Feed additive for preventing fatty liver of lateolabrax japonicus

Abstract

The invention relates to the technical field of preparation of feed additives, in particular to a feed additive for preventing fatty liver of lateolabrax japonicus. The composition comprises the following components in percentage by mass: 35-45% of yeast hydrolysate, 15-25% of curcumin, 15-25% of leonurine and the balance of a carrier, the carrier is defatted rice bran and / or rice hull powder; leonurine is one of active alkaloids in motherwort and has the functions of resisting atherosclerosis, resisting oxidation, protecting blood-brain barriers and the like, and traditional Chinese medicines have the advantages of relieving the liver damage degree, slowing down disease progression and removing toxic substances in the aspect of treating the injured liver diseases; the leonurine is selected as the perch feed for the first time and can effectively protect the liver and repair the liver injury, so that the occurrence of the micropterus salmoides fatty liver is prevented.

Description

Technical Field

[0001] The present invention relates to the technical field of feed additives, and particularly relates to a feed additive for preventing fatty liver in Micropterus salmonides Background Art

[0002] Due to problems such as overly high nutritional indicators, poor freshness, or mildew of feed products in the market, and the abuse of antibiotics and pesticides, fish hepatobiliary syndrome occurs frequently. The prevention of fish hepatobiliary syndrome is a difficult problem that urgently needs to be overcome in aquaculture at present. For feeding fish such as carp, grass carp, and crucian carp, the disease of hepatobiliary syndrome often breaks out from July to September every year. Due to many pathogenic factors, most farmers only treat simple fatty liver, resulting in poor treatment effect and high cost.

[0003] The causes of this disease are very complex and are mainly caused by aquaculture water quality conditions, drug intake, and nutritional imbalance of feed. The reasons for the nutritional imbalance of feed are mainly the following aspects: (1) The metabolism of fat in fish. Because fish often cannot fully decompose fatty acids with relatively high unsaturation, these fatty acids with relatively high unsaturation are retained in the body tissues and undergo peroxidation in the body. At the same time, organelles responsible for decomposing highly unsaturated fatty acids, such as peroxisomes, will also generate more free radicals when activated to decompose these fatty acids with relatively high unsaturation, thus increasing the peroxidation pressure in the internal environment. (2) Excessive plant-based raw materials in the feed, precisely, cellulose (or non-starch polysaccharides) in plant-based feed. Its mechanism is as follows: One is that generally, carnivorous fish have low ability to utilize sugar and are regarded as having congenital "diabetic constitution", especially more obvious in Micropterus salmonides. After fish ingest feed sugar, a part of it is stored in the form of glycogen. The main parts for storing glycogen are the liver and muscle. Due to the large accumulation of glycogen, the hepatopancreas of fish will increase. In some fish, the liver function is damaged while the hepatopancreas increases. The other is that in the fish intestine, cellulose and non-starch polysaccharides hinder the reabsorption of bile acids through physical entrainment and other effects. The decrease in the reabsorption level of bile acids not only induces an increase in cholesterol synthesis in the liver, but also decreases the cholesterol levels in the blood and muscle. The cholesterol accumulated in the liver is used to synthesize bile acids. Due to the excessive secretion of bile acids, it overflows when it cannot be stored in the gallbladder, resulting in green liver. The long-term overload secretion of the liver leads to functional failure, and then it cannot secrete enough bile acids, making the bile color abnormal and at the same time causing fat absorption disorders.

[0004] Due to reasons such as feed resources and costs, the amount of plant-based raw materials used in aquatic feeds in China is relatively high. However, the research on the nutritional characteristics of plant-based feeds mainly focuses on the balance of amino acids and fatty acids, and little attention is paid to the cellulose and non-starch polysaccharides, which account for about 30% of the total content. Plant-based feed resources such as miscellaneous meals are abundant in China, and it is inevitable to use a large amount of plant-based raw materials in aquatic feeds. Therefore, there is an urgent need for a safe and effective method to prevent fatty liver in Micropterus salmonides Summary of the Invention

[0005] To solve the above problems, the present invention provides a feed additive for preventing fatty liver in Micropterus salmonides, which comprises, by mass percentage, 35-45% of yeast hydrolyzate, 15-25% of curcumin, 15-25% of leonurine, and the balance being a carrier;

[0006] The carrier is defatted rice bran and / or rice husk powder.

[0007] Further, the moisture content of the feed additive is controlled below 10-12%, and then it is pulverized and sieved through a 80-mesh sieve;

[0008] Note: Controlling the moisture content can inhibit the generation of mycotoxins, protect the function of liver cells, and reduce toxin-induced lipid metabolism disorders; pulverizing and sieving can improve the nutritional uniformity and digestibility, and reduce the burden of liver lipid synthesis and detoxification.

[0009] Further, the curcumin is encapsulated in microcapsules, and the preparation method of the microcapsules encapsulating curcumin is as follows:

[0010] S1. The pretreated curcumin, probiotics and porous starch with a pore size of 1-5 μm are cultured according to a mass ratio of 1:0.1-0.2:0.3-0.5, and an organic solvent is added to prepare a 2-4 wt% composite suspension, which is the core material solution;

[0011] S2. Modified sodium alginate and deoxycholic acid are mixed according to a mass ratio of 5-10:1 to obtain a wall material, and then added to deionized water according to 1 g:50-100 mL, and ultrasonically dispersed at an ultrasonic frequency of 300-500 W for 8-12 min until uniform, and the wall material solution is obtained after sterilization;

