High-fat feed as well as preparation method and application thereof

Abstract

The invention discloses a high-fat feed as well as a preparation method and application thereof. Belongs to the technical field of breeding. The high-fat feed comprises a basic high-fat feed, a microbial inoculum, glucurolactone and an enzyme preparation. The high-fat feed provided by the invention solves the adverse effects on fish health caused by inhibition of fish growth, liver toxicity deposition, inflammation induction, ferroptosis and the like due to long-term feeding of the high-fat feed, ensures the fish health, and effectively improves the growth performance of the fish.

Description

Technical Field

[0001] The present invention relates to the technical field of breeding, and in particular to a high-fat feed and a preparation method and application thereof. Background Art

[0002] High-fat diets (HFDs) are widely used in aquaculture. High-fat diets can promote fish growth, improve protein utilization, save protein, and reduce feed costs. However, long-term intake of high-fat diets will have adverse effects on the health of fish. It will not only inhibit the growth of fish, but also induce inflammatory responses, damage immune function, and lead to frequent fish diseases. In addition, high-fat diets can also cause liver toxicity deposition, inhibit antioxidant capacity and GPX4 antioxidant system, induce ROS generation and iron deposition, and ultimately cause ferroptosis, which seriously threatens the sustainable development of large-scale aquaculture. Therefore, there is an urgent need for a high-fat diet that is beneficial to fish health to promote the healthy and sustainable development of the aquaculture industry. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a high-fat feed and a preparation method and application thereof, so as to overcome the problem in the prior art that high-fat feed damages fish health and induces ferroptosis.

[0004] In a first aspect, the present invention provides a high-fat feed comprising a basic high-fat feed, a bacterial agent, glucuronolactone and an enzyme preparation.

[0005] Compared with the prior art, the high-fat feed of the present invention contains glucuronolactone and enzyme preparation, and glucuronolactone can effectively prevent the adverse effects of high-fat feed on fish, improve its growth performance, reduce liver toxicity and inflammatory response caused by high-fat feed, improve the antioxidant capacity of cells, and prevent iron death. The enzyme preparation can promote the decomposition and absorption of glucuronolactone by the organism, accelerate the metabolic process of glucuronolactone in the fish body, and further weaken the adverse effects of high-fat feed on fish health. In addition, the present invention also adds a bacterial agent, which degrades complex carbohydrates in the feed, changes the nutritional components in the feed, provides small molecule beneficial substances, and thus improves the utilization rate of the feed in the organism.

[0006] Furthermore, in the high-fat feed, based on the mass of each kilogram of basic high-fat feed, the amount of bacterial agent added is 10wt‰~20wt‰ of the basic high-fat feed addition, the amount of glucuron-lactone added is 0.1wt‰~0.8wt‰ of the basic high-fat feed addition, and the amount of enzyme preparation added is 0.5wt%~5wt% of the amount of glucuron-lactone added.

[0007] The above technical scheme defines the content of each component in the high-fat feed. Within the above content range, glucuron lactone can better participate in metabolic reactions in the body, improve liver toxicity, enhance the antioxidant capacity of cells, and prevent ferroptosis; the addition of an enzyme preparation with an addition amount of 0.5wt% to 5wt% of glucuron lactone can more efficiently catalyze the metabolic reaction of glucuron lactone in the body; the addition of the above-mentioned specific amount of bacterial agent can fully ferment the high-fat feed, so that the basic high-fat feed, glucuron lactone, and enzyme preparation work together during the fermentation process, making it easier to be digested and absorbed by the organism.

[0008] Furthermore, the enzyme preparation includes any one or more of glucuronolactone dehydrogenase, acetaldehyde dehydrogenase or acetyl-CoA synthetase.

[0009] The three enzyme preparations in the above technical solution can all participate in the metabolic process of glucuron lactone in the body. Glucuron lactone dehydrogenase can catalyze the conversion of glucuron lactone into acetaldehyde, and is one of the key enzymes in the glucuron lactone metabolic pathway. Acetaldehyde dehydrogenase can further oxidize acetaldehyde into acetic acid. Acetyl-CoA synthetase can catalyze the combination of acetaldehyde and coenzyme A to form acetyl-CoA, and is another key enzyme in the glucuron lactone metabolic pathway. These three enzymes improve the decomposition and absorption efficiency of glucuron lactone in the body by promoting the metabolic pathway of glucuron lactone.

[0010] Furthermore, the basic high-fat feed includes the following components, calculated by weight: 740-760 parts of protein substances, 105-120 parts of oil substances, 115-125 parts of starch, 5-8 parts of amino acids and 0-25 parts of nutritional additives.

[0011] In the above technical scheme, the components and proportions of the basic high-fat feed are defined. The reasonable proportions of protein substances, oil substances and starch in the above components provide basic nutrients for the growth and reproduction of fish. At the same time, the basic high-fat feed can improve protein utilization and reduce feed costs, while amino acids and nutritional additives can provide other nutrients for fish, further improving the growth performance and feeding efficiency of fish.

[0012] Further, the protein material includes any one or more of fish meal, chicken meal, soybean meal, cottonseed protein concentrate and gluten;

[0013] Oils include fish oil and soybean oil;

[0014] The starch is tapioca starch;

[0015] Amino acids include methionine and lysine;

[0016] The nutritional additives include any one or more of multivitamin, calcium dihydrogen phosphate, and choline chloride.

[0017] The above technical scheme further defines the specific components of the basic high-fat feed. Fish meal, chicken meal, soybean meal, cottonseed protein concentrate and gluten powder are all rich in high-quality protein; fish oil and soybean oil are rich in a variety of important nutrients. In addition to providing unsaturated fatty acids, they also contain a variety of vitamins, which can enhance immunity and promote fish growth.

[0018] Cassava starch is a high-energy feed ingredient; methionine and lysine are both essential amino acids in fish. Appropriate addition of methionine and lysine helps to achieve a balanced amino acid spectrum in the feed, ensuring that fish can fully utilize the protein in the feed; any one or more of multivitamin, dicalcium phosphate, and choline chloride as nutritional additives can supplement nutrition for fish, promote digestion and absorption of basic feed, and increase fish growth rate.

[0019] Furthermore, the crude fat content in the basic high-fat feed is 14% to 18wt%, the crude protein content is 47.4% to 48.6wt%, the water content is 4.1% to 4.9wt%, and the remainder is carbohydrates.

[0020] The above technical scheme defines the content range of crude fat, crude protein, and water in the basic high-fat feed. It can be seen that the crude protein content in the basic high-fat feed of the present invention is relatively high, which is suitable for fish breeding, but the crude fat with a higher content in the feed improves protein utilization and also causes liver poisoning, induces reactive oxygen species (ROS) generation and iron deposition, and ultimately causes cell ferroptosis, which damages fish health. Although the crude fat content is relatively high in the basic high-fat feed of the present invention, it is combined with glucuron-lactone, enzyme preparations, and bacterial agents, and each raw material synergizes, so that the high crude fat in the feed of the present invention not only does not affect the health of fish, but also improves the growth performance of fish.

