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

Abstract

The present application relates to the technical field of aquaculture feed, and provides a hermetia illucens protein peptide and a preparation method thereof.The hermetia illucens protein peptide prepared by using the technical solution of the present application can be used as a feed protein source, replace most of the fish meal in the feed, reduce the use of fish meal in the aquaculture feed, and alleviate the problem of fish meal shortage and insufficient supply.The present application also provides application of the hermetia illucens protein peptide in preparation of functional feed for improving the growth performance of fish and improving the muscle quality of fish.The functional feed for fish prepared by using the hermetia illucens protein peptide of the present application can not only significantly improve the growth of rainbow trout, but also has no negative effect on the antioxidant capacity of the liver and back muscle of animals, and can also improve the muscle quality of rainbow trout.

Description

Technical Field

[0001] This invention relates to the field of aquaculture feed technology, and in particular to the preparation of a black soldier fly protein peptide and its application in functional feeds that improve fish growth and meat quality. Background Technology

[0002] The black soldier fly, also known as the gleaming flat-horned soldier fly, belongs to the family Typhaeidae in the order Diptera. Its larvae are highly efficient at degrading organic solid waste and are now widely used in the treatment of kitchen waste and livestock manure. During this degradation process, the black soldier fly accumulates useful nutrients, transforming them into high-value organic matter, making it a valuable raw material for aquatic animal feed and other products.

[0003] However, existing experimental results have shown that adding high levels of black soldier fly larvae meal to feed will harm the health of aquatic animals. This is because the high fat content of black soldier fly larvae limits their use in aquatic animal feed, and the chitin in their exoskeleton significantly reduces their digestibility. The feed industry is the foundation for the development of modern aquaculture, but the limited supply and high price of fishmeal, the main protein source in aquatic feed, restricts the healthy development of aquaculture. Therefore, developing new, green black soldier fly protein sources can alleviate the shortage and insufficient supply of fishmeal. However, reducing the fishmeal content in aquatic feed often leads to decreased palatability, lower amino acid levels, and the presence of anti-nutritional factors, further resulting in reduced animal growth performance, oxidative stress damage, and decreased muscle quality.

[0004] In summary, it is essential to obtain a black soldier fly protein source that can replace fishmeal in aquatic feed, and to use this black soldier fly protein source to prepare aquatic functional feed with good palatability that can ensure the growth performance and meat quality of aquatic animals. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing black soldier fly protein peptides and their application in functional feeds that improve fish growth and meat quality.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing black soldier fly protein peptides, comprising the following steps:

[0008] (1) Black soldier fly larvae were inactivated and then subjected to subcritical extraction to obtain defatted black soldier fly powder;

[0009] (2) The defatted black soldier fly powder was mixed with protease and enzymatically hydrolyzed to obtain black soldier fly protease hydrolysate, which was then filtered and dried to obtain black soldier fly protein peptides.

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

[0011] Preferably, the solvent for subcritical extraction in step (1) is butane, the mass-to-volume ratio of the subcritical extract to the liquid is 1 mg:(1-5) mL, the temperature of the subcritical extraction is 30-55℃, 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, acidic protease, neutral protease, papain, and bromelain.

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

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

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

[0016] The present invention also provides the application of the black soldier fly protein peptide in the preparation of functional feeds that improve the growth performance of fish.

[0017] The present invention also provides the application of the black soldier fly protein peptide in the preparation of functional feeds that improve the muscle quality of fish.

[0018] The present invention also provides a functional fish feed containing the aforementioned black soldier fly protein peptide, comprising the following components in parts by weight: 34.6-97 parts protein source, 12-18.9 parts carbohydrate source, 14-18 parts fat source, 0.8-1.2 parts calcium dihydrogen phosphate, 0.4-0.6 parts compound vitamins, 0.4-0.6 parts compound minerals, 0.8-1.2 parts single vitamins, 0.14-0.8 parts essential amino acids, and 0.08-0.12 parts antifungal agent.

[0019] Preferably, the protein source includes 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 includes fish oil and / or lecithin.

