Feed for eel broodstock and method for preparing and using the same

Abstract

The application discloses eel broodstock feed and a preparation method and application thereof, and relates to the technical field of eel feed. The eel broodstock feed comprises white fish meal, peach gold beetle enzymatic protein, egg yolk, squid liver, pre-gelatinized starch, euphausia, a mixture containing yeast and B vitamins, minerals, choline chloride, compound multivitamin, vitamin E, soybean lecithin, probiotics, frozen fish oil and arachidonic acid. The peach gold beetle enzymatic protein is obtained by subjecting peach gold beetle larvae to EM bacteria fermentation and acid protease enzymolysis. The peach gold beetle larvae are subjected to EM bacteria fermentation to generate organic acids and flavor substances, improve palatability, and enhance the feeding willingness of broodstock; and the peach gold beetle larvae are subjected to acid protease enzymolysis to generate a large amount of small molecular peptides and free amino acids, so as to guarantee the supply of nutrients when the feeding amount of the broodstock decreases. Compared with common eel post-smolt feed, the present feed can significantly improve the development level of eel broodstock, and then improve the fertilization rate and hatching rate.

Description

Technical Field

[0001] This application relates to the field of eel feed technology, specifically designing an eel broodstock feed and its preparation method and application. Background Technology

[0002] Currently, eels can be artificially bred, but many problems remain, and wild fry are still the primary source. Overfishing, environmental changes, and the natural decline of eel fry resources have led to continuously rising fry prices, becoming a bottleneck restricting the sustainable development of the industry. Achieving fully artificial eel breeding, that is, obtaining fertilized eggs through artificial breeding of parent fish and cultivating them into glass eels, is the fundamental way to solve the problem of fry sourcing.

[0003] Broodstock eels refer to adult eels used for reproduction. Currently, farmed eel broodstock are mostly fed fresh miscellaneous fish, fish paste, or ordinary adult eel feed, resulting in an unbalanced nutritional structure. This leads to: (1) The ovaries of female eels are often in the early stage before the yolk sac develops, and they cannot complete the accumulation of yolk sac and the final maturation of oocytes on their own. Even with the use of hormones to induce spawning, there are often cases of low oocyte maturation rate, unsmooth ovulation, or the production of immature oocytes, which in turn leads to problems such as abnormal embryonic development, low hatching rate, and high rate of larval deformities.

[0004] (2) The development of testes in male eels also lags behind that in the natural state. Although artificial maturation can produce semen, it generally suffers from low sperm concentration, weak initial motility, and short duration of activity. This makes it difficult for sperm to effectively penetrate the egg membrane during artificial insemination, which is one of the important reasons for the low fertilization rate.

[0005] In artificial breeding environments, procedures such as catching, inspecting, and injecting hormones cause severe stress to both male and female parent fish. This stress can lead to ovulation failure or decreased egg quality, as well as premature sperm release or a sharp drop in sperm motility.

[0006] The above problems restrict the industrialization of artificial breeding of eels. Summary of the Invention

[0007] To address the problem that, in artificial breeding environments, the ovarian development of female eels and the testis development of male eels lag behind their natural state, resulting in poor physical condition and low stress resistance, this application proposes a broodstock eel feed and its preparation method. This broodstock eel feed is then applied to feed broodstock eels to accelerate ovarian development in female eels and testis development in male eels, thereby enhancing the physical condition of the broodstock eels and improving their stress resistance. To this end, this application adopts the following solution.

[0008] In the first aspect, this application proposes a feed for eel broodstock and adopts the following technical solution.

[0009] A type of eel broodstock feed includes white fish meal, pine jelly hydrolyzed protein, egg yolk, squid liver, pregelatinized starch, krill, a mixture containing yeast and B vitamins, minerals, choline chloride, multivitamins, vitamin E, soybean lecithin, probiotics, frozen fish oil, and arachidonic acid. The enzymatically hydrolyzed protein from the larvae of the bougainvillea is a product obtained by fermenting bougainvillea larvae with EM bacteria and hydrolyzing them with acidic protease.

[0010] By employing the above technical solutions, the larvae of the Eel broodstock undergo EM (Effective Microorganisms) fermentation to produce organic acids and flavor substances, improving palatability and enhancing the feeding willingness of broodstock. Acidic protease hydrolysis produces a large amount of small-molecule peptides and free amino acids, ensuring nutritional supply when broodstock feed intake decreases. White fish meal and Eel broodstock hydrolyzed protein provide a dual protein source of macromolecular proteins and small-molecule peptides. White fish meal provides slow-release amino acids, while hydrolyzed protein provides directly absorbable small-molecule peptides, reducing the digestive burden on broodstock during the breeding season and improving protein utilization. Pregelatinized starch provides slow-release energy and also acts as a feed binder, forming stable clumps in the water after kneading. Egg yolk, squid liver, and krill are natural functional ingredients, rich in lecithin, cholesterol, taurine, astaxanthin, and growth factors, synergistically promoting sex hormone synthesis and yolk deposition in broodstock. A mixture containing yeast and B vitamins provides B vitamins to promote energy metabolism and yeast cell protein to enhance immunity. Minerals and multivitamins provide the trace elements necessary for basal metabolism and reproduction. Choline chloride prevents fatty liver caused by high-fat diets and protects the liver function of broodstock. Vitamin E acts as an antioxidant to protect reproductive cell membranes, soybean lecithin participates in the assembly of vitellin lipoproteins, and arachidonic acid, a prostaglandin precursor, directly promotes ovulation and sperm maturation. Probiotics regulate the gut microbiota, enhance stress resistance, and reduce the occurrence of diseases during the breeding season. Frozen fish oil provides EPA, DHA, and other n-3 long-chain polyunsaturated fatty acids, improving the fluidity of egg and sperm cell membranes and increasing fertilization rate. Compared with ordinary adult eel feed, this feed can significantly improve the gonadal index (GSI), egg quality, and sperm motility of broodstock eels, thereby increasing fertilization and hatching rates.

