Preparation method of microbial compound microbial agent and chinese herbal medicine compound microbial agent and application thereof in offshore fish culture

Abstract

The application discloses a microbial compound microbial agent and a preparation method and application of Chinese herbal medicine compound microbial agent in offshore fish culture, and belongs to the technical field of microbial engineering. The microbial compound microbial agent comprises bacillus megaterium and rhodotorula mucilaginosa. In addition, the application further provides a preparation method of the Chinese herbal medicine compound microbial agent, which comprises the following steps: mixing the microbial compound microbial agent, Chinese herbal medicine and base fluid and then fermenting. In addition, the application further provides a Chinese herbal medicine compound microbial agent prepared by the preparation method. In addition, the application further provides a feed, and the mass addition amount of the microbial compound microbial agent prepared by the preparation method in the feed is 5%-10%. In addition, the application further provides application of the Chinese herbal medicine compound microbial agent prepared by the preparation method in offshore fish culture. The microbial compound microbial agent can be used for Chinese herbal medicine fermentation, and after being fed to fish, the fish obesity problem caused by artificial sweeteners can be effectively improved.

Description

Technical Field

[0001] This invention relates to the field of microbial engineering technology, specifically to a method for preparing a microbial compound agent and a traditional Chinese medicine compound agent, and their application in nearshore fish farming. Background Technology

[0002] With increasing demand for marine fish products and the development of aquaculture technology, near-shore fish farming has developed rapidly in my country and has become an important part of my country's agriculture. Seafood is a vital component of the global food system, and its contribution to food security and nutrition supply in the 21st century is increasingly significant. It is estimated that by 2050, 12–25% of the increased demand for high-quality protein will come from marine aquaculture products. Artificial sweeteners are now considered a new type of pollutant, characterized by their poor metabolism and degradation in the human body, as well as their ability to persist and accumulate in the environment. Currently, acesulfame potassium is present in seawater worldwide (1.9 × 10⁻⁶). -4 –9.9μg L -1 The presence of acesulfame potassium in the liver of carp poses a threat to the health of aquatic ecosystems and inhibits the development of nearshore fish aquaculture. Previous studies have indicated that acesulfame potassium can accumulate in the liver of carp.

[0003] Currently, in nearshore fish farming, the addition of artificial sweeteners leads to excessive obesity in fish. How to improve the obesity problem in fish is a technical problem that needs to be solved by existing technologies. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide a method for preparing microbial compound inoculants and traditional Chinese medicine compound inoculants and their application in nearshore fish farming, thereby solving the technical problem of how to effectively improve obesity caused by artificial sweeteners in nearshore fish in the prior art.

[0005] To achieve the above-mentioned technical objectives, the present invention provides a microbial compound inoculant, comprising Bacillus megaterium and Rhodotorula glutinis.

[0006] In any embodiment, the mass ratio of the Bacillus megaterium and the Rhodotorula glutinis powder is (5-15):(15-30), and the powder is prepared by the following steps:

[0007] The number of Bacillus megaterium and Rhodotorula glutinis cultured in the culture medium to the bacterial culture volume were 1×10⁻⁶. 8 -1×10 9 CFU / mL and 1×10 8 -3×10 8 The concentration was CFU / mL, then purified and freeze-dried to obtain the final product.

[0008] In any embodiment, the Bacillus megaterium or the Rhodotorula glutinis is cultured in a culture medium containing artificial sweeteners.

[0009] In addition, the present invention also proposes a method for preparing a compound microbial agent of traditional Chinese medicine, comprising the following steps: mixing the above-mentioned compound microbial agent, traditional Chinese medicine and base liquid and then fermenting, wherein the amount of the compound microbial agent added is 2.5%-7.5% of the mass of the base liquid, the fermentation temperature is 25-37℃, and the pH value is 6.5-7.5;

[0010] The Chinese herbal medicines mentioned include seaweed powder, prepared rehmannia root, pomegranate peel, scutellaria baicalensis, hawthorn, licorice, and dried tangerine peel;

[0011] The amount of the traditional Chinese medicine added to the base solution is 50-75 g / L.

[0012] In any embodiment, the base liquid comprises 30-50 g / L sucrose, 10-15 g / L casein, 5-15 g / L cellulose, 0.2-0.5 g / L calcium chloride, 0.1-0.3 g / L potassium dihydrogen phosphate, and 1-3 g / L urea.

[0013] In any embodiment, the traditional Chinese medicine, calculated by weight, includes 5-10 parts of seaweed powder, 5-10 parts of prepared rehmannia root, 5-10 parts of pomegranate peel, 5-10 parts of scutellaria root, 5-10 parts of hawthorn, 5-10 parts of licorice root, and 5-10 parts of dried tangerine peel.

[0014] In any embodiment, the fermentation time is 48-96 hours; and / or, the viable counts of both Bacillus megaterium and Rhodotorula glutinis after fermentation are ≥5×10⁻⁶. 3 CFU / mL.

[0015] In addition, the present invention also proposes a compound microbial agent of traditional Chinese medicine, which is prepared by the above preparation method.

[0016] In addition, the present invention also proposes a feed in which the microbial compound agent prepared by the above preparation method or the above microbial compound agent is added to the feed at a mass ratio of 5%-10%.

