Method for extracting chitin and biological peptide based on waste insects

Abstract

The invention provides a method for extracting chitin and biological peptide based on waste insects, and relates to the technical field of biological enzymolysis. The method comprises the following specific steps: S1, pretreatment: removing impurities from waste insects, drying, and carrying out superfine grinding to obtain insect powder; s2, separation and purification: adding the insect powder in the S1 into deionized water, adding a compound enzyme, carrying out enzymolysis and solid-liquid separation, collecting a liquid phase, and washing a solid phase for later use; s3, preparing chitin: adding the solid phase in the S2 into an ethylenediamine tetraacetic acid solution, carrying out constant-temperature microwave-ultrasonic synergistic treatment, adjusting the pH value of the solution, adding chitin compound enzyme, and continuing constant-temperature microwave-ultrasonic synergistic treatment and solid-liquid separation to obtain the chitin as the solid phase; s4, preparing the biological peptide: concentrating the liquid phase in S2, separating through an ultrafiltration membrane to obtain a concentrated solution, and freeze-drying to obtain the biological peptide. The preparation method disclosed by the invention is mild in process conditions, does not need strong acid and strong alkali, does not cause pollution to the environment, and has good economic benefits and social benefits.

Description

Technical Field

[0001] This invention relates to the field of bioenzymatic hydrolysis technology, and in particular to a method for extracting chitin and bioactive peptides from waste insects. Background Technology

[0002] Against the backdrop of dwindling resources and increasingly stringent environmental standards, the effective utilization of waste insect resources has become a research hotspot. As one of the most biodiverse and numerous biological groups on Earth, insects produce enormous amounts of waste every year, with common species such as mealworms and black soldier flies generating significant amounts. Improper disposal of these waste insects can not only lead to resource depletion but also potentially cause environmental problems.

[0003] Chitin, widely found in insect exoskeletons, is a natural high-molecular-weight polysaccharide with excellent biocompatibility, biodegradability, and antibacterial properties, showing great potential for application in multiple fields such as medicine, food, agriculture, and environmental protection. Bioactive peptides are a class of small-molecule proteins with diverse biological activities, demonstrating significant value in the food, health product, and cosmetic industries. For example, antioxidant peptides can be used for food preservation, while antibacterial peptides can enhance the antibacterial efficacy of skincare products.

[0004] Currently, traditional preparation processes for chitin and bioactive peptides face numerous challenges. Extracting chitin from conventional raw materials such as shrimp and crab shells is not only limited by raw material supply, but also requires the use of large quantities of highly corrosive chemicals such as hydrochloric acid and sodium hydroxide, resulting in high energy consumption, heavy pollution, and the waste of precious freshwater due to frequent cleaning and washing. Furthermore, it can reduce the molecular weight and acetylation degree of chitin, damaging its structure and affecting product quality. For example, patent CN102603921A discloses a comprehensive insect processing method and its application, which involves crushing insects and then performing supercritical extraction to fully extract and separate chitin and antimicrobial peptides. This method suffers from high cost, long processing time, and low efficiency. Patent CN118063645A discloses an ultrasonic-assisted bioenzymatic extraction method for insect chitin and chitosan, utilizing dual-frequency synchronous ultrasound combined with intermittent ultrasound to enzymatically hydrolyze proteins. This method also uses acid and alkaline solutions, which may potentially affect the quality of chitin, and it does not specify the preparation method for bioactive peptides.

[0005] Therefore, developing new methods for preparing chitin and bioactive peptides based on waste insect resources can not only realize the resource utilization of waste and reduce environmental pressure, but also open up new raw material sources for these two high-value products. The preparation process is environmentally friendly and pollution-free, reduces production costs, and improves product quality, thus having significant economic and environmental benefits and practical significance. Summary of the Invention

[0006] Therefore, this invention proposes a method for extracting chitin and bioactive peptides from waste insects.