[0011] The preferred formulation of this eel broodstock feed is as follows: the components are formulated in the following proportions by weight: 39-44 parts white fish meal, 16-25 parts enzymatically hydrolyzed protein from bouillon, 1-5 parts egg yolk, 1-4 parts squid liver, 20-22 parts pregelatinized starch, 1-8 parts krill, 1-4 parts a mixture containing yeast and B vitamins, 1-4 parts minerals, 0.2-0.5 parts choline chloride, 0.2-0.5 parts multivitamins, 0.1-0.3 parts vitamin E, 1-3 parts soybean lecithin, 0.1-0.4 parts probiotics, 1-5 parts frozen fish oil, and 0.1-1 parts arachidonic acid.

[0012] By adopting the above technical solutions, feed with this ratio range can significantly improve the reproductive performance of eel broodstock.

[0013] Secondly, this application proposes a method for preparing enzymatically hydrolyzed protein from bouillon powder, and adopts the following technical solution.

[0014] A method for preparing enzymatically hydrolyzed protein from bouillon, comprising: S1. Grind the larvae of the bougainvillea to obtain insect porridge, then defatt it to obtain defatted insect porridge.

[0015] S2, mix carbon source, nitrogen source, phosphorus source, magnesium source and water evenly, adjust the temperature to 30-35℃, add EM bacterial stock solution, disperse evenly, and incubate in a sealed container at 30-35℃ for 2-3 days to obtain EM bacterial culture solution; wherein, the initial mass ratio of each raw material is, EM bacterial stock solution: carbon source: nitrogen source: phosphorus source: magnesium source: water = 10: (8-12): (0.1-0.2): (0.05-0.1): (0.01-0.02): (70-90).

[0016] S3, take the defatted insect sludge prepared in step S1, the EM bacterial culture prepared in step S2, carbon source, water, acidic protease, and chitinase and mix them evenly in a mass ratio of (200-300):(100-200):(30-60):(600-700):(0.5-2.5):(0.1-0.3). Adjust the initial pH to 5.5-6.5 and incubate at 30-35℃ for 3-5 days. Then adjust the pH to 3-4 and raise the temperature to 40-50℃ for enzymatic hydrolysis for 1-2 days. Continue to raise the temperature to inactivate the acidic protease. Remove the insoluble residue and remove the water from the remaining liquid to obtain the enzymatic hydrolysate of bougainvillea.

[0017] By adopting the above technical solution, step S1, defatting, reduces the influence of fat on microbial fermentation and enzymatic hydrolysis; step S2 establishes an EM (Effective Microorganisms) culture system to increase the viable cell count; step S3 involves initial fermentation, where EM bacteria degrade large-molecule proteins at pH 5.5-6.5 and 30-35℃ to produce flavor compounds. Then, the pH is adjusted to 3-4 and the temperature is raised to 40-50℃, where acidic proteases efficiently hydrolyze the remaining protein into small-molecule peptides. Finally, enzyme inactivation terminates the reaction, preventing excessive hydrolysis and the production of bitter peptides. Adding chitinase degrades insect chitin, releasing bound proteins and simultaneously producing chitin oligosaccharides (prebiotics). The enzymatically hydrolyzed protein prepared by this method is rich in small-molecule peptides, free amino acids, and chitin oligosaccharides, exhibiting digestive-promoting and immune-enhancing effects.

[0018] The preferred embodiment of the preparation method of the enzymatic hydrolysate of the bougainvillea is as follows: in step S1, the bougainvillea larvae are first cleaned, then blanched in boiling water to kill the microorganisms on the surface of the bougainvillea larvae, and then the bougainvillea larvae are minced.

[0019] By adopting the above technical solution, blanching is used to kill miscellaneous bacteria on the surface of the insects, creating an advantageous environment for subsequent pure EM fermentation.

[0020] The preferred embodiment of the preparation method of the enzymatic hydrolysate of the peach jelly is that step S1 uses a pressing method to degrease the insect sludge, including loading the insect sludge into a filter bag, applying pressure to the filter bag to allow liquid to flow out, and the residue in the filter bag is the degreased insect sludge.

[0021] By adopting the above technical solution, this physical degreasing method that does not require organic solvents removes most of the free fat, and the remaining fat does not affect fermentation, while retaining some fat-soluble nutrients.

[0022] The preferred embodiment of the preparation method of the peach ginseng enzymatic hydrolysate protein is as follows: in step S2, the carbon source is brown sugar, the nitrogen source is urea, the phosphorus source is potassium dihydrogen phosphate, and the magnesium source is magnesium sulfate; in step S3, the carbon source is brown sugar, and the acidic protease is an acidic protease obtained by fermentation and refining of Aspergillus niger.

