Preparation of hermetia illucens protein peptide and application of hermetia illucens protein peptide in functional feed for improving fish growth and meat quality

Abstract

The invention relates to the technical field of aquaculture feed. According to the hermetia illucens protein peptide and the preparation method thereof, the hermetia illucens protein peptide prepared by adopting the technical scheme can be used as a feed protein source to replace most fish meal in feed, so that the use of the fish meal in aquatic feed is reduced, and the problems of shortage and insufficient supply of the fish meal are solved. The invention also provides application of the hermetia illucens protein peptide in preparation of functional feed for improving fish growth performance and fish muscle and meat quality. Fish functional feed prepared from the hermetia illucens protein peptide not only can significantly improve growth of rainbow trout, but also has no negative effect on oxidation resistance of animal livers and dorsal muscles, and can also improve muscle meat quality of rainbow trout.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture feed, and in particular to the preparation of a black soldier fly protein peptide and the application of the peptide in a functional feed for increasing fish growth and improving meat quality. Background Art

[0002] The black soldier fly, commonly known as the black soldier fly, belongs to the family Schizophoridae of the order Diptera. Because its larvae can efficiently degrade organic solid waste, it has been widely used in the treatment of food waste and livestock and poultry manure. In this degradation process, the black soldier fly enriches useful nutrients and converts them into organic matter with high added value. It has high nutritional value and can therefore be used as a raw material for products such as aquatic animal feed.

[0003] However, experimental results have shown that adding high levels of black soldier fly larvae meal to feed will damage the health of aquatic animals. The reason is that the high fat content of black soldier fly larvae limits its use in aquatic animal feed, and the chitin in the black soldier fly exoskeleton seriously reduces the digestibility of aquatic animals. The feed industry is the basis for the development of modern aquaculture, and the supply restrictions and high prices of fish meal, the main protein source of aquatic feed, restrict the healthy development of aquaculture. Therefore, the development of new green black soldier fly protein sources can alleviate the shortage and insufficient supply of fish meal. However, the reduction of fish meal content in aquatic feed often leads to a decrease in feed palatability, a decrease in amino acid levels, and the presence of anti-nutritional factors, which further leads to reduced growth performance, oxidative stress damage and decreased muscle quality of animals.

[0004] In summary, it is very necessary to obtain a black soldier fly protein source that can replace fish meal in aquatic feed, and to use the black soldier fly protein source to prepare aquatic functional feed with good palatability and capable of ensuring the growth performance and meat quality of aquatic animals. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method of a black soldier fly protein peptide and its application in a functional feed for improving fish growth and meat quality.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a black soldier fly protein peptide, comprising the following steps:

[0008] (1) inactivating black soldier flies and performing subcritical extraction to obtain defatted black soldier fly powder;

[0009] (2) The defatted black soldier fly powder is mixed with protease for enzymatic hydrolysis to obtain a black soldier fly protease hydrolysate, which is then filtered and dried to obtain a black soldier fly protein peptide.

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

[0011] Preferably, the solvent of the subcritical extraction in step (1) is butane, the mass-to-volume ratio of the feed liquid of the subcritical extraction is 1 mg:(1-5) mL, the temperature of the subcritical extraction is 30-55° C., the time of the subcritical extraction is 40-55 min / time, and the number of subcritical extractions is 4-6 times.

[0012] Preferably, the protease in step (2) is any two or more of keratinase, alkaline protease, flavor protease, acid protease, neutral protease, papain and bromelain.

[0013] Preferably, the mass ratio of the defatted black soldier fly powder and the protease mixed in step (2) is 100:(0.5-3).

[0014] Preferably, when the proteases are keratinase and alkaline protease, the mass ratio of keratinase to alkaline protease is (1-5): (1-5).

[0015] The invention also provides the black soldier fly protein peptide prepared by the preparation method.

[0016] The present invention also provides the use of the black soldier fly protein peptide in preparing a functional feed for improving the growth performance of fish.

[0017] The present invention also provides the use of the black soldier fly protein peptide in preparing a functional feed for improving the muscle quality of fish.

[0018] The present invention also provides a functional fish feed containing the black soldier fly protein peptide, comprising the following components in parts by weight: 34.6-97 parts of a protein source, 12-18.9 parts of a sugar source, 14-18 parts of a fat source, 0.8-1.2 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, 0.14-0.8 parts of essential amino acids and 0.08-0.12 parts of a mildew preventer.

[0019] Preferably, the protein source comprises fish meal, soybean meal, soy protein isolate and the black soldier fly protein peptide in a mass ratio of (10-36):(8-12):(10-14):(6.6-35).

[0020] Preferably, the fat source comprises fish oil and / or lecithin.

