Bile acid composition for fish and shrimp feed and preparation method thereof

Abstract

The invention belongs to the technical field of fish and shrimp breeding feed additives, and particularly relates to a bile acid composition for fish and shrimp feed and a preparation method of the bile acid composition, and the composition comprises the following components in parts by weight: 50-60 parts of hyocholic acid, 40-55 parts of chenodeoxycholic acid, 40-60 parts of hyodeoxycholic acid, 30-45 parts of methionine glycine dipeptide and 60-115 parts of a compound plant extract. According to the invention, the methionine glycine dipeptide is used for improving the lipid metabolism capability of fishes and shrimps; the composite plant extract is used for enhancing the immunity and the anti-stress capability of the fish and the shrimps.

Description

Technical Field

[0001] This invention belongs to the technical field of fish and shrimp aquaculture feed additives, specifically relating to a bile acid composition for fish and shrimp feed and its preparation method. Background Technology

[0002] Bile acids, primarily cholic acid with deoxycholic acid as a secondary component, play important physiological roles in promoting fat digestion and absorption and maintaining liver and gallbladder health. With the widespread use of high-energy feeds in aquaculture and rising raw material costs, improving energy digestibility and utilization has become an industry demand. Bile acids can effectively improve fish growth performance by increasing lipase activity and promoting fat metabolism.

[0003] The invention patent with publication number CN104041678A uses pig bile extract as raw material and employs a mature extraction process involving saponification, decolorization, acidification, and purification to retain core components such as porcine deoxycholic acid, chenodeoxycholic acid, and porcine cholic acid. However, when the bile acid composition obtained by this patent was used as a feed additive for fish and shrimp, it resulted in low immunity, weak stress resistance, and low lipid metabolism levels in the fish and shrimp. Summary of the Invention

[0004] The purpose of this invention is to provide a bile acid composition for fish and shrimp feed and its preparation method, so as to solve the above-mentioned technical problems.

[0005] To achieve the above-mentioned technical objectives, the technical solution of the present invention is as follows: A bile acid composition for fish and shrimp feed, comprising, by weight, 50-60 parts of porcine cholic acid, 40-55 parts of chenodeoxycholic acid, 40-60 parts of porcine deoxycholic acid, 30-45 parts of methionylglycine dipeptide, and 60-115 parts of compound plant extract.

[0006] As a further improvement, the preparation method of the methionylglycine dipeptide is as follows: S1: Dissolve Boc-Met-Gly-OBzl in dichloromethane, slowly add trifluoroacetic acid dichloromethane solution at 0℃, stir the reaction at room temperature for 60~90min, concentrate under reduced pressure to obtain H2N-Met-Gly-OBzl trifluoroacetate; S2: Dissolve H2N-Met-Gly-OBzl trifluoroacetate in deionized water, adjust the pH to 7-8, extract with ethyl acetate, dry and concentrate the organic phase to obtain H2N-Met-Gly-OBzl; S3: Dissolve H2N-Met-Gly-OBzl in anhydrous ethyl acetate, add palladium on carbon, and purge with hydrogen gas, replacing the air in the system with hydrogen gas three times; react for 2-6 hours in a hydrogen atmosphere at room temperature and 2500 rpm, then replace the hydrogen gas with nitrogen gas; filter, concentrate the filtrate under reduced pressure, purify, and freeze-dry to obtain methionylglycine dipeptide; The mass ratio of Boc-Met-Gly-OBzl to dichloromethane is 1:13.25; the volume fraction of the trifluoroacetic acid-dichloromethane solution is 30%.

[0007] The reaction equation for the synthesis of methionylglycine dipeptide is as follows: ; As a further improvement, the preparation method of the Boc-Met-Gly-OBzl is as follows: S11: Dissolve Boc-Met-OH and 1-hydroxybenzotriazole in anhydrous DMF, add N,N'-dicyclohexylcarbodiimide anhydrous DMF solution under stirring at 0℃ and stir for 30 min to obtain an activated ester mixture; S12: Dissolve H2N-Gly-OBzl p-toluenesulfonate in anhydrous DMF, add N,N-diisopropylethylamine to obtain a free amine solution; S13: Add the free amine solution dropwise to the activated ester mixture, stir in an ice bath for 2 hours, raise to room temperature and stir for 12-24 hours, filter, concentrate the filtrate under reduced pressure to obtain the residue; S14: Dissolve the residue in ethyl acetate, and wash it once each with 10% citric acid aqueous solution, saturated NaHCO3 aqueous solution and saturated saline solution; separate the liquid and dry the organic phase, filter, concentrate and purify to obtain Boc-Met-Gly-OBzl.

[0008] As a further improvement, the mass ratio of Boc-Met-OH to 1-hydroxybenzotriazole was 0.48:0.32; and the concentration of the anhydrous DMF solution of N,N'-dicyclohexylcarbodiimide was 0.09 g / mL.

[0009] As a further improvement, the preparation method of Boc-Met-OH is as follows: methionine is dissolved in a mixture of 1,4-dioxane and 1 mol / L NaOH aqueous solution. Under stirring at 0°C, di-tert-butyl dicarbonate and 4-dimethylaminopyridine are slowly added. The mixture is then raised to room temperature and reacted for 2-6 hours. The mixture is washed three times with diethyl ether, separated, and the organic phase is removed. The aqueous phase is cooled in an ice bath, and the pH is adjusted to 2-3. The aqueous phase is extracted with ethyl acetate, and the organic phase is collected, washed, dried, filtered, concentrated under reduced pressure, and recrystallized to obtain Boc-Met-OH.

