selected from at least one of Bacillus subtilis culture and Aspergillus oryzae culture.

[0008] Preferably, the anaerobic fermentation agent is obtained by mixing a plant complex extract composed of Bacillus amyloliquefaciens bacterial solution, Lactobacillus plantarum bacterial solution, and Rubia cordifolia extract and Ulva prolifera extract.

[0009] Further explanation of this invention: The *Bacillus amyloliquefaciens* and *Lactobacillus plantarum* in the anaerobic fermentation agent can produce various enzymes such as proteases, amylases, and cellulases. These enzymes can decompose proteins, carbohydrates, and cellulose in the fermentation substrate, significantly shortening fermentation time and increasing the yield and quality of the fermentation broth, while also generating more amino acids and peptides. Simultaneously, the *Rubia cordifolia* extract in the anaerobic fermentation agent contains various antioxidant phenolic compounds and antibacterial flavonoids, which can not only scavenge free radicals generated during fermentation and protect the nutrients in the fermentation broth from oxidative damage, but also inhibit the growth of some harmful microorganisms in the fermentation broth, reducing contamination and thus helping to maintain the nutritional value and improve the quality of the fermentation broth. Meanwhile, the *Ulva prolifera* extract is rich in polysaccharides, which can promote the growth and reproduction of beneficial microorganisms in the fermentation broth, regulate the microbial community structure, and make the fermentation process more stable. It also contains a certain amount of vitamins and minerals, which can supplement the nutrients in the fermentation broth, making it more comprehensive and meeting the needs of animals for various nutrients, thus improving the nutritional value and functionality of the fermentation broth.

[0010] Preferably, the black soldier fly powder is obtained by blanching black soldier fly larvae in boiling water for 1-2 minutes to kill them, drying them at 60-65℃, pulverizing them, and then passing them through a 100-200 mesh sieve.

[0011] More preferably, the method for preparing the enzymatically hydrolyzed Miscanthus powder is as follows, by weight: After drying the Miscanthus stalks, crush them and pass them through a 10-200 mesh sieve to obtain Miscanthus powder. Dry the Miscanthus powder at 60-80°C until the moisture content is <5% to obtain dried Miscanthus powder for later use. Add 1-3 parts of dried Miscanthus powder to 40-80 parts of sodium acetate buffer solution with a pH of 4-6, preheat in a constant temperature shaking incubator at 45-55°C for 20 minutes, add 0.1-0.3% of the total weight of dried Miscanthus powder with a compound enzyme, and enzymatically hydrolyze for 24-48 hours at a reaction temperature of 40-60°C and 200-400 rpm. After enzymatic hydrolysis, boil in a boiling water bath for 10-20 minutes to inactivate residual cellulase and obtain enzymatically hydrolyzed Miscanthus powder. Alternatively, the Miscanthus stalks can be dried, pulverized, and passed through a 100-200 mesh sieve to obtain Miscanthus powder. The Miscanthus powder is then dried at 60-80°C until the moisture content is <5%, yielding dried Miscanthus powder for later use. The dried Miscanthus powder is then mixed with a pretreatment solvent at a solid-liquid ratio of 1:5-10 (g:mL), and reacted at 90-120°C for 1-3 hours under a nitrogen atmosphere. After cooling to room temperature, the solid residue is separated by vacuum filtration. The solid residue is washed with water until neutral, and then dried to obtain the pretreated Miscanthus powder. Prepare Miscanthus sinensis powder; add 1-3 parts of pretreated Miscanthus sinensis powder to 40-80 parts of sodium acetate buffer solution with a pH of 4-6, preheat in a constant temperature shaking incubator at 45-55℃ for 10-20 min, add 0.1-0.3% of compound enzyme by weight of the total weight of dried Miscanthus sinensis powder, and enzymatically hydrolyze for 24-48 h at a reaction temperature of 40-60℃ and 200-400 rpm; after enzymatic hydrolysis, boil in a boiling water bath for 10-20 min to inactivate residual cellulase, and obtain enzymatically hydrolyzed Miscanthus sinensis powder.

[0012] Preferably, the pretreatment solvent is a water-γ-valerolactone mixed solvent obtained by mixing water and γ-valerolactone at a volume ratio of 1:2-5, or a p-toluenesulfonic acid-γ-valerolactone mixed solvent obtained by mixing p-toluenesulfonic acid aqueous solution with a concentration of 20-60 g / L and γ-valerolactone at a volume ratio of 1:2-5, or a lactate-γ-valerolactone mixed solvent obtained by mixing ethyl lactate and γ-valerolactone at a volume ratio of 1:2-5.

[0013] Preferably, the composite enzyme is composed of ligninase, cellulase and hemicellulase mixed in a weight ratio of 0.5-1:1-2:1-2.