[0023] By adopting the above technical solutions, brown sugar, in addition to providing a carbon source, also contains various trace elements and B vitamins, making it superior to pure sucrose, and promoting more vigorous growth of EM bacteria. Urea is inexpensive and readily available, and EM bacteria utilize it efficiently. Potassium dihydrogen phosphate provides both phosphorus and potassium, and has a strong pH buffering capacity. Magnesium sulfate provides magnesium ions, which are cofactors for proteases. Acidic proteases derived from Aspergillus niger exhibit stable degradation efficiency.

[0024] Thirdly, this application proposes a method for preparing eel broodstock feed, and adopts the following technical solution.

[0025] A method for preparing eel broodstock feed involves taking 5-10% of the total amount of white fish meal, adding minerals, choline chloride, compound multivitamins, vitamin E, and probiotics, and stirring evenly to obtain a premix.

[0026] The remaining white fish meal, the prepared bougainvillea hydrolyzed protein, pregelatinized starch, and krill powder are added to a mixer and stirred evenly. Then, egg yolk powder, squid liver powder, a mixture containing yeast and B vitamins, and soybean lecithin powder are added and stirred evenly. Next, the premix is ​​added and stirred evenly. Finally, frozen fish oil and arachidonic acid are sprayed in and stirred evenly to obtain the eel broodstock feed.

[0027] By adopting the above technical solution, trace components are first mixed on a small scale to obtain a premix, so that components with an addition amount of less than 1% such as minerals, vitamins, and probiotics are evenly distributed in the feed.

[0028] Fourthly, this application proposes an application of eel broodstock feed and adopts the following technical solution.

[0029] An application of eel broodstock feed involves mixing the eel broodstock feed, fish paste, and water in a mass ratio of 100:(15-25):(80-120), kneading the mixture into balls, and then feeding it to the eel broodstock.

[0030] By adopting the above technical solutions, fish paste provides fresh feed-attracting components and myofiber protein, enhancing the viscoelasticity of the feed pellets; the pelleted feed conforms to the feeding habits of eels, which involve holding and tearing food, reducing feed loss and lowering water pollution.

[0031] In summary, the eel broodstock feed, its preparation method, and its application of this application have the following beneficial effects: This application scientifically combines white fish meal, enzymatically hydrolyzed protein from bougainvillea, pregelatinized starch, egg yolk, squid liver, krill, yeast, B vitamins, minerals, choline chloride, multivitamins, vitamin E, soybean lecithin, probiotics, fish oil, and arachidonic acid to form a reproductive fortified feed specifically for the yolk accumulation and spermatogenesis periods of eel broodstock. The components synergistically enhance each other, simultaneously promoting ovarian development in female eels and sperm development in male eels.

[0032] This application uses enzymatically hydrolyzed balsam protein as one of the core protein sources, forming a dual protein supply system with white fish meal. The enzymatically hydrolyzed balsam protein is rich in small molecule peptides and free amino acids, which can be directly absorbed and utilized by broodstock, significantly reducing the digestive burden caused by gonadal pressure during the breeding season and improving protein utilization. The enzymatically hydrolyzed protein, fermented with EM bacteria, also contains organic acids, flavor substances, and bacterial metabolites, exhibiting excellent appetite-stimulating effects and effectively solving the technical problem of decreased feed intake in broodstock during the breeding season. Attached Figure Description

[0033] Figure 1 The flowchart is a reference diagram for the raw material example 1. Detailed Implementation

[0034] The technical solutions in the embodiments are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the following embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0035] The following is information on some of the raw materials used in each embodiment and comparative example.

[0036] White fish meal: The supplier is Guangzhou Muchang Import & Export Trading Co., Ltd.

[0037] A mixture containing yeast and B vitamins: provided by digestive enzyme tablets, which are a compound preparation composed of dry yeast, vitamin B1, vitamin B2, niacin and excipients.

[0038] The minerals are mixed in the following proportions by weight: 100 parts of dicalcium phosphate, 40 parts of potassium dihydrogen phosphate, 15 parts of sodium chloride, 15 parts of magnesium chloride, 3 parts of ferrous sulfate, 1 part of zinc sulfate, 1 part of manganese sulfate, 0.1 parts of copper sulfate, 0.01 parts of potassium iodide, 0.001 parts of sodium selenite, and 0.001 parts of cobalt chloride.

[0039] Each kilogram of the multivitamin contains: Vitamin A 1,200,000 IU, Vitamin D3 240,000 IU, Vitamin E 15,000 mg, Vitamin K3 4,000 mg, Vitamin B1 5,000 mg, Vitamin B2 10,000 mg, Vitamin B6 5,000 mg, Vitamin B12 25 mg, Niacin 30,000 mg, Calcium Pantothenate 15,000 mg, Folic Acid 1,500 mg, Biotin 100 mg, Vitamin C Phosphate 60,000 mg, Inositol 50,000 mg, with the remainder being defatted corn gluten powder carrier.

[0040] The probiotics are EM bacteria powder, supplied by Shandong Xinyi Biotechnology Co., Ltd., with a live bacteria count ≥100 billion / g.

[0041] Frozen fish oil: Sardinian oil is refined through degumming, deacidification, dehydration, decolorization, and degreasing. It has the effect of "not freezing in winter" and is rich in natural eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

[0042] EM stock solution: The supplier is Shandong Infinida Biotechnology Co., Ltd., and the product name is "Aquatic EM Stock Solution". The effective live bacteria count is ≥20 billion / g.

