A fermented traditional chinese medicine feed additive for catfish and its preparation method and application

Abstract

A fermented traditional Chinese medicine feed additive for catfish, its preparation method, and its application are disclosed, relating to the field of aquatic feed additives. The preparation method involves activating and expanding the culture of *Saccharomyces cerevisiae*, *Bacillus subtilis*, and *Lactobacillus plantarum*; pulverizing *Paeonia suffruticosa* leaves, *Smilax china*, *Scutellaria baicalensis*, *Rhus chinensis*, hawthorn, *Polygonum cuspidatum*, ginkgo, and forsythia suspensa* to obtain traditional Chinese medicine powders; preparing a solid fermentation medium using the traditional Chinese medicine powders, soybean meal powder, corn cob granules, and wheat bran; inoculating *Saccharomyces cerevisiae* seed culture and *Bacillus subtilis* seed culture for aerobic fermentation; and inoculating *Lactobacillus plantarum* seed culture for anaerobic fermentation to obtain the fermented traditional Chinese medicine feed additive. The resulting fermented traditional Chinese medicine feed additive promotes the absorption and metabolic elimination of enrofloxacin in catfish, shortens the withdrawal period, has preventive and adjunctive therapeutic effects on bacterial diseases in catfish, promotes growth, reduces feed conversion ratio, and improves survival rate.

Description

Technical Field

[0001] This invention relates to the field of aquatic feed additives, specifically to a fermented herbal feed additive for catfish, its preparation method, and its application. Background Technology

[0002] The Egyptian catfish, also known as the Egyptian barbel catfish, originates from Africa and is a tropical and subtropical fish. It is highly nutritious, high in protein, and has a delicious flavor, possessing both tonic and medicinal value. It can breathe air, tolerates fertile water, low oxygen levels, and long-distance transport, exhibiting strong environmental adaptability, rapid growth, and a short breeding cycle. It is an omnivorous fish that primarily feeds on animal matter, resulting in high yields. It is farmed throughout China. With the widespread adoption of high-density intensive farming of Egyptian catfish, various diseases and pests have become frequent, particularly bacterial diseases, posing a significant threat to the aquaculture industry. Antibiotics, with their advantages of low dosage, high bactericidal effect, and significant therapeutic efficacy, are the preferred drugs for treating bacterial diseases in Egyptian catfish. Enrofloxacin belongs to the fluoroquinolone class of antibiotics. It is rapidly and completely absorbed after oral administration, has a long half-life, and boasts advantages such as wide distribution in the body, no cross-resistance with other antibacterial drugs, and low toxicity. It is highly effective against bacterial diseases such as hemorrhagic septicemia and enteritis in barnyard catfish caused by harmful bacteria like *Aeromonas hydrophila* and *Aeromonas tempera*. The use of enrofloxacin as a treatment drug in barnyard catfish farming is very common. However, enrofloxacin also poses problems such as drug residues and drug resistance, posing risks to the quality of barnyard catfish and the safe use of enrofloxacin. According to Chinese aquatic product standards, enrofloxacin residue levels must be below 0.1 mg / kg for the product to be marketable. Generally, after oral administration of enrofloxacin, it takes 12-15 days for barnyard catfish to reach a safe level before they are ready for market. Barnyard catfish are tropical fish, and in most parts of my country, their farming cycle is short, meaning their market availability is often closely related to their price. Therefore, effective and rapid methods and formulations to reduce antibiotic residues in barnyard catfish are urgent problems that the aquaculture industry needs to solve.

[0003] There are reports in the prior art of using fermented Chinese medicine for animal applications. For example, Chinese invention patent application number 201410015670.0 discloses a compound microbial active additive and its application. It involves pulverizing Chinese medicines such as hawthorn, angelica, and schisandra and adding microorganisms such as Bacillus licheniformis and Bacillus subtilis for fermentation. The fermentation product can be used simultaneously with antibiotics, which can protect internal organs from damage and accelerate the excretion of metabolic toxins, reducing drug residue problems. However, it is mainly used for livestock and poultry and is not specific, which may pose potential drug risks.

[0004] Existing technologies also disclose preparations containing traditional Chinese medicine raw materials that can be directly used to accelerate the metabolism of antibiotics in fish, as well as their preparation methods. For example, Chinese invention patent application number 201710885611.2 discloses a catfish feed that can safely use antibiotics and its preparation method. Active ingredients such as Eucommia ulmoides, Acer palmatum, oxalic acid, and alanine, as well as the antibiotic gentamicin, are added to fish meal, soybean meal, cottonseed meal, and other excipients. The resulting animal feed can accelerate and reduce the metabolism of the antibiotic gentamicin in catfish, thus reducing its residue. However, this technology has several drawbacks. First, directly adding pulverized Chinese herbal medicines to feed results in low utilization rates, especially for carnivorous fish. First, most active ingredients in Chinese herbal medicines reside within plant cells or intercellular matrix, encased in cell walls and pectin. Since fish intestines generally lack the enzymes necessary to break down these cell walls and intercellular matrix barriers, the active ingredients are not readily available. Second, most Chinese herbal medicines have a distinctly bitter taste, making them unpalatable to fish. Third, this method is only effective for catfish when gentamicin is administered orally; it has no significant effect on other fish or other types of antibiotics. Summary of the Invention

[0005] The present invention aims to provide a fermented Chinese herbal medicine feed additive for catfish, its preparation method and application, in order to solve the technical problems of enrofloxacin drug residue and long withdrawal period in the breeding of catfish.