[0021] Preferably, the essential amino acid includes 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 peptides prepared in this invention have undergone defatting treatment, significantly reducing their fat content. They can be used as a feed protein source, replacing most of the fishmeal in feed, thus reducing the use of fishmeal in aquatic feed and alleviating the shortage and insufficient supply of fishmeal. The functional fish feed prepared using the black soldier fly protein peptides described in this invention not only significantly improves the growth of rainbow trout but also has no negative impact on the antioxidant capacity of the animal's liver and back muscles, and also improves the muscle quality of rainbow trout. Attached Figure Description

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

[0025] Figure 2 Enzymatic hydrolysis of defatted black soldier fly powder under different liquid-to-solid ratios;

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

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

[0028] Figure 5 The effect of different proportions of black soldier fly protein peptides replacing fish meal on the weight gain rate of rainbow trout;

[0029] Figure 6 Effects of different black soldier fly protein peptide substitution ratios on the textural properties of rainbow trout muscle ( Figure 6 In this context, A represents adhesiveness, B represents elasticity, and C represents chewiness. Detailed Implementation

[0030] This invention provides a method for preparing black soldier fly protein peptides, comprising the following steps:

[0031] (1) Black soldier fly larvae were inactivated and then subjected to subcritical extraction to obtain defatted black soldier fly powder;

[0032] (2) The defatted black soldier fly powder was mixed with protease and enzymatically hydrolyzed to obtain black soldier fly protease hydrolysate, which was then filtered and dried to obtain black soldier fly protein peptides.

[0033] In this invention, black soldier fly larvae are selected as raw materials. 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 this invention, black soldier fly larvae are inactivated, dried, and pulverized. The preferred inactivation method is hot water inactivation, with a preferred temperature of 90-100℃, more preferably 92-98℃, and even more preferably 95℃; the preferred inactivation time is 5-15 minutes, more preferably 7-12 minutes, and even more preferably 10 minutes. The drying method preferably includes oven drying or sun drying; the degree of drying is preferably such that the moisture content of the black soldier fly larvae is reduced to 5-10%. When oven drying is used, the preferred drying temperature is 105℃. The dried black soldier fly larvae are pulverized, filtered, and the sieve residue is collected for later use. The mesh size of the sieve is preferably 150-250 mesh, more preferably 180-220 mesh, and even more preferably 200 mesh.

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

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

[0037] In this 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 this invention, the preferred mass ratio of defatted black soldier fly powder to protease is 100:(0.5-3), more preferably 100:(1-2.5), and even more preferably 100:2; the preferred liquid-to-solid ratio (liquid-to-solid mass ratio) of water to defatted black soldier fly powder is (5-20):1, more preferably (8-15):1, and even more preferably 10:1. The preferred enzymatic hydrolysis temperature is 50-60℃, more preferably 52-58℃, and even more preferably 55℃; the preferred hydrolysis time is 100-240 min, more preferably 150-200 min, and even more preferably 180 min. During the enzymatic hydrolysis process, stirring is preferably performed, with a stirring speed preferably 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, more preferably 9.

[0039] In this invention, the black soldier fly larvae enzymatic hydrolysate is subjected to inactivation treatment. The preferred method of inactivation treatment is water bath heating inactivation. The preferred inactivation temperature is 90-100℃, further preferably 92-98℃, and even more preferably 95℃. The preferred inactivation time is 5-15 min, further preferably 7-12 min, and even more preferably 10 min.

[0040] In this invention, the inactivated black soldier fly larvae enzymatic hydrolysate is filtered, the supernatant is collected, and then dried to obtain solid black soldier fly larvae protein peptides. The filtration is preferably performed using silk gauze, preferably with a mesh size of 80-150 mesh, more preferably 100-130 mesh, and even more preferably 120 mesh. The drying method is preferably vacuum drying, and the drying temperature is preferably 50-85℃, more preferably 60-82℃, and even more preferably 80℃.

[0041] The present invention also provides the application of the black soldier fly protein peptide in the preparation of functional feeds that improve the growth performance of fish.

[0042] The present invention also provides the application of the black soldier fly protein peptide in the preparation of functional feeds that improve the muscle quality of fish.

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

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

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

[0046] In this invention, the protein source preferably includes fish meal, soybean meal, soy protein isolate, and black soldier fly protein peptide. The mass ratio of the fish meal, soybean meal, soy protein isolate, and 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 even more preferably 10.5:10:12:33.