[0043] Acidic protease: The supplier is Taian Xindeli Bioengineering Co., Ltd. The acidic protease is refined from the fermentation of superior strains of Aspergillus niger. It is a light yellow powder with a specification of 100,000 U / g.

[0044] Raw Material Example 1 Preparation of enzymatically hydrolyzed protein from bouillon, refer to Figure 1 The implementation steps are as follows.

[0045] S1. Clean the larvae of the bougainvillea, blanch them in boiling water for 2 minutes, drain the water, and mince them to obtain bougainvillea larvae porridge. Put the porridge into a 200-mesh nylon filter bag and press it with a press at a pressure of 5 MPa until no liquid flows out. The residue in the filter bag is defatted porridge.

[0046] S2, take 40℃ warm water, add brown sugar, and stir to dissolve; then add urea, potassium dihydrogen phosphate, and magnesium sulfate, and stir until completely dissolved; then adjust the temperature to 32℃, add EM stock solution, and stir evenly; finally, pour the liquid into a fermentation tank, seal it, and ferment at 32℃ for 2.5 days to obtain EM inoculum. The initial mass ratio of materials in step S2 is EM stock solution: brown sugar: urea: potassium dihydrogen phosphate: magnesium sulfate: water = 10:10:0.15:0.07:0.015:80.

[0047] In step S3, the defatted insect sludge prepared in step S1 is transferred to another fermenter. Water is added and stirred until a paste is formed. The pH is adjusted to 6.0 using sodium carbonate / lactic acid. Brown sugar is added and stirred until homogeneous. The EM inoculum prepared in step S2 is added and stirred until homogeneous. Acidic protease and chitinase are added. The fermenter in this step is kept semi-sealed to facilitate the degassing of the EM bacteria. The mixture is incubated at 32°C for 4 days. Then, the pH is adjusted to 3.5 using lactic acid, and the temperature is raised to 45°C for enzymatic hydrolysis for 1.5 days. Finally, the temperature is raised to 90°C and held for 20 minutes to inactivate the acidic protease, thus terminating the enzymatic hydrolysis reaction. The mixture is filtered through a 100-mesh sieve to remove insoluble residues. The remaining liquid is spray-dried to obtain a powder of boudin hydrolyzed protein. In step S3, the defatted insect sludge, EM inoculum, brown sugar, water, acidic protease, and chitinase are mixed in a mass ratio of 250:150:45:650:1.5:0.2.

[0048] Raw Material Example 2 The steps for preparing enzymatically hydrolyzed protein from bouillon are as follows.

[0049] S1. Clean the larvae of the bougainvillea, blanch them in boiling water for 2 minutes, drain the water, and mince them to obtain bougainvillea larvae porridge. Put the porridge into a 200-mesh nylon filter bag and press it with a press at a pressure of 5 MPa until no liquid flows out. The residue in the filter bag is defatted porridge.

[0050] S2, take 40℃ warm water, add brown sugar, and stir to dissolve; then add urea, potassium dihydrogen phosphate, and magnesium sulfate, and stir until completely dissolved; then adjust the temperature to 30℃, add EM stock solution, and stir evenly; finally, pour the liquid into a fermentation tank, seal it, and ferment at 30℃ for 3 days to obtain EM inoculum. The initial mass ratio of materials in step S2 is EM stock solution: brown sugar: urea: potassium dihydrogen phosphate: magnesium sulfate: water = 10:8:0.2:0.05:0.02:70.

[0051] In step S3, the defatted insect sludge prepared in step S1 is added to another fermenter, water is added, and the mixture is stirred until it becomes a paste. The pH is adjusted to 5.5 using sodium carbonate / lactic acid. Brown sugar is added and stirred until well combined. The EM inoculum prepared in step S2 is added and stirred until well combined. Acidic protease and chitinase are added. The fermenter in this step is kept semi-sealed to facilitate the degassing of the EM bacteria. The mixture is incubated at 30°C for 5 days. Then, the pH is adjusted to 3 using lactic acid, and the temperature is raised to 40°C for enzymatic hydrolysis for 2 days. Finally, the temperature is raised to 90°C and held for 20 minutes to inactivate the acidic protease, thus terminating the enzymatic hydrolysis reaction. The mixture is filtered through a 100-mesh sieve to remove insoluble residues. The remaining liquid is spray-dried to obtain a powder of peach jelly hydrolyzed protein. In step S3, the defatted insect sludge, EM inoculum, brown sugar, water, acidic protease, and chitinase are mixed in a mass ratio of 200:200:30:700:0.5:0.3.

[0052] Raw Material Example 3 The steps for preparing enzymatically hydrolyzed protein from bouillon are as follows.

[0053] S1. Clean the larvae of the bougainvillea, blanch them in boiling water for 2 minutes, drain the water, and mince them to obtain bougainvillea larvae porridge. Put the porridge into a 200-mesh nylon filter bag and press it with a press at a pressure of 5 MPa until no liquid flows out. The residue in the filter bag is defatted porridge.

[0054] S2, take 40℃ warm water, add brown sugar, and stir to dissolve; then add urea, potassium dihydrogen phosphate, and magnesium sulfate, and stir until completely dissolved; then adjust the temperature to 35℃, add EM stock solution, and stir evenly; finally, pour the liquid into a fermentation tank, seal it, and ferment at 35℃ for 2 days to obtain EM inoculum. The initial mass ratio of materials in step S2 is EM stock solution: brown sugar: urea: potassium dihydrogen phosphate: magnesium sulfate: water = 10:12:0.1:0.1:0.01:90.

