Continuous extraction method of melanin and chitin of hermetia illucens and application of melanin and chitin in preparation of functional feed for improving fish growth and metabolic immunity

Abstract

The invention relates to the technical field of aquaculture feed. The invention provides a continuous extraction method of black soldier fly melanin and chitin, the black soldier fly melanin prepared by adopting the technical scheme can be used as a feed additive, and the negative influence on lipid metabolism after fish oil in feed is replaced is effectively improved. The invention also provides an application of the black soldier fly melanin in preparation of a fish feed additive or preparation of a functional feed for improving fish growth and metabolic immunity, and the fish functional feed prepared by adding the black soldier fly melanin can improve fish growth, enhance non-specific immune enzyme activity of fish and improve the immunity of the fish. And the feed additive has no negative influence on the antioxidant capacity of animals, improves the health condition of the animals, and enables the fish culture industry to be sustainable and green in development.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture feeds, and particularly relates to a continuous extraction method of black soldier fly melanin and chitin and its application in the preparation of functional feeds for improving fish growth and metabolism immunity. Background Art

[0002] Rainbow trout are widely cultured globally due to their rich nutritional value and high aquaculture economic benefits. Data in 2022 showed that the aquaculture output of rainbow trout in China reached 37,300 tons. As a cold-water carnivorous fish, rainbow trout have relatively high requirements for feeds, and the feed issue has always been an important factor restricting the development of the aquaculture rainbow trout industry in China. Rainbow trout prefer high-lipid feeds. Research shows that when the crude fat level in the feed reaches 26.06%, the growth performance of rainbow trout is optimal. Traditionally, the fat source in rainbow trout feeds is mainly fish oil. However, due to the high cost of fish oil, to reduce aquaculture costs, a variety of alternative fat sources have been developed. However, when fish oil in the feed is replaced by other fat sources, it will change the fatty acid composition of the feed. In particular, the increase in the content of saturated fatty acids in the feed will cause fatty liver and lipid metabolism disorders in fish, which may have a negative impact on the growth performance and metabolism immunity of rainbow trout.

[0003] As a biological pigment, melanin is widely present in animals, plants, and protists. Melanin not only has good free radical scavenging activity but also has multiple functions such as antibacterial, anti-inflammatory, and lipid-lowering effects. Previous studies have shown that the exoskeleton of black soldier flies contains melanin and chitin. However, current studies can often only extract melanin-chitin complexes and it is difficult to isolate single melanin and chitin. Generally speaking, applying melanin to fish feeds has the potential to improve fish health and enhance animal growth, which is of great significance for improving resource utilization and creating economic value. Summary of the Invention

[0004] The purpose of the present invention is to provide a continuous extraction method of black soldier fly melanin and chitin and its application in the preparation of functional feeds for improving fish growth and metabolism immunity. By adding black soldier fly melanin, it can not only improve fish growth, enhance the non-specific immune enzyme activity of fish, but also have no negative impact on the antioxidant capacity of animals, and improve the negative impact on lipid metabolism caused by the replacement of fish oil in the feed, enabling the sustainable and green development of the fish aquaculture industry.

[0005] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a continuous extraction method of black soldier fly melanin and chitin, comprising the following steps:

[0007] (1) Inactivate the black soldier fly larvae, and then obtain black soldier fly residues after subcritical butane extraction and enzymatic hydrolysis.

[0008] (2) Mix the black soldier fly residues with lye for extraction, filter to obtain the supernatant and then precipitate to obtain black soldier fly melanin.

[0009] (3) Demineralize and decolorize the filter residue obtained by filtering in step (2) to obtain black soldier fly chitin.

[0010] Preferably, the temperature of inactivation in step (1) is 90 - 100 °C, and the time of inactivation is 5 - 15 min.

[0011] Preferably, the temperature of subcritical butane extraction in step (1) is 32 - 38 °C, the extraction time is 45 - 55 min / time, the number of extractions is 3 - 6 times, and the extraction pressure is 0.1 - 0.3 MPa.

[0012] Preferably, the proteases used for enzymatic hydrolysis in step (1) are keratinase and alkaline protease, and the mass ratio of keratinase to alkaline protease is 1:(0.5 - 1.5).

[0013] Preferably, the time of enzymatic hydrolysis is 150 - 240 min, and the temperature of enzymatic hydrolysis is 50 - 60 °C.

[0014] Preferably, the lye in step (2) is sodium hydroxide solution, and the concentration of the lye is 1 M - 3 M.

[0015] Preferably, the mass - volume ratio of black soldier fly residues to lye is (10 - 25) mg:1 mL; the extraction time is 30 - 180 min, and the extraction temperature is 60 - 90 °C.

[0016] Preferably, 1 - 8 M hydrochloric acid solution is used for precipitation in step (2).

[0017] Preferably, 0.5 - 3 M hydrochloric acid solution is used for demineralization in step (3), and sodium hypochlorite solution with a volume concentration of 0.1% - 1% or hydrogen peroxide solution with a volume concentration of 1% - 3.5% is used for decolorization.

[0018] The present invention also provides the black soldier fly melanin obtained by the continuous extraction method.

[0019] The present invention also provides the application of the black soldier fly melanin in the preparation of fish feed additives.

[0020] The present invention also provides the application of the black soldier fly melanin in the preparation of functional feeds for improving fish growth and metabolism immunity.

[0021] The present invention also provides a functional fish feed containing the black soldier fly melanin, which comprises the following components in parts by weight: 58 - 70 parts of protein source, 16 - 22 parts of sugar source, 12 - 19 parts of fat source, 0.5 - 1.5 parts of calcium dihydrogen phosphate, 0.4 - 0.6 parts of compound vitamin, 0.4 - 0.6 parts of compound mineral, 0.8 - 1.2 parts of monomer vitamin, 0.05 - 0.15 parts of mold inhibitor, and 0.01 - 0.5 parts of the black soldier fly melanin; the fat source comprises black soldier fly oil and lecithin with a mass ratio of (10 - 14):(2 - 5).

