Composition for inhibiting PNPLA3 expression and improving nitrogen-retaining and phosphorus-fixing capabilities of micropterus salmoides as well as preparation method and application of composition

Abstract

The invention belongs to the technical field of aquaculture, and discloses a composition for inhibiting PNPLA3 expression and improving nitrogen and phosphorus fixation capacity of micropterus salmoides and a preparation method and application thereof.The composition is prepared by the steps that capsaicin, sulforaphane and litchi anthocyanin are smashed, sieved and fully mixed, and a coarse composition is prepared; adding the crude composition into a methanol solution of sodium alginate and a dispersing agent, and homogenizing; and stirring at 50-85 DEG C, cooling to room temperature, standing, carrying out suction filtration, washing, and drying to obtain the product. According to the invention, scientific compatibility is carried out by fully utilizing different biological structures of the raw materials to play a synergistic effect, and the bioavailability of the composition is remarkably improved by adopting a micro-encapsulation method. According to the composition, by inhibiting PNPLA3 expression and enhancing the glycolipid metabolism function of fish bodies, the nitrogen conservation and phosphorus fixation capacity of the micropterus salmoides is improved, the culture economic and ecological benefits are increased, and the composition can be applied to the field of micropterus salmoides culture.

Description

Technical Field

[0001] The invention belongs to the field of aquaculture, and in particular relates to a composition for inhibiting PNPLA3 expression and improving the nitrogen and phosphorus retention capacity of largemouth bass, as well as a preparation method and application thereof. Background Art

[0002] As the amount of fish meal used increases year by year, its price is also increasing. In order to reduce the amount of fish meal in feed, high-energy, especially high-sugar feeds are gradually being used in aquaculture. However, largemouth bass, a freshwater carnivorous economic fish fed with high-sugar feeds, often show significant increases in blood sugar, blood lipid levels and liver fat content, causing liver metabolic dysfunction, resulting in a significant increase in nitrogen and phosphorus emissions in the aquaculture water, which in turn leads to serious eutrophication of the water body and the death of a large number of farmed fish. In view of this, the development of metabolic regulators with nitrogen and phosphorus fixation functions has important practical application value in reducing feed costs and improving aquaculture ecology and economic benefits.

[0003] The patatin-like phospholipase domain 3 (PNPLA3) protein is considered to be a key molecular target for the treatment of fatty liver disease in patients with type II diabetes. Inhibiting its expression can reduce the body's blood lipid and liver lipid content, thereby improving the lipid metabolism function of patients with type II diabetes. At present, a variety of Western medicines have been developed for this gene to improve the body's lipid metabolism function. However, no metabolic regulators developed around this gene have been found in fish. In addition, Western medicines are prone to drug resistance and drug residues in aquatic animals during use, which restricts their widespread use in feed. In recent years, with the rise of the application of natural active substances in the fields of food and medicine, the research on active ingredients of medicinal plants has received increasing attention. Capsaicin is a vanilloid alkaloid in peppers that improves the body's lipid metabolism function and increases appetite; sulforaphane is an isothiocyanate found in radish with strong antioxidant capacity; litchi peel anthocyanin is a flavonoid widely found in litchi peel, which has the effects of preventing diabetes and losing weight. However, there are still few studies on the effects of the three on the metabolic functions of aquatic animals, and there are no reports on related products that utilize the different biological structure characteristics of the three substances to scientifically combine and exert synergistic effects to regulate the metabolic functions of aquatic animals and improve the ability to retain nitrogen and phosphorus. Summary of the invention

[0004] In order to solve the above-mentioned deficiencies and disadvantages of the prior art, the object of the present invention is to provide a composition for inhibiting the expression of PNPLA3 and improving the nitrogen and phosphorus retention capacity of largemouth bass. The composition improves the nitrogen and phosphorus retention capacity of largemouth bass by inhibiting the expression of PNPLA3.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned composition for improving the nitrogen and phosphorus retention capacity of largemouth bass. The method uses microencapsulation technology to target PNPLA3, and based on the pharmacological synergistic relationship between capsaicin, sulforaphane and anthocyanins, a composition for improving the nitrogen and phosphorus retention capacity of largemouth bass after feeding high-sugar feed is prepared.