[0006] To solve the above technical problems, the specific solution adopted by this invention is as follows: a method for preparing a fermented traditional Chinese medicine feed additive for catfish, comprising the following steps: S1: Bacillus subtilis, Lactobacillus plantarum, and Cytomegalovirus are activated and cultured separately to obtain Bacillus subtilis seed liquid, Lactobacillus plantarum seed liquid, and Cytomegalovirus seed liquid, for later use; S2: Take 70-90 parts by weight of peony leaves, 50-70 parts of Scutellaria baicalensis, 30-50 parts of pigweed, 20-40 parts of gallnut, 20-40 parts of hawthorn, 20-40 parts of Polygonum cuspidatum, 20-40 parts of ginkgo, 20-40 parts of forsythia, 20-40 parts of rhubarb, 15-25 parts of Atractylodes macrocephala, and 15-25 parts of pomegranate peel, pulverize, mix evenly, and obtain traditional Chinese medicine powder for later use; S3: Take 60-80 parts by weight of soybean meal powder and 60-80 parts by weight of corn cob pellets. 80 parts of wheat bran, 100-120 parts of mineral salt solution, 100 parts of the Chinese herbal powder obtained in step S2, 450-490 parts of tap water or distilled water, mixed evenly, and sterilized by steam to obtain a solid fermentation culture medium for later use; S4: Add 12-15 parts of the above-mentioned *Bacillus subtilis* seed liquid and 24-30 parts of *Bacillus subtilis* seed liquid to the above solid fermentation culture medium according to the weight ratio, mix evenly, control the temperature at 28-32℃, and ferment for 16-22 hours by introducing sterilized air or oxygen; Stop introducing sterilized air or oxygen, add 48-60 parts of *Lactobacillus plantarum* seed liquid, stir evenly, control the temperature at 30-36℃, and continue fermentation for 36-48 hours to obtain fermented Chinese herbal medicine; S5: Dry the fermented Chinese herbal medicine by introducing hot air at 40-50℃, pulverize it, and obtain the fermented Chinese herbal medicine feed additive for *Catfish spp.*

[0007] As a further optimization of the above technical solution, the number of Bacillus subtilis in the Bacillus subtilis seed solution, the number of Lactobacillus plantarum in the Lactobacillus plantarum seed solution, and the number of Saccharomyces cerevisiae in the Saccharomyces cerevisiae seed solution are all 3-8 × 10⁻⁶. 8 per mL.

[0008] As a further optimization of the above technical solution, the inoculation ratio of *Saccharomyces cerevisiae*, *Bacillus subtilis*, and *Lactobacillus plantarum* is 1:2:4.

[0009] As a further optimization of the above technical solution, the method for preparing corn cob particles is as follows: select clean, dry corn cobs free from mold contamination, and crush them into small pieces with a diameter of 5-10 mm in a pulverizer to obtain corn cob particles.

[0010] As a further optimization of the above technical solution, the method for preparing the mineral salt solution is as follows: weigh 30-40 parts of potassium dihydrogen phosphate, 60-70 parts of dipotassium hydrogen phosphate, 40-50 parts of sodium chloride and 6-8 parts of magnesium sulfate according to the weight ratio, put them into a container, pour in 100 parts of tap water or distilled water, and dissolve them completely to obtain the mineral salt solution.

[0011] As a further optimization of the above technical solution, the method for activating and scaling up the Bacillus subtilis culture is as follows:

[0012] Activation culture: Bacillus subtilis culture was placed in a 37℃ incubator for 4-6 hours, then inoculated into beef extract peptone medium and cultured at 37℃ for 24 hours on a shaker at 200 rpm / min to obtain activated Bacillus subtilis. The beef extract peptone medium contained 5g beef extract, 10g peptone, and 5g sodium chloride per liter, with the remainder being water, and a pH of 7.2-7.5.

[0013] Expanded culture: Activated Bacillus subtilis cultured medium was inoculated into Bacillus subtilis expansion medium. The medium was shaken at 200 rpm / min and incubated at 37°C for 24 hours. Bacillus subtilis expansion medium was then added to adjust the viable count to 3-8 × 10⁻⁸ Bacillus subtilis. 8 The seed culture of Bacillus subtilis is obtained by measuring 10g / mL of Bacillus subtilis. The Bacillus subtilis expansion culture medium contains 10g corn flour, 5g glucose, 20g soybean meal, 5g calcium carbonate, 1g ammonium sulfate, 0.3g dipotassium hydrogen phosphate, 0.2g magnesium sulfate heptahydrate, 0.2g manganese sulfate hydrate, and the balance is water, with a pH of 7.0-7.5.

[0014] As a further optimization of the above technical solution, the method for activating and expanding the culture of *Lactobacillus plantarum* is as follows:

[0015] Activation culture: Lactobacillus plantarum was cultured at 34℃ for 4-6 hours, then inoculated into MRS medium and incubated statically at 34℃ for 18-24 hours; the MRS medium contained 10g of protein powder, 5g of beef extract, 4g of yeast powder, 20g of glucose, 1.0mL of Tween 80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of triammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, and the remainder was water;

[0016] Expanded culture: The activated Lactobacillus plantarum culture was inoculated into Lactobacillus plantarum expansion medium and incubated statically at 34℃ for 24 h. Lactobacillus plantarum expansion medium was then added to adjust the viable count to 3-8 × 10⁻⁶ cells / year. 8 The number of cells / mL is the seed liquid of Lactobacillus plantarum; the mass percentage of each component in the Lactobacillus plantarum expansion culture medium is as follows: 5% bean sprout juice, 10% tomato juice, 5% potato, 2% sucrose, 4% salt, and the remainder is water.