[0017] Furthermore, the present invention also proposes a compound microbial agent of traditional Chinese medicine prepared by the above preparation method or the application of the above compound microbial agent of traditional Chinese medicine in nearshore fish farming.

[0018] Compared with existing technologies, the beneficial effects of this invention include: the microbial compound inoculant proposed in this invention can be used for the fermentation of traditional Chinese medicine, and subsequent feeding to fish can effectively improve the obesity problem in fish caused by artificial sweeteners. Furthermore, it has been found to effectively alleviate the effect of acesulfame potassium on reduced fish schooling behavior and can increase the protein content of fish. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the acesulfame K exposure experiment design of the present invention.

[0020] Figure 2 This invention describes the effects of different treatments on the weight gain rate of black sea bream.

[0021] Figure 3 This invention describes the effects of different treatments on the specific growth rate of black sea bream.

[0022] Figure 4 This invention describes the effects of different treatments on the fatness of black sea bream.

[0023] Figure 5 This invention describes the effect of different treatments on the nearest neighbor distance of black sea bream.

[0024] Figure 6 This invention relates to the effect of different treatments on the synchronization of swimming speed in black sea bream.

[0025] Figure 7 This invention describes the effect of different treatments on the protein content of black sea bream meat. Detailed Implementation

[0026] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60–120 and 80–110 are listed for a specific parameter, it is understood that ranges of 60–110 and 80–120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0027] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0028] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0029] This specific embodiment provides a microbial compound inoculant, comprising Bacillus megaterium and Rhodotorula glutinis, wherein the mass ratio of the corresponding inoculant powders of Bacillus megaterium and Rhodotorula glutinis is (5-15):(15-30), and the inoculant powder is prepared by the following steps:

[0030] Bacillus megaterium and Rhodotorula glutinis were cultured in culture medium at 25-35℃ and pH 5.5-8.5 for 8-36 hours, respectively, until the bacterial count in the culture solution reached 1×10⁻⁶. 8 -1×10 9 CFU / mL and 1×10 8 -3×10 8 The concentration was CFU / mL, then purified and freeze-dried to obtain the final product.

[0031] In some embodiments, the Bacillus megaterium or the Rhodotorula glutinis is cultured in a culture medium containing artificial sweeteners.

[0032] This specific embodiment provides a method for preparing a compound microbial agent of traditional Chinese medicine, including the following steps: mixing the compound microbial agent, traditional Chinese medicine, and base liquid, and then fermenting for 48-96 hours; the amount of the compound microbial agent added is 2.5%-7.5% of the mass of the base liquid; the fermentation temperature is 25-37℃; and the pH value is 6.5-7.5; the viable counts of Bacillus megaterium and Rhodotorula glutinis after fermentation are both ≥5×10⁻⁶. 3 CFU / mL;

[0033] The Chinese herbal medicines mentioned include seaweed powder, prepared rehmannia root, pomegranate peel, scutellaria baicalensis, hawthorn, licorice, and dried tangerine peel;

[0034] The amount of the traditional Chinese medicine added to the base solution is 50-75 g / L.

[0035] In some embodiments, the base solution comprises 30-50 g / L sucrose, 10-15 g / L casein, 5-15 g / L cellulose, 0.2-0.5 g / L calcium chloride, 0.1-0.3 g / L potassium dihydrogen phosphate, and 1-3 g / L urea.

[0036] This specific embodiment also proposes a compound herbal microbial agent, which is prepared by the above preparation method.

[0037] This specific embodiment also proposes a feed in which the herbal compound microbial agent prepared by the above preparation method or the herbal compound microbial agent is added to the feed at a mass ratio of 5%-10%.

[0038] This specific embodiment also proposes a compound microbial agent of traditional Chinese medicine prepared by the above preparation method or the application of the above compound microbial agent of traditional Chinese medicine in nearshore fish farming.

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] In this invention, the terms "some embodiments," "this embodiment," and examples are used to describe a subset of all possible embodiments. However, it is understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with each other without conflict.

[0041] If the application documents contain similar descriptions such as "first / second", the following explanation shall be added: In the following description, the terms "first / second / third" are used only to distinguish similar objects and do not represent a specific ordering of objects. It is understood that "first / second / third" may be interchanged in a specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0042] In this embodiment, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, object A and / or object B can represent three situations: object A exists alone, object A and object B exist simultaneously, and object B exists alone.

[0043] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0044] The sources of Bacillus megaterium and Rhodotorula glutinis in the following examples or comparative examples are as follows:

[0045] Bacillus megaterium PP84, deposited by China General Microbiological Culture Collection Center (CGMCC) on July 20, 2016, accession number: CGMCC No. 12798.

[0046] Rhodotorula mucilaginosa OP11, deposited by China General Microbiological Culture Collection Center (CGMCC) on January 6, 2017, with accession number CGMCC No. 13540.

[0047] Example 1

[0048] This embodiment proposes a microbial compound agent, including Bacillus megaterium and Rhodotorula glutinis.

[0049] Bacillus megaterium and Rhodotorula glutinis are prepared by the following steps:

[0050] Bacillus megaterium was cultured in the corresponding culture medium at 30°C and pH 6.5 for 20 hours.