[0022] Preferably, the protein source includes one or more of fish meal, soybean meal, and soy protein isolate; the sugar source includes tapioca starch and / or flour; the monomer vitamin includes choline chloride and / or vitamin C.

[0023] By adopting the above technical solution, the present invention has the following beneficial effects:

[0024] 1. After fish oil in the technical solution of the present invention is replaced by black soldier fly oil, the addition of black soldier fly melanin can significantly improve the growth performance of rainbow trout, without affecting the survival rate, feed coefficient, and food intake of rainbow trout; it can also shorten the breeding cycle, save breeding costs, and increase the yield.

[0025] 2. The addition of black soldier fly melanin in the technical solution of the present invention to the feed can significantly restore the total cholesterol content in the serum, significantly increase the activity of alkaline phosphatase in the serum, without affecting the activities of glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase. It can restore the lipid metabolism of rainbow trout and improve the non-specific immune ability of rainbow trout, thereby improving the stress resistance and disease resistance of rainbow trout. The rainbow trout fed with the feed containing black soldier fly melanin will not cause oxidative stress in the liver and muscle, without affecting the antioxidant stress ability of rainbow trout. It can also reduce the increase in crude fat in the liver of rainbow trout caused by the replacement of fish oil with black soldier fly oil in the feed and relieve lipid metabolism disorders.

[0026] 3. The technical solution of the present invention can not only improve fish growth, enhance the non-specific immune enzyme activity of fish, but also have no negative impact on the antioxidant ability of animals, improve the negative impact on lipid metabolism caused by the replacement of fish oil in the feed, and enable the sustainable green development of the fish farming industry.

[0027] 4. Based on the fact that the black soldier fly larvae already have rich nutritional value through the technical solution of the present invention, single melanin and chitin can be further separated from the black soldier fly, which can effectively improve the utilization efficiency of resources and achieve a double improvement in environmental and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a graph showing the influence results of different extraction conditions on the yield of black soldier fly melanin (Figure 1 In which, A represents the influence of liquid-solid ratio, B represents the influence of NaOH concentration, C represents the influence of extraction time, and D represents the influence of extraction temperature);

[0029] Figure 2 is the ultraviolet-visible light spectrum of black soldier fly melanin;

[0030] Figure 3 is the Fourier transform infrared spectrum of black soldier fly melanin;

[0031] Figure 4 is the thermogravimetric analysis chart of black soldier fly melanin;

[0032] Figure 5 is the high performance liquid chromatography analysis chart of black soldier fly melanin;

[0033] Figure 6 is the result chart of the influence of different decolorization methods on chitin purification ( Figure 6 the left side in which represents the action of sodium hypochlorite solution, and the right side represents the action of hydrogen peroxide solution);

[0034] Figure 7 is the result chart of the comparison of Fourier transform infrared spectra of black soldier fly chitin and shrimp shell;

[0035] Figure 8 is the thermogravimetric analysis chart of black soldier fly chitin;

[0036] Figure 9 is the relationship chart between the proportion of different black soldier fly replacing fish meal and the weight gain rate of rainbow trout;

[0037] Figure 10 is the influence chart of different feeds on the liver morphology of rainbow trout. Specific implementation mode

[0038] The present invention provides a continuous extraction method for black soldier fly melanin and chitin, which comprises the following steps:

[0039] (1) Inactivate black soldier fly larvae, and then obtain black soldier fly residues after subcritical butane extraction and enzymatic hydrolysis;

[0040] (2) Mix the black soldier fly residues with an alkali solution for extraction, filter to obtain a supernatant, and then carry out precipitation to obtain black soldier fly melanin;

[0041] (3) Carry out demineralization and decolorization on the filter residue obtained by filtering in step (2) to obtain black soldier fly chitin.

[0042] In the present invention, black soldier fly larvae are selected for inactivation. The preferred age of the black soldier fly larvae is 6 - 13 days old, and more preferably 12 days old; the preferred inactivation temperature is 90 - 100 °C, more preferably 92 - 98 °C, and even more preferably 95 °C; the preferred inactivation time is 5 - 15 min, more preferably 8 - 12 min, and even more preferably 10 min.

[0043] In the present invention, the inactivated black soldier fly larvae are defatted to obtain black soldier fly larvae powder. Preferably, the subcritical butane extraction method is used. The preferred temperature for subcritical butane extraction is 32 - 38 °C, more preferably 33 - 37 °C, and even more preferably 35 °C; the preferred extraction time is 45 - 55 min / time, more preferably 48 - 53 min / time, and even more preferably 50 min / time; the preferred number of extractions is 3 - 6 times, and more preferably 5 times. The preferred pressure for subcritical butane extraction in the present invention is 0.1 - 0.3 MPa, more preferably 0.15 - 0.25 MPa, and even more preferably 0.2 MPa; the preferred liquid - solid ratio for subcritical butane extraction is (1 - 3):1, more preferably (1.5 - 2.5):1, and even more preferably 2:1.

[0044] In the present invention, after defatting, deproteinization treatment is carried out to obtain black soldier fly residue. Preferably, the protease enzymatic hydrolysis method is used. The proteases used for enzymatic hydrolysis are keratinase and alkaline protease. The keratinase is derived from Bacillus licheniformis JR - 101, and the alkaline protease is derived from Bacillus subtilis WB - 600; the preferred mass ratio of keratinase to alkaline protease is 1:(0.5 - 1.5), more preferably 1:(0.8 - 1.2), and even more preferably 1:1. In the present invention, the preferred enzymatic hydrolysis time is 150 - 240 min, more preferably 170 - 200 min, and even more preferably 180 min; the preferred enzymatic hydrolysis temperature is 50 - 60 °C, more preferably 50 - 58 °C, and even more preferably 55 °C; the preferred enzyme - substrate ratio is 1% - 3.5%, more preferably 2% - 3.2%, and even more preferably 3%; the liquid - solid ratio (liquid - solid mass ratio) of water to black soldier fly larvae powder is preferably (5 - 10):1, more preferably (6 - 8):1, and even more preferably 7:1. In the present invention, stirring treatment is preferably carried out during the enzymatic hydrolysis process. The preferred stirring speed is 150 - 250 rpm, more preferably 180 - 220 rpm, and even more preferably 200 rpm. The pH value is maintained at 8 - 10 during the enzymatic hydrolysis process in the present invention, and more preferably 9.