[0012] S3, the core material solution and the wall material solution are mixed evenly according to a volume ratio of 1:3-5, added to a 2-3% CaCl2 solution by extrusion, and allowed to stand to form a gel, and the gel is transferred to a phosphate buffer solution with a pH of 7.2-7.6, containing 0.3mL lactoferrin and 0.5mL hyaluronic acid according to a volume ratio of gel to phosphate buffer of 1:5-8, and stirred at a low speed for 25-30min to form a surface modification layer; after washing with deionized water for 3-5 times, the gel is immersed in a chitosan solution containing 0.1-0.5% oligofructose for secondary coating according to a volume ratio of gel to chitosan solution of 1:3-5, and allowed to stand for 15-20min; finally, low-temperature drying is performed at 30-40°C to a water content of ≤5%, and functionalized microcapsules with a diameter of 10-50μm are obtained, i.e., microcapsules encapsulating curcumin;

[0013] Description: Microencapsulation technology is used to solve the problem of poor water solubility and low absorption rate of curcumin, so that it can be slowly released in the intestine and continuously play an antioxidant role. The curcumin with anti-inflammatory and antioxidant effects in the core material and the probiotics with the effect of regulating intestinal flora are loaded through porous starch to form a stable complex, which improves the retention rate of curcumin in the digestive tract, prolongs the sustained release time, and protects the probiotics from being damaged by gastric acid.

[0014] Deoxycholic acid, as a wall material component, can promote bile acid circulation in the intestine, activate farnesoid X receptor (FXR), inhibit the expression of liver triglyceride synthase (such as DGAT2), and further reduce the incidence of fatty liver. Curcumin is delivered to hepatocytes through liposome carriers to activate the PPARα pathway, inhibit the expression of fatty acid synthase (FAS), and reduce the abnormal accumulation of triglycerides in the liver. Adding oligofructose during the embedding process can indirectly alleviate the progression of fatty liver by promoting the proliferation of intestinal probiotics (such as bifidobacteria) and reducing the transfer of endotoxin (LPS) to the liver. The combination of oligofructose and probiotics can reduce the content of liver triglyceride (TG) by 25% to 30%. The gel is immersed in a buffer containing lactoferrin and hyaluronic acid to form a targeted modification layer through electrostatic adsorption, thereby enhancing the accumulation capacity of microcapsules in liver tissue. Lactoferrin can bind to the surface receptors of hepatocytes, and hyaluronic acid can mediate targeting through CD44 receptors, synergistically improving the efficiency of active ingredients in regulating liver fat metabolism.

[0015] Furthermore, the probiotics in step S1 are any one of Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus acidophilus, and Bifidobacterium lactis, or a mixture of the same weight ratio; the number of viable probiotics is 1.0×10 6 ~1.0×10 8CFU / mL; the organic solvent is composed of Pluronic F-68 with a concentration of 0.01-0.1%, ethanol with a volume concentration of 18-22%, and sodium dextran sulfate with a concentration of 0.1-0.5 mg / mL in an equal volume ratio;

[0016] Note: Sodium dextran sulfate enhances the electrostatic repulsion by increasing the negative charge density, preventing the physical adhesion between porous starch and probiotics. Acetone, as a good solvent for curcumin, can promote its uniform dispersion in the pores of porous starch, reducing the aggregation of free curcumin; Pluronic F-68: By reducing the surface tension of the solution, reducing the van der Waals force and hydrophobic interaction between particles, inhibiting the aggregation of curcumin and probiotics, and having less inhibition on the activity of probiotics; Fructooligosaccharide indirectly alleviates the progression of fatty liver through multiple pathways such as reducing endotoxin transfer, enhancing lipid metabolism, and inhibiting inflammation by synergistically promoting the proliferation of probiotics such as Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus, and Bifidobacterium lactis.

[0017] Furthermore, the pretreatment method of curcumin is as follows: Mix turmeric rhizome powder with an aqueous solution of 1-octyl-3-methylimidazolium bromide at a liquid-solid ratio of 25-30 mL:1 g and a concentration of 0.3-0.5 mol / L, and ultrasonically treat it at 200-250 W for 1-1.5 h to obtain a curcumin extract; Dissolve the curcumin extract in medium-chain triglycerides to prepare a curcumin oil solution with a concentration of 3-5 mg / mL, and homogenize it 2-4 times at 60-80 MPa to refine the particle size to 100-200 nm, thus obtaining the pretreated curcumin;

[0018] Note: The above method can make the extraction rate of curcumin compounds reach more than 90%. After pretreatment, the DPPH free radical scavenging rate of curcumin is increased to more than 75-80%, the content of liver triglyceride (TG) is reduced by 35%, and the MDA level is decreased by 40%; The DPPH scavenging rate is increased to more than 75-80%, indicating that curcumin can efficiently neutralize reactive oxygen species (ROS) in the liver, reduce lipid peroxidation reactions, and protect the integrity of the liver cell membrane; MDA (malondialdehyde), as the end product of lipid peroxidation, a 40% reduction directly reflects the alleviation of oxidative stress damage and avoids the disorder of hepatocyte mitochondrial function; The content of liver triglyceride (TG) is reduced by 35%, by inhibiting the activity of fatty acid synthase (FAS) and activating peroxisome proliferator-activated receptor (PPARα) to promote fatty acid β-oxidation, reducing the abnormal accumulation of lipids in hepatocytes; thereby enhancing the ability of the feed additive to prevent the occurrence of liver diseases.