[0051] Rhodotorula glutinis was cultured in the corresponding culture medium at 30°C and pH 6.5 for 25 hours.

[0052] The culture medium for *Bacillus megaterium* consisted of: 10 g / L glucose, 5 g / L soybean meal, 2 g / L corn flour, 0.2 g / L magnesium sulfate, 5.0 g / L bovine peptone, 5 g / L NaCl, 0.5 g / L dipotassium hydrogen phosphate, 0.3 g / L calcium carbonate, and 1 mg / L acesulfame potassium (artificial sweetener). The pH was natural. The addition of acesulfame potassium facilitated symbiosis and served to acclimatize the strain.

[0053] The Rhodotorula glutinis culture medium consisted of: 20 g / L malt extract, 20 g / L glucose, 1.5 g / L tryptone, 0.5 g / L soybean peptone, and 1 mg / L acesulfame potassium (artificial sweetener), with a natural pH. The addition of acesulfame potassium facilitated symbiosis and served to acclimatize the strain.

[0054] Strain propagation: The viability of the cultured *Bacillus megaterium* and *Rhodotorula glutinis* was tested, and strains with high viability were selected for propagation. The propagation medium was the same as described above. The viable cell counts of the propagated *Bacillus megaterium* and *Rhodotorula glutinis* cultures were 2 × 10⁻⁶. 8 CFU / mL and 6×10 7 CFU / mL;

[0055] Preparation of bacterial powder: Bacillus megaterium and Rhodotorula glutinis colonies were purified, and the obtained Bacillus megaterium and Rhodotorula glutinis bacterial liquids were freeze-dried to obtain solid Bacillus megaterium and Rhodotorula glutinis bacterial powders. Mixed bacterial powders were prepared at a mass ratio of Bacillus megaterium and Rhodotorula glutinis bacterial powders of 5:15.

[0056] This embodiment also proposes a method for preparing a compound microbial agent of traditional Chinese medicine, including the following steps: The compound microbial agent, traditional Chinese medicine, and base liquid are mixed and fermented in a fermenter at 30℃, pH 7, and for 60 hours. The traditional Chinese medicine, by weight, includes: 12 parts seaweed powder, 12 parts prepared Rehmannia glutinosa, 8 parts pomegranate peel, 6 parts Scutellaria baicalensis, 6 parts hawthorn, 6 parts licorice, and 6 parts dried tangerine peel. The herbal powder is dried, pulverized, and passed through a 20-mesh sieve. The viable count of Bacillus megaterium and Rhodotorula glutinis after fermentation is ≥5×10⁻⁶. 3 CFU / mL;

[0057] The amount of Chinese herbal medicine added to the base solution is 55 g / L; the base solution includes 30 g / L sucrose, 11 g / L casein, 7 g / L cellulose, 0.2 g / L calcium chloride, 0.1 g / L potassium dihydrogen phosphate and 1 g / L urea.

[0058] This embodiment also proposes to mix the compound microbial agent of traditional Chinese medicine with the feed at a mass ratio of 5:95 and then put it into the experimental fish tank with an artificial sweetener acesulfame potassium concentration of 1 mg / L.

[0059] Example 2

[0060] This embodiment proposes a microbial compound agent, including Bacillus megaterium and Rhodotorula glutinis.

[0061] Bacillus megaterium and Rhodotorula glutinis are prepared by the following steps:

[0062] Bacillus megaterium was cultured in the corresponding culture medium at 30°C and pH 6.5 for 20 hours.

[0063] Rhodotorula glutinis was cultured in the corresponding culture medium at 30°C and pH 6.5 for 25 hours.

[0064] The culture medium for *Bacillus megaterium* consisted of: 10 g / L glucose, 5 g / L soybean meal, 2 g / L corn flour, 0.2 g / L magnesium sulfate, 5.0 g / L bovine peptone, 5 g / L NaCl, 0.5 g / L dipotassium hydrogen phosphate, 0.3 g / L calcium carbonate, and 1 mg / L acesulfame potassium (artificial sweetener). The pH was natural. The addition of acesulfame potassium facilitated symbiosis and served to acclimatize the strain.

[0065] The Rhodotorula glutinis culture medium consisted of: 20 g / L malt extract, 20 g / L glucose, 1.5 g / L tryptone, 0.5 g / L soybean peptone, and 1 mg / L acesulfame potassium (artificial sweetener), with a natural pH. The addition of acesulfame potassium facilitated symbiosis and served to acclimatize the strain.

[0066] Strain propagation: The viability of the cultured *Bacillus megaterium* and *Rhodotorula glutinis* was tested, and strains with high viability were selected for propagation. The propagation medium was the same as described above. The viable cell counts in the propagated *Bacillus megaterium* and *Rhodotorula glutinis* cultures were 4 × 10⁻⁶. 8 CFU / mL and 1.2×10 8 CFU / mL;

[0067] Preparation of bacterial powder: Bacillus megaterium and Rhodotorula glutinis colonies were purified, and the obtained Bacillus megaterium and Rhodotorula glutinis bacterial liquids were freeze-dried to obtain solid Bacillus megaterium and Rhodotorula glutinis bacterial powders. Mixed bacterial powders were prepared at a mass ratio of Bacillus megaterium to Rhodotorula glutinis bacterial powders of 7:18.