[0021] Furthermore, the bacterial agent includes any one or more of Enterococcus faecalis, Bacillus lysinicola, and Lactobacillus buchneri.

[0022] The above technical solution defines the specific components of the microbial agent. Using the microbial agent with the above specific components to ferment high-fat feed can more fully decompose the macromolecular raw materials in the feed that are difficult to absorb into small molecules, further promoting the digestion and absorption of the feed in the body.

[0023] In a second aspect, the present invention provides a method for preparing a high-fat feed, which is applied to the above-mentioned high-fat feed, comprising the following steps:

[0024] The raw materials in the basic high-fat feed are crushed and sieved, and then uniformly mixed with glucuronolactone and the enzyme preparation to obtain a first mixture;

[0025] Adding the bacterial agent to the first mixture and mixing them evenly, mixing the obtained second mixture with water until the water content of the second mixture is 30wt% to 50wt%;

[0026] placing the second mixture into a fermentation tank for fermentation;

[0027] After the fermentation is completed, the fermented high-fat feed is dried and crushed to obtain the high-fat feed.

[0028] Compared with the prior art, the beneficial effects of the high-fat feed preparation method of the present invention are the same as the beneficial effects of the high-fat feed described in the above technical solution, and will not be described in detail here.

[0029] Furthermore, during fermentation, the fermentation temperature is controlled to be 20°C to 35°C, and the fermentation time is 12h to 72h.

[0030] In a third aspect, the present invention provides an application of a high-fat feed, wherein the high-fat feed is applied to fish breeding.

[0031] Compared with the prior art, the beneficial effects of the application of the high-fat feed of the present invention are the same as the beneficial effects of the high-fat feed described in the above technical solution, and will not be described in detail here. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the quadratic curve equation of the addition amount of glucuronolactone in high-fat diet and specific growth rate (SGR).

[0033] Figure 2 The histological analysis results of the liver staining of largemouth bass in Test Example 2.

[0034] Figure 3 The activity of antioxidant-related enzymes and the total antioxidant capacity level in the liver tissue of largemouth black bass in Test Example 4.

[0035] Figure 4 It is the expression levels of the antioxidant-related genes sod1, cat, gpx1, nrf2, and keap1 in largemouth black bass in test example 4.

[0036] Figure 5 It is the quadratic curve equation of the added amount of glucuronolactone in high-fat diet and the total antioxidant capacity (T-AOC).

[0037] Figure 6 It is the quadratic curve equation of the added amount of glucuronolactone in high-fat diet and the activity of glutathione peroxidase (GPX).

[0038] Figure 7 The expression levels of iron metabolism-related genes ferritin 1, ferritin 2, and hepcidin in the largemouth black bass in test example 5 and the iron content in the liver tissue.

[0039] Figure 8 The expression levels of the iron death defense system related genes gpx4, slc7a11, and p53 in the largemouth black bass in test example 6.

[0040] Fig. 9 The expression levels of genes casp3, casp8, casp9, and bcl2 related to the iron death defense system in largemouth black bass in test example 6.

[0041] Fig.10 The expression levels of inflammatory response-related genes nf-kb, tnf-α, il-1β, and il15 in largemouth black bass in test example 7 and the contents of inflammatory cytokines IL-1β and TNF-α in plasma. DETAILED DESCRIPTION

[0042] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0043] In a first aspect, an embodiment of the present invention provides a high-fat feed, comprising a basic high-fat feed, a bacterial agent, glucuronolactone and an enzyme preparation.

[0044] The high-fat feed in the above technical scheme is composed of a basic high-fat feed, a bacterial agent, glucuron-lactone and an enzyme preparation. The crude fat and crude protein contents in the basic high-fat feed are relatively high. A high content of crude protein can provide sufficient nutrients for fish growth, and a high content of crude fat helps to optimize energy supply, improve amino acid utilization, and reduce protein loss. However, long-term continuous intake of a basic high-fat feed can induce an inflammatory response and inhibit growth performance. Therefore, the present application adds glucuron-lactone and an enzyme preparation to the basic high-fat feed, so that fish can take in sufficient glucuron-lactone from the high-fat feed. Glucuron-lactone enters the body to reduce the inflammatory response, improve the antioxidant capacity of the liver, and inhibit the apoptosis and hepatotoxicity of liver cells. The enzyme preparation participates in the metabolic process of glucuron-lactone in the body, can accelerate the decomposition and absorption of glucuron-lactone, and help glucuron-lactone to play an efficient role. At the same time, the bacterial agent in the high-fat feed can decompose the macromolecular substances that are not easily absorbed into small molecules, further improving the utilization rate of the feed.

[0045] Furthermore, based on the above embodiments, in the high-fat feed of the embodiments of the present invention, the amount of the bacterial agent added is 10wt‰~20wt‰ of the basic high-fat feed addition amount, the amount of glucuron-lactone added is 0.1wt‰~0.8wt‰ of the basic high-fat feed addition amount, and the amount of the enzyme preparation added is 0.5wt%~5wt% of the glucuron-lactone added amount per kilogram of the basic high-fat feed mass. For example, the amount of bacterial agent added can be selected as 10wt‰, 12wt‰, 14wt‰, 16wt‰, 18wt‰ or 20wt‰ of the basic high-fat feed addition, preferably 12wt‰~18wt‰ of the basic high-fat feed addition; the amount of glucuron-lactone added can be selected as 0.1wt‰, 0.2wt‰, 0.4wt‰, 0.6wt‰ or 0.8wt‰ of the basic high-fat feed addition, preferably 0.4wt‰~0.6wt‰ of the basic high-fat feed addition; the amount of enzyme preparation added can be selected as 0.5wt%, 1wt%, 2wt%, 3wt%, 4wt% or 5wt% of glucuron-lactone added, preferably 1wt%~4wt% of glucuron-lactone added.

[0046] In the above embodiments, the inventors found that when the above high-fat feed contains 0.1wt‰~0.8wt‰, especially 0.4wt‰~0.6wt‰ of glucuronolide, which is the amount added to the basic high-fat feed, the oxidative stress and inflammation induced by the basic high-fat feed can be significantly reduced, and the growth performance of fish can be significantly improved. The inventors used the quadratic curve equation of the specific growth rate (SGR) and the glucuronolide (Glu) content to determine that the optimal addition amount of glucuronolide in the high-fat feed is 583.29 mg / kg.

[0047] Furthermore, on the basis of the above embodiment, the basic high-fat feed of the embodiment of the present invention includes the following components, calculated by weight of the basic high-fat feed: 740 to 760 parts of protein substances, 105 to 120 parts of oil substances, 115 to 125 parts of starch, 5 to 8 parts of amino acids and 0 to 25 parts of nutritional additives. For example, protein substances can be selected from 740 parts, 745 parts, 750 parts, 755 parts or 760 parts, etc., preferably 745-755 parts; oil substances can be selected from 105 parts, 110 parts, 115 parts or 120 parts, etc., preferably 105-110 parts; starch can be selected from 115 parts, 118 parts, 120 parts, 122 parts or 125 parts, etc., preferably 118-12 parts; amino acids can be selected from 5 parts, 6 parts, 7 parts or 8 parts, etc., preferably 5-6 parts; nutritional additives can be selected from 0 parts, 5 parts, 10 parts, 15 parts, 20 parts or 25 parts, etc., preferably 15-25 parts.