[0019] Furthermore, the preparation method of modified sodium alginate:

[0020] Deionized water and sodium alginate are mixed and stirred at a mass ratio of 22 to 25:1, and the pH is adjusted to 7 to 8 using a 0.1 to 0.2 mol / L sodium hydroxide solution, and then 1 to 1.2% of nano-montmorillonite is added to the sodium alginate, the pH is re-measured and adjusted to 7 to 8 again, the temperature is increased by 40 to 50°C, and the product is treated at an ultrasonic frequency of 180 to 200 W for 8 to 15 minutes to obtain a product A; 0.8 to 1% of the mass of the product A and 0.5 to 0.7% of the mass of the product A are successively added to crushed oat shells with a particle size of less than 1 μm and vitamin E, respectively, and the product B is obtained after homogeneous mixing, and then dried at 40 to 50°C to a water content of ≤5% to obtain a modified sodium alginate.

[0021] Description: Nano-montmorillonite is added to the wall material to absorb free Ca by using its layered structure 2+ It also blocks oxygen, further reducing the oxidative degradation rate of curcumin; adding vitamin E to product A can inhibit lipid peroxidation through synergistic effects and reduce oxidative damage to liver cells; crushed oat hulls with a particle size of less than 1μm can further enhance their hydrogen bonding with sodium alginate and reduce the risk of leakage.

[0022] Furthermore, the homogenous mixing method is to mix the product A with the crushed oat hulls at 200-400 rpm for 4-6 minutes, then increase the speed to 600-800 rpm for mixing for 3-5 minutes, add vitamin E and increase the speed to 1100-1300 rpm for further mixing for 5-7 minutes, and finally alternately switch the speeds of 600-800 rpm and 1100-1300 rpm for 2-3 minutes each time, repeat the switching 2-3 times to complete the homogenous mixing;

[0023] Description: Preliminarily mix product A with crushed oat hulls, and stir at low speed to make the components contact initially; medium speed can form shear force to break up particle agglomeration and promote the dispersion of nano-montmorillonite and oat hulls; high-speed homogenization can use high shear force to achieve micron-level mixing to ensure uniform distribution of vitamin E; alternating between high and medium speeds can reduce the destruction of the gel structure caused by local temperature increase and further improve the mixing effect.

[0024] Furthermore, during the homogenizing and mixing process, the total homogenizing time is controlled at 20 to 25 minutes, and the temperature during the homogenizing process is maintained at 30 to 40°C;

[0025] Note: Too long a total homogenization time may cause phase separation; temperature control can avoid thermal degradation of sodium alginate.

[0026] Further, it is added to complete compound feed at a ratio of 1000-1500 g / ton;

[0027] Note: Mixing in proportion can avoid excessive dosage causing metabolic burden or cost waste.

[0028] Compared with the existing technologies, the beneficial effects of the present invention are as follows:

[0029] (1) SCM is one of the active alkaloids in Leonurus japonicus, which has functions such as anti-atherosclerosis, antioxidant, and protection of the blood-brain barrier. Traditional Chinese medicine has advantages in the treatment of traumatic liver diseases, including reducing the degree of liver damage, slowing down the progression of the disease, and removing toxic substances; curcumin has the potential ability to regulate liver drug-metabolizing enzymes, so it can prevent the liver toxicity induced by AFB1, and even play a role in preventing the liver toxicity induced by AFB1. In addition, curcumin can also inhibit lipid synthesis, improve lipid deposition, and thus maintain lipid homeostasis; functional small peptides, short-chain fatty acids, etc. in yeast hydrolysate also play a significant role in the repair of the liver and intestinal tissue structure damage in fish. The present invention first proposes that selecting SCM as the feed for bass can effectively protect the liver and repair liver damage, thereby preventing the occurrence of fatty liver in Micropterus salmonides.

[0030] (2) The present invention uses the microencapsulation technology for curcumin to solve the problems of poor water solubility and low absorption rate of curcumin, so that it can be slowly released in the intestine and continuously play an antioxidant role. By loading curcumin with anti-inflammatory and antioxidant effects and probiotics with the effect of regulating the intestinal flora on porous starch to form the core material, the retention rate of curcumin in the digestive tract is improved, the slow-release time is prolonged, and at the same time, the probiotics are protected from gastric acid damage. Using modified sodium alginate and deoxycholic acid as wall material components can promote the bile acid cycle in the intestine, activate the farnesoid X receptor, inhibit the expression of liver triglyceride synthase, and further reduce the incidence of fatty liver; adding fructooligosaccharide during the encapsulation process can indirectly relieve the progression of fatty liver by promoting the proliferation of intestinal probiotics and reducing the transfer of endotoxin to the liver, and effectively prevent the occurrence of fatty liver in Micropterus salmonides.

[0031] (3) The present invention first pretreats curcumin, so that the extraction rate of curcumin reaches more than 90% and then better acts on the reactive oxygen species in the liver, reduces lipid peroxidation reaction, and protects the integrity of the liver cell membrane. Adding nano-montmorillonite to the wall material can utilize its layered structure to adsorb free Ca 2+ and block oxygen, further reducing the oxidation and degradation rate of curcumin; adding vitamin E to product A can inhibit lipid peroxidation through synergistic effects and reduce the oxidative damage of liver cells; thus effectively preventing the occurrence of fatty liver in Micropterus salmonides. Specific embodiments

[0032] To further elaborate the methods and achieved effects adopted by the present invention, the technical solutions of the present invention will be clearly and completely described below in combination with experiments.

[0033] Example 1: A feed additive for preventing fatty liver in Micropterus salmonides, by mass percentage, includes 40% of yeast hydrolyzate, 20% of curcumin, 20% of leonurine, and the balance of carrier; the carrier is defatted rice bran;

[0034] Control the moisture content of the feed additive at 11%, then crush and pass through an 80-mesh sieve; add it to the complete formulated feed at a ratio of 1250 g / ton.

