Method for extracting fly maggot chitin with high efficiency and low cost

Abstract

The invention discloses a high-efficiency and low-cost method for extracting fly maggot chitin. The method comprises fly maggot shell pretreatment, deproteinization treatment, demineralization treatment, purification and drying. According to the method, enzyme-alkali synergistic step-by-step deproteinization is adopted; crude protein is preliminarily removed by optimizing a compound enzyme ratio (chitinase: cellulase) and an enzymolysis condition; and performing secondary deproteinization by adopting a low-alkali NaOH solution to thoroughly remove residual protein. By means of the mild demineralization technology, damage of strong acid to the fly maggot chitin structure is avoided, then EDTA is added to assist demineralization, and the demineralization efficiency is improved. And finally, performing low-temperature drying, so that the fly maggot chitin maintains a three-dimensional porous structure, the subsequent derivatization activity is enhanced, and the extraction of the high-purity fly maggot chitin is realized. The whole process reduces use of strong acid and strong alkali, and reduces production cost and environmental pollution.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological resource utilization, and specifically relates to a method for efficiently and low-cost extracting chitin from fly maggot shells. The method is suitable for the fields of environmental protection and agriculture, and can realize efficient recycling of resources. BACKGROUND

[0002] Chitin is the second largest renewable polysaccharide in nature, and fly maggot chitin is a natural high-molecular polysaccharide extracted from fly maggot shells. It is the only active natural polysaccharide in chitin, and its chemical structure is similar to that of chitin in shrimp and crab shells. It is mainly composed of N-acetylglucosamine connected by β-1, 4-glycosidic bond. Its chitin content and activity are much higher than those of chitin extracted from shrimp and crab shells. The advantages of fly maggot are: fly larvae reproduce fast, and the protein content is high. The cuticle is rich in chitin (10%-15% of dry weight), and the structure is loose and easy to extract. The cost of fly maggot raw material is 50% lower than that of shrimp and crab shells, which is suitable for large-scale production. Fly maggot chitin has wide application in the fields of medicine, chemical industry, environmental protection, agriculture and other fields. In the field of agriculture, it can be used as seed treatment agent, seed coating agent, fruit preservative, induced fungicide, crop stress resistance agent, plant growth regulator, biological pesticide, biological fertilizer and soil conditioner, to promote plant growth and improve disease resistance.

[0003] At present, the extraction methods of fly maggot chitin mainly include traditional acid-base method, enzymatic method and microbial fermentation method. The following are several existing technologies and their defects: Document 1: Strong acid (such as HCl) and strong base (such as NaOH) are used to treat fly maggot shells alternately to extract chitin. The extraction rate of this method is about 75%, but there are the following problems: the use of strong acid and strong base leads to the partial degradation of chitin structure, and the product purity is low (≤80%), and the discharge of acid-base waste liquid causes serious pollution to the environment. The process is complex, the energy consumption is high, and the production cost is high. Document 2: Single chitinase is used to treat fly maggot shells to extract chitin. The extraction rate of this method is about 80%, but there are the following problems: the efficiency of single enzyme treatment is low, and it is difficult to fully degrade cellulose and other impurities in fly maggot shells; the reaction time is long (≥8 hours) because the enzyme reaction conditions (such as pH, temperature) are not optimized; the product contains high content of residual protein and inorganic salt, and the purity is low (≤92%). Document 3: Microbial fermentation method is used to extract fly maggot chitin. The extraction rate of this method is about 85%, but there are the following problems: the fermentation period is long (≥72 hours), and the production efficiency is low. The microbial culture conditions are complex, and the cost is high; the product contains high content of impurities, and the subsequent purification steps are complex.

[0004] Therefore, the fly maggot chitin extraction methods in the prior art generally have the following defects:

[0005] Low extraction efficiency: the extraction rate of traditional acid-base method and single enzymatic method is usually less than 85%.

[0006] Low product purity: Due to imperfect process, residual protein and inorganic salt content in product is high, purity is usually less than 92%.

[0007] High cost: Use of strong acid, strong base or microbial fermentation increases raw material cost and process complexity.

[0008] Environmental pollution: Discharge of acid, alkali waste liquid or fermentation waste liquid pollutes the environment.

[0009] Complex process: Existing methods usually require multi-step processing, complex process and high energy consumption. SUMMARY:

[0010] In order to solve the defects in the prior art, the present application discloses a high-efficiency and low-cost fly larva chitin extraction method, which optimizes the steps of enzymatic hydrolysis, deproteinization and demineralization, and solves the above problems:

[0011] A method for efficiently and low-cost extracting fly larva chitin, characterized in that it comprises the following steps:

[0012] Step 1, fly larva shell pretreatment: live fly larvae are starved for 24 hours, washed with water, inactivated in boiling water, dried at 60°C, and ground to 30-60 mesh to obtain fly larva shell powder.

[0013] Step 2, enzymatic hydrolysis: mix fly larva shell powder with complex enzyme (chitinase: cellulase 3:1-2:1) at a ratio of 1:10 (w / v), adjust pH to 5.5-6.0, and react at 50-55°C for 4-6 hours.

[0014] Step 3, deproteinization: after centrifugal separation of the enzymatic hydrolysis product, the precipitate is treated with 1-2% NaOH solution at 50-60°C for 1.5-2 hours to remove residual protein.

[0015] Step 4, demineralization: the deproteinized product is treated with 0.5-1% HCl solution at room temperature for 1-1.5 hours to remove inorganic salts, and 0.05-0.1M EDTA is added to assist demineralization to improve demineralization efficiency.

[0016] Step 5, purification and drying: the demineralized product is washed with deionized water until neutral, dried at low temperature 60°C, and high purity (≥99%) chitin is obtained.

[0017] Step 6, environmental protection: enzyme hydrolysis waste liquid is discharged after inactivation, EDTA is recovered by calcium salt precipitation, and protein precipitate is converted into organic fertilizer.

[0018] Preferably, the fly larva shell powder is ground to 40 mesh.

[0019] Preferably, the ratio of the enzyme-alkali synergistic stepwise deproteinization complex enzyme is chitinase: cellulase = 4:1.5 mixed at a ratio of 1:10 (w / v); the amount of complex enzyme added is 10% of the weight of fly maggot shell powder.

[0020] Preferably, the enzymatic protein decomposition treatment is performed at a pH of 5.8, a reaction temperature of 52°C, and a reaction time of 5 hours.

[0021] Preferably, the concentration of the NaOH solution in the deproteinization treatment is 1%, the treatment temperature is 60°C, and the treatment time is 2 hours.

[0022] Preferably, the concentration of the HCl solution in the demineralization treatment is 1%, the treatment temperature is room temperature, and the treatment time is 1 hour; 0.05M EDTA is added to assist in demineralization.