[0048] In the above embodiment, the crude fat content of the basic high-fat feed is 14% to 18wt%, the crude protein content is 47.4% to 48.6wt%, the water content is 4.1% to 4.9wt%, and the remainder is carbohydrates. It should be understood that the above basic high-fat feed may also contain other nutrients beneficial to fish health, such as minerals and vitamins.

[0049] From the content range of crude fat, crude protein and water in the above-mentioned basic high-fat feed, it can be known that the crude protein content in the basic high-fat feed of the present invention is relatively high, which is suitable for fish breeding, but the crude fat with a higher content in the feed improves protein utilization and also causes liver poisoning, induces reactive oxygen species (ROS) generation and iron deposition, and ultimately causes cell iron death, which damages fish health. Although the crude fat content is relatively high in the basic high-fat feed of the present invention, it is combined with glucuron-lactone, enzyme preparations, and bacterial agents, and each raw material works synergistically, so that the high crude fat in the feed of the present invention not only does not affect the health of fish, but also can improve the growth performance of fish.

[0050] In some embodiments, the protein material can be selected from any one or more of fish meal, chicken meal, soybean meal, cottonseed protein concentrate and gluten, wherein the fish meal can be selected from Peruvian fish meal and / or domestic fish meal. For example, the added mass ratio of Peruvian fish meal, domestic fish meal, chicken meal, soybean meal, cottonseed protein concentrate and gluten can be 1:1:(0.75-0.85):(0.7-0.8):(0.7-0.8):(0.4-0.5).

[0051] In some embodiments, the oily substance can be selected from fish oil and soybean oil. For example, the added mass ratio of fish oil and soybean oil can be 1:1.

[0052] In some embodiments, the starch may be selected from tapioca starch.

[0053] In some embodiments, the amino acids may be selected from methionine and lysine, wherein the methionine may be DL-methionine. For example, the mass ratio of methionine to lysine added may be (2-2.5):(3-4).

[0054] In some embodiments, the nutritional additive can be selected from any one or more of multivitamin, calcium dihydrogen phosphate, and choline chloride. The specific types and proportions of the multivitamins and minerals contained in multivitamin are not limited here. For example, per kilogram of high-fat feed, the vitamin mixture in multivitamin can be: riboflavin, 45.0 mg; thiamine, 25.0 mg; vitamin K3, 10.0 mg; inositol, 800.0 mg; pyridoxine hydrochloride, 20.0 mg; vitamin B12, 0.1 mg; calcium pantothenate, 60.0 mg; biotin, 1.2 mg; vitamin A, 32.0 mg; vitamin D, 5.0 mg; niacin, 200.0 mg; folic acid, 20.0 mg; vitamin E, 120.0 mg.

[0055] For example, the mineral mixture in Multivitamin per kilogram of high-fat feed can be: KI, 0.8 mg; sodium fluoride, 2.0 mg; Fe2(SO4)3, 80.0 mg; ZnSO4, 50.0 mg; CoCl2·6H2O, 50.0 mg; CuSO4·5H2O, 10.0 mg; magnesium sulfate, 200.0 mg; sodium chloride, 100.0 mg; Ca(H2PO4)2, 3000.0 mg.

[0056] The basic high-fat feed in the above embodiment has a reasonable ratio of each component, which can provide sufficient and balanced nutrition for fish and ensure the growth performance of fish. At the same time, the above specific types of protein substances, oil substances, starch, amino acids, and nutritional additives have better synergistic performance after being matched, which is more conducive to the growth of fish.

[0057] Further, based on the above embodiment, the enzyme preparation in the embodiment of the present invention includes any one or more of glucuronolactone dehydrogenase, acetaldehyde dehydrogenase or acetyl-CoA synthetase. The above enzyme preparations can all participate in the metabolic reaction of glucuronolactone in the body, and can further improve the detoxification and antioxidant effects of glucuronolactone in the body.

[0058] In some embodiments, the enzyme preparation may be selected from glucuronolactone dehydrogenase.

[0059] In some embodiments, the enzyme preparation can be selected from glucuronolactone dehydrogenase and acetaldehyde dehydrogenase. For example, the mass ratio of glucuronolactone dehydrogenase to acetaldehyde dehydrogenase can be (0.8-1.2): (0.8-1.2).

[0060] In some embodiments, the enzyme preparation can be selected from glucuronolactone dehydrogenase and acetyl-CoA synthetase. For example, the mass ratio of glucuronolactone dehydrogenase and acetyl-CoA synthetase added can be (0.8-1.2): (0.8-1.2).

[0061] In some embodiments, the enzyme preparation can be selected from glucuronolactone dehydrogenase, acetaldehyde dehydrogenase and acetyl-CoA synthetase. For example, the mass ratio of glucuronolactone dehydrogenase, acetaldehyde dehydrogenase and acetyl-CoA synthetase added can be (1.8-2.2): (0.8-1.2): (0.8-1.2).

[0062] Further, based on the above embodiment, the bacterial agent in the embodiment of the present invention includes any one or more of Enterococcus faecalis, Bacillus lysinicus, and Lactobacillus buchneri. The above three bacterial agents make the entire high-fat feed easier to digest and absorb in the fish body, thereby improving the utilization rate of the feed.

[0063] In a second aspect, an embodiment of the present invention provides a method for preparing a high-fat feed, which is applied to the above-mentioned high-fat feed, and comprises the following steps:

[0064] S1. Grind and sieve the raw materials in the basic high-fat feed, and evenly mix them with glucuronolactone and enzyme preparation to obtain a first mixture.

[0065] S2, adding the bacterial agent to the first mixture, mixing evenly, and mixing the obtained second mixture with water until the water content of the second mixture is 30wt% to 50wt%. For example, the water content of the second mixture can be 30wt%, 35wt%, 40wt%, 45wt% or 50wt%, preferably 35wt% to 45wt%. It should be understood that the corresponding water content is selected according to the fermentation bacterial agent, fermentation time and fermentation temperature.

[0066] S3, placing the second mixture into a fermentation tank for fermentation.

[0067] During fermentation, the fermentation temperature is controlled to be 20°C to 35°C, and the fermentation time is 12h to 72h. For example, the fermentation temperature can be 20°C, 25°C, 30°C or 35°C, preferably 25°C to 30°C; the fermentation time can be 12h, 24h, 36h, 48h, 60h or 72h, preferably 24h to 48h. It should be understood that different fermentation temperatures and fermentation times are selected according to different types of bacterial agents.

[0068] S4, after the fermentation is finished, the fermented high-fat feed is dried and crushed to obtain a high-fat feed. The drying method or processing technology is not limited here. For example, a twin-screw extruder can be used to prepare a granular high-fat feed with a particle size of about 3 mm, and the obtained granular high-fat feed is dried at 70° C. and stored at 4° C.

[0069] In a third aspect, an embodiment of the present invention provides an application of a high-fat feed, wherein the high-fat feed is applied to fish breeding.