[0006] Another object of the present invention is to provide the use of the composition for improving the nitrogen and phosphorus retention ability of largemouth bass by inhibiting the expression of PNPLA3. The composition can be used as a highly efficient and environmentally friendly aquatic feed additive to greatly reduce feed costs and improve aquaculture economic and environmental benefits.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A composition for inhibiting PNPLA3 expression and improving the nitrogen and phosphorus retention capacity of largemouth bass is disclosed. The composition is prepared by first crushing capsaicin, sulforaphane and litchi anthocyanin respectively, sieving and fully mixing them to obtain a crude composition; adding the crude composition to a methanol solution of sodium alginate and a dispersant for homogenization; stirring at 50 to 85° C., cooling to room temperature and standing, filtering and washing, and drying.

[0009] Preferably, the mass ratio of capsaicin, sulforaphane, litchi anthocyanin, sodium alginate and dispersant is (1-4):(1-4):(1-5):(20-80):(80-150); the volume ratio of the total mass of sodium alginate and dispersant to the methanol solution is (500-1150) g:(1-3) L.

[0010] Preferably, the dispersant is a mixture of sodium polyacrylate and sodium methylene bis(methyl naphthalene sulfonate).

[0011] More preferably, the mass ratio of the sodium polyacrylate to sodium methylene bis(methyl naphthalene sulfonate) is (1-4):(1-7).

[0012] Preferably, the volume ratio of methanol to distilled water in the methanol solution is (1-4):(1-7).

[0013] The preparation method of the composition for inhibiting PNPLA3 expression and improving the nitrogen and phosphorus retention capacity of largemouth bass comprises the following specific steps:

[0014] S1. After capsaicin, sulforaphane and litchi anthocyanins are crushed separately, sieved and fully mixed to prepare a crude composition;

[0015] S2. adding sodium alginate and a dispersant to the methanol solution and homogenizing to obtain a solution A;

[0016] S3. Add the crude composition to solution A to a concentration of 10 to 35 wt %, stir at 50 to 85° C., cool to room temperature and let stand, filter and wash, then dry to obtain a composition for improving the nitrogen and phosphorus retention capacity of largemouth bass.

[0017] Preferably, the capsaicin, sulforaphane and litchi anthocyanin in step S1 are all crushed at a temperature lower than 40° C., and the aperture of the sieve is 120-150 meshes.

[0018] Preferably, the homogenization rate in step S2 is 10000-25000 r / min, and the homogenization time is 10-30 min.

[0019] Preferably, the stirring rate in step S3 is 1000-3000 r / min, the stirring time is 12-24 h, and the standing time is 8-24 h.

[0020] The composition is used in largemouth bass breeding, and the addition amount of the composition in largemouth bass feed is 0.2-1.2wt%.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The composition of the present invention uses capsaicin, sulforaphane and anthocyanin as raw materials, and makes full use of the different characteristics of their biological structures to carry out scientific compatibility in order to exert synergistic effects.

[0023] 2. The present invention adopts a microencapsulation method, selects PNPLA3 as the target, and inhibits the expression of PNPLA3 to enhance the glucose and lipid metabolism function of largemouth bass, improve the feed utilization rate and nitrogen and phosphorus retention capacity of largemouth bass, increase the economic and ecological benefits of breeding, and can be applied to the field of largemouth bass breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The expression of PNPLA3 gene in liver cells of largemouth bass by the composition of Example 1 and different treatment groups;

[0025] Figure 2 The results are as follows: The effects of the composition of Example 1 and different treatment groups on the content of triglyceride in hepatocytes of largemouth bass. DETAILED DESCRIPTION

[0026] The content of the present invention is further described below in conjunction with specific examples, but it should not be construed as limiting the present invention. If not specifically indicated, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0027] Example 1

[0028] 1. 5 g of capsaicin, 5 g of sulforaphane and 25 g of anthocyanins, which were crushed at below 40° C. and passed through a 120-mesh sieve, were fully mixed to ensure that the mixture was uniform and stable, to obtain 35 g of a crude composition.

[0029] 2. Add 100g sodium alginate and 400g dispersant (sodium polyacrylate and sodium methylenebis(methyl)naphthalene sulfonate in a mass ratio of 2:1) to 1L methanol solution (methanol and distilled water in a volume ratio of 1:3) and homogenize at 12000r / min for 10min.