[0010] As a further improvement, the mass ratio of methionine, di-tert-butyl carbonate, and 4-dimethylaminopyridine is (0.75~1):1.15:(0.061~0.1); the volume ratio of 1,4-dioxane to 1 mol / L NaOH aqueous solution is 1:1.

[0011] As a further improvement, the preparation method of H2N-Gly-OBzl p-toluenesulfonate is as follows: glycine, benzyl alcohol, p-toluenesulfonic acid and anhydrous toluene are uniformly mixed and refluxed at 120°C for 6-12 h. After cooling to room temperature, the acidic substances are neutralized with saturated NaHCO3 aqueous solution, and then extracted with ethyl acetate. The organic phase is collected, washed, and dehydrated. Dry hydrogen chloride gas is then introduced, filtered, the precipitate is collected, washed, and dried to obtain H2N-Gly-OBzl p-toluenesulfonate.

[0012] As a further improvement, the mass ratio of glycine to p-toluenesulfonic acid is (0.38~0.5):(0.095~0.1); the volume ratio of benzyl alcohol to anhydrous toluene is 2.7:30.

[0013] As a further improvement, the compound plant extract is composed of a uniform mixture of Dendrobium officinale extract, ginger extract and Astragalus extract; the mass ratio of Dendrobium officinale extract, ginger extract and Astragalus extract is (5~15):(10~20):(10~30).

[0014] The present invention also provides a bile acid composition for fish and shrimp feed, which is prepared by taking the porcine cholic acid, chenodeoxycholic acid, porcine deoxycholic acid, methionylglycine dipeptide and compound plant extract in proportion and mixing them evenly.

[0015] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows: This invention utilizes a bile acid composition composed of methionine dipeptide, porcine cholic acid, chenodeoxycholic acid, and porcine deoxycholic acid as a feed additive, which has multiple effects: the bile acid components can significantly improve the digestibility and absorption of fat and protein, reduce the feed conversion ratio and promote animal growth by emulsifying fat, activating lipase, and regulating intestinal flora balance; at the same time, it protects liver and gallbladder health, reduces toxin accumulation, and enhances stress resistance; methionine dipeptide, as a small molecule peptide, enhances intestinal enzyme activity, improves feed protein digestibility, and reduces fat deposition; it can promote protein synthesis and deposition in fish and shrimp, improve muscle growth performance, and participate in the regulation of the insulin-like growth factor pathway; methionine dipeptide decomposes into methionine, which participates in protein synthesis and fat metabolism, and provides active methyl groups to promote the production of choline and phosphatidylcholine, thereby protecting the liver and detoxifying, and supporting immune function and tissue repair.

[0016] When used as a feed additive, a bile acid composition consisting of Dendrobium officinale extract, ginger extract, and Astragalus extract, along with methionine glycine dipeptide, porcine cholic acid, chenodeoxycholic acid, and porcine deoxycholic acid, can significantly improve the health and growth performance of fish and shrimp. The bile acid composition, synergistically with ginger extract, effectively regulates lipid metabolism in rainbow trout: bile acids promote fat absorption and utilization by activating lipase and lipoprotein lipase activity; while ginger extract reduces excessive lipid accumulation in the liver caused by high-fat diets, preventing fatty liver disease; the polysaccharides and alkaloids of Dendrobium officinale, combined with astragaloside A and astragalus polysaccharides in Astragalus extract, enhance the disease resistance of fish and shrimp; the anti-inflammatory and antioxidant properties of ginger extract alleviate liver oxidative stress and protect hepatobiliary function; the yin-nourishing and liver-protecting effects of Dendrobium officinale, combined with the metabolic regulatory function of bile acids, reduce the risk of liver damage; this bile acid composition can improve the digestion, immunity, and stress resistance of fish and shrimp, while also improving meat quality. Attached Figure Description

[0017] Figure 1 The results show the detection results of TC, HDL and LDL concentrations in the liver and serum of rainbow trout in the control group and experimental group. Among them, a is the total cholesterol (TC) content in the liver of rainbow trout, b is the concentration of high-density lipoprotein cholesterol (HDL) and low-density lipoprotein cholesterol (LDL) in the liver of rainbow trout, c is the total cholesterol (TC) content in the serum of rainbow trout, and d is the concentration of high-density lipoprotein cholesterol (HDL) and low-density lipoprotein cholesterol (LDL) in the serum of rainbow trout. Figure 2 The images show the liver tissue morphology staining of rainbow trout in the experimental and control groups, where a represents the experimental group and b represents the control group. Figure 3 The graph shows the relative expression levels of mRNAs related to lipid synthesis and metabolism in the liver, as well as the results of TG concentration detection in the liver and serum. In the graph, a is the relative expression level of mRNAs related to lipid synthesis and metabolism in the liver, b is the triglyceride content in the liver of rainbow trout, c is the relative expression level of mRNAs related to lipid catabolism and metabolism in the liver, and d is the triglyceride content in the serum of rainbow trout. Detailed Implementation

[0018] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0019] Example 1: A bile acid composition for fish and shrimp feed, comprising, by weight, 55 parts of porcine cholic acid, 48 parts of chenodeoxycholic acid, 50 parts of porcine deoxycholic acid, 38 parts of methionylglycine dipeptide, and 88 parts of a compound plant extract; wherein the compound plant extract is composed of 10 parts of Dendrobium officinale extract, 15 parts of ginger extract, and 20 parts of Astragalus membranaceus extract, all mixed uniformly.

[0020] The preparation method of the above-mentioned bile acid composition for fish and shrimp feed is as follows: take the porcine cholic acid, chenodeoxycholic acid, porcine deoxycholic acid, methionylglycine dipeptide and compound plant extract in proportion and mix them evenly.

