A composition for preventing fatty liver of aquatic animals and a method for preparing the same

Abstract

This invention relates to a composition for preventing fatty liver in aquatic animals and its preparation method, belonging to the technical field of aquatic feed additives. The composition comprises the following components in parts by weight: 20-50 parts of a fermentation composition, 5-15 parts of bile acids, 20-50 parts of taurine, and 10-30 parts of a carrier; wherein the fermentation composition is prepared by fermenting licorice, Atractylodes macrocephala, and Schizochytrium powder using Clostridium butyricum. Animal experiments of this invention show that adding 0.5% of the composition of this invention to a high-soybean meal feed for grass carp, after continuous feeding for 8 weeks, resulted in better liver-to-body ratio, liver triglycerides, serum ALT, and AST activity data compared to the control group. The composition of this invention, through fermentation and synergistic compounding, improves the interference of non-starch polysaccharides on the enterohepatic circulation of bile acids through multiple pathways such as intestinal repair, exogenous supplementation, and promotion of fat transport. The raw materials are widely available, the preparation process is simple, and it is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of aquatic feed additive technology, and relates to a composition for preventing fatty liver in aquatic animals and its preparation method. Background Technology

[0002] Fatty liver disease in aquatic animals is a common metabolic disease characterized by abnormal fat accumulation in the liver, falling under the category of hepatobiliary syndrome. It has become a prevalent disease in aquaculture over the past decade, with the peak season from June to October. It affects a variety of fish, including grass carp, tilapia, largemouth bass, large yellow croaker, and grouper, and can affect both juvenile and adult fish. Fatty liver often disrupts various metabolic functions, leading to decreased immunity and stress resistance, which in turn triggers bacterial and viral infections, resulting in hepatobiliary syndrome and causing large-scale, widespread mortality, resulting in significant losses to the aquaculture industry.

[0003] In the early stages of fish hepatobiliary syndrome, there are no obvious external symptoms. Upon dissection, the liver is found to be significantly enlarged, and in some cases, parts or most of the liver may turn green. As the disease progresses, the liver turns yellow or white, or exhibits a patchy yellow-red-white "flowery liver" symptom. The liver is easily broken upon light touch, and the gallbladder is significantly enlarged. Due to severe lesions in the major organs, the body's resistance to disease decreases, often accompanied by symptoms such as hemorrhage, gill rot, and enteritis.

[0004] Studies have shown that excessive non-starch polysaccharides in feed are a major cause of hepatobiliary syndrome. Non-starch polysaccharides hinder the reabsorption of bile acids in the intestine through physical encapsulation, thereby inducing the liver to continuously synthesize bile acids. Excessive secretion of bile acids leads to excessive storage in the gallbladder, and even overflow, resulting in "green liver." Simultaneously, the absorption rate of dietary fat decreases, and the protective effect of fat in hepatocytes increases, manifesting as fatty liver. Prolonged overload of liver secretion leads to functional failure and various secondary diseases, and in severe cases, death. Therefore, the prevention and treatment of hepatobiliary syndrome should begin with eliminating the interference of non-starch polysaccharides on the bile acid cycle.

[0005] In the prior art, patent CN106923082A discloses a method for adding bile acids or taurine to feed, but this solution only compensates by directly adding exogenous bile acids and fails to address the problem from the perspective of intestinal repair and promoting endogenous reabsorption. Patent CN113383874A discloses a compound glycolipid metabolism activator, which uses Lactobacillus and does not involve the process of fermenting and transforming traditional Chinese medicine.

[0006] Existing technologies do not yet provide a comprehensive solution that can simultaneously improve intestinal repair, promote endogenous bile acid reabsorption, supplement bile acids exogenously, and reduce hepatic fat deposition. Summary of the Invention

[0007] To address the shortcomings of existing technologies that only compensate by adding bile acids exogenously without considering intestinal repair and promoting endogenous reabsorption, this invention provides a composition for preventing fatty liver in aquatic animals and its preparation method.