[0007] The technical solution of this invention is implemented as follows: A method for extracting chitin and bioactive peptides from waste insects, comprising the following steps: S1. Pretreatment: Remove impurities from waste insects, dry them, and pulverize them into insect powder; S2. Separation and purification: Add the insect powder from S1 to deionized water, add the compound enzyme, perform enzymatic hydrolysis, separate the solid and liquid phases, collect the liquid phase, and wash the solid phase for later use. S3. Preparation of chitin: The solid phase in S2 is added to an ethylenediaminetetraacetic acid solution, subjected to constant temperature microwave and ultrasonic combined treatment, the pH of the solution is adjusted, chitin complex enzyme is added, and constant temperature microwave and ultrasonic combined treatment is continued to separate the solid and liquid phases. The solid phase is chitin. S4. Preparation of bioactive peptides: The liquid phase of S2 is concentrated, separated by ultrafiltration membrane, and the concentrate is freeze-dried to obtain bioactive peptides.

[0008] Furthermore, in step S1, the removal of impurities from the waste insects specifically involves washing with deionized water 1-2 times, followed by ultrasonic cleaning with 100-200W for 8-12 minutes; the particle size of the ultrafine pulverization is less than 10μm.

[0009] Further, in step S2, the ratio of insect powder to deionized water is 1:10-20 g / mL; the compound enzyme, by weight, consists of 20-30 parts papain, 5-15 parts lipase, and the remainder Bacillus subtilis neutral protease per 100 parts of compound enzyme; the amount of compound enzyme added is 0.1%-0.2% w / v; the enzymatic hydrolysis is carried out at 50-60℃ and 150-200 rpm for 8-12 hours.

[0010] After solid-liquid separation by enzymatic hydrolysis, the liquid phase mainly contains protein hydrolysis products and enzyme solution, while the solid phase mainly consists of insect shells and other residues containing chitin. The solid phase is washed with deionized water and then used for later use.

[0011] Furthermore, in step S3, the solid phase to ethylenediaminetetraacetic acid solution ratio is 1:10-20 g / mL; the concentration of the ethylenediaminetetraacetic acid solution is 0.5-1.5 mol / L; and the pH of the solution is 5-7.

[0012] Furthermore, in step S3, the temperature of the constant-temperature microwave-ultrasound co-processing is 30-40℃, the microwave frequency is 2300-2500MHz, the ultrasonic power is 150-200W, and the time is 20-30min; the duration of the continued constant-temperature microwave-ultrasound co-processing is 1-2h.

[0013] In step S3, solid-liquid separation is performed. The solid phase is washed with deionized water until no calcium ions are detected in the washing liquid. The washed solid phase is dried at 40-50℃ to constant weight, which is chitosan.

[0014] Furthermore, in step S3, the chitin complex enzyme is extracted from Bacillus subtilis bacterial solution, and the enzyme activity ratio of endonuclease to exonuclease is 1:1.5-2.5; the amount of chitin complex enzyme added is 2%-3% w / v.

[0015] Furthermore, the extraction steps for the chitin complex enzyme are as follows: (1) Pick a single colony from the slant of Bacillus subtilis, inoculate it into a liquid culture medium and culture it under constant temperature and shaking. Centrifuge and take out the supernatant a, which is the crude enzyme solution containing the target enzyme. (2) Add ammonium sulfate powder to the crude enzyme solution to 30% saturation, stir, centrifuge, retain supernatant b, increase the ammonium sulfate saturation in supernatant b to 55%, stir, centrifuge, collect the precipitate, retain supernatant c, the precipitate is the preliminarily purified exonuclease, increase the ammonium sulfate saturation in supernatant c to 80%, stir, centrifuge, collect the precipitate, which is the preliminarily purified endonuclease; (3) Add the pre-purified exonuclease and the pre-purified endonuclease to phosphate buffer, respectively, and put them into dialysis bags for dialysis. Collect the solution in the bag, which is the target exonuclease and endonuclease. (4) Using colloidal chitin as a substrate, the activity of endonuclease was determined by DNS method, and the activity of exonuclease was determined by spectrophotometry using p-NAG as a substrate. The chitin complex enzyme was obtained by mixing endonuclease and exonuclease in a ratio of 1:1.5-2.5 of enzyme activity units.