[0021] The preparation method of methionylglycine dipeptide includes the following steps: S1. Dissolve 0.75 g of methionine in a mixture of 15 mL of 1,4-dioxane and 15 mL of 1 mol / L NaOH aqueous solution. Cool to 0 °C in an ice bath. While stirring at 300 rpm, slowly add 1.15 g of di-tert-butyl dicarbonate and 61 mg of 4-dimethylaminopyridine. Raise to room temperature and react for 2 h. After the reaction is complete, a protected amino reaction solution is obtained. Wash the protected amino reaction solution with 50 mL of diethyl ether, separate the liquid and remove the organic phase. Cool the aqueous phase in an ice bath and adjust the pH to 2 with 10% HCl to obtain an acidified aqueous phase. Extract the acidified aqueous phase three times with 50 mL of ethyl acetate. Combine the three organic phases and wash with 50 mL of saturated brine. Dry with 2 g of anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and finally recrystallize from ethyl acetate to obtain pure Boc-Met-OH.

[0022] S2. Mix 0.38 g glycine, 2.7 mL benzyl alcohol, 95 mg p-toluenesulfonic acid, and 30 mL anhydrous toluene thoroughly. Reflux at 120 °C for 6 h. Remove the water generated during the reaction using a water separator. Cool to room temperature to obtain a protected carboxyl group reaction solution. Neutralize the acidic substances in the protected carboxyl group reaction solution with 20 mL saturated NaHCO3 aqueous solution, and then extract three times with 50 mL ethyl acetate. Combine the organic phases and wash with 50 mL saturated brine. Dry with 2 g anhydrous magnesium sulfate, and then pass dry hydrogen chloride gas into the dried organic phase until no more precipitate forms. Collect the white precipitate by filtration, wash with ethyl acetate at 0 °C, and dry under vacuum to obtain H2N-Gly-OBzl p-toluenesulfonate.

[0023] S3. Dissolve 0.48 g Boc-Met-OH and 0.32 g 1-hydroxybenzotriazole in 20 mL of anhydrous N,N-dimethylformamide (DMF), cool to 0 °C in an ice bath, and add 0.45 g while stirring at 300 rpm. N,N'-Dicyclohexylcarbodiimide was dissolved in 5 mL of anhydrous DMF and stirred at 0 °C for 30 min. A white precipitate of N,N'-dicyclohexylurea was formed, indicating in-situ formation of the "activated ester," yielding an activated ester mixture. 0.36 g of H₂N-Gly-OBzl p-toluenesulfonate was dissolved in 5 mL of anhydrous DMF, and 0.77 mL of N,N-diisopropylethylamine was added to neutralize the sulfonate, releasing free amine and yielding a free amine solution. This free amine solution was added dropwise to the activated ester mixture, and the mixture was stirred in an ice bath for 2 h to obtain a reaction mixture. The reaction mixture was then brought to room temperature and stirred for another 12 h. After the reaction was complete, the white precipitate of N,N'-dicyclohexylurea was removed by filtration, and the precipitate was removed using 10 mL of [a specific solution / method / applied]. The filter cake was washed three times with DMF; the combined filtrates were concentrated under reduced pressure until dry to obtain the residue; the residue was dissolved in 50 mL of ethyl acetate and washed once each with 50 mL of 10% citric acid aqueous solution, 50 mL of saturated NaHCO3 aqueous solution, and 50 mL of saturated saline solution; the organic phase was dried with anhydrous magnesium sulfate, filtered, concentrated, and then purified by silica gel column chromatography to obtain Boc-Met-Gly-OBzl.

[0024] S4. Dissolve 1g of Boc-Met-Gly-OBzl in 13.25g of dichloromethane to obtain a Boc-Met-Gly-OBzl solution. Cool the Boc-Met-Gly-OBzl solution to 0℃ in an ice bath. Slowly add 10mL of a 30% (v / v) trifluoroacetic acid solution in dichloromethane. Bring to room temperature and stir for 60min. After the reaction is complete, obtain the reaction solution. Concentrate the reaction solution under reduced pressure to remove most of the trifluoroacetic acid and solvent. Add 5mL of dichloromethane and concentrate again. Repeat this process three times to completely remove the trifluoroacetic acid, obtaining H2N-Met-Gly-OBzl trifluoroacetate. Dissolve the H2N-Met-Gly-OBzl trifluoroacetate in... The pH of 10 mL of deionized water was adjusted to 7 with saturated NaHCO3 solution, and then extracted with ethyl acetate. The organic phase was dried and concentrated to obtain H2N-Met-Gly-OBzl. H2N-Met-Gly-OBzl was then dissolved in 20 mL of anhydrous ethyl acetate, and 0.1 g of 20% palladium on carbon was added. Hydrogen gas was introduced, and the air in the system was replaced with hydrogen gas three times. The reaction was carried out under a hydrogen atmosphere and stirred vigorously at room temperature for 2 h. After the reaction was completed, the hydrogen gas was replaced with nitrogen gas. The palladium on carbon catalyst was removed by filtration through a diatomaceous earth pad, and the filter cake was thoroughly washed with methanol. The combined filtrate was concentrated under reduced pressure, purified by reversed-phase C18 column chromatography, and freeze-dried to obtain methionine dipeptide.

[0025] Example 2: A bile acid composition for fish and shrimp feed, comprising, by weight, 50 parts of porcine cholic acid, 40 parts of chenodeoxycholic acid, 40 parts of porcine deoxycholic acid, 30 parts of methionine glycine dipeptide, and 60 parts of a compound plant extract; wherein the compound plant extract is composed of 5 parts of Dendrobium officinale extract, 10 parts of ginger extract, and 10 parts of Astragalus membranaceus extract, all mixed uniformly.