[0068] This embodiment also proposes a method for preparing a compound microbial agent of traditional Chinese medicine, including the following steps: The compound microbial agent, traditional Chinese medicine, and base liquid are mixed and fermented in a fermenter at a temperature of 25℃, pH 6.5, and a fermentation time of 96 hours. The traditional Chinese medicine, by weight, includes: 10 parts seaweed powder, 10 parts prepared Rehmannia glutinosa, 10 parts pomegranate peel, 5 parts Scutellaria baicalensis, 5 parts hawthorn, 5 parts licorice, and 5 parts dried tangerine peel. The traditional Chinese medicine powder is dried, pulverized, and passed through a 20-mesh sieve. The viable count of Bacillus megaterium and Rhodotorula glutinis after fermentation is ≥5×10⁻⁶. 3 CFU / mL.

[0069] The amount of Chinese herbal medicine added to the base solution is 60 g / L; the base solution includes 35 g / L sucrose, 12 g / L casein, 9 g / L cellulose, 0.25 g / L calcium chloride, 0.15 g / L potassium dihydrogen phosphate and 1.5 g / L urea.

[0070] This embodiment also proposes to mix the compound herbal microbial agent with the feed at a mass ratio of 6:94 and then add it to the experimental fish tank with an artificial sweetener acesulfame potassium concentration of 1 mg / L.

[0071] Example 3

[0072] This embodiment proposes a microbial compound agent, including Bacillus megaterium and Rhodotorula glutinis.

[0073] Bacillus megaterium and Rhodotorula glutinis are prepared by the following steps:

[0074] Bacillus megaterium was cultured in the corresponding culture medium at 30°C and pH 6.5 for 20 hours.

[0075] Rhodotorula glutinis was cultured in the corresponding culture medium at 30°C and pH 6.5 for 25 hours.

[0076] The culture medium for *Bacillus megaterium* consisted of: 10 g / L glucose, 5 g / L soybean meal, 2 g / L corn flour, 0.2 g / L magnesium sulfate, 5.0 g / L bovine peptone, 5 g / L NaCl, 0.5 g / L dipotassium hydrogen phosphate, 0.3 g / L calcium carbonate, and 1 mg / L acesulfame potassium (artificial sweetener). The pH was natural. The addition of acesulfame potassium facilitated symbiosis and served to acclimatize the strain.

[0077] The Rhodotorula glutinis culture medium consisted of: 20 g / L malt extract, 20 g / L glucose, 1.5 g / L tryptone, 0.5 g / L soybean peptone, and 1 mg / L acesulfame potassium (artificial sweetener), with a natural pH. The addition of acesulfame potassium facilitated symbiosis and served to acclimatize the strain.

[0078] Strain propagation: The viability of the cultured *Bacillus megaterium* and *Rhodotorula glutinis* was tested, and strains with high viability were selected for propagation. The propagation medium was the same as described above. The viable cell counts in the propagated *Bacillus megaterium* and *Rhodotorula glutinis* cultures were 6 × 10⁻⁶. 8 CFU / mL and 1.8×10 8 CFU / mL;

[0079] Preparation of bacterial powder: Bacillus megaterium and Rhodotorula glutinis colonies were purified, and the obtained Bacillus megaterium and Rhodotorula glutinis bacterial liquids were freeze-dried to obtain solid Bacillus megaterium and Rhodotorula glutinis bacterial powders. Mixed bacterial powders were prepared at a mass ratio of Bacillus megaterium to Rhodotorula glutinis bacterial powders of 9:21.

[0080] This embodiment also proposes a method for preparing a compound microbial agent made from traditional Chinese medicine, including the following steps:

[0081] The microbial compound inoculant, traditional Chinese medicine, and base liquid were mixed and fermented in a fermenter at 37℃, pH 7.5, for 48 hours. The traditional Chinese medicine, by weight, included: 5 parts seaweed powder, 5 parts prepared Rehmannia root, 5 parts pomegranate peel, 10 parts Scutellaria baicalensis, 10 parts hawthorn, 10 parts licorice root, and 10 parts dried tangerine peel. The herbal powder was dried, pulverized, and passed through a 20-mesh sieve. The viable count of Bacillus megaterium and Rhodotorula glutinis after fermentation was ≥5×10⁻⁶. 3 CFU / mL.

[0082] The amount of Chinese herbal medicine added to the base solution is 65 g / L; the base solution includes 40 g / L sucrose, 13 g / L casein, 11 g / L cellulose, 0.3 g / L calcium chloride, 0.2 g / L potassium dihydrogen phosphate and 2 g / L urea.

[0083] This embodiment also proposes to mix the compound microbial agent of traditional Chinese medicine with the feed at a mass ratio of 7:93 and then put it into the experimental fish tank with an artificial sweetener acesulfame potassium concentration of 1 mg / L.

[0084] Example 4

[0085] This embodiment proposes a microbial compound agent, including Bacillus megaterium and Rhodotorula glutinis.