[0021] Preferably, the essential amino acids include any one or more of DL-methionine, L-lysine hydrochloride and L-threonine.

[0022] By adopting the above technical solution, the present invention has the following beneficial effects:

[0023] The black soldier fly protein peptide prepared in the technical solution of the present invention has been defatted, which significantly reduces its fat content, and can be used as a feed protein source to replace most of the fish meal in the feed, reduce the use of fish meal in aquatic feed, and alleviate the shortage and insufficient supply of fish meal. The fish functional feed prepared using the black soldier fly protein peptide of the present invention can not only significantly improve the growth of rainbow trout, but also has no negative impact on the antioxidant capacity of animal liver and back muscle, and also has the effect of improving the muscle quality of rainbow trout. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The enzymatic hydrolysis of defatted black soldier fly powder by different types of proteases;

[0025] Figure 2 The enzymatic hydrolysis of defatted black soldier fly powder at different liquid-solid ratios;

[0026] Figure 3 The enzymatic hydrolysis of defatted black soldier fly powder at different enzymatic hydrolysis times;

[0027] Figure 4 The molecular weight distribution of the black soldier fly protein peptide;

[0028] Figure 5 To study the effect of different black soldier fly protein peptide replacement ratios on the weight gain rate of rainbow trout.

[0029] Figure 6 Effects of different black soldier fly protein peptide replacement ratios on the texture properties of rainbow trout muscle Figure 6 A represents adhesion, B represents elasticity, and C represents chewiness). DETAILED DESCRIPTION

[0030] The present invention provides a method for preparing a black soldier fly protein peptide, comprising the following steps:

[0031] (1) inactivating black soldier flies and performing subcritical extraction to obtain defatted black soldier fly powder;

[0032] (2) The defatted black soldier fly powder is mixed with protease for enzymatic hydrolysis to obtain a black soldier fly protease hydrolysate, which is then filtered and dried to obtain a black soldier fly protein peptide.

[0033] In the present invention, black soldier fly larvae are selected as raw materials, and the age of the black soldier fly larvae is preferably 6-14 days old, more preferably 8-13 days old, and even more preferably 12 days old.

[0034] In the present invention, the black soldier fly larvae are inactivated and then dried and crushed. The inactivation method in the present invention is preferably hot water inactivation, and the inactivation temperature is preferably 90-100°C, further preferably 92-98°C, and further preferably 95°C; the inactivation time is preferably 5-15min, further preferably 7-12min, and further preferably 10min. The drying method of the present invention preferably includes drying or airing; the degree of drying is preferably that the moisture content of the black soldier fly larvae is reduced to 5-10%. When the drying method of drying is adopted, the drying temperature is preferably 105°C. The dried black soldier fly larvae are crushed and filtered, and the sieve is collected for standby use. The mesh number of the sieve is preferably 150-250 mesh, further preferably 180-220 mesh, and further preferably 200 mesh.

[0035] In the present invention, the collected undersize (black soldier fly larvae powder) is subjected to subcritical extraction to obtain defatted black soldier fly powder. The solvent of the subcritical extraction in the present invention is preferably butane, and the mass volume ratio of the feed liquid (black soldier fly larvae powder and butane) of the subcritical extraction is preferably 1 mg: (1-5) mL, further preferably 1: (2-4), and further preferably 1: 2. The temperature of the subcritical extraction is preferably 30-55°C, further preferably 32-45°C, and further preferably 35°C; the extraction time of the subcritical extraction is preferably 40-55 min / time, further preferably 45-52 min / time, and further preferably 50 min / time; the number of subcritical extractions is preferably 4-6 times, and further preferably 5 times; the pressure of the subcritical extraction is preferably 0.1-0.3 MPa, and further preferably 0.2 MPa.

[0036] In the present invention, defatted black soldier fly powder is mixed with protease for enzymolysis to obtain black soldier fly enzymolysis liquid. The protease of the present invention is preferably any two or more of keratinase, alkaline protease, flavor protease, acid protease, neutral protease, papain and bromelain. The keratinase described in the present invention is derived from Bacillus licheniformis JR-101, the alkaline protease is derived from Bacillus subtilis WB-600, the flavor protease is derived from Aspergillus oryzae, the acid protease is derived from Aspergillus niger, the neutral protease is derived from Bacillus subtilis, the papain is derived from Pseudocydonia sinensis, and the bromelain is derived from Ananas comosus.

[0037] In the present invention, when the protease is preferably keratinase and alkaline protease, the mass ratio of keratinase to alkaline protease is preferably (1-5):(1-5), and more preferably 1:1.

