Enzymatic hydrolysis compound feed capable of reducing stress of fish fries

Abstract

The invention belongs to the technical field of aquatic feeds, and particularly relates to an enzymolysis compound feed capable of reducing stress of fish fries. The cordyceps militaris, the Chinese wolfberry fruits, the ginseng and the rhizoma bletillae are subjected to specific compound enzymolysis and anaerobic fermentation, so that deep utilization and biological activity of the raw materials are realized, and active substances of the raw materials can improve the oxidation resistance of cells so as to regulate an immune system; the shrimp meal, the rice flour, the barley flour and the red bean flour are mixed with the compound fermentation liquor, so that sufficient nutrients are provided for the growth of the fish fries, and the function release of the compound fermentation liquor is assisted. Therefore, the invention provides a high-quality feed beneficial to healthy growth of trachidermus fasciatus, and the feed can reduce stress response, enhance immunity of trachidermus fasciatus bodies and promote healthy growth of fry. Meanwhile, the enzymolysis compound feed provided by the invention consists of natural substances, does not contain chemical components, is safe and reliable, and provides a good water quality environment for survival of fish fries.

Description

Technical Field

[0001] This invention belongs to the field of aquatic feed technology, specifically relating to an enzymatically hydrolyzed compound feed that reduces stress in fish fry. Background Technology

[0002] Sea bass are carnivorous fish, known for their ferocity and voracious appetite. They primarily feed on small fish, shrimp, benthic mollusks, and aquatic insects. Sea bass meat is delicious and nutritious, and there is a high market demand. Therefore, sea bass farming can not only meet consumer demand and enrich the aquatic product market, but also drive the development of related industries, promote regional economic development, alleviate the pressure of wild sea bass fishing, and protect wild resources.

[0003] There are existing reports on feeds specifically for sea bass. For example, Chinese Patent CN 1833534 B discloses a feed for spotted sea bass, which includes imported fishmeal, soybean meal, corn gluten meal, cuttlefish meal, cottonseed meal No. 4 powder, fish oil, soybean oil, soybean lecithin, calcium dihydrogen phosphate, compound vitamins, compound minerals, immunopolysaccharides, enzyme preparations, choline chloride, and betaine as additives. This feed has a low coefficient of performance, high nutritional value, low production cost, and is easy to promote. Furthermore, using this feed for aquaculture effectively protects near-shore resources, significantly reduces disease transmission and pollution of aquaculture waters, and is safe and reliable to use. For example, Chinese patent CN111418705 A discloses a perch feed prepared by mixing corn flour, fermented soybean meal, imported white fish meal, pork liver powder, water shield powder, brine shrimp powder, castor oil residue, mineral salt mixture, multivitamin premix, earthworm protein extract, conjugated linoleic acid, yeast extract, zinc oxide, coating material, citric acid, and probiotics. This feed not only promotes gastrointestinal digestion and absorption but also reduces the incidence of perch diseases and improves survival rate and fish meat quality. However, no reports have been found on feeds that reduce stress in Songjiang perch fry. Summary of the Invention

[0004] The purpose of this invention is to provide an enzymatically hydrolyzed compound feed that reduces stress in fish fry, thereby reducing stress in Songjiang perch and improving their immune performance.

[0005] This invention provides an enzymatically hydrolyzed compound feed to reduce stress in fish fry. By weight, the enzymatically hydrolyzed compound feed comprises the following raw materials: 20-25 parts shrimp powder, 15-18 parts rice powder, 10-12 parts barley powder, 6-8 parts red bean powder, and 7-12 parts compound fermentation liquid. The compound fermentation liquid includes the following raw materials: Cordyceps militaris, wolfberry, ginseng and Bletilla striata.

[0006] Preferably, the compound fermentation liquid comprises the following raw materials in parts by weight: 10-12 parts of Cordyceps militaris, 1.5-2 parts of Lycium barbarum, 3.5-5.5 parts of ginseng, and 0.5-1 parts of Bletilla striata.