[0016] Furthermore, in step (1), the liquid culture medium consists of 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 0.8-1.2 g / L colloidal chitin, 55-65 mg / L MgSO4 and 10-20 mg / L MnSO4. The isothermal shaking culture is first cultured in 5 mL of liquid culture medium at 37-39°C and 200-250 rpm for 16-18 hours, and then inoculated into 50 mL of liquid culture medium at 1%-2% at 37-39°C and 200-250 rpm for 35-40 hours.

[0017] Furthermore, in step (3), the pH of the phosphate buffer is 7.2±0.1 and the concentration is 0.02-0.03mol / L; the dialysis is performed at 4-6℃ with a pH of 7.2±0.1 and a concentration of 0.02-0.03mol / L in the phosphate buffer for 6-12 hours, during which the dialysis solution is changed every 2-4 hours.

[0018] Furthermore, in step S4, the molecular weight cutoff of the ultrafiltration membrane is 3000-5000 Da.

[0019] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention makes rational use of waste insect resources as raw materials, improves the comprehensive utilization rate of insect resources, reduces dependence on traditional chitin raw materials such as shrimp and crab shells, and reduces the pressure of waste insects on the environment.

[0020] 2. The product obtained by the method of the present invention is of high quality. As verified by the examples, the purity of chitin can reach 96.57%. The present invention uses ultrasonic and microwave synergistic treatment to achieve a specific ratio of enzyme activity units of endonuclease and exonuclease in the chitin complex enzyme, which significantly increases the purity, extraction rate and extraction amount of the obtained chitin.

[0021] 3. The process conditions for extracting chitin and bioactive peptides in this invention are mild, do not require strong acids or alkalis, will not cause environmental pollution, have good economic and social benefits, and have good application prospects. Detailed Implementation

[0022] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0023] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0025] The waste insects of this invention include, but are not limited to, mealworms and black soldier flies.

[0026] Example 1 A method for extracting chitin and bioactive peptides from waste insects, comprising the following steps: S1. Pretreatment: Collect waste insect resources and wash them once with deionized water. Then, clean them with ultrasound at 100W for 8 minutes. Then, dry them at 60℃ to constant weight. Use an ultra-micro pulverizer to pulverize the dried insects into powder with a particle size of less than 10μm to obtain insect powder. S2. Separation and purification: According to the material-liquid ratio of 1:10 g / mL, add the insect powder from S1 to deionized water, add 0.1% w / v compound enzyme, which, by weight percentage, consists of 20% papain, 5% lipase and 75% Bacillus subtilis neutral protease. Place in a constant temperature shaking incubator at 50℃ and perform enzymatic hydrolysis at 150 rpm for 8 hours. Perform solid-liquid separation, collect the liquid phase, which mainly contains protein hydrolysis products and enzyme solution, while the solid phase mainly contains insect shells and other residues containing chitin. The solid phase is washed with deionized water and then used for later use. S3. Preparation of Chitosan: According to the material-to-liquid ratio of 1:10 g / mL, the solid phase in S2 was added to a 0.5 mol / L ethylenediaminetetraacetic acid solution and subjected to microwave-ultrasonic co-treatment at 30℃ for 20 min. The microwave frequency was 2300 MHz and the ultrasonic power was 150 W. The pH of the solution was adjusted to 5. Then, 2% w / v chitin complex enzyme was added. The chitin complex enzyme was extracted from Bacillus subtilis bacterial solution, and the ratio of endonuclease to exonuclease activity was 1:1.5. The mixture was subjected to microwave-ultrasonic co-treatment at 30℃ for 1 h to separate the solid and liquid phases. The solid phase was chitosan. It was washed with deionized water until no calcium ions were detected in the washing solution. The washed solid phase was dried at 40℃ to constant weight to obtain the chitosan product. S4. Preparation of bioactive peptides: The liquid phase of S2 is concentrated and separated by an ultrafiltration membrane with a molecular weight cutoff of 3000 Da to obtain a concentrated solution containing bioactive peptides. The concentrated solution is then freeze-dried to obtain bioactive peptides.