[0014] Further explanation of this invention: Enzymatic hydrolysis can transform the complex cellulose structure of Miscanthus sinensis powder, releasing its nutrients, such as polysaccharides, oligosaccharides, and some proteins. These components can be further decomposed and transformed by microorganisms during fermentation, generating more amino acids, polypeptides, and organic acids. This improves the nutritional composition and value of the black soldier fly fermentation broth, while also increasing the digestibility of nutrients. Furthermore, the oligosaccharides and polysaccharides produced during the enzymatic hydrolysis of Miscanthus sinensis powder have a certain sweetness, which can improve the flavor of the fermentation broth, reduce unpleasant odors that may occur during fermentation, and increase animal feed intake. Simultaneously, the organic acids in the hydrolysis products can adjust the pH of the fermentation broth, making it closer to the digestive environment of animals and improving palatability.

[0015] Preferably, the effective viable bacteria content of the Bacillus subtilis bacterial solution is 2×10⁻⁶. 7 -6×10 7 CFU / mL; the effective viable bacteria content of the Aspergillus oryzae solution is 2×10⁻⁶. 6 -6×10 6 CFU / m.

[0016] Preferably, the effective viable bacteria content of the *Lactobacillus plantarum* bacterial solution is 2 × 10⁻⁶. 7 -6×10 7 CFU / mL; the effective viable bacterial content of the Bacillus amyloliquefaciens is 2×10⁻⁶. 6 -6×10 7 CFU / mL.

[0017] The present invention also provides a black soldier fly larvae fermentation broth, which is prepared by the above method.

[0018] The present invention also provides the application of the above-mentioned black soldier fly fermentation broth in the preparation of feed.

[0019] The beneficial effects of this invention are: 1. Compared with the prior art, the present invention introduces Bacillus subtilis and Aspergillus oryzae liquid as aerobic fermentation agents, and co-fers black soldier fly with papain. Combining the advantages of microorganisms and enzyme preparations, it can not only improve the fermentation efficiency, but also reduce the production of bitterness during the fermentation process, and prepare a black soldier fly fermentation broth with high nutritional value and high quality.

[0020] 2. Compared with existing technologies, the present invention employs a two-stage fermentation process in the preparation of black soldier fly fermentation broth. The primary fermentation is aerobic fermentation. Under aerobic conditions, microorganisms can multiply rapidly, decompose carbohydrates and proteins in the substrate, produce organic acids and other metabolites, and lower the pH value, creating conditions for subsequent anaerobic fermentation. The secondary fermentation is anaerobic fermentation. Under anaerobic conditions, microorganisms can further decompose organic matter in the substrate, producing more polypeptides, amino acids, and lactic acid, thereby improving the nutritional value and flavor of the fermentation broth.

[0021] 3. This invention introduces an anaerobic fermentation agent prepared by combining enzymatically hydrolyzed Miscanthus powder with a mixed bacterial solution of Bacillus amyloliquefaciens and Lactobacillus plantarum, along with a plant complex extract. This synergistic effect enhances the value and efficacy of the black soldier fly aerobic fermentation agent in feed applications. The introduction of enzymatically hydrolyzed Miscanthus powder and the anaerobic fermentation agent not only improves the nutritional value of the black soldier fly aerobic fermentation agent, increasing the content of crude protein, protein peptides, umami amino acids, essential amino acids, and non-essential amino acids, but also improves the flavor and palatability of the aerobic fermentation agent, reduces potential adverse effects during feeding, and increases the digestibility of nutrients. Detailed Implementation