[0007] This invention provides a method for preparing the enzymatically hydrolyzed compound feed described above, comprising the following steps: Cordyceps militaris, wolfberry, ginseng, and Bletilla striata were pulverized separately and then mixed to obtain a mixture; The mixture, cellulase and water were mixed and then subjected to ultrasonic enzymatic hydrolysis and enzyme inactivation in sequence to obtain the enzymatic hydrolysate; The enzymatic hydrolysate, energy pack, and Bacillus licheniformis were mixed and then subjected to anaerobic fermentation to obtain a compound fermentation broth. The shrimp powder, rice powder, barley powder, red bean powder, and compound fermentation liquid are combined to obtain an enzymatically hydrolyzed compound feed. The energy pack contains glucose, potassium dihydrogen phosphate, sodium chloride, and vitamin C.

[0008] Preferably, during ultrasonic enzymatic hydrolysis, the mass ratio of the mixture, cellulase, and water is 1:0.015-0.025:10-12.

[0009] Preferably, the conditions for ultrasonic enzymatic hydrolysis include: a temperature of 65-67℃, a time of 3-4 hours, and a power of 500-650W.

[0010] Preferably, in the energy pack, the mass ratio of glucose, potassium dihydrogen phosphate, sodium chloride and vitamin C is 5-6:2-3:1-1.5:0.5-0.8.

[0011] Preferably, the mass ratio of the enzymatic hydrolysate, energy pack, and Bacillus licheniformis is 100:10-13:2.5-3; The effective viable count of the Bacillus licheniformis is 1×10⁻⁶. 8 -1.5×10 8 cfu / g.

[0012] Preferably, the anaerobic fermentation conditions include a temperature of 40-45℃ and a time of 24-26h.

[0013] This invention provides the application of the enzymatically hydrolyzed compound feed described above in reducing fish fry stress and / or improving fish fry immunity.

[0014] Preferably, the species of fish fry includes: Songjiang perch.

[0015] Beneficial effects: This invention provides an enzymatically hydrolyzed compound feed to reduce stress in fish fry. By weight, the enzymatically hydrolyzed compound feed comprises the following ingredients: 20-25 parts shrimp powder, 15-18 parts rice powder, 10-12 parts barley powder, 6-8 parts red bean powder, and 7-12 parts compound fermentation liquid. The compound fermentation liquid comprises the following ingredients: Cordyceps militaris, wolfberry, ginseng, and Bletilla striata. This invention specifically combines Cordyceps militaris, wolfberry, ginseng, and Bletilla striata, whose active substances can enhance the antioxidant capacity of cells, thereby regulating the immune system. Furthermore, by mixing shrimp powder, rice powder, barley powder, and red bean powder with the above-mentioned compound fermentation liquid, this invention provides sufficient nutrients for the growth of fish fry and assists in the release of the functions of the compound fermentation liquid. Therefore, the enzymatically hydrolyzed compound feed provided by this invention can reduce stress-induced damage and promote the healthy growth of fish fry.

[0016] Based on the aforementioned technical advantages, this invention also provides a method for preparing enzymatically hydrolyzed compound feed. This invention involves the enzymatic hydrolysis of Cordyceps militaris, wolfberry, ginseng, and Bletilla striata, which facilitates the extraction of effective active substances, thereby achieving deep utilization of plant raw materials and synergistically enhancing the bioactivity of each ingredient, providing high-quality feed for the healthy growth of Songjiang perch. Furthermore, the enzymatically hydrolyzed compound feed provided by this invention consists entirely of natural substances, free of chemical components, making it safe and reliable and providing a favorable aquatic environment for the survival of fish fry. Detailed Implementation

[0017] Unless otherwise specified, the raw materials and equipment used in this invention are all purchased conventionally.

[0018] To further illustrate the present invention, the 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.

