Method for extracting melanin and melanin / chitin compound from hermetia illucens

Abstract

The invention provides a method for extracting melanin and a melanin / chitin compound from hermetia illucens, and belongs to the technical field of separation and extraction. The deep-eutectic solvent extraction technology is applied to extraction of the melanin and the melanin / chitin compound in the hermetia illucens pupae shells, inorganic salt and protein in the hermetia illucens can be rapidly removed, the natural melanin / chitin compound is obtained in one step, then the melanin is extracted from the natural melanin / chitin compound through alkali extraction and acid precipitation, and the melanin content of the melanin / chitin compound is greatly improved. The separation of the melanin and the melanin / chitin compound is realized, so that melanin and melanin / chitin compound products with high added values are obtained. According to the extraction method disclosed by the invention, protease is prevented from being used, and the extraction cost is greatly reduced; meanwhile, the deep eutectic solvent method avoids the use of extreme and highly concentrated acid and alkali reagents, and compared with the traditional acid-alkali method, the method has more advantages in the yield of melanin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of separation and extraction, and particularly relates to a method for extracting melanin and melanin / chitin composite from black soldier fly. BACKGROUND

[0002] Chitin (beta-1, 4-poly-N-acetylglucosamine) widely exists in the exoskeletons of insects and crustaceans and the cell walls of fungi, has a rigid crystal structure and biocompatibility, but poor mechanical properties. Melanin (indole-phenolic polymer) is a common biological pigment, which is distributed in the tissues such as hair, skin and eyes of organisms. Melanin is a biological macromolecule polymerized by indole and phenol, which is easy to combine with protein, oil and polysaccharide, and is insoluble in water, acidic solution and most organic solvents. Melanin has the characteristics of photoprotection and free radical quenching, but is difficult to be formed alone.

[0003] The pupal case of black soldier fly contains melanin and chitin as natural biological resources. On the one hand, melanin has physiological activities such as anti-tumor, anti-radiation and improving immunity, and has wide application value in medicine, cosmetics and biological materials, and belongs to high value-added products. On the other hand, after the combination of melanin and chitin, a complementary melanin / chitin composite can be formed, which provides structural support by chitin and antioxidant function by melanin, and has great application potential in the field of medical materials. However, the pupal case of black soldier fly is mostly biological waste material, and there is no research on extracting melanin and melanin / chitin composite from the pupal case of black soldier fly in the prior art. SUMMARY

[0004] Therefore, the present application aims to provide a method for extracting melanin and melanin / chitin composite from black soldier fly. The method provided by the present application can effectively separate melanin and melanin / chitin composite in the pupal case of black soldier fly, and can convert the pupal case waste of black soldier fly into high value-added products.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions: The present application provides a method for extracting melanin and melanin / chitin composite from black soldier fly, comprising the following steps: dehydration, crushing and degreasing treatment of black soldier fly to obtain dehydrated and degreased pupal case powder; mixing the dehydrated and degreased pupal case powder with a eutectic solvent, performing eutectic solvent extraction, and collecting a first solid component; mixing the first solid component with lye, performing alkali extraction, and obtaining an alkali extraction liquid and a second solid component after solid-liquid separation, wherein the second solid component is a melanin / chitin composite; Mixing the alkaline extract with an acid solution to perform acid precipitation to obtain melanin.

[0006] Preferably, the defatting reagent used in the defatting treatment comprises one or more of petroleum ether, ethanol and ethyl acetate. The defatting treatment is performed at a temperature of 50-90℃ for 6-12h.

[0007] Preferably, the components of the deep eutectic solvent comprise a hydrogen bond donor and a hydrogen bond acceptor, the hydrogen bond donor comprises an organic acid, and the hydrogen bond acceptor comprises an organic base. The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1-3:1.

[0008] Preferably, the organic acid comprises one or more of lactic acid, malic acid, acetic acid and citric acid, and the organic base comprises choline chloride and / or betaine.

[0009] Preferably, the mass of the dehydrated and defatted chitin powder to the volume of the deep eutectic solvent is 1g:10-30mL. The deep eutectic solvent extraction is performed at a temperature of 70-110℃ for 2-6h.