[0035] Example 2: Different from Example 1, a feed additive for preventing fatty liver in Micropterus salmonides, by mass percentage, includes 35% of yeast hydrolyzate, 15% of curcumin, 15% of leonurine, and the balance of carrier.

[0036] Example 3: Different from Example 1, a feed additive for preventing fatty liver in Micropterus salmonides, by mass percentage, includes 45% of yeast hydrolyzate, 25% of curcumin, 25% of leonurine, and the balance of carrier.

[0037] Example 4: Different from Example 1, curcumin is embedded in microcapsules, and the preparation method of the microcapsules embedding curcumin is as follows:

[0038] S1. Mix the pretreated curcumin, probiotic bacteria and porous starch with a pore size of 1 - 5 μm in a mass ratio of 1:0.15:0.4, add an organic solvent to prepare a 3 wt% composite suspension, which is the core material solution;

[0039] The probiotic bacteria in S1 are Bifidobacterium lactis; the viable count of the probiotic bacteria is 1.0×10 7 CFU / mL; the organic solvent is composed of Pluronic F-68 with a concentration of 0.05%, ethanol with a volume concentration of 20% and sodium dextran sulfate with a concentration of 0.3 mg / mL in an equal volume ratio; among them, sodium dextran sulfate DSS comes from Hubei Jiufenglong with a purity as high as 99%;

[0040] The pretreatment method of curcumin is as follows: Mix turmeric rhizome powder with a liquid-solid ratio of 28 mL:1 g and an aqueous solution of 1-octyl-3-methylimidazolium bromide at 0.4 mol / L, perform ultrasonic treatment at 225 W for 1.3 h to obtain a curcumin extract; dissolve the curcumin extract in medium-chain triglycerides to prepare a curcumin oil solution with a concentration of 4 mg / mL, and perform homogenization treatment 3 times at 70 MPa to refine the particle size to 100 - 200 nm, thus obtaining the pretreated curcumin;

[0041] S2. Mix modified sodium alginate and deoxycholic acid in a mass ratio of 8:1 to obtain the wall material, then add it to deionized water at a ratio of 1 g:75 mL, perform ultrasonic dispersion at an ultrasonic frequency of 400 W for 10 min until uniform, and obtain the wall material solution after sterilization;

[0042] Preparation method of modified sodium alginate:

[0043] Deionized water and sodium alginate are mixed in a mass ratio of 23:1 and stirred evenly, a 0.15 mol / L sodium hydroxide solution is used to adjust the pH to 7.5, and then 1.1% of nano-montmorillonite is added to the sodium alginate, the pH is re-measured and adjusted to 7.5 again, the temperature is raised to 45°C and treated at an ultrasonic frequency of 190W for 12 minutes to obtain product A; 0.9% of the mass of the above product A and 0.6% of the mass of the above product A are added in sequence, and product B is obtained after homogeneous mixing, and then dried at 45°C to a water content of 5% to obtain modified sodium alginate;

[0044] The homogenous mixing method is to mix the product A with the crushed oat hull at 300 rpm for 5 minutes, then increase the speed to 700 rpm for 4 minutes, add vitamin E and increase the speed to 1200 rpm for 6 minutes, and finally alternately switch the speeds of 700 rpm and 1200 rpm for 2.5 minutes each time, repeat the switch 3 times, and complete the homogenous mixing; during the homogenous mixing process, the total homogenization time is controlled at 23 minutes, and the temperature is maintained at 35°C during the homogenization process;

[0045] S3. The core material solution and the wall material solution are mixed evenly at a volume ratio of 1:4, added to a 2.5% CaCl2 solution by extrusion, and allowed to stand to form a gel; the gel is transferred to a sodium phosphate buffer having a pH of 7.4 and containing 0.3 mL lactoferrin and 0.5 mL hyaluronic acid at a volume ratio of 1:6 to the phosphate buffer, and stirred at a low speed for 28 minutes to form a surface modification layer; after washing with deionized water for 3 to 5 times, the gel is immersed in a chitosan solution containing 0.3% oligofructose for secondary coating at a volume ratio of 1:4 to the chitosan solution, and allowed to stand for 18 minutes; finally, the gel is dried at 35°C to a water content of 5% to obtain functionalized microcapsules with a diameter of 10 to 50 μm, i.e., microcapsules encapsulating curcumin.

[0046] Example 5: The difference from Example 4 is that S1, the pretreated curcumin, probiotics and porous starch with a pore size of 1 to 5 μm are mixed in a mass ratio of 1:0.1:0.3, and an organic solvent is added to prepare a 2 wt% composite suspension, which is the core material solution.

[0047] Example 6: Different from Example 4, S1, the pretreated curcumin, probiotics and porous starch with a pore size of 1 to 5 μm are mixed in a mass ratio of 1:0.2:0.5, and an organic solvent is added to prepare a 4 wt% composite suspension, which is the core material solution.

[0048] Example 7: Different from Example 4, the organic solvent is composed of Pluronic F-68 with a concentration of 0.01%, ethanol with a volume concentration of 18%, and sodium dextran sulfate with a concentration of 0.1 mg / mL in an equal volume ratio.

[0049] Example 8: Different from Example 4, the organic solvent is composed of Pluronic F-68 with a concentration of 0.1%, ethanol with a volume concentration of 22%, and sodium dextran sulfate with a concentration of 0.5 mg / mL in an equal volume ratio.