[0017] As a further optimization of the above technical solution, the activation and scale-up culture method of the capsule-coated yeast is as follows:

[0018] Activation culture: The *Saccharomyces cerevisiae* was cultured at 30℃ for 4-6 hours, then inoculated into YPD liquid medium and cultured at 30℃ with a shaking speed of 180 rpm / min for 18-24 hours to obtain activated *Saccharomyces cerevisiae* culture. The YPD liquid medium contained 20g glucose, 20g peptone, 10g yeast extract, and the remainder was water per liter.

[0019] Expanded culture: Inoculate the activated culture of *Saccharomyces cerevisiae* into expanded liquid culture medium, then incubate at 180 rpm / min and 30℃ for 18-24 h on a shaker. Afterward, add more expanded liquid culture medium to adjust the concentration to achieve a *Saccharomyces cerevisiae* count of 3-8 × 10⁻⁶. 8 The seed culture of *Saccharomyces cerevisiae* was obtained by measuring 1000 cells / mL. The expanded liquid culture medium for *Saccharomyces cerevisiae* consisted of 50g glucose, 10g ammonium sulfate, 5g urea, 20g yeast extract, 1g dipotassium hydrogen phosphate, 0.5g potassium dihydrogen phosphate, 1.2g magnesium sulfate, and the remainder being water. The pH was adjusted to 5.5, and 1000mL of distilled water was added.

[0020] A fermented herbal feed additive for catfish prepared by the above method.

[0021] The application of a fermented Chinese herbal medicine feed additive for catfish in reducing enrofloxacin drug residues, preventing hemorrhagic septicemia in catfish, and treating hemorrhagic septicemia in catfish, is described, with the dosage in the feed being 2-3% of the feed weight.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. During the development of a disease-resistant and growth-promoting additive for the barbel catfish, it was unexpectedly discovered that the fermented traditional Chinese medicine feed additive described in this invention accelerates the metabolism of enrofloxacin in the barbel catfish, removes enrofloxacin drug residues, and shortens the withdrawal period. It showed no significant effect on other fish species or antibiotics, demonstrating specificity. When the fermented traditional Chinese medicine feed additive was administered after enrofloxacin withdrawal, the time it took for the enrofloxacin content in the muscle to reach the withdrawal period standard was shortened by 40% compared to normal enrofloxacin metabolism.

[0024] Drug transformation and clearance in organisms are primarily facilitated by drug-metabolizing enzymes. Cytochrome P450 (CYP450) is widely distributed in animals, plants, fish, and other organisms. It is a superfamily of heme-containing isoenzymes and the most important metabolic enzyme system in drug transformation, often representing the rate-limiting step in drug elimination. Therefore, CYP450 enzyme activity is directly related to drug clearance rates. Cytochrome P450 is a class of proteins composed of varying numbers of amino acids. To date, over 2000 CYP450 enzymes have been discovered, and the subtypes and expression levels of CYP450 enzymes vary significantly among different biological species. The metabolic rate and residual time of drugs are often related to the induction and inhibition of CYP450. Different drugs activate or inhibit specific CYP450 enzymes. Different biological species often exhibit significant differences in the specific types and expression levels of CYP450 enzymes. Using different drugs on the same organism can activate entirely different CYP450 enzymes, and the same drug used on different organisms can also activate or inhibit significantly different types of specific enzymes. Enrofloxacin inhibits the CYP450 activity of the barbel catfish. On the one hand, enrofloxacin has a long-lasting effect, providing good preventative and therapeutic benefits. On the other hand, enrofloxacin is slowly eliminated from the fish's body, leaving significant residues that require a considerable amount of time to reduce to below safe concentrations. The compound traditional Chinese medicine used in this invention, containing active ingredients such as baicalin and gallic acid, has an inducing effect on the CYP450 activity of the barbel catfish, significantly accelerating the metabolism of enrofloxacin within the fish. After fermentation, these active ingredients are more easily dissolved from the herbal cells, inducing efficient and rapid expression of genes related to the CYP450 enzyme that degrades enrofloxacin. This significantly increases CYP450 enzyme activity, allowing enrofloxacin to be rapidly degraded and excreted from the body. Due to the differences in the types and expression levels of CYP450 enzymes among different fish species, the specific types and effects of the same traditional Chinese medicine or compound traditional Chinese medicine on activating or inhibiting CYP450 enzymes vary considerably among different fish species. As a result, fermented traditional Chinese medicine additives have not shown significant effects in removing enrofloxacin residues or shortening withdrawal periods for other common farmed fish species such as carp and crucian carp. Furthermore, no significant degradation effect has been found for other antibiotics to date.

[0025] 2. The fermented traditional Chinese medicine feed additive for catfish described in this invention has a significant preventive effect on diseases such as hemorrhagic septicemia in catfish. After using the fermented traditional Chinese medicine additive, the survival rate of catfish increased by 65%. After fermentation, the "cell wall breaking effect" of microorganisms makes the nutrients and beneficial components in the traditional Chinese medicine cells easily available to fish, promoting the absorption, transport, and metabolism of the effective components in the fish's body, and improving the efficacy of the traditional Chinese medicine. Rhubarb and Scutellaria baicalensis in the compound have a direct killing effect on various intestinal bacteria and fish pathogens, such as Aeromonas hydrophila and Staphylococcus aureus, improving the intestinal environment and facilitating the colonization of lactic acid bacteria and Bacillus subtilis in the intestine. On the other hand, Bacillus subtilis and lactic acid bacteria exert "biological oxygen depletion," "spatial competition," and "food competition" to inhibit the reproduction of harmful bacteria. Furthermore, the microbial fermentation process produces a large number of small peptides, organic acids, and various vitamins, which also play a certain role in promoting the immune system.