[0010] Preferably, the alkaline solution is an aqueous solution of an alkali metal hydroxide, and the molar concentration of the alkaline solution is 0.5-2mol / L. The alkaline extraction is performed at a temperature of 50-90℃ for 1.5-3h.

[0011] Preferably, the acid solution is one or more of hydrochloric acid, lactic acid or acetic acid, and the pH value of the obtained mixture after mixing the alkaline extract with the acid solution is 1-2.

[0012] Preferably, the process further comprises repeating the alkaline extraction and acid precipitation on the obtained precipitate, and the number of repetitions is 2-3.

[0013] The present application provides the melanin / chitin composite obtained by the above method.

[0014] The application provides a method for extracting melanin and melanin / chitin complex from black soldier fly, comprising the following steps: dehydrating, crushing and defatting the black soldier fly to obtain dehydrated and defatted pupa shell powder; mixing the dehydrated and defatted pupa shell powder with a deep eutectic solvent, performing deep eutectic solvent extraction, and collecting a first solid component; mixing the first solid component with an alkali solution, performing alkali extraction, and obtaining an alkali extraction liquid and a second solid component after solid-liquid separation, wherein the second solid component is a melanin / chitin complex; mixing the alkali extraction liquid with an acid solution, performing acid precipitation, and obtaining melanin. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is an alkali concentration optimization curve used for extracting melanin in the application; Figure 2 is an alkali extraction reaction time optimization curve for extracting melanin in the application; Figure 3 is an alkali extraction reaction temperature optimization curve for extracting melanin in the application; Figure 4 is a precipitation time optimization curve for acid precipitation of melanin in the application; Figure 5 is a comparison diagram of ultraviolet-visible spectra of melanin extracted by two methods in the application; Figure 6 is a Fourier transform infrared spectrum of melanin extracted in the application; Figure 7 is a nuclear magnetic resonance hydrogen spectrum of melanin extracted in the application; Figure 8 is an X-ray photoelectron spectrum of melanin extracted in the application; Figure 9 is a Fourier transform infrared spectrum of melanin / chitin complex extracted in the application. DETAILED DESCRIPTION

[0016] The application provides a method for extracting melanin and melanin / chitin complex from black soldier fly, comprising the following steps: The pupa shell of the black soldier fly is subjected to dehydration, crushing and defatting treatment to obtain dehydrated and defatted pupa shell powder; The dehydrated and defatted pupa shell powder is mixed with a eutectic solvent to perform eutectic solvent extraction, and a first solid component is collected; The first solid component is mixed with lye to perform alkali extraction, and after solid-liquid separation, an alkali extraction liquid and a second solid component are obtained, and the second solid component is a melanin / chitin complex; The alkali extraction liquid is mixed with acid to perform acid precipitation to obtain melanin.

[0017] In the application, the foreign matters on the inner and outer surfaces of the black soldier fly are removed, and the black soldier fly is repeatedly washed with water and then soaked in an ethanol solution for standby. In the application, the dehydration mode is preferably drying; the crushing mode is preferably grinding and sieving, and the sieving is preferably passing through a 60-mesh sieve, and the particle size of the dehydrated pupa shell powder obtained after sieving is preferably 180-250 mu m, and more preferably 200-220 mu m.

[0018] In the application, the defatting reagent used in the defatting treatment includes one or more of petroleum ether, ethanol and ethyl acetate, and the mass ratio of the dehydrated pupa shell powder to the defatting reagent is preferably 1:5-15, and more preferably 1:10. In the application, the defatting treatment is preferably performed by a Soxhlet extraction mode, the temperature of the defatting treatment is preferably 50-90 DEG C, and specifically can be 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C, 85 DEG C or 90 DEG C, and the time is preferably 6-12 h, and more preferably 6-8 h.

[0019] After the defatting treatment, the application preferably performs filtration to obtain a filtrate and a filter residue. In the application, the filtrate is subjected to desolventization at 40-60 DEG C to obtain black soldier fly oil, and the removed solvent can be recycled for defatting treatment, thereby reducing the cost of extracting melanin and melanin / chitin complex from the black soldier fly. In the application, the filter residue is dehydrated and defatted pupa shell powder.