[0070] Compared with the prior art, the beneficial effects of the application of the high-fat feed of the present invention are the same as the beneficial effects of the high-fat feed described in the above technical solution, and will not be described in detail here.

[0071] In order to better illustrate the technical solution of the present invention, the present invention also provides the following specific examples. It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.

[0072] Example 1

[0073] The present embodiment provides a high-fat feed, and the raw materials of the high-fat feed are as follows: a basic high-fat feed, a bacterial agent, glucuronolide and an enzyme preparation, wherein the basic high-fat feed includes the following components: 156.5g Peruvian fish meal, 156.5g domestic fish meal, 125g chicken meal, 119.8g soybean meal, 120g cottonseed protein concentrate, 70g gluten powder, 53g fish oil, 53g soybean oil, 120.2g cassava starch, 2.1g methionine, 3.6g lysine, 10g multivitamin, 10g calcium dihydrogen phosphate, and 0.3g choline chloride. The added amount of the bacterial agent Enterococcus faecalis is 15g, the added amount of glucuronolide is 0.1g, and the added amount of the enzyme preparation glucuronolide dehydrogenase is 0.5wt% of the mass of glucuronolide.

[0074] The preparation method of the high-fat feed in this embodiment is as follows:

[0075] S1. Grind the raw materials in the above-mentioned basic high-fat feed according to the above-mentioned proportions, pass through an 80-mesh sieve, and evenly mix with glucuronolactone and glucuronolactone dehydrogenase to obtain a first mixture.

[0076] S2. Add Enterococcus faecalis to the first mixture and mix them evenly. Mix the obtained second mixture with water until the water content of the second mixture is 30 wt %.

[0077] S3. Put the second mixture into a fermentation tank for fermentation, and control the fermentation temperature to be 20° C. and the fermentation time to be 12 h.

[0078] S4. After the fermentation is completed, a granular high-fat feed with a particle size of about 3 mm is prepared using a twin-screw extruder, and the obtained granular high-fat feed is dried at 70° C. and stored at 4° C.

[0079] Example 2

[0080] The present embodiment provides a high-fat feed, and the raw materials of the high-fat feed are as follows: a basic high-fat feed, a bacterial agent, glucuronolide and an enzyme preparation, wherein the basic high-fat feed includes the following components: 156.5g Peruvian fish meal, 156.5g domestic fish meal, 125g chicken meal, 119.8g soybean meal, 120g cottonseed protein concentrate, 70g gluten powder, 53g fish oil, 53g soybean oil, 120.2g cassava starch, 2.1g methionine, 3.6g lysine, 10g multivitamin, 10g calcium dihydrogen phosphate, and 0.3g choline chloride. The added amount of the bacterial agent Bacillus lysine is 20g, the added amount of glucuronolide is 0.2g, the added amount of the enzyme preparation glucuronolide dehydrogenase is 0.5wt% of the mass of glucuronolide, and the added amount of acetaldehyde dehydrogenase is 0.5wt% of the mass of glucuronolide.

[0081] The preparation method of the high-fat feed in this embodiment is as follows:

[0082] S1. Grind the raw materials in the above-mentioned basic high-fat feed according to the above-mentioned proportions, pass through an 80-mesh sieve, and evenly mix with glucuron-lactone and acetaldehyde dehydrogenase to obtain a first mixture.

[0083] S2. Adding Bacillus lysinin to the first mixture and mixing them evenly, the obtained second mixture is mixed with water until the water content of the second mixture is 40%.

[0084] S3. Put the second mixture into a fermentation tank for fermentation, and control the fermentation temperature to be 25° C. and the fermentation time to be 24 h.

[0085] S4. After the fermentation is completed, a granular high-fat feed with a particle size of about 3 mm is prepared using a twin-screw extruder, and the obtained granular high-fat feed is dried at 70° C. and stored at 4° C.

[0086] Example 3

[0087] The present embodiment provides a high-fat feed, and the raw materials of the high-fat feed are as follows: a basic high-fat feed, a bacterial agent, glucuronolide and an enzyme preparation, wherein the basic high-fat feed includes the following components: 156.5g Peruvian fish meal, 156.5g domestic fish meal, 125g chicken meal, 119.8g soybean meal, 120g cottonseed protein concentrate, 70g gluten, 53g fish oil, 53g soybean oil, 120.2g cassava starch, 2.1g methionine, 3.6g lysine, 10g multivitamin, 10g calcium dihydrogen phosphate, and 0.3g choline chloride. The added amount of the bacterial agent Lactobacillus buchneri is 17g, the added amount of glucuronolide is 0.4g, the added amount of the enzyme preparation glucuronolide dehydrogenase is 2.5wt% of the glucuronolide content, and the added amount of acetyl-CoA synthetase is 2.5wt% of the glucuronolide content.

[0088] The preparation method of the high-fat feed in this embodiment is as follows:

[0089] S1. Grind the raw materials in the above-mentioned basic high-fat feed according to the above-mentioned proportions, pass through an 80-mesh sieve, and evenly mix with glucuronolactone and acetyl-CoA synthetase to obtain a first mixture.

[0090] S2. Add Lactobacillus buchneri to the first mixture and mix well. Mix the obtained second mixture with water until the water content of the second mixture is 50%.

[0091] S3. Put the second mixture into a fermentation tank for fermentation, and control the fermentation temperature to be 30° C. and the fermentation time to be 36 hours.

[0092] S4. After the fermentation is completed, a granular high-fat feed with a particle size of about 3 mm is prepared using a twin-screw extruder, and the obtained granular high-fat feed is dried at 70° C. and stored at 4° C.

[0093] Example 4

[0094] The present embodiment provides a high-fat feed, and the raw materials of the high-fat feed are as follows: a basic high-fat feed, a bacterial agent, glucuronolide and an enzyme preparation, wherein the basic high-fat feed includes the following components: 156.5g Peruvian fish meal, 156.5g domestic fish meal, 125g chicken meal, 119.8g soybean meal, 120g cottonseed protein concentrate, 70g gluten, 53g fish oil, 53g soybean oil, 120.2g cassava starch, 2.1g methionine, 3.6g lysine, 10g multivitamin, 10g calcium dihydrogen phosphate, 0.3g choline chloride, the bacterial agent Lactobacillus buchneri added in an amount of 12g, the glucuronolide added in an amount of 0.6g, the enzyme preparation glucuronolide dehydrogenase added in an amount of 2wt% of the glucuronolide added, the acetaldehyde dehydrogenase added in an amount of 1wt% of the glucuronolide added, and the acetyl-CoA synthetase added in an amount of 1wt% of the glucuronolide content.

[0095] The preparation method of the high-fat feed in this embodiment is as follows:

[0096] S1. Grind the raw materials in the above-mentioned basic high-fat feed according to the above-mentioned proportions, pass through an 80-mesh sieve, and evenly mix with glucuronolactone and glucuronolactone dehydrogenase to obtain a first mixture.

[0097] S2. Add Lactobacillus buchneri to the first mixture and mix well. Mix the obtained second mixture with water until the water content of the second mixture is 35%.