[0019] Example 1 The steps for preparing an enzymatically hydrolyzed compound feed to reduce stress in fish fry are as follows: (1) By weight, Cordyceps militaris ( Cordyceps militaris 10 servings of goji berries Lycia barbarian 1.5 portions of ginseng ( Panax ginseng 5.5 portions and Bletilla striata ( Bletilla striata (Thunb.) Reichb. f. Weigh one portion for later use.

[0020] (2) Preparation of compound fermentation broth, the process is as follows: Cordyceps militaris, wolfberry, ginseng, and Bletilla striata were washed, dried, and pulverized separately to obtain Cordyceps militaris pulverized material, wolfberry pulverized material, ginseng pulverized material, and Bletilla striata pulverized material (each pulverized material passed through a 100-mesh sieve). The powdered Cordyceps militaris, powdered wolfberry, powdered ginseng, and powdered Bletilla striata are mixed evenly to obtain a mixture; The mixture, cellulase and water were mixed evenly at a mass ratio of 1:0.015:10, and then enzymatically hydrolyzed by sonication at 67℃ and 650W for 4 hours. The enzyme was then inactivated by heating at 90℃ for 1 minute to obtain the enzymatic hydrolysate. The enzymatic hydrolysate, the energy pack (made by mixing glucose, potassium dihydrogen phosphate, sodium chloride, and vitamin C in a mass ratio of 5:2:1:0.5), and Bacillus licheniformis (Latin: Bacillus licheniformis, The effective viable count is 1×10 8 The mixtures (cfu / g) were thoroughly mixed at a mass ratio of 100:10:2.5 and then anaerobic fermented at 40℃ for 24 hours to obtain the compound fermentation broth.

[0021] (3) Mix 20 parts shrimp powder, 15 parts rice powder, 10 parts barley powder, 6 parts red bean powder and 10 parts compound fermentation liquid evenly to obtain enzymatically hydrolyzed compound feed.

[0022] Example 2 The steps for preparing an enzymatically hydrolyzed compound feed to reduce stress in fish fry are as follows: (1) Weigh 10 parts of Cordyceps militaris, 2 parts of wolfberry, 4 parts of ginseng and 1 part of Bletilla striata by mass and set aside.

[0023] (2) Preparation of compound fermentation broth, the process is as follows: Cordyceps militaris, wolfberry, ginseng, and Bletilla striata were washed, dried, and pulverized separately to obtain Cordyceps militaris pulverized material, wolfberry pulverized material, ginseng pulverized material, and Bletilla striata pulverized material (each pulverized material passed through a 100-mesh sieve). The powdered Cordyceps militaris, powdered wolfberry, powdered ginseng, and powdered Bletilla striata are mixed evenly to obtain a mixture; The mixture, cellulase and water were mixed evenly at a mass ratio of 1:0.020:12 and ultrasonically hydrolyzed at 67℃ and 650W for 4 hours. Then, the enzyme was inactivated by heating at 90℃ for 1 minute to obtain the hydrolysate. The enzymatic hydrolysate, the energy pack (made by mixing glucose, potassium dihydrogen phosphate, sodium chloride, and vitamin C in a mass ratio of 5:2:1:0.5), and Bacillus licheniformis (Latin: Bacillus licheniformis, The effective viable count is 1×10 8 The mixtures (cfu / g) were thoroughly mixed at a mass ratio of 100:10:2.5 and then anaerobic fermented at 40℃ for 24 hours to obtain the compound fermentation broth.

[0024] (3) is the same as step (3) in Example 1.

[0025] Comparative Example 1 The steps for preparing an enzymatically hydrolyzed compound feed to reduce stress in fish fry are as follows: (1) Weigh 10 parts of Cordyceps militaris, 1.5 parts of wolfberry, 5.5 parts of ginseng and 3 parts of Bletilla striata by mass and set aside.

[0026] (2) Same as step (2) in Example 1.