[0020] After obtaining the dehydrated and defatted chitin powder, the dehydrated and defatted chitin powder is mixed with a deep eutectic solvent to perform deep eutectic solvent extraction, and a first solid component is collected. In the present application, the components of the deep eutectic solvent include a hydrogen bond donor and a hydrogen bond acceptor, the hydrogen bond donor preferably includes an organic acid, and the organic acid preferably includes one or more of lactic acid, malic acid, acetic acid and citric acid; the hydrogen bond acceptor includes an organic base, and the organic base preferably includes choline chloride and / or betaine. In the present application, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is preferably 1-3:1, and more preferably 2:1. As a specific embodiment of the present application, the components of the deep eutectic solvent are choline chloride and lactic acid with a molar ratio of 1:2; or choline chloride and malonic acid with a molar ratio of 1:2; or choline chloride and acetic acid with a molar ratio of 1:2; or choline chloride and oxalic acid with a molar ratio of 1:2; or choline chloride and citric acid with a molar ratio of 1:2. In the present application, the preparation method of the deep eutectic solvent preferably includes the following steps: mixing the organic acid and the organic base, and the mixing temperature is preferably 70-90°C, and more preferably 80°C.

[0021] In the present application, the mass of the dehydrated and defatted chitin powder to the volume of the deep eutectic solvent is preferably 1g:10-30mL, and more preferably 1g:20mL. In the present application, the deep eutectic solvent extraction is preferably performed under stirring, and the temperature of the deep eutectic solvent extraction is preferably 70-110°C, and more preferably 80-100°C, and the time is preferably 2-6h, and more preferably 3-5h. Within this temperature range, the viscosity of the deep eutectic solvent can be reduced to enhance the penetration and dissolution capacity, promote the dissociation of inorganic salts and the dissolution of proteins, and at the same time, avoid the decomposition of the solvent or the denaturation of proteins caused by excessively high temperature, while reducing energy consumption and volatile loss; the appropriate time can ensure that the target components are fully dissolved, and avoid insufficient extraction rate in a short time or excessive dissolution of impurities and protein degradation in a long time.

[0022] The present application preferably adopts the way of centrifugation to collect the first solid component. In the present application, the rate of centrifugation is preferably 4000r / min, and the time is preferably 5min.

[0023] After obtaining the first solid component, the first solid component is mixed with an alkali solution to perform alkali extraction, and after solid-liquid separation, an alkali extraction liquid and a second solid component are obtained, and the second solid component is a melanin / chitin composite. In the present application, the alkali solution is preferably an aqueous solution of alkali metal hydroxide, and the alkali metal hydroxide is preferably one or more of lithium hydroxide, sodium hydroxide and potassium hydroxide; and the molar concentration of the alkali solution is preferably 0.5-2mol / L, and more preferably 1-1.2mol / L. In the present application, the mass of the first solid component to the volume of the alkali solution is preferably 1g:10-15mL.

[0024] In the present application, the alkaline extraction is preferably carried out under stirring, the temperature of the alkaline extraction is preferably 50-90℃, more preferably 60-80℃, and the time is 1.5-3h, more preferably 2-2.5h. In the present application, the alkaline extraction ionizes the polar functional groups such as phenolic hydroxyl and carboxyl in the melanin molecules, thereby enhancing the water solubility of the melanin, making it more easily dissolved from the originally insoluble solid matrix into the alkaline solution phase; at the same time, the alkaline environment destroys the combination of melanin with proteins, polysaccharides and the like in the biomass, so that more melanin is dissolved; in addition, the alkaline environment makes the melanin negatively charged, which inhibits its self-aggregation and precipitation by electrostatic repulsion, reduces the loss and improves the extraction rate. In the present application, the solid-liquid separation is preferably carried out by centrifugation, and the centrifugal rate of the centrifugation is preferably 15000-20000r / min, more preferably 16000-18000r / min, and the time is preferably 15-20min, more preferably 16-18min.

[0025] After obtaining the alkaline extract, the present application mixes the alkaline extract with an acid solution to carry out acid precipitation to obtain melanin. In the present application, the acid solution is preferably one or more of hydrochloric acid, lactic acid or acetic acid, and the concentration of the acid solution is preferably 3-6mol / L; in the present application, after the alkaline extract is mixed with the acid solution, the pH value of the obtained mixed solution is 1-2. In the present application, the acid precipitation makes the polar functional groups such as phenolic hydroxyl and carboxyl in the melanin combine with hydrogen ions under acidic conditions, so that the hydrophilicity of the melanin is suddenly reduced, and the melanin is precipitated from the solution; at the same time, the acidic environment destroys the intermolecular electrostatic repulsion and promotes aggregation. In the present application, the temperature of the acid precipitation is preferably room temperature, and the time is preferably 1-48h, more preferably 2-24h, and further preferably 4-12h.