[0055] S3: The defatted insect sludge prepared in step S1 is transferred to another fermenter. Water is added and stirred until a paste is formed. The pH is adjusted to 6.5 using sodium carbonate / lactic acid. Brown sugar is added and stirred until homogeneous. The EM inoculum prepared in step S2 is added and stirred until homogeneous. Acidic protease and chitinase are added. The fermenter is kept semi-sealed to facilitate EM degassing. The mixture is incubated at 35°C for 3 days. Then, the pH is adjusted to 4 using lactic acid, and the temperature is raised to 50°C for enzymatic hydrolysis for 1 day. Finally, the temperature is raised to 90°C and maintained for 20 minutes to inactivate the acidic protease, thus terminating the enzymatic hydrolysis reaction. Insoluble residues are removed by filtration through a 100-mesh sieve. The remaining liquid is spray-dried to obtain a powder of peach jelly hydrolyzed protein. In step S3, the defatted insect sludge, EM inoculum, brown sugar, water, acidic protease, and chitinase are mixed in a mass ratio of 300:100:60:600:2.5:0.1.

[0056] Feed Example 1 To prepare eel broodstock feed, the following ingredients were formulated in the indicated weight proportions: 40 parts white fish meal, 20 parts enzymatically hydrolyzed protein from bouillon powder, 2 parts egg yolk powder, 2 parts squid liver powder, 21 parts pregelatinized starch, 5 parts Antarctic krill powder, 2.4 parts digestive enzyme tablets, 2 parts minerals, 0.3 parts choline chloride, 0.4 parts multivitamins, 0.2 parts vitamin E, 2 parts soybean lecithin powder, 0.2 parts probiotics, 3 parts frozen fish oil, and 0.5 parts arachidonic acid.

[0057] The specific steps for preparing eel broodstock feed are as follows.

[0058] Take 10% of the total amount of white fish meal as a carrier, add minerals, choline chloride, multivitamins, vitamin E and probiotics, stir well to obtain a premix.

[0059] The remaining white fish meal, the enzymatically hydrolyzed protein of bougainvillea prepared in Example 1, the pregelatinized starch, and the Antarctic krill powder were put into a mixer and stirred evenly. Then, egg yolk powder, squid liver powder, gluten tablets, and soybean lecithin powder were added and stirred evenly. Then, the premix was added and stirred evenly. Finally, frozen fish oil and arachidonic acid were sprayed in and stirred evenly to obtain powdered eel broodstock feed.

[0060] Feed Example 2 To prepare eel broodstock feed, the following ingredients were formulated in the indicated weight proportions: 39 parts white fish meal, 25 parts enzymatically hydrolyzed protein from bouillon powder, 1 part egg yolk powder, 4 parts squid liver powder, 20 parts pregelatinized starch, 8 parts Antarctic krill powder, 1 part digestive enzyme tablets, 4 parts minerals, 0.2 parts choline chloride, 0.5 parts multivitamins, 0.1 parts vitamin E, 3 parts soybean lecithin powder, 0.1 parts probiotics, 5 parts frozen fish oil, and 0.1 parts arachidonic acid.

[0061] The specific steps for preparing eel broodstock feed are as follows.

[0062] Take 5% of the total amount of white fish meal as a carrier, add minerals, choline chloride, multivitamins, vitamin E and probiotics, stir well to obtain a premix.

[0063] The remaining white fish meal, the enzymatically hydrolyzed protein of bougainvillea prepared in Example 2, the pregelatinized starch, and the Antarctic krill meal were put into a mixer and stirred evenly. Then, egg yolk powder, squid liver powder, gluten tablets, and soybean lecithin powder were added and stirred evenly. Then, the premix was added and stirred evenly. Finally, frozen fish oil and arachidonic acid were sprayed in and stirred evenly to obtain powdered eel broodstock feed.

[0064] Feed Example 3 To prepare eel broodstock feed, the following ingredients were formulated in the indicated weight proportions: 44 parts white fish meal, 16 parts pine nut hydrolysate protein, 5 parts egg yolk powder, 1 part squid liver powder, 22 parts pregelatinized starch, 1 part Antarctic krill powder, 4 parts digestive enzyme tablets, 1 part minerals, 0.5 parts choline chloride, 0.2 parts compound multivitamins, 0.3 parts vitamin E, 1 part soybean lecithin powder, 0.4 parts probiotics, 1 part frozen fish oil, and 1 part arachidonic acid.

[0065] The specific steps for preparing eel broodstock feed are as follows.

[0066] Take 8% of the total amount of white fish meal as a carrier, add minerals, choline chloride, multivitamins, vitamin E and probiotics, stir well to obtain a premix.

[0067] The remaining white fish meal, the enzymatically hydrolyzed protein of bougainvillea prepared in Example 3, the pregelatinized starch, and the Antarctic krill powder were put into a mixer and stirred evenly. Then, egg yolk powder, squid liver powder, gluten tablets, and soybean lecithin powder were added and stirred evenly. Then, the premix was added and stirred evenly. Finally, frozen fish oil and arachidonic acid were sprayed in and stirred evenly to obtain powdered eel broodstock feed.

[0068] Raw material comparison example 1 The preparation of enzymatically hydrolyzed protein from *Pterocarya stenoptera* differs from that in Example 1 in that only the larvae of *Pterocarya stenoptera* are defatted, without fermentation or enzymatic hydrolysis. The preparation steps for this comparative example are as follows.

