Preparation method of traditional Chinese medicine leavening agent for promoting health of livers of aquatic animals

Abstract

The invention relates to a preparation method of a traditional Chinese medicine leavening agent for promoting the health of livers of aquatic animals, which is characterized in that the traditional Chinese medicine leavening agent for promoting the health of livers of aquatic animals is prepared by fermenting traditional Chinese medicine extracts by adopting a conventional culture medium and culture conditions, the content of beneficial metabolites such as short-chain fatty acid in the liver-protecting traditional Chinese medicine extract is increased. According to the present invention, the cetella sobolifera provided by the invention has been preserved in the China General Microbiological Culture Collection Center (CGMCC), and the registration number of the cetella sobolifera is CGMCC NO.31637; the traditional Chinese medicine extract mainly comprises astragalus extract and liquorice extract. Animal experiments show that the leavening agent can effectively promote animal liver health when being used as a feed additive for aquaculture. In conclusion, the invention provides a production technology of the traditional Chinese medicine fermented feed additive with the healthy livers of the aquaculture animals, the hepatobiliary syndrome of the aquatic animals is effectively prevented, and the traditional Chinese medicine fermented feed additive has positive significance in promoting the sustainable development of the aquaculture industry.

Description

Technical Field

[0001] The invention belongs to the technical field of fermented feed, and particularly relates to a method for preparing a traditional Chinese medicine fermentation agent for promoting liver health of aquatic animals. Background Art

[0002] Hepatobiliary syndrome is one of the fish diseases with a high incidence in recent years, causing great losses to the aquaculture industry. This type of disease mainly harms grass carp, crucian carp, carp, black carp, tilapia, spotted forktail, black snakehead, rainbow trout, and bighead bream. It can occur in fish from fingerlings to adult fish, and the mortality rate can be as high as 50% to 90%, causing heavy economic losses to the aquaculture industry. It is generally believed that water quality deterioration, excessive harmful substances in water bodies, infection with pathogenic microorganisms, abuse or excessive use of disinfectants, pesticides, antibiotics or hormone drugs, and excessive feeding are the main causes of fish hepatobiliary syndrome. Single feed formula and improper storage, feed fat oxidation, vitamin deficiency, and excessive carbohydrates are also considered to be the causes of hepatobiliary syndrome; some studies have pointed out that this disease is closely related to lipid metabolism in fish. There is no specific drug for the treatment of hepatobiliary syndrome, so new treatment methods need to be found.

[0003] In recent years, studies have found that short-chain fatty acids are produced by intestinal microorganisms fermenting dietary fiber in food, including acetic acid, propionic acid, and butyric acid. They play an important role in energy metabolism and help maintain the integrity and stability of the intestine, intestinal barrier, and gut-liver axis. Supplementation of short-chain fatty acids can reduce the expression of proinflammatory cytokines in the liver and adipose tissue and improve intestinal permeability. The regulatory effect of short-chain fatty acids on intestinal flora can increase the number of beneficial bacteria and reduce the number of toxin-producing bacteria. In addition, short-chain fatty acids can also improve liver steatosis and inflammation, and increasing the concentration of fecal butyrate can reduce systemic obesity, inflammation, and liver disease in rats fed a high-fat diet.

[0004] Therefore, the present invention ferments the liver-protecting Chinese medicine extract with a strain of native bacteria from the healthy fish intestine that has the ability to regulate lipid metabolism, thereby increasing beneficial ingredients such as short-chain fatty acids, promoting liver health, reducing the occurrence of hepatobiliary syndrome, and promoting the healthy development of aquaculture. Summary of the invention

[0005] The present invention provides a method for preparing a Chinese medicinal fermentation agent for promoting liver health of aquatic animals, aiming to use a Chinese medicinal extract with liver protection effect as a new additive for protecting fish liver health through fermentation products of fish intestinal native bacteria, thereby reducing the hepatobiliary syndrome problem with a high incidence rate in the aquatic industry.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a Chinese medicine fermentation agent for promoting liver health of aquatic animals, comprising healthy fish intestinal native bacteria, GSM culture medium and Chinese medicine extract, wherein the native bacteria is Cetobacterium somerae, and the preparation method of the Chinese medicine fermentation agent for promoting liver health of aquatic animals is:

[0007] The Bacillus sordellii and the Chinese medicine extract are placed in a GSM culture medium and fermented in a fermenter to increase the content of beneficial metabolites such as short-chain fatty acids in the liver-protecting Chinese medicine extract. The fermentation liquid obtained after fermentation is the Chinese medicine fermentation additive.