[0086] Bacillus megaterium and Rhodotorula glutinis are prepared by the following steps:

[0087] Bacillus megaterium was cultured in the corresponding culture medium at 30°C and pH 6.5 for 20 hours.

[0088] Rhodotorula glutinis was cultured in the corresponding culture medium at 30°C and pH 6.5 for 25 hours.

[0089] The culture medium for *Bacillus megaterium* consisted of: 10 g / L glucose, 5 g / L soybean meal, 2 g / L corn flour, 0.2 g / L magnesium sulfate, 5.0 g / L bovine peptone, 5 g / L NaCl, 0.5 g / L dipotassium hydrogen phosphate, 0.3 g / L calcium carbonate, and 1 mg / L acesulfame potassium (artificial sweetener). The pH was natural. The addition of acesulfame potassium facilitated symbiosis and served to acclimatize the strain.

[0090] The Rhodotorula glutinis culture medium consisted of: 20 g / L malt extract, 20 g / L glucose, 1.5 g / L tryptone, 0.5 g / L soybean peptone, and 1 mg / L acesulfame potassium (artificial sweetener), with a natural pH. The addition of acesulfame potassium facilitated symbiosis and served to acclimatize the strain.

[0091] Strain propagation: The viability of the cultured *Bacillus megaterium* and *Rhodotorula glutinis* was tested, and strains with high viability were selected for propagation. The propagation medium was the same as described above. The viable cell counts of the propagated *Bacillus megaterium* and *Rhodotorula glutinis* cultures were 8 × 10⁻⁶. 8 CFU / mL and 2.4×10 8 CFU / mL;

[0092] Preparation of bacterial powder: Bacillus megaterium and Rhodotorula glutinis colonies were purified, and the obtained Bacillus megaterium and Rhodotorula glutinis bacterial liquids were freeze-dried to obtain solid Bacillus megaterium and Rhodotorula glutinis bacterial powders. Mixed bacterial powders were prepared at a mass ratio of Bacillus megaterium to Rhodotorula glutinis bacterial powders of 11:24.

[0093] This embodiment also proposes a method for preparing a compound microbial agent made from traditional Chinese medicine, including the following steps:

[0094] The microbial compound inoculant, traditional Chinese medicine, and base liquid were mixed and fermented in a fermenter at 30℃, pH 7, and for 60 hours. The traditional Chinese medicine, by weight, included: 9 parts seaweed powder, 9 parts prepared Rehmannia root, 10 parts pomegranate peel, 10 parts Scutellaria baicalensis, 7 parts hawthorn, 7 parts licorice root, and 7 parts dried tangerine peel. The herbal powder was dried, pulverized, and passed through a 20-mesh sieve. The viable count of Bacillus megaterium and Rhodotorula glutinis after fermentation was ≥5×10⁻⁶. 3 CFU / mL.

[0095] The amount of Chinese herbal medicine added to the base solution is 70 g / L; the base solution includes 45 g / L sucrose, 14 g / L casein, 13 g / L cellulose, 0.35 g / L calcium chloride, 0.25 g / L potassium dihydrogen phosphate and 2.5 g / L urea.

[0096] This embodiment also proposes to mix the compound herbal microbial agent with the feed at a mass ratio of 8:92 and then add it to the experimental fish tank with an artificial sweetener acesulfame potassium concentration of 1 mg / L.

[0097] Example 5

[0098] This embodiment proposes a microbial compound agent, including Bacillus megaterium and Rhodotorula glutinis.

[0099] Bacillus megaterium and Rhodotorula glutinis are prepared by the following steps:

[0100] Bacillus megaterium was cultured in the corresponding culture medium at 30°C and pH 6.5 for 20 hours.

[0101] Rhodotorula glutinis was cultured in the corresponding culture medium at 30°C and pH 6.5 for 25 hours.

[0102] The culture medium for *Bacillus megaterium* consisted of: 10 g / L glucose, 5 g / L soybean meal, 2 g / L corn flour, 0.2 g / L magnesium sulfate, 5.0 g / L bovine peptone, 5 g / L NaCl, 0.5 g / L dipotassium hydrogen phosphate, 0.3 g / L calcium carbonate, and 1 mg / L acesulfame potassium (artificial sweetener). The pH was natural. The addition of acesulfame potassium facilitated symbiosis and served to acclimatize the strain.

[0103] The Rhodotorula glutinis culture medium consisted of: 20 g / L malt extract, 20 g / L glucose, 1.5 g / L tryptone, 0.5 g / L soybean peptone, and 1 mg / L acesulfame potassium (artificial sweetener), with a natural pH. The addition of acesulfame potassium facilitated symbiosis and served to acclimatize the strain.

[0104] Strain propagation: The viability of the cultured *Bacillus megaterium* and *Rhodotorula glutinis* was tested, and strains with high viability were selected for propagation. The propagation medium was the same as described above. The viable cell counts of the propagated *Bacillus megaterium* and *Rhodotorula glutinis* cultures were 1 × 10⁻⁶. 9 CFU / mL and 3×10 8 CFU / mL;

[0105] Preparation of bacterial powder: Bacillus megaterium and Rhodotorula glutinis colonies were purified, and the obtained Bacillus megaterium and Rhodotorula glutinis bacterial liquids were freeze-dried to obtain solid Bacillus megaterium and Rhodotorula glutinis bacterial powders. Mixed bacterial powders were prepared at a mass ratio of Bacillus megaterium and Rhodotorula glutinis bacterial powders of 15:30.