[0098] S3. Put the second mixture into a fermentation tank for fermentation, and control the fermentation temperature to be 35° C. and the fermentation time to be 48 hours.

[0099] S4. After the fermentation is completed, a granular high-fat feed with a particle size of about 3 mm is prepared using a twin-screw extruder, and the obtained granular high-fat feed is dried at 70° C. and stored at 4° C.

[0100] Example 5

[0101] The present embodiment provides a high-fat feed, and the raw materials of the high-fat feed are as follows: a basic high-fat feed, a bacterial agent, glucuronolide and an enzyme preparation, wherein the basic high-fat feed includes the following components: 156.5g Peruvian fish meal, 156.5g domestic fish meal, 125g chicken meal, 119.8g soybean meal, 120g cottonseed protein concentrate, 70g gluten powder, 53g fish oil, 53g soybean oil, 120.2g cassava starch, 2.1g methionine, 3.6g lysine, 10g multivitamin, 10g calcium dihydrogen phosphate, and 0.3g choline chloride. The added amount of the bacterial agent Bacillus lysine is 10g, the added amount of glucuronolide is 0.8g, and the added amount of the enzyme preparation glucuronolide dehydrogenase is 2% of the added amount of glucuronolide.

[0102] The preparation method of the high-fat feed in this embodiment is as follows:

[0103] S1. Grind the raw materials in the above-mentioned basic high-fat feed according to the above-mentioned proportions, pass through an 80-mesh sieve, and evenly mix with glucuron-lactone and acetaldehyde dehydrogenase to obtain a first mixture.

[0104] S2. Adding Bacillus lysinin to the first mixture and mixing them evenly, the obtained second mixture is mixed with water until the water content of the second mixture is 45%.

[0105] S3. Put the second mixture into a fermentation tank for fermentation, and control the fermentation temperature to be 35° C. and the fermentation time to be 72 h.

[0106] S4. After the fermentation is completed, a granular high-fat feed with a particle size of about 3 mm is prepared using a twin-screw extruder, and the obtained granular high-fat feed is dried at 70° C. and stored at 4° C.

[0107] Comparative Example 1

[0108] The high-fat feed was prepared according to the method of Example 4, except that an equal amount of glucuronolactone in the high-fat feed was replaced by Lactobacillus buchneri.

[0109] Comparative Example 2

[0110] A high-fat feed was prepared according to the method of Example 4, except that the content of glucuronolactone in the high-fat feed was 4 g.

[0111] Comparative Example 3

[0112] The high-fat feed was prepared according to the method of Example 4, except that an equal amount of glucuronolactone dehydrogenase in the high-fat feed was replaced by Lactobacillus buchneri.

[0113] Comparative Example 4

[0114] The high-fat feed was prepared according to the method of Example 4, except that an equal amount of Lactobacillus buchneri in the high-fat feed was replaced with glucuronolactone dehydrogenase.

[0115] Test Case

[0116] Preparation of fish for experiments:

[0117] Largemouth bass from Hubei Zhenghao Aquaculture Company were selected and fed with ordinary feed for one week, twice a day, with the feeding amount controlled at 1% of the fish body weight each time, and the feeding was stopped 24 h before the formal experiment.

[0118] The ingredients of this common feed are as follows:

[0119] Peruvian fish meal 156.5g / kg, domestic fish meal 156.5g / kg, chicken meal 125g / kg, soybean meal 119.8g / kg, cottonseed protein concentrate 120g / kg, gluten 70g / kg, cassava starch 120.2g / kg, fish oil 25g / kg, soybean oil 25g / kg, multivitamin 10g / kg, calcium dihydrogen phosphate 10g / kg, choline chloride 0.3g / kg, lysine 3.6g / kg, DL-methionine 2.1g / kg, cellulose 56g / kg. This feed is a low-fat feed (LFD) with a crude fat content of 10.2%.

[0120] Experimental methods:

[0121] A control group, 5 experimental groups and 4 comparative groups were set up to carry out feeding experiments using the above-mentioned ordinary feed (i.e., low-fat feed), the high-fat feed in Examples 1 to 5 and Comparative Examples 1 to 4, respectively. Three fish tanks were set up in each group, and 23 largemouth black bass were placed in each fish tank. The largemouth black bass were selected from the above-mentioned experimental fish, and the individual size was required to be uniform [initial body weight (45.81±0.15) g], and the feeding experiment was carried out for 8 weeks. The experiment used an indoor temperature-controlled circulating water culture system, and the specific experimental settings are shown in the following table:

[0122]

[0123] Test Example 1

[0124] The plasma biochemical analysis of the largemouth bass in the above test example was performed as follows:

[0125] Three fish were randomly selected from each fish tank for anesthesia, and blood was collected after anesthesia. After blood collection, the blood was centrifuged at 4000 r / min at 4°C for 10 min to obtain plasma.

[0126] The contents of alkaline phosphatase (ALP), blood glucose (GLU) and urea nitrogen (UREA) in the above plasma were detected using a fully automatic biochemical analyzer, which was from Shenzhen Mindray Biopharmaceutical Co., Ltd.

[0127] The activities of glutamate aminotransferase (ALT, C009-2) and aspartate aminotransferase (AST, C010-2) in the above plasma were measured, and the detection kits used were from Nanjing Jiancheng Bioengineering Institute.

[0128] The test results are shown in Table 1.

[0129] Table 1 Plasma biochemical indexes

[0130] Experimental group number ALP(U / L) AST(U / gprot) ALT(U / gprot) GLU (mmol / L) UREA (mmol / L) Control group 1 <![CDATA[79.06±7.21 d ]]> <![CDATA[97.22±5.62 b ]]> <![CDATA[12.29±1.17 de ]]> <![CDATA[9.07±0.68 b ]]> <![CDATA[0.47±0.04 c ]]> Experimental Group 1 <![CDATA[125.29±4.32 ab ]]> <![CDATA[147.25±3.58 a ]]> <![CDATA[22.16±2.24 ab ]]> <![CDATA[9.89±0.17 b ]]> <![CDATA[0.69±0.23 ab ]]> Experimental Group 2 <![CDATA[113.07±7.21 bc ]]> <![CDATA[145.68±11.44 a ]]> <![CDATA[20.61±2.26 a ]]> <![CDATA[10.40±1.24 b ]]> <![CDATA[0.69±0.83 ab ]]> Experimental Group 3 <![CDATA[98.53±4.72 cd ]]> <![CDATA[138.19±14.77 a ]]> <![CDATA[16.55±2.05 cd ]]> <![CDATA[9.67±0.63 b ]]> <![CDATA[0.65±0.07 a ]]> Experimental Group 4 <![CDATA[92.33±5.68 cd ]]> <![CDATA[71.11±5.06 b ]]> <![CDATA[8.35±1.08 e ]]> <![CDATA[10.40±0.99 b ]]> <![CDATA[0.56±0.03 bc ]]> Experimental Group 5 <![CDATA[111.80±3.16 bc ]]> <![CDATA[138.97±11.75 a ]]> <![CDATA[18.34±1.42 bc ]]> <![CDATA[9.37±0.49 b ]]> <![CDATA[0.68±0.05 a ]]> Comparison Group 1 <![CDATA[159.53±10.95 a ]]> <![CDATA[167.64±11.78 a ]]> <![CDATA[23.74±1.98 ab ]]> <![CDATA[16.09±2.83 a ]]> <![CDATA[0.97±0.14 a ]]> Comparison Group 2 <![CDATA[133.07±8.62 ab ]]> <![CDATA[148.15±9.46 a ]]> <![CDATA[23.28±2.84 ab ]]> <![CDATA[9.23±0.61 b ]]> <![CDATA[0.77±0.03 ab ]]> Comparison group 3 <![CDATA[132.41±3.41 ab ]]> <![CDATA[152.23±8.76 a ]]> <![CDATA[23.46±1.82 ab ]]> <![CDATA[9.48±0.32 b ]]> <![CDATA[0.77±0.08 ab ]]> Comparison Group 4 <![CDATA[130.28±7.92 ab ]]> <![CDATA[155.27±5.86 a ]]> <![CDATA[23.72±3.89 ab ]]> <![CDATA[9.77±0.41 b ]]> <![CDATA[0.78±0.32 ab ]]>