[0023] Preferably, the drying temperature in the purification and drying steps is 60°C.

[0024] Beneficial effects

[0025] High efficiency: Extraction rate ≥ 90% (Basis for achieving this extraction rate: the high efficiency of the complex enzyme in the enzyme-alkali synergistic stepwise deproteinization process, assisting the secondary deproteinization process with low-alkalinity NaOH to ensure full degradation of chitin; optimized conditions for deproteinization and demineralization processes to minimize product loss), product purity ≥ 99% (Basis for achieving this purity: the high efficiency of chitinase: cellulase 4:1.5 mixed enzymatic hydrolysis in the enzyme-alkali synergistic stepwise deproteinization process, followed by the high efficiency of 1% NaOH solution to assist in deproteinization; the high efficiency of 1% HCl solution combined with 0.05MEDTA in the demineralization process; and the thorough removal of residual impurities in the purification and drying steps).

[0026] Low cost: The enzyme-alkali synergistic stepwise deproteinization process and the acid-EDTA mild demineralization process replace strong acid and strong alkali treatment, reducing the use of chemicals and lowering production costs.

[0027] Environmental friendliness: No strong acid or alkali waste liquid is discharged, which meets the requirements of green process production and reduces environmental pollution. Attached Figure Description

[0028] Figure 1 The production process flow chart of this invention is as follows (raw material pretreatment → enzyme-alkali synergistic stepwise deproteinization → demineralization → purification → finished product). Detailed Implementation

[0029] This invention discloses a method for efficiently and cost-effectively extracting chitin from fly larvae, comprising the following steps:

[0030] Step 1, fly chitin pretreatment: live fly larvae were starved for 24 h, then inactivated by boiling water, dried at 60°C, and ground to 30-60 mesh to obtain fly chitin powder.

[0031] Particle size optimization scheme

[0032] The optimization of raw material particle size directly affects the efficiency of deproteinization / mineral removal, product purity, and yield. The following is an explanation of the particle size optimization scheme:

[0033] Particle size optimization range

[0034]

[0035] Specific effects and mechanisms of particle size on each link

[0036] 1. Enzymatic deproteinization stage (core limiting step) Particle size too small (<0.2 mm): Advantages: significantly improved enzyme hydrolysis rate (specific surface area ↑ 300%). Risks: fly chitin microfibrils are broken, molecular weight decreases (subsequent mineral removal degree is difficult to control); filtration is difficult (forming a colloidal layer).

[0037] Particle size too large (>0.6 mm): Enzymes are difficult to penetrate to the core, and residual protein ↑ (actual measurement: residual protein is 2-3 times higher when particle size is 0.8 mm than when particle size is 0.4 mm).

[0038] 2. Mild deproteinization stage with low alkali

[0039] 0.3-1.0 mm is reasonable: Alkali (1-2% NaOH) can penetrate into the interior of the particles and remove residual hydrophobic proteins from enzyme hydrolysis. Avoiding <0.3 mm when alkali excessively erodes chitin chains (unexpectedly increases mineral removal degree, affecting subsequent applications).

[0040] 3. Acid mineral removal stage

[0041] When particle size is ≤0.8 mm, 1% HCl can remove >95% ash in 1 h at room temperature; >0.8 mm requires extension to 90 min or increasing acid concentration (↑ risk of chitin hydrolysis).

[0042] 4. Conclusion: optimal solution and optimization range

[0043] Golden particle size: 0.4 mm (40 mesh) → balance enzyme hydrolysis efficiency, alkali treatment safety, and acid mineral removal speed. Preferred range: 0.3-0.6 mm (30-50 mesh), forbidden line: avoid >0.8 mm (mineral removal is not complete) or <0.2 mm (yield ↓, molecular weight ↓).

[0044] Pulverization to 30-50 mesh can increase the surface area of the raw material, improve the efficiency of subsequent enzymatic reaction, the residual protein can be controlled in <1.0%, ash <0.5%, yield >90% in this interval. If the pulverization is too coarse (such as <20 mesh), the enzymatic reaction is insufficient, and the demineralization is not complete; if it is too fine (such as >80 mesh), the energy consumption increases and the enzymatic product is easily lost, the yield decreases, and the molecular weight decreases. 0.3-0.6mm (30-50 mesh) is the preferred range of the present application, and 0.4mm (40 mesh) is the golden particle size.

[0045] Step 2, enzyme-alkali synergistic stepwise deproteinization treatment:

[0046] In the stepwise deproteinization process of complex enzymes (chitinase + cellulase) and alkali, high-efficiency deproteinization (residual protein <1%) and protection of chitin structure integrity need to be achieved through systematic optimization.

[0047] The pretreated fly maggot shell powder was mixed with a complex enzyme solution at a weight volume ratio (w / v) of 1:10 (i.e. 10 mL of complex enzyme solution was added for every 1 g of fly maggot shell powder), wherein the complex enzyme was composed of chitinase and cellulase at a mass ratio of 4:1.5, and the enzyme preparation activity requirements were as follows:

[0048] Chitinase activity definition: 1 unit (U) of enzyme activity is defined as the amount of enzyme required to release 1 μmol of N-acetylglucosamine per minute using carboxymethyl chitin as the substrate at pH 5.8 and 50°C;

[0049] Chitinase activity detection: 0.1 mL of enzyme solution was mixed with 0.9 mL of 1% carboxymethyl chitin solution (pH 5.8), and the reaction was carried out at 50°C for 30 minutes. Then, 1 mL of DNS reagent was added to terminate the reaction. After boiling in a water bath for 5 minutes and cooling, the absorbance was measured at a wavelength of 540 nm. The enzyme activity (U / mg) was calculated according to the N-acetylglucosamine standard curve.

[0050] Cellulase activity definition: 1 unit (U) of enzyme activity is defined as the amount of enzyme required to release 1 μmol of glucose per minute using carboxymethyl cellulose sodium as the substrate at pH 5.0 and 50°C;

[0051] Cellulase activity detection: 0.1 mL of enzyme solution was mixed with 0.9 mL of 1% carboxymethyl cellulose sodium solution (pH 5.0), and the reaction was carried out at 50°C for 30 minutes. Then, 1 mL of DNS reagent was added to terminate the reaction. After boiling in a water bath for 5 minutes and cooling, the absorbance was measured at a wavelength of 540 nm. The enzyme activity (U / mg) was calculated according to the glucose standard curve.

[0052] Chitinase: ≥100 U / mg (recommended: Sigma-Aldrich, C8241, or equivalent active commercial enzyme);

[0053] Cellulase: ≥ 50 U / mg (recommended: Novozymes Cellic, Ctec2, or equivalent active commercial enzyme).