[0008] The objective of this invention can be achieved through the following technical solutions: In a first aspect, the present invention provides a composition for preventing fatty liver in aquatic animals, comprising the following components in parts by weight: 20-50 parts of fermentation composition; Bile acids 5-15 parts; Taurine 20-50 parts; And 10-30 copies of the carrier; The fermentation composition is prepared by fermenting licorice, Atractylodes macrocephala and Schizochytrium powder with Clostridium butyricum.

[0009] The fermentation composition refers to a complex obtained by anaerobic fermentation and drying of Clostridium butyricum as the fermentation strain and licorice, Atractylodes macrocephala and Schizochytrium powder as fermentation substrates. It contains Clostridium butyricum cells and its metabolites (butyric acid, enzymes, vitamins, etc.), as well as hepatoprotective active ingredients such as glycyrrhetinic acid and hydroxyatractylyl lactone that may be produced or released after biotransformation.

[0010] Clostridium butyricum is an anaerobic, Gram-positive spore-forming bacillus that produces metabolites such as butyric acid and butyric acid clostridium, which have functions in regulating intestinal flora, repairing the intestinal barrier, and promoting nutrient absorption. The Clostridium butyricum used in this invention can be commercially available feed-grade live c. butyricum powder or spore powder with a live bacteria content ≥1×10⁻⁶. 8 CFU / g.

[0011] Preferably, the weight ratio of bile acid to taurine is 1:4 to 1:6.

[0012] Bile acids are steroidal compounds produced by the metabolism of cholesterol in the liver. They have the functions of emulsifying fats, promoting the absorption of fat-soluble vitamins, and regulating lipid metabolism.

[0013] Taurine is a sulfur-containing amino acid that combines with bile acids in the body to form taurocholic acid, one of the main forms of bile acids. It promotes fat transport, protects liver cells, and also has independent antioxidant and growth-promoting functions.

[0014] Preferably, the carrier is selected from at least one of zeolite powder, wheat bran, corn cob powder, and starch.

[0015] Non-starch polysaccharides hinder the reabsorption of bile acids in the intestine through physical encapsulation, leading to interruption of the enterohepatic circulation. Upon receiving the false signal of "insufficient bile acids," the liver continuously synthesizes bile acids in a compensatory manner, causing the liver to work overtime, the gallbladder to become overfilled, and even bile reflux (green liver). Simultaneously, insufficient bile acids lead to decreased absorption of dietary fat and increased fat protection in hepatocytes, resulting in fatty liver.

[0016] To address the above problems, the composition of the present invention is obtained by fermenting licorice, Atractylodes macrocephala and Schizochytrium powder with Clostridium butyricum, and then combined with bile acids and taurine to work synergistically through the following three pathways: (1) Clostridium butyricum metabolites repair the intestinal barrier and promote the active reabsorption of bile acids; (2) exogenous bile acids are directly supplemented to reduce the synthetic burden on the liver; (3) taurine promotes fat transport and bile secretion.

[0017] In addition, Clostridium butyricum can secrete cellulase, amylase and protease during fermentation, which is beneficial to increasing the content of glycyrrhetinic acid active ingredients in the composition and to the release or structural modification of atractylodes lactone components. At the same time, docosahexaenoic acid in Schizochytrium powder, as a polyunsaturated fatty acid, plays a role in reducing liver fat deposition and improving stress resistance and immunity.

[0018] Secondly, the present invention provides a method for preparing a composition for preventing fatty liver in aquatic animals, comprising the following steps: (1) Glycyrrhiza and Atractylodes macrocephala are pulverized and mixed with Schizochytrium powder, carbon source and nitrogen source, and water is added to prepare a fermentation culture medium; after sterilization, Clostridium butyricum seed liquid is inoculated and anaerobic fermentation is carried out at 30-37℃ for 48-72 hours; the fermentation liquid is dried to obtain the fermentation composition; (2) Mix bile acids, taurine and carrier evenly to obtain a premix; (3) Mix the fermentation composition of step (1) with the premix of step (2) for 10-20 minutes to obtain the composition.

[0019] Preferably, the amount of *Schizochytrium* powder added is 5-20 g / L; the carbon source is glucose or corn flour, and the amount added is 10-30 g / L; the nitrogen source is yeast powder or ammonium sulfate, and the amount added is 5-15 g / L.

[0020] Preferably, the weight ratio of licorice to Atractylodes macrocephala is 1:1 to 2:1.