[0008] Furthermore, the fermentation conditions of the Chinese herbal fermentation agent for promoting liver health of aquatic animals are: rotation speed 150-200rpm, temperature 25-35°C, tank pressure 0.01-0.15MPa, ventilation volume 1:0.5-1, and fermentation time 50-100 hours.

[0009] Furthermore, the preferred fermentation conditions of the traditional Chinese medicine fermentation agent for promoting liver health of aquatic animals are: rotation speed 180 rpm, temperature 28°C, tank pressure 0.10 MPa, ventilation volume 1:0.9, and fermentation time 78 hours.

[0010] Furthermore, the Chinese herbal medicine extract includes astragalus extract, the main component of which is astragalus polysaccharide.

[0011] Furthermore, the Chinese herbal medicine extract includes liquorice extract, the main components of which are glycyrrhizic acid and glycyrrhizin.

[0012] The Cetobacterium somerae provided by the present invention ferments a Chinese medicine extract under conventional culture medium and culture conditions to increase the content of beneficial metabolites such as short-chain fatty acids. Animal experiments show that the fermentation agent can effectively promote animal liver health when used as a feed additive in aquaculture. In summary, the present invention provides a production technology of a Chinese medicine fermented feed additive with liver health for aquaculture animals, effectively prevents hepatobiliary syndrome in aquatic animals, and has positive significance for promoting the sustainable development of the aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a crucian carp serum metabolism parameter diagram of the present invention;

[0014] Figure 2 It is the crucian carp liver pathological staining result of the present invention;

[0015] Figure 3 It is a graph of short-chain fatty acid levels in the intestinal tract of crucian carp of the present invention. DETAILED DESCRIPTION

[0016] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application.

[0017] The present invention is based on Cetobacterium somerae RSDMS001 The strain is a production strain, which has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, and the registration number is CGMCC NO.31637. Cetobacterium somerae is a highly abundant indigenous bacterium in the intestinal flora of many fish. The bacterium participates in multiple metabolisms, including carbohydrate metabolism, amino acid metabolism, membrane transport, coenzyme / vitamin metabolism and energy metabolism, and is of great significance in regulating host lipid metabolism. The bacterium is a new generation of probiotics in aquaculture.

[0018] The present invention uses GSM medium as the basic medium; GSM medium is a standard growth medium, which is used for microbial culture and counting in some studies to evaluate microbial changes in composting processes, etc. The specific components of GSM medium may vary depending on the specific research and application scenarios, but generally include carbon sources (such as sugars such as glucose, which provide energy for microbial growth), nitrogen sources (such as amino acids, proteins, nitrates, etc., which are used for microbial synthesis of biomacromolecules such as proteins), phosphorus sources (such as phosphates, which participate in cell energy metabolism, nucleic acid synthesis, etc.), and sulfur sources (such as sulfates, which are components of certain proteins and biomolecules).

[0019] The liver-protecting Chinese medicines are astragalus and licorice; astragalus and licorice are important Chinese medicines. Astragalus is a qi-tonifying medicine that can tonify qi and raise yang, benefit the stomach and consolidate the exterior, detoxify and promote muscle growth, promote diuresis and reduce swelling, and can improve some symptoms of patients with liver disease. Astragalus extract is extracted from the dried root of astragalus, and the main component is astragalus polysaccharide, which usually contains 70%. Licorice is a common Chinese medicinal material, which generally contains glycyrrhizic acid, liquiritin, licorice flavonoids, polysaccharides, alkaloids and other ingredients. Some of these ingredients, such as glycyrrhizic acid and glycyrrhizin, do have a certain liver-protecting effect in appropriate amounts, can help the liver detoxify and resist inflammation, and even have a certain therapeutic effect on certain liver diseases. Therefore, astragalus and licorice extracts have an auxiliary effect on hepatobiliary syndrome. Chinese medicine polysaccharides can be used as an important source of prebiotics, and can produce short-chain fatty acids after intestinal microbial fermentation.