[0022] The parameters and sources of some substances in the examples are as follows: Black soldier fly larvae, sourced from Jiangsu Shengjiu Biotechnology Co., Ltd.; Wheat bran, 100 mesh, commercially available; Miscanthus stalks, commercially available; Preparation of madder extract: Wash, dry and pulverize madder (commercially available) through an 80-mesh sieve. Then mix madder with 95% ethanol at a ratio of 1:10. Heat and reflux at 85°C twice for 2 hours each time. Combine the filtrates from the two extractions, recover the solvent under reduced pressure, and dry to obtain madder extract. Preparation of Ulva prolifera extract: Ulva prolifera (commercially available) was desanded, dried, and pulverized through an 80-mesh sieve to obtain Ulva prolifera powder; then 12 kg of water was added to 1 kg of Ulva prolifera powder, the pH was adjusted to 9.5 with potassium hydroxide, and then the mixture was stirred and extracted at 85℃ for 5 h; after filtration, the supernatant was concentrated under vacuum to a specific gravity of 1.2 g / mL to obtain Ulva prolifera extract; Papain, enzyme activity: 50,000U, sourced from Shandong Fengtai Biotechnology Co., Ltd. Ligninase, enzyme activity: 10000U, sourced from Shandong Nuojie Biotechnology Co., Ltd. Cellulase, enzyme activity: 100,000 U, sourced from Shandong Nuojie Biotechnology Co., Ltd. Hemicellulase, enzyme activity: 100,000U, sourced from Shandong Nuojie Biotechnology Co., Ltd. The bacterial solutions involved in the embodiments and comparative examples of this invention were prepared by the following method: Bacillus subtilis, ATCC 6051, sourced from Beijing Bio-Bio Biotechnology Co., Ltd., platform number: Bio-72808; Lactobacillus plantarum, ATCC 8014, sourced from Beijing Bio-Bio Biotechnology Co., Ltd., platform number: Bio-67374; Aspergillus oryzae, ATCC 42149, sourced from Beijing Bio-Bio Biotechnology Co., Ltd., platform number: Bio-52980; Bacillus amyloliquefaciens, CGMCC 1.0504, sourced from Beijing Bio-Bio Biotechnology Co., Ltd., platform number: Bio-53060; Bacillus subtilis culture: Inoculate the bacterial strain onto LB slant and incubate at 37°C for 12-16 hours; pick a single colony and transfer it to 5 mL LB liquid medium, and incubate at 37°C with shaking at 200 rpm for 8-12 hours; transfer it to 500 mL LB liquid medium (concentrated flask volume ≤ 1 / 3) at a 1% inoculation rate (v / v), and incubate at 37°C with shaking at 200 rpm for 12-16 hours until the logarithmic growth phase; then serially dilute with sterile physiological saline to obtain Bacillus subtilis culture, and store at 4°C for later use.

[0023] Lactobacillus plantarum culture: Inoculate the bacterial strain onto MRS slant and anaerobic culture at 37°C (candle jar method or anaerobic jar) for 18-24 hours; pick a single colony and transfer it to 5 mL of MRS liquid medium and incubate at 37°C for 12-16 hours; transfer it to 500 mL of MRS liquid medium at a 1% inoculation rate and incubate at 37°C for 18-24 hours; then dilute with sterile physiological saline to obtain Lactobacillus plantarum culture and store at 4°C.

[0024] Aspergillus oryzae suspension: Transfer the strain to PDA slant and incubate at 30℃ for 3-5 days until conidia are produced. Scrape the spores and wash them with sterile physiological saline to prepare a spore suspension. Transfer the suspension to 500 mL of Czapek's medium at a 1% inoculum and incubate at 28℃ and 150 rpm for 3-5 days with shaking. Then dilute the spore suspension with sterile physiological saline to obtain Aspergillus oryzae suspension and store at 4℃ in the dark.

[0025] Bacillus amyloliquefaciens bacterial suspension: Inoculate the bacterial strain onto LB slant and incubate at 37°C for 12-16 hours; pick a single colony and transfer it to 5 mL LB liquid medium, and incubate at 37°C with shaking at 200 rpm for 8-12 hours; transfer 1% inoculum to 500 mL LB liquid medium (container in an Erlenmeyer flask with liquid volume ≤1 / 3), and incubate at 37°C with shaking at 200 rpm for 12-16 hours until the logarithmic growth phase; then serially dilute with sterile physiological saline to obtain Bacillus amyloliquefaciens bacterial suspension, and store at 4°C for later use.

[0026] Example 1 A method for preparing black soldier fly larvae fermentation broth includes the following steps: (1) Mix 25 parts by weight of black soldier fly powder, 5 parts by weight of wheat bran, 4 parts by weight of enzymatic hydrolyzed cogon grass powder and 52 parts by weight of sterile water evenly to obtain a mixture. Then add 1.5% of the total weight of the mixture of aerobic fermentation agent and 0.2% of the total weight of the mixture of papain and mix evenly. Then ferment aerobically at 36℃ for 2 days to obtain the primary fermentation material of black soldier fly. (2) Mix the primary fermentation material of black soldier fly with the anaerobic fermentation agent at a weight ratio of 100:1.5, and then seal and anaerobic ferment at 36℃ for 5 days to obtain the black soldier fly fermentation liquid.

[0027] The black soldier fly powder is obtained by blanching black soldier fly larvae in boiling water for 2 minutes to kill them, drying them at 63°C, pulverizing them, and then passing them through a 100-mesh sieve.

[0028] The aerobic fermentation agent is composed of Bacillus subtilis bacterial solution (effective live bacteria content 5×10⁻⁶). 7 CFU / mL), Aspergillus oryzae solution (effective viable count 5×10⁻⁶) 6 The mixture (CFU / mL) was prepared by mixing the same volume of the mixture.

[0029] The anaerobic fermentation agent consists of Bacillus amyloliquefaciens bacterial solution (effective live bacteria content 5×10⁻⁶). 7 CFU / mL), Lactobacillus plantarum bacterial suspension (effective live bacteria content 5×10⁻⁶) 7 The mixture (CFU / mL) was prepared by mixing the same volume of the mixture.