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

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

[0049] In this invention, the preferred components of the compound vitamins include vitamin B12 (0-40 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, further preferred components include 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, with further preferred values ​​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 B1 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 this invention, the preferred composite minerals 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. 0.5-1.5 mg / kg, further preferred components include MgSO4·H2O 1050-1095 mg / kg, KH2PO4 920-940 mg / kg, NaH2PO4·2H2O 420-440 mg / kg, FeC6H5O7·5H2O 175-190 mg / kg, ZnCl2 75-85 mg / kg, CuSO4·5H2O 61-65 mg / kg, AlCl3·6H2O 50-55 mg / kg, MnSO4·H2O 28-33 mg / kg, KI 27-30 mg / kg, CoCl2·6H2O 5-7 mg / kg, and Na2SeO3·H2O 0.7-1.0 mg / kg, with MgSO4·H2O being even more preferred. KI 28mg / kg, CoCl2·6H2O 6mg / kg and Na2SeO3·H2O 0.8mg / kg.

[0051] In this invention, the monomeric vitamin preferably includes choline chloride and / or vitamin C. When the monomeric vitamin of this invention is choline chloride and vitamin C, the mass ratio of choline chloride to 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.

[0052] In this invention, the essential amino acid includes any one or more of DL-methionine, L-lysine hydrochloride, and L-threonine. When the essential amino acid in this invention is DL-methionine, L-lysine hydrochloride, and L-threonine, the mass ratio of 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 even more preferably 0.29:0.29:0.12.

[0053] In this invention, the antifungal agent is preferably calcium propionate.

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

[0055] Example 1

[0056] A black soldier fly protein peptide, prepared by the following method:

[0057] (1) Select 12-day-old black soldier fly larvae, remove impurities and inactivate them in boiling water at 95℃ for 10 minutes, then dry them at 105℃ until the moisture content of the dried larvae is reduced to 5-10%; remove impurities again, then crush the black soldier fly larvae and filter them using a 200-mesh filter bag to obtain black soldier fly larvae powder.

[0058] (2) Black soldier fly larvae powder was placed in an extraction tank for subcritical extraction. The vacuum was evacuated to a vacuum degree of 0.2 MPa. Butane with a mass twice that of the material was added, and the extraction was carried out at 35°C for 50 min / time. The extraction was repeated 5 times to obtain defatted black soldier fly powder.

[0059] (3) The liquid-solid ratio of water to defatted black soldier fly powder is 10:1, and the defatted black soldier fly powder and protease are mixed at a mass ratio of 100:2 for enzymatic hydrolysis. The protease is keratinase and alkaline protease in a mass ratio of 1:1. The enzymatic hydrolysis is carried out 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 black soldier fly enzymatic hydrolysate.

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

[0061] Example 2

[0062] A black soldier fly protein peptide, prepared by the following method:

[0063] (1) Select 13-day-old black soldier fly larvae, remove impurities and inactivate them in boiling water at 90℃ for 15 minutes, then dry them outdoors until the moisture content of the dried insects is reduced to 5-10%; remove impurities again, then crush the black soldier fly larvae and filter them using a 150-mesh filter bag to obtain black soldier fly larvae powder.

[0064] (2) Black soldier fly larvae powder was placed in an extraction tank for subcritical extraction. The vacuum was evacuated to a vacuum degree of 0.2 MPa. Butane was added at four times the mass of the material and extracted at 30°C for 55 min / time. The extraction was repeated 4 times to obtain defatted black soldier fly powder.

[0065] (3) The liquid-solid ratio of water to defatted black soldier fly powder is 5:1, and the defatted black soldier fly powder and protease are mixed at a mass ratio of 100:3 for enzymatic hydrolysis. The protease is keratinase and alkaline protease at a mass ratio of 1:5. The enzymatic hydrolysis is carried out at 50°C for 240 min. During the enzymatic hydrolysis, the mixture is stirred at a speed of 150 rpm and the pH value is maintained at 9 to obtain black soldier fly enzymatic hydrolysate.