[0030] 3. Add the crude composition to the solution obtained in step 2 to a concentration of 10 wt %, stir at 80°C and 1000 r / min for 12 h, cool naturally to room temperature and then stand for 12 h. Filter and wash with a Buchner funnel, cool and dry for 12 h to obtain 15 g of a composition for improving the nitrogen and phosphorus retention capacity of largemouth bass.

[0031] Due to high sugar culture, lipid deposition occurs in liver cells. In order to verify that the composition can reduce the PNPLA3 gene expression of liver cells and reduce the lipid deposition of liver cells after high sugar culture medium treatment. Primary liver cells of largemouth bass were cultured to 1.5×10 6 cFU / mL, and then transferred to a 6-well cell culture plate and cultured for 24 h, and then inoculated with high-glucose culture medium (the glucose concentration in the culture medium was 20 mmol / L, purchased from Gibco's DMEM / F12, model 11320033), high-glucose culture medium + capsaicin, high-glucose culture medium + sulforaphane, high-glucose culture medium + anthocyanin, high-glucose culture medium + the composition of Example 1 (corresponding to Figure 1 and 2 The high sugar, high sugar + capsaicin, high sugar + sulforaphane, high sugar + anthocyanin, high sugar + control composition 1) were cultured for 24 hours and then tested for relevant indicators. The results were as follows Figure 1 and 2 shown. Figure 1 The expression of PNPLA3 gene in liver cells of largemouth bass by the composition of Example 1 and different treatment groups. Figure 1 It can be seen that the PNPLA3 gene expression level of fish liver cells in the high-glucose culture medium group was significantly higher than that in other experimental groups, but the PNPLA3 gene expression level of fish liver cells in the high-glucose culture medium + composition of Example 1 group was significantly lower than that in other experimental groups. Figure 2 The composition of Example 1 and different treatment groups have an effect on the content of triglyceride in the liver cells of largemouth bass. Figure 2It can be seen that the triglyceride content of fish liver cells in the high-sugar culture medium group was significantly higher than that in other experimental groups, but the triglyceride content of fish liver cells in the high-sugar culture medium + composition of Example 1 group was significantly lower than that in other experimental groups. This shows that the composition of Example 1 can reduce the triglyceride content of high-sugar treated liver cells by downregulating the expression of the PNPLA3 gene, thereby preventing excessive lipid deposition.

[0032] Example 2

[0033] 1. 7 g of capsaicin, 12 g of sulforaphane and 16 g of anthocyanins, which were crushed at less than 40° C. and passed through a 120-mesh sieve, were mixed thoroughly to ensure that the mixture was uniform and stable, to obtain 35 g of a crude composition.

[0034] 2. Add 150 g of sodium alginate and 450 g of dispersant (sodium polyacrylate and sodium methylenebis(methyl)naphthalene sulfonate in a mass ratio of 2:1) to 1.5 L of methanol solution (methanol and distilled water in a volume ratio of 1:2) and homogenize at 10,000 r / min for 20 min.

[0035] 3. Add the crude composition to the solution obtained in step 2 to a concentration of 20 wt %, stir at 85 ° C and 1500 r / min for 10 h, cool naturally to room temperature and let stand for 12 h, filter and wash with a Buchner funnel, and cool and dry for 12 h to obtain 15 g of a composition for improving the nitrogen and phosphorus retention capacity of largemouth bass.

[0036] Example 3

[0037] 1. 8 g of capsaicin, 10 g of sulforaphane and 17 g of anthocyanins, which were crushed at less than 40° C. and passed through a 120-mesh sieve, were mixed thoroughly to ensure that the mixture was uniform and stable, to obtain 35 g of a crude composition.

[0038] 2. Add 200 g of sodium alginate and 500 g of dispersant (sodium polyacrylate and sodium methylenebis(methyl)naphthalene sulfonate in a mass ratio of 1:2) to 1.0 L of methanol solution (methanol and distilled water in a volume ratio of 1:1) and homogenize at 12,000 r / min for 25 min.