[0030] The method for preparing the enzymatically hydrolyzed Miscanthus powder is as follows: After drying the Miscanthus stalks, they were crushed and passed through a 100-mesh sieve to obtain Miscanthus powder. The Miscanthus powder was dried at 65°C until the moisture content was <5% to obtain dried Miscanthus powder for later use. 1.5g of dried Miscanthus powder was added to 60mL of 50mmol / L sodium acetate buffer solution with a pH of 5 and preheated in a constant temperature shaking incubator at 50°C for 20min. 0.2% of the total weight of the dried Miscanthus powder was added as a compound enzyme, and the mixture was enzymatically hydrolyzed at 50°C and 300rpm for 48h. After the enzymatic hydrolysis was completed, the mixture was boiled in a boiling water bath for 20min to inactivate the residual enzyme, thus obtaining enzymatically hydrolyzed Miscanthus powder.

[0031] The complex enzyme is composed of ligninase, cellulase, and hemicellulase mixed in a weight ratio of 0.6:1.2:1.2.

[0032] Example 2 A method for preparing black soldier fly larvae fermentation broth includes the following steps: (1) Mix 25 parts by weight of black soldier fly powder, 5 parts by weight of wheat bran, 4 parts by weight of enzymatic hydrolyzed cogon grass powder and 52 parts by weight of sterile water evenly to obtain a mixture. Then add 1.5% of the total weight of the mixture of aerobic fermentation agent and 0.2% of the total weight of the mixture of papain and mix evenly. Then ferment aerobically at 36℃ for 2 days to obtain the primary fermentation material of black soldier fly. (2) Mix the primary fermentation material of black soldier fly with the anaerobic fermentation agent at a weight ratio of 100:1.5, and then seal and anaerobic ferment at 36℃ for 5 days to obtain the black soldier fly fermentation liquid.

[0033] The black soldier fly powder is obtained by blanching black soldier fly larvae in boiling water for 2 minutes to kill them, drying them at 63°C, pulverizing them, and then passing them through a 100-mesh sieve.

[0034] The aerobic fermentation agent is composed of Bacillus subtilis bacterial solution (effective live bacteria content 5×10⁻⁶). 7 CFU / mL), Aspergillus oryzae solution (effective viable count 5×10⁻⁶) 6 The mixture (CFU / mL) was prepared by mixing the same volume of the mixture.

[0035] The anaerobic fermentation agent consists of Bacillus amyloliquefaciens bacterial solution (effective live bacteria content 5×10⁻⁶). 7 CFU / mL), Lactobacillus plantarum bacterial suspension (effective live bacteria content 5×10⁻⁶) 7 The mixture (CFU / mL) was prepared by mixing the same volume of the mixture.

[0036] The method for preparing the enzymatically hydrolyzed Miscanthus powder is as follows: After drying the Miscanthus stalks, they were crushed and passed through a 100-mesh sieve to obtain Miscanthus powder. The Miscanthus powder was dried at 65°C until the moisture content was <5% to obtain dried Miscanthus powder for later use. Then, the dried Miscanthus powder was mixed with a water-γ-valerol mixed solvent at a solid-liquid ratio of 1:8 (g:mL) and reacted at 110°C for 2 hours under a nitrogen atmosphere. After cooling to room temperature, the solid residue was separated by vacuum filtration. The solid residue was washed with water until neutral and then dried to obtain pretreated Miscanthus powder. 1.5g of pretreated Miscanthus powder was added to 60mL of 50mmol / L sodium acetate buffer solution with a pH of 5 and preheated in a constant temperature shaking incubator at 50°C for 20 minutes. 0.2% of the total weight of the dried Miscanthus powder was added as a complex enzyme, and the mixture was enzymatically hydrolyzed at 50°C and 300rpm for 48 hours. After the enzymatic hydrolysis was completed, the mixture was boiled in a boiling water bath for 20 minutes to inactivate the residual enzyme, obtaining enzymatically hydrolyzed Miscanthus powder.

[0037] The water-γ-valerol mixed solvent is obtained by mixing water and γ-valerol at a volume ratio of 1:4.

[0038] The complex enzyme is composed of ligninase, cellulase, and hemicellulase mixed in a weight ratio of 0.6:1.2:1.2.