[0069] S1. Clean the larvae of the bougainvillea, blanch them in boiling water for 2 minutes, drain the water, and mince them to obtain bougainvillea larvae porridge. Put the porridge into a 200-mesh nylon filter bag and press it with a press at a pressure of 5 MPa until no liquid flows out. The residue in the filter bag is defatted porridge.

[0070] Raw material comparison ratio 2 The preparation of enzymatically hydrolyzed protein from *Pterocarya stenoptera* differs from that in Example 1 in that step S3 only involves fermenting the larval stenoptera of *Pterocarya stenoptera* without enzymatic hydrolysis, i.e., no acidic protease or chitinase is added. The preparation steps for this comparative example are as follows.

[0071] S1. Clean the larvae of the bougainvillea, blanch them in boiling water for 2 minutes, drain the water, and mince them to obtain bougainvillea larvae porridge. Put the porridge into a 200-mesh nylon filter bag and press it with a press at a pressure of 5 MPa until no liquid flows out. The residue in the filter bag is defatted porridge.

[0072] S2, take 40℃ warm water, add brown sugar, and stir to dissolve; then add urea, potassium dihydrogen phosphate, and magnesium sulfate, and stir until completely dissolved; then adjust the temperature to 32℃, add EM stock solution, and stir evenly; finally, pour the liquid into a fermentation tank, seal it, and ferment at 32℃ for 2.5 days to obtain EM inoculum. The initial mass ratio of materials in step S2 is EM stock solution: brown sugar: urea: potassium dihydrogen phosphate: magnesium sulfate: water = 10:10:0.15:0.07:0.015:80.

[0073] S3: The defatted insect sludge prepared in step S1 is transferred to another fermenter. Water is added and stirred until a paste is formed. The pH is adjusted to 6.0 using sodium carbonate / lactic acid. Brown sugar is added and stirred until homogeneous. The EM inoculum prepared in step S2 is added and stirred until homogeneous. The fermenter in this step is kept semi-sealed to facilitate EM degassing. The mixture is incubated at 32℃ for 4 days. Finally, the temperature is raised to 90℃ and maintained for 20 minutes. Insoluble residues are removed by filtration through a 100-mesh sieve. The remaining liquid is spray-dried to obtain a powdered product, namely, peach kernel jelly fermented protein. In step S3, the defatted insect sludge, EM inoculum, brown sugar, and water are mixed in a mass ratio of 250:150:45:650.

[0074] Raw material comparison ratio 3 The preparation of enzymatically hydrolyzed protein from *Pterocarya stenoptera* differs from that in Example 1 in that step S3 only involves enzymatic hydrolysis of *Pterocarya stenoptera* larvae without fermentation; that is, step S2 is omitted, and EM bacteria are not added in step S3. The preparation steps of this comparative example are as follows.

[0075] S1. Clean the larvae of the bougainvillea, blanch them in boiling water for 2 minutes, drain the water, and mince them to obtain bougainvillea larvae porridge. Put the porridge into a 200-mesh nylon filter bag and press it with a press at a pressure of 5 MPa until no liquid flows out. The residue in the filter bag is defatted porridge.

[0076] In step S3, the defatted insect sludge prepared in step S1 is placed into a fermentation tank, water is added, and the mixture is stirred until it becomes a paste. Acidic protease and chitinase are added, and the pH is adjusted to 3.5 with lactic acid. The mixture is then heated to 45°C and enzymatically hydrolyzed for 1.5 days. Finally, the temperature is raised to 90°C and maintained for 20 minutes to inactivate the acidic protease, thus terminating the enzymatic hydrolysis reaction. The mixture is filtered through a 100-mesh sieve to remove insoluble residues, and the remaining liquid is spray-dried to obtain a powder of peach kernel jelly hydrolyzed protein. In step S3, the defatted insect sludge, brown sugar, water, acidic protease, and chitinase are mixed in a mass ratio of 250:45:650:1.5:0.2.

[0077] Raw material comparison ratio 4 The only difference between the preparation of longhorn beetle enzymatic hydrolysate and the raw material in Example 1 is that the larvae of the bougainvillea were replaced with the same mass of longhorn beetle larvae, and the longhorn beetle enzymatic hydrolysate was finally prepared.

[0078] Raw material comparison ratio 5 The only difference between the preparation of sardine enzymatic hydrolysate and the raw material in Example 1 is that the larvae of the bougainvillea are replaced with an equal mass of sardines, and the final product is sardine enzymatic hydrolysate.

[0079] Feed Comparison 1 The only difference between the eel broodstock feed and feed example 1 is that the enzymatically hydrolyzed protein of bougainvillea was replaced with an equal mass of defatted insect sludge from raw material comparison example 1.

[0080] Feed Comparison Ratio 2 The only difference between the eel broodstock feed and feed example 1 is that the enzymatic hydrolysate of bougainvillea is replaced with an equal mass of fermented bougainvillea protein from raw material comparison example 2.

[0081] Feed Comparison Ratio 3 The only difference between the eel broodstock feed and feed example 1 is that the cinnamon hydrolysate was replaced with an equal mass of the cinnamon hydrolysate from raw material comparison example 3.

[0082] Feed Comparison Ratio 4 The only difference between the eel broodstock feed and feed example 1 is that the pine needle enzymatic hydrolysate was replaced with an equal mass of the longhorn beetle enzymatic hydrolysate from raw material comparison example 4.