[0027] (3) is the same as step (3) in Example 1.

[0028] Comparative Example 2 The steps for preparing an enzymatically hydrolyzed compound feed to reduce stress in fish fry are as follows: (1) Same as step (1) in Example 1.

[0029] (2) Preparation of the complex enzymatic hydrolysate, the process is as follows: Cordyceps militaris, wolfberry, ginseng, and Bletilla striata were washed, dried, and pulverized separately to obtain Cordyceps militaris pulverized material, wolfberry pulverized material, ginseng pulverized material, and Bletilla striata pulverized material (each pulverized material passed through a 100-mesh sieve). The powdered Cordyceps militaris, powdered wolfberry, powdered ginseng, and powdered Bletilla striata are mixed evenly to obtain a mixture; The mixture, cellulase and water were mixed evenly at a mass ratio of 1:0.015:10, and then enzymatically hydrolyzed by sonication at 67℃ and 650W for 4 hours. The enzyme was then inactivated by heating at 90℃ for 1 minute to obtain the enzymatic hydrolysate. (3) Mix 20 parts shrimp powder, 15 parts rice powder, 10 parts barley powder, 6 parts red bean powder and 10 parts compound enzymatic hydrolysate evenly to obtain enzymatically hydrolyzed compound feed.

[0030] Comparative Example 3 The steps for preparing an enzymatically hydrolyzed compound feed to reduce stress in fish fry are as follows: (1) Same as step (1) in Example 1.

[0031] (2) Preparation of compound fermentation broth, the process is as follows: Cordyceps militaris, wolfberry, ginseng, and Bletilla striata were washed, dried, and pulverized separately to obtain Cordyceps militaris pulverized material, wolfberry pulverized material, ginseng pulverized material, and Bletilla striata pulverized material (each pulverized material passed through a 100-mesh sieve). The powdered Cordyceps militaris, powdered wolfberry, powdered ginseng, and powdered Bletilla striata are mixed evenly to obtain a mixture; The mixture, cellulase and water were mixed evenly at a mass ratio of 1:0.015:10, and then enzymatically hydrolyzed by sonication at 67℃ and 650W for 4 hours. The enzyme was then inactivated by heating at 90℃ for 1 minute to obtain the enzymatic hydrolysate. The enzymatic hydrolysate and Bacillus licheniformis (Latin: Bacillus licheniformis, The effective viable count is 1×108 The mixtures (cfu / g) were thoroughly mixed at a mass ratio of 100:2.5, and then anaerobic fermented at 40℃ for 24 hours to obtain the compound fermentation broth.

[0032] (3) is the same as step (3) in Example 1.

[0033] Comparative Example 4 The steps for preparing an enzymatically hydrolyzed compound feed to reduce stress in fish fry are as follows: (1) Same as step (1) in Example 1.

[0034] (2) Preparation of compound fermentation broth, the process is as follows: Cordyceps militaris, wolfberry, ginseng, and Bletilla striata were washed, dried, and pulverized separately to obtain Cordyceps militaris pulverized material, wolfberry pulverized material, ginseng pulverized material, and Bletilla striata pulverized material (each pulverized material passed through a 100-mesh sieve). The powdered Cordyceps militaris, powdered wolfberry, powdered ginseng, and powdered Bletilla striata were mixed evenly to obtain mixture 1; Mixture 1, cellulase, and water are mixed evenly at a mass ratio of 1:0.015:10 to obtain mixture 2; Mixture 2, the energy pack (made by mixing glucose, potassium dihydrogen phosphate, sodium chloride, and vitamin C in a mass ratio of 5:2:1:0.5), and Bacillus licheniformis (Latin: Bacillus licheniformis, The effective viable count is 1×10 8 The mixtures (cfu / g) were thoroughly mixed at a mass ratio of 100:10:2.5 and then anaerobic fermented at 40℃ for 24 hours to obtain the compound fermentation broth.

[0035] (3) is the same as step (3) in Example 1.