[0039] Example 3 A method for preparing black soldier fly larvae fermentation broth differs from Example 2 in that the method for preparing the enzymatically hydrolyzed Miscanthus sinensis powder is as follows: After drying the Miscanthus stalks, they were crushed and passed through a 100-mesh sieve to obtain Miscanthus powder. The Miscanthus powder was dried at 65°C until the moisture content was <5% to obtain dried Miscanthus powder for later use. Then, the dried Miscanthus powder was mixed with a mixed solvent of p-toluenesulfonic acid-γ-valerol at a solid-liquid ratio of 1:8 (g:mL) and reacted at 110°C for 2 hours under a nitrogen atmosphere. After cooling to room temperature, the solid residue was separated by vacuum filtration. The solid residue was washed with water until neutral and then dried to obtain pretreated Miscanthus powder. 1.5g of pretreated Miscanthus powder was added to 60mL of 50mmol / L sodium acetate buffer solution with a pH of 5 and preheated in a constant temperature shaking incubator at 50°C for 20 minutes. 0.2% of the total weight of the dried Miscanthus powder was added as a compound enzyme, and the mixture was enzymatically hydrolyzed at 50°C and 300rpm for 48 hours. After the enzymatic hydrolysis was completed, the mixture was boiled in a boiling water bath for 20 minutes to inactivate the residual enzyme, obtaining enzymatically hydrolyzed Miscanthus powder.

[0040] The p-toluenesulfonic acid-γ-valerol mixed solvent is obtained by mixing a 50 g / L p-toluenesulfonic acid aqueous solution with γ-valerol at a volume ratio of 1:4.

[0041] The complex enzyme is composed of ligninase, cellulase, and hemicellulase mixed in a weight ratio of 0.6:1.2:1.2.

[0042] Example 4 A method for preparing black soldier fly larvae fermentation broth differs from Example 2 in that the method for preparing the enzymatically hydrolyzed Miscanthus sinensis powder is as follows: After drying the Miscanthus stalks, they were crushed and passed through a 100-mesh sieve to obtain Miscanthus powder. The Miscanthus powder was dried at 65°C until the moisture content was <5% to obtain dried Miscanthus powder for later use. Then, the dried Miscanthus powder was mixed with a mixed solvent of ethyl lactate-γ-valerol at a solid-liquid ratio of 1:8 (g:mL) and reacted at 110°C for 2 hours under a nitrogen atmosphere. After cooling to room temperature, the solid residue was separated by vacuum filtration. The solid residue was washed with water until neutral and then dried to obtain pretreated Miscanthus powder. 1.5g of pretreated Miscanthus powder was added to 60mL of 50mmol / L sodium acetate buffer solution with a pH of 5 and preheated in a constant temperature shaking incubator at 50°C for 20 minutes. 0.2% of the total weight of the dried Miscanthus powder was added as a compound enzyme, and the mixture was enzymatically hydrolyzed at 50°C and 300rpm for 48 hours. After the enzymatic hydrolysis was completed, the mixture was boiled in a boiling water bath for 20 minutes to inactivate the residual enzyme, obtaining enzymatically hydrolyzed Miscanthus powder.

[0043] The ethyl lactate-γ-valerol mixed solvent is obtained by mixing ethyl lactate and γ-valerol at a volume ratio of 1:4.

[0044] The complex enzyme is composed of ligninase, cellulase, and hemicellulase mixed in a weight ratio of 0.6:1.2:1.2.

[0045] Example 5 A method for preparing black soldier fly fermentation broth, differing from Example 4 in that the anaerobic fermentation agent is composed of Bacillus amyloliquefaciens bacterial solution (effective viable bacteria content 5 × 10⁻⁶). 7 CFU / mL), Lactobacillus plantarum bacterial suspension (effective live bacteria content 5×10⁻⁶) 7 The mixed bacterial solution (CFU / mL) was prepared by mixing the same volume of bacteria and the plant compound extract in a weight ratio of 10:2.

[0046] The plant compound extract is composed of madder extract and seaweed extract mixed in equal quantities.

[0047] Comparative Example 1 A method for preparing black soldier fly larvae fermentation broth includes the following steps: (1) Mix 25 parts by weight of black soldier fly powder, 5 parts by weight of wheat bran and 52 parts by weight of sterile water evenly to obtain a mixture. Then add 1.5% of the total weight of the mixture of aerobic fermentation agent and 0.2% of the total weight of the mixture of papain and mix evenly. Then ferment aerobically at 36°C for 2 days to obtain the primary fermentation material of black soldier fly. (2) Mix the primary fermentation material of black soldier fly with the anaerobic fermentation agent at a weight ratio of 100:1.5, and then seal and anaerobic ferment at 36℃ for 5 days to obtain the black soldier fly fermentation liquid.

[0048] The black soldier fly powder is obtained by blanching black soldier fly larvae in boiling water for 2 minutes to kill them, drying them at 63°C, pulverizing them, and then passing them through a 100-mesh sieve.

[0049] The aerobic fermentation agent is composed of Bacillus subtilis bacterial solution (effective live bacteria content 5×10⁻⁶). 7 CFU / mL), Aspergillus oryzae solution (effective viable count 5×10⁻⁶) 6 The mixture (CFU / mL) was prepared by mixing the same volume of the mixture.

[0050] The anaerobic fermentation agent is the same as that in Example 5.