[0027] The extraction steps for the chitin complex enzyme in step S3 are as follows: (1) Pick a single colony from the slant of Bacillus subtilis and inoculate it into a 5 mL liquid culture medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 0.8 g / L colloidal chitin, 55 mg / L MgSO4 and 10 mg / L MnSO4). Incubate at 37°C and 200 rpm for 16 h with shaking. Then, inoculate the culture into a 50 mL liquid culture medium at a 1% inoculation rate and incubate at 37°C and 200 rpm for 35 h with shaking. Transfer the culture to a centrifuge tube and centrifuge at 10000 g for 20 min at 4°C. Take the supernatant a, which is the crude enzyme solution containing the target enzyme. (2) Add ammonium sulfate powder to the crude enzyme solution to 30% saturation, stir at 4℃ for 30 min, centrifuge at 10000 g for 10 min, retain supernatant b, then increase the ammonium sulfate saturation in supernatant b to 55%, stir at 4℃ for 30 min, centrifuge at 10000 g for 10 min, collect the precipitate, retain supernatant c, the precipitate is the preliminarily purified exonuclease, increase the ammonium sulfate saturation in supernatant c to 80%, stir at 4℃ for 30 min, centrifuge at 10000 g for 10 min, collect the precipitate, which is the preliminarily purified endonuclease; (3) The preliminarily purified exonuclease and preliminarily purified endonuclease were added to 0.02 mol / L phosphate buffer (pH 7.2±0.1) respectively, and placed into dialysis bags. Dialysis was performed at 4°C using 0.02 mol / L phosphate buffer for 6 h, with the dialysis solution changed every 2 h. The solution in the bag was collected, which is the target exonuclease and endonuclease. (4) Using colloidal chitin as a substrate, the activity of endonuclease was determined by DNS method, and the activity of exonuclease was determined by spectrophotometry using p-NAG as a substrate. The chitin complex enzyme was obtained by mixing endonuclease and exonuclease in a ratio of 1:1.5 of enzyme activity units.

[0028] Example 2 A method for extracting chitin and bioactive peptides from waste insects, comprising the following steps: S1. Pretreatment: Collect waste insect resources and wash them once with deionized water. Then, clean them with ultrasound at 150W for 10 minutes. Then, dry them at 70℃ to constant weight. Use an ultra-micro pulverizer to pulverize the dried insects into powder with a particle size of less than 10μm to obtain insect powder. S2. Separation and purification: According to the material-liquid ratio of 1:15 g / mL, add the insect powder from S1 to deionized water, add 0.15% w / v compound enzyme, which, by weight percentage, consists of 25% papain, 10% lipase and 65% Bacillus subtilis neutral protease. Place in a constant temperature shaking incubator at 55℃ and perform enzymatic hydrolysis at 175 rpm for 10 h. Perform solid-liquid separation, collect the liquid phase, which mainly contains protein hydrolysis products and enzyme solution, while the solid phase mainly contains insect shells and other residues containing chitin. The solid phase is washed with deionized water and then used for later use. S3. Preparation of Chitosan: According to the material-to-liquid ratio of 1:15 g / mL, the solid phase in S2 was added to a 1 mol / L ethylenediaminetetraacetic acid solution and subjected to microwave-ultrasonic co-treatment at 35℃ for 25 min. The microwave frequency was 2400 MHz and the ultrasonic power was 175 W. The pH of the solution was adjusted to 6. Then, 2.5% w / v chitin complex enzyme was added. The chitin complex enzyme was extracted from Bacillus subtilis bacterial solution, and the ratio of endonuclease to exonuclease activity was 1:2. The mixture was then subjected to microwave-ultrasonic co-treatment at 35℃ for 1.5 h to separate the solid and liquid phases. The solid phase was chitosan. It was washed with deionized water until no calcium ions were detected in the washing solution. The washed solid phase was dried at 45℃ to constant weight to obtain the chitosan product. S4. Preparation of bioactive peptides: The liquid phase of S2 is concentrated and separated by an ultrafiltration membrane with a molecular weight cutoff of 4000 Da to obtain a concentrated solution containing bioactive peptides. The concentrated solution is then freeze-dried to obtain bioactive peptides.