[0026] 3. The fermented traditional Chinese medicine feed additive for catfish described in this invention has a certain therapeutic effect on diseases such as hemorrhagic septicemia in catfish. The therapeutic effect is significant when used in combination with enrofloxacin. Adding 2.5% of the fermented traditional Chinese medicine additive to the feed for treating catfish infected with Aeromonas hydrophila increased the survival rate by 25% compared to the positive control group without added traditional Chinese medicine. When enrofloxacin and fermented traditional Chinese medicine were used in combination, the survival rate increased by 80% compared to the positive control group and by 22.5% compared to the enrofloxacin group. This may be because the fermented traditional Chinese medicine itself has a certain therapeutic effect on hemorrhagic septicemia, and the fermented traditional Chinese medicine additive further promotes the absorption of enrofloxacin by the catfish.

[0027] 4. The fermented herbal feed additive for catfish described in this invention has good palatability, significantly promotes the growth of catfish, improves survival rate, and reduces feed conversion ratio. After fermentation with mixed strains, a certain amount of microbial protein and a large amount of vitamins, lactic acid, and other microbial metabolites are produced. Large molecules such as proteins and starches in plant materials and herbal medicines are broken down into small peptides, amino acids, glucose, and other small molecules. Cellulose and hemicellulose, which make up plant cell walls, are converted into components that can be utilized by catfish. The herbal medicines contain flavonoids, glycosides, and other active substances. These components promote appetite, enhance metabolic activity, and promote the secretion of digestive enzymes, accelerating the growth and development of aquatic animals. After fermentation, these substances are more easily utilized by fish.

[0028] 5. The fermented Chinese herbal feed additive for catfish described in this invention is safe for catfish and has no toxic side effects. Even if used in excess (≤5%), no toxic side effects will be produced.

[0029] 6. In the preparation method of the fermented Chinese herbal medicine feed additive for catfish described in this invention, the Bacillus subtilis, Lactobacillus plantarum, and Cytomegalovirus are commonly used strains. The method does not rely on specific strains; after multiple experiments, strains purchased from different preservation institutions and biological product companies have all achieved the desired results. The Chinese herbal medicines used are all common varieties available on the market, green and natural, inexpensive and readily available. When added to feed, they do not accumulate, leave residues, or pollute water bodies. Attached Figure Description

[0030] Figure 1 The effect of fermented traditional Chinese medicine additives on drug residues in the muscle of catfish after oral administration of florfenicol;

[0031] Figure 2 The effect of fermented traditional Chinese medicine additives on drug residues in the muscle of catfish after oral administration of thiamphenicol;

[0032] Figure 3 The effect of fermented traditional Chinese medicine additives on drug residues in the muscle of catfish after oral administration of vancomycin;

[0033] Figure 4 The effect of compound microbial active additives on drug residues in muscle of catfish after oral administration of enrofloxacin. Detailed Implementation

[0034] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. Parts not described or disclosed in detail in the following embodiments of the present invention should be understood as prior art known or should be known by those skilled in the art.

[0035] To avoid repetition, the sources of raw materials and processing techniques involved in the specific embodiments of the present invention will be explained in a unified manner as follows:

[0036] In the following embodiments, the Chinese herbal medicines used are all common Chinese herbal medicine varieties on the market, and the microorganisms used for fermentation are all common probiotics in fish feed. They can all be purchased from the China Center for Type Culture Collection or the China General Microbiological Culture Collection Center, and there are no special requirements.

[0037] In the following embodiments, the methods for activating and scaling up the Bacillus subtilis are as follows:

[0038] (1) Activation culture: Take out the preserved Bacillus subtilis strain, put it in a 37℃ incubator for 4-6 hours, and inoculate it into a 250mL Erlenmeyer flask containing 50mL beef extract peptone medium. The inoculation amount is 1-3 loops. Culture it in a shaker at 200rpm / min and 37℃ for 24 hours to obtain activated Bacillus subtilis.

[0039] The beef extract peptone culture medium contains 5g of beef extract, 10g of peptone, and 5g of sodium chloride per liter of culture medium, with the remainder being water, and a pH value of 7.2-7.5.

[0040] (2) Expanded culture: Activated Bacillus subtilis cultured medium was inoculated into several 250mL Erlenmeyer flasks containing 50mL of Bacillus subtilis expansion medium at a mass ratio of 1:100. The flasks were shaken at 200rpm / min and incubated at 37℃ for 24 hours. The resulting culture contained approximately 1×10⁻⁶ Bacillus subtilis. 9 CFU / mL; Add Bacillus subtilis expansion medium to adjust the viable count to 3-8 × 10⁶ CFU / mL. 8 Bacillus subtilis seed solution is obtained by obtaining Bacillus subtilis seed solution at a concentration of 1000

[0041] The Bacillus subtilis expansion culture medium contains 10g corn flour, 5g glucose, 20g soybean meal, 5g calcium carbonate, 1g ammonium sulfate, 0.3g dipotassium hydrogen phosphate, 0.2g magnesium sulfate heptahydrate, 0.2g manganese sulfate hydrate, and the remainder is water, with a pH of 7.0-7.5.