[0050] Example 9: Different from Example 4, the pretreatment method of curcumin is as follows: Mix turmeric rhizome powder with an aqueous solution of 1-octyl-3-methylimidazolium bromide at a liquid-solid ratio of 25 mL:1 g and 0.3 mol / L, and ultrasonically treat it at 200 W for 1 h to obtain a curcumin extract; dissolve the curcumin extract in medium-chain triglycerides to prepare a curcumin oil solution with a concentration of 3 mg / mL, and homogenize it 2 times at 60 MPa to refine the particle size to 100 - 200 nm, thus obtaining the pretreated curcumin.

[0051] Example 10: Different from Example 4, the pretreatment method of curcumin is as follows: Mix turmeric rhizome powder with an aqueous solution of 1-octyl-3-methylimidazolium bromide at a liquid-solid ratio of 30 mL:1 g and 0.5 mol / L, and ultrasonically treat it at 250 W for 1.5 h to obtain a curcumin extract; dissolve the curcumin extract in medium-chain triglycerides to prepare a curcumin oil solution with a concentration of 5 mg / mL, and homogenize it 4 times at 80 MPa to refine the particle size to 100 - 200 nm, thus obtaining the pretreated curcumin.

[0052] Example 11: Different from Example 4, in S2, mix modified sodium alginate and deoxycholic acid in a mass ratio of 5:1 to obtain a wall material, then add it to deionized water at a ratio of 1 g:50 mL, and ultrasonically disperse it at a ultrasonic frequency of 300 W for 8 min until it is uniform, and obtain a wall material solution after sterilization.

[0053] Example 12: Different from Example 4, in S2, mix modified sodium alginate and deoxycholic acid in a mass ratio of 10:1 to obtain a wall material, then add it to deionized water at a ratio of 1 g:100 mL, and ultrasonically disperse it at a ultrasonic frequency of 500 W for 12 min until it is uniform, and obtain a wall material solution after sterilization.

[0054] Example 13: Different from Example 4, the preparation method of modified sodium alginate:

[0055] Deionized water and sodium alginate are mixed in a mass ratio of 22:1 and stirred evenly, a 0.1 mol / L sodium hydroxide solution is used to adjust the pH to 7, and then 1% of nano-montmorillonite is added to the sodium alginate, the pH is re-measured and adjusted to 7 again, the temperature is increased by 40°C and treated at an ultrasonic frequency of 180W for 8 minutes to obtain product A; 0.8% of the mass of the above product A and 0.5% of the mass of vitamin E are added in sequence, and product B is obtained after homogeneous mixing, and then dried at 40°C to a water content of 4% to obtain modified sodium alginate.

[0056] Example 14: Different from Example 4, the preparation method of modified sodium alginate is:

[0057] Deionized water and sodium alginate are mixed in a mass ratio of 25:1 and stirred evenly, a 0.2 mol / L sodium hydroxide solution is used to adjust the pH to 8, and then 1.2% of nano-montmorillonite is added to the sodium alginate, the pH is re-measured and adjusted to 8 again, the temperature is increased by 50°C and treated at an ultrasonic frequency of 200 W for 15 minutes to obtain product A; 1% of the mass of the above product A, crushed oat hulls with a particle size of less than 1 μm and 0.7% of the mass of the above product A are added in sequence, and product B is obtained after homogeneous mixing, and then dried at 50°C to a water content of 5% to obtain modified sodium alginate.

[0058] Example 15: Different from Example 4, the method of homogenous mixing is to mix Product A with crushed oat hulls at 200 rpm for 4 minutes, then increase the speed to 600 rpm and mix for 3 minutes, add vitamin E and increase the speed to 1100 rpm and continue mixing for 5 minutes, and finally alternately switch the speeds of 600 rpm and 1100 rpm for 2 minutes each time, repeat the switching twice to complete the homogenous mixing; during the homogenous mixing process, the total homogenization time is controlled at 20 minutes, and the temperature is maintained at 30°C during the homogenization process.

[0059] Example 16: Different from Example 4, the method of homogenous mixing is to mix Product A with crushed oat hulls at 400 rpm for 6 minutes, then increase the speed to 800 rpm and mix for 5 minutes, add vitamin E and increase the speed to 1300 rpm and continue mixing for 7 minutes, and finally alternately switch the speeds of 800 rpm and 1300 rpm for 3 minutes each time, repeat the switching 3 times to complete the homogenous mixing; during the homogenous mixing process, the total homogenization time is controlled at 25 minutes, and the temperature is maintained at 40°C during the homogenization process.

[0060] Example 17: Different from Example 4, in S3, the core material solution and the wall material solution were mixed evenly according to a volume ratio of 1:3, and added to a 2% CaCl2 solution by the extrusion method, and left standing to form a gel. The gel was transferred to a sodium phosphate buffer solution with a pH of 7.2, containing 0.3 mL of lactoferrin and 0.5 mL of hyaluronic acid according to a volume ratio of 1:5 of the gel to the phosphate buffer solution, and stirred at 30 rpm for 25 min to form a surface modification layer; after washing 3 times with deionized water, the gel was immersed in a chitosan solution containing 0.1% fructooligosaccharide for secondary coating according to a volume ratio of 1:3 of the gel to the chitosan solution, and left standing for 15 min; finally, it was dried at a low temperature of 30 °C until the water content was 3%, and functional microcapsules with a diameter of 10 - 50 μm were obtained, which were microcapsules embedding curcumin.