[0038] In the present invention, the mass ratio of the defatted black soldier fly powder and the protease is preferably 100:(0.5-3), further preferably 100:(1-2.5), further preferably 100:2; the liquid-solid ratio (liquid-solid mass ratio) of water to defatted black soldier fly powder is preferably (5-20):1, further preferably (8-15):1, further preferably 10:1. The temperature of the enzymolysis of the present invention is preferably 50-60°C, further preferably 52-58°C, further preferably 55°C; the time of the enzymolysis is preferably 100-240min, further preferably 150-200min, further preferably 180min. The enzymolysis process of the present invention is preferably stirred, and the stirring speed is preferably 150-250rpm, further preferably 180-220rpm, further preferably 200rpm. The pH value of the enzymolysis process of the present invention is maintained at 8-10, further preferably 9.

[0039] In the present invention, the black soldier fly enzymatic hydrolysate is subjected to an inactivation treatment, and the inactivation treatment method is preferably a water-insulated heating inactivation, and the inactivation temperature is preferably 90-100°C, further preferably 92-98°C, and further preferably 95°C; the inactivation time is preferably 5-15min, further preferably 7-12min, and further preferably 10min.

[0040] In the present invention, the inactivated black soldier fly enzymatic hydrolysate is filtered, the supernatant is collected, and then dried to obtain solid black soldier fly protein peptide. The filtration of the present invention is preferably carried out using yarn, and the mesh number of the yarn is preferably 80-150 mesh, further preferably 100-130 mesh, and further preferably 120 mesh. The drying method of the present invention is preferably vacuum drying, and the drying temperature is preferably 50-85°C, further preferably 60-82°C, and further preferably 80°C.

[0041] The present invention also provides the use of the black soldier fly protein peptide in preparing a functional feed for improving the growth performance of fish.

[0042] The present invention also provides the use of the black soldier fly protein peptide in preparing a functional feed for improving the muscle quality of fish.

[0043] In the present invention, the fish is preferably rainbow trout.

[0044] The present invention also provides a functional fish feed containing the black soldier fly protein peptide, the components of which include a protein source, a sugar source, a fat source, calcium dihydrogen phosphate, complex vitamins, complex minerals, monomeric vitamins, essential amino acids and a mildew preventer.

[0045] In the present invention, the weight portion of the protein source is preferably 34.6-97 parts, further preferably 45-85 parts, and further preferably 65.5 parts; the weight portion of the sugar source is preferably 12-18.9 parts, further preferably 13.5-15.5 parts, and further preferably 13.95 parts; the weight portion of the fat source is preferably 14-18 parts, further preferably 15-17 parts, and further preferably 16.75 parts; the weight portion of the calcium dihydrogen phosphate is preferably 0.8-1.2 parts, further preferably 0.9-1.1 parts, and further preferably 1 part; the weight portion of the multivitamin is preferably 0.4-0.6 parts, and further preferably The weight portion of the composite mineral is preferably 0.4-0.6 parts, further preferably 0.45-0.55 parts, further preferably 0.5 parts; the weight portion of the monomer vitamin is preferably 0.8-1.2 parts, further preferably 0.8-1.1 parts, further preferably 1 part; the weight portion of the essential amino acid is preferably 0.14-0.8 parts, further preferably 0.5-0.75 parts, further preferably 0.7 parts; the weight portion of the antifungal agent is preferably 0.08-0.12 parts, further preferably 0.09-0.11 parts, further preferably 0.1 parts.

[0046] In the present invention, the protein source preferably includes fish meal, soybean meal, soy protein isolate and the black soldier fly protein peptide, and the mass ratio of the fish meal, soybean meal, soy protein isolate and the black soldier fly protein peptide is preferably (10-36):(8-12):(10-14):(6.6-35), further preferably (10.2-25):(9-11):(11-13):(20-34), and further preferably 10.5:10:12:33.

[0047] In the present invention, the sugar source is preferably tapioca starch and / or flour. When the sugar source of the present invention is tapioca starch and flour, the mass ratio of tapioca starch to flour is preferably (4-6):(8-12.9), further preferably (4.5-5.5):(8.5-10), and further preferably 5:8.95.

[0048] In the present invention, the fat source is preferably fish oil and / or lecithin. When the fat source of the present invention is fish oil and lecithin, the mass ratio of the fish oil to the lecithin is preferably (12-14):(2-4), further preferably (12.8-13.8):(2.5-3.5), and further preferably 13.75:3.

[0049] In the present invention, the complex vitamin preferably includes vitamin B120-40mg / kg, vitamin B250-70mg / kg, vitamin B610-30mg / kg, niacin 180-220mg / kg, calcium pantothenate 80-120mg / kg, inositol 80-120mg / kg, biotin 2-3mg / kg, folic acid 8-12mg / kg, vitamin B120.05-0.15mg / kg, vitamin K330-50mg / kg, vitamin A 9000-11000IU / kg, vitamin D31800-2200IU / kg and vitamin E 150-170 IU / kg, further preferably including vitamin B12 5-35 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, further preferably including 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.