[0036] Comparative Example 5 The steps for preparing an enzymatically hydrolyzed compound feed to reduce stress in fish fry are as follows: (1) Same as step (1) in Example 1.

[0037] (2) Preparation of compound fermentation broth, the process is as follows: Cordyceps militaris, wolfberry, ginseng, and Bletilla striata were washed, dried, and pulverized separately to obtain Cordyceps militaris pulverized material, wolfberry pulverized material, ginseng pulverized material, and Bletilla striata pulverized material (each pulverized material passed through a 100-mesh sieve). The powdered Cordyceps militaris, powdered wolfberry, powdered ginseng, and powdered Bletilla striata are mixed evenly to obtain a mixture; The mixture, cellulase and water were mixed evenly at a mass ratio of 1:0.015:10, and then enzymatically hydrolyzed by sonication at 67℃ and 650W for 4 hours. The enzyme was then inactivated by heating at 90℃ for 1 minute to obtain the enzymatic hydrolysate. The enzymatic hydrolysate, the energy pack (made by mixing glucose, potassium dihydrogen phosphate, and vitamin C in a mass ratio of 5:2:0.5), and Bacillus licheniformis (Latin: Bacillus licheniformis, The effective viable count is 1×10 8 The mixtures (cfu / g) were thoroughly mixed at a mass ratio of 100:10:2.5 and then anaerobic fermented at 40℃ for 24 hours to obtain the compound fermentation broth.

[0038] (3) is the same as step (3) in Example 1.

[0039] Application Example 1 Songjiang perch ( Trachyderm banded Fish fry were used as the test subjects.

[0040] Fish fry (3-4 cm in length) were fed in net cages. Each net cage was a treatment, with 10,000 fish fry in each treatment. They were managed in a conventional manner (the experimental period was 150 days). The daily feeding amount of each feed was about 9% of the fish fry's body weight.

[0041] Group 1: Feeding with the enzymatically hydrolyzed compound feed prepared in Example 1; Group 2: Feeding with the enzymatically hydrolyzed compound feed prepared in Example 2; Group 3: Feeding with the enzymatically hydrolyzed compound feed prepared in Comparative Example 1; Group 4: Feeding with the enzymatically hydrolyzed compound feed prepared in Comparative Example 2; Group 5: Feeded with the enzymatically hydrolyzed compound feed prepared in Comparative Example 3; Group 6: Feeded with the enzymatically hydrolyzed compound feed prepared in Comparative Example 4; Group 7: Feeding with the enzymatically hydrolyzed compound feed prepared in Comparative Example 5; Group 8: Feed with commercially available bass feed.

[0042] After the experimental results were obtained, three Songjiang perch were randomly selected from each group, and the average serum lysozyme (LZM) and average serum immunoglobulin (IgM) content in each treatment were determined according to the operation instructions of the ELISA kit. The results are shown in Table 1.

[0043] Table 1. Immunization status of Songjiang perch under different treatments

[0044] As can be seen from the data in Table 1, compared with the comparative example, the fish fry fed with the enzymatically hydrolyzed compound feed prepared according to Examples 1 and 2 of the present invention had higher serum lysozyme and serum immunoglobulin contents. This indicates that the technical solution of the present invention is beneficial to improving the immune performance of fish fry, reducing pathogen infection, and improving stress damage in fish fry.

[0045] Application Example 2 Based on the results of Application Example 1, Group 1 (Example 1), Group 2 (Example 2), Group 7 (Comparative Example 5) and Group 8 (conventionally purchased bass feed) were selected for the following experiment, and the results are shown in Table 2.

[0046] Stress group: 30 healthy Songjiang perch of 25-28cm in length were selected from each of the above groups and placed in a plastic box. The box was tapped once every 10 minutes to startle the fish. After the 10th tapping, the fish were left to stand for 10 minutes. Three Songjiang perch were randomly selected from each treatment, and indicators that can be used to measure oxidative stress in the muscle were measured.