[0051] Comparative Example 2 A method for preparing black soldier fly larvae fermentation broth includes the following steps: Mix 25 parts by weight of black soldier fly powder, 5 parts by weight of wheat bran, 4 parts by weight of enzymatically hydrolyzed Miscanthus sinensis powder, and 52 parts by weight of sterile water to obtain a mixture. Then add 1.5% by weight of aerobic fermentation agent and 0.2% by weight of papain to the mixture and mix well. Then ferment aerobically at 36°C for 2 days to obtain black soldier fly fermentation broth.

[0052] The black soldier fly powder is obtained by blanching black soldier fly larvae in boiling water for 2 minutes to kill them, drying them at 63°C, pulverizing them, and then passing them through a 100-mesh sieve.

[0053] The preparation method of the enzymatic hydrolysis of Miscanthus powder is the same as that in Example 4.

[0054] The aerobic fermentation agent is composed of Bacillus subtilis bacterial solution (effective live bacteria content 5×10⁻⁶). 7 CFU / mL), Aspergillus oryzae solution (effective viable count 5×10⁻⁶) 6 The mixture (CFU / mL) was prepared by mixing the same volume of the mixture.

[0055] Test Example 1 The black soldier fly broth prepared in Examples 1-5 and Comparative Examples 1-2 of this invention was used as the experimental group sample in this test example. Meanwhile, unfermented black soldier fly broth (prepared by uniformly mixing 25 parts by weight of black soldier fly powder, 5 parts by weight of wheat bran, 4 parts by weight of enzymatically hydrolyzed Miscanthus sinensis powder, and 52 parts by weight of sterile water; the preparation method of the enzymatically hydrolyzed Miscanthus sinensis powder is the same as in Example 4) was used as the control group sample. The nutritional components of the experimental group sample and the control group sample were tested separately, and the specific testing methods are as follows: Crude protein content determination: Referring to GB / T 6432-2018 Determination of crude protein in feed - Kjeldahl method, the crude protein content in the control group and experimental group samples was determined by the Kjeldahl method. Protein peptide content determination: Each group of samples was dried at 105℃ for 1 hour and at 65℃ for 4 hours, then pulverized and passed through an 80-mesh sieve to obtain sample powder; then 4g of sample powder was accurately weighed, added to 50 mL of trichloroacetic acid (15%) solution, mixed evenly, allowed to stand for 5 min, filtered through medium-speed qualitative filter paper, the filtrate was transferred to a 50 mL centrifuge tube, centrifuged at 4000 r / min for 10 min, 10 mL of supernatant was accurately transferred to a digestion tube, and its peptide content was determined according to the total crude protein determination method. The specific operation and calculation refer to "GB / T 6432-2018 Determination of Crude Protein in Feed - Kjeldahl Method"; The specific results are shown in Table 1.

[0056]

[0057] As shown in Table 1, compared with the control group, the crude protein and protein peptide contents of Examples 1-5 and Comparative Examples 1-2 increased significantly, indicating that the nutritional components of the black soldier fly fermentation broth were improved after fermentation. Comparing Examples 1-5 and Comparative Examples 1-2, the crude protein and protein peptide contents of Examples 1-5 were higher than those of Comparative Examples 1-2, indicating that the synergistic use of enzymatically hydrolyzed Miscanthus powder and anaerobic fermentation agent is more beneficial in improving the nutritional components of the black soldier fly fermentation broth. Furthermore, compared with the use of a mixed bacterial culture of Bacillus amyloliquefaciens and Lactobacillus plantarum alone as an anaerobic fermentation agent, the synergistic use of a mixed bacterial culture of Bacillus amyloliquefaciens and Lactobacillus plantarum with plant compound extracts to prepare an anaerobic fermentation agent is more effective in improving the nutritional components of the black soldier fly fermentation broth. Comparing Examples 1-5, it was found that Example 5, which introduced enzymatic hydrolysis of Miscanthus sinensis powder and used in conjunction with an anaerobic fermentation agent composed of a mixed bacterial solution of Bacillus amyloliquefaciens and Lactobacillus plantarum and plant compound extracts, had the highest crude protein and protein peptide content, significantly higher than other examples, and had a better effect on improving the nutritional components of black soldier fly fermentation liquid.

[0058] Test Example 2 The black soldier fly larvae fermentation broths prepared in Examples 1-5 and Comparative Examples 1-2 of this invention were used as the experimental group samples in this test example. Meanwhile, unfermented black soldier fly larvae broth (obtained by uniformly mixing 25 parts by weight of black soldier fly powder, 5 parts by weight of wheat bran, 4 parts by weight of enzymatically hydrolyzed Miscanthus powder, and 52 parts by weight of sterile water; the preparation methods of the black soldier fly powder and enzymatically hydrolyzed Miscanthus powder are the same as in Example 4) was selected as the control group sample. The nutritional components of the experimental group samples and the control group samples were tested separately, and the specific testing methods are as follows: Amino acid content determination: The amino acid content in the control group and experimental group samples was detected using an automatic amino acid analyzer, referring to the method in GB / T 18246-2019 Determination of Amino Acids in Feed; the specific test results are shown in Table 2.