[0050] In the present invention, the composite minerals preferably include MgSO4·H2O 1000-1100 mg / kg, KH2PO4 900-950 mg / kg, NaH2PO4·2H2O 400-450 mg / kg, FeC6H5O7·5H2O 170-200 mg / kg, ZnCl2 70-90 mg / kg, CuSO4·5H2O 60-70 mg / kg, AlCl3·6H2O 45-60 mg / kg, MnSO4·H2O 25-35 mg / kg, KI 25-35 mg / kg, CoCl2·6H2O 4-9 mg / kg and Na2SeO3·H2O 0.5-1.5mg / kg, further preferably including MgSO4·H2O 1050-1095mg / kg, KH2PO4 920-940mg / kg, NaH2PO4·2H2O 420-440mg / kg, FeC6H5O7·5H2O 175-190mg / kg, ZnCl2 75-85mg / kg, CuSO4·5H2O 61-65mg / kg, AlCl3·6H2O 50-55mg / kg, MnSO4·H2O 28-33mg / kg, KI 27-30mg / kg, CoCl2·6H2O 5-7mg / kg and Na2SeO3·H2O 0.7-1.0mg / kg, further preferably including MgSO4·H2O KI 28mg / kg, CoCl2·6H2O 6mg / kg and Na2SeO3·H2O 0.8mg / kg.

[0051] In the present invention, the monomeric vitamin preferably includes choline chloride and / or vitamin C. When the monomeric vitamins of the present invention are choline chloride and vitamin C, the mass ratio of choline chloride to vitamin C is preferably (0.4-0.6):(0.4-0.6), further preferably (0.45-0.58):(0.45-0.55), and further preferably 0.5:0.5.

[0052] In the present invention, the essential amino acids include any one or more of DL-methionine, L-lysine hydrochloride and L-threonine. When the essential amino acids described in the present invention are DL-methionine, L-lysine hydrochloride and L-threonine, the mass ratio of the DL-methionine, L-lysine hydrochloride and L-threonine is preferably (0.06-0.32):(0.06-0.33):(0.02-0.15), more preferably (0.1-0.30):(0.1-0.30):(0.08-0.13), and further preferably 0.29:0.29:0.12.

[0053] In the present invention, the mildew inhibitor is preferably calcium propionate.

[0054] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0055] Example 1

[0056] A black soldier fly protein peptide, the preparation method is as follows:

[0057] (1) selecting 12-day-old black soldier fly larvae, removing impurities and inactivating them in 95° C. boiling water for 10 min, and then drying them at 105° C. until the moisture content of the dried insects is reduced to 5-10%; removing impurities again, and then crushing the black soldier fly larvae and filtering them with a 200-mesh filter bag to obtain black soldier fly larvae powder;

[0058] (2) placing the black soldier fly larvae powder in an extraction tank for subcritical extraction, evacuating to a vacuum degree of 0.2 MPa, adding butane twice the mass of the material, extracting at 35° C. for 50 min / time, extracting 5 times, and obtaining defatted black soldier fly powder;

[0059] (3) mixing water and defatted black soldier fly powder at a liquid-solid ratio of 10:1, and defatted black soldier fly powder and protease at a mass ratio of 100:2 for enzymatic hydrolysis, wherein the protease is keratinase and alkaline protease at a mass ratio of 1:1, and performing enzymatic hydrolysis at 55° C. for 180 min. During the enzymatic hydrolysis, the mixture is stirred at a speed of 200 rpm and the pH value is maintained at 9 to obtain a black soldier fly enzymatic hydrolyzate;

[0060] (4) The black soldier fly enzymatic hydrolysate was inactivated in 95°C boiling water for 10 min, then filtered using 120-mesh gauze, the supernatant was collected, and then vacuum dried at 80°C to obtain solid black soldier fly protein peptide.

[0061] Example 2

[0062] A black soldier fly protein peptide, the preparation method is as follows:

[0063] (1) selecting 13-day-old black soldier fly larvae, removing impurities and inactivating them in 90° C. boiling water for 15 min, and then drying them outdoors until the moisture content of the insects is reduced to 5-10%; removing impurities again, and then crushing the black soldier fly larvae and filtering them with a 150-mesh filter bag to obtain black soldier fly larvae powder;

[0064] (2) placing the black soldier fly larvae powder in an extraction tank for subcritical extraction, evacuating to a vacuum degree of 0.2 MPa, adding butane with a volume four times the mass of the material, extracting at 30° C. for 55 min / time, extracting 4 times, and obtaining defatted black soldier fly powder;