[0042] In the following embodiments, the methods for activating and expanding the culture of Lactobacillus plantarum are as follows:

[0043] (1) Activation culture: Take out the preserved Lactobacillus plantarum, put it in a 34℃ incubator for 4-6 hours, and then inoculate it into a 250mL Erlenmeyer flask containing 200mL MRS medium. The inoculation amount is 1-3 loops, and it is placed at 34℃ for static culture for 18-24 hours.

[0044] The MRS medium contains 10g of protein powder, 5g of beef extract, 4g of yeast powder, 20g of glucose, 1.0mL of Tween 80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of triammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, and the remainder is water per liter.

[0045] (2) Expanded culture: The activated Lactobacillus plantarum cultured in vitro was inoculated into several Lactobacillus plantarum expansion culture media at a mass ratio of Lactobacillus plantarum bacterial suspension to Lactobacillus plantarum expansion culture medium of 1:50. The culture was incubated at 34℃ for 24 hours. The viable count of Lactobacillus plantarum was adjusted by adding more Lactobacillus plantarum expansion culture medium to make the Lactobacillus plantarum count 3-8×10⁶. 8 The concentration of Lactobacillus plantarum seed culture is 100 cells / mL.

[0046] The mass percentages of each component in the Lactobacillus plantarum expanded culture medium are as follows: 5% bean sprout juice, 10% tomato juice, 5% potato, 2% sucrose, 4% salt, and the remainder is water.

[0047] In the following embodiments, the activation and scale-up culture methods of the *Saccharomycopsis fibuligera* are as follows:

[0048] (1) Activation culture: Take out the cladomycin and place it in a 30℃ incubator for 4-6 hours. Inoculate it into a 250mL Erlenmeyer flask containing 50mL LYPD liquid medium. The inoculation amount is 1-3 inoculation loops. Then place it at 30℃ and shake it at 180rpm / min for 18-24 hours to obtain the activated cladomycin liquid.

[0049] The YPD liquid culture medium contains 20g of glucose, 20g of peptone, 10g of yeast extract, and the remainder is water per liter of culture medium;

[0050] (2) Expanded culture: The activated cultured *Saccharomyces cerevisiae* was inoculated into several 250 mL Erlenmeyer flasks containing 50 mL of *Saccharomyces cerevisiae* expanded liquid culture medium. The inoculation amount was 2% of the volume of the *Saccharomyces cerevisiae* expanded liquid culture medium. Then, the flasks were placed on a shaker at 180 rpm / min and 30℃ and cultured for 18-24 h. After that, *Saccharomyces cerevisiae* expanded liquid culture medium was added and the concentration was adjusted to make the number of *Saccharomyces cerevisiae* cells 3-8 × 10⁸ / mL to obtain *Saccharomyces cerevisiae* seed culture.

[0051] The expanded liquid culture medium for the capsule-forming yeast comprises: 50g glucose, 10g ammonium sulfate, 5g urea, 20g yeast extract, 1g dipotassium hydrogen phosphate, 0.5g potassium dihydrogen phosphate, 1.2g magnesium sulfate, and the remainder is water, with the pH adjusted to 5.5 and 1000mL distilled water.

[0052] In the following embodiments, the corn cob particles are prepared by selecting clean, dry corn cobs free from mold contamination and crushing them into small pieces with a diameter of 5-10 mm in a pulverizer to obtain corn cob particles.

[0053] In the following embodiments, the fermented Chinese herbal medicine feed additive for catfish is a microbial fermented Chinese herbal medicine additive for catfish.

[0054] Example 1

[0055] Step 1: Take 70 parts by weight of peony leaves, 70 parts of scutellaria baicalensis, 30 parts of pigweed, 40 parts of gallnut, 20 parts of hawthorn, 40 parts of polygonum cuspidatum, 20 parts of ginkgo, 40 parts of forsythia, 15 parts of atractylodes macrocephala, 20 parts of rhubarb, and 25 parts of pomegranate peel. Remove impurities from the Chinese herbs, dry them in the sun, pulverize them through an 80-mesh sieve, mix them evenly, and obtain Chinese herbal powder for later use.

[0056] Step 2: Weigh out 30 parts of potassium dihydrogen phosphate, 70 parts of dipotassium hydrogen phosphate, 40 parts of sodium chloride and 8 parts of magnesium sulfate according to the weight ratio, put them into a container, pour in 100 parts of distilled water, and dissolve them completely to obtain a mineral salt solution.

[0057] Step 3: According to the weight proportions, take 60 parts of soybean meal powder, 60 parts of corn cob granules, 120 parts of wheat bran, 100 parts of mineral salt solution and 450 parts of the prepared Chinese medicine powder, add 340 parts of distilled water, mix evenly and put into a solid aerobic fermentation tank. After steam sterilization for 15 minutes, the temperature is reduced to 40℃ to obtain solid fermentation culture medium for later use.

[0058] Step 4: Load the above solid fermentation culture medium into a solid fermentation tank, add 15 parts by weight of the above *Saccharomyces cerevisiae* seed liquid and 30 parts by weight of *Bacillus subtilis* seed liquid, mix well, control the temperature at 28°C, and ferment in the fermentation tank by purging sterile air or oxygen for 22 hours; stop purging sterile air or oxygen, add 60 parts by weight of *Lactobacillus plantarum* seed liquid, stir well, control the temperature at 30°C, and continue fermentation for 48 hours; the inoculation ratio of *Saccharomyces cerevisiae*, *Bacillus subtilis*, and *Lactobacillus plantarum* is 1:2:4;

[0059] Step 5: Spread the obtained fermented Chinese medicine out to a thickness of about 1-2 cm, and air dry it with hot air at 40-50℃ for 2-3 hours until the moisture content does not exceed 7%. Stop drying, crush it, and pass it through a 40-mesh sieve to obtain the fermented Chinese medicine feed additive for catfish.