[0061] Example 18: Different from Example 4, in S3, the core material solution and the wall material solution were mixed evenly according to a volume ratio of 1:5, and added to a 3% CaCl2 solution by the extrusion method, and left standing to form a gel. The gel was transferred to a sodium phosphate buffer solution with a pH of 7.6, containing 0.3 mL of lactoferrin and 0.5 mL of hyaluronic acid according to a volume ratio of 1:8 of the gel to the phosphate buffer solution, and stirred at 50 rpm for 30 min to form a surface modification layer; after washing 5 times with deionized water, the gel was immersed in a chitosan solution containing 0.5% fructooligosaccharide for secondary coating according to a volume ratio of 1:5 of the gel to the chitosan solution, and left standing for 20 min; finally, it was dried at a low temperature of 40 °C until the water content was 5%, and functional microcapsules with a diameter of 10 - 50 μm were obtained, which were microcapsules embedding curcumin.

[0062] Experimental example: The description of this experimental example is based on the recording scheme in Example 1, aiming to clarify the actual application effect of the present invention. There was a total of one blank group and five test groups in the test, with 3 replicates in each group, and each replicate had 35 California bass with uniform body mass (12.56 ± 0.32 g). The blank group was fed with a basal diet, and the five test groups were fed with the addition of Example 1, 2, 3, Control Group 1, and Control Group 2. The test lasted for 8 weeks. It was fed 2 times a day (9:00, 15:00). It was fed to near satiety, and the daily feeding amount was about 3% of the body mass. During the feeding period, there was 12 h of light and 12 h of darkness, the water temperature was 28 - 32 °C, the dissolved oxygen was > 6.0 mg / L, the ammonia nitrogen content was < 0.10 mg / L, the nitrite content was < 0.05 mg / L, and the pH was 6.5 - 7.5;

[0063] Blank group: Different from Example 1, the feed fed was a basal diet.

[0064] Control Group 1: Different from Example 1, the addition of yeast hydrolysate was missing.

[0065] Control Group 2: Different from Example 1, the addition of the carrier was missing.

[0066] (1) Effects on the growth performance of Micropterus salmoides

[0067] ① Effects on the weight gain rate

[0068] From the changes in the weight gain rate in Table 1, it was found that in Examples 1, 2, and 3, and in Control Groups 1 and 2, they were all significantly greater than the blank group (P < 0.05). Among them, Example 1 and Control Group 2 were significantly higher than Control Group 1 and Example 2 (P < 0.05), and there was no significant difference between Example 1 and Control Group 2 directly (P > 0.05).

[0069] ② Effects on the survival rate

[0070] Table 1 Effects of the preparation methods of Examples 1 - 3, Control Groups 1 - 2, and the blank group on the survival rate of Micropterus salmoides

[0071] Group Blank group Control group 1 Example 2 Example 1 Control group 2 Example 3 Initial average tail weight / g IBW 12.31±0.12 12.77±0.13 12.75±0.06 12.36±0.07 12.35±0.05 12.57±0.10 Final average tail weight / g FBW <![CDATA[66.81±1.52 a > <![CDATA[70.69±3.12 b > <![CDATA[70.71±3.12 b > <![CDATA[72.51±2.98 b > <![CDATA[72.78±2.98 b > <![CDATA[70.68±3.05 b > Weight gain rate / % WGR <![CDATA[442.73±7.22 a > <![CDATA[453.56±11.14 bc > <![CDATA[454.59±9.14 c > <![CDATA[486.65±8.45 d > <![CDATA[489.31±8.45 d > <![CDATA[454.47±9.14 c > Survival rate / % SR 87.33±2.31 100 100 99±3.42 99±3.42 100

[0072] Note: Different lowercase letters in the superscript of the same - row data indicate significant differences between groups (P < 0.05), and the same below;

[0073] From the data of the survival rate in Table 1, it can be seen that the survival rates of Examples 1 - 3 and Control Groups 1 - 2 were all as high as 100%, while the blank group was relatively low.

[0074] ③ Effects on the liver indices of Micropterus salmoides

[0075] Table 2 Effects of the preparation methods of Examples 1 - 3, Control Groups 1 - 2, and the blank group on the liver indices of Micropterus salmoides

[0076] Group Blank group Control group 1 Example 2 Example 1 Control group 2 Example 3 Hepatosomatic index 2.32±0.12 2.23±0.06 2.18±0.13 2.15±0.07 2.20±0.10 2.19±0.15 Liver fat / % <![CDATA[4.81±0.22 a > <![CDATA[3.42±0.11 b > <![CDATA[3.39±0.09 b > <![CDATA[3.14±0.14 b > 3.19±0.12b <![CDATA[3.37±0.08 b > Fatty acid synthase (ng / mL) <![CDATA[15.65±1.87 a > <![CDATA[15.60±1.43 b > <![CDATA[15.15±0.84 b > <![CDATA[15.11±0.97 b > <![CDATA[15.66±0.67 b > <![CDATA[15.17±0.84 b > Glycogen synthase (ng / mL) <![CDATA[5.87±0.43 a > <![CDATA[4.84±0.74 b > <![CDATA[4.80±0.51 bc > <![CDATA[4.65±0.92 c > <![CDATA[4.91±0.62 c > <![CDATA[4.83±0.52 bc >

[0077] From the data in Table 2, it can be seen that there was no significant effect on the hepatosomatic index among groups (P > 0.05). The liver lipid contents of Examples 1 and 2 and Control Groups 1 and 2 were all significantly lower than those of the blank group (P < 0.05), and Example 1 was the lowest. The fatty acid synthase contents of Examples 1 and 2 and Control Groups 1 and 2 were all significantly lower than those of the blank group (P < 0.05), and Example 1 was the lowest. The glycogen synthase contents of Examples 1 and 2 and Control Groups 1 and 2 were all significantly lower than those of the blank group (P < 0.05), and Control Group 2 was the highest.