[0045] In the present invention, black soldier fly larvae residues are mixed with an alkali solution for extraction, and then filtered to obtain a supernatant and residues. The supernatant is precipitated to obtain black soldier fly melanin. The alkali solution in the present invention is a sodium hydroxide solution, and the concentration of the alkali solution is preferably 1M - 3M, more preferably 1.5 - 2.5M, and even more preferably 2.2M; the mass - volume ratio of the black soldier fly larvae residues to the alkali solution is preferably (10 - 25)mg:1mL, more preferably (12 - 20)mg:1mL, and even more preferably 15mg:1mL. The extraction time in the present invention is preferably 30 - 180min, more preferably 60 - 150min, and even more preferably 90min; the extraction temperature is preferably 60 - 90°C, more preferably 65 - 80°C, and even more preferably 70°C.

[0046] In the present invention, after the extraction is completed, filtration is carried out. The filtration preferably uses a gauze, and the mesh number of the gauze is preferably 80 - 150 meshes, more preferably 100 - 150 meshes, and even more preferably 120 meshes. After filtration, the supernatant and residues are collected separately. The present invention preferably uses a hydrochloric acid solution to precipitate the supernatant, and the concentration of the hydrochloric acid solution is preferably 1 - 8M, more preferably 3 - 7M, and even more preferably 6M.

[0047] In the present invention, the residues are demineralized and decolorized to obtain black soldier fly chitin. The present invention preferably uses a hydrochloric acid solution for demineralization treatment, and the concentration of the hydrochloric acid solution is preferably 0.5 - 3M, more preferably 0.8 - 2M, and even more preferably 1M; the demineralization treatment time is preferably 20 - 30h, more preferably 22 - 26h, and even more preferably 24h. The present invention preferably uses a sodium hypochlorite solution or a hydrogen peroxide solution for decolorization treatment. The volume concentration of the sodium hypochlorite solution is preferably 0.1% - 1%, more preferably 0.1 - 0.5%, and even more preferably 0.4%; the volume concentration of the hydrogen peroxide solution is preferably 1% - 3.5%, more preferably 2% - 3.2%, and even more preferably 3%.

[0048] The present invention also provides the black soldier fly melanin obtained by the continuous extraction method.

[0049] The present invention also provides the application of the black soldier fly melanin in the preparation of fish feed additives.

[0050] The present invention also provides the application of the black soldier fly melanin in the preparation of functional feeds for improving fish growth and metabolism immunity.

[0051] The present invention also provides a functional fish feed containing the black soldier fly melanin, which comprises the following components: protein source, sugar source, fat source, calcium dihydrogen phosphate, compound vitamins, compound minerals, monomer vitamins, mold inhibitor, and the black soldier fly melanin.

[0052] In the present invention, the weight parts of the protein source are preferably 58 - 70 parts, more preferably 60 - 68 parts, and even more preferably 64 parts; the weight parts of the sugar source are preferably 16 - 22 parts, more preferably 17 - 20 parts, and even more preferably 17.9 parts; the weight parts of the fat source are preferably 12 - 19 parts, more preferably 13 - 17 parts, and even more preferably 15 parts; the weight parts of the calcium dihydrogen phosphate are preferably 0.5 - 1.5 parts, more preferably 0.8 - 1.2 parts, and even more preferably 1 part; the weight parts of the compound vitamins are preferably 0.4 - 0.6 parts, more preferably 0.45 - 0.55 parts, and even more preferably 0.5 parts; the weight parts of the compound minerals are preferably 0.4 - 0.6 parts, more preferably 0.45 - 0.55 parts, and even more preferably 0.5 parts; the weight parts of the monomer vitamins are preferably 0.8 - 1.2 parts, more preferably 0.8 - 1.1 parts, and even more preferably 1 part; the weight parts of the mold inhibitor are preferably 0.05 - 0.15 parts, more preferably 0.08 - 0.12 parts, and even more preferably 0.1 part; the weight parts of the black soldier fly melanin are preferably 0.01 - 0.5 parts, more preferably 0.1 - 0.45 parts, and even more preferably 0.4 parts.

[0053] In the present invention, the fat source preferably includes black soldier fly oil and lecithin, and the mass ratio of the black soldier fly oil to lecithin is preferably (10 - 14):(2 - 5), more preferably (11 - 13):(2.5 - 4), and even more preferably 12:3.

[0054] In the present invention, the protein source preferably includes one or more of fish meal, soybean meal, and soy protein isolate. When the protein source of the present invention is fish meal, soybean meal, and soy protein isolate, the mass ratio of the fish meal, soybean meal, and soy protein isolate is preferably (40 - 44):(7 - 13):(11 - 13), more preferably (41 - 43):(8 - 12):(11.5 - 12.5), and even more preferably 42:10:12.

[0055] In the present invention, the sugar source preferably includes tapioca starch and / or flour. When the sugar source in the present invention is tapioca starch and flour, the mass ratio of the tapioca starch to the flour is preferably (4 - 8):(12 - 14), more preferably (4.5 - 6):(12 - 13), and even more preferably 5:12.5.

[0056] In the present invention, the monomeric vitamins preferably include choline chloride and / or vitamin C. When the monomeric vitamins in the present invention are choline chloride and vitamin C, the mass ratio of the choline chloride to the vitamin C is preferably (0.4 - 0.6):(0.4 - 0.6), more preferably (0.45 - 0.58):(0.45 - 0.55), and even more preferably 0.5:0.5.