[0026] After the acid precipitation, the present application preferably carries out centrifugation to separate the melanin precipitate. In the present application, the centrifugal rate of the centrifugation is preferably 3000-5000r / min, more preferably 4000r / min, and the time is preferably 10-20min, more preferably 15min.

[0027] In the present application, after the acid precipitation, the process further preferably comprises repeating the alkaline extraction and acid precipitation of the precipitate obtained by the acid precipitation reaction, and the number of repetitions is 2-3 times.

[0028] The present application provides the melanin / chitin complex separated as described above. In the present application, the mass content of the melanin in the melanin / chitin complex is preferably 34.64%-59.47%.

[0029] The method for extracting melanin and melanin / chitin complex from black soldier flies provided by the present application will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0030] Example 1 The following steps were used to extract melanin and melanin / chitin complex from black soldier fly pupa shell by using a deep eutectic solvent method: (1) Degreasing: First, remove the foreign matter on the inside and outside of the black soldier fly, rinse repeatedly with water, then soak in an ethanol solution, rinse with water, and then dry, grind, and sieve to obtain a 60-mesh dehydrated pupa shell powder for use; Take 10 g of dehydrated pupa shell powder for degreasing, use Soxhlet extraction method, and use petroleum ether as solvent for degreasing treatment, the mass of dehydrated pupa shell powder to the volume of petroleum ether is 1 g: 10 mL, the degreasing temperature is 87°C, and the time is 6 h, to obtain degreased pupa shell powder and oil, the oil yield is 5%.

[0031] (2) One-step removal of protein and inorganic salt in pupa shell by deep eutectic solvent: Mix choline chloride and lactic acid according to a molar ratio of 1:2, the total volume of the mixture is 30 mL, and then add it into a round-bottom flask, place it in a 90°C water bath with constant stirring for 0.5 h to form a transparent liquid, which is the deep eutectic solvent; take 1.5 g of degreased pupa shell powder and add it into the natural deep eutectic solvent prepared above, and react in a 90°C water bath for 5 h, then collect the first solid component by centrifugation.

[0032] (3) Alkaline extraction of melanin: Take 2M NaOH solution and the above-mentioned first solid component according to the volume / weight ratio of 15 mL: 1 g to react in a water bath to obtain an extraction liquid, the extraction time is 90 min, and the water bath temperature is 70°C; centrifuge the obtained extraction liquid at a speed of 15000 r / min for 15 min to obtain the supernatant, and the solid component is the melanin / chitin complex, the yield is 27.5%.

[0033] (4) Acid precipitation of melanin: Take the supernatant and add dilute hydrochloric acid to adjust the pH value of the solution to pH=1, then stand at room temperature for 24 h, and then centrifuge at a speed of 4000 r / min for 10 min to obtain the precipitate, filter the fine residue, and then centrifuge the extraction liquid under acidic conditions to obtain the preliminary melanin extract, the yield is 3.3%.

[0034] Example 2 Extraction of melanin / chitin complex by choline chloride-acetic acid deep eutectic solvent system: The eutectic solvent system in step (2) of Example 1 was changed to choline chloride and acetic acid solution mixed in a molar ratio of 1:2, the total volume of the mixed solution was 30 mL, and stirring was carried out in a water bath at 80°C until it became a transparent colorless solution. 0.3 g of defatted chitin powder was dissolved in the above-mentioned 9 mL eutectic solvent, and after ultrasonic treatment for 20 min, the water bath at 80°C was continued for 4 h, and then filtration and drying were carried out. The remaining steps were the same as in Example 1. The melanin / chitin complex obtained by the experiment was 0.0781 g, with a yield of 26%.