[0106] This embodiment also proposes a method for preparing a compound microbial agent made from traditional Chinese medicine, including the following steps:

[0107] The microbial compound inoculant, traditional Chinese medicine, and base liquid were mixed and fermented in a fermenter at 30℃, pH 6.5, for 48 hours. The traditional Chinese medicine, by weight, included: 8 parts seaweed powder, 8 parts prepared Rehmannia root, 5 parts pomegranate peel, 5 parts Scutellaria baicalensis, 5 parts hawthorn, 8 parts licorice root, and 8 parts dried tangerine peel. The herbal powder was dried, pulverized, and passed through a 20-mesh sieve. The viable counts of Bacillus megaterium and Rhodotorula glutinis after fermentation were ≥5×10⁻⁶. 3 CFU / mL.

[0108] The amount of Chinese herbal medicine added to the base solution is 75 g / L; the base solution includes 50 g / L sucrose, 15 g / L casein, 15 g / L cellulose, 0.4 g / L calcium chloride, 0.3 g / L potassium dihydrogen phosphate and 3 g / L urea.

[0109] This embodiment also proposes to mix the compound herbal microbial agent with the feed at a mass ratio of 9:91 and then add it to the experimental fish tank with an artificial sweetener acesulfame potassium concentration of 1 mg / L.

[0110] Comparative Example 1

[0111] Unfermented Chinese herbal medicine powder was mixed with feed at a mass ratio of 5:95 and then added to an experimental fish tank with an artificial sweetener acesulfame potassium concentration of 1 mg / L.

[0112] Comparative Example 2

[0113] Solid Bacillus megaterium powder was prepared by mixing it with traditional Chinese medicine and a prepared base liquid according to the preparation method in Example 1, and then fermenting it to prepare a compound microbial agent of traditional Chinese medicine.

[0114] After mixing the herbal compound microbial agent with the feed at a mass ratio of 5:95, the mixture was added to the experimental fish tank with an artificial sweetener acesulfame potassium concentration of 1 mg / L.

[0115] Comparative Example 3

[0116] Solid red yeast rice powder was prepared separately, and then mixed with traditional Chinese medicine and prepared base liquid according to the preparation method in Example 1 to prepare a traditional Chinese medicine compound microbial agent by fermentation.

[0117] After mixing the herbal compound microbial agent with the feed at a mass ratio of 5:95, the mixture was added to the experimental fish tank with an artificial sweetener acesulfame potassium concentration of 1 mg / L.

[0118] Comparative Example 4

[0119] Commercially available Bacillus subtilis was mixed with feed at a mass ratio of 5:95 and then added to an experimental fish tank with an artificial sweetener acesulfame potassium concentration of 1 mg / L.

[0120] Comparative Example 5

[0121] Commercially available Bacillus licheniformis was mixed with feed at a mass ratio of 5:95 and then added to an experimental fish tank with an artificial sweetener acesulfame potassium concentration of 1 mg / L.

[0122] Comparative Example 6

[0123] Commercially available brewer's yeast was mixed with feed at a mass ratio of 5:95 and then added to an experimental fish tank with an artificial sweetener acesulfame potassium concentration of 1 mg / L.

[0124] Comparative Example 7

[0125] Commercially available marine red yeast was mixed with feed at a mass ratio of 5:95 and then added to an experimental fish tank with an artificial sweetener acesulfame potassium concentration of 1 mg / L.

[0126] Comparative Example 8

[0127] The feed was directly added to the experimental fish tank where the concentration of the artificial sweetener acesulfame potassium was 1 mg / L.

[0128] Acesulfame K exposure experiment

[0129] This study employed an aqueous exposure method to conduct an acesulfame K exposure experiment, based on the predicted worst-case environmental concentration of acesulfame K in seawater: 1 mg / L. -1 Exposure experiment design such as Figure 1As shown. After 7 days of domestication, 585 healthy juvenile black sea bream were randomly selected and evenly distributed into 39 aquariums (25×30×30cm). The weight and length of the juveniles were measured. The experimental groups consisted of Examples 1, 2, 3, 4, and 5, and Comparative Examples 1, 2, 3, 4, 5, 6, 7, and 8, with three replicates per group. Each aquarium contained 12L of UV-sterilized natural seawater (pH 8.1±0.1, salinity 32±1‰, water temperature 16±1℃, DO 7.5±0.5mg L⁻¹), with a light (12h) : dark (12h) lighting condition. During the exposure period, fish food was fed daily from 8:00 AM to 8:30 AM at a rate of 3% of the total body weight of the juveniles in each tank (this daily feeding amount met the nutritional needs of the juveniles while avoiding water pollution caused by overfeeding). 80% of the exposure solution in the tanks was replaced daily from 2:00 PM to 3:00 PM. After the 20-day exposure period, behavioral observation and analysis were conducted on the juveniles. After the behavioral analysis experiment, the juveniles were euthanized using an ice-water mixture, and their muscles were dissected.