[0059] Table 2 shows that, compared with the control group, the contents of umami amino acids aspartic acid and glutamic acid, essential amino acids, and non-essential amino acids in Examples 1-5 and Comparative Examples 1-2 increased significantly, indicating that fermentation is beneficial to improving the amino acid content of the black soldier fly fermentation broth. Comparing Examples 1-5 and Comparative Examples 1-2, the amino acid content of Examples 1-5 was higher than that of Comparative Examples 1-2, indicating that the synergistic use of enzymatically hydrolyzed Miscanthus powder and anaerobic fermentation agent is more beneficial to improving the amino acid content of the black soldier fly fermentation broth. Furthermore, compared with the use of a mixed bacterial culture of Bacillus amyloliquefaciens and Lactobacillus plantarum as an anaerobic fermentation agent, the synergistic use of this mixed bacterial culture and plant compound extract to prepare an anaerobic fermentation agent is more effective in improving the amino acid content of the black soldier fly fermentation broth. Comparing Examples 1-5, Example 5, which uses enzymatically hydrolyzed Miscanthus powder in synergy with an anaerobic fermentation agent composed of a mixed bacterial culture of Bacillus amyloliquefaciens and Lactobacillus plantarum and plant compound extract, showed the highest amino acid content and a significant improvement effect.

[0060] Test Example 3 Aquaculture experiment Commercial feed (32.0% crude protein, 7.8% crude fat) was used as the base feed. Control group: fed with basal feed; Experimental group: Before feeding, the black soldier fly broth prepared in Examples 1-5 and Comparative Examples 1-2 of this invention was added to the basic feed at a mass percentage of 3%, stirred evenly, and air-dried before use. These were recorded as Examples 1-5 and Comparative Examples 1-2, respectively. The fish fry were pearl grouper, sourced from Hainan Chenhai Aquatic Products Co., Ltd. After 27 days of hatching, the fry were raised on commercial feed for one week. Healthy fry of uniform size were selected as experimental fish. A total of 720 fry were randomly divided into 24 600L rearing tanks, with 30 fry per tank. The 24 tanks were then randomly divided into 8 treatments, with 3 replicates per treatment, and each treatment was fed one of the 8 different feeds. The rearing tanks were aerated 24 hours a day, and the experiment lasted 30 days.

[0061] Water quality in each tank was tested daily, and water and air temperatures were recorded twice daily, at 9:00 AM and 5:00 PM. The number of dead fish was recorded daily. Fish were fed three times daily, at 8:00 AM, 12:00 PM, and 5:00 PM, stopping each feeding while a small number of fish were still competing for food. After 30 days, the weight of each tank of fish was weighed and divided by the number of surviving fish to calculate the average weight of each fish. The average survival rate of the farmed fish was calculated based on the total number of surviving fish after 30 days and the initial number of fish.

[0062]

[0063] As shown in Table 3, compared with the control group, the experimental groups supplemented with black soldier fly larvae fermentation liquid showed increased average weight gain and average survival rate. Comparing Examples 1-5 and Comparative Examples 1-2, the average weight gain and average survival rate of Examples 1-5 were higher than those of Comparative Examples 1-2. Specifically, Example 5 achieved an average weight gain of 9.26g and an average survival rate of 96.2%, demonstrating the best performance among all feeds, resulting in excellent fry growth.

Claims

1. A method for preparing black soldier fly larvae fermentation broth, characterized in that, Includes the following steps, by weight: (1) Mix 20-30 parts of black soldier fly powder, 3-6 parts of wheat bran, 3-5 parts of enzymatic hydrolyzed Miscanthus powder, and 45-60 parts of sterile water evenly to obtain a mixture. Then add 1-2% of the total weight of the mixture of aerobic fermentation agent and 0.1-0.3% of the total weight of the mixture of papain and mix evenly. Then ferment aerobically at 35-37℃ for 1-3 days to obtain the primary fermentation material of black soldier fly. (2) Mix the primary fermentation material of black soldier fly with the anaerobic fermentation agent at a weight ratio of 100:1-2, and then seal and anaerobic ferment at 35-37℃ for 3-5 days to obtain the black soldier fly fermentation liquid. The enzymatically hydrolyzed Miscanthus powder is either Miscanthus powder that has been treated with a compound enzyme or Miscanthus powder that has been pretreated with a water-γ-valerol mixed solvent, a p-toluenesulfonic acid-γ-valerol mixed solvent, or an ethyl lactate-γ-valerol mixed solvent as pretreatment solvents before being treated with a compound enzyme. The aerobic fermentation agent is selected from at least one of Bacillus subtilis bacterial liquid and Aspergillus oryzae bacterial liquid; The anaerobic fermentation agent is obtained by mixing Bacillus amyloliquefaciens bacterial solution, Lactobacillus plantarum bacterial solution, and a plant compound extract composed of Rubia cordifolia extract and Ulva prolifera extract.