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

[0067] Example 3

[0068] A black soldier fly protein peptide, prepared by the following method:

[0069] (1) Select 6-day-old black soldier fly larvae, remove impurities and inactivate them in boiling water at 100℃ for 5 minutes, then dry them at 105℃ until the moisture content of the dried larvae is reduced to 5-10%; remove impurities again, then crush the black soldier fly larvae and filter them using a 150-mesh filter bag to obtain black soldier fly larvae powder.

[0070] (2) Black soldier fly larvae powder was placed in an extraction tank for subcritical extraction. The vacuum was drawn to a vacuum degree of 0.2 MPa. Butane with a mass of five times the volume of the material was added and extracted at 55°C for 40 min / time. The extraction was repeated 6 times to obtain defatted black soldier fly powder.

[0071] (3) The liquid-solid ratio of water to defatted black soldier fly powder is 20:1, and the defatted black soldier fly powder and protease are mixed at a mass ratio of 100:0.5 for enzymatic hydrolysis. The protease is keratinase and alkaline protease at a mass ratio of 5:1. The enzymatic hydrolysis is carried out 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 black soldier fly enzymatic hydrolysate.

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

[0073] Example 4

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

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

[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 K3 40 mg / kg, Vitamin A 10000 IU / kg, Vitamin D3 2000 IU / kg, and Vitamin E 160 IU / kg;

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

[0078] Example 5

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

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

[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 K3 40 mg / kg, Vitamin A 10000 IU / kg, Vitamin D3 2000 IU / kg, and Vitamin E 160 IU / kg;

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

[0083] Example 6

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

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

[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 K3 40 mg / kg, Vitamin A 10000 IU / kg, Vitamin D3 2000 IU / kg, and Vitamin E 160 IU / kg;

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

[0088] Experimental Example 1

[0089] Black soldier fly larvae were fed with kitchen waste. Larvae aged 6-13 days were collected, impurities were removed, and the larvae were inactivated in boiling water above 90℃ for 5-15 minutes. They were then dried at 105℃ or air-dried outdoors until the moisture content decreased to 5-10%. After drying, impurities were removed again, and the larvae were initially crushed. An appropriate amount of the crushed larvae was placed in a 200-mesh filter bag and then placed in an extraction tank. The extraction tank was sealed, and a vacuum was drawn to -0.1 MPa. Butane, twice the mass of the material, was then injected into the extraction tank until the extraction pressure reached 0.2 MPa. The extraction conditions were 35-55℃, 40-50 minutes per extraction, for 3-5 extractions. The extraction results are shown in Table 1.

[0090] Table 1. Extraction results under different extraction conditions.

[0091]

[0092]

[0093] The results showed that the optimal extraction conditions for subcritical fluid extraction of black soldier fly larvae oil were: extraction temperature of 35℃, extraction time of 50 minutes, and extraction times of 5 times. Under these conditions, the extraction rate of black soldier fly larvae oil reached 100.61%, which was higher than that under 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 by organic solvent extraction was 89.29%, and the extraction rate by wet extraction was 75.91%.

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

[0096] Deproteinizing black soldier fly larvae powder was performed using keratinase (from Bacillus licheniformis JR-101) and alkaline protease (from Bacillus subtilis WB-600). The protein content was compared with commonly available proteases, including flavor protease (from Aspergillus oryzae), acidic protease (from Aspergillus niger), neutral protease (from Bacillus subtilis), papain (from Pseudocydonia sinensis), and bromelain (from Ananascomosus). The enzymatic hydrolysis conditions were: temperature 55℃, enzyme-to-solid ratio 0.5%-3%, hydrolysis time 30-240 minutes, water-to-degreased black soldier fly larvae powder liquid-to-solid ratio 5-20:1, and pH 9. After enzymatic hydrolysis, the mixture was heated to 95°C for inactivation. After cooling, it was filtered through 120-mesh silk and the supernatant was collected. The supernatant was then vacuum-dried at 80°C and the black soldier fly protein peptides were collected.