[0054] After mixing, the pH of the system is adjusted to 5.5-6.0 using a pH meter (such as Mettler FE28), and the reaction system is placed in a constant temperature water bath shaker, set at a temperature of 50-55°C and a shaking speed of 150 rpm, and the reaction is continued for 4-6 hours. After the reaction is completed, the supernatant (containing degradation products) and the precipitate (containing chitin crude product) are separated by centrifugation at 8000 rpm for 10 minutes.

[0055] The ratio of the complex enzyme: The ratio of chitinase to cellulase is 4:1.5, which is optimized based on the ratio of chitin to cellulose in fly maggot shells. Chitinase mainly degrades chitin, while cellulase assists in degrading cellulose to avoid interference of cellulose with chitin extraction. If the ratio is unbalanced (e.g., too much chitinase), the enzymatic hydrolysis efficiency will decrease; if there is too much cellulase, the chitin structure may be damaged;

[0056] The amount of complex enzyme added is 10% of the weight of fly maggot shell powder; this amount of enzyme ensures that the enzymatic reaction proceeds sufficiently. If the amount of enzyme is too low (e.g., <5%), the reaction efficiency will decrease; if it is too high (e.g., >15%), the cost will increase and side reactions may occur;

[0057] pH value: The optimal activity range of the complex enzyme is pH 5.5-6.0, which ensures that the enzymatic reaction proceeds efficiently. If the pH is too low (e.g., <5.0) or too high (e.g., >6.5), the enzyme activity will decrease significantly, resulting in a decrease in reaction efficiency.

[0058] Reaction temperature and time: The optimal temperature range of the complex enzyme is 50-55°C, and the reaction is continued for 4-6 hours to ensure that chitin is sufficiently degraded into low molecular weight chitooligosaccharides. If the temperature is too low (e.g., <45°C), the reaction rate will be too slow; if it is too high (e.g., >60°C), the enzyme will be easily inactivated. If the reaction time is too short (e.g., <4 hours), chitin will not be sufficiently degraded; if it is too long (e.g., >6 hours), the energy consumption will increase and the product may be decomposed.

[0059] This step is the core step of the present application, and the selection of the ratio of the complex enzyme, pH value, temperature, and time directly affects the extraction efficiency and product purity.

[0060] Step 3, mild alkaline deproteinization: After deproteinization by the complex enzyme, mild alkaline treatment is needed to remove stubborn proteins that cannot be completely hydrolyzed by the enzyme method. The specific operation is as follows: after centrifugation of the enzymatic product, the precipitate is treated with 1% NaOH solution at 60°C for 2 hours to remove residual proteins.

[0061] NaOH concentration and temperature: 1% NaOH solution at 60°C for 2 hours can effectively remove proteins while avoiding the destruction of chitin structure; if the concentration of NaOH is too high (such as >1%), it may lead to chitin degradation, and if the concentration is too low (such as <0.5%), it cannot be fully degraded; if the temperature is too high (such as >70°C), it may cause chitin denaturation.

[0062] NaOH in this step can be replaced by other basic compounds such as KOH, but NaOH is more cost-effective and has better results. In addition, the deproteinization process is a key step to ensure product purity, and 1% NaOH solution at 60°C for 2 hours is the preferred condition of the present application.

[0063] Step 4, demineralization treatment: The deproteinized product is treated with 0.5-1% HCl solution at room temperature for 1 hour to remove inorganic salts; combined with 0.5% EDTA to assist in demineralization.

[0064] HCl concentration and treatment time: 1% HCl solution at room temperature for 1 hour can effectively remove inorganic salts while avoiding chitin hydrolysis. If the concentration of HCl is too low (such as <0.5%), the removal efficiency of inorganic salts is low, and if the concentration of HCl is too high (such as >1%), it may lead to chitin degradation; if the treatment time is too long (such as >2 hours), it increases energy consumption and may cause product loss; EDTA as a chelating agent can effectively remove metal ions such as calcium and magnesium, further improving the demineralization efficiency to 98%; if EDTA is not added, the demineralization efficiency may be reduced to below 95%. HCl can be replaced by other weak acids (such as acetic acid), but HCl has higher demineralization efficiency and lower cost.

[0065] Demineralization treatment is an important step to ensure product purity, and 1% HCl solution combined with 0.5% EDTA is the preferred condition of the present application.

[0066] Step 5, purification and drying: The demineralized product is washed with deionized water until neutral, and dried to obtain high-purity fly maggot chitin.

[0067] Washing and drying conditions: Washing with deionized water until neutral can remove residual acid and base substances, and drying at 60°C can ensure the structural stability of fly maggot chitin. If the drying temperature is too high (such as >70°C), it may cause fly maggot chitin denaturation. Purification and drying are the last step to ensure product purity and stability, and drying at 60°C is the preferred condition of the present application.

[0068] The demineralized product needs to be washed to neutral by the following steps:

[0069] Primary washing: Mix the demineralized product with deionized water at 1:20 (w / v) (i.e. 1 g product added to 20 mL water), magnetically stir for 10 minutes, centrifuge (8000 rpm, 10 minutes) to separate the supernatant from the precipitate;

[0070] pH detection: Take the supernatant and detect the pH with precision pH paper (such as Whatman pH 4.0-7.0) or a pH meter: if the pH is in the range of 6.5-7.5, it is determined to be neutral; if the pH is out of range, repeat steps 1-2 until the pH of the supernatant meets the standard;

[0071] Conductivity verification: Use a conductivity meter (such as METTLER TOLEDO SevenExcellence) to detect the conductivity of the last washing liquid, which requires a conductivity of ≤10 μS / cm, if it does not meet the standard, repeat the washing until it meets the requirements to ensure that the inorganic salt residue meets the standard.

[0072] Step 6: Residual liquid treatment

[0073] Enzymatic waste liquid treatment: Heat the enzymatic waste liquid to 100°C and maintain for 10 minutes to inactivate the enzyme activity, adjust the pH to 6.5-7.5, and then discharge;

[0074] EDTA recovery operation:

[0075] 1. CaCl 22 Addition: Add CaCl2 to the demineralized waste liquid to a final concentration of 1% (w / v) (i.e. add 1 g CaCl2 per 100 mL waste liquid), magnetically stir for 30 minutes;

[0076] 2. Centrifugation conditions: centrifuge at 8000 rpm for 10 minutes, collect the precipitate (EDTA-Ca complex), and wash with deionized water 3 times;

[0077] 3. Drying: dry at 60°C to recover EDTA, with a recovery rate of ≥90%.

[0078] Waste residue utilization: The protein precipitate after centrifugation (step 3) can be used as an organic fertilizer raw material after drying.