[0039] 3. Add the crude composition to the solution obtained in step 2 to a concentration of 15 wt %, stir at 70 ° C and 1500 r / min for 12 h, cool naturally to room temperature and let stand for 8 h, then filter and wash with a Buchner funnel, and cool and dry for 10 h to obtain 15 g of a composition for improving the nitrogen and phosphorus retention capacity of largemouth bass.

[0040] Example 4

[0041] 1. 10 g of capsaicin, 11 g of sulforaphane and 14 g of anthocyanins, which were crushed at below 40° C. and passed through a 150-mesh sieve, were fully mixed to ensure that the mixture was uniform and stable, to obtain 35 g of a crude composition.

[0042] 2. Add 220 g of sodium alginate and 550 g of dispersant (sodium polyacrylate and sodium methylenebis(methyl)naphthalene sulfonate in a mass ratio of 1:1) to 2.0 L of methanol solution (methanol and distilled water in a volume ratio of 3:1) and homogenize at 12,000 r / min for 25 min.

[0043] 3. Add the crude composition to the solution obtained in step 2 to a concentration of 12 wt %, stir at 65 ° C and 2000 r / min for 12 h, cool naturally to room temperature and let stand for 10 h, then filter and wash with a Buchner funnel, and cool and dry for 18 h to obtain 15 g of a composition for improving the nitrogen and phosphorus retention capacity of largemouth bass.

[0044] Example 5

[0045] 1. 12 g of capsaicin, 12 g of sulforaphane and 11 g of anthocyanins, which were crushed at below 40° C. and passed through a 150-mesh sieve, were fully mixed to ensure that the mixture was uniform and stable, to obtain 35 g of a crude composition.

[0046] 2. Add 220 g of sodium alginate and 600 g of a dispersant (a mixture of sodium polyacrylate and sodium methylenebis(methyl)naphthalene sulfonate in a weight ratio of 1:3) to 2.0 L of a methanol aqueous solution (a mixed solution of methanol and distilled water in a volume ratio of 1:3) and homogenize at 12,000 r / min for 30 min.

[0047] 3. Add the crude composition to the solution obtained in step 2 to a concentration of 12 wt %, stir at 3000 r / min at 60°C for 12 h, cool naturally to room temperature and let stand for 10 h, then filter and wash with a Buchner funnel, and cool and dry for 18 h to obtain 15 g of a composition for improving the nitrogen and phosphorus retention capacity of largemouth bass.

[0048] Example 6

[0049] 1. 14 g of capsaicin, 15 g of sulforaphane and 6 g of anthocyanins, which were crushed at below 40° C. and passed through a 150-mesh sieve, were fully mixed to ensure that the mixture was uniform and stable, to obtain 35 g of a crude composition.

[0050] 2. Add 250 g of sodium alginate and 600 g of dispersant (sodium polyacrylate and sodium methylenebis(methyl)naphthalene sulfonate in a mass ratio of 2:3) to 2.5 L of methanol aqueous solution (methanol and distilled water in a volume ratio of 1:4) and homogenize at 20,000 r / min for 30 min.

[0051] 3. Add the crude composition to the solution obtained in step 2 to a concentration of 15 wt %, stir at 65 ° C and 2000 r / min for 12 h, cool naturally to room temperature and let stand for 12 h, then filter and wash with a Buchner funnel, and cool and dry for 24 h to obtain 15 g of a composition for improving the nitrogen and phosphorus retention capacity of largemouth bass.

[0052] Example 7

[0053] 1. 15 g of capsaicin, 15 g of sulforaphane and 5 g of anthocyanins, which were crushed at below 40° C. and passed through a 150-mesh sieve, were fully mixed to ensure that the mixture was uniform and stable, to obtain 35 g of a crude composition.

[0054] 2. Add 300g of sodium alginate and 500g of dispersant (sodium polyacrylate and sodium methylenebis(methyl)naphthalene sulfonate in a mass ratio of 2:5) to 3L of methanol solution (methanol and distilled water in a volume ratio of 1:5) and homogenize at 20,000r / min for 15min.

[0055] 3. Add the crude composition to the solution obtained in step 2 to a concentration of 20 wt %, stir at 55 ° C and 2000 r / min for 12 h, cool naturally to room temperature and let stand for 24 h, then filter and wash with a Buchner funnel, and cool and dry for 24 h to obtain 15 g of a composition for improving the nitrogen and phosphorus retention capacity of largemouth bass.