[0020] In one embodiment, the fermentation conditions are: rotation speed 150-200 rpm, temperature 25-35° C., tank pressure 0.01-0.15 MPa, ventilation volume 1:0.5-1, fermentation time 50-100 hours, fermentation 50-108 hours. The fermentation liquid obtained after fermentation is the traditional Chinese medicine fermentation additive.

[0021] In one embodiment, the fermentation conditions are preferably: rotation speed 180 rpm, temperature 28° C., tank pressure 0.10 MPa, ventilation volume 1:0.9, and fermentation time 78 hours. The fermentation liquid obtained after fermentation is the traditional Chinese medicine fermentation additive.

[0022] The ventilation rate in the present invention refers to the amount of gas passing through a unit volume of fermentation liquid per minute.

[0023] Example 1 In vitro fermentation of Astragalus polysaccharide by Bacillus sordellii

[0024] (1) Experimental groups:

[0025] Blank control group: 4.5 ml basal medium, 0.5 ml 2.0×10 8 CFU / mL of Bacillus sordellii culture solution;

[0026] Astragalus polysaccharide group (APS): 4.5 ml basal medium, 25 mg APS, 0.5 ml 2.0×10 8 CFU / mL of Bacillus sordellii culture solution;

[0027] Fructooligosaccharide group (FOS, control): 4.5 ml basal medium, 25 mg FOS, 0.5 ml 2.0×10 8 CFU / mL of Bacillus sordellii culture solution;

[0028] (2) Take 1 ml of sample at 4h and 8h respectively to measure SCFAs.

[0029] Sample treatment: Take 800μl of fermentation broth, centrifuge at 12000rpm for 15min, transfer the supernatant, add 25% metaphosphoric acid at a ratio of v:v=9:1, shake for 3h, centrifuge at 12000rpm for 15min, filter through a 0.45μm filter to remove impurities, take 1ml of supernatant into a sample bottle, perform GC analysis, and calculate the concentrations of various short-chain fatty acids according to the standard curve.

[0030] The results are shown in Table 1:

[0031]

[0032] Table 1 Concentrations of various short-chain fatty acids at 4h and 8h

[0033] Note: - indicates not detected.

[0034] The results in Table 1 show that the in vitro fermentation of Astragalus polysaccharide by Bacillus sordellii increased the content of total short-chain fatty acids in vitro.

[0035] Example 2 Astragalus polysaccharide-Bacillus sordellii fermentation agent alleviates liver damage caused by trichlorfon

[0036] After two weeks of adaptation, 60 crucian carps were randomly distributed into five 50L aquariums, with 12 in each group, for a total of 5 groups. They were randomly divided into control group (Con), model group (MG), cetacean group (Cet), astragalus polysaccharide group (APS) and fermentation culture group (CT).

[0037] The control group (Con) and the model group (MG) were fed with basic feed, the Cetacean group (Cet) was fed with feed containing bacteria, the APS group (AP) was fed with basic feed containing APS, and the starter group (CT) was fed with feed containing starter.

[0038] Feed for 30 days, feed slowly during feeding to ensure that crucian carp can quickly eat the feed and reduce the loss of Bacillus cereus and astragalus polysaccharide in the water. In addition, MG, Cet, AP and CT groups were exposed to 2 mg / L trichlorfon solution, and trichlorfon was calculated and supplemented after daily water change. After 30 days of feeding, crucian carp were sampled 12 hours after the last feeding.

[0039] refer to Figure 1, the levels of total cholesterol (TC) (Figure a), triglycerides (TG) (Figure b), low-density lipoprotein cholesterol (LDL-C) (Figure c), high-density lipoprotein cholesterol (HDL-C) (Figure d), alanine aminotransferase (ALT) (Figure e) and aspartate aminotransferase (AST) (Figure f) in serum were detected by kits, among which *P<0.05, **P<0.01. Compared with the control group, trichlorfon significantly increased the concentrations of TC, TG, and LDL-C in the serum of crucian carp, and significantly decreased the concentration of HDL-C. The three treatment groups alleviated these abnormal changes to varying degrees. This shows that the fermentation agent improves the lipid metabolism of crucian carp. Exposure to trichlorfon led to an increase in the levels of AST and ALT in the serum of crucian carp, while the fermentation agent decreased these two indicators.