[0021] Preferably, the inoculation amount of the Clostridium butyricum seed solution is 5%-10%.

[0022] Preferably, the drying method in step (1) is spray drying. Spray drying has the characteristics of fast drying speed, short material heating time, and suitability for continuous industrial production, which can ensure that the strain has a high survival rate.

[0023] Preferably, cellulase is added to the fermentation medium in step (1), and the amount added is 0.1%-1% of the total substrate.

[0024] Thirdly, the present invention provides the application of a composition for preventing fatty liver in aquatic animals in aquatic animal feed: the composition is added to the feed at an amount of 0.1%-1.0% of the total weight of the feed.

[0025] The beneficial effects of this invention are as follows: This invention utilizes *Clostridium butyricum* anaerobic fermentation of licorice, *Atractylodes macrocephala*, and *Schizochytrium* powder to obtain a fermented composition. This composition is then combined with bile acids and taurine to synergistically improve the interference of non-starch polysaccharides on the enterohepatic circulation of bile acids through three pathways: intestinal repair, exogenous supplementation, and promotion of fat transport, thereby preventing fatty liver in aquatic animals. Animal experiments have verified that the fermented composition, combined with bile acids and taurine, can significantly reduce the liver-to-body ratio, liver triglyceride content, and serum ALT and AST activities. The raw materials for this composition are widely available, the preparation process is simple and feasible, suitable for industrial production, and can be widely used in aquatic animal feed. Detailed Implementation

[0026] To further illustrate the technical means and effects of the present invention in achieving the intended purpose, the following detailed description of the specific implementation methods, structures, features and effects of the present invention, in conjunction with preferred embodiments, is provided below.

[0027] Example 1 A method for preparing a composition for preventing fatty liver in aquatic animals includes the following steps: (1) Take 20 parts of licorice and 10 parts of Atractylodes macrocephala, grind them to 100 mesh, mix them with 10 parts of Schizochytrium globosum powder, 20 parts of corn flour and 10 parts of yeast powder, and add water at a solid-liquid ratio of 1:10 (parts by weight:parts by volume); adjust the pH to 7.0 with sodium hydroxide solution. Put the fermentation medium into an anaerobic fermenter, sterilize at 121℃ for 20 minutes, and cool to 35℃. Inoculate with Clostridium butyricum seed liquid, the inoculation amount is 10% of the fermentation medium volume (live bacteria content 1×10⁻⁶). 8 The fermentation broth (CFU / mL) was fermented at 35°C under anaerobic conditions for 60 hours. After fermentation, the fermentation broth was spray-dried (inlet air temperature 100°C, outlet air temperature 60°C) to obtain the fermentation composition.

[0028] (2) Mix 10 parts of bile acid, 40 parts of taurine and 20 parts of zeolite powder evenly to obtain a premix; (3) Take 30 parts of the fermentation composition from step (1) and mix it with the premix from step (2). Mix the mixture in a three-dimensional mixer for 15 minutes to obtain the composition. Vacuum package the mixture to obtain the finished product.

[0029] Example 2 A method for preparing a composition for preventing fatty liver in aquatic animals includes the following steps: (1) Take 25 parts of licorice and 15 parts of Atractylodes macrocephala, grind them to 100 mesh, mix them with 15 parts of Schizochytrium powder, 25 parts of glucose and 10 parts of ammonium sulfate, and add water at a solid-liquid ratio of 1:10 (parts by weight:parts by volume); adjust the pH to 6.8 with sodium hydroxide solution. Put the fermentation medium into an anaerobic fermenter, sterilize at 121℃ for 20 minutes, and cool to 37℃. Inoculate with Clostridium butyricum seed liquid, the inoculation amount is 6% of the fermentation medium volume (live bacteria content 1×10⁻⁶). 8 The fermentation broth (CFU / mL) was fermented at 37°C under anaerobic conditions for 72 hours. After fermentation, the fermentation broth was spray-dried (inlet air temperature 100°C, outlet air temperature 60°C) to obtain the fermentation composition.