[0057] In the present invention, the mildew preventive is preferably calcium propionate.

[0058] In the present invention, the compound vitamins preferably include vitamin B1 20 - 40 mg / kg, vitamin B2 50 - 70 mg / kg, vitamin B6 10 - 30 mg / kg, niacin 180 - 220 mg / kg, calcium pantothenate 80 - 120 mg / kg, inositol 80 - 120 mg / kg, biotin 2 - 3 mg / kg, folic acid 8 - 12 mg / kg, vitamin B12 0.05 - 0.15 mg / kg, vitamin K3 30 - 50 mg / kg, vitamin A 9000 - 11000 IU / kg, vitamin D3 1800 - 2200 IU / kg, and vitamin E 150 - 170 IU / kg. More preferably, they include vitamin B1 25 - 35 mg / kg, vitamin B2 55 - 65 mg / kg, vitamin B6 15 - 25 mg / kg, niacin 190 - 210 mg / kg, calcium pantothenate 90 - 110 mg / kg, inositol 90 - 110 mg / kg, biotin 2.2 - 2.8 mg / kg, folic acid 9 - 11 mg / kg, vitamin B12 0.08 - 0.12 mg / kg, vitamin K3 35 - 45 mg / kg, vitamin A 9500 - 10500 IU / kg, vitamin D3 1900 - 2100 IU / kg, and vitamin E 155 - 165 IU / kg. Even more preferably, they include vitamin B1 30 mg / kg, vitamin B2 60 mg / kg, vitamin B6 20 mg / kg, niacin 200 mg / kg, calcium pantothenate 100 mg / kg, inositol 100 mg / kg, biotin 2.5 mg / kg, folic acid 10 mg / kg, vitamin B12 0.1 mg / kg, vitamin K3 40 mg / kg, vitamin A 10000 IU / kg, vitamin D3 2000 IU / kg, and vitamin E 160 IU / kg.

[0059] In the present invention, the composite mineral preferably includes MgSO 4 ·H 2 O 1000 - 1100 mg / kg, KH 2 PO 4 900 - 950 mg / kg, NaH 2 PO 4 ·2H 2 O 400 - 450 mg / kg, FeC 6 H 5 O 7 ·5H 2 O 170 - 200 mg / kg, ZnCl 2 70 - 90 mg / kg, CuSO 4 ·5H 2 O 60 - 70 mg / kg, AlCl 3 ·6H 2 O 45 - 60 mg / kg, MnSO 4 ·H 2 O 25 - 35 mg / kg, KI 25 - 35 mg / kg, CoCl 2 ·6H 2 O 4 - 9 mg / kg and Na 2 SeO 3 ·H 2 O 0.5 - 1.5 mg / kg, and more preferably includes MgSO 4 ·H 2 O 1050 - 1095 mg / kg, KH 2 PO 4 920 - 940 mg / kg, NaH 2 PO 4 ·2H 2 O 420 - 440 mg / kg, FeC 6 H 5 O 7 ·5H 2 O 175 - 190 mg / kg, ZnCl 2 75 - 85 mg / kg, CuSO 4 ·5H 2 O 61 - 65 mg / kg, AlCl 3 ·6H 2 O 50 - 55 mg / kg, MnSO 4 ·H 2 O 28 - 33 mg / kg, KI 27 - 30 mg / kg, CoCl 2 ·6H 2O 5 - 7 mg / kg and Na 2 SeO 3 ·H 2 O 0.7 - 1.0 mg / kg, more preferably including MgSO 4 ·H 2 O 1090 mg / kg, KH 2 PO 4 932 mg / kg, NaH 2 PO 4 ·2H 2 O 432 mg / kg, FeC 6 H 5 O 7 ·5H 2 O 181 mg / kg, ZnCl 2 80 mg / kg, CuSO 4 ·5H 2 O 63 mg / kg, AlCl 3 ·6H 2 O 51 mg / kg, MnSO 4 ·H 2 O 31 mg / kg, KI 28 mg / kg, CoCl 2 ·6H 2 O 6 mg / kg and Na 2 SeO 3 ·H 2 O 0.8 mg / kg.

[0060] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0061] Example 1

[0062] A kind of black soldier fly melanin and chitin, the preparation method is as follows:

[0063] (1) Select 12-day-old black soldier fly larvae, inactivate them at 95 °C for 10 min, and then extract and degrease them with subcritical butane to obtain black soldier fly larvae powder. The extraction conditions are 35 °C, 50 min / time, extract 5 times, extraction pressure 0.2 MPa, liquid-solid ratio 2:1; carry out enzymatic hydrolysis according to the liquid-solid mass ratio of water to black soldier fly larvae powder of 7:1 and enzyme-substrate ratio of 3%. The protease is keratinase and alkaline protease with a mass ratio of 1:1, carry out enzymatic hydrolysis at 55 °C for 180 min, and stir at a speed of 200 rpm during the enzymatic hydrolysis process to keep the pH value at 9;

[0064] (2) Mix the black soldier fly larvae residue with 2.2 M sodium hydroxide solution at a ratio of 15 mg:1 mL, then extract at 70 °C for 90 min, and filter using a 120-mesh silk screen to obtain the supernatant and residue; precipitate the supernatant with 6 M hydrochloric acid solution to obtain black soldier fly melanin;

[0065] (3) Carry out demineralization treatment on the residue with 1 M hydrochloric acid solution for 24 h, and then carry out decolorization treatment with 0.4% sodium hypochlorite solution to obtain black soldier fly chitin.