[0065] (3) mixing water and defatted black soldier fly powder at a liquid-solid ratio of 5:1, and defatted black soldier fly powder and protease at a mass ratio of 100:3 for enzymatic hydrolysis, wherein the protease is keratinase and alkaline protease at a mass ratio of 1:5, and performing enzymatic hydrolysis at 50° C. for 240 min. During the enzymatic hydrolysis, stirring was performed at a speed of 150 rpm, and the pH value was maintained at 9 to obtain a black soldier fly enzymatic hydrolyzate;

[0066] (4) The black soldier fly enzymatic hydrolysate was inactivated in 90°C boiling water for 10 min, then filtered using 150 mesh gauze, the supernatant was collected, and then vacuum dried at 80°C to obtain solid black soldier fly protein peptide.

[0067] Example 3

[0068] A black soldier fly protein peptide, the preparation method is as follows:

[0069] (1) selecting 6-day-old black soldier fly larvae, removing impurities and inactivating them in 100° C. boiling water for 5 min, and then drying them at 105° C. until the moisture content of the dried insects is reduced to 5-10%; removing impurities again, and then crushing the black soldier fly larvae and filtering them with a 150-mesh filter bag to obtain black soldier fly larvae powder;

[0070] (2) placing the black soldier fly larvae powder in an extraction tank for subcritical extraction, evacuating to a vacuum degree of 0.2 MPa, adding butane with a volume five times the mass of the material, extracting at 55° C. for 40 min / time, extracting 6 times, and obtaining defatted black soldier fly powder;

[0071] (3) mixing water and defatted black soldier fly powder at a liquid-to-solid ratio of 20:1, and defatted black soldier fly powder and protease at a mass ratio of 100:0.5 for enzymatic hydrolysis, wherein the protease is keratinase and alkaline protease at a mass ratio of 5:1, and enzymolysis is performed at 60° C. for 100 min. During the enzymatic hydrolysis, the mixture is stirred at a speed of 250 rpm and the pH value is maintained at 9 to obtain a black soldier fly enzymatic hydrolyzate;

[0072] (4) The black soldier fly enzymatic hydrolysate was inactivated in 100° C. boiling water for 5 min, and then filtered using 120-mesh gauze to collect the supernatant, which was then vacuum dried at 80° C. to obtain solid black soldier fly protein peptide.

[0073] Example 4

[0074] A functional fish feed containing the black soldier fly protein peptide described in Example 1:

[0075] 10.5 parts of fish meal, 10 parts of soybean meal, 12 parts of soy protein isolate, 33 parts of black soldier fly protein peptide, 5 parts of tapioca starch, 8.95 parts of flour, 13.75 parts of fish oil, 3 parts of lecithin, 1 part of calcium dihydrogen phosphate, 0.5 parts of multivitamins, 0.5 parts of multiminerals, 0.5 parts of choline chloride, 0.5 parts of vitamin C, 0.29 parts of DL-methionine, 0.29 parts of L-lysine hydrochloride, 0.12 parts of L-threonine, 0.1 parts of calcium propionate;

[0076] The compound vitamins are: 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 K 340 mg / kg, vitamin A 10000 IU / kg, vitamin D 3 2000 IU / kg and vitamin E 160 IU / kg;

[0077] The composite minerals include MgSO4·H2O 1090mg / kg, KH2PO4 932mg / kg, NaH2PO4·2H2O 432mg / kg, FeC6H5O7·5H2O 181mg / kg, ZnCl2 80mg / kg, CuSO4·5H2O 63mg / kg, AlCl3·6H2O 51mg / kg, MnSO4·H2O 31mg / kg, KI 28mg / kg, CoCl2·6H2O 6mg / kg and Na2SeO3·H2O 0.8mg / kg.

[0078] Example 5

[0079] A functional fish feed containing the black soldier fly protein peptide described in Example 2:

[0080] 10.5 parts of fish meal, 10 parts of soybean meal, 12 parts of soy protein isolate, 33 parts of black soldier fly protein peptide, 5 parts of tapioca starch, 8.95 parts of flour, 13.75 parts of fish oil, 3 parts of lecithin, 1 part of calcium dihydrogen phosphate, 0.5 parts of multivitamins, 0.5 parts of multiminerals, 0.5 parts of choline chloride, 0.5 parts of vitamin C, 0.29 parts of DL-methionine, 0.29 parts of L-lysine hydrochloride, 0.12 parts of L-threonine, 0.1 parts of calcium propionate;

[0081] The compound vitamins are: 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 K 340 mg / kg, vitamin A 10000 IU / kg, vitamin D 3 2000 IU / kg and vitamin E 160 IU / kg;

[0082] The composite minerals include MgSO4·H2O 1090mg / kg, KH2PO4 932mg / kg, NaH2PO4·2H2O 432mg / kg, FeC6H5O7·5H2O 181mg / kg, ZnCl2 80mg / kg, CuSO4·5H2O 63mg / kg, AlCl3·6H2O 51mg / kg, MnSO4·H2O 31mg / kg, KI 28mg / kg, CoCl2·6H2O 6mg / kg and Na2SeO3·H2O 0.8mg / kg.