2. The method for preparing black soldier fly fermentation broth according to claim 1, characterized in that: The black soldier fly powder is obtained by blanching black soldier fly larvae in boiling water for 1-2 minutes to kill them, drying them at 60-65℃, pulverizing them, and then passing them through a 100-200 mesh sieve.

3. The method for preparing black soldier fly fermentation broth according to claim 1, characterized in that: The method for preparing the enzymatically hydrolyzed Miscanthus powder is as follows, by weight: After drying the Miscanthus stalks, crush them and pass them through a 100-200 mesh sieve to obtain Miscanthus powder. Dry the Miscanthus powder at 60-80°C until the moisture content is <5% to obtain dried Miscanthus powder for later use. Add 1-3 parts of dried Miscanthus powder to 40-80 parts of sodium acetate buffer solution with a pH of 4-6, preheat in a constant temperature shaking incubator at 45-55°C for 20 minutes, add 0.1-0.3% of the total weight of the dried Miscanthus powder with a compound enzyme, and enzymatically hydrolyze for 24-48 hours at a reaction temperature of 40-60°C and 200-400 rpm. After enzymatic hydrolysis, boil in a boiling water bath for 10-20 minutes to inactivate residual cellulase and obtain enzymatically hydrolyzed Miscanthus powder. Alternatively, the Miscanthus stalks can be dried, pulverized, and passed through a 100-200 mesh sieve to obtain Miscanthus powder. The Miscanthus powder is then dried at 60-80°C until the moisture content is <5%, yielding dried Miscanthus powder for later use. The dried Miscanthus powder is then mixed with a pretreatment solvent at a solid-liquid ratio of 1:5-10 (g:mL), and reacted at 90-120°C for 1-3 hours under a nitrogen atmosphere. After cooling to room temperature, the solid residue is separated by vacuum filtration. The solid residue is washed with water until neutral, and then dried to obtain the pretreated Miscanthus powder. Prepare Miscanthus sinensis powder; add 1-3 parts of pretreated Miscanthus sinensis powder to 40-80 parts of sodium acetate buffer solution with a pH of 4-6, preheat in a constant temperature shaking incubator at 45-55℃ for 10-20 min, add 0.1-0.3% of compound enzyme by weight of the total weight of dried Miscanthus sinensis powder, and enzymatically hydrolyze for 24-48 h at a reaction temperature of 40-60℃ and 200-400 rpm; after enzymatic hydrolysis, boil in a boiling water bath for 10-20 min to inactivate residual cellulase, and obtain enzymatically hydrolyzed Miscanthus sinensis powder.

4. The method for preparing black soldier fly fermentation broth according to claim 3, characterized in that: The pretreatment solvent is a water-γ-valerolactone mixed solvent obtained by mixing water and γ-valerolactone at a volume ratio of 1:2-5, or a p-toluenesulfonic acid-γ-valerolactone mixed solvent obtained by mixing p-toluenesulfonic acid aqueous solution with a concentration of 20-60 g / L and γ-valerolactone at a volume ratio of 1:2-5, or a lactate-γ-valerolactone mixed solvent obtained by mixing ethyl lactate and γ-valerolactone at a volume ratio of 1:2-5.

5. The method for preparing black soldier fly fermentation broth according to claim 3, characterized in that: The complex enzyme is composed of ligninase, cellulase, and hemicellulase mixed in a weight ratio of 0.5-1:1-2:1-2.

6. The method for preparing black soldier fly fermentation broth according to claim 1, characterized in that: The effective viable bacteria content of the Bacillus subtilis bacterial solution is 2×10⁻⁶. 7 -6×10 7 CFU / mL; the effective viable bacteria content of the Aspergillus oryzae solution is 2×10⁻⁶. 6 -6×10 6 CFU / mL.

7. The method for preparing black soldier fly fermentation broth according to claim 1, characterized in that: The effective viable bacteria content of the *Lactobacillus plantarum* bacterial solution is 2 × 10⁻⁶. 7 -6×10 7 CFU / mL; the effective viable bacterial count of the Bacillus amyloliquefaciens is 2×10⁻⁶. 6 -6×10 7 CFU / mL.

8. A black soldier fly larvae fermentation broth, characterized in that: Prepared by the method described in any one of claims 1-7.

9. The application of the black soldier fly fermentation broth according to claim 8 in the preparation of feed.

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