[0066] Example 2

[0067] A kind of black soldier fly melanin and chitin, the preparation method is as follows:

[0068] (1) Select 12-day-old black soldier fly larvae, inactivate them at 90 °C for 15 min, and then extract fat with subcritical butane to obtain black soldier fly larvae powder. The extraction conditions are 32 °C, 55 min / time, extract 6 times, extraction pressure 0.1 MPa, liquid-solid ratio 2:1; carry out enzymatic hydrolysis by mixing according to the liquid-solid mass ratio of water to black soldier fly larvae powder of 7:1 and enzyme-substrate ratio of 1%, and the protease is keratinase and alkaline protease with a mass ratio of 1:0.5, carry out enzymatic hydrolysis at 50 °C for 240 min, and stir at a speed of 200 rpm during the enzymatic hydrolysis process to keep the pH value at 9;

[0069] (2) Mix the black soldier fly larvae residue with 2.2 M sodium hydroxide solution at a ratio of 10 mg:1 mL, then extract at 60 °C for 180 min, and filter using an 80-mesh silk screen to obtain the supernatant and residue; precipitate the supernatant with 6 M hydrochloric acid solution to obtain black soldier fly melanin;

[0070] (3) Carry out demineralization treatment on the residue with 1 M hydrochloric acid solution for 24 h, and then carry out decolorization treatment with 0.4% sodium hypochlorite solution to obtain black soldier fly chitin.

[0071] Example 3

[0072] A kind of black soldier fly melanin and chitin, the preparation method is as follows:

[0073] (1) Select 12-day-old black soldier fly larvae, inactivate them at 100 °C for 5 min, and then extract fat with subcritical butane to obtain black soldier fly larvae powder. The extraction conditions are 38 °C, 45 min / time, extract 3 times, extraction pressure 0.3 MPa, liquid-solid ratio 3:1; carry out enzymatic hydrolysis by mixing according to the liquid-solid mass ratio of water to black soldier fly larvae powder of 10:1 and enzyme-substrate ratio of 3%, and the protease is keratinase and alkaline protease with a mass ratio of 1:1.5, carry out enzymatic hydrolysis at 60 °C for 150 min, and stir at a speed of 220 rpm during the enzymatic hydrolysis process to keep the pH value at 9;

[0074] (2) Mix the black soldier fly larvae residue with 2.2 M sodium hydroxide solution at a ratio of 25 mg: 1 mL, then extract at 90 °C for 60 min, and filter through 80-mesh silk screen to obtain the supernatant and residue; precipitate the supernatant with 1 M hydrochloric acid solution to obtain black soldier fly melanin;

[0075] (3) Carry out demineralization treatment on the residue with 1 M hydrochloric acid solution for 24 h, and then carry out decolorization treatment with 3% hydrogen peroxide solution to obtain black soldier fly chitin.

[0076] Example 4

[0077] A functional fish feed containing the black soldier fly melanin described in Example 1, with the following components:

[0078] 42 parts of fish meal, 10 parts of soybean meal, 12 parts of soy protein isolate, 5 parts of tapioca starch, 12.5 parts of flour, 12 parts of black soldier fly oil, 3 parts of lecithin, 1 part of monocalcium phosphate, 0.5 part of compound vitamin, 0.5 part of compound mineral, 0.5 part of choline chloride, 0.5 part of vitamin C, 0.1 part of calcium propionate, and 0.4 part of the black soldier fly melanin described in Example 1;

[0079] The compound vitamin is: 30 mg / kg of vitamin B1, 60 mg / kg of vitamin B2, 20 mg / kg of vitamin B6, 200 mg / kg of niacin, 100 mg / kg of calcium pantothenate, 100 mg / kg of inositol, 2.5 mg / kg of biotin, 10 mg / kg of folic acid, 0.1 mg / kg of vitamin B12, 40 mg / kg of vitamin K3, 10000 IU / kg of vitamin A, 2000 IU / kg of vitamin D3, and 160 IU / kg of vitamin E;

[0080] The compound mineral is MgSO 4 ·H 2 O 1090 mg / kg, KH 2 PO 4 932 mg / kg, NaH 2 PO 4 ·2H 2 O 432 mg / kg, FeC 6 H 5 O 7 ·5H 2 O 181 mg / kg, ZnCl 2 80 mg / kg, CuSO 4 ·5H 2 O 63 mg / kg, AlCl 3 ·6H 2 O 51 mg / kg, MnSO 4 ·H 2O 31 mg / kg, KI 28 mg / kg, CoCl 2 ·6H 2 O 6 mg / kg and Na 2 SeO 3 ·H 2 O 0.8 mg / kg.

[0081] Example 5

[0082] A functional fish feed containing the black soldier fly melanin described in Example 2, with the following components:

[0083] Fish meal 42 parts, soybean meal 10 parts, soy protein isolate 12 parts, cassava starch 5 parts, flour 12.5 parts, black soldier fly oil 12 parts, lecithin 3 parts, calcium dihydrogen phosphate 1 part, compound vitamin 0.5 part, compound mineral 0.5 part, choline chloride 0.5 part, vitamin C 0.5 part, calcium propionate 0.1 part, and the black soldier fly melanin described in Example 2 0.4 part;

[0084] The compound vitamin is: vitamin B1 30 mg / kg, vitamin B2 60 mg / kg, vitamin B6 20 mg / kg, niacin 200 mg / kg, calcium pantothenate 100 mg / kg, inositol 100 mg / kg, biotin 2.5 mg / kg, folic acid 10 mg / kg, vitamin B12 0.1 mg / kg, vitamin K3 40 mg / kg, vitamin A 10000 IU / kg, vitamin D3 2000 IU / kg, and vitamin E 160 IU / kg;

[0085] The compound mineral is MgSO 4 ·H 2 O 1090 mg / kg, KH 2 PO 4 932 mg / kg, NaH 2 PO 4 ·2H 2 O 432 mg / kg, FeC 6 H 5 O 7 ·5H 2 O 181 mg / kg, ZnCl 2 80 mg / kg, CuSO 4 ·5H 2 O 63 mg / kg, AlCl 3 ·6H 2 O 51 mg / kg, MnSO 4 ·H 2 O 31 mg / kg, KI 28 mg / kg, CoCl 2 ·6H 2O 6 mg / kg and Na 2 SeO 3 ·H 2 O 0.8 mg / kg.