[0040] These results indicate that exposure to trichlorfon can lead to abnormalities in serum metabolic parameters in crucian carp. Increased AST and ALT are sensitive indicators of hepatocellular damage, suggesting that trichlorfon can cause liver damage in crucian carp. The fermentation agent improved serum metabolic parameters and alleviated hepatocellular damage.

[0041] refer to Figure 2 , the liver tissue was sectioned and embedded using the hematoxylin and eosin H&E staining method (Figure a, ×100), and the damage to the liver tissue was confirmed under an optical microscope. The results showed that the hepatocytes of the control group were richly and tightly arranged. After 30 days of dipterex immersion, the crucian carp showed obvious liver cavitation (indicated by the arrow in Figure a). After feeding the fermentation agent, the size and frequency of liver cavitation were significantly reduced. Oil red O staining of liver tissue (Figure b, ×200) further showed that after exposure to dipterex, a large amount of lipids were deposited in the liver of crucian carp. After treatment, the abnormal accumulation of liver lipids was reduced. Among them, the CT group was closest to the Con group, with neatly arranged liver cell structures, the greatest reduction in lipid accumulation, and improved liver damage.

[0042] The pathological staining results of this example showed that exposure to trichlorfon caused liver damage and abnormal lipid accumulation in crucian carp, while fermentation agents improved liver damage and reduced liver lipid accumulation.

[0043] refer to Figure 3 , the content of short-chain fatty acids in the intestine of crucian carp was determined, among which Figure a shows the acetic acid level of each group, Figure b shows the propionic acid level, and Figure c shows the butyric acid level, *P<0.05, **P<0.01. Exposure to trichlorfon caused a decrease in the acetic acid level in the intestinal contents. Compared with the MG group, the acetic acid levels in the Cet group and the AP group increased, and the fermentation agent group treatment significantly increased the acetic acid level in the intestine. In addition, compared with the control group, there was no significant change in the propionic acid and butyric acid levels in the MG group. Compared with the MG group, the butyric acid levels in the CT group and the AP group increased.

[0044] The results showed that after exposure to trichlorfon, the content of short-chain fatty acids in the intestine of crucian carp did not change significantly. However, the use of fermentation agents could increase the content of short-chain fatty acids in the intestine, especially acetic acid and butyric acid.

[0045] Animal experiments have shown that the fermentation agent has a protective effect on the liver. The fermentation agent can be used as a feed additive in aquaculture to effectively promote animal liver health.

[0046] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing a Chinese medicinal fermentation agent for promoting liver health of aquatic animals, characterized in that: The native bacteria and the Chinese medicine extract are placed in a GSM culture medium and fermented in a fermenter. The fermentation liquid obtained after fermentation is the Chinese medicine fermentation additive. The native bacteria is Bacillus sordellii.

2. The method for preparing a Chinese medicinal fermentation agent for promoting liver health of aquatic animals according to claim 1, characterized in that: The temperature of the fermentation tank is 25-35° C., the rotation speed is 150-200 rpm, the tank pressure is 0.01-0.15 MPa, the ventilation volume is 1:0.5-1, and the fermentation time is 50-108 hours.

3. The method for preparing a Chinese medicinal fermentation agent for promoting liver health of aquatic animals according to claim 2, characterized in that: The fermentation tank has a rotation speed of 180 rpm, a temperature of 28° C., a tank pressure of 0.10 MPa, a ventilation volume of 1:0.9, and a fermentation time of 78 hours.

4. The method for preparing a Chinese medicinal fermentation agent for promoting liver health of aquatic animals according to claim 1, characterized in that: The Chinese medicine extract comprises astragalus extract, the main component of which is astragalus polysaccharide.

5. The method for preparing a Chinese medicinal fermentation agent for promoting liver health of aquatic animals according to claim 1, characterized in that: The traditional Chinese medicine extract comprises liquorice extract, and the main components are glycyrrhizic acid and glycyrrhizin.

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