[0035] Example 3 Extraction of melanin / chitin complex using choline chloride-citric acid eutectic solvent system: The eutectic solvent system in step (2) of Example 1 was changed to choline chloride and citric acid solution mixed in a molar ratio of 1:2, the total volume of the mixed solution was 50 mL, and stirring was carried out in a water bath at 80°C until it became a transparent colorless solution, and then 5 g of defatted chitin powder was added and stirring was continued in a water bath at 80°C for 2 h, and then filtration and drying were carried out. The remaining steps were the same as in Example 1. The melanin / chitin complex obtained by the experiment was 1.291 g, with a yield of 25.8%.

[0036] Example 4 Optimization of alkali concentration for melanin extraction in step (3) of Example 1: 2.5 g of the first solid component was weighed and reacted with 0.5 mol / L, 1 mol / L, and 2 mol / L NaOH solution, respectively, in a volume / weight ratio of 15 mL / 1 g at 80°C for 3 h, and the solid component was collected by centrifugation. In order to determine the effect of reaction alkali concentration on the extraction of melanin, the optimal alkali concentration was determined by the yield of melanin. The results are shown in Table 1. Figure 1 As can be seen from Table 1, the yield of melanin extracted using 2 mol / L NaOH was the highest.

[0037] Example 5 Optimization of reaction time for alkali extraction of melanin in step (3) of Example 1: The same mass of defatted chitin powder was reacted with 2 mol / L NaOH solution in a volume / weight ratio of 15 mL / 1 g at 70°C for 60, 90, 120, and 150 min, respectively, and the solid component was collected by centrifugation. In order to determine the effect of reaction time on the extraction of melanin, the optimal reaction time was determined by the yield of melanin. The results are shown in Table 2. Figure 2 As can be seen from Table 2, the yield of melanin extracted by reaction with alkali for 90 min was the highest.

[0038] Example 6 Optimization of reaction temperature for alkali extraction of melanin in step (3) of Example 1: The same mass of defatted chitin powder and 2 mol / L NaOH solution were reacted at 60°C, 70°C, 80°C for 90 min at a volume / weight ratio of 15 mL / 1 g, and the solid components were collected by centrifugation. In order to determine the effect of reaction temperature on the extraction of melanin, the optimal reaction temperature was determined by the yield of melanin. The results are shown in Table 1. Figure 3 As shown in Table 1, the yield of melanin extracted at 70°C was the highest.

[0039] Example 7 Step (3) in Example 1, the solid-liquid ratio of the alkaline melanin extraction reaction solution was optimized: The same mass of defatted chitin powder and 2 mol / L NaOH solution were reacted at 70°C for 90 min at a volume / weight ratio of 15 mL / 1 g and 10 mL / 1 g, and the solid components were collected by centrifugation. In order to determine the effect of the solid-liquid ratio on the extraction of melanin, the optimal solid-liquid ratio was determined by the yield of melanin. When the volume / weight ratio was 10, 0.0115 g of melanin was obtained, and when the volume / weight ratio was 15, 0.0798 g of melanin was obtained.

[0040] Example 8 Step (4) in Example 1, the precipitation time of the acid precipitated melanin was optimized: A certain amount of defatted chitin powder and 2 mol / L NaOH solution were reacted at 70°C for 90 min at a volume / weight ratio of 15 mL / 1 g, and the supernatant was collected by centrifugation. The obtained supernatant was divided into six equal parts, and an equal amount of 6 mol / L dilute hydrochloric acid was added to make the solution pH = 1, and precipitated at room temperature for 1, 2, 3, 4, 24, and 48 h, respectively. In order to determine the effect of acid precipitation time on the extraction of melanin, the optimal acid precipitation time was determined by the yield of melanin. The results are shown in Table 2. Figure 4 As shown in Table 2, the yield of melanin precipitated for 2 h was the highest.

[0041] Comparative Example 1 The acid-base method for extracting melanin and melanin / chitin composite from black soldier fly pupa shells includes the following steps: (1) Oil removal: First, remove the foreign matter on the inside and outside of the black soldier fly, rinse repeatedly with water, then soak in an ethanol solution, rinse with water, and then dry, grind, and sieve to obtain 60-mesh dehydrated pupa shell powder for use; Take 10 g of dehydrated pupa shell powder for oil removal, use Soxhlet extraction method, use petroleum ether as solvent for oil removal treatment, the mass of dehydrated pupa shell powder to the volume of petroleum ether is 1 g:10 mL, the oil removal temperature is 87°C, and the time is 4 h, to obtain dehydrated pupa shell powder and oil, the oil yield is 5%.