[0056] Example 8

[0057] 1. 20 g of capsaicin, 10 g of sulforaphane and 5 g of anthocyanins, which were crushed at below 40° C. and passed through a 150-mesh sieve, were fully mixed to ensure that the mixture was uniform and stable, to obtain 35 g of a crude composition.

[0058] 2. Add 300g of sodium alginate and 650g of dispersant (sodium polyacrylate and sodium methylenebis(methyl)naphthalene sulfonate in a mass ratio of 3:5) to 2L of methanol solution (methanol and distilled water in a volume ratio of 2:5) and homogenize at 20,000r / min for 20min.

[0059] 3. Add the crude composition to the solution obtained in step 2 to a concentration of 25 wt %, stir at 50°C and 2000 r / min for 24 h, cool naturally to room temperature and let stand for 12 h, then filter and wash with a Buchner funnel, and cool and dry for 24 h to obtain 15 g of a composition for improving the nitrogen and phosphorus retention capacity of largemouth bass.

[0060] Example 9

[0061] 1. 10 g of capsaicin, 20 g of sulforaphane and 5 g of anthocyanins, which were crushed at below 40° C. and passed through a 150-mesh sieve, were fully mixed to ensure that the mixture was uniform and stable, to obtain 35 g of a crude composition.

[0062] 2. Add 350 g of sodium alginate and 650 g of dispersant (sodium polyacrylate and sodium methylenebis(methyl)naphthalene sulfonate in a mass ratio of 4:5) to 2 L of methanol solution (methanol and distilled water in a volume ratio of 3:7) and homogenize at 15,000 r / min for 20 min.

[0063] 3. Add the crude composition to the solution obtained in step 2 to a concentration of 30 wt %, stir at 55 ° C and 2500 r / min for 24 hours, cool naturally to room temperature and let stand for 10 hours, then filter and wash with a Buchner funnel, and cool and dry for 24 hours to obtain 15 g of a composition for improving the nitrogen and phosphorus retention capacity of largemouth bass.

[0064] Example 10

[0065] 1. 10 g of capsaicin, 17 g of sulforaphane and 8 g of anthocyanins, which were crushed at less than 40° C. and passed through a 150-mesh sieve, were fully mixed to ensure that the mixture was uniform and stable, to obtain 35 g of a crude composition.

[0066] 2. Add 400 g of sodium alginate and 750 g of dispersant (sodium polyacrylate and sodium methylenebis(methyl)naphthalene sulfonate in a mass ratio of 4:7) to 3 L of methanol solution (methanol and distilled water in a volume ratio of 4:7) and homogenize at 25,000 r / min for 30 min.

[0067] 3. Add the crude composition to the solution obtained in step 2 to a concentration of 35 wt %. Stir at 3000 r / min at 45 ° C for 24 hours, naturally cool to room temperature and then stand for 12 hours. Then filter and wash with a Buchner funnel, cool and dry for 12 hours to obtain 15 g of a composition for improving the nitrogen and phosphorus retention capacity of largemouth bass.

[0068] Application Example 1

[0069] Breeding experiment and result analysis of juvenile largemouth black bass (about 100g): This breeding experiment was carried out at the teaching and research base of the College of Oceanography of South China Agricultural University, and the juvenile fish used were purchased from a fishery breeding farm in Guangzhou. 360 juvenile fish of uniform size (initial weight: 103.02±2.80 g) were randomly placed in 20 cages (specifications: 2.0 m×1.0 m×1.0 m), with 30 fish in each cage. The juvenile fish were cultured for 12 weeks, during which the feeding and growth conditions of the juvenile fish were observed. The feed formula is shown in Table 1: (1) Basic group: fed with feed ingredients (40 wt% fish meal, 26 wt% soybean meal, 5 wt% fish oil, 5 wt% soybean oil, 20 wt% corn starch, 2 wt% calcium dihydrogen phosphate, 2 wt% premix); (2) Basic + Composition 1 group: fed with high sugar feed + 1 wt% of the composition of Example 1; (3) Basic + Composition 2 group: fed with high sugar feed + 1 wt% of the composition of Example 2; (4) Basic + Composition 3 group: fed with high sugar feed + 1 wt% of the composition of Example 3; (5) Basic + Composition 4 group: fed with basic feed + 1 wt% of the composition of Example 4. After the breeding, blood and tissue samples were collected for subsequent index analysis. Table 2 shows the effects of different treatment groups on the growth, physiological and biochemical indicators of largemouth bass. As shown in Table 2, the basic + combination group 4 significantly increased the weight gain rate, nitrogen retention rate and phosphorus retention rate of largemouth bass fed high-sugar feed, but the liver fat content, blood sugar level and PNPLA3 gene expression were significantly reduced. This shows that the basic + combination group 4 is better in promoting fish growth, lowering blood sugar and blood lipids, and fixing nitrogen and phosphorus.