[0131] Different letters indicate statistically significant differences (P<0.05).

[0132] From the results in Table 1, it can be seen that in terms of plasma biochemical indicators, the ALP, AST, ALT and UREA levels in the plasma of the largemouth black bass in the experimental groups 1 to 5 were greatly reduced by feeding with the high-fat diet in Examples 1 to 5, while the ALP, AST, ALT and UREA levels in the plasma of the largemouth black bass in the comparative groups 1 to 4 fed with the high-fat diet in Comparative Examples 1 to 4 were greatly increased. In particular, the ALP (U / L) in the plasma of the largemouth black bass in the experimental group fed with the high-fat diet in Example 4 can be reduced to 92.33±5.68 cd , AST can be reduced to 71.11±5.06 b , ALT (U / gprot) can be reduced to 8.35±1.08 e , which was significantly reduced (P<0.05).

[0133] ALP, ALT, AST, GLU and UREA are markers of liver damage and liver function, and the reduction of their levels indicates that the negative impact on liver health is alleviated. Table 1 shows that feeding largemouth black bass with the high-fat diet in Examples 1 to 5 can reduce the negative impact on liver health.

[0134] Test Example 2

[0135] The histological analysis of the largemouth bass in the above test case was performed as follows:

[0136] Three fish were randomly selected from each fish tank and anesthetized. After anesthesia, the liver tissue was dissected out and collected in a centrifuge tube containing RNAlater solution (R0118, Beyotime, China) and stored at -80°C for subsequent analysis.

[0137] The liver tissue was fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin and cut into 5 μm liver sections. The liver sections were dewaxed and rehydrated with alcohol for Prussian blue staining. The sections were immediately transferred to Prussian blue working staining solution, stained at room temperature for 20 minutes, rinsed with distilled water, and then counterstained with nuclear fast red. Finally, the sections were dehydrated with ethanol, washed with xylene, and mounted. The stained sections were observed under an optical microscope.

[0138] The results of histological analysis were Figure 2 As shown, Figure 2 In the figure, LFD is a liver staining diagram of the largemouth black seabass in the control group fed with a low-fat diet for histological analysis; HFD is a liver staining diagram of the largemouth black seabass in the comparison group 1 fed with the high-fat diet in Comparative Example 1 for histological analysis; 200mg / kg, 400mg / kg, 600mg / kg, and 800mg / kg are liver staining diagrams of the largemouth black seabass in the experimental groups 2 to 5 fed with the high-fat diet in Examples 2 to 5, respectively; 4000mg / kg is a liver staining diagram of the largemouth black seabass in the comparison group 2 fed with the high-fat diet in Comparative Example 2 for histological analysis.

[0139] Depend on Figure 2 The results shown show that: compared with the control group LFD figure, the liver tissue of the largemouth black bass in the HFD figure of the comparison group 1 is vacuolated, and the nuclei in the liver tissue are reduced, indicating that the conventional high-fat diet without glucuron lactone is used for feeding, even under the action of bacterial agents and enzymes, it will cause the liver tissue structure of the largemouth black bass to be abnormal. In the experimental groups 2 to 5 fed with high-fat diets of Examples 2 to 5, the number of nuclei in the liver tissue of the largemouth black bass increased, the vacuolar structure in the liver tissue gradually decreased, and the liver tissue structure tended to be normal, indicating that the synergistic effect of adding 200 to 800 mg / kg of glucuron lactone, specific enzymes and bacterial agents to the conventional high-fat diet can weaken the adverse effects of conventional high-fat diets on the liver tissue structure. In addition, the largemouth black bass in the comparison group 2 fed with the high-fat diet of Comparative Example 2 has the most vacuolar structures in the liver tissue, and the liver structure is more abnormal. At the same time, the number of nuclei in the liver tissue is further reduced, indicating that excessive addition of glucuron lactone in the high-fat diet will also have an adverse effect on the liver tissue, further exacerbating the abnormality of the liver tissue structure.

[0140] Test Example 3

[0141] The growth performance of the largemouth black bass in the above test example is tested as follows:

[0142] Two fish were selected from each fish tank, and after measuring their body length and weight, the viscera and liver were dissected out and weighed.

[0143] The specific growth rate (SGR), feed conversion factor (FCR), condition factor (CF), hepatosomatic index (HSI) and visceral index (VSI) were calculated as follows:

[0144] Specific growth rate SGR (% / d) = 100 × (Ln (FBW) - Ln (IBW)) / day;

[0145] Feed coefficient FCR = dry feed intake (g) / weight gain (g);

[0146] Condition coefficient CF (g / cm 3 ) = 100 × (weight) / (height) 3 ;

[0147] Hepatosomatic index HSI (%) = 100 × liver weight / total body weight;

[0148] Visceral index VSI (%) = 100 × visceral weight / total body weight;

[0149] Note: FBW is the final body weight, IBW is the initial body weight.

[0150] SPSS 22.0 was used for data analysis. One-way analysis of variance (ANOVA) combined with Tukey's test (P < 0.05) was used to determine the mean differences of the data. All data are expressed as mean ± standard error of the mean (SEMs). The results are shown in Table 2.

[0151] Table 2 Growth performance of largemouth bass

[0152]

[0153] As shown in Table 2, compared with the control group 1, the control groups 1 to 4 significantly reduced the specific growth rate (SGR) of the largemouth black bass, while the experimental groups 1 to 5 increased the specific growth rate (SGR) of the largemouth black bass compared with the control group 1 to 4, especially the experimental groups 3 and 4, which significantly increased the specific growth rate (SGR) of the largemouth black bass. This shows that the use of the high-fat diet of Examples 1 to 5 to feed largemouth black bass can increase the growth rate of largemouth black bass.

[0154] Compared with the control group 1, the condition coefficient (CF) of the largemouth black bass was reduced in the control groups 1 to 4, while the condition coefficient (CF) of the largemouth black bass was increased in the experimental groups 1 to 5 compared with the control groups 1 to 4, indicating that the use of the high-fat feed of embodiments 1 to 5 to feed largemouth black bass can make the fish fatter and have a better nutritional status.