[0042] (2) Removal of inorganic salts: The 1.4M dilute hydrochloric acid and the above-mentioned dehydrated and degreased chitin powder are weighed at a volume / weight ratio of 12 mL:1 g, stirred at room temperature for 45 minutes to remove inorganic salts, and then centrifuged at a centrifugal rate of 4000 r / min for 15 minutes to collect the first solid component.

[0043] (3) Removal of protein by dilute alkali: The 0.5M NaOH and the above-mentioned first solid component are weighed at a volume / weight ratio of 10 mL / 1 g, reacted in a water bath at 55°C for 2 hours, and then centrifuged at a centrifugal rate of 4000 r / min for 15-20 minutes to obtain the second solid component and the supernatant. The second solid component is dried in an oven at 60°C, 6M dilute hydrochloric acid is added thereto to adjust the pH to 4, and then the precipitate is collected by centrifugation to obtain the protein, and the protein yield is 3.2%.

[0044] (4) Extraction of melanin by alkali solution: The 1M NaOH solution and the above-mentioned second solid component are taken at a volume / weight ratio of 15 mL:1 g, reacted in a water bath to obtain an extraction solution, the extraction time is 1.5 hours, and the water bath temperature is 70°C. The obtained extraction solution is centrifuged at a centrifugal rate of 17000 r / min for 15 minutes to obtain the supernatant, and the solid component is the melanin / chitin compound, and the yield is 12.95%.

[0045] (5) Precipitation of melanin by acid solution: The supernatant is added with dilute hydrochloric acid to adjust the pH of the solution to 1, and then the solution is centrifuged at a centrifugal rate of 4000 r / min for 15 minutes after being placed at room temperature for 24 hours. The extraction solution after filtering the fine residue is centrifuged under acidic conditions to obtain the preliminary melanin extract, and the melanin yield is 1.4%.

[0046] Structural characterization Figure 5 is a comparison chart of the ultraviolet-visible spectra of the melanin extracted in Example 1 and Comparative Example 1 of the present application, which is obtained by Figure 5 It can be seen that the obtained melanin has no maximum absorption peak in the visible light range, and has a maximum absorption peak at a wavelength of about 222 nm in the ultraviolet-visible light region, which is consistent with the typical ultraviolet absorption spectrum of true black melanin reported previously. There is no obvious absorption peak at 260 nm and 280 nm, indicating that the content of nucleic acid, protein and fat in the melanin is less.

[0047] Figure 6 is a Fourier transform infrared spectrum of the melanin extracted in Example 1 of the present application, which is obtained by Figure 6 It can be seen that the melanin has a relatively wide absorption peak at 3400 cm -1 , which is generated by O-H and N-H stretching vibration of indole, and is a characteristic absorption peak of melanin. The melanin sample has a peak at 2960-2850 cm-1 There is a relatively weak absorption peak near 1646 cm -1 , which is the stretching of aromatic C=C bond and the symmetric stretching vibration of -COOH or the bending vibration of N-H of indole system and the stretching vibration of C-N (secondary amine). The absorption at 1532 cm -1 is caused by the deformation vibration of COO- or C=C / C=N deformation vibration. The absorption peak at 1446 cm -1 is caused by the deformation of aliphatic C-H. The absorption peak at 1232 cm -1 is caused by the stretching of C=O in -COOH and the deformation of O-H and the stretching of C-O bond of phenol. From the absorption peak of melanin, it is shown that the black water beetle-derived melanin has a very typical indole structure.

[0048] Figure 7 The nuclear magnetic resonance hydrogen spectrum of the melanin proposed in Example 1 of the present application is shown in Figure 2. Figure 7 It can be seen that the absorption peak at δ = 1.23 ppm is mainly the characteristic peak of the terminal methyl of aliphatic hydrocarbon; the peak at δ = 2.50 ppm is the DMSO solvent peak; the spectrum peak at δ = 3.00-5.00 ppm is the hydrogen on the carbon connected with the group having strong electronegativity, and the weak broad peak in the low field region of 6.00-8.40 ppm is the absorption peak of aromatic hydrogen, which shows that the black water beetle melanin has indole structure and amide structure.