[0030] (2) Mix 8 parts of bile acid, 48 parts of taurine and 20 parts of corn cob powder evenly to obtain a premix; (3) Take 40 parts of the fermentation composition from step (1) and mix it with the premix from step (2). Mix the mixture in a three-dimensional mixer for 15 minutes to obtain the composition. Vacuum package the mixture to obtain the finished product.

[0031] Example 3 A method for preparing a composition for preventing fatty liver in aquatic animals includes the following steps: (1) Take 20 parts of licorice and 10 parts of Atractylodes macrocephala, grind them to 100 mesh, mix them with 10 parts of Schizochytrium globosum powder, 20 parts of corn flour, 10 parts of yeast powder, and 0.5 parts of cellulase, and add water at a solid-liquid ratio of 1:10 (parts by weight:parts by volume); adjust the pH to 7.0 with sodium hydroxide solution. Put the fermentation medium into an anaerobic fermenter, sterilize at 121℃ for 20 minutes, and cool to 35℃. Inoculate with Clostridium butyricum seed liquid, the inoculation amount is 8% of the fermentation medium volume (live bacteria content 1×10⁻⁶). 8 The fermentation broth (CFU / mL) was fermented at 35°C under anaerobic conditions for 60 hours. After fermentation, the fermentation broth was spray-dried (inlet air temperature 100°C, outlet air temperature 60°C) to obtain the fermentation composition.

[0032] (2) Mix 10 parts of bile acid, 40 parts of taurine and 20 parts of zeolite powder evenly to obtain a premix; (3) Take 30 parts of the fermentation composition from step (1) and mix it with the premix from step (2). Mix the mixture in a three-dimensional mixer for 15 minutes to obtain the composition. Vacuum package the mixture to obtain the finished product.

[0033] Comparative Example 1 Mix 10 parts bile acid, 40 parts taurine, and 50 parts zeolite powder evenly in a three-dimensional mixer for 15 minutes to obtain the composition. Vacuum package to obtain the finished product.

[0034] Comparative Example 2 (1) Take 20 parts of licorice and 10 parts of Atractylodes macrocephala, grind them to 100 mesh, mix them with 10 parts of Schizochytrium globosum powder, 20 parts of corn flour and 10 parts of yeast powder, and add water at a solid-liquid ratio of 1:10 (parts by weight:parts by volume); adjust the pH to 7.0 with sodium hydroxide solution. Put the fermentation medium into an anaerobic fermenter, sterilize at 121℃ for 20 minutes, and cool to 35℃. Inoculate with Clostridium butyricum seed liquid, the inoculation amount is 10% of the fermentation medium volume (live bacteria content 1×10⁻⁶). 8 The fermentation broth (CFU / mL) was fermented at 35°C under anaerobic conditions for 60 hours. After fermentation, the fermentation broth was spray-dried (inlet air temperature 100°C, outlet air temperature 60°C) to obtain the fermentation composition.

[0035] (2) Take 30 parts of the fermentation composition from step (1), mix it evenly with 20 parts of zeolite powder and 50 parts of corn starch to obtain the composition; vacuum pack it to obtain the finished product.

[0036] Comparative Example 3 (1) Take 20 parts of licorice and 10 parts of Atractylodes macrocephala, grind them to 100 mesh, mix them with 10 parts of Schizochytrium powder and 1 part of Clostridium butyricum powder, and spray dry them directly without fermentation (inlet air temperature 100℃, outlet air temperature 60℃) to obtain a mixture. (2) Mix 10 parts of bile acid, 40 parts of taurine and 20 parts of zeolite powder evenly to obtain a premix; (3) Take 30 parts of the mixture from step (1) and mix it with the premix from step (2). Mix in a three-dimensional mixer for 15 minutes to obtain the composition; vacuum package to obtain the finished product.

[0037] animal experiments Grass carp (initial weight approximately 50±5g) were used as experimental subjects and randomly divided into groups of 3 replicates each, with 30 fish per replicate. The experimental diet consisted of a high soybean meal diet, with different combinations added at 0.5% of the total feed weight. A blank control group was also included. The rearing period was 8 weeks.