[0155] Compared with the control group 1, the liver-body index (HSI) of the largemouth black bass was significantly increased in the control groups 1 to 4, while the liver-body index (HSI) of the largemouth black bass was reduced in the experimental groups 1 to 4 compared with the control groups 1 to 4. It is shown that feeding largemouth black bass with the high-fat diet of Examples 1 to 4 can reduce the accumulation of liver fat in fish.

[0156] Figure 1 The horizontal axis is the amount of glucuronolactone added in the high-fat diet, and the vertical axis is the specific growth rate (SGR). Excel was used to fit the quadratic curve equation.

[0157] Test Example 4

[0158] The antioxidant parameters of the largemouth bass in the above test example were tested as follows:

[0159] Three fish were randomly selected from each fish tank and anesthetized. After anesthesia, the liver tissue was dissected out and collected in a centrifuge tube containing RNAlater solution (R0118, Beyotime, China) and stored at -80°C for subsequent analysis.

[0160] Kits were used to detect the activities of superoxide dismutase (SOD, A001-3), catalase (CAT, A007-1), glutathione peroxidase (GPX, A005-1) and total antioxidant capacity (T-AOC, A015-2) in the above liver tissues.

[0161] The results are as follows Figure 3 As shown: Figure 3 In the SOD graph, the 7 columns from left to right represent the activities of superoxide dismutase (SOD) in the liver tissue of largemouth black bass in control group 1, comparison group 1, experimental groups 2 to 5 and comparison group 2 respectively.

[0162] The 7 columns in the CAT graph represent the activity of catalase (CAT) in the liver tissue of largemouth black bass in control group 1, comparative group 1, experimental groups 2 to 5 and comparative group 2 from left to right.

[0163] The 7 columns in the GPx graph represent the activity of glutathione peroxidase (GPx) in the liver tissue of largemouth black bass in control group 1, comparative group 1, experimental groups 2 to 5 and comparative group 2 from left to right.

[0164] The 7 bars in the T-AOC graph represent the total antioxidant capacity (T-AOC) of liver tissue of largemouth black bass in control group 1, comparison group 1, experimental groups 2 to 5 and comparison group 2 from left to right.

[0165] from Figure 3It can be seen that compared with the control group one, the activities of superoxide dismutase (SOD) and catalase (CAT) in the liver tissue of largemouth black bass in comparison group one, comparison group two and experimental group two were significantly decreased, while the activities of superoxide dismutase (SOD) and catalase (CAT) in the liver tissue of largemouth black bass in experimental groups three to five were significantly increased compared with comparison groups one and two.

[0166] Compared with control group one, the activity of glutathione peroxidase (GPx) in the liver tissue of largemouth black bass in control groups one and two was significantly decreased, while the activity of glutathione peroxidase (GPx) in the liver tissue of largemouth black bass in experimental groups two to five was significantly increased compared with that in the control group. Compared with control group two, the activity of glutathione peroxidase (GPx) in the liver tissue of largemouth black bass in experimental groups two to five was increased, among which the activity of glutathione peroxidase (GPx) in the liver tissue of largemouth black bass in the experimental group was significantly increased.

[0167] Compared with control group one, the total antioxidant capacity (T-AOC) of the liver tissue of largemouth black bass in control groups one and two was significantly decreased, while the total antioxidant capacity (T-AOC) of the liver tissue of largemouth black bass in experimental groups two to five was increased compared with that in control groups one and two, among which the total antioxidant capacity (T-AOC) of the liver tissue of largemouth black bass in experimental groups three and four experimental groups was significantly increased.

[0168] Figure 3 The results show that feeding fish with the high-fat diet in Examples 2 to 5 of the present application can improve their antioxidant capacity.

[0169] Figure 5 The horizontal axis is the amount of glucuronolactone added in the high-fat diet, and the vertical axis is the total antioxidant capacity (T-AOC). The quadratic curve equation was fitted using Excel.

[0170] Figure 6 The horizontal axis is the amount of glucuronolactone added in the high-fat diet, and the vertical axis is the activity of glutathione peroxidase (GPX). Excel was used to fit the quadratic curve equation.

[0171] Total RNA was extracted from largemouth black bass by RIzol method and reverse transcribed into cDNA. qRT-PCR was used to detect the expression levels of antioxidant-related genes sod1, cat, gpx1, nrf2, and keap1 in largemouth black bass.

[0172] Test results such as Figure 4 As shown: Figure 4In the expression of nrf2, sod1, cat, and gpx1 are superoxide dismutase 1 gene, catalase gene, and glutathione peroxidase 1 gene, respectively. The expression of nrf2 can activate the expression of downstream antioxidant genes, while keap1 can control the expression of nrf2.

[0173] LFD represents the expression level of related antioxidant genes in one large mouth black sea bass in the control group, HFD represents the expression level of related antioxidant genes in one large mouth black sea bass in the comparison group, and 600 mg / kg Glu represents the expression level of related antioxidant genes in one large mouth black sea bass in the experimental group.

[0174] from Figure 4 It can be seen that compared with the control group 1, the expression levels of sod1 and gpx1 in the largemouth black sea bass in the control group decreased significantly, and the expression level of keap1 increased significantly. Compared with the control group, the expression levels of sod1, gpx1, and nrf2 in the largemouth black sea bass in the experimental group 4 increased significantly, and the expression level of keap1 decreased significantly. This shows that the high-fat diet with added glucuronolactone can improve the antioxidant capacity of largemouth black sea bass.

[0175] Test Example 5

[0176] The iron metabolism in the largemouth black bass in the above test example was tested as follows:

[0177] Total RNA was extracted from largemouth black bass by RIzol method and reverse transcribed into cDNA. qRT-PCR was used to detect the expression levels of iron metabolism-related genes ferritin 1, ferritin 2, and hepcidin in largemouth black bass.

[0178] The liver tissue of the largemouth black sea bass in Test Example 4 was selected, and the iron (A039-2) content of the liver tissue was detected by a kit, and the homogenate protein content in the liver tissue was detected by a BCA protein detection kit (P0012S, Beyotime, China), so as to calculate the iron content per gram of protein in the liver tissue.

[0179] Test results such as Figure 7 As shown: ferritin 1 is the expression of ferritin 1 gene, ferritin 2 is the expression of ferritin 2 gene, hepcidin is the expression of iron regulatory hormone gene, lron is the iron content in liver tissue of largemouth black bass. LFD represents control group 1, HFD represents comparison group 1, and 600 represents experimental group 4.

[0180] from Figure 7It can be seen that compared with the control group 1, the expression levels of ferritin 1 and ferritin 2 in the control group were significantly decreased, and the expression levels of ferritin 1 and ferritin 2 in the experimental group 4 were significantly increased compared with the control group. The expression of ferritin 1 and ferritin 2 can store iron ions and regulate the homeostasis of intracellular iron.