[0130] Group behavior test

[0131] After a 20-day exposure period, 10 juvenile fish were randomly selected from each tank for an open field experiment. A circular white acrylic tank (100cm in diameter) was placed inside a stainless steel bracket (1.5m in diameter, 1.8m high) fitted with LED light strips. A camera was fixed to the top using a bracket for filming. Before each filming session, the juvenile fish were placed in the open field for 5 minutes to acclimatize, followed by continuous filming for 20 minutes. Subsequently, based on the positional coordinates of the 10 juvenile fish in the group, parameters related to school behavior, including nearest neighbor distance and swimming speed synchronization, were calculated.

[0132] Protein content determination

[0133] The protein content was determined using the Kjeldahl method in accordance with GB5009.5—2016, "National Food Safety Standard - Determination of Protein in Food".

[0134] The effects of different treatments on the weight gain rate, specific growth rate, and condition factor of black sea bream are shown in the figure. Figure 2-4Compared with Comparative Example 8, the weight gain rates of Examples 1, 2, 3, 4, 5 and Comparative Examples 1, 2, 3, 4, 5, 6, 7 were significantly reduced by 19.39%, 31.76%, 38.23%, 34.94%, 40.73% and 2.27%, 18.78%, 19.88%, 8.33%, 12.34%, 12.22%, 11.88%, respectively, and the specific growth rates were significantly reduced by 20.04%, 21.77%, 26.99%, 32.98%, respectively. The percentages of fatness in the black sea bream were 36.69%, 5.60%, 18.01%, 18.70%, 13.07%, 11.28%, 9.34%, and 10.88%, respectively, with significant reductions in fatness of 43.76%, 51.24%, 50.80%, 52.05%, and 60.66% and 6.09%, 28.32%, 30.92%, 20.02%, 17.51%, 17.97%, and 16.79%, respectively. This indicates that the other treatment groups can alleviate the excessive obesity of the black sea bream. The treatment group consisting of Bacillus megaterium and Rhodotorula glutinis alone showed significantly better results than the treatment group consisting of commercially available Bacillus subtilis, Bacillus licheniformis, Saccharomyces cerevisiae, and Rhodotorula glutinis. In addition, the microbial compound agent in the other embodiment had a more significant effect on black sea bream than the treatment group consisting of Bacillus megaterium and Rhodotorula glutinis alone. This indicates that the microbial compound agent consisting of Bacillus megaterium and Rhodotorula glutinis effectively alleviates the effect of acesulfame potassium on excessive weight gain in fish. This may be because Bacillus megaterium and Rhodotorula glutinis promote the growth of beneficial bacteria and inhibit the reproduction of harmful bacteria, thereby maintaining intestinal health and promoting normal physiological metabolism in fish.

[0135] The effects of different treatments on nearest neighbor distance and swimming speed synchronization in black sea bream are shown in the figure. Figure 5 and Figure 6Compared with Comparative Example 8, the nearest neighbor distances of Examples 1, 2, 3, 4, 5 and Comparative Examples 1, 2, 3, 4, 5, 6, 7 were significantly reduced by 11.00%, 11.98%, 18.29%, 18.32%, 23.56% and 2.44%, 9.00%, 8.78%, 3.96%, 4.38%, 5.22%, 5.58%, respectively, while swimming speed synchronization was significantly increased by 181.25%, 182.59%, 182.25%, 169.35%, 188.39% and 67.66%, 129.75%, 113.92%, 69.62%, 62.10%, 63.78%, 64.58%, respectively. These results indicate that the other treatment groups can promote fish schooling behavior. The treatment group consisting of Bacillus megaterium and Rhodotorula glutinis alone showed significantly better results than the group consisting of commercially available Bacillus subtilis, Bacillus licheniformis, Saccharomyces cerevisiae, and Rhodotorula glutinis. In addition, the herbal compound microbial agent in the other example had a more significant effect on black sea bream than the treatment group consisting of Bacillus megaterium and Rhodotorula glutinis alone. This indicates that the microbial compound microbial agent consisting of Bacillus megaterium and Rhodotorula glutinis effectively alleviates the effect of acesulfame potassium on the reduced schooling behavior of fish. The reason for this phenomenon may be that Bacillus megaterium and Rhodotorula glutinis maintain intestinal health and affect the schooling behavior of fish through brain-gut axis signaling, thereby improving aquaculture efficiency.

[0136] The effects of different treatments on the protein content of black sea bream are shown in the figure. Figure 7 Compared with Comparative Example 8, the protein content of Examples 1, 2, 3, 4, 5 and Comparative Examples 1, 2, 3, 4, 5, 6, 7 increased significantly by 26.32%, 28.65%, 28.77%, 37.67%, 50.03% and 12.55%, 20.33%, 20.25%, 8.58%, 12.06%, 10.63%, 10.92%, respectively. The results indicate that the other treatment groups can increase the protein content in fish meat and improve the nutritional quality of fish meat. Among them, the treatment group with Bacillus megaterium and Rhodotorula glutinis alone was significantly more effective than the group with commercially available Bacillus subtilis, Bacillus licheniformis, Saccharomyces cerevisiae, and Rhodotorula glutinis. In addition, the herbal compound microbial agent in the other examples had a more significant effect on black sea bream than the treatment with Bacillus megaterium and Rhodotorula glutinis alone. The microbial compound microbial agent in Example 5 was the most significant. This indicates that high doses of the microbial compound microbial agent with Bacillus megaterium and Rhodotorula glutinis can effectively increase the protein content of fish meat. The reason for this phenomenon may be that Bacillus megaterium and Rhodotorula glutinis promote the reproduction of beneficial bacteria and maintain intestinal health. At the same time, the microbial compound microbial agent feed is easier for fish to utilize and absorb, thus improving feed utilization.