[0086] Example 6

[0087] A functional fish feed containing the black soldier fly melanin described in Example 3, with the following components:

[0088] 42 parts of fish meal, 10 parts of soybean meal, 12 parts of soy protein isolate, 5 parts of cassava starch, 12.5 parts of flour, 12 parts of black soldier fly oil, 3 parts of lecithin, 1 part of monocalcium phosphate, 0.5 part of compound vitamin, 0.5 part of compound mineral, 0.5 part of choline chloride, 0.5 part of vitamin C, 0.1 part of calcium propionate, and 0.4 part of the black soldier fly melanin described in Example 3;

[0089] The compound vitamin is: 30 mg / kg of vitamin B1, 60 mg / kg of vitamin B2, 20 mg / kg of vitamin B6, 200 mg / kg of niacin, 100 mg / kg of calcium pantothenate, 100 mg / kg of inositol, 2.5 mg / kg of biotin, 10 mg / kg of folic acid, 0.1 mg / kg of vitamin B12, 40 mg / kg of vitamin K3, 10000 IU / kg of vitamin A, 2000 IU / kg of vitamin D3, and 160 IU / kg of vitamin E;

[0090] The compound mineral is MgSO 4 ·H 2 O 1090 mg / kg, KH 2 PO 4 932 mg / kg, NaH 2 PO 4 ·2H 2 O 432 mg / kg, FeC 6 H 5 O 7 ·5H 2 O 181 mg / kg, ZnCl 2 80 mg / kg, CuSO 4 ·5H 2 O 63 mg / kg, AlCl 3 ·6H 2 O 51 mg / kg, MnSO 4 ·H 2 O 31 mg / kg, KI 28 mg / kg, CoCl 2 ·6H 2 O 6 mg / kg and Na 2 SeO 3 ·H2 O 0.8 mg / kg.

[0091] Test Example 1

[0092] (I) Extraction of black soldier fly melanin

[0093] Select 12-day-old black soldier fly larvae, inactivate them at 95 °C for 10 min, and then obtain black soldier fly larvae powder by subcritical butane extraction for defatting. The extraction conditions are 35 °C, 50 min / time, extract 5 times, extraction pressure 0.2 MPa, liquid-solid ratio 2:1; carry out enzymatic hydrolysis according to the liquid-solid mass ratio of water to black soldier fly larvae powder being 7:1 and enzyme-substrate ratio 3%. The protease is keratinase and alkaline protease with a mass ratio of 1:1, carry out enzymatic hydrolysis at 55 °C for 180 min, stir at a speed of 200 rpm during the enzymatic hydrolysis process, and keep the pH value at 9. Mix the black soldier fly insect residue with 0.05 - 3 M sodium hydroxide solution according to the ratio of (15 - 40) mg:1 mL, then extract at 40 - 90 °C for 30 - 180 min, and filter with a 120-mesh silk screen to obtain the supernatant and the filter residue; precipitate the supernatant with 6 M hydrochloric acid solution to obtain black soldier fly melanin.

[0094] Table 1 Effects of different extraction conditions on the yield of black soldier fly melanin

[0095]

[0096] Through the optimization of the conditions of sodium hydroxide concentration, extraction time, extraction temperature and liquid-solid ratio, the results are as Figure 1 shown in Table 1. The yield of black soldier fly melanin increases with the increase of sodium hydroxide concentration, extraction time and extraction temperature, and decreases with the increase of liquid-solid ratio; at the same time, the purity of melanin increases with the increase of liquid-solid ratio, sodium hydroxide concentration and extraction temperature, and decreases with the prolongation of extraction time. Taking the highest yield as the goal, the optimized extraction conditions are liquid-solid ratio 15:1, NaOH concentration 2.2 M, extraction time 90 min, extraction temperature 70 °C, and the yield is 8.03% (percentage of insect residue).

[0097] (II) Characterization of black soldier fly melanin

[0098] Identify black soldier fly melanin by technical methods such as ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, elemental analysis, thermogravimetric analysis, high performance liquid chromatography, etc.

[0099] The results are as Figure 2 shown. Black soldier fly melanin has a strong absorption peak at 210 nm ( Figure 2 the black line in Figure 2(red line in). In addition, no absorbance peak was observed at 260 or 280 nm, indicating the absence of impurities such as proteins and lipids.

[0100] The results of Fourier transform infrared spectroscopy showed that the melanin of Hermetia illucens had the functional group characteristics of its structural monomers 5,6-dihydroxyindole (DHI) and 5,6-dihydroxy-2-indolecarboxylic acid (DHICA). As Figure 3 shown, the broad absorption band observed near 3349 cm -1 belongs to the hydroxyl group (ROH), and the absorption bands at 2923 - 2852 cm -1 are for the asymmetric and symmetric stretching vibrations of methylene (CH 2 ), the peak at 1710 cm -1 is for the stretching of the carbonyl group (C=O), the peak at 1655 cm -1 is the characteristic peak for the vibration of the carbon-carbon double bond (C=C) on the amide I aromatic ring and the symmetric stretching of the acyl group (RCO), the band at 1541 cm -1 points to the bending vibration of the N-H group, indicating the presence of an indole structure in the melanin of Hermetia illucens, the peak at 1464 cm -1 is for the strong shear bending of methylene, the absorption peaks at 1246 - 1046 cm -1 are for phenolic bond vibration, and the absorption peak at 898 cm -1 is for the out-of-plane bending of O-H (δ-OH).