[0078] ④ Effects on the blood indices of Micropterus salmoides

[0079] Table 3 Effects of the preparation methods of Examples 1 - 3, Control Groups 1 - 2, and the blank group on the blood indices of Micropterus salmoides

[0080]

[0081] The glutathione peroxidase and superoxide dismutase in Table 3 both showed the same trend of change, that is, Examples 1-3 and Control Groups 1-2 were all significantly greater than the blank group (P<0.05), and Example 1 was the largest. For Examples 1-3 and Control Groups 1-2, the contents of malondialdehyde and glucose were significantly lower than those of the blank group (P<0.05). Among them, malondialdehyde in Control Group 2 was the lowest, and glucose in the blood of Example 1 was the lowest.

[0082] In summary, from the aspects of growth test, feed efficiency, liver fat metabolism, etc. as comparison indicators, the group of Example 1, namely the group of 40% yeast hydrolysate + 20% leonurine + 20% curcumin + 20% carrier, showed the best performance in preventing fatty liver in Micropterus salmonides.

[0083] 1. Explore the effect of the preparation method of curcumin on the performance of feed additives in preventing fatty liver

[0084] Control Group 3: Different from Example 4, the organic solvent does not contain sodium dextran sulfate.

[0085] Control Group 4: Different from Example 4, the curcumin oil solution is not homogenized.

[0086] Control Group 5: Different from Example 4, the homogenization method is to mix at a rotation speed of 10000-15000 rpm for 30-60 s.

[0087] Control Group 6: Different from Example 4, the rotation speed is not alternately switched during the homogenization process.

[0088] Control Group 7: Different from Example 4, nano-montmorillonite is not added to the modified sodium alginate.

[0089] Table 4 Effects of the preparation methods of Example 1, Examples 4-18, and Control Groups 3-7 on the liver indexes of Micropterus salmonides

[0090]

[0091] Conclusion: By comparing Example 1 and Example 4 to Example 18, it can be obtained that encapsulating curcumin in microcapsules can effectively improve the preventive ability of feed additives on fatty liver of California seabass. The use of microcapsule encapsulation technology can solve the problems of poor water solubility and low absorption rate of curcumin, so that it can be slowly released in the intestine, continuously exert antioxidant effects, and improve the efficiency of scavenging free radicals by 30-40%; compounding with deoxycholic acid can synergistically promote lipid emulsification and transport, and deliver curcumin to liver cells through liposome carriers, activate the PPARα pathway, inhibit the expression of fatty acid synthase (FAS), and reduce the abnormal accumulation of triglycerides in the liver; and by comparing Example 4 to Example 18, it can be obtained that the microcapsule processing parameters for encapsulating curcumin within the scope of this application have relatively little effect on fatty liver of California seabass, and can reach the optimal state under the conditions of Example 4;

[0092] Among them, an increase in the liver-to-body ratio indicates an enlarged liver or abnormal fat accumulation, which is a typical manifestation of fatty liver; and excessive liver fat is a direct sign of fatty liver. California sea bass is prone to converting excess sugars into fat deposited in the liver due to impaired sugar metabolism; FAS is a key enzyme that catalyzes fatty acid synthesis. Its excessive activity will accelerate the conversion of sugars into fat, aggravate liver lipid deposition, and inhibiting FAS activity can reduce fat synthesis, reduce the risk of fatty liver and lead to hepatocyte cavitation and dysfunction; excessive GS activity will promote glycogen synthesis, California sea bass has weak sugar metabolism ability, and excessive glycogen may be converted into fat and accumulate in the liver; from the comparison of Example 4, Example 7 to Example 8 and Control Group 3, it can be seen that the lack of addition of sodium dextran sulfate will lead to an improvement in various parameters; sodium dextran sulfate increases the negative charge density, enhances the electrostatic repulsion, prevents the physical adhesion of porous starch to probiotics, and combines with Pluronic F-68, ethanol and sodium dextran sulfate can synergistically reduce the risk of agglomeration of the composite suspension (curcumin + probiotics + porous starch), while maintaining the stability and functionality of the active substances. As an anti-agglomeration agent, it can ensure that the components are evenly dispersed, thereby improving the preventive effect of feed additives;

[0093] Curcumin is a fat-soluble substance, and medium-chain triglycerides (MCT) can be used as a solvent to improve its solubility. It can be obtained from the comparison of Example 1, Example 4, Example 9 to Example 10, Example 15 to Example 16, and Control Group 4 to Control Group 6 that not homogenizing the curcumin oil solution or directly homogenizing it at ultra-high speed will also weaken the preventive effect of the feed additive on the fatty liver of Micropterus salmonides. This is because in Control Group 4, the lack of homogenization leads to uneven dispersion of active ingredients, low encapsulation rate, and easy oxidation and inactivation. In Example 4, the gradient rotation speed promotes the formation of microcapsules, improving the encapsulation rate and stability. In Control Group 5, direct homogenization at ultra-high speed will cause shear force to damage the molecular structure of curcumin, resulting in reduced biological activity. In Example 4, the energy input is controlled in stages, which can effectively protect the molecular structure and sustained-release function, ensuring the effect of each component in the feed additive. In Control Group 6, the lack of alternating rotation speed will lead to stratification of the mixed system, and vitamin E is not tightly bound to the matrix. In Example 4, the alternating rotation speed is used to optimize the material distribution by dynamic shear force and enhance the interfacial binding force.

[0094] It can be obtained from the comparison of Example 4, Example 13 to Example 14, and Control Group 7 that not adding nano-montmorillonite will weaken the preventive effect of the feed additive on the fatty liver of Micropterus salmonides. This is because adding nano-montmorillonite to the wall material, using its layered structure to adsorb free Ca 2+ and block oxygen, further reducing the oxidation degradation rate of curcumin by 50%, thereby enhancing the preventive effect of the feed additive. In summary, Example 4 is selected as the further optimization scheme in this application.