[0079] Example 1

[0080] 1. Fly shell pretreatment: Take 100 g of fly shell, wash and dry, crush through a 40 mesh sieve, and obtain fly shell powder.

[0081] 2. Enzyme-alkali synergistic stepwise deproteinization:

[0082] (1) Enzymatic treatment: The fly chitin shell powder was mixed with a complex enzyme (chitinase: cellulase = 4: 1.5) at a ratio of 1:10 (w / v), the pH was adjusted to 5.8, and the reaction was carried out at 52°C for 5 hours, wherein: chitinase uses Sigma-Aldrich C8241 or Novozymes CTec2, without limitation.

[0083] (2) Low-alkalinity NaOH solution treatment: After centrifugal separation of the enzymatic product, the precipitate was treated with 1% NaOH solution at 60°C for 2 hours.

[0084] 3. Demineralization treatment: The deproteinized product was treated with 1% HC1 solution at room temperature for 1 hour. By adding 0.05M EDTA to assist demineralization, the demineralization efficiency was improved.

[0085] 4. Purification and drying: The demineralized product was washed with deionized water to neutral, and dried at 60°C to obtain high-purity chitin.

[0086] 5. Environmental treatment: After inactivation of the enzymatic waste liquid, the pH was adjusted to 6.5-7.5, and EDTA was recovered by CaCl2 precipitation (recovery rate 92%), and the protein precipitate was dried as an organic fertilizer.

[0087] The following data results were obtained after testing:

[0088] Extraction rate: 92.6%

[0089] Product purity: 99.5% (protein residue 0.2%, ash 0.3%)

[0090] Deproteinization efficiency: 98.0%

[0091] Demineralization efficiency: 96.0%

[0092] Conductivity 9.2 μS / cm

[0093] Example 2

[0094] 1. Fly chitin shell pretreatment: 100g of fly chitin shell was cleaned and dried, and then crushed to 30 mesh to obtain fly chitin shell powder.

[0095] 2. Enzyme-alkali synergistic deproteinization:

[0096] (1) Enzymatic treatment: The fly chitin shell powder was mixed with a complex enzyme (chitinase: cellulase = 3: 1) at a ratio of 1:10 (w / v), the pH was adjusted to 6.0, and the reaction was carried out at 55°C for 6 hours, wherein: chitinase uses Sigma-Aldrich C8241 or Novozymes CTec2, without limitation.

[0097] (2) Low alkalinity NaOH solution treatment: after centrifugal separation of the enzymatic product, the precipitate is treated with 1.0% NaOH solution at 60°C for 2 hours.

[0098] 3. Demineralization treatment: after deproteinization, the product is treated with 0.5% HC1 solution at room temperature for 1 hour, and 0.03M EDTA is added to assist demineralization.

[0099] 4. Purification and drying: the demineralized product is washed with deionized water until neutral, and dried at 60°C to obtain high-purity chitin.

[0100] 5. Environmental treatment: after inactivation of the enzymatic waste liquid, the pH is adjusted to 6.5-7.5, and EDTA is recovered by CaCl2 precipitation (recovery rate 92%), and the protein precipitate is dried as an organic fertilizer.

[0101] After testing, the following data results are obtained:

[0102] Extraction rate: 91.3%

[0103] Product purity: 99.1% (protein residue 0.4%, ash 0.5%)

[0104] Deproteinization efficiency: 94.0%

[0105] Demineralization efficiency: 93.0%

[0106] Conductivity 9.7 μS / cm

[0107] Example 3

[0108] 1. Fly larva shell pretreatment: 100g of fly larva shell is cleaned and dried, crushed to 50 mesh to obtain fly larva shell powder.

[0109] 2. Enzyme-alkali synergistic stepwise deproteinization:

[0110] (1) Enzymatic treatment: the fly larva shell powder is mixed with a complex enzyme (chitinase: cellulase = 5:2.0) at a ratio of 1:10 (w / v), the pH is adjusted to 5.5, and the reaction is carried out at 60°C for 5 hours, wherein: chitinase uses Sigma-Aldrich C8241 or Novozymes CTec2, without limitation.

[0111] (2) Low alkalinity NaOH solution treatment: after centrifugal separation of the enzymatic product, the precipitate is treated with 1% NaOH solution at 60°C for 2 hours.

[0112] 3. Demineralization treatment: after deproteinization, the product is treated with 1% HC1 solution at room temperature for 1 hour, and 0.05M EDTA is added to assist demineralization.

[0113] 4. Purification and drying: The demineralized product is washed with deionized water until neutral, and dried at 60°C to obtain high-purity chitin.

[0114] 5. Environmental treatment: After inactivation of the enzymatic hydrolysis effluent, the pH is adjusted to 6.5-7.5, and EDTA is recovered by precipitation with CaCl2(recovery rate 92%), and the protein precipitate is dried as an organic fertilizer.

[0115] The following data were obtained after testing:

[0116] Extraction yield: 91.5%

[0117] Product purity: 99.1% (protein residue 0.5%, ash 0.4%)

[0118] Deproteinization efficiency: 93.0%

[0119] Demineralization efficiency: 96.0%

[0120] Conductivity 9.5 μS / cm

[0121] Example 4

[0122] 1. Fly larvae shell pretreatment: as in Example 1

[0123] 2. Enzyme-alkali synergistic deproteinization:

[0124] (1) Enzymatic treatment: the fly larvae shell powder is mixed with a complex enzyme (chitinase: cellulase = 4: 1.5) at a ratio of 1:10 (w / v), the pH is adjusted to 5.5, and the reaction is carried out at 55°C for 5 hours, wherein: chitinase is not limited to Sigma-Aldrich C8241 or Novozymes CTec2.

[0125] (2) Low-alkalinity NaOH solution treatment: after centrifugation of the enzymatic hydrolysate, the precipitate is treated with 0.5% NaOH solution at 60°C for 2 hours.

[0126] 3-5 steps: as in Example 1

[0127] The following data were obtained after testing:

[0128] Extraction yield: 91.6%

[0129] Purity: 99.2% (protein residue 0.4%, ash 0.4%)

[0130] Deproteinization efficiency: 93.5%

[0131] Demineralization efficiency: 96.5%

[0132] Conductivity 9.5 μS / cm

[0133] Example 5 (Enzymolysis time boundary value: 4 hours)

[0134] 1-2 step: Same as Example 1 (adjust the enzymolysis time to 4 hours).

[0135] 3-5 step: Same as Example 1.

[0136] The following data results were obtained through testing:

[0137] Extraction rate: 91.8%

[0138] Purity: 99.0% (protein residue 0.6%, ash residue 0.4%)

[0139] Deproteinization efficiency: 90.5%

[0140] Demineralization green: 96.2%

[0141] Conductivity: 9.3 μS / cm

[0142] Example 6 (EDTA auxiliary concentration optimization: 0.1 M)

[0143] 1-3 step: Same as Example 1, 0.1 M EDTA (originally 0.05 M) was added in the demineralization treatment.