[0083] Feed ratio 5 The only difference between the eel broodstock feed and feed example 1 is that the enzymatically hydrolyzed sardine protein from raw material comparison example 5 was replaced with an equal mass of enzymatically hydrolyzed sardine protein.

[0084] Feed ratio 6 The only difference between the eel broodstock feed and the feed in Example 1 is that no pine jelly hydrolysate protein is added.

[0085] Experimental Example 1 The raw materials prepared in Examples 1-3 and Comparative Examples 1-5 were used to determine the contents of chitosan oligosaccharides, small molecule peptides and total organic acids. The results are shown in Table 1.

[0086] Table 1. Data on the determination of nutrient content

[0087] Experimental Example 2 Select female and male eels from the same batch. Characteristics of the selected females: weight 500-800g / fish, body length 45-60cm, 4-6 years old (gonads developed to stage II-III), no external injuries, active swimming, normal feeding, and no parasites. Characteristics of the selected males: weight 200-400g / fish, body length 35-50cm, 3-5 years old (testes developed to stage II), no external injuries, active swimming, normal feeding, and no parasites.

[0088] The female fish were divided into 9 groups of 30 each. The male fish were divided into 9 groups of 20 each. All fish were fed in the same water conditions with a temperature of 18-22℃.

[0089] Eel broodstock feed, cod surimi, and water were mixed evenly at a mass ratio of 100:20:100, kneaded into balls, and then fed to the eel broodstock under the same feeding conditions. The eel broodstock feed was taken from the eel broodstock feeds prepared in Feed Examples 1-3 and Feed Comparison Examples 1-6, respectively. The cod surimi was made from fresh cod meat after removing the head, internal organs, bones, and scales, and then ground into a paste. Nine groups of female eels were fed the eel broodstock feeds prepared in Feed Examples 1-3 and Feed Comparison Examples 1-6, respectively. Nine groups of male eels were fed the eel broodstock feeds prepared in Feed Examples 1-3 and Feed Comparison Examples 1-6, respectively. The feeding conditions are shown in Table 2.

[0090] Table 2 Feeding conditions

[0091] Daily feeding rate for females = Daily feed weight / Average weight of all females * 100%. Daily feeding rate for males = Daily feed weight / Average weight of all males * 100%. Feeding is stopped during the spawning period after 22 weeks and post-spawning.

[0092] Artificial insemination was performed on female and male fish fed with the same feed prepared in Example 1, for example, female and male fish fed with the same feed prepared in Example 1 were artificially inseminated, and so on.

[0093] Artificial insemination was performed on female and male fish fed the same feed as those prepared in the same proportion. For example, artificial insemination was performed on female and male fish fed the same feed as those prepared in proportion 1, and so on.

[0094] The development of the ovaries of female fish and the testes of male fish were examined. The fertilization rate, hatching rate and fry survival rate after artificial insemination were also measured, as shown in Table 3 below.

[0095] Table 3. Statistics on the development and reproductive performance of eels

[0096] In Table 3, the female gonadal index (GSI) = (ovarian weight / body weight) × 100%, the male gonadal index (GSI) = (testis weight / body weight) × 100%, the fertilization rate = (number of fertilized eggs / total number of eggs) × 100%, the hatching rate (number of hatched larvae / number of fertilized eggs) × 100%, and the larval survival rate (7 days old) = (number of 7-day-old larvae / number of newly hatched larvae) × 100%.

[0097] Combining the data from Experiments 1 and 2, it can be seen that the enzymatically hydrolyzed protein prepared by *Echinochloa crus-galli* larvae in Examples 1-3, using *Echinochloa crus-galli* larvae as raw material, undergoes fermentation and enzymatic hydrolysis. The resulting enzymatically hydrolyzed protein is rich in chitin oligosaccharides, small peptides, and amino acids. Feeding this protein to both female and male eel broodstock improves gonadal development in both sexes, ultimately increasing fertilization rate, hatching rate, and larval survival rate. In contrast, the feed in Comparative Example 1, which contains defatted *Echinochloa crus-galli* larvae without fermentation and enzymatic hydrolysis, has lower levels of chitin oligosaccharides, small peptides, and amino acids. Feeding this feed to eel broodstock resulted in lower gonadal development in both female and male eels, leading to significantly lower fertilization rate, hatching rate, and larval survival rate compared to Examples 1-3. In raw material comparison example 2, defatted insect sludge was fermented without enzymatic hydrolysis; in raw material comparison example 3, defatted insect sludge was enzymatically hydrolyzed without fermentation; in raw material comparison example 4, longhorn beetle larvae were replaced with longhorn beetle larvae; in raw material comparison example 5, sardines were replaced with sardines; and in feed comparison example 6, no sardine hydrolysate was added. When these products were fed to eel broodstock, compared with the products in feed examples 1-3, the development level of eel broodstock was reduced to varying degrees, and the fertilization rate, hatching rate, and larval survival rate were also reduced to varying degrees.

[0098] This application describes the process of processing *Echinochloa crus-galli* larvae into enzymatically hydrolyzed protein through synergistic bacterial and enzyme treatment. This protein serves as the core protein source for eel broodstock feed, enhancing the feed's growth-promoting effects. The application employs a process involving defatting, EM bacteria fermentation, and enzymatic hydrolysis with acidic protease and chitinase. This results in feed rich in chitin oligosaccharides, small peptides, and amino acids, which promote gonadal development in eel broodstock and ultimately improve reproductive performance.