[0097] The results of enzymatic hydrolysis are as follows Figure 1 As shown, keratinase and alkaline protease exhibit the best enzymatic hydrolysis effect on defatted black soldier fly powder. At a temperature of 50-60℃, an enzyme-to-substrate ratio of 1-3%, and a liquid-to-solid ratio of 10:1, after 60 minutes of enzymatic hydrolysis, they can reduce substrate mass by 71.67%-74.56% and 74.50%-77.39%, respectively. Under the same conditions, flavor protease, acidic protease, neutral protease, papain, and bromelain show poor enzymatic hydrolysis effects on defatted black soldier fly powder. Further preferred enzymatic hydrolysis of defatted black soldier fly powder involves adding keratinase and alkaline protease in equal proportions, achieving a maximum substrate mass reduction of 81.05%, which is 69.84% higher than without enzymes. A further preferred enzyme-to-substrate ratio is 1%-3%, and even more preferred is 2%.

[0098] The effect of liquid-to-solid ratio (liquid-to-solid mass ratio) on the enzymatic hydrolysis effect of defatted black soldier fly larvae is as follows: Figure 2 As shown, the enzymatic hydrolysis effect is best when the liquid-to-solid ratio reaches 15:1. However, an excessively high liquid-to-solid ratio will result in an excessively large liquid volume during the enzymatic hydrolysis process, which will increase the volume of the liquid to be dried and thus increase the cost of water and electricity. Therefore, the enzymatic hydrolysis conditions are further optimized to a liquid-to-solid ratio of 7-10:1, and even more optimized to 7:1.

[0099] The effect of enzymatic hydrolysis time on the enzymatic hydrolysis efficiency of defatted black soldier fly larvae is as follows: Figure 3 As shown, the enzymatic hydrolysis time reaches a plateau at 180 minutes, and the substrate quality no longer changes significantly. Considering the heating cost and time cost, the enzymatic hydrolysis time was further optimized to 180 minutes.

[0100] Experimental Example 3: Nutritional Characteristics and Molecular Weight Identification of Black Soldier Fly Protein Peptides

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

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

[0103]

[0104]

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

[0106] Furthermore, the molecular weight of the black soldier fly protein peptides was determined by high-performance liquid chromatography, and the results are as follows: Figure 4 As shown, 88.30% of the peptides have a molecular weight of less than 1000 Da, indicating that the black soldier fly protein peptides produced by this method are small peptide protein products with good digestibility and absorption properties for aquatic animals.

[0107] Experiment 4: Black soldier fly protein peptides as a protein source in rainbow trout feed (one)

[0109] An 8-week culture experiment was designed to evaluate the effects of black soldier fly larvae protein peptides (BSFP) as a dietary protein source on rainbow trout. The control group (CD) had BSFP15, BSFP30, BSFP45, BSFP60, and BSFP75 diets prepared by replacing 15%, 30%, 45%, 60%, and 75% of the fishmeal in their feed, respectively. The experimental diet formulations are shown in Tables 3 and 4. During the 8-week culture experiment, the fish were fed to satiety levels at 7:00 AM and 5:00 PM daily.

[0110] Table 3 Experimental feed formulation (%)

[0111]

[0112]

[0113] Table 4. Nutritional composition (%) of the experimental diet

[0114] (two)

[0116] After the 8-week culture 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.

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

[0118]

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

[0120] The results showed that the feed described in this invention can significantly improve the growth performance of rainbow trout while maintaining their nutritional requirements (Table 5). Figure 5 This method can shorten the breeding cycle and save breeding costs without affecting the feeding and survival rate of rainbow trout, thereby increasing production and economic income. (three)

[0122] After an 8-week culture experiment, all fish were fasted for 24 hours. Then, the liver and dorsal muscle of rainbow trout were harvested, and the antioxidant enzyme activity was measured using kits from Beyotime Biotechnology Co., Ltd. and Nanjing Jiancheng Biotechnology Research Institute. The results are shown in Table 6.

[0123] Table 6. Effects of black soldier fly protein peptides on antioxidant enzyme activity in the liver and dorsal muscle of rainbow trout.

[0124]

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

[0126] Table 6 shows that feeding rainbow trout with feed containing black soldier fly protein peptides does not cause oxidative stress in the liver and muscles. The results indicate that the black soldier fly protein peptides prepared by the present invention can be used as a substitute for fishmeal in rainbow trout feed without affecting the rainbow trout's ability to resist external environmental stress, demonstrating significant advantages over other animal and plant protein sources.