[0047] Non-stress group: Three Songjiang perch were randomly selected from the above groups, and indicators in the muscle that can be used to measure oxidative stress in the body were measured.

[0048] Table 2. Indicators of oxidative stress in fish under different treatments

[0049] As shown in Table 2, after the fish were startled, their superoxide dismutase (SOD) decreased while their glutathione peroxidase (GPO) increased, indicating that the fish experienced oxidative stress. Compared to the non-stress group, the reduction rate of SOD in the fry after feeding them with the enzymatically hydrolyzed compound feed provided by this invention was only 7.06%-9.15%, while in groups 7 and 8, the reduction rate of SOD was as high as 45.6%-53.8%, but the increase in GPO was much higher. Therefore, the technical solution of this invention results in lower oxidative stress in the fry, meaning that the enzymatically hydrolyzed compound feed prepared by this invention helps reduce stress responses in the fry.

[0050] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An enzymatically hydrolyzed compound feed for reducing stress in fish fry, characterized in that, By weight, the enzymatically hydrolyzed compound feed comprises the following ingredients: 20-25 parts shrimp powder, 15-18 parts rice powder, 10-12 parts barley powder, 6-8 parts red bean powder, and 7-12 parts compound fermentation liquid. The compound fermentation liquid includes the following raw materials: Cordyceps militaris, wolfberry, ginseng and Bletilla striata.

2. The enzymatically hydrolyzed compound feed according to claim 1, characterized in that, The compound fermentation liquid comprises the following raw materials by mass: 10-12 parts Cordyceps militaris, 1.5-2 parts wolfberry, 3.5-5.5 parts ginseng, and 0.5-1 parts Bletilla striata.

3. The method for preparing enzymatically hydrolyzed compound feed according to claim 1 or 2, characterized in that, Includes the following steps: Cordyceps militaris, wolfberry, ginseng, and Bletilla striata are pulverized separately and then mixed to obtain a mixture; The mixture, cellulase and water were mixed and then subjected to ultrasonic enzymatic hydrolysis and enzyme inactivation in sequence to obtain the enzymatic hydrolysate; The enzymatic hydrolysate, energy pack, and Bacillus licheniformis were mixed and then subjected to anaerobic fermentation to obtain a compound fermentation broth. The shrimp powder, rice powder, barley powder, red bean powder, and compound fermentation liquid are combined to obtain an enzymatically hydrolyzed compound feed. The energy pack contains glucose, potassium dihydrogen phosphate, sodium chloride, and vitamin C.

4. The preparation method according to claim 3, characterized in that, When performing ultrasonic enzymatic hydrolysis, the mass ratio of the mixture, cellulase and water is 1:0.015-0.025:10-12.

5. The preparation method according to claim 3 or 4, characterized in that, The conditions for ultrasonic enzymatic hydrolysis include: a temperature of 65-67℃, a time of 3-4 hours, and a power of 500-650W.

6. The preparation method according to claim 3, characterized in that, In the energy pack, the mass ratio of glucose, potassium dihydrogen phosphate, sodium chloride, and vitamin C is 5-6:2-3:1-1.5:0.5-0.

8.

7. The preparation method according to claim 3 or 5, characterized in that, The mass ratio of the enzymatic hydrolysate, energy pack, and Bacillus licheniformis is 100:10-13:2.5-3; The effective viable count of the Bacillus licheniformis is 1×10⁻⁶. 8 -1.5×10 8 cfu / g.

8. The preparation method according to claim 3, characterized in that, The conditions for anaerobic fermentation include: a temperature of 40-45℃ and a time of 24-26 hours.

9. The application of the enzymatically hydrolyzed compound feed according to claim 1 or 2 in reducing fish fry stress and / or improving fish fry immunity.

10. The application according to claim 9, characterized in that, The types of fish fry mentioned include: Songjiang perch.

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