[0029] The extraction steps for the chitin complex enzyme in step S3 are as follows: (1) Pick a single colony from the slant of Bacillus subtilis and inoculate it into a 5 mL liquid culture medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 1 g / L colloidal chitin, 60 mg / L MgSO4 and 15 mg / L MnSO4). Incubate at 38°C and 225 rpm for 17 h with shaking. Then, inoculate the culture into a 50 mL liquid culture medium at a 1% inoculation rate and incubate at 38°C and 225 rpm for 38 h with shaking. Transfer the culture to a centrifuge tube and centrifuge at 11000 g for 20 min at 4°C. Take the supernatant a, which is the crude enzyme solution containing the target enzyme. (2) Add ammonium sulfate powder to the crude enzyme solution to 30% saturation, stir at 4℃ for 350 min, centrifuge at 10000 g for 10 min, retain supernatant b, then increase the ammonium sulfate saturation in supernatant b to 55%, stir at 4℃ for 35 min, centrifuge at 10000 g for 10 min, collect the precipitate, retain supernatant c, the precipitate is the preliminarily purified exonuclease, increase the ammonium sulfate saturation in supernatant c to 80%, stir at 4℃ for 35 min, centrifuge at 10000 g for 10 min, collect the precipitate, which is the preliminarily purified endonuclease; (3) The preliminarily purified exonuclease and preliminarily purified endonuclease were added to 0.02 mol / L phosphate buffer (pH 7.2 ± 0.1) respectively, and placed into dialysis bags. Dialysis was performed at 4°C using 0.02 mol / L phosphate buffer for 9 h, with the dialysis solution changed every 3 h. The solution in the bag was collected, which is the target exonuclease and endonuclease. (4) Using colloidal chitin as a substrate, the activity of endonuclease was determined by DNS method, and the activity of exonuclease was determined by spectrophotometry using p-NAG as a substrate. The chitin complex enzyme was obtained by mixing endonuclease and exonuclease in a ratio of 1:2 of enzyme activity units.

[0030] Example 3 A method for extracting chitin and bioactive peptides from waste insects, comprising the following steps: S1. Pretreatment: Collect waste insect resources and wash them twice with deionized water. Then, clean them with ultrasound at 200W for 12 minutes. Then, dry them at 80℃ to constant weight. Use an ultra-micro pulverizer to pulverize the dried insects into powder with a particle size of less than 10μm to obtain insect powder. S2. Separation and purification: According to the material-liquid ratio of 1:20 g / mL, add the insect powder from S1 to deionized water, add 0.2% w / v compound enzyme, which, by weight percentage, consists of 30% papain, 15% lipase and 55% Bacillus subtilis neutral protease. Place in a constant temperature shaking incubator at 60℃ and perform enzymatic hydrolysis at 200 rpm for 12 hours. Perform solid-liquid separation, collect the liquid phase, which mainly contains protein hydrolysis products and enzyme solution, while the solid phase mainly contains insect shells and other residues containing chitin. The solid phase is washed with deionized water and then used for later use. S3. Preparation of Chitosan: According to the material-to-liquid ratio of 1:20 g / mL, the solid phase in S2 was added to a 1.5 mol / L ethylenediaminetetraacetic acid solution and subjected to microwave-ultrasonic co-treatment at 40℃ for 30 min. The microwave frequency was 2500 MHz and the ultrasonic power was 200 W. The pH of the solution was adjusted to 7. Then, 3% w / v chitin complex enzyme was added. The chitin complex enzyme was extracted from Bacillus subtilis bacterial solution, and the enzyme activity ratio of endonuclease to exonuclease was 1:2.5. The mixture was subjected to microwave-ultrasonic co-treatment at 40℃ for 2 h to separate the solid and liquid phases. The solid phase was chitosan. It was washed with deionized water until no calcium ions were detected in the washing solution. The washed solid phase was dried at 50℃ to constant weight to obtain the chitosan product. S4. Preparation of bioactive peptides: The liquid phase of S2 is concentrated and separated by an ultrafiltration membrane with a molecular weight cutoff of 5000 Da to obtain a concentrated solution containing bioactive peptides. The concentrated solution is then freeze-dried to obtain bioactive peptides.