[0101] Table 2 Elemental composition of the melanin of Hermetia illucens

[0102]

[0103] The results of elemental analysis showed that, as shown in Table 2, the melanin of Hermetia illucens was composed of 57.73% C, 8.21% H, 3.77% N, 0.3% S, and 29.97% O, and the C / N ratio was 15.29%. The results of thermogravimetric analysis showed three weight loss peaks ( Figure 4 ), the peak at 55 °C was for weakly bound water, the peak at 282 °C was for the decomposition of aliphatic components, and the peak at 435 to 530 °C was for the decomposition of aromatic compounds. After 550 °C and higher temperatures, about 25% of the mass was retained. The results of high performance liquid chromatography showed that the melanin sample of Hermetia illucens was oxidized in an alkaline environment and thus transformed into different biomarkers, namely 2,3-pyrroledicarboxylic acid (PDCA) and 2,3,5-pyrroletricarboxylic acid (PTCA), and the results were as Figure 5 shown. The obvious separation of the PDCA peak and the PTCA peak in the melanin sample of Hermetia illucens.

[0104] Test Example 2

[0105] (I) Extraction and purification of chitin from Hermetia illucens

[0106] The filter residue remaining after filtration in Test Example 1 was subjected to demineralization treatment with 1 M hydrochloric acid solution for 24 h, and then decolorization treatment was carried out using 0.4% sodium hypochlorite solution or 3% hydrogen peroxide solution respectively to obtain black soldier fly chitin. Two other different treatment groups were set up. In one group, the filter residue was only not subjected to acid treatment, and the other treatments were the same; in the other group, only decolorization treatment was not carried out, and the other treatments were the same.

[0107] Table 3 Extraction and purification effects of black soldier fly chitin

[0108]

[0109] The results are shown in Table 3 (sodium hypochlorite treatment results). After acid treatment, the crude ash content of the insect residue decreased from 20.09% to 1.17%, and the total nitrogen increased from 5.58% to 7.08%, indicating that acid treatment can effectively reduce the minerals in the insect residue. Subsequently, decolorization treatment was carried out on the crude chitin, and the results are as Figure 6 shown ( Figure 6 the left side in represents sodium hypochlorite, and the right side represents hydrogen peroxide). The whiter the color, the better the decolorization effect. Visually observing, the color of the sodium hypochlorite solution treatment group is whiter, indicating that the sodium hypochlorite solution can effectively decolorize the crude chitin.

[0110] (II) Characterization of black soldier fly chitin

[0111] The black soldier fly chitin was identified by Fourier transform infrared spectroscopy, elemental analysis and thermogravimetric analysis. The results of Fourier transform infrared spectroscopy showed (as Figure 7 ) that the black soldier fly chitin had the typical characteristic absorption bands of α-chitin ( Figure 7 the black line in), which was the same as the chitin derived from shrimp shells ( Figure 7 the blue line in), and had absorption peaks at 1650, 1619 and 1551 cm -1 . Among them, the bands at 1650 and 1619 cm -1 belonged to the stretching vibration of the secondary amide of the carbonyl group, and the band at 1551 cm -1 belonged to the bending of N-H and the stretching of the C-N group. The band at 2924 cm -1 indicated the symmetric stretching of methylene. The obvious peak at 1376 cm -1 was due to the bending of C-H and the symmetric deformation of the methyl group, while the obvious peak at 1306 cm -1 was due to the amide vibration of the C-N group. The continuous peaks at 1206, 1155, 1113 and 1062 cm -1 were for the in-plane bending of the hydroxyl group, the stretching of the C-O group, the stretching of the asymmetric bridging C-O-C and C-O groups.

[0112] Table 4 Elemental composition of black soldier fly chitin

[0113]

[0114] The results of elemental analysis showed that as shown in Table 4, black soldier fly chitin was composed of 44.19% C, 6.76% H, 6.22% N, and 42.83% O, and the C / N ratio was 7.10%. The results of thermogravimetric analysis showed (as Figure 8 ) that as the temperature increased, black soldier fly chitin exhibited two distinct degradation stages. The initial stage occurred at 30 - 120 °C with a weight loss of approximately 2%, corresponding to the evaporation of weakly bound water. The second weight loss occurred at 387 °C, mediated by the dehydration polymerization of sugar rings and the decomposition of chitin acetylation and deacetylation units. After 550 °C and higher temperatures, approximately 19.85% of the mass remained.

[0115] Test Example 3

[0116] An 8 - week feeding experiment was designed to evaluate the effect of black soldier fly melanin as a feed additive on rainbow trout.

[0117] In the control group (CD), all fish oil in CD was replaced with black soldier fly oil, and then 0%, 0.01%, 0.05%, 0.1%, 0.2%, and 0.4% of melanin were supplemented respectively to prepare NC, Mela1, Mela2, Mela3, Mela4, and Mela5 feeds. The experimental feed formulations are shown in Tables 5 and 6. During the 8 - week feeding experiment, satiation feeding was carried out at 7:00 am and 5:00 pm every day.

[0118] Table 5 Experimental feed formulation (%)

[0119]

[0120] Table 6 Nutritional composition of experimental feeds

[0121]

[0122] (I)

[0124] After the 8 - week feeding experiment, the fish in each net cage were counted and weighed to calculate the weight gain rate, specific growth rate, feed conversion ratio, survival rate, and feed intake.

[0125] The results showed that the present invention could significantly improve the growth performance of rainbow trout (as shown in Table 7 and Figure 9 ), without affecting the survival rate, feed conversion ratio, and feed intake of rainbow trout, which could not only shorten the breeding cycle, save breeding costs, but also increase the yield.

[0126] Table 7 Effect of black soldier fly melanin on the growth performance of rainbow trout

[0127]

[0128] Note: Different letters represent significant differences (P < 0.05). (II)

[0130] After the 8-week breeding experiment, all fish were fasted for 24 hours, and then the livers and dorsal muscles of rainbow trout were taken to measure the antioxidant enzyme activities.

[0131] Table 8 Effects of black soldier fly melanin on antioxidant enzyme activities in the liver and dorsal muscle of rainbow trout

[0132]

[0133]

[0134] Note: Different letters represent significant differences (P < 0.05).

[0135] The results are shown in Table 8. Feeding rainbow trout with feed containing black soldier fly protein peptides does not cause oxidative stress in the liver and muscle. The present invention can be used as a feed additive in rainbow trout feed without affecting the antioxidant stress ability of rainbow trout. (III)

[0137] The serum of rainbow trout was taken for analysis of blood lipid and metabolic immune enzyme activities.