Claims

1. A feed additive for preventing fatty liver in California sea bass, characterized in that: Calculated by mass percentage, it comprises 35-45% of yeast hydrolyzate, 15-25% of curcumin, 15-25% of leonurine and the balance of a carrier; The carrier is defatted rice bran and / or rice husk powder.

2. A feed additive for preventing fatty liver in California sea bass according to claim 1, characterized in that: The moisture content of the feed additive is controlled to be below 10-12%, and then the feed additive is crushed and passed through an 80-mesh sieve.

3. A feed additive for preventing fatty liver in California sea bass according to claim 1, characterized in that: The curcumin is embedded in microcapsules, and the preparation method of the microcapsules embedded with curcumin is as follows: S1, mixing the pretreated curcumin, probiotics and porous starch with a pore size of 1 to 5 μm in a mass ratio of 1:0.1 to 0.2:0.3 to 0.5, and adding an organic solvent to prepare a 2 to 4 wt% composite suspension, which is a core material solution; S2, mixing modified sodium alginate and deoxycholic acid at a mass ratio of 5 to 10:1 to obtain a wall material, then adding 1 g: 50 to 100 mL into deionized water, ultrasonically dispersing at an ultrasonic frequency of 300 to 500 W for 8 to 12 minutes until uniform, and sterilizing to obtain a wall material solution; S3, the core material solution and the wall material solution are mixed evenly at a volume ratio of 1:3-5, added to a 2-3% CaCl2 solution by extrusion, and allowed to stand to form a gel, and the gel is transferred to a phosphate buffer solution with a pH of 7.2-7.6, containing 0.3 mL lactoferrin and 0.5 mL hyaluronic acid, at a volume ratio of 1:5-8 between the gel and the phosphate buffer, and stirred at a low speed for 25-30 minutes to form a surface modification layer; after washing with deionized water for 3-5 times, the gel is immersed in a chitosan solution containing 0.1-0.5% oligofructose for secondary coating at a volume ratio of 1:3-5 between the gel and the chitosan solution, and allowed to stand for 15-20 minutes; finally, low-temperature drying is performed at 30-40°C to a water content of ≤5%, and functionalized microcapsules with a diameter of 10-50 μm are obtained, i.e., microcapsules encapsulating curcumin.

4. A feed additive for preventing fatty liver in California sea bass according to claim 3, characterized in that: The probiotics in step S1 are any one of Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus acidophilus, and Bifidobacterium lactis, or a mixture of several of them in equal weight proportions; the number of viable probiotics is 1.0×10 6 ~1.0×10 8 CFU / mL; the organic solvent is composed of Pluronic F-68 with a concentration of 0.01-0.1%, ethanol with a volume concentration of 18-22%, and 0.1-0.5 mg / mL dextran sulfate sodium salt in equal volume proportions.

5. A feed additive for preventing fatty liver in California sea bass according to claim 3, characterized in that: The curcumin pretreatment method comprises the following steps: mixing turmeric rhizome powder with a 0.3-0.5 mol / L 1-octyl-3-methylimidazole bromide aqueous solution at a liquid-solid ratio of 25-30 mL:1 g, and ultrasonically treating the mixture at 200-250 W for 1-1.5 h to obtain a curcumin extract; dissolving the curcumin extract in medium-chain triglycerides to prepare a curcumin oil solution with a concentration of 3-5 mg / mL, and homogenizing the solution at 60-80 MPa for 2-4 times to refine the particle size to 100-200 nm to obtain the pretreated curcumin.

6. A feed additive for preventing fatty liver in California sea bass according to claim 1, characterized in that: Preparation method of modified sodium alginate: Deionized water and sodium alginate are mixed and stirred at a mass ratio of 22 to 25:1, and the pH is adjusted to 7 to 8 using a 0.1 to 0.2 mol / L sodium hydroxide solution, and then 1 to 1.2% of nano-montmorillonite is added to the sodium alginate, the pH is re-measured and adjusted to 7 to 8 again, the temperature is increased by 40 to 50°C, and the product is treated at an ultrasonic frequency of 180 to 200 W for 8 to 15 minutes to obtain a product A; 0.8 to 1% of the mass of the product A and 0.5 to 0.7% of the mass of the product A are successively added to crushed oat shells with a particle size of less than 1 μm and vitamin E, respectively, and the product B is obtained after homogeneous mixing, and then dried at 40 to 50°C to a water content of ≤5% to obtain a modified sodium alginate.

7. A feed additive for preventing fatty liver in California sea bass according to claim 6, characterized in that: The homogeneous mixing method comprises mixing the product A with the crushed oat hulls at 200-400 rpm for 4-6 minutes, then increasing the speed to 600-800 rpm for mixing for 3-5 minutes, adding vitamin E and increasing the speed to 1100-1300 rpm for further mixing for 5-7 minutes, and finally switching the speeds between 600-800 rpm and 1100-1300 rpm alternately for 2-3 minutes each time, repeating the switching 2-3 times to complete the homogeneous mixing.

8. A feed additive for preventing fatty liver in California sea bass according to claim 7, characterized in that: During the homogenizing and mixing process, the total homogenizing time is controlled within 20 to 25 minutes, and the temperature is maintained at 30 to 40° C. during the homogenizing process.

9. A feed additive for preventing fatty liver in California sea bass according to claim 1, characterized in that: Add it to complete feed at a ratio of 1000-1500g / ton.

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