[0060] Example 2

[0061] Step 1: Take 80 parts of peony leaves, 60 parts of scutellaria baicalensis, 40 parts of pigweed, 30 parts of gallnut, 30 parts of hawthorn, 30 parts of polygonum cuspidatum, 30 parts of ginkgo, 30 parts of forsythia, 20 parts of atractylodes macrocephala, 30 parts of rhubarb, and 20 parts of pomegranate peel according to the weight proportions. Remove impurities from the Chinese herbs, dry them in the sun, pulverize them through an 80-mesh sieve, mix them evenly, and obtain Chinese herbal powder for later use.

[0062] Step 2: Weigh out 35 parts of potassium dihydrogen phosphate, 65 parts of dipotassium hydrogen phosphate, 45 parts of sodium chloride, and 7 parts of magnesium sulfate according to the weight ratio, put them into a container, pour in 100 parts of distilled water, and dissolve them completely to obtain a mineral salt solution.

[0063] Step 3: According to the weight proportions, take 70 parts of soybean meal powder, 70 parts of corn cob granules, 110 parts of wheat bran, 100 parts of mineral salt solution and 470 parts of the prepared Chinese medicine powder, add 370 parts of distilled water, mix evenly and put into a solid aerobic fermentation tank. After steam sterilization for 15 minutes, the temperature is reduced to 40℃ to obtain solid fermentation culture medium for later use.

[0064] Step 4: Load the above solid fermentation culture medium into a solid fermentation tank, and add 14 parts by weight of the above *Saccharomyces cerevisiae* seed liquid and 27 parts by weight of *Bacillus subtilis* seed liquid. Mix well, control the temperature at 30°C, and ferment in the fermentation tank by purging sterile air or oxygen for 19 hours. Stop purging sterile air or oxygen, add 54 parts by weight of *Lactobacillus plantarum* seed liquid, stir well, control the temperature at 34°C, and continue fermentation for 42 hours. The inoculation ratio of *Saccharomyces cerevisiae*, *Bacillus subtilis*, and *Lactobacillus plantarum* is 1:2:4.

[0065] Step 5: Spread the obtained fermented Chinese medicine out to a thickness of about 1-2 cm, and air dry it with hot air at 40-50℃ for 2-3 hours until the moisture content does not exceed 7%. Stop drying, crush it, and pass it through a 40-mesh sieve to obtain the fermented Chinese medicine feed additive for catfish.

[0066] Example 3

[0067] Step 1: Take 90 parts by weight of peony leaves, 50 parts of scutellaria baicalensis, 50 parts of pigweed, 20 parts of gallnut, 40 parts of hawthorn, 20 parts of polygonum cuspidatum, 40 parts of ginkgo, 20 parts of forsythia, 25 parts of atractylodes macrocephala, 40 parts of rhubarb, and 15 parts of pomegranate peel. Remove impurities from the Chinese herbs, dry them in the sun, pulverize them through an 80-mesh sieve, mix them evenly, and obtain Chinese herbal powder for later use.

[0068] Step 2: Weigh out 40 parts of potassium dihydrogen phosphate, 60 parts of dipotassium hydrogen phosphate, 50 parts of sodium chloride, and 6 parts of magnesium sulfate according to the weight ratio, put them into a container, pour in 100 parts of tap water, and dissolve them completely to obtain a mineral salt solution.

[0069] Step 3: According to the weight proportions, take 80 parts of soybean meal powder, 80 parts of corn cob granules, 100 parts of wheat bran, 100 parts of mineral salt solution and 490 parts of the prepared Chinese medicine powder, add 400 parts of tap water, mix evenly and put into a solid aerobic fermentation tank, steam sterilize for 15 minutes, and then lower the temperature to 40℃ to obtain solid fermentation culture medium for later use.

[0070] Step 4: Load the above solid fermentation culture medium into a solid fermentation tank, add 12 parts by weight of the above *Saccharomyces cerevisiae* seed liquid and 24 parts by weight of *Bacillus subtilis* seed liquid, mix well, control the temperature at 32°C, and ferment in the fermentation tank by purging sterile air or oxygen for 16 hours; stop purging sterile air or oxygen, add 48 parts by weight of *Lactobacillus plantarum* seed liquid, stir well, control the temperature at 36°C, and continue fermentation for 36 hours; the inoculation ratio of *Saccharomyces cerevisiae*, *Bacillus subtilis*, and *Lactobacillus plantarum* is 1:2:4.

[0071] Step 5: Spread the obtained fermented Chinese medicine out to a thickness of about 1-2 cm, and air dry it with hot air at 40-50℃ for 2-3 hours until the moisture content does not exceed 7%. Stop drying, crush it, and pass it through a 40-mesh sieve to obtain the fermented Chinese medicine feed additive for catfish.

[0072] Comparative Example 1

[0073] The steps of this comparative example are basically the same as those of Example 2, except that:

[0074] Step 4: Load the above solid fermentation culture medium into a solid-state fermentation tank, mix thoroughly, control the temperature at 30℃, and circulate sterile air in the fermentation tank for 19 hours; stop circulating sterile air, stir thoroughly, control the temperature at 34℃, and continue for 42 hours. The result is a traditional Chinese medicine additive that has not undergone microbial fermentation.