[0038] The experiment was conducted in a recirculating aquaculture system, with the water temperature controlled at 28±2℃, dissolved oxygen ≥5.0mg / L, and pH 7.0-8.0. Fish were fed three times daily (8:00, 12:00, and 16:00) at a rate of 4% of their body weight, with the feeding amount adjusted weekly.

[0039] After the aquaculture experiment, the fish were fasted for 24 hours. Ten fish were randomly selected from each replicate, their body weight was measured, blood was collected from the tail vein, serum was separated, and stored at -20℃ for later use. The liver was dissected, weighed, and stored at -80℃ for biochemical index detection.

[0040] Detection indicators and methods:

[0041] Experimental results:

[0042] Compared with the blank control group, the compositions of Examples 1-3 of this invention significantly reduced the liver-to-body ratio, liver triglyceride content, and serum ALT and AST activities in grass carp (liver-to-body ratio decreased from 3.25% to 1.96-2.11%, liver triglycerides decreased from 46.2 mg / g to 23.8-25.2 mg / g, serum ALT decreased from 68.5 U / L to 28.6-34.1 U / L, and serum AST decreased from 132.3 U / L to 68.2-81.3 U / L), indicating that the compositions of this invention can effectively prevent fatty liver induced by high non-starch polysaccharide plant protein feed in aquatic animals. Among them, Example 3 showed better results, indicating that the addition of cellulase can further improve fermentation efficiency and preventive effect. At the same time, the effects of Examples 1-3 were significantly better than those of the comparative example, indicating that there is a synergistic effect between the fermentation composition and bile acids and taurine, and that the fermentation process makes an important contribution to improving the effect of the composition.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A composition for preventing fatty liver in aquatic animals, characterized in that, It contains the following components in parts by weight: 20-50 parts of fermentation composition; Bile acids 5-15 parts; Taurine 20-50 parts; And 10-30 copies of the carrier; The fermentation composition is prepared by fermenting licorice, Atractylodes macrocephala and Schizochytrium powder with Clostridium butyricum.

2. The composition for preventing fatty liver in aquatic animals according to claim 1, characterized in that, The weight ratio of bile acids to taurine is 1:4 to 1:

6.

3. The composition for preventing fatty liver in aquatic animals according to claim 1, characterized in that, The carrier is selected from at least one of zeolite powder, wheat bran, corn cob powder, and starch.

4. A method for preparing the composition for preventing fatty liver in aquatic animals as described in claim 1, characterized in that, Includes the following steps: (1) Glycyrrhiza and Atractylodes macrocephala are pulverized and mixed with Schizochytrium powder, carbon source and nitrogen source, and water is added to prepare a fermentation culture medium; after sterilization, Clostridium butyricum seed liquid is inoculated and anaerobic fermentation is carried out at 30-37℃ for 48-72 hours; the fermentation liquid is dried to obtain the fermentation composition; (2) Mix bile acids, taurine and carrier evenly to obtain a premix; (3) Mix the fermentation composition of step (1) with the premix of step (2) for 10-20 minutes to obtain the composition.

5. The method for preparing the composition for preventing fatty liver in aquatic animals according to claim 4, characterized in that, The amount of *Schizochytrium* powder added is 5-20 g / L; the carbon source is glucose or corn flour, and the amount added is 10-30 g / L; the nitrogen source is yeast powder or ammonium sulfate, and the amount added is 5-15 g / L.

6. The method for preparing the composition for preventing fatty liver in aquatic animals according to claim 4, characterized in that, The weight ratio of licorice to Atractylodes macrocephala is 1:1 to 2:

1.

7. The method for preparing the composition for preventing fatty liver in aquatic animals according to claim 4, characterized in that, The inoculation amount of the Clostridium butyricum seed solution is 5%-10%.

8. The method for preparing the composition for preventing fatty liver in aquatic animals according to claim 4, characterized in that, The drying method in step (1) is spray drying.

9. The method for preparing the composition for preventing fatty liver in aquatic animals according to claim 4, characterized in that, Cellulase is also added to the fermentation medium in step (1), and the amount added is 0.1%-1% of the total substrate.

10. The use of the composition for preventing fatty liver in aquatic animals according to any one of claims 1-3 in aquatic animal feed, characterized in that, The composition is added to the feed at a rate of 0.1%-1.0% of the total feed weight.

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