[0083] Example 6

[0084] A functional fish feed containing the black soldier fly protein peptide described in Example 3:

[0085] 10.5 parts of fish meal, 10 parts of soybean meal, 12 parts of soy protein isolate, 33 parts of black soldier fly protein peptide, 5 parts of tapioca starch, 8.95 parts of flour, 13.75 parts of fish oil, 3 parts of lecithin, 1 part of calcium dihydrogen phosphate, 0.5 parts of multivitamins, 0.5 parts of multiminerals, 0.5 parts of choline chloride, 0.5 parts of vitamin C, 0.29 parts of DL-methionine, 0.29 parts of L-lysine hydrochloride, 0.12 parts of L-threonine, 0.1 parts of calcium propionate;

[0086] The compound vitamins are: 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 K 340 mg / kg, vitamin A 10000 IU / kg, vitamin D 3 2000 IU / kg and vitamin E 160 IU / kg;

[0087] The composite minerals include MgSO4·H2O 1090mg / kg, KH2PO4 932mg / kg, NaH2PO4·2H2O 432mg / kg, FeC6H5O7·5H2O 181mg / kg, ZnCl2 80mg / kg, CuSO4·5H2O 63mg / kg, AlCl3·6H2O 51mg / kg, MnSO4·H2O 31mg / kg, KI 28mg / kg, CoCl2·6H2O 6mg / kg and Na2SeO3·H2O 0.8mg / kg.

[0088] Test Example 1

[0089] Feed the black soldier fly larvae with kitchen waste, collect the black soldier fly larvae of 6-13 days old, remove impurities and inactivate them in boiling water above 90℃ for 5-15min, then dry them at 105℃ or dry them outdoors until the moisture content of the insects is reduced to 5-10%. After drying, remove impurities again, and then crush the black soldier fly larvae preliminarily. Put an appropriate amount of crushed black soldier fly larvae into a 200-mesh filter bag, put it into an extraction tank, and then seal the extraction tank and evacuate it until the vacuum degree reaches -0.1MPa. Then inject butane twice the mass of the material into the extraction tank until the extraction pressure is 0.2MPa, the extraction conditions are 35-55℃, 40-50min / time, and the number of extractions is 3-5 times. The extraction results are shown in Table 1.

[0090] Table 1 Extraction results corresponding to different extraction conditions

[0091]

[0092]

[0093] The results show that the optimal extraction conditions for subcritical fluid extraction of black soldier fly larvae oil are: extraction temperature 35℃, extraction time 50 minutes, and extraction times 5 times. Under this condition, the extraction rate of black soldier fly larvae oil can reach 100.61%, which is higher than other conditions or other extraction methods.

[0094] Under the same material conditions, the extraction rate of black soldier fly larvae oil by mechanical pressing was 70.32%, the extraction rate of black soldier fly larvae oil by organic solvent extraction was 89.29%, and the extraction rate of black soldier fly larvae oil by wet extraction was 75.91%.

[0095] Experimental Example 2 Preparation of Black Soldier Fly Protein Peptide

[0096] Keratinase (from Bacillus licheniformis JR-101) and alkaline protease (from Bacillus subtilis WB-600) were used to deproteinize defatted black soldier fly powder, and compared with common proteases on the market, including flavor protease (from Aspergillus oryzae), acid protease (from Aspergillus niger), neutral protease (from Bacillus subtilis), papain (from Pseudocydonia sinensis) and bromelain (from Ananascomosus). The enzymolysis reaction conditions are temperature 55°C, enzyme substrate ratio 0.5%-3%, enzymolysis time 30-240 minutes, liquid-solid ratio of water to defatted black soldier fly powder 5-20:1, pH 9. After enzymatic hydrolysis, the temperature was raised to 95°C for inactivation, and after cooling, the supernatant was filtered with 120-mesh gauze and collected. The supernatant was vacuum dried at 80°C and the black soldier fly protein peptide was collected.