[0049] Figure 8 The X-ray photoelectron spectrum of the melanin proposed in the present application is shown in Figure 3. Figure 8 It can be seen that the melanin contains C, O and N elements, and does not contain S element, which shows that the melanin in the pupal shell of black water beetle is true melanin, which is consistent with the result of ultraviolet.

[0050] Figure 9 The Fourier transform infrared spectrum of the melanin / chitin proposed in Example 1 of the present application is shown in Figure 4. Figure 9 It can be seen that the broad peak near 3448 cm -1 is the superposition peak of the stretching vibration of hydroxyl O-H and amino N-H. Chitin contains a large amount of hydroxyl and amino groups, and the melanin also contains polar groups such as hydroxyl, and the hydroxyl and amino groups of the two jointly contribute to the broad peak in this region. The peak near 2926 cm -1 corresponds to the sp 3 C-H stretching vibration of saturated hydrocarbon (such as the alkyl chain in chitin), which shows that there is saturated carbon hydrogen structure in the complex. The peak near 1661 cm -1 corresponds to the C=O stretching vibration of amide I band, which is the typical characteristic of amide group in chitin, and the peak at 1554 cm -1Nearby: the peak at 1074 cm-1corresponds to the superposition of N-H bending vibration and C-N stretching vibration of amide II, further proving the existence of amide structure of chitin. The peak at 1074 cm -1 corresponds to the C-O stretching vibration in chitin, and the heterocyclic ring (such as indole ring) vibration of melanin may also contribute to the peak in this region. From the above, the characteristic functional groups of chitin in the complex are clearly present, and at the same time the polar groups of melanin form hydrogen bonds with chitin, indicating that melanin and chitin are complexed while retaining their respective key structural features. Figure 9

[0051] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.​

Claims

1. A method for extracting melanin and melanin / chitoxin complexes from black soldier flies, characterized in that, Includes the following steps: The black soldier fly pupa shells were dehydrated, crushed, and degreased to obtain dehydrated and degreased pupa shell powder. The dehydrated and defatted pupal shell powder was mixed with a eutectic solvent and extracted using the eutectic solvent to collect the first solid component. The first solid component is mixed with an alkaline solution for alkaline extraction. After solid-liquid separation, an alkaline extract and a second solid component are obtained. The second solid component is a melanin / chitosan complex. The alkaline extract was mixed with an acid solution and subjected to acid precipitation to obtain melanin.

2. The method according to claim 1, characterized in that, The degreasing agents used in the degreasing treatment include one or more of petroleum ether, ethanol, and ethyl acetate. The degreasing treatment is carried out at a temperature of 50~90℃ for 6~12 hours.

3. The method according to claim 1, characterized in that, The eutectic solvent comprises hydrogen bond donors and hydrogen bond acceptors, wherein the hydrogen bond donors comprise organic acids and the hydrogen bond acceptors comprise organic bases; The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1 to 3:

1.

4. The method according to claim 3, characterized in that, The organic acid includes one or more of lactic acid, malic acid, acetic acid and citric acid; the organic base includes choline chloride and / or betaine.

5. The method according to claim 1 or 3, characterized in that, The mass ratio of the dehydrated and defatted pupa shell powder to the volume ratio of the eutectic solvent is 1g:10~30mL; The extraction temperature of the eutectic solvent is 70~110℃, and the time is 2~6h.

6. The method according to claim 1, characterized in that, The alkaline solution is an aqueous solution of an alkali metal hydroxide, and the molar concentration of the alkaline solution is 0.5~2 mol / L; The alkaline extraction temperature is 50~90℃, and the time is 1.5~3h.

7. The method according to claim 1, characterized in that, The acid solution is one or more of hydrochloric acid, lactic acid, or acetic acid; after the alkaline extract is mixed with the acid solution, the pH value of the resulting mixture is 1 to 2.

8. The method according to claim 1 or 7, characterized in that, It also includes the process of repeatedly performing alkaline extraction and acid precipitation on the precipitate obtained from the acid precipitation reaction, and the number of repetitions is 2 to 3 times.

9. The melanin / chitosan complex obtained by the method according to any one of claims 1 to 8.

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