[0144] 4-5 step: Same as Example 1.

[0145] The following data results were obtained through testing:

[0146] Extraction rate: 91.2%

[0147] Purity: 99.1% (protein residue 0.2%, ash residue 0.7%)

[0148] Deproteinization rate: 9%

[0149] Demineralization efficiency: 93.0%

[0150] Conductivity: 9.6 μS / cm

[0151] Comparative Example 1 (Enzyme-alkali synergistic stepwise deproteinization composite enzyme ratio imbalance)

[0152] 1. Fly shell pretreatment: Take 100 g of fly shell, clean and dry, crush to 40 mesh to obtain fly shell powder.

[0153] 2. Enzyme-alkali synergistic stepwise deproteinization

[0154] (1) Enzymolysis treatment: Mix fly shell powder with composite enzyme (chitinase: cellulase = 2:1) at a ratio of 1:10 (w / v), adjust pH to 5.8, and react at 52°C for 5 hours.

[0155] (2) Low alkalinity NaOH solution treatment: after centrifugal separation of the enzymatic product, the precipitate was treated with 1% NaOH solution at 60°C for 2 hours.

[0156] 3-5 steps were the same as in Example 1

[0157] The following data results were obtained after testing:

[0158] Extraction rate: 85.2%

[0159] Product purity: 95.1% (protein residue 2.9%, ash 2.0%)

[0160] Deproteinization efficiency: 88.6%

[0161] Demineralization efficiency: 92.5%

[0162] Conductivity: 10.9 μS / cm

[0163] Conclusion: Enzyme imbalance leads to a decrease in enzymatic efficiency, and significantly reduces the extraction rate and product purity.

[0164] Comparative Example 2 (pH is not suitable in the process of enzyme-alkali synergistic deproteinization enzymolysis)

[0165] 1. Fly shell pretreatment: 100 g of fly shell was cleaned and dried, crushed to 40 mesh, and fly shell powder was obtained.

[0166] 2. Enzyme-alkali synergistic stepwise deproteinization:

[0167] (1) Enzymolysis treatment: the fly shell powder was mixed with the complex enzyme (chitinase: cellulase = 4: 1.5) at a ratio of 1:10 (w / v), the pH was adjusted to 4.5, and the reaction was carried out at 52°C for 5 hours.

[0168] (2) Deproteinization treatment: after centrifugal separation of the enzymatic product, the precipitate was treated with 1% NaOH solution at 60°C for 2 hours.

[0169] 3-5 steps were the same as in Example 1

[0170] The following data results were obtained after testing:

[0171] Extraction rate: 80.6%

[0172] Product purity: 92.5% (protein residue 4.5%, ash 3.0%)

[0173] Deproteinization efficiency: 86.2%

[0174] Demineralization efficiency: 93.1%

[0175] Conductivity: 10.8 μS / cm

[0176] Conclusion: Too low pH value in the enzymatic process leads to the decrease of enzyme activity, extraction rate and product purity.

[0177] Comparative Example 3 (Too high temperature in the enzymatic process in enzyme-alkali synergistic stepwise deproteinization)

[0178] 1. Fly chitin pretreatment: 100 g of fly chitin was cleaned, dried, and ground to 40 mesh to obtain fly chitin powder.

[0179] 2. Enzyme-alkali synergistic stepwise deproteinization:

[0180] (1) Enzymatic treatment: The fly chitin powder was mixed with a complex enzyme (chitinase: cellulase = 4: 1.5) at a ratio of 1:10 (w / v), the pH was adjusted to 5.8, and the reaction was carried out at 70°C for 5 hours.

[0181] (2) Low-alkalinity NaOH solution treatment: After centrifugal separation of the enzymatic product, the precipitate was treated with 1% NaOH solution at 60°C for 2 hours.

[0182] Steps 3-5 are the same as in Example 1

[0183] The following data results were obtained after testing:

[0184] Extraction rate: 76.0%

[0185] Product purity: 88.0% (8.0% protein residue, 4.0% ash)

[0186] Deproteinization efficiency: 82.0%

[0187] Demineralization efficiency: 90.8%

[0188] Conductivity: 10.8 μS / cm.

[0189] Conclusion: Too high temperature in the enzymatic process leads to enzyme inactivation, significant decrease in extraction rate and product purity, and deformation of fly chitin structure (infrared spectrum shows that β-glycosidic bonds are broken).

[0190] Comparative Example 4 (No EDTA added in the demineralization process)

[0191] Steps 1-2 are the same as in Example 1

[0192] 3. Demineralization treatment: The deproteinized product was treated with 1% HCl solution at room temperature for 1 hour (without adding EDTA).

[0193] Steps 4-5 are the same as in Example 1.

[0194] The following data results were obtained after testing:

[0195] Extraction rate: 89.1%

[0196] Product purity: 98.7% (protein residue 0.5%, ash content 0.8%)

[0197] Deproteinization efficiency: 90.3%

[0198] Demineralization efficiency: 90.8%

[0199] Electrical conductivity: 11.3 μS / cm.

[0200] Conclusion: The absence of EDTA resulted in reduced demineralization efficiency, and a slight decrease in extraction rate and product purity.

[0201] Comparative Example 5 (Acid concentration exceeded the standard during demineralization process: 2% HCl)

[0202] Steps 1-3: Same as in Example 1, but with demineralization treatment to increase the HCl concentration to 2% (originally 1%).

[0203] Steps 4-5: Same as in Example 1.

[0204] The following data results were obtained after testing:

[0205] Extraction rate: 80%

[0206] Purity: 90% (6.0% ash residue)

[0207] Product molecular weight decreased by >30% (GPC detection)

[0208] Comparative Example 6 (Purified water temperature exceeded the standard: 90℃)

[0209] Steps 1-4: Same as in Example 1, but the temperature of the purified washing water is raised to 90°C (originally room temperature).

[0210] 5 steps: Same as Example 1.

[0211] The following data results were obtained after testing:

[0212] Extraction rate: 82%

[0213] Purity: 91% (5.5% protein residue, 3.5% ash)

[0214] The chitin in fly larvae turns yellow (due to thermal degradation).

[0215] Through comparative analysis of Examples 1-6 and Comparative Examples 1-6, the following conclusions are drawn:

[0216] 1. The technical solutions of the present invention (Examples 1-6) maintain an extraction rate of ≥90% and a product purity of ≥99% within the range of key parameters (enzyme ratio 3:1-2:1, pH 5.5-6.0, temperature 50-55℃, NaOH solution 0.5-1%, 60℃, 2h, HCl solution 0.5-1%, EDTA 0.05-0.1M), verifying the universality and stability of the solution.