[0031] The extraction steps for the chitin complex enzyme in step S3 are as follows: (1) Pick a single colony from the slant of Bacillus subtilis and inoculate it into a 5 mL liquid culture medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 1.2 g / L colloidal chitin, 65 mg / L MgSO4 and 20 mg / L MnSO4). Incubate at 39 °C and 250 rpm for 18 h with shaking. Then, inoculate the culture into a 50 mL liquid culture medium at a 2% inoculation rate and incubate at 39 °C and 250 rpm for 40 h with shaking. Transfer the culture to a centrifuge tube and centrifuge at 12000 g for 20 min at 4 °C. Take the supernatant a, which is the crude enzyme solution containing the target enzyme. (2) Add ammonium sulfate powder to the crude enzyme solution to 30% saturation, stir at 4℃ for 40 min, centrifuge at 10000 g for 10 min, retain supernatant b, then increase the ammonium sulfate saturation in supernatant b to 55%, stir at 4℃ for 40 min, centrifuge at 10000 g for 10 min, collect the precipitate, retain supernatant c, the precipitate is the preliminarily purified exonuclease, increase the ammonium sulfate saturation in supernatant c to 80%, stir at 4℃ for 40 min, centrifuge at 10000 g for 10 min, collect the precipitate, which is the preliminarily purified endonuclease; (3) The preliminarily purified exonuclease and preliminarily purified endonuclease were added to 0.02 mol / L phosphate buffer (pH 7.2 ± 0.1) and placed into dialysis bags. Dialysis was performed at 4°C using 0.02 mol / L phosphate buffer for 12 h, with the dialysis solution changed every 4 h. The solutions in the bags were collected, which were the target exonuclease and endonuclease. (4) Using colloidal chitin as a substrate, the activity of endonuclease was determined by DNS method, and the activity of exonuclease was determined by spectrophotometry using p-NAG as a substrate. The chitin complex enzyme was obtained by mixing endonuclease and exonuclease in a ratio of 1:2.5 of enzyme activity units.

[0032] Comparative Example 1-1 The difference from Example 1 is that ultrasonic treatment is not used in step S3, that is, only microwave treatment is used, while the rest is the same as in Example 1.

[0033] Comparative Examples 1-2 The difference from Example 2 is that ultrasonic treatment is not used in step S3, that is, only microwave treatment is used, while the rest is the same as Example 2.

[0034] Comparative Examples 1-3 The difference from Example 3 is that ultrasonic treatment is not used in step S3, that is, only microwave treatment is used, while the rest is the same as Example 3.

[0035] Comparative Example 2-1 The difference from Example 1 is that microwave processing is not used in step S3, that is, only ultrasonic processing is used, while the rest is the same as in Example 1.

[0036] Comparative Example 2-2 The difference from Example 2 is that microwave treatment is not used in step S3, that is, only ultrasonic treatment is used, while the rest is the same as Example 2.

[0037] Comparative Examples 2-3 The difference from Example 3 is that microwave treatment is not used in step S3, that is, only ultrasonic treatment is used, while the rest is the same as Example 3.

[0038] Comparative Example 3-1 The difference from Example 1 is that microwave processing and ultrasonic processing are not used in step S3, but otherwise it is the same as Example 1.

[0039] Comparative Example 3-2 The difference from Example 2 is that microwave processing and ultrasonic processing are not used in step S3, but otherwise the same as in Example 2.

[0040] Comparative Example 3-3 The difference from Example 3 is that microwave processing and ultrasonic processing are not used in step S3, but otherwise the same as in Example 3.

[0041] Comparative Example 4-1 The difference from Example 1 is that in step S3, the chitin complex enzyme is replaced with chitinase (analytical grade), otherwise it is the same as Example 1.

[0042] Comparative Example 4-2 The difference from Example 2 is that in step S3, the chitin complex enzyme is replaced with chitinase (analytical grade), otherwise it is the same as Example 2.

[0043] Comparative Example 4-3 The difference from Example 3 is that in step S3, the chitin complex enzyme is replaced with chitinase (analytical grade), otherwise it is the same as Example 3.