[0075] Comparative Example 2

[0076] The composite microbial active additive is prepared according to the method described in the patent "A Composite Microbial Active Additive and Its Application" (application number: 201410015670.0).

[0077] Example of effect

[0078] <The Effects of Fermented Traditional Chinese Medicine Additives on the Growth of Catfish>

[0079] (1) Basic feed for catfish: fish meal 5%, soybean meal 29.1%, wheat flour 15%, cottonseed meal 5%, rice bran 25%, fish oil 3%, corn gluten meal 5%, blood meal 5%, meat and bone meal 5%, vitamin and mineral premix 1%, salt 0.4%, calcium dihydrogen phosphate 1.5%.

[0080] (2) Selected catfish weighing (5.54±0.21) grams were placed in aquariums equipped with temperature and circulating water control systems. During the experiment, the water temperature was maintained at 28–33℃. After the catfish adapted to the aquarium environment, 720 catfish of uniform size and without injury or disease were selected. The control group was fed a basic diet daily. The traditional Chinese medicine group, the 2.5% fermented traditional Chinese medicine group, and the 5% fermented traditional Chinese medicine group had 2.5% traditional Chinese medicine and 2.5% and 5% fermented traditional Chinese medicine added to their basic diet, replacing the same amount of soybean meal in the basic diet. Each group had 60 fish, with 3 replicates per group, and was fed 3 times a day.

[0081] (3) Survival rate (%) = (last number / first number) × 100;

[0082] Specific growth rate (%) / d = [Ln(average final weight of the experiment) - Ln(average initial weight of the experiment)] / number of days of the experiment × 100;

[0083] Feed conversion ratio = Feeding amount / Weight gain;

[0084] The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in liver and plasma were determined using a kit.

[0085] Table 1. Effects of fermented traditional Chinese medicine additives on growth and liver function of catfish.

[0086]

[0087] Note: Different shoulder note letters in the same column indicate significant differences (P<0.05), and any identical shoulder note number indicates no significant difference (P>0.05). The data in the table are (mean ± standard error).

[0088] Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are synthesized in the liver. Liver damage or abnormalities can lead to elevated serum AST and ALT levels. In all experimental groups, serum AST and ALT levels were lower than in the control group, indicating that the use of fermented traditional Chinese medicine additives resulted in normal hepatopancreatic function and no significant damage to hepatopancreatic cells. Dissection of the *Catfish simonii* in each experimental group revealed no significant differences in the appearance, color, elasticity, or thickness of the liver, spleen, intestines, and other internal organs. Even when the amount of fermented traditional Chinese medicine added exceeded the normal usage by 100% (5% fermented traditional Chinese medicine group), there were no significant changes in the internal organs. This demonstrates that the obtained fermented traditional Chinese medicine feed additive is safe, reliable, and has no toxic side effects.

[0089] The 2.5% fermented traditional Chinese medicine group showed significantly higher specific growth rate and survival rate than the control group, while its feed conversion ratio was significantly lower (P<0.05). There were no significant differences in specific growth rate, survival rate, feed conversion ratio, and the control group between the traditional Chinese medicine group and the control group (P>0.05). The 5% fermented traditional Chinese medicine group showed a significantly higher survival rate than the control group (P<0.05), while there were no significant differences in specific growth rate and feed conversion ratio. This indicates that adding 2.5% fermented traditional Chinese medicine to the feed can significantly promote the growth of *Catfish simonii*, improve survival rate, and reduce feed conversion ratio, which is superior to the effects of the same amount of traditional Chinese medicine and 5% fermented traditional Chinese medicine. After fermentation with mixed strains, although some energy substances are consumed during the fermentation process, a certain amount of microbial protein and a large amount of vitamins, lactic acid, and other microbial metabolites are produced. Large molecules such as proteins and starches in plant raw materials and traditional Chinese medicine are decomposed into small molecules such as peptides, amino acids, and glucose. Cellulose and hemicellulose, which make up the plant cell walls, are converted into components that can be utilized by *Catfish simonii*. Traditional Chinese medicine contains active substances such as flavonoids and glycosides. These components can promote appetite, enhance the body's metabolic activities, promote the secretion of digestive enzymes, and accelerate the growth and development of aquatic animals. After fermentation, these substances are more easily utilized by fish.

[0090] <The Effects of Fermented Traditional Chinese Medicine Additives on CYP450 Enzyme Activity and Enrofloxacin Residue in Catfish>

[0091] (1) Select catfish weighing (70±5) grams and raise them in aquariums with temperature control and circulating water control systems. The basic feed was the basic feed described in (1) of the experiment on the effect of fermented Chinese medicine additive on the growth of catfish. The control group was fed enrofloxacin daily at a dose of 50 mg / (kg·d) for 5 consecutive days. After 5 days, the basic feed was fed until the end of the experiment. The enrofloxacin feeding method for the Chinese medicine group and the fermented Chinese medicine group was the same as that for the control group. On the 5th day, 2.5% of Chinese medicine or fermented Chinese medicine was added to the feed. After 4 consecutive days, the basic feed was fed until the end of the experiment. Chinese medicine group: the Chinese medicine additive prepared by the method of Comparative Example 1, with 2.5% of the Chinese medicine additive replacing an equal amount of soybean meal in the basic feed of catfish; fermented Chinese medicine group: the microbial fermented Chinese medicine additive prepared by the method of Example 2, with 2.5% of the microbial fermented Chinese medicine additive replacing an equal amount of soybean meal in the basic feed of catfish.