[0217] 2. Deviation of any core parameter in Comparative Examples 1-6 (such as enzyme ratio imbalance, pH / temperature exceeding limits, acid / alkali concentration exceeding limits, EDTA deficiency, or process exceeding standards) will result in:

[0218] Extraction rate decreased by more than 10% (down to a minimum of 76%).

[0219] Purity decrease >8% (minimum 88%)

[0220] The residual amount of key impurities (protein / ash) surged by more than 300%.

[0221] 3. Data gradient proof:

[0222] The necessity of a mild process: strong acids / strong alkalis / high temperatures directly destroy the chitin structure (comparative examples 3, 5, and 6);

[0223] The irreplaceable nature of synergistic effects: Imbalance in enzyme ratios reduces extraction rate by 5-10% (Comparative Example 1);

[0224] Synergistic effect of EDTA: Demineralization efficiency decreased by >5% when EDTA was not added (Comparative Example 4).

[0225] In summary, the technical solution of the present invention is fully disclosed, feasible, and can solve the problems of low extraction efficiency, low product purity, high cost, and environmental pollution in the prior art.

[0226] This invention employs a stepwise enzymatic-alkali synergistic deproteinization process. By optimizing the ratio of the compound enzyme (chitinase:cellulase = 4:1.5) and the enzymatic hydrolysis conditions (pH 5.5-6.0, 50-55℃, hydrolysis for 4-6 hours), crude protein is removed. A second deproteinization process using low-alkali NaOH (1%, 60℃, 2 hours) completely removes residual protein (protein residue <1%). A gentle demineralization process (1% HCl, room temperature, 1 hour) avoids the damage to the chitin structure of fly larvae caused by strong acids. The addition of 0.05% MEDTA further enhances the demineralization efficiency to 98%, achieving an extraction efficiency ≥90% and a product purity ≥99%. This reduces the use of strong acids and alkalis, lowering production costs and environmental pollution. Finally, low-temperature drying maintains the three-dimensional porous structure of the fly larvae chitin, enhancing its subsequent derivatization activity.

[0227] The detection data results in the above embodiments and comparative examples, including the detection methods for extraction rate, product purity, deproteinization efficiency, demineralization efficiency, and conductivity, are as follows:

[0228] Extraction rate detection method:

[0229] Extraction rate = (Factory product weight of fly larvae chitin / Theoretical weight of raw fly larvae chitin) × 100%

[0230] Specific measurement process:

[0231] Theoretical quality of raw fly larvae chitin: The total nitrogen content of fly larvae shells was determined by the Kjeldahl method (GB / T35818-2018), an internationally recognized method for the quantitative determination of proteins and nitrogen-containing compounds. Then, the nitrogen content was determined based on the molecular formula of fly larvae chitin (C8H). 13 The nitrogen content of NO5 (approximately 6.9%) is used to calculate the theoretical chitin content of fly maggots.

[0232] Quality of fly larvae chitin finished product: The quality of the purified and dried fly larvae chitin finished product is directly weighed.

[0233] Product purity testing methods:

[0234] Purity = 100% - Protein Residue - Inorganic Salt Residue (Ash)

[0235] Specific measurement process:

[0236] Protein residues: Kjeldahl nitrogen determination method (GB / T35818-2018), an internationally recognized method for quantifying proteins and nitrogen-containing compounds.

[0237] Take 0.5g of fly larvae chitin sample, add potassium sulfate-copper sulfate catalyst, digest until clear at 420℃; after distillation, absorb ammonia with boric acid, and titrate with standard hydrochloric acid; calculate the protein content according to the formula: protein content (%) = nitrogen content × 6.25.

[0238] Inorganic salt residue (ash): Determination of ash content (GB / T 35818-2018).

[0239] Take 0.5g of fly larvae chitin sample, place it in a crucible, and ashing it in a muffle furnace at 550℃ for 4 hours; after cooling, weigh the residue and calculate the mass according to the formula:

[0240] Ash content (%) = (Mass of residue after ashing / Mass of sample) × 100%

[0241] Deproteinization efficiency testing method:

[0242] Deproteinization efficiency is calculated by the percentage reduction in protein content before and after treatment:

[0243] Deproteinization efficiency = [(Protein content before treatment - Protein content after treatment) / Protein content before treatment] × 100%

[0244] Specific measurement process:

[0245] The protein content before and after treatment was determined by the Kjeldahl method (same method as above).

[0246] Demineralization efficiency testing methods:

[0247] Demineralization efficiency is calculated by the percentage reduction in inorganic salt content before and after treatment:

[0248] Demineralization efficiency = [(Inorganic salt content before treatment - Inorganic salt content after treatment) / Inorganic salt content before treatment] × 100%

[0249] Specific measurement process:

[0250] The inorganic salt content before and after treatment was indirectly assessed using known methods of ash determination (as above) or conductivity method.

[0251] Conductivity method: Use a conductivity meter (such as METTLER TOLEDO Seven Excellence) to test the conductivity of the washing liquid. The final conductivity of the washing liquid should be ≤10μS / cm.

[0252] The above-mentioned detection methods (Kjeldahl nitrogen determination, ash content determination, and conductivity method) are all internationally recognized standard methods, conforming to national standards such as GB / T 35818-2018, and are considered well-known technologies. Of course, other well-known detection methods can also be used to detect the above results. The appendix further ensures the scientific validity and reliability of the data through experimental design (such as triple replication and one-way ANOVA).

[0253] The following provides a complete experimental design, data, and verification process example for particle size optimization experiments and enzyme ratio optimization verification experiments in the enzyme-alkali synergistic deproteinization process, to further enhance the scientific basis for parameter selection:

[0254] 1. Verify the effects of different particle sizes (0.21mm, 0.30mm, 0.42mm, 0.60mm, 0.85mm) on the extraction rate and purity of chitin from fly larvae, and determine the optimal particle size range.

[0255] 1. Raw material grouping: Take 500g of fly larvae shells and divide them into 5 groups (100g each). Crush them through 70 mesh, 60 mesh, 40 mesh, 30 mesh and 20 mesh sieves respectively.

[0256] 2. Subsequent processing: The enzyme-base combined synergistic stepwise method for deproteinization, demineralization and purification in Example 1 was used uniformly.

[0257] 4. Testing indicators: extraction rate and product purity (determined according to GB / T 35818-2018).

[0258] Experimental results

[0259] Pulverization particle size (mesh) Extraction rate (%) Deproteinization efficiency (%) Demineralization efficiency (%) Product purity (%) 20 75.3±0.9 88.6±0.3 93.2±0.5 92.5±0.4 30 90.5±0.7 95.8±0.6 95.5±0.6 99.3±0.1 40 92.6±0.5 98.0±0.3 96.0±0.3 99.5±0.2 60 90.3±0.8 97.3±0.2 94.8±0.6 99.1±0.3 70 83.1±0.9 86.6±0.5 91.6±0.4 89.2±0.5

[0260] In the process of extracting high-purity chitin from fly larvae using a synergistic stepwise deproteinization and acid demineralization process combining enzyme and alkali, the optimization of raw material particle size directly affects the deproteinization and demineralization efficiency, product purity, and yield. During the deproteinization stage using a compound enzyme method, excessively small particle sizes (<0.21 mm, 70 mesh) result in a significantly faster enzymatic hydrolysis rate, while excessively large particle sizes (>0.6 mm, 20 mesh) make it difficult for the enzyme to penetrate the core, leading to increased residual protein.

[0261] The above experimental results demonstrate that 40 mesh is the optimal particle size: the extraction rate (92.6%) and purity (99.5%) are significantly higher than other particle sizes (p<0.05), because 40 mesh balances surface area and energy consumption, resulting in a more complete enzymatic hydrolysis reaction. The defects of 20 mesh and 70 mesh are: 20 mesh particles are too coarse, resulting in insufficient contact area for enzymatic hydrolysis and a decrease in extraction rate; 70 mesh particles are too fine, easily lost during centrifugation, and increase energy consumption during grinding.

[0262] II. Enzyme ratio verification experiment: to verify the effect of different enzyme ratios (chitinase:cellulase = 3:1, 4:1.5, 5:2, 7:3, 2:1) on the chitin extraction effect of fly maggots, and to determine the optimal enzyme ratio.

[0263] 1. Enzyme ratio grouping:

[0264] Group 1: Chitinase: Cellulase = 3:1 (total enzyme content 10%);

[0265] Group 2: Chitinase: Cellulase = 4:1.5 (total enzyme content 10%);

[0266] Group 3: Chitinase: Cellulase = 5:2 (Total enzyme content 10%)

[0267] Group 4: Chitinase: Cellulase = 7:3 (Total enzyme content 10%)

[0268] Group 5: Chitinase: Cellulase = 2:1 (Total enzyme content 10%)

[0269] 2. Uniform conditions: 40-mesh fly larvae shell powder, enzymatic hydrolysis at pH 5.8, temperature 52℃, time 5 hours, and subsequent steps are the same as in Example 1.

[0270] 3. Testing indicators: extraction rate, product purity, and deproteinization efficiency.

[0271] Experimental results

[0272] Enzyme ratio Extraction rate (%) Product purity (%) Deproteinization efficiency (%) 3:1 86.8±0.7 99.3±0.3 99.7±0.2 4:1.5 92,6±0.5 99.5±0.2 99.5±0.3 5:2 91.3±0.6 99.3±0.3 99.4±0.3 7:3 90.1±0.5 99.1±0.1 99.3±0.2 2:1 86,3±0.6 98.2±0.3 89.5±0.3

[0273] The above experimental results demonstrate that the extraction rate of 4:1.5 (91.0%) is significantly higher than that of 3:1, 5:2, 7:3, and 2:1 (p<0.05). This ratio matches the natural content of chitin and cellulose in fly maggot shells, resulting in the best synergistic effect of enzymes. Imbalances in enzyme ratios (such as 3:1 or 2:1) lead to insufficient degradation of cellulose (2:1 group) or excessive hydrolysis of chitin (3:1 group), affecting purity.

[0274] III. Basis for Experimental Data Processing

[0275] 1. Data source: All experiments were repeated 3 times. Data are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS 26.0 (p < 0.05 was considered statistically significant).

[0276] 2. Testing standards and methods:

[0277] Extraction rate calculation method: Extraction rate = (finished product mass of fly larvae chitin / theoretical mass of raw fly larvae chitin) × 100%, where the theoretical mass of raw fly larvae chitin is calculated by determining the total nitrogen content of fly larvae shells using the Kjeldahl method;

[0278] Purity testing: According to GB / T 35818-2018, the specific procedures are as follows:

[0279] Protein residue determination: Take 0.5g of fly larvae chitin sample and determine the nitrogen content using the Kjeldahl method (digestion temperature 420℃, potassium sulfate-copper sulfate catalyst). Calculate the nitrogen content using the formula "protein content (%) = nitrogen content × 6.25".

[0280] Inorganic salt residue determination: Take 0.5g of fly larvae chitin sample and ashing it in a muffle furnace at 550℃ for 4 hours. Calculate the ash content according to the formula "ash content (%) = (mass of residue after ashing / mass of sample) × 100%".

[0281] Purity = 100% - protein residue - inorganic salt residue.

[0282] 3. Environmental control: All experiments were conducted in a constant temperature laboratory at 25℃ with humidity ≤60% to avoid environmental interference.

[0283] IV. Preparation method of fly maggot shells

[0284] 1. Inactivation and initial treatment of fly larvae

[0285] Live fly larvae (such as black soldier fly larvae) are immersed in boiling water for 8-10 minutes, or rapidly frozen to below -20°C for 24 hours to completely terminate their life activities. The purpose is to prevent soft tissue decay during subsequent processing and ensure the integrity of the shell structure.

[0286] After inactivation, rinse the fly larvae three times with clean water, stirring for 5 minutes each time to remove surface slime, feed residue, and impurities. Water quality requirements: Use deionized water or water sources that meet the "Standards for Drinking Water Quality".

[0287] 2. Mechanical crushing and shell-soft tissue separation

[0288] Crushing process:

[0289] Equipment: High-speed homogenizer (recommended speed 5000 rpm) or ball mill (grinding ball diameter ≤ 2 mm).

[0290] Parameters: Crushing time 5 minutes, intermittent operation (run for 1 minute, pause for 30 seconds) to avoid overheating and damage to the shell structure.

[0291] Centrifugal separation:

[0292] The crushed mixture was centrifuged at 10,000 rpm for 15 minutes to separate the supernatant (containing soft tissues such as fat and protein) from the precipitate (shell fragments).

[0293] Repeat centrifugation: After resuspending the precipitate in deionized water, centrifuge again (8000 rpm, 10 minutes) to further remove residual soft tissue.

[0294] Enzyme-assisted purification:

[0295] Add 0.5% alkaline protease solution (pH 8.5, 50℃) to the precipitate and stir for 1.5 hours. Effect: Degrade residual protein and significantly reduce ash residue (experimental data show that ash content can be reduced by ≥1.2%).

[0296] 3. Deep cleaning and flotation purification

[0297] Flotation medium: Prepare a 5% NaCl solution with a density of 1.03 g / cm³. 3 Separation is achieved by utilizing the density difference between the shell and soft tissue.