[0044] Comparative Example 5-1 The difference from Example 1 is that the ratio of endonuclease to exonuclease activity units in the chitin complex enzyme in step S3 is less than 1:1.5, while the rest is the same as in Example 1.

[0045] Comparative Example 5-2 The difference from Example 2 is that the ratio of endonuclease to exonuclease activity units in the chitin complex enzyme in step S3 is less than 1:1.5, while the rest is the same as in Example 2.

[0046] Comparative Example 5-3 The difference from Example 3 is that the ratio of endonuclease to exonuclease activity units in the chitin complex enzyme in step S3 is less than 1:1.5, while the rest is the same as in Example 3.

[0047] Comparative Example 6-1 The difference from Example 1 is that the ratio of endonuclease to exonuclease activity in the chitin complex enzyme in step S3 is greater than 1:2.5, while the rest is the same as in Example 1.

[0048] Comparative Example 6-2 The difference from Example 2 is that the ratio of endonuclease to exonuclease activity in the chitin complex enzyme in step S3 is greater than 1:2.5, while the rest is the same as in Example 2.

[0049] Comparative Example 6-3 The difference from Example 3 is that the ratio of endonuclease to exonuclease activity in the chitin complex enzyme in step S3 is greater than 1:2.5, while the rest is the same as in Example 3.

[0050] Application examples 100g (fresh weight) of black soldier fly larvae were used as experimental material. Chitin was obtained according to the preparation methods of the examples and comparative examples. The results of its purity, extraction rate and extraction amount are shown in Table 1.

[0051] Table 1

[0052] Note: Different uppercase letters in the table indicate significant differences between treatments (P<0.05), and different lowercase letters indicate significant differences within treatments (P<0.05).

[0053] As can be seen from Table 1, compared with Comparative Examples 1-1 to 1-3 which only used microwave treatment without ultrasonic treatment, Comparative Examples 2-1 to 2-3 which only used ultrasonic treatment without microwave treatment, and Comparative Examples 3-1 to 3-3 which did not use ultrasonic or microwave treatment, the chitin purity, extraction rate, and extraction amount obtained by the preparation method of the present invention are significantly increased. It can also be seen that ultrasonic and microwave treatment have a synergistic effect in increasing chitin purity, extraction rate, and extraction amount.

[0054] Furthermore, compared to chitin complex enzymes in Comparative Examples 4-1 to 4-3, when chitinase was replaced with chitinase, the ratio of endonuclease to exonuclease activity units in the complex chitinases of Comparative Examples 5-1 to 5-3 was less than 1:1.5, while the ratio of endonuclease to exonuclease activity units in the complex chitinases of Comparative Examples 6-1 to 6-3 was greater than 1:2.5. The chitin purity, extraction rate, and extraction amount obtained by the preparation method of the present invention were significantly increased.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting chitin and bioactive peptides from waste insects, characterized in that, The specific steps include: S1. Pretreatment: Remove impurities from waste insects, dry them, and pulverize them into insect powder; S2. Separation and purification: Add the insect powder from S1 to deionized water, add the compound enzyme, perform enzymatic hydrolysis, separate the solid and liquid phases, collect the liquid phase, and wash the solid phase for later use. S3. Preparation of chitin: The solid phase in S2 is added to an ethylenediaminetetraacetic acid solution, subjected to constant temperature microwave and ultrasonic combined treatment, the pH of the solution is adjusted, chitin complex enzyme is added, and constant temperature microwave and ultrasonic combined treatment is continued to separate the solid and liquid phases. The solid phase is chitin. S4. Preparation of bioactive peptides: The liquid phase of S2 is concentrated, separated by ultrafiltration membrane, and the concentrate is freeze-dried to obtain bioactive peptides.

2. The method for extracting chitin and bioactive peptides from waste insects as described in claim 1, characterized in that, In step S1, the removal of impurities from the waste insects specifically involves washing with deionized water 1-2 times, followed by ultrasonic cleaning with 100-200W for 8-12 minutes; the particle size of the ultrafine pulverization is less than 10μm.