[0070] Table 1 Feed formula for different treatment groups

[0071]

[0072] Premix: Each kg of premix contains the following minerals (g / kg) and vitamins (IU ormg / kg): CuSO4·5H2O, 2.0g; FeSO4·7H2O, 25g; ZnSO4·7H2O, 22g; MnSO4·4H2O, 7g; Na2SeO3, 0.04g; KI, 0.026g; CoCl2·6H2O, 0.1g; VA, 900,000IU; VD, 200,000IU; VE, 4500mg; VK3, 220mg; VB1, 320mg; VB2, 1090mg; niacin 2800mg; VB5, 2000mg; VB6, 500mg; VB12, 1.6mg, VC, 5000mg, pantothenic acid; 1000mg; folic acid, 165m; choline, 60000mg; inositol, 15000mg.

[0073] Table 2 Effects of different treatment groups of feed on growth and physiological and biochemical parameters of largemouth bass

[0074]

[0075] Note: The same lowercase letters in the same row indicate no significant difference

[0076] Application Example 2

[0077] Largemouth bass fry (about 50g) breeding experiment and result analysis: This breeding experiment was carried out at the teaching and research base of the College of Oceanography, South China Agricultural University. The fry used were purchased from a breeding farm in Guangzhou. 360 fry of uniform size (initial weight: 54.01±1.36g) were randomly placed in 20 cages (specifications: 2.0m×1.0m×1.0m), 30 fish in each cage, and cultured for 12 weeks. During this period, the feeding and growth conditions of the fry were observed. (1) Basic + Composition 4 Group: fed with high sugar feed + 1wt% of the composition of Example 4; (2) Basic + Composition 5 Group: fed with high sugar feed + 1wt% of the composition of Example 5; (3) Basic + Composition 6 Group: fed with high sugar feed + 1wt% of the composition of Example 6; (4) Basic + Composition 7 Group: fed with high sugar feed + 1wt% of the composition of Example 7; (5) Basic + Composition 8 Group: fed with high sugar feed + 1wt% of the composition shown in Example 8. After the breeding, blood and tissue samples were collected for subsequent index analysis. Table 3 shows the effects of different feeds on the growth, physiological and biochemical indicators of largemouth bass. As shown in Table 3, the basic + combination group 8 significantly increased the weight gain rate, nitrogen retention rate, and phosphorus retention rate of largemouth bass fed high-sugar feed, but the liver fat content, blood sugar level, and PNPLA3 gene expression were significantly reduced. This shows that the basic + combination group 8 is better in promoting fish growth, lowering blood sugar and blood lipids, and fixing nitrogen and phosphorus.

[0078] Table 3 Effects of different feeds on growth and physiological and biochemical parameters of largemouth bass

[0079]

[0080] Application Example 3

[0081] Largemouth bass juveniles (about 10g) culture experiment and result analysis: This culture experiment was carried out at the teaching and research base of the College of Oceanography, South China Agricultural University. The juveniles used were purchased from a breeding farm in Guangzhou. 360 juveniles of uniform size (initial weight: 11.21±1.02g) were randomly placed in 12 cages (specifications: 2.0m×1.0m×1.0m), with 30 fish in each cage, and cultured for 12 weeks, during which the feeding and growth of the juveniles were observed. (1) Basic + Composition 8 group: fed with high sugar feed + 1wt% of the composition of Example 8; (2) Basic + Composition 9 group: fed with high sugar feed + 1wt% of the composition of Example 9; (3) Basic + Composition 10 group: fed with high sugar feed + 1wt% of the composition of Example 10. After the culture was completed, blood and tissue samples were collected for subsequent index analysis. Table 4 shows the effects of feeds in different treatment groups on the growth and physiological and biochemical indicators of largemouth bass. As shown in Table 4, the basic + combination 10 group significantly increased the weight gain rate, nitrogen retention rate, and phosphorus retention rate of largemouth bass fed high-sugar feed, but significantly reduced liver fat content, blood sugar level, and PNPLA3 gene expression. This shows that the basic + combination 10 is better in promoting fish growth, lowering blood sugar and blood lipids, and fixing nitrogen and phosphorus.

[0082] Table 4 Effects of different treatment groups' diets on growth and physiological and biochemical parameters of largemouth bass

[0083] Related indicators Basic + Composition 8 sets Basics + Composition 9 Groups Basics + Composition 10 sets Weight gain rate (%) <![CDATA[529.04±3.01 c ]]> <![CDATA[832.33±21.10 b ]]> <![CDATA[1213.98±4.29 a ]]> Liver fat content (g / kg) <![CDATA[4.28±0.01 a ]]> <![CDATA[3.29±0.02 b ]]> <![CDATA[3.01±0.01 c ]]> <![CDATA[Blood glucose level ( mmol / L)]]> <![CDATA[4.39±0.01 a ]]> <![CDATA[3.89±0.01 b ]]> <![CDATA[3.27±0.01 c ]]> PNPLA3 gene expression <![CDATA[1.02±0.01 a ]]> <![CDATA[0.76±0.01 b ]]> <![CDATA[0.43±0.01 c ]]> Nitrogen retention rate <![CDATA[73.19±3.21 b ]]> <![CDATA[74.28±2.11 b ]]> <![CDATA[78.33±4.13 a ]]> Phosphorus retention rate <![CDATA[65.29±2.22 b ]]> <![CDATA[68.21±5.21 b ]]> <![CDATA[70.98±3.13 a ]]>

[0084] Application Example 4

[0085] The breeding experiment and result analysis of largemouth bass juveniles (about 40g): This breeding experiment was carried out at the teaching and research base of the College of Oceanography of South China Agricultural University, and the juveniles used were purchased from a breeding farm in Guangzhou. 360 juveniles of uniform size (initial weight: 43.01±3.80g) were randomly placed in 24 cages (specifications: 2.0m×1.0m×1.0m), with 30 fish in each cage, and cultured for 12 weeks, during which the feeding and growth of the juveniles were observed. (1) Basic group: fed with high-sugar feed (sugar level of 20%); (2) Basic + capsaicin group: fed with high-sugar feed + 1.0% capsaicin; (3) Basic + sulforaphane group: fed with high-sugar feed + 1.0% sulforaphane; (4) Basic + anthocyanin group: fed with high-sugar feed + 1.0% anthocyanin; (5) Basic + Composition 10 group: fed with high-sugar feed + 1.0% composition shown in Example 10, and the feed formula components are shown in Table 5. After the end of the breeding, blood and tissue samples were collected for subsequent index analysis. Table 6 shows the effects of feeds of different treatment groups on the growth and blood biochemical indicators of largemouth bass. As shown in Table 6, the basic + composition 10 significantly increased the weight gain rate, nitrogen retention rate, and phosphorus retention rate of largemouth bass fed with high-sugar feed, but significantly reduced the liver fat content, blood sugar level, and PNPLA3 gene expression. It further shows that the basic + composition 10 is better in promoting fish growth, lowering blood sugar and blood lipids, and fixing nitrogen and phosphorus.

[0086] Table 5 Feed formula for different treatment groups

[0087]

[0088] Premix: Each kg of premix contains the following minerals (g / kg) and vitamins (IU ormg / kg): CuSO4·5H2O, 2.0g; FeSO4·7H2O, 25g; ZnSO4·7H2O, 22g; MnSO4·4H2O, 7g; Na2SeO3, 0.04g; KI, 0.026g; CoCl2·6H2O, 0.1g; VA, 900,000IU; VD, 200,000IU; VE, 4500mg; VK3, 220mg; VB1, 320mg; VB2, 1090mg; niacin 2800mg; VB5, 2000mg; VB6, 500mg; VB12, 1.6mg, VC, 5000mg, pantothenic acid; 1000mg; folic acid, 165m; choline, 60000mg; inositol, 15000mg. .

[0089] Table 6 Effects of different treatment groups of feed on growth and blood biochemical parameters of largemouth bass

[0090]

[0091] Note: The same lowercase letters in the same row indicate no significant difference

[0092] The present invention combines the growth and physical and chemical index results of relevant breeding experiments, confirming that the composition can enhance the glucose and lipid metabolism function of fish by inhibiting the expression of PNPLA3, and has a strong pertinence in regulating the metabolic function and nitrogen and phosphorus fixation ability of largemouth bass, thereby improving the nitrogen and phosphorus fixation ability of largemouth bass and increasing the economic and ecological benefits of breeding.

[0093] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A composition for inhibiting the expression of PNPLA3 and improving the nitrogen and phosphorus retention capacity of largemouth bass, characterized in that: The composition is prepared by first crushing capsaicin, sulforaphane and litchi anthocyanin respectively, sieving and fully mixing to obtain a crude composition; adding the crude composition to a methanol solution of sodium alginate and a dispersant for homogenization; stirring at 50-85° C., cooling to room temperature and standing, filtering, washing and drying.

2. The composition for inhibiting PNPLA3 expression and improving the nitrogen and phosphorus retention ability of largemouth bass according to claim 1, characterized in that: The mass ratio of capsaicin, sulforaphane, litchi anthocyanin, sodium alginate and dispersant is (1-4):(1-4):(1-5):(20-80):(80-150); the volume ratio of the total mass of sodium alginate and dispersant to the methanol solution is (500-1150) g:(1-3) L.

3. The composition for inhibiting PNPLA3 expression and improving the nitrogen and phosphorus retention ability of largemouth bass according to claim 1, characterized in that: The dispersant is a mixture of sodium polyacrylate and sodium methylene bis(methyl naphthalene sulfonate).

4. The composition for inhibiting PNPLA3 expression and improving the nitrogen and phosphorus retention ability of largemouth bass according to claim 3, characterized in that: The mass ratio of the sodium polyacrylate to sodium methylene bis(methyl naphthalene)sulfonate is (1-4):(1-7).

5. The composition for inhibiting PNPLA3 expression and improving the nitrogen and phosphorus retention ability of largemouth bass according to claim 1, characterized in that: The volume ratio of methanol to distilled water in the methanol solution is (1-4):(1-7).

6. The method for preparing the composition for inhibiting PNPLA3 expression and improving the nitrogen and phosphorus retention capacity of largemouth bass according to any one of claims 1 to 5, characterized in that: The specific steps include: S1. After capsaicin, sulforaphane and litchi anthocyanins are crushed separately, sieved and fully mixed to prepare a crude composition; S2. adding sodium alginate and a dispersant to the methanol solution and homogenizing to obtain a solution A; S3. Add the crude controlled composition to solution A to a concentration of 10 to 35 wt %, stir at 50 to 85° C., cool to room temperature and let stand, filter and wash, and then dry to obtain a composition for improving the nitrogen and phosphorus retention capacity of largemouth bass.

7. The method for preparing the composition for inhibiting PNPLA3 expression and improving the nitrogen and phosphorus retention capacity of largemouth bass according to claim 6, characterized in that: In step S1, the capsaicin, sulforaphane and litchi anthocyanin are all crushed at a temperature lower than 40° C., and the aperture of the sieve is 120-150 meshes.

8. The method for preparing the composition for inhibiting PNPLA3 expression and improving the nitrogen and phosphorus retention capacity of largemouth bass according to claim 6, characterized in that: The homogenization rate in step S2 is 10000-25000 r / min, and the homogenization time is 10-30 min.

9. The method for preparing the composition for inhibiting PNPLA3 expression and improving the nitrogen and phosphorus retention capacity of largemouth bass according to claim 6, characterized in that: The stirring rate in step S3 is 1000-3000 r / min, the stirring time is 12-24 h, and the standing time is 8-24 h.

10. Use of the composition according to any one of claims 1 to 5 in largemouth bass breeding, characterized in that: The addition amount of the composition in largemouth bass feed is 0.2-1.2 wt%.

Images