[0092] (2) Timing began after the last administration of enrofloxacin-medicated feed. Samples were taken at 1, 3, 5, 10, 20, 60, 120, 180, 240, and 300 hours. Three fish were taken as one sample from each group. Blood samples were collected from the tail vein and placed in centrifuge tubes containing heparin sodium anticoagulant. The blood was centrifuged, and the supernatant was collected. After the fish were euthanized, liver and muscle tissue samples were taken for testing.

[0093] (3) Assay of CYP1A1 and CYP3A activity in liver of catfish

[0094] Liver tissue was rinsed with physiological saline pre-cooled at 4℃ to remove blood cells. After wiping away excess liquid, the tissue was weighed and added to 0.1mol / L PBS homogenate at pH 4 in a 1:4 ratio. The tissue was minced, homogenized at low temperature, and then centrifuged at 15000r / min for 30min. The supernatant was collected. The activities of CYP1A1 and CYP3A were measured according to the instructions of the cytochrome P450 subenzyme CYP1A1 (MROD) activity quantitative detection kit and the cytochrome P450 subenzyme CYP3A (END) activity colorimetric quantitative detection kit, respectively. The blank control group was fed only the basic feed, and other aspects were the same as those in group (1).

[0095] Table 2. Effects of enrofloxacin and fermented traditional Chinese medicine additives on CYP450 enzymes in the liver of *Catfish simonii*.

[0096]

[0097] Note: Different shoulder notes in the same row indicate significant differences (P<0.05), and any identical shoulder note number indicates no significant difference (P>0.05). The data in the table are (mean ± standard error).

[0098] CYP1A1 and CYP3A are important CYP450 enzymes that catalyze the metabolism of various drugs. Compared with the blank control group, the activities of CYP1A1 and CYP3A in the liver of *Catfish simonii* in the enrofloxacin group (control group) were significantly reduced (P<0.05), decreasing by 19.23% and 21.36%, respectively, indicating that oral enrofloxacin can reduce the activities of CYP1A1 and CYP3A in the liver of *Catfish simonii*. After administration of traditional Chinese medicine, compared with the blank control group, the activity of CYP1A1 was significantly increased (P<0.05), while the activity of CYP3A increased but not significantly. After administration of fermented traditional Chinese medicine, compared with the blank control group, the activities of CYP1A1 and CYP3A were significantly increased (P<0.05), increasing by 43.59% and 25.24%, respectively, indicating that administration of fermented traditional Chinese medicine can increase the activities of CYP1A1 and CYP3A in the liver of *Catfish simonii*. The active ingredients such as baicalin and gallic acid in the compound traditional Chinese medicine have an inducing effect on CYP450 of catfish, which significantly accelerates the metabolism of enrofloxacin in catfish. After fermentation, the active ingredients such as gallic acid and baicalin are more easily dissolved from the cells of traditional Chinese medicine, which induces the related genes of CYP450 enrofloxacin degradation enzyme to be expressed more efficiently and rapidly, resulting in a significant increase in CYP450 enzyme activity.

[0099] Table 3. Effects of fermented traditional Chinese medicine additives on enrofloxacin residues in the liver and blood of *Catfish simonii*.

[0100]

[0101] Note: Different shoulder notes in the same row indicate significant differences (P<0.05), and any identical shoulder note number indicates no significant difference (P>0.05). The data in the table are (mean ± standard error).

[0102] Table 4. Effects of fermented traditional Chinese medicine additives on enrofloxacin residues in the muscle of *Catfish simonii*.

[0103]

[0104] Note: Different shoulder notes in the same row indicate significant differences (P<0.05), and any identical shoulder note number indicates no significant difference (P>0.05). The data in the table are (mean ± standard error).

[0105] The enrofloxacin content in the liver of the control group, the traditional Chinese medicine (TCM) group, and the fermented TCM group all reached their peak at 3 hours. The highest peak content was in the fermented TCM group, followed by the TCM group, and then the control group, indicating that both TCM and fermented TCM can promote the absorption of enrofloxacin. The peak enrofloxacin content in the fermented TCM group was significantly higher than that in the control group (P<0.05). After reaching the peak, the enrofloxacin content in all three experimental groups gradually decreased over time, with the fermented TCM group showing the fastest decrease, followed by the TCM group. At all sampling points from 10 to 300 hours, the enrofloxacin content in both the TCM and fermented TCM groups was significantly lower than that in the control group (P<0.05), indicating that both TCM and fermented TCM can accelerate the metabolism of enrofloxacin in the liver. The situation in the blood was similar to that in the liver. The enrofloxacin content in the fermented TCM group fell below the safety standard (0.1 mg / kg) after 60 hours, while that in the TCM group fell below the safety standard after 180 hours. The highest levels of enrofloxacin in muscle tissue were observed at 1 hour in all three groups. In the fermented traditional Chinese medicine group, the enrofloxacin level was below the safety standard at 180 hours, while in the control group it was below the safety standard at 300 hours. Compared to the control group administered enrofloxacin, the time to reach the withdrawal period standard for enrofloxacin in muscle tissue was shortened by 40% after adding the fermented traditional Chinese medicine additive; compared to the traditional Chinese medicine group, the time was shortened by 25%. This indicates that both traditional Chinese medicine and the fermented traditional Chinese medicine additive can promote the absorption and metabolic elimination of enrofloxacin, with the fermented traditional Chinese medicine showing superior efficacy. Enrofloxacin is primarily metabolized in the liver. Both traditional Chinese medicine and fermented traditional Chinese medