Multi-component resource utilization process of hermetia illucens worm pulp

Abstract

This invention provides a multi-component resource utilization process for black soldier fly larvae larvae slurry, relating to the field of insect resource utilization technology. The process sequentially includes raw material pretreatment, compound enzymatic hydrolysis, lactic acid bacteria fermentation, stratified separation, and targeted treatment of each component. The compound enzymatic hydrolysis uses a combined enzyme system of trypsin and papain, and the lactic acid bacteria fermentation uses *Lactobacillus plantarum*. The fermentation parameters are: carbon-to-nitrogen ratio of 20:1, material-to-liquid ratio of 4:1 (w / v), pH of 6.5, fermentation temperature of 37°C, and fermentation time of 24 hours. This process achieves the synergistic recovery and high-value utilization of all components of black soldier fly larvae. A single process can simultaneously produce four types of products: insect oil, water-soluble fertilizer, antimicrobial peptide additives, and antibiotic-free aquatic feed ingredients, significantly improving resource utilization and industrial added value. The entire process involves no organic solvent addition, employs low-temperature enzyme inactivation to reduce energy consumption, is environmentally friendly and energy-saving, and avoids antibiotic resistance issues.

Description

Technical Field

[0001] This invention relates to the field of resource utilization technology of insect resources, specifically a multi-component resource utilization process for black soldier fly larvae slurry. Background Technology

[0002] In the current livestock farming and food industry, the problem of antibiotic resistance caused by overuse is becoming increasingly prominent. Developing safe, efficient, and residue-free natural antibacterial agents, as well as green and environmentally friendly antibiotic-free feed, has become a core demand for industry development. The high-value utilization of resource insects provides an important solution to this demand. Black soldier flies, as a typical resource insect, possess significant advantages such as rapid reproduction, low feeding costs, and high bioconversion rates. Their larvae are rich in 40%–60% protein and 20%–30% fat, and also contain various bioactive substances such as antimicrobial peptides. This makes them an excellent carrier for preparing natural antibacterial agents and feed ingredients. Furthermore, black soldier flies can be fed using organic waste such as kitchen waste and livestock manure, achieving resource utilization of waste while reducing breeding costs, which highly aligns with the industrial concepts of green development and circular economy.

[0003] Currently, the industry's utilization of black soldier fly larvae is mainly based on direct powdering as a feed ingredient, lacking in-depth development of its rich active ingredients and nutritional components. The overall resource utilization rate is at a low level. Existing processing technologies not only fail to achieve the synergistic recovery of insect oil, antimicrobial peptides, small molecule peptides, and antibiotic-free feed ingredients, but also generally suffer from problems such as excessively high oil content leading to decreased feed palatability and digestibility, incomplete degradation of large protein molecules affecting absorption and utilization, and serious loss of bioactive substances. Furthermore, some processes rely on organic solvent extraction and high-temperature and high-pressure pretreatment, which not only easily cause environmental pollutant emissions but also have drawbacks such as high energy consumption and damage to product activity. These factors severely restrict the high-value development and industrialization of black soldier fly resources. Therefore, it is urgent to develop a green, energy-saving, efficient, and high-value multi-component resource utilization process for black soldier flies to achieve targeted recovery and comprehensive utilization of its various components, thereby enhancing industrial added value and market competitiveness. Summary of the Invention

[0004] Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a multi-component resource utilization process for black soldier fly larvae slurry. This process solves the problems of low resource utilization rate, inability to synergistically recover multiple components, excessive oil content, low protein absorption rate, and environmental and energy consumption issues related to the use of organic solvents and high temperature and energy consumption in existing black soldier fly utilization processes.

[0005] Technical solution To achieve the above objectives, the present invention provides the following technical solution: a multi-component resource utilization process for black soldier fly larvae slurry, comprising the following steps: S1 raw material pretreatment involves selecting fresh black soldier fly larvae, washing, blanching, freezing, and low-temperature crushing to obtain black soldier fly larvae porridge; S2 complex enzymatic hydrolysis involves adding deionized water to the black soldier fly larvae and adjusting the pH, adding a complex enzyme system of trypsin and papain, pre-treating with ultrasound, hydrolyzing at a constant temperature, and then inactivating the enzyme at low temperature and cooling to obtain the larvae hydrolysate. S3 lactic acid bacteria fermentation involves adding glucose to the insect mollusc hydrolysate to adjust the carbon-nitrogen ratio, adjusting the pH, inoculating with a suspension of Lactobacillus plantarum, and then performing anaerobic fermentation at a constant temperature to obtain the fermentation product. S4 layer separation: The fermentation product is naturally separated into layers after high-speed centrifugation to obtain upper layer insect oil, middle layer small molecule peptide aqueous solution, and lower layer insect residue. S5 involves targeted processing of each component, specifically processing the insect oil, small molecule peptide aqueous solution, and insect residue to prepare corresponding finished products; The material-to-liquid ratio of the compound enzymatic hydrolysis is 4:1 (w / v), the carbon-to-nitrogen ratio of the lactic acid bacteria fermentation is 20:1, the pH is 6.5, the inoculum amount is 3% (v / w), the fermentation temperature is 37℃, and the fermentation time is 24h.

[0006] Preferably, the raw material pretreatment steps are as follows: select fresh black soldier fly larvae with a body length of 2-3 cm, rinse with clean water, blanch at 100℃ for 2 min, freeze at -30℃ for 30 min, and crush at 20000 r / min for 1 min at ≤45℃ to obtain black soldier fly larvae dregs.

[0007] Preferably, the steps of the compound enzymatic hydrolysis are as follows: adding deionized water to black soldier fly larvae at a material-to-liquid ratio of 4:1 (w / v), adjusting the pH to 7.5, adding a compound enzyme system with an enzyme activity of 580 U / g, ultrasonic pretreatment for 5 min, enzymatic hydrolysis for 2 h at 48℃ and 1500 r / min, followed by enzyme inactivation in a boiling water bath at 90~100℃ for 10 min, and cooling to room temperature; the compound enzyme system contains trypsin and papain in a 1:1 ratio.

[0008] Preferably, before the lactic acid bacteria fermentation, glucose is added to the enzyme-inactivated and cooled insect molasses hydrolysate to adjust the carbon-nitrogen ratio to 20:1, the solution is stirred and dissolved, the pH is adjusted to 6.5, and then a 3% (v / v) suspension of *Lactobacillus plantarum* is inoculated. After sealing, fermentation is carried out.

[0009] Preferably, the step of layer separation is as follows: the fermented material is centrifuged at 8000 r / min for 10 min, and the material naturally separates into upper layer insect oil, middle layer small molecule peptide aqueous solution, and lower layer insect residue.

[0010] Preferably, in the targeted processing of each component, the insect oil is processed by placing the stratified material at 4°C for refrigeration, and then taking it out and recycling it after the upper layer of insect oil has solidified.

[0011] Preferably, in the targeted treatment of each component, the small molecule peptide aqueous solution is treated as follows: after filtration through a 0.45μm organic phase filter membrane, it is directly used as a small molecule peptide water-soluble fertilizer, or after freeze-drying at -50℃ for 24h, antimicrobial peptide dry powder is obtained; the proportion of components with a molecular weight ≤10kDa in the antimicrobial peptide dry powder is ≥90%.

[0012] Preferably, in the targeted treatment of each component, the insect residue is treated as follows: dried at 80°C for 24 hours or frozen at -18°C and then vacuum freeze-dried for 24 hours, and then pulverized through an 80-mesh sieve to obtain black soldier fly powder; the black soldier fly powder has an oil content of 6% to 12% and a protein content of 42.89% to 53.95%.

[0013] Beneficial effects This invention provides a multi-component resource utilization process for black soldier fly larvae slurry, which has the following beneficial effects: 1. This invention provides a multi-component resource utilization process for black soldier fly larvae larvae. This invention achieves targeted recovery and high-value utilization of all components of black soldier fly larvae. A single process can simultaneously yield four types of products: insect oil, small molecule peptide water-soluble fertilizer, antimicrobial peptide additives, and antibiotic-free aquatic feed ingredients. This significantly improves the comprehensive utilization rate of black soldier fly resources, effectively taps into the industrial value of black soldier fly resources, and promotes the large-scale development of the insect resource utilization industry.

[0014] 2. This invention provides a multi-component resource utilization process for black soldier fly larvae slurry. The process of this invention does not use any additional organic solvents throughout, thus avoiding the environmental pollution problems caused by organic solvents. At the same time, it adopts a low-temperature enzyme inactivation process instead of the traditional high-pressure sterilization process, significantly reducing the overall energy consumption of the process and optimizing its environmental friendliness and energy efficiency, aligning with green development policies and industry development needs. Detailed Implementation

[0015] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0016] This embodiment uses 1 kg of fresh black soldier fly larvae as raw material to carry out the process. Among the experimental materials used, healthy black soldier fly larvae with a body length of 2-3 cm and a fresh weight of about 0.1 g / head were selected. Lactobacillus plantarum and Lactobacillus acidophilus were purchased from the China Microbiological Culture Collection Center, and Escherichia coli and Staphylococcus aureus were purchased from the China General Microbiological Culture Collection Center. The enzyme activities of trypsin and papain were both 580 U / g. Glucose and other reagents were of analytical grade. The multi-functional crusher, constant temperature shaker, high-speed centrifuge, freeze dryer, Soxhlet extractor, Kjeldahl nitrogen analyzer and other instruments and equipment were all commercially available standard products without special restrictions.

[0017] Take 1 kg of the above-mentioned fresh black soldier fly larvae, rinse them repeatedly with clean water to remove surface impurities and dirt, and then blanch them in a boiling water bath at 100℃ for 2 minutes to quickly inactivate pathogenic bacteria and other bacteria on the surface. After blanching, quickly transfer the larvae to a -30℃ low-temperature environment and freeze them for 30 minutes to break down the cell walls of the larvae by forming ice crystals. Then, put the frozen larvae into a multi-functional crusher and crush them at a speed of 20,000 r / min for 1 minute at a low temperature of ≤45℃. The temperature is controlled throughout the process to avoid protein denaturation and oil oxidation, and finally homogenized black soldier fly larvae dregs are obtained, which provides sufficient reaction contact area for subsequent enzymatic hydrolysis. The prepared black soldier fly larvae were transferred into a reaction vessel, and 4 L of deionized water was added at a material-to-liquid ratio of 4:1 (w / v). After thorough mixing, the pH of the system was adjusted to 7.5. A complex enzyme system of trypsin and papain was added at a dosage of 580 U / g, with a 1:1 ratio of the two enzymes. After adding the enzyme system, the system was subjected to ultrasonic pretreatment for 5 min to further disperse the system and reduce mass transfer resistance. The reaction vessel was then placed in a constant temperature shaker at 48℃ and 1500 r / min for 2 h of constant temperature enzymatic hydrolysis. During the enzymatic hydrolysis, the synergistic effect of the two enzymes was used to achieve the directional degradation of macromolecular proteins. After the enzymatic hydrolysis reaction was completed, the reaction vessel was placed in a 95℃ boiling water bath for 10 min to inactivate the enzyme protein and terminate the enzymatic hydrolysis reaction, avoiding excessive enzymatic hydrolysis that would lead to the degradation of small molecule peptides. After the enzyme inactivation was completed, the system was naturally cooled to room temperature to obtain the black soldier fly larvae enzymatic hydrolysate.

[0018] Add glucose to the cooled black soldier fly larvae enzymatic hydrolysate and stir continuously until the glucose is completely dissolved. Adjust the carbon-to-nitrogen ratio of the system to 20:1, then adjust the pH of the system to 6.5. Inoculate the system with activated *Lactobacillus plantarum* suspension at a rate of 3% (v / w). The *Lactobacillus plantarum* suspension is activated with MRS liquid medium to a bacterial concentration of 1×10⁻⁶. 9After inoculation with CFU / mL, the mixture was thoroughly stirred and sealed with sterile sealing film. The container was then placed in a 37℃ incubator for anaerobic fermentation for 24 hours. During fermentation, the metabolic activity of *Lactobacillus plantarum* further degraded the lipid components in the system, while simultaneously promoting the enrichment of small molecule peptides and the synthesis of antimicrobial peptides, achieving targeted regulation of the system's nutritional and active components. After fermentation, the fermentation product of *Black Soldier Fly* was obtained. The fermentation product was transferred to a high-speed centrifuge at 8000 r / min for 10 min, utilizing the density differences of the components for efficient separation. After centrifugation, the fermentation product naturally separated into three layers: a pale yellow insect oil phase on top, a light brown small molecule peptide aqueous solution phase in the middle, and a grayish-brown insect residue solid phase on the bottom. Each layer was then collected separately for targeted processing and product preparation.

[0019] The material containing the insect oil phase was placed in a 4°C refrigerated environment. After the upper layer of insect oil completely solidified, the small amount of residual aqueous solution in the lower layer was removed. The solidified insect oil was collected and purified by vacuum filtration to remove trace impurities, yielding refined insect oil. The relevant test indicators of this refined insect oil are shown in Table 1. All indicators meet the standards for cosmetic-grade raw materials and can be directly used as raw materials in cosmetics, light industry, and other fields. The middle layer of small molecule peptide aqueous solution was vacuum filtered using a 0.45μm organic phase filter membrane to remove suspended impurities and bacteria, yielding purified small molecule peptide aqueous solution. This aqueous solution can be directly used as a small molecule peptide water-soluble fertilizer, and its relevant test indicators are shown in Table 2. It is suitable for the cultivation of various crops such as fruits and vegetables. When used in tomato cultivation, it can increase the growth rate of tomatoes by 15% to 20%. Part of the purified small molecule peptide aqueous solution was placed in a freeze dryer and vacuum dried at -50°C for 24 hours to obtain antimicrobial peptide powder. The performance indicators of this antimicrobial peptide powder are shown in Table 3. It can be used as an antimicrobial peptide additive for aquatic feed, etc., and can completely replace antibiotics. The insect residue collected from the lower layer was divided into two equal portions. The first portion was placed in an electric thermostatic drying oven and dried at 80°C for 24 hours. The second portion was first frozen at -18°C for 6 hours, and then transferred to a vacuum freeze dryer for 24 hours. The insect residues treated by the two drying methods were then pulverized using a high-speed pulverizer and passed through an 80-mesh standard sieve to obtain two types of black soldier fly powder, which were used as raw materials for antibiotic-free aquatic feed. The main nutritional indicators of the two black soldier fly powders are shown in Table 4. There were no pathogenic bacteria residues, and they fully met the nutritional standards for aquatic feed.

[0020] The black soldier fly powder prepared in this embodiment was used to replace fishmeal in the preparation of antibiotic-free shrimp feed. The optimized shrimp feed formula is as follows: 1.5% aquatic compound multivitamin, 7% wheat flour, 0.5% sea salt crystals, 2% microcrystalline cellulose, 15% soybean meal, 4% dicalcium phosphate, 1.5% sodium carboxymethyl cellulose, 2% corn flour, 0.8% betaine, 1.2% fish oil, 3% soybean lecithin, 35.3% fishmeal, 25.2% black soldier fly powder, and 1% yeast cell wall. The relevant nutritional indicators of the shrimp feed prepared by this formula are shown in Table 5. The oil content and protein content meet the nutritional standards of aquatic feed. Using this feed can reduce the cost of feed raw materials by 20%, and in the feeding trial, the survival rate of shrimp increased by 12% to 15% compared with traditional antibiotic-containing feed. In this embodiment, 1 kg of fresh black soldier fly larvae, after being processed by the above process, can simultaneously produce 85 g of refined insect oil, approximately 3.8 L of small molecule peptide water-soluble fertilizer, approximately 12 g of antibacterial peptide dry powder, and approximately 280 g of black soldier fly powder, achieving synergistic recovery of four types of products. The comprehensive utilization rate of resources is ≥90%. No additional organic solvents are used in the entire process. A 90~100℃ low-temperature enzyme inactivation process is used to replace the traditional high-pressure sterilization process, which reduces energy consumption by approximately 40% compared to the traditional high-temperature and high-pressure process. There are no pollutant emissions throughout the process, which combines the characteristics of green environmental protection and energy efficiency. Moreover, the parameters of each step of the process have been optimized and determined through multiple pre-experiments. Within the scope of protection defined by the claims, the process parameters can be reasonably fine-tuned according to the actual production scale, and the fine-tuned process can still achieve the same technical effect.

[0021] Table 1. Detection results of refined insect oil ; Table 2. Detection results of small molecule peptide water-soluble fertilizer ; Table 3. Detection results of antimicrobial peptide dry powder ; Table 4 Results of Black Soldier Fly Powder Detection ; Table 5. Results of Nutritional Index Testing in Antibiotic-Free Shrimp Feed ; Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A multi-component resource utilization process for black soldier fly larvae slurry, characterized in that, Includes the following steps: S1 raw material pretreatment involves selecting fresh black soldier fly larvae, washing, blanching, freezing, and low-temperature crushing to obtain black soldier fly larvae porridge; S2 complex enzymatic hydrolysis involves adding deionized water to the black soldier fly larvae and adjusting the pH, adding a complex enzyme system of trypsin and papain, pre-treating with ultrasound, hydrolyzing at a constant temperature, and then inactivating the enzyme at low temperature and cooling to obtain the larvae hydrolysate. S3 lactic acid bacteria fermentation involves adding glucose to the insect mollusc hydrolysate to adjust the carbon-nitrogen ratio, adjusting the pH, inoculating with a suspension of Lactobacillus plantarum, and then performing anaerobic fermentation at a constant temperature to obtain the fermentation product. S4 layer separation: The fermentation product is naturally separated into layers after high-speed centrifugation to obtain upper layer insect oil, middle layer small molecule peptide aqueous solution, and lower layer insect residue. S5 involves targeted processing of each component, specifically processing the insect oil, small molecule peptide aqueous solution, and insect residue to prepare corresponding finished products; The material-to-liquid ratio of the compound enzymatic hydrolysis is 4:1 (w / v), the carbon-to-nitrogen ratio of the lactic acid bacteria fermentation is 20:1, the pH is 6.5, the inoculum amount is 3% (v / w), the fermentation temperature is 37℃, and the fermentation time is 24h.

2. The multi-component resource utilization process for black soldier fly larvae slurry according to claim 1, characterized in that, The pretreatment steps for the raw materials are as follows: Select fresh black soldier fly larvae with a body length of 2-3 cm, rinse with clean water, blanch at 100℃ for 2 minutes, freeze at -30℃ for 30 minutes, and crush at 20000 r / min for 1 minute at ≤45℃ to obtain black soldier fly larvae dregs.

3. The multi-component resource utilization process for black soldier fly larvae slurry according to claim 1, characterized in that, The steps of the compound enzymatic hydrolysis are as follows: Deionized water is added to black soldier fly larvae at a material-to-liquid ratio of 4:1 (w / v), the pH is adjusted to 7.5, a compound enzyme system with an enzyme activity of 580 U / g is added, and after ultrasonic pretreatment for 5 min, enzymatic hydrolysis is carried out at 48℃ and 1500 r / min for 2 h, followed by enzyme inactivation in a boiling water bath at 90~100℃ for 10 min, and then cooled to room temperature; the compound enzyme system contains trypsin and papain in a compound ratio of 1:

1.

4. The multi-component resource utilization process for black soldier fly larvae slurry according to claim 1, characterized in that, Before the lactic acid bacteria fermentation, glucose is added to the enzyme-inactivated and cooled insect molasses hydrolysate to adjust the carbon-nitrogen ratio to 20:

1. After stirring and dissolving, the pH is adjusted to 6.5, and then a 3% (v / v) suspension of *Lactobacillus plantarum* is inoculated. After sealing, fermentation is carried out.

5. The multi-component resource utilization process for black soldier fly larvae slurry according to claim 1, characterized in that, The stratification separation step is as follows: the fermented material is centrifuged at 8000 r / min for 10 min, and the material naturally separates into upper layer insect oil, middle layer small molecule peptide aqueous solution, and lower layer insect residue.

6. The multi-component resource utilization process for black soldier fly larvae slurry according to claim 5, characterized in that, In the targeted processing of each component, the insect oil is processed as follows: the layered material is placed in a 4°C refrigerator, and the upper layer of insect oil is taken out and recycled after solidification.

7. The multi-component resource utilization process for black soldier fly larvae slurry according to claim 5, characterized in that, In the targeted treatment of each component, the small molecule peptide aqueous solution is treated as follows: after filtration through a 0.45μm organic phase filter membrane, it is directly used as a small molecule peptide water-soluble fertilizer, or it is freeze-dried at -50℃ for 24h to obtain antimicrobial peptide dry powder; the proportion of components with a molecular weight ≤10kDa in the antimicrobial peptide dry powder is ≥90%.

8. The multi-component resource utilization process for black soldier fly larvae slurry according to claim 5, characterized in that, In the targeted treatment of each component, the insect residue is treated as follows: after drying at 80℃ for 24 hours or freezing at -18℃, it is vacuum freeze-dried for 24 hours, then pulverized and passed through an 80-mesh sieve to obtain black soldier fly powder; the black soldier fly powder has an oil content of 6% to 12% and a protein content of 42.89% to 53.95%.