[0097] The enzymatic hydrolysis results are as follows Figure 1 As shown, it can be seen that keratinase and alkaline protease have the best enzymatic hydrolysis effect on defatted black soldier fly powder. At a temperature of 50-60°C, an enzyme-substrate ratio of 1-3%, a liquid-solid ratio of 10:1, and after 60 minutes of enzymatic hydrolysis, the substrate mass can be reduced by 71.67%-74.56% and 74.50%-77.39%, respectively. Under the same conditions, flavor protease, acid protease, neutral protease, papain and bromelain have poor enzymatic hydrolysis effects on defatted black soldier fly powder. It is further preferred that keratinase and alkaline protease are added in equal proportions to enzymatic hydrolyze defatted black soldier fly powder, which can reduce the substrate mass by up to 81.05%, which is 69.84% higher than not using enzymes. It is further preferred that the enzyme-substrate ratio is 1%-3%, and it is further preferred that it is 2%.

[0098] Effect of liquid-to-solid ratio (liquid-to-solid mass ratio) on enzymatic hydrolysis of defatted black soldier fly larvae Figure 2 As shown, it can be seen that when the liquid-to-solid ratio reaches 15:1, the enzymatic hydrolysis effect is the best, but too high a liquid-to-solid ratio will lead to too large a liquid volume during the enzymatic hydrolysis process, and the volume of liquid to be dried will increase, which will increase the cost of water and electricity. Therefore, the enzymatic hydrolysis condition is further optimized to a liquid-to-solid ratio of 7-10:1, and further optimized to 7:1.

[0099] Effect of enzymatic hydrolysis time on the enzymatic hydrolysis of defatted black soldier fly larvae Figure 3 As shown, it can be seen that the enzymolysis time reaches a plateau when it reaches 180 minutes, and the substrate mass no longer changes significantly. Considering the heating cost and time cost, the enzymolysis time is further optimized to be 180 minutes.

[0100] Test Example 3 Nutritional properties and peptide molecular weight identification of black soldier fly protein peptide

[0101] The above enzymatic hydrolysate was filtered and dried to form solid black soldier fly protein peptides, and then its nutritional properties were studied. Crude protein was determined by Dumas combustion nitrogen determination method (SN / T 2115-2008), crude fat was determined by oil weight method (GB / T 6433-2006), crude ash was determined by combustion method (GB / T6438-2007), and amino acid content was determined by conventional acid hydrolysis method (GB / T 18246-2019).

[0102] Table 2 Nutritional characteristics of full-fat black soldier flies, defatted black soldier flies and black soldier fly protein peptides

[0103]

[0104]

[0105] The results are shown in Table 2. The crude protein content of the black soldier fly protein peptide further increased to nearly 62%, and the total amino acid content increased to 54.09%, which was 8.50% higher than that of the defatted black soldier fly, and the proportion of total amino acids in crude protein increased by 7.50% compared with the defatted black soldier fly. In addition, defatting and enzymatic hydrolysis can increase total nitrogen by 17.86% and 13.17%, respectively, which is mainly caused by the increase in the proportion of amino acid nitrogen, indicating that the protein quality of the black soldier fly protein peptide is improved after two-step treatment.

[0106] Furthermore, the molecular weight of the black soldier fly protein peptide was tested by high performance liquid chromatography. Figure 4 It can be seen that 88.30% of the polypeptides have a molecular weight of less than 1000Da, indicating that the black soldier fly protein peptide produced by this method is a small peptide protein product and has good digestion and absorption properties for aquatic animals.

[0107] Test Example 4 Black soldier fly protein peptide as a protein source for rainbow trout feed (one)

[0109] An 8-week breeding experiment was designed to evaluate the effect of black soldier fly protein peptide as a feed protein source on rainbow trout. In the control group (CD), black soldier fly protein peptide was used to replace 15%, 30%, 45%, 60% and 75% of fish meal in the feed to make BSFP15, BSFP30, BSFP45, BSFP60, and BSFP75 feeds. The experimental feed formulas are shown in Tables 3 and 4. During the 8-week breeding experiment, the fish were fed at 7:00 am and 17:00 pm every day.

[0110] Table 3 Experimental feed formula (%)

[0111]

[0112]

[0113] Table 4 Nutrient composition of experimental feed (%)

[0114] (two)

[0116] At the end of the 8-week culture experiment, the fish in each cage were counted and weighed for calculation of weight gain rate, specific growth rate, feed coefficient, survival rate and food intake.

[0117] Table 5 Effects of black soldier fly protein peptide on growth performance of rainbow trout

[0118]

[0119] Note: Different superscript letters represent significant differences (P<0.05)

[0120] The results showed that the feed of the present invention can significantly improve the growth performance of rainbow trout while maintaining its nutritional requirements (Table 5, Figure 5 ) without affecting the feeding and survival rate of rainbow trout, which can not only shorten the breeding cycle and save breeding costs, but also increase production and increase economic income. (three)

[0122] After the 8-week breeding experiment, all fish were fasted for 24 h, and then the liver and back muscle of rainbow trout were taken and the antioxidant enzyme activity was determined using the kits of Biyuntian Biotechnology Co., Ltd. and Nanjing Jiancheng Bioengineering Institute. The results are shown in Table 6.