[0137] Compared with the prior art, the present invention has the following beneficial effects:

[0138] (1) Bacillus megaterium and Rhodotorula glutinis adhere to the intestinal cells of nearshore fish without damaging the cells, which is conducive to their symbiotic relationship with the intestinal flora of farmed fish. Bacillus megaterium and Rhodotorula glutinis can maintain intestinal health by regulating the balance of intestinal microorganisms, promoting the growth of beneficial bacteria and inhibiting the reproduction of harmful bacteria, thereby alleviating the excessive obesity of black sea bream caused by the artificial sweetener acesulfame potassium and promoting normal physiological metabolism in fish. In addition, they may also promote fish schooling behavior and improve aquaculture efficiency through brain-gut axis signaling.

[0139] (2) The microbial herbal compound preparation obtained by the method of the present invention is simple to prepare. It utilizes the fermentation of Bacillus megaterium and Rhodotorula glutinis to transform some beneficial macromolecular substances in traditional Chinese medicine that are not easily absorbed into small molecules. When this microbial herbal compound preparation is used to formulate feed, the feed is used to raise farmed fish, which is conducive to the absorption and utilization of the fish and improves the fish's immunity.

[0140] (3) Bacillus megaterium and Rhodotorula glutinis can reproduce normally in water bodies polluted by artificial sweeteners, which helps to repair the marine aquaculture environment polluted by artificial sweetener acesulfame, increase the protein content of black sea bream, improve feed utilization, and increase the income of nearshore fish farmers.

[0141] 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 preparation method of a Chinese herbal medicine complex microbial agent, characterized by, The method comprises the following steps: fermenting the mixture of the microbial complex microbial agent, Chinese herbal medicine and base solution, wherein the microbial complex microbial agent is added in an amount of 2.5%-7.5% of the mass of the base solution, the fermentation temperature is 25-37℃, the fermentation time is 48-96 h, and the pH value is 6.5-7.5; the Chinese herbal medicine comprises seaweed powder, Radix Rehmanniae Preparata, pomegranate peel, Scutellaria, hawthorn, liquorice and dried tangerine or orange peel; the Chinese herbal medicine is added in an amount of 50-75 g / L in the base solution; The microbial complex microbial agent comprises Bacillus megaterium (Bacillus megaterium) Bacillus megaterium ) PP84, the preservation number is CGMCC No. 12798; and Rhodotorula mucilaginosa (Rhodotorula mucilaginosa) Rhodotorula mucilaginosa ) OP11, the preservation number is CGMCC No. 13540; the mass ratio of the bacterial powder corresponding to the Bacillus megaterium and the Rhodotorula mucilaginosa is (5-15):(15-30). The bacterial powder is prepared by the following steps: Bacillus megaterium and Rhodotorula mucilaginosa were respectively cultured in the medium to the number of 1 x 10 8 -1 x 10 9 CFU / mL and 1 x 10 8 -3 x 10 8 CFU / mL in the bacterial liquid, and then purified and freeze-dried to obtain; the Bacillus megaterium and the Rhodotorula mucilaginosa were respectively cultured in the medium added with an artificial sweetener Acesulfame-K; the base solution comprises sucrose 30-50 g / L, casein 10-15 g / L, cellulose 5-15 g / L, calcium chloride 0.2-0.5 g / L, potassium dihydrogen phosphate 0.1-0.3 g / L and urea 1-3 g / L; the Chinese herbal medicine comprises seaweed powder 5-10 parts, Radix Rehmanniae Preparata 5-10 parts, pomegranate peel 5-10 parts, Scutellaria 5-10 parts, hawthorn 5-10 parts, liquorice 5-10 parts and dried tangerine or orange peel 5-10 parts, by weight.

2. The preparation method of the Chinese herbal medicine complex microbial agent according to claim 1, characterized in that, The viable cell counts of Bacillus megaterium and Rhodotorula mucilaginosa after fermentation were both ≥5×10 3 CFU / mL.

3. A Chinese herbal medicine complex microbial agent, characterized by, The Chinese herbal medicine complex microbial agent prepared by the preparation method of any one of claims 1-2 or the Chinese herbal medicine complex microbial agent of claim 3.

4. A feed, characterized in that, The mass addition amount of the Chinese herbal medicine complex microbial agent prepared by the preparation method of any one of claims 1-2 or the Chinese herbal medicine complex microbial agent of claim 3 in the feed is 5%-10%.

5. The Chinese herbal medicine complex microbial agent prepared by the preparation method of any one of claims 1-2 or the Chinese herbal medicine complex microbial agent of claim 3 is applied in the mariculture of near-shore fish.

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