[0181] Compared with the control group 1, the expression of hepcidin in the black sea bass in the control group was significantly increased, and the expression of hepcidin in the black sea bass in the experimental group 4 was significantly decreased compared with the control group. The decrease in hepcidin expression will promote the release and absorption of iron.

[0182] Compared with the control group one, the iron content in the liver tissue of a large black sea bass in the control group was significantly increased, and the iron content in the liver tissue of a large black sea bass in the experimental group four was significantly decreased compared with the control group.

[0183] Figure 7 This indicates that high-fat feed supplemented with glucuronolactone can improve iron deposition caused by iron metabolism disorders in fish.

[0184] Test Example 6

[0185] The genes related to the iron death defense system of the largemouth black bass in the above test example were detected as follows:

[0186] Total RNA was extracted from largemouth black bass by RIzol method and reverse transcribed into cDNA. qRT-PCR was used to detect the expression levels of ferroptosis defense system-related genes gpx4, slc7a11, p53, casp3, casp8, casp9, and bcl2 in largemouth black bass.

[0187] Test results such as Figure 8 , Fig. 9 Shown: LFD represents control group 1, HFD represents comparative group 1, and 600 mg / kg Glu represents experimental group 4.

[0188] Depend on Figure 8 It can be seen that compared with the control group one, the expression levels of gpx4 and p53 in the large mouth black sea bass in the control group were significantly increased, and the expression level of slc7a11 was significantly decreased; compared with the control group, the expression levels of gpx4 and p53 in the large mouth black sea bass in the experimental group four were significantly decreased, and the expression level of slc7a11 was significantly increased.

[0189] gpx4 is the glutathione peroxidase 4 gene, and the expression of gpx4 is activated during ferroptosis. Downregulation of slc7a11 can lead to intracellular glutathione depletion, a reduction in gpx4 antioxidant reaction substrates, and then lead to lipid peroxide accumulation, ultimately leading to ferroptosis. The expression of slc7a11 is affected by p53, and the expression of p53 will reduce the level of slc7a11, ultimately inducing ferroptosis. Therefore, Figure 8 This indicates that high-fat diet supplemented with glucuronolactone can reduce cellular iron death.

[0190] Depend on Fig. 9 It can be seen that compared with the control group 1, the expression of the pro-apoptotic genes casp3, casp8, and casp9 in the control group of black bass increased significantly, and the expression of the anti-apoptotic gene bcl2 decreased significantly; compared with the control group, the expression of the pro-apoptotic genes casp3, casp8, and casp9 in the experimental group of black bass decreased significantly, and the expression of the anti-apoptotic gene bcl2 increased significantly. This shows that the high-fat diet with added glucuronolactone can reduce cell apoptosis.

[0191] Test Example 7

[0192] The genes related to the inflammatory response in the largemouth black bass in the above test example and the inflammatory cytokines in the plasma were detected as follows:

[0193] Total RNA was extracted from largemouth black bass by RIzol method and reverse transcribed into cDNA. qRT-PCR was used to detect the expression levels of inflammatory response-related genes nf-kb, tnf-α, il-1β, and il15 in largemouth black bass.

[0194] The plasma of largemouth black bass in Test Example 1 was taken, and the levels of IL-1β (KP-9845) and TNF-α (KP-9678) in the plasma were determined according to the ELISA kit recommended by Wuhan Keep Biotechnology Co., Ltd.

[0195] Test results such as Fig.10 Shown: LFD represents control group 1, HFD represents comparative group 1, and 600 mg / kg Glu represents experimental group 4.

[0196] Depend on Fig.10 As shown in (a), compared with the control group one, the expression levels of nf-kb, tnf-α, il-1β, and il15 in the large mouth black sea bass in the comparison group were greatly increased, while the expression levels of nf-kb, tnf-α, il-1β, and il15 in the large mouth black sea bass in the experimental group four were significantly decreased compared with the control group.

[0197] Depend on Fig.10As shown in (b), compared with the control group one, the levels of inflammatory cytokines IL-1β and TNF-α in the plasma of largemouth black sea bass in the comparison group were significantly increased, while the levels of inflammatory cytokines IL-1β and TNF-α in the plasma of largemouth black sea bass in the experimental group four were significantly decreased compared with the control group.

[0198] Fig.10 This indicates that feeding largemouth black bass with the high-fat feed in Comparative Example 1 will induce an inflammatory response in the body of the largemouth black bass, while adding glucuronolactone can alleviate the inflammatory response of the largemouth black bass.

[0199] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A high-fat feed, characterized in that: Including basic high-fat feed, bacterial agents, glucuron-lactone and enzyme preparations.

2. The high-fat feed according to claim 1, characterized in that In the high-fat feed, based on the mass of each kilogram of basic high-fat feed, the added amount of the bacterial agent is 10wt‰ to 20wt‰ of the basic high-fat feed, the added amount of the glucuron-lactone is 0.1wt‰ to 0.8wt‰ of the basic high-fat feed, and the added amount of the enzyme preparation is 0.5wt% to 5wt% of the added amount of the glucuron-lactone.

3. The high-fat feed according to claim 1 or 2, characterized in that: The enzyme preparation comprises any one or more of glucuronolactone dehydrogenase, acetaldehyde dehydrogenase or acetyl-CoA synthetase.

4. The high-fat feed according to claim 1, characterized in that The basic high-fat feed includes the following components, calculated by weight: 740-760 parts of protein substances, 105-120 parts of oil substances, 115-125 parts of starch, 5-8 parts of amino acids and 0-25 parts of nutritional additives.

5. The high-fat feed according to claim 4, characterized in that The protein material includes any one or more of fish meal, chicken meal, soybean meal, cottonseed protein concentrate and gluten powder; The oil substances include fish oil and soybean oil; The starch is tapioca starch; The amino acids include methionine and lysine; The nutritional additives include any one or more of multivitamin, calcium dihydrogen phosphate, and choline chloride.

6. The high-fat feed according to claim 1, characterized in that The basic high-fat feed has a crude fat content of 14% to 18wt%, a crude protein content of 47.4% to 48.6wt%, a water content of 4.1% to 4.9wt%, and the remainder is carbohydrates.

7. The high-fat feed according to claim 1, characterized in that: The bacterial agent includes any one or more of Enterococcus faecalis, Bacillus lysine, and Lactobacillus buchneri.

8. A method for preparing a high-fat feed, applied to the high-fat feed according to any one of claims 1 to 7, characterized in that: The steps include: The raw materials in the basic high-fat feed are crushed and sieved, and then uniformly mixed with glucuronolactone and an enzyme preparation to obtain a first mixture; Adding the bacterial agent to the first mixture and mixing them evenly, and mixing the obtained second mixture with water until the water content of the second mixture is 30wt% to 50wt%; placing the second mixture into a fermentation tank for fermentation; After the fermentation is completed, the fermented high-fat feed is dried and crushed to obtain the high-fat feed.

9. The preparation method according to claim 8, characterized in that: During fermentation, the fermentation temperature is controlled at 20°C ~ 35°C and the fermentation time is 12h ~ 72h.

10. An application of a high-fat feed, characterized in that: The high-fat feed according to any one of claims 1 to 7 is used for feeding fish.

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