[0099] In the feed of this application: arachidonic acid, vitamin E, egg yolk powder, and squid liver powder synergistically promote the development of eggs and sperm; frozen fish oil, Antarctic krill powder, and soybean lecithin powder provide high-quality phospholipids and fatty acids, resulting in more complete yolk deposition in female eels; enzymatically hydrolyzed protein from bouillon, squid liver powder, and probiotics enhance appetite and utilization; compound multivitamins, minerals, probiotic tablets, and vitamin E enhance immunity and reduce mortality during the reproductive period. The synergistic effect of these components improves the development of various systems in broodstock eels, enhances their stress resistance, and ultimately improves fertility, making the eel broodstock feed of this application have significant economic benefits and application prospects.

[0100] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A feed for eel broodstock, characterized in that, Including white fish meal, tau jelly hydrolyzed protein, egg yolk, squid liver, pregelatinized starch, krill, a mixture containing yeast and B vitamins, minerals, choline chloride, multivitamins, vitamin E, soy lecithin, probiotics, frozen fish oil, and arachidonic acid; The enzymatically hydrolyzed protein from the larvae of the bougainvillea is a product obtained by fermenting bougainvillea larvae with EM bacteria and hydrolyzing them with acidic protease.

2. The eel broodstock feed according to claim 1, characterized in that, The components are proportioned as follows by weight: 39-44 parts white fish meal, 16-25 parts enzymatically hydrolyzed protein, 1-5 parts egg yolk, 1-4 parts squid liver, 20-22 parts pregelatinized starch, 1-8 parts krill, 1-4 parts a mixture containing yeast and B vitamins, 1-4 parts minerals, 0.2-0.5 parts choline chloride, 0.2-0.5 parts multivitamins, 0.1-0.3 parts vitamin E, 1-3 parts soy lecithin, 0.1-0.4 parts probiotics, 1-5 parts frozen fish oil, and 0.1-1 parts arachidonic acid.

3. A method for preparing enzymatically hydrolyzed protein of bouillon as described in claim 1 or 2, characterized in that, include: S1, mince the larvae of the bougainvillea to obtain insect porridge, then defatt it to obtain defatted insect porridge; S2, mix carbon source, nitrogen source, phosphorus source, magnesium source and water evenly, adjust the temperature to 30-35℃, add EM bacterial stock solution, disperse evenly, and incubate in a sealed container at 30-35℃ for 2-3 days to obtain EM bacterial inoculum; wherein, the initial mass ratio of each raw material is, EM bacterial stock solution: carbon source: nitrogen source: phosphorus source: magnesium source: water = 10: (8-12): (0.1-0.2): (0.05-0.1): (0.01-0.02): (70-90); S3, take the defatted insect sludge prepared in step S1, the EM bacterial culture prepared in step S2, carbon source, water, acidic protease, and chitinase and mix them evenly in a mass ratio of (200-300):(100-200):(30-60):(600-700):(0.5-2.5):(0.1-0.3). Adjust the initial pH to 5.5-6.5 and incubate at 30-35℃ for 3-5 days. Then adjust the pH to 3-4 and raise the temperature to 40-50℃ for enzymatic hydrolysis for 1-2 days. Continue to raise the temperature to inactivate the acidic protease. Remove the insoluble residue and remove the water from the remaining liquid to obtain the enzymatic hydrolysate of bougainvillea.

4. The method for preparing enzymatically hydrolyzed protein of bougainvillea according to claim 3, characterized in that, In step S1, the larvae of the bougainvillea are first cleaned, then blanched in boiling water to kill microorganisms on the surface of the larvae, and then the larvae are minced.

5. The method for preparing enzymatically hydrolyzed protein from bouillon according to claim 3, characterized in that, Step S1 uses a pressing method to degrease the insect sludge, which includes loading the insect sludge into a filter bag, applying pressure to the filter bag to allow liquid to flow out, and leaving the degreased insect sludge in the filter bag.

6. The method for preparing enzymatically hydrolyzed protein from bougainvillea according to claim 3, characterized in that, In step S2, the carbon source is brown sugar, the nitrogen source is urea, the phosphorus source is potassium dihydrogen phosphate, and the magnesium source is magnesium sulfate; in step S3, the carbon source is brown sugar, and the acidic protease is an acidic protease obtained by fermentation and refining of Aspergillus niger.

7. A method for preparing eel broodstock feed, characterized in that, Take 5-10% of the total amount of white fish powder, add minerals, choline chloride, compound multivitamins, vitamin E and probiotics, stir evenly to obtain a premix; The remaining white fish meal, the enzymatically hydrolyzed protein of bougainvillea prepared according to any one of claims 3-6, the pregelatinized starch, and the krill powder are put into a mixer and stirred evenly. Then, egg yolk powder, squid liver powder, a mixture containing yeast and B vitamins, and soybean lecithin powder are added and stirred evenly. Then, the premix is ​​added and stirred evenly. Finally, frozen fish oil and arachidonic acid are sprayed in and stirred evenly to obtain the eel broodstock feed.

8. An application of the eel broodstock feed as described in claim 1 or 2, characterized in that, Mix the eel broodstock feed, fish paste and water in a mass ratio of 100:(15-25):(80-120), knead into a ball and feed to the eel broodstock.

Images