[0127] Meanwhile, analysis of the muscle texture properties of rainbow trout dorsal muscles revealed that black soldier fly larvae protein peptides significantly reduced the adhesiveness of the dorsal muscles and significantly increased their elasticity and chewability (e.g., ...). Figure 6(As shown). Therefore, black soldier fly protein peptides help improve the muscle texture of rainbow trout, increasing its appeal to consumers. Using other protein sources to replace fishmeal in aquaculture feed often leads to increased muscle adhesion and decreased elasticity and chewiness, resulting in looser meat and a decline in the palatability of the fish, thus reducing its appeal to consumers. The feed described in this invention significantly alleviates the problem of decreased muscle palatability in rainbow trout caused by existing low-fishmeal formulated feeds on the market. Black soldier fly protein peptides, as a novel feed protein source, have significant economic value.

[0128] In summary, the black soldier fly protein peptides prepared by the technical solution of this invention contain 61.86% crude protein, of which 87.44% of the nitrogen is amino acid nitrogen, and more than 88% of the polypeptides have a molecular weight of less than 1000 Da. They can serve as a major protein source in aquatic feed, improving the growth performance and accelerating the growth rate of rainbow trout, while having no negative impact on the antioxidant capacity of rainbow trout, maintaining their ability to resist external environmental stress during aquaculture. They can also improve the muscle texture of rainbow trout, reduce adhesion, and significantly increase elasticity and chewiness. Furthermore, they can replace most of the fishmeal in feed, reducing the use of fishmeal in aquatic feed and enabling the sustainable and green development of the rainbow trout farming industry.

[0129] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of black soldier fly protein peptides in the preparation of functional feeds that reduce the adhesion of fish dorsal muscles, characterized in that, The black soldier fly protein peptide was prepared by the following method, with the following steps: (1) Black soldier fly larvae were inactivated and then subjected to subcritical extraction to obtain defatted black soldier fly powder; (2) The defatted black soldier fly powder was mixed with protease and enzymatically hydrolyzed to obtain black soldier fly protease hydrolysate, which was then filtered and dried to obtain black soldier fly protein peptides. The solvent for subcritical extraction in step (1) is butane, the mass-to-volume ratio of the subcritical extract is 1 mg: (1-5) mL, the temperature of the subcritical extraction is 30-55℃, the time of the subcritical extraction is 40-55 min / time, and the number of subcritical extractions is 4-6 times. In step (2), the mass ratio of defatted black soldier fly powder to protease is 100:(0.5-3); The protease is keratinase and alkaline protease, and the mass ratio of keratinase to alkaline protease is (1-5):(1-5).

2. The application according to claim 1, characterized in that, The inactivation temperature in step (1) is 90-100℃, and the inactivation time is 5-15 min.

3. A functional fish feed containing black soldier fly protein peptides, characterized in that, It comprises the following components in parts by weight: protein source 34.6-97 parts, carbohydrate source 12-18.9 parts, fat source 14-18 parts, calcium dihydrogen phosphate 0.8-1.2 parts, complex vitamins 0.4-0.6 parts, complex minerals 0.4-0.6 parts, single vitamins 0.8-1.5 parts, essential amino acids 0.14-0.8 parts, and antifungal agent 0.08-0.12 parts; The protein sources include fish meal, soybean meal, soy protein isolate, and black soldier fly protein peptides in a mass ratio of (10-36):(8-12):(10-14):(6.6-35). The black soldier fly protein peptide was prepared by the following method, with the following steps: (1) Black soldier fly larvae were inactivated and then subjected to subcritical extraction to obtain defatted black soldier fly powder; (2) The defatted black soldier fly powder was mixed with protease and enzymatically hydrolyzed to obtain black soldier fly protease hydrolysate, which was then filtered and dried to obtain black soldier fly protein peptides. The solvent for subcritical extraction in step (1) is butane, the mass-to-volume ratio of the subcritical extract is 1 mg: (1-5) mL, the temperature of the subcritical extraction is 30-55℃, the time of the subcritical extraction is 40-55 min / time, and the number of subcritical extractions is 4-6 times. In step (2), the mass ratio of defatted black soldier fly powder to protease is 100:(0.5-3); The protease is keratinase and alkaline protease, and the mass ratio of keratinase to alkaline protease is (1-5):(1-5).

4. The functional fish feed according to claim 3, 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.

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