[0298] Operating procedures:

[0299] 1. Mix the centrifuged shell fragments with NaCl solution at a ratio of 1:10 (w / v) and stir magnetically for 10 minutes;

[0300] 2. After settling and separating, collect the lower precipitate (purification shell) and repeat the flotation process twice.

[0301] 4. Drying and pulverizing

[0302] Drying conditions:

[0303] Equipment: Vacuum drying oven, temperature 60℃, vacuum degree ≤0.09MPa, drying to moisture content ≤5% (testing according to GB5009.3-2016).

[0304] Advantages: Low-temperature vacuum drying avoids thermal degradation of chitin and maintains the integrity of the shell structure.

[0305] Crushing and sieving:

[0306] The dried shells were pulverized to 80 mesh (particle size ≤ 180 μm) using an ultra-micro pulverizer, and the substandard particles were removed after sieving.

[0307] Screening equipment: Vibrating screen (amplitude 2mm, frequency 50Hz), screen mesh size conforms to GB / T 6003.1-2012 standard.

[0308] 5. Quality Control and Testing

[0309] Ash content detection:

[0310] According to GB / T 35818-2018, take 1.0g of shell powder sample and ashing it in a muffle furnace at 550℃ for 4 hours. The ash residue should be ≤0.7%.

[0311] Handling of non-compliance: If the ash content is >0.7%, the enzyme-assisted purification and flotation steps need to be repeated.

[0312] Protein residue detection:

[0313] The nitrogen content is determined by the Kjeldahl method (GB 5009.5-2016), and the protein residue should be ≤0.5%.

[0314] Particle size verification:

[0315] Laser particle size analyzer (such as Malvern Mastersizer 3000) test results show that ≥95% of particles are 80 mesh.

[0316] The chitin extraction method from fly larvae provided by this invention has advantages such as high efficiency, low cost, high purity, and environmental friendliness, and can be widely applied in agriculture, environmental protection, and other fields. Through large-scale production, it can achieve efficient recycling of resources, combining economic and environmental benefits.

[0317] The table below compares the extraction efficiency of chitin from fly larvae using the enzyme-alkali synergistic stepwise method of this invention, the enzymatic hydrolysis method, and the traditional acid-alkali method (extraction rate of enzyme-alkali synergistic method ≥90%, enzymatic hydrolysis method ≤85%, and extraction rate of traditional method ≤80%).

[0318]

[0319] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for efficiently and cost-effectively extracting chitin from fly larvae, characterized in that, Includes the following steps: (1) Pretreatment: The fly maggot shells are washed, dried and crushed to obtain fly maggot shell powder; (2) Enzyme-alkali synergistic stepwise deproteinization treatment: fly maggot shell powder and compound enzyme solution are mixed at a weight-volume ratio to remove crude protein; then a second treatment is carried out by passing it through a low-concentration NaOH solution at a certain temperature; (3) Mild demineralization treatment: The deproteinized product is treated with a low concentration of HCl solution at room temperature, and EDTA is added to assist in demineralization; (4) Purification and drying: The demineralized product was washed with deionized water until neutral and dried at low temperature to obtain fly larvae chitin; (5) Environmental treatment: The enzymatic hydrolysis waste liquid is discharged after heating and inactivation. CaCl2 is added to the demineralized waste liquid to recover EDTA. The protein precipitate is dried and used as organic fertilizer.

2. The method for efficiently and cost-effectively extracting chitin from fly larvae according to claim 1, characterized in that: The pretreatment in step (1) includes the following: live fly larvae are starved and detoxified for 24 hours, rinsed and then inactivated by boiling water, dried at a low temperature of 60°C, and pulverized through a 30-60 mesh sieve.

3. The method for efficiently and cost-effectively extracting chitin from fly larvae according to claim 2, characterized in that: The amount of the compound enzyme added is 10% of the weight of the fly maggot shell powder; the particle size of the powder is 40 mesh.

4. The method for efficiently and cost-effectively extracting chitin from fly larvae according to claim 1, characterized in that: Step (2) includes the following: Enzyme-alkali synergistic stepwise deproteinization: First, by optimizing the ratio of compound enzymes: chitinase: cellulase 3:1-2:1 and the enzymatic hydrolysis conditions: pH 5.5-6.0, 50-55℃, 4-6h, crude protein is removed. Then, a second deproteinization is performed using a low-concentration NaOH solution of 1-2% at 60℃ for 2h to remove residual protein. The protein residue rate is <1%.

5. The method for efficiently and cost-effectively extracting chitin from fly larvae according to claim 4, characterized in that: The enzymatic hydrolysis treatment had a pH of 5.8, a reaction temperature of 52°C, and a reaction time of 5 hours; the low-concentration NaOH solution had a concentration of 1%, a reaction temperature of 60°C, and a reaction time of 2 hours.

6. The method for efficiently and cost-effectively extracting chitin from fly larvae according to claim 4, characterized in that: The composite enzyme contains chitinase activity ≥100 U / mg and cellulase activity ≥50 U / mg. The activity unit is defined as follows: Chitinase: the amount of enzyme required to release 1 μmol N-acetylglucosamine per minute using carboxymethyl chitin as a substrate at pH 5.8 and 50℃; Cellulase: the amount of enzyme required to release 1 μmol glucose per minute using sodium carboxymethyl cellulose as a substrate at pH 5.0 and 50℃.

7. The method for efficiently and cost-effectively extracting chitin from fly larvae according to claim 1, characterized in that: Step (3) includes the following: a mild demineralization process, followed by the addition of 0.05-0.1M EDTA to assist in demineralization.

8. The method for efficiently and cost-effectively extracting chitin from fly larvae according to claim 7, characterized in that: The demineralization process uses 1% HCl at room temperature for 1 hour, followed by the addition of 0.05M EDTA to assist in demineralization; the conductivity of the product after demineralization is ≤10μS / cm.

9. The method for efficiently and cost-effectively extracting chitin from fly larvae according to claim 1, characterized in that: Step (4) includes the following: the demineralized product is washed with deionized water until neutral and dried at a low temperature of 60°C to obtain fly larvae chitin.

10. The fly larvae chitin prepared by the efficient and low-cost method for extracting fly larvae chitin according to claim 1, characterized in that: Step (5) includes the following: the enzymatic hydrolysis waste liquid is heated and inactivated and then discharged; CaCl2 is added to the demineralized waste liquid to recover EDTA; and the protein precipitate is dried and used as organic fertilizer.

11. The maggot chitin prepared by the efficient and low-cost method for extracting maggot chitin according to claim 1, characterized in that: (a) Purity ≥ 99%; (b) Protein residue rate <1%; (c) Ash residue rate < 0.5%.

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