3. The method for extracting chitin and bioactive peptides from waste insects as described in claim 1, characterized in that, In step S2, the ratio of insect powder to deionized water is 1:10-20 g / mL; the compound enzyme is composed of 20-30 parts papain, 5-15 parts lipase and the remainder Bacillus subtilis neutral protease per 100 parts by weight; the amount of compound enzyme added is 0.1%-0.2% w / v; the enzymatic hydrolysis is carried out at 50-60℃ and 150-200 rpm for 8-12 hours.

4. The method for extracting chitin and bioactive peptides from waste insects as described in claim 1, characterized in that, In step S3, the solid phase to ethylenediaminetetraacetic acid solution ratio is 1:10-20 g / mL; the concentration of the ethylenediaminetetraacetic acid solution is 0.5-1.5 mol / L; and the pH of the solution is 5-7.

5. The method for extracting chitin and bioactive peptides from waste insects as described in claim 1, characterized in that, In step S3, the temperature of the constant temperature microwave-ultrasound co-processing is 30-40℃, the microwave frequency is 2300-2500MHz, the ultrasonic power is 150-200W, and the time is 20-30min; the duration of the continued constant temperature microwave-ultrasound co-processing is 1-2h.

6. The method for extracting chitin and bioactive peptides from waste insects as described in claim 1, characterized in that, In step S3, the chitin complex enzyme is extracted from Bacillus subtilis bacterial culture, and the enzyme activity ratio of endonuclease to exonuclease is 1:1.5-2.5; the amount of chitin complex enzyme added is 2%-3% w / v.

7. The method for extracting chitin and bioactive peptides from waste insects as described in claim 6, characterized in that, The extraction steps of the chitin complex enzyme are as follows: (1) Pick a single colony from the slant of Bacillus subtilis, inoculate it into a liquid culture medium and culture it under constant temperature and shaking. Centrifuge and take out the supernatant a, which is the crude enzyme solution containing the target enzyme. (2) Add ammonium sulfate powder to the crude enzyme solution to 30% saturation, stir, centrifuge, retain supernatant b, increase the ammonium sulfate saturation in supernatant b to 55%, stir, centrifuge, collect the precipitate, retain supernatant c, the precipitate is the preliminarily purified exonuclease, increase the ammonium sulfate saturation in supernatant c to 80%, stir, centrifuge, collect the precipitate, which is the preliminarily purified endonuclease; (3) Add the pre-purified exonuclease and the pre-purified endonuclease to phosphate buffer, respectively, and put them into dialysis bags for dialysis. Collect the solution in the bag, which is the target exonuclease and endonuclease. (4) Using colloidal chitin as a substrate, the activity of endonuclease was determined by DNS method, and the activity of exonuclease was determined by spectrophotometry using p-NAG as a substrate. The chitin complex enzyme was obtained by mixing endonuclease and exonuclease in a ratio of 1:1.5-2.5 of enzyme activity units.

8. The method for extracting chitin and bioactive peptides from waste insects as described in claim 7, characterized in that, In step (1), the liquid culture medium consists of 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 0.8-1.2 g / L colloidal chitin, 55-65 mg / L MgSO4 and 10-20 mg / L MnSO4. The isothermal shaking culture is first cultured in 5 mL of liquid culture medium at 37-39°C and 200-250 rpm for 16-18 hours, and then inoculated into 50 mL of liquid culture medium at 1%-2% at 37-39°C and 200-250 rpm for 35-40 hours.

9. The method for extracting chitin and bioactive peptides from waste insects as described in claim 7, characterized in that, In step (3), the pH of the phosphate buffer is 7.2±0.1 and the concentration is 0.02-0.03mol / L; the dialysis is performed at 4-6℃ with a pH of 7.2±0.1 and a concentration of 0.02-0.03mol / L in the phosphate buffer for 6-12 hours, during which the dialysis solution is changed every 2-4 hours.

10. The method for extracting chitin and bioactive peptides from waste insects as described in claim 1, characterized in that, In step S4, the molecular weight cutoff of the ultrafiltration membrane is 3000-5000 Da.

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