[0138] Table 9 Effects of black soldier fly melanin on blood lipid and metabolic enzyme activities in the serum of rainbow trout

[0139]

[0140] Note: Different letters represent significant differences (P < 0.05).

[0141] The results are shown in Table 9. Black soldier fly melanin can significantly restore the total cholesterol content in the serum to the control group level, and significantly increase the alkaline phosphatase activity in the serum without affecting the activities of aspartate aminotransferase and alanine aminotransferase. The above results indicate that after the fish oil in the feed is replaced by black soldier fly oil, supplementing black soldier fly melanin can restore the lipid metabolism of rainbow trout to the control group level and improve the non-specific immune ability of rainbow trout, thereby enhancing the stress resistance and disease resistance of rainbow trout. (IV)

[0143] The whole fish, dorsal muscle and liver of rainbow trout were taken for component analysis.

[0144] Table 10 Effects of black soldier fly melanin on the components of the whole fish, dorsal muscle and liver of rainbow trout

[0145]

[0146] Note: Different letters represent significant differences (P<0.05).

[0147] The results are shown in Table 10. Hermetia illucens melanin does not affect the moisture and crude protein contents of whole fish and dorsal muscle of rainbow trout, and significantly reduces the crude fat content of the liver. After the fish oil in the feed is replaced by other oils, the content of saturated fatty acids in the feed often increases while the content of unsaturated fatty acids decreases, resulting in lipid metabolism disorders in animals, promoting lipid synthesis and inhibiting fat decomposition, and further causing liver fat accumulation and fatty degeneration, resulting in fatty liver in fish, which seriously damages animal health. The present invention can reduce the increase in crude fat in the liver of rainbow trout caused by the replacement of fish oil in the feed with Hermetia illucens oil and alleviate lipid metabolism disorders (such as Figure 10 ).

[0148] In summary, the technical solution of the present invention provides a continuous extraction method of Hermetia illucens melanin and chitin and a functional feed prepared therefrom. The Hermetia illucens melanin of the present invention has the characteristics of eumelanin, and the Hermetia illucens chitin has the characteristics of α-chitin. The melanin yield can reach 1.01%, and the chitin yield can reach 6.92% (accounting for the proportion of dry Hermetia illucens insects). After the fish oil in the feed is replaced by other oils, feeding the functional feed containing Hermetia illucens melanin can improve the growth performance of rainbow trout without affecting the feeding and survival rate of rainbow trout. In addition, Hermetia illucens melanin can improve the non-specific immune ability of animals without having a negative impact on the antioxidant ability of rainbow trout; it can improve fat accumulation, lipid metabolism disorders and fatty liver caused by high saturated fatty acids, improve the health status of animals, and enable the sustainable green development of the rainbow trout aquaculture industry.

[0149] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for continuously extracting black soldier fly melanin and chitin, characterized in that: The steps include: (1) inactivating black soldier fly larvae, and then extracting and enzymolyzing them with subcritical butane to obtain black soldier fly residue; (2) extracting the black soldier fly residue by mixing it with an alkali solution, filtering the supernatant and then precipitating it to obtain black soldier fly melanin; (3) Demineralizing and decolorizing the filter residue obtained by filtering in step (2) to obtain black soldier fly chitin.

2. The continuous extraction method according to claim 1, characterized in that The inactivation temperature in step (1) is 90-100° C. and the inactivation time is 5-15 min. The temperature of the subcritical butane extraction in step (1) is 32-38° C., the extraction time is 45-55 min / time, the number of extractions is 3-6 times, and the extraction pressure is 0.1-0.3 MPa.

3. The continuous extraction method according to claim 1, characterized in that The proteases used in the enzymatic hydrolysis in step (1) are keratinase and alkaline protease, and the mass ratio of the keratinase to the alkaline protease is 1:(0.5-1.5); The enzymolysis time is 150-240 min, and the enzymolysis temperature is 50-60°C.

4. The continuous extraction method according to claim 1, characterized in that The alkali solution in step (2) is a sodium hydroxide solution, and the concentration of the alkali solution is 1M-3M; The mass volume ratio of the mixture of black soldier fly slag and alkali solution is (10-25) mg: 1 mL; The extraction time is 30-180 min, and the extraction temperature is 60-90° C.; In step (2), 1-8M hydrochloric acid solution is used for precipitation.

5. The continuous extraction method according to claim 1, characterized in that In step (3), 0.5-3M hydrochloric acid solution is used for demineralization, and sodium hypochlorite solution with a volume concentration of 0.1%-1% or hydrogen peroxide solution with a volume concentration of 1%-3.5% is used for decolorization.

6. Black soldier fly melanin obtained by the continuous extraction method according to any one of claims 1 to 5.

7. Use of the black soldier fly melanin according to claim 6 in the preparation of fish feed additives.

8. Use of the black soldier fly melanin according to claim 6 in preparing a functional feed for increasing fish growth and improving metabolic immunity.

9. A functional feed for fish containing the black soldier fly melanin according to claim 6, characterized in that: The invention comprises the following components in parts by weight: 58-70 parts of a protein source, 16-22 parts of a sugar source, 12-19 parts of a fat source, 0.5-1.5 parts of calcium dihydrogen phosphate, 0.4-0.6 parts of complex vitamins, 0.4-0.6 parts of complex minerals, 0.8-1.2 parts of monomeric vitamins, 0.05-0.15 parts of a mildew preventer, and 0.01-0.5 parts of the black soldier fly melanin according to claim 6; The fat source comprises black soldier fly oil and lecithin in a mass ratio of (10-14):(2-5).

10. The functional feed for fish according to claim 9, characterized in that: The protein source includes one or more of fish meal, soybean meal, and soy protein isolate; The sugar source includes tapioca starch and / or flour; The monomeric vitamins include choline chloride and / or vitamin C.

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