[0026] The preparation method of the above-mentioned bile acid composition for fish and shrimp feed is as follows: take the porcine cholic acid, chenodeoxycholic acid, porcine deoxycholic acid, methionylglycine dipeptide and compound plant extract in proportion and mix them evenly.

[0027] The preparation method of methionylglycine dipeptide includes the following steps: S1. Dissolve 0.88 g of methionine in a mixture of 15 mL of 1,4-dioxane and 15 mL of 1 mol / L NaOH aqueous solution. Cool to 0 °C in an ice bath. While stirring at 300 rpm, slowly add 1.15 g of di-tert-butyl dicarbonate and 80 mg of 4-dimethylaminopyridine. Raise to room temperature and react for 4 h. After the reaction is complete, a protected amino reaction solution is obtained. Wash the protected amino reaction solution with 50 mL of diethyl ether, separate the liquid and remove the organic phase. Cool the aqueous phase in an ice bath and adjust the pH to 2.5 with 10% HCl to obtain an acidified aqueous phase. Extract the acidified aqueous phase three times with 50 mL of ethyl acetate. Combine the three organic phases and wash with 50 mL of saturated brine. Dry with 2 g of anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and finally recrystallize from ethyl acetate to obtain pure Boc-Met-OH.

[0028] S2. Mix 0.44 g glycine, 2.7 mL benzyl alcohol, 98 mg p-toluenesulfonic acid, and 30 mL anhydrous toluene thoroughly. Reflux at 120 °C for 9.5 h. Remove the water produced by the reaction using a water separator. Cool to room temperature to obtain a protected carboxyl group reaction solution. Neutralize the acidic substances in the protected carboxyl group reaction solution with 20 mL saturated NaHCO3 aqueous solution, and then extract three times with 50 mL ethyl acetate. Combine the organic phases and wash with 50 mL saturated brine. Dry with 2 g anhydrous magnesium sulfate, and then pass dry hydrogen chloride gas into the dried organic phase until no more precipitate forms. Collect the white precipitate by filtration, wash with ethyl acetate at 0 °C, and dry under vacuum to obtain H2N-Gly-OBzl p-toluenesulfonate.

[0029] S3. Dissolve 0.48 g Boc-Met-OH and 0.32 g 1-hydroxybenzotriazole in 20 mL anhydrous N,N-dimethylformamide (DMF), cool to 0 °C in an ice bath, and add 0.45 g N,N'-dicyclohexylcarbodiimide dissolved in 5 mL anhydrous DMF while stirring at 300 rpm. Keep stirring at 0 °C for 30 min. At this time, a white precipitate of N,N'-dicyclohexylurea is formed, which is the in-situ formation of "activated ester", and an activated ester mixture is obtained. Dissolve 0.36 g H2N-Gly-OBzl p-toluenesulfonate in 5 mL anhydrous DMF, and add 0.77 mL N,N-diisopropylethylamine to neutralize its sulfonate, releasing free amine, and obtaining a free amine solution. The free amine solution was added dropwise to the activated ester mixture, and the mixture was stirred in an ice bath for 2 hours to obtain a reaction mixture. The reaction mixture was then brought to room temperature and stirred for another 18 hours. After the reaction was complete, the white precipitate of N,N'-dicyclohexylurea generated in the reaction was removed by filtration. The filter cake was washed three times with 10 mL of LDM. The combined filtrate was concentrated under reduced pressure until dry to obtain the residue. The residue was dissolved in 50 mL of ethyl acetate and washed once each with 50 mL of 10% citric acid aqueous solution, 50 mL of saturated NaHCO3 aqueous solution, and 50 mL of saturated saline solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and then purified by silica gel column chromatography to obtain Boc-Met-Gly-OBzl.

[0030] S4. Dissolve 1g of Boc-Met-Gly-OBzl in 13.25g of dichloromethane to obtain a Boc-Met-Gly-OBzl solution. Cool the Boc-Met-Gly-OBzl solution to 0°C in an ice bath. Slowly add 10mL of a 30% trifluoroacetic acid solution in dichloromethane. Raise to room temperature and stir for 75min. After the reaction is complete, obtain the reaction solution. Concentrate the reaction solution under reduced pressure to remove most of the trifluoroacetic acid and solvent. Add 5mL of dichloromethane and concentrate again. Repeat this process 3 times to remove all trifluoroacetic acid, obtaining H2N-Met-Gly-OBzl trifluoroacetate. Dissolve the H2N-Met-Gly-OBzl trifluoroacetate in 1... In 0 mL of deionized water, the pH was adjusted to 7.5 with saturated NaHCO3 solution, and then extracted with ethyl acetate. The organic phase was dried and concentrated to obtain H2N-Met-Gly-OBzl. H2N-Met-Gly-OBzl was then dissolved in 20 mL of anhydrous ethyl acetate, and 0.1 g of 20% palladium on carbon was added. Hydrogen gas was introduced, and the air in the system was replaced with hydrogen gas three times. The reaction was carried out under a hydrogen atmosphere with vigorous stirring at room temperature for 4 h. After the reaction was completed, the hydrogen gas was replaced with nitrogen gas. The palladium on carbon catalyst was removed by filtration through a diatomaceous earth pad, and the filter cake was thoroughly washed with methanol. The combined filtrate was concentrated under reduced pressure, purified by reversed-phase C18 column chromatography, and freeze-dried to obtain methionine dipeptide.

[0031] Example 3: A bile acid composition for fish and shrimp feed, comprising, by weight, 60 parts of porcine cholic acid, 55 parts of chenodeoxycholic acid, 60 parts of porcine deoxycholic acid, 45 parts of methionylglycine dipeptide, and 115 parts of a compound plant extract; wherein the compound plant extract is composed of 15 parts of Dendrobium officinale extract, 20 parts of ginger extract, and 30 parts of Astragalus membranaceus extract, all mixed uniformly.

[0032] The preparation method of the above-mentioned bile acid composition for fish and shrimp feed is as follows: take the porcine cholic acid, chenodeoxycholic acid, porcine deoxycholic acid, methionylglycine dipeptide and compound plant extract in proportion and mix them evenly.

[0033] The preparation method of methionylglycine dipeptide includes the following steps: S1. Dissolve 1g of methionine in a mixture of 15mL of 1,4-dioxane and 15mL of 1mol / L NaOH aqueous solution. Cool to 0℃ in an ice bath. While stirring at 300rpm, slowly add 1.15g of di-tert-butyl dicarbonate and 100mg of 4-dimethylaminopyridine. Raise to room temperature and react for 6h. After the reaction is complete, a protected amino reaction solution is obtained. Wash the protected amino reaction solution with 50mL of diethyl ether, separate the liquid and remove the organic phase. Cool the aqueous phase in an ice bath and adjust the pH to 3 with 10% HCl to obtain an acidified aqueous phase. Extract the acidified aqueous phase three times with 50mL of ethyl acetate. Combine the three organic phases and wash with 50mL of saturated brine. Dry with 2g of anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and finally recrystallize with ethyl acetate to obtain pure Boc-Met-OH.

[0034] S2. Mix 0.5g glycine, 2.7mL benzyl alcohol, 100mg p-toluenesulfonic acid, and 30mL anhydrous toluene thoroughly. Reflux at 120℃ for 12h. Remove the water generated during the reaction using a water separator. Cool to room temperature to obtain a protected carboxyl group reaction solution. Neutralize the acidic substances in the protected carboxyl group reaction solution with 20mL saturated NaHCO3 aqueous solution, and then extract three times with 50mL ethyl acetate. Combine the organic phases and wash with 50mL saturated brine. Dry with 2g anhydrous magnesium sulfate, and then pass dry hydrogen chloride gas into the dried organic phase until no more precipitate forms. Collect the white precipitate by filtration, wash with ethyl acetate at 0℃, and dry under vacuum to obtain H2N-Gly-OBzl p-toluenesulfonate.

[0035] S3. Dissolve 0.48 g Boc-Met-OH and 0.32 g 1-hydroxybenzotriazole in 20 mL of anhydrous N,N-dimethylformamide (DMF), cool to 0 °C in an ice bath, and add 0.45 g N,N'-dicyclohexylcarbodiimide dissolved in 5 mL of anhydrous DMF while stirring at 300 rpm. Keep stirring at 0 °C for 30 min. At this time, a white precipitate of N,N'-dicyclohexylurea is formed, which is the in-situ formation of the "activated ester", and an activated ester mixture is obtained; add 0.36 g H 2N-Gly-OBzl p-toluenesulfonate was dissolved in 5 mL of anhydrous DMF, and 0.77 mL of N,N-diisopropylethylamine was added to neutralize its sulfonate, releasing free amine to obtain a free amine solution. The free amine solution was added dropwise to the activated ester mixture, and the mixture was stirred in an ice bath for 2 h to obtain a reaction mixture. The reaction mixture was then brought to room temperature and stirred for another 24 h. After the reaction was completed, the white precipitate of N,N'-dicyclohexylurea generated in the reaction was removed by filtration. The filter cake was washed three times with 10 mL of DMF. The combined filtrates were concentrated under reduced pressure until dry to obtain the residue. The residue was dissolved in 50 mL of ethyl acetate and washed once each with 50 mL of 10% citric acid aqueous solution, 50 mL of saturated NaHCO3 aqueous solution, and 50 mL of saturated saline solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and then purified by silica gel column chromatography to obtain Boc-Met-Gly-OBzl.

[0036] S4. Dissolve 1g of Boc-Met-Gly-OBzl in 13.25g of dichloromethane to obtain a Boc-Met-Gly-OBzl solution. Cool the Boc-Met-Gly-OBzl solution to 0℃ in an ice bath. Slowly add 10mL of a 30% (v / v) trifluoroacetic acid solution in dichloromethane. Heat to room temperature and stir for 90min. After the reaction is complete, obtain the reaction solution. Concentrate the reaction solution under reduced pressure to remove most of the trifluoroacetic acid and solvent. Add 5mL of dichloromethane and concentrate again. Repeat this process three times to completely remove the trifluoroacetic acid, obtaining H2N-Met-Gly-OBzl trifluoroacetate. Dissolve the H2N-Met-Gly-OBzl trifluoroacetate in... The pH of 10 mL of deionized water was adjusted to 8 with saturated NaHCO3 solution, and then extracted with ethyl acetate. The organic phase was dried and concentrated to obtain H2N-Met-Gly-OBzl. H2N-Met-Gly-OBzl was then dissolved in 20 mL of anhydrous ethyl acetate, and 0.1 g of 20% palladium on carbon was added. Hydrogen gas was introduced, and the air in the system was replaced with hydrogen gas three times. The reaction was carried out under a hydrogen atmosphere with vigorous stirring at room temperature for 6 h. After the reaction was completed, the hydrogen gas was replaced with nitrogen gas. The palladium on carbon catalyst was removed by filtration through a diatomaceous earth pad, and the filter cake was thoroughly washed with methanol. The combined filtrate was concentrated under reduced pressure, purified by reversed-phase C18 column chromatography, and freeze-dried to obtain methionine dipeptide.

[0037] Comparative Example 1: Compared with Example 1, Comparative Example 1 did not add 38 parts of methionine-glycine dipeptide to the bile acid composition for fish and shrimp feed. Specifically, by weight, it included 55 parts of porcine cholic acid, 48 parts of chenodeoxycholic acid, 50 parts of porcine deoxycholic acid, and 88 parts of compound plant extract. The compound plant extract was composed of 10 parts of Dendrobium officinale extract, 15 parts of ginger extract, and 20 parts of Astragalus membranaceus extract, which were uniformly mixed.

[0038] Comparative Example 2: Compared with Example 1, Comparative Example 2 did not add 88 parts of compound plant extract to the bile acid composition for fish and shrimp feed. Specifically, by weight, it included 55 parts of porcine cholic acid, 48 parts of chenodeoxycholic acid, 50 parts of porcine deoxycholic acid, and 38 parts of methionine dipeptide.

[0039] Comparative Example 3: Compared with Example 1, Comparative Example 3 did not add 38 parts of methionylglycine dipeptide and 88 parts of compound plant extract to the bile acid composition for fish and shrimp feed. Specifically, by weight, it included 55 parts of porcine cholic acid, 48 parts of chenodeoxycholic acid and 50 parts of porcine deoxycholic acid.

[0040] The ability of methionylglycine dipeptide to affect lipid metabolism in rainbow trout liver: Healthy rainbow trout of uniform size (500.0±5.0g) were selected and cultured in flowing spring water. They were housed in cylindrical tanks 3m in diameter and 3m high, divided into two groups: a control group and an experimental group. Each group consisted of three tanks, with 25 fish per tank. Fish were fed three times daily (morning, noon, and evening). The control group was fed normal rainbow trout feed plus 3% (w / w) of the bile acid composition from Comparative Example 3. The experimental group was fed normal rainbow trout feed plus 3% (w / w) of the bile acid composition from Example 1. After four weeks of continuous feeding, eight fish were randomly selected from each tank for sampling. Dissolved oxygen was measured daily, and ammonia nitrogen and other water quality indicators were measured every other day.

[0041] To investigate the effect of dietary supplementation with methionylglycine dipeptide on lipid metabolism in rainbow trout livers, liver and serum samples were collected from both the control and experimental groups. The concentrations of total cholesterol (TC), high-density lipoprotein cholesterol (HDL), and low-density lipoprotein cholesterol (LDL) in the liver and serum of both groups were measured using a kit. The results are shown below. Figure 1 As shown.

[0042] The results showed that the assessment of liver health status indicated that... Figure 1 -a and Figure 1 In the liver of the -b group, the concentrations of TC and LDL were significantly decreased, while the concentrations of HDL showed no difference; Figure 1 -c and Figure 1In the serum of the experimental group, the concentration of TC was significantly reduced, the concentration of HDL was significantly increased, while the concentration of LDL was not different.

[0043] The liver tissue morphology was observed by HE staining according to the hematoxylin-eosin (HE) staining method.

[0044] Preparation of paraffin sections of liver tissue: Rainbow trout in both the control and experimental groups were subjected to a 24-hour fasting period. The fish were then anesthetized with ethyl m-aminobenzoate methanesulfonate (MS-222). Liver tissues from both groups were harvested, gently rinsed with physiological saline, and then rapidly immersed in 4% paraformaldehyde fixative to ensure complete immersion. The fixed tissues were then placed in a 4°C refrigerator or at room temperature for 24 hours. The fixed liver tissues were then dehydrated for 1 hour in 70% ethanol, transferred to 85% ethanol for 1 hour, then to 95% ethanol I for 1 hour, then to 95% ethanol II for 1 hour, then to anhydrous ethanol I for 1 hour, and finally to anhydrous ethanol II for 45 minutes. After dehydration, the liver tissues were first soaked in a 1:1 mixture of anhydrous ethanol and xylene for 30 minutes, then in xylene I for 7 minutes, and finally in xylene II for 7 minutes to obtain clear liver tissues. After clearing, liver tissue was immersed three times in paraffin at 60°C. First, it was immersed in a 1:1 xylene-paraffin mixture for 30 minutes, then in paraffin I for 1.5 hours, and finally in paraffin II for 1 hour. The paraffin-immersed liver tissue was placed in a molten embedding machine, carefully removed with forceps, and immersed on an embedding stage. Molten paraffin was injected into a pre-assembled metal embedding frame, and the tissue was then placed sequentially into the center of the frame according to the desired cut surface. The paraffin block was allowed to solidify and stored at room temperature. The cut surface was then trimmed according to experimental requirements and fixed onto a microtome until the desired tissue section was obtained, with a section thickness of 5 μm. The section was placed in a slide warmer at approximately 41°C until the cut surface was no longer wrinkled. The slide was then placed in a 65°C oven for 2 hours. The processed slides were then stored at 4°C.

[0045] Hematoxylin-eosin (HE) staining procedure: Place the slides to be stained in a 65℃ constant temperature oven for 10 minutes. First, dewax with xylene I for 20 minutes, then with xylene II for 15 minutes. Immerse in anhydrous ethanol I for 2 minutes, then in anhydrous ethanol II for 2 minutes, then in 95% ethanol for 2 minutes, then in 85% ethanol for 2 minutes, then in 70% ethanol for 2 minutes, and finally in 50% ethanol for 2 minutes. Wash with deionized water for 3 minutes to displace the xylene. Stain with hematoxylin and eosin for 6 minutes, then wash with deionized water for 3 minutes. Immerse the slides in differentiation solution (99 mL of 70% ethanol + 1 mL of concentrated HCl) for 5 seconds, then rinse in water for 5 seconds. Wipe dry and examine under a microscope. If the staining is too dark, continue rinsing in water for a period of time to fully remove HCl. If differentiation is incomplete, immerse again in the differentiation solution. After rinsing, immerse in a blueing solution (99 mL of 70% ethanol + 1 mL of concentrated ammonia) for 5 seconds, then rinse under distilled water and immerse again in distilled water for 3 minutes. Soak the slide in 85% ethanol for 5 minutes, then in 95% ethanol for 5 minutes, and finally in eosin solution (1 g of water-soluble eosin dissolved in 100 mL of 85% ethanol) for 3 minutes. If staining is difficult, add 1-2 drops of glacial acetic acid to each 100 mL of staining solution to facilitate staining and prevent fading. After rinsing with deionized water for 3 minutes, dehydrate with 95% ethanol I for 5 minutes, then with 95% ethanol II for 2 minutes, then with anhydrous ethanol I for 2 minutes, then with anhydrous ethanol II for 2 minutes, then soak in xylene I for 5 minutes, and finally soak in xylene II for 5 minutes. Staining is now complete. Remove the completely transparent sections from the xylene and remove the xylene from the back of the slide. Apply one drop of neutral resin to one end of the slide using a glass rod. Immediately afterward, heat a coverslip over an alcohol lamp flame to remove moisture. Place one end of the coverslip in contact with the resin at a 30° angle to the slide and slowly lower it to seal the slide. After sealing, place the slide flat in a 37°C oven for approximately 2 hours before microscopic examination.

[0046] Test results as follows Figure 2 As shown in the figure; further, the relative mRNA expression levels of lipid synthesis and degradation-related genes in the liver were determined using conventional qRT-PCR, including: steroid regulatory element-binding protein 1 (Srebp-1), acetyl-CoA carboxylase α (ACCα), fatty acid synthase (FAS), fatty acid dehydrogenase (Fads2), and stearoyl-CoA desaturase (Scd) related to lipid synthesis; and low-density lipoprotein cholesterol (LPL), carnitine palmitoyltransferase 1α (Cpt1α), and peroxisome proliferator-activated receptor α (PPARα) related to lipid degradation. The triglyceride (TG) levels in the liver and serum of different treatment groups were measured using a kit. The results are shown in the figure. Figure 3 As shown.

[0047] The results showed that, based on HE staining, the number of white fat granules in the liver of the experimental group was reduced, and quantitative results showed that... Figure 3 In the -a group, the expression levels of genes related to lipid synthesis, such as Srebp-1, ACCα, FAS, and Fads2, were significantly downregulated, and Scd expression also showed a decreasing trend; while Figure 3 In the -c group, LPL expression, which is associated with lipid breakdown, was downregulated, while Cpt1α and PPARα expression were significantly upregulated. Correspondingly, the TG content in the liver of the experimental group was significantly reduced, while the TG content in serum showed no difference. These results indicate that methionylglycine dipeptide enhances lipid catabolism in the liver, thereby reducing lipid deposition in the liver.

[0048] Effects of compound plant extracts on serum immunoenzyme activity in rainbow trout: Healthy rainbow trout of uniform size (500.0±5.0g) were selected and cultured in flowing spring water. They were housed in cylindrical tanks 3m in diameter and 3m high, divided into two groups: a control group and an experimental group. Each group consisted of three tanks, with 25 fish per tank. Fish were fed three times daily (morning, noon, and evening). The control group was fed normal rainbow trout feed plus 3% (w / w) of the bile acid composition from Comparative Example 3. The experimental group was fed normal rainbow trout feed plus 3% (w / w) of the bile acid composition from Example 1. After four weeks of continuous feeding, eight fish were randomly selected from each tank for sampling. Dissolved oxygen was measured daily, and ammonia nitrogen and other water quality indicators were measured every other day.

[0049] Two groups of rainbow trout were fasted at 13–18°C for 24 h before sampling. Eight fish from each group were randomly selected and rapidly transferred to MS-222 (50 mg / L) for deep anesthesia. Blood was collected from the tail vein and allowed to stand at 4°C for 2 h. Serum was then prepared by centrifugation at 3000 r / min for 10 min at 4°C. The serum was collected, flash-frozen in liquid nitrogen, and stored in an ultra-low temperature freezer (-80°C) for later use.

[0050] The activities of lysozyme (LZM), acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), and total antioxidant capacity (T-AOC) in rainbow trout serum were determined using a kit, and the assay methods were performed according to the kit instructions.

[0051] Table 1. Effects of compound plant extracts on serum immunoenzyme activity in rainbow trout (N=8)

[0052] Note: Different lowercase letters indicate significant differences (P < 0.05), and different uppercase letters indicate highly significant differences (P < 0.01).

[0053] Compared with the control group, the addition of compound plant extracts to the feed increased the activity of six immune enzymes and total antioxidant capacity in rainbow trout serum, with the high-dose group generally showing higher levels than the low-dose group. Lysozyme activity in the experimental groups was significantly higher than that in the control group. In the study of antioxidant enzymes, the activities of superoxide dismutase and catalase in the high-dose group were significantly higher than those in the control group; the activity of glutathione peroxidase in all experimental groups was significantly higher than that in the control group; and the determination of total antioxidant activity showed that all experimental groups were significantly higher than the control group. These data indicate that the addition of compound plant extracts to the feed can improve the activity of immune enzymes and total antioxidant capacity in rainbow trout serum, enhance non-specific immunity in rainbow trout, and thus improve the fish's disease resistance.

[0054] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A fish and shrimp feed bile acid composition, characterized by, The composition comprises 50-60 parts of porcine cholic acid, 40-55 parts of chenodeoxycholic acid, 40-60 parts of hyodeoxycholic acid, 30-45 parts of methionylglycine dipeptide and 60-115 parts of complex plant extract by weight.

2. The fish and shrimp feed bile acid composition according to claim 1, characterized by, The preparation method of the methionylglycine dipeptide is as follows: S1: Boc-Met-Gly-OBzl is dissolved in dichloromethane, and a dichloromethane solution of trifluoroacetic acid is slowly added at 0℃, and the reaction is stirred at room temperature for 60-90 min, and concentrated under reduced pressure to obtain H2N-Met-Gly-OBzl trifluoroacetate; S2: H2N-Met-Gly-OBzl trifluoroacetate is dissolved in deionized water, and the pH is adjusted to 7-8, and then extracted with ethyl acetate, and the organic phase is dried and concentrated to obtain H2N-Met-Gly-OBzl; S3: H2N-Met-Gly-OBzl is dissolved in anhydrous ethyl acetate, and palladium-carbon is added, and hydrogen is introduced, and the air in the system is replaced with hydrogen for 3 times; the reaction is carried out under hydrogen atmosphere at room temperature at 2500 rpm for 2-6 h, and the hydrogen is replaced with nitrogen; the filtrate is concentrated under reduced pressure, purified, freeze-dried to obtain methionylglycine dipeptide; The mass ratio of Boc-Met-Gly-OBzl to dichloromethane is 1:13.25; the volume fraction of the dichloromethane solution of trifluoroacetic acid is 30%.

3. The fish and shrimp feed bile acid composition according to claim 2, characterized in that, The preparation method of the Boc-Met-Gly-OBzl is as follows: S11: Boc-Met-OH and 1-hydroxybenzotriazole are dissolved in anhydrous DMF, and N,N'-dicyclohexyl carbodiimide anhydrous DMF solution is added under stirring at 0℃ for 30 min to obtain an activated ester mixture; S12: H2N-Gly-OBzl p-toluenesulfonic acid salt is dissolved in anhydrous DMF, and N,N-diisopropyl ethylamine is added to obtain a free amine solution; S13: The free amine solution is added dropwise to the activated ester mixture, stirred in an ice bath for 2 h, and then stirred at room temperature for 12-24 h, and the filtrate is concentrated under reduced pressure to obtain a residue; S14: The residue is dissolved in ethyl acetate, and then washed with 10% citric acid aqueous solution, saturated NaHCO3 aqueous solution and saturated brine respectively; the organic phase is separated, dried, filtered, concentrated and purified to obtain Boc-Met-Gly-OBzl.

4. The fish and shrimp feed bile acid composition according to claim 3, characterized by, The mass ratio of Boc-Met-OH to 1-hydroxybenzotriazole is 0.48:0.32; the concentration of N,N'-dicyclohexyl carbodiimide anhydrous DMF solution is 0.09 g / mL.

5. The fish and shrimp feed bile acid composition according to claim 3, characterized in that, The preparation method of the Boc-Met-OH is as follows: methionine is dissolved in a mixture of 1,4-dioxane and 1 mol / L NaOH aqueous solution, and di-tert-butyl dicarbonate and 4-dimethylamino pyridine are slowly added under stirring at 0℃, and the reaction is carried out at room temperature for 2-6 h, and washed with ether for 3 times, and the organic phase is removed, and the water phase is cooled with ice bath, and the pH is adjusted to 2-3, and extracted with ethyl acetate, and the organic phase is collected, washed, dried, filtered, concentrated under reduced pressure, and recrystallized to obtain Boc-Met-OH.

6. The fish and shrimp feed bile acid composition according to claim 5, characterized in that, The mass ratio of the methionine, di-tert-butyl carbonate and 4-dimethylamino pyridine is (0.75-1):1.15:(0.061-0.1); the volume ratio of 1,4-dioxane and 1 mol / L NaOH aqueous solution is 1:

1.

7. The fish and shrimp feed bile acid composition according to claim 3, characterized in that, The preparation method of the H2N-Gly-OBzl p-toluenesulfonate is: uniformly mixing glycine, benzyl alcohol, p-toluenesulfonic acid and anhydrous toluene, refluxing at 120 DEG C for 6-12 hours, reducing to room temperature, neutralizing the acidic substance with saturated NaHCO3 aqueous solution, then extracting with ethyl acetate, collecting the organic phase, washing, drying, then passing in dry hydrogen chloride gas, filtering, collecting the precipitate, washing, drying, to obtain the H2N-Gly-OBzl p-toluenesulfonate.

8. The fish and shrimp feed bile acid composition according to claim 7, characterized in that, The mass ratio of the glycine and p-toluenesulfonic acid is (0.38-0.5):(0.095-0.1); the volume ratio of the benzyl alcohol and anhydrous toluene is 2.7:

30.

9. The fish and shrimp feed bile acid composition according to claim 1, characterized in that, The composite plant extract is composed of Dendrobium officinale extract, ginger extract and Astragalus extract; the mass ratio of the Dendrobium officinale extract, ginger extract and Astragalus extract is (5-15):(10-20):(10-30).

10. A process for the preparation of a fish and shrimp feed bile acid composition according to any one of claims 1 to 9, characterized in that, The pig gallate, chenodeoxycholic acid, hyodeoxycholic acid, methionyl glycine dipeptide and composite plant extract are taken in proportion, mixed uniformly, and then used.

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