[0123] Table 6 Effects of black soldier fly protein peptide on antioxidant enzyme activities in liver and back muscle of rainbow trout

[0124]

[0125] Note: Different superscript letters represent significant differences (P<0.05)

[0126] It can be seen from Table 6 that the ingestion of feed containing black soldier fly protein peptide by rainbow trout does not cause oxidative stress in liver and muscle. The results show that the black soldier fly protein peptide prepared by the technical solution of the present invention can be used as a substitute for fish meal in rainbow trout feed, and does not affect the ability of rainbow trout to resist external environmental pressure, and has significant advantages over other animal and plant protein sources.

[0127] At the same time, the back muscle of rainbow trout was taken for muscle texture analysis. The results showed that black soldier fly protein peptide can significantly reduce the adhesion of the back muscle and significantly increase the elasticity and chewiness of the back muscle (such as Figure 6As shown). Therefore, black soldier fly protein peptide helps to improve the muscle quality of rainbow trout and increase its appeal to consumers. Using other protein sources to replace fish meal in aquatic feed often leads to increased muscle adhesion, reduced elasticity and chewiness, which makes the meat loose and reduces the taste of fish meat, thereby reducing its appeal to consumers. The feed of the present invention greatly alleviates the problem of reduced taste of rainbow trout muscle caused by low-fish meal artificial feed on the market. Black soldier fly protein peptide has important economic value as a new feed protein source.

[0128] In summary, it can be seen that the black soldier fly protein peptide prepared by the technical solution of the present invention contains 61.86% crude protein, and 87.44% of the nitrogen is amino acid nitrogen, of which more than 88% of the polypeptides have a molecular weight of less than 1000Da. It can be used as the main protein source in aquatic feed, and can improve the growth performance of rainbow trout and accelerate the growth rate. At the same time, it has no negative impact on the antioxidant capacity of rainbow trout, and maintains its ability to resist external environmental pressure during breeding; it can improve the muscle quality of rainbow trout, reduce adhesion, and significantly increase elasticity and chewiness; it can replace most of the fish meal in feed, reduce the use of fish meal in aquatic feed, and enable the sustainable and green development of the rainbow trout breeding industry.

[0129] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a black soldier fly protein peptide, characterized in that: The steps include: (1) inactivating black soldier flies and performing subcritical extraction to obtain defatted black soldier fly powder; (2) The defatted black soldier fly powder is mixed with protease for enzymatic hydrolysis to obtain a black soldier fly protease hydrolysate, which is then filtered and dried to obtain a black soldier fly protein peptide.

2. The preparation 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.

3. The preparation method according to claim 1, characterized in that: The solvent of the subcritical extraction in step (1) is butane, the feed-liquid mass volume ratio of the subcritical extraction is 1 mg: (1-5) mL, the temperature of the subcritical extraction is 30-55° C., the time of the subcritical extraction is 40-55 min / time, and the number of subcritical extractions is 4-6 times.

4. The preparation method according to claim 1, characterized in that: The protease in step (2) is any two or more of keratinase, alkaline protease, flavor protease, acid protease, neutral protease, papain and bromelain.

5. The preparation method according to claim 1, characterized in that: The mass ratio of the defatted black soldier fly powder and the protease mixed in step (2) is 100:(0.5-3); When the protease is keratinase and alkaline protease, the mass ratio of keratinase to alkaline protease is (1-5): (1-5).

6. Black soldier fly protein peptide obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the black soldier fly protein peptide according to claim 6 in preparing a functional feed for improving the growth performance of fish.

8. Use of the black soldier fly protein peptide according to claim 6 in preparing a functional feed for improving the muscle quality of fish.

9. A functional fish feed containing the black soldier fly protein peptide according to claim 6, characterized in that: The invention comprises the following components in parts by weight: 34.6-97 parts of protein source, 12-18.9 parts of sugar source, 14-18 parts of fat source, 0.8-1.2 parts of calcium dihydrogen phosphate, 0.4-0.6 parts of complex vitamins, 0.4-0.6 parts of complex minerals, 0.8-1.5 parts of monomer vitamins, 0.14-0.8 parts of essential amino acids and 0.08-0.12 parts of antifungal agent; The protein source comprises fish meal, soybean meal, soy protein isolate and the black soldier fly protein peptide according to claim 6 in a mass ratio of (10-36):(8-12):(10-14):(6.6-35).

10. The functional feed for fish according to claim 9, characterized in that: The fat source includes fish oil and / or lecithin; The essential amino acids include any one or more of DL-methionine, L-lysine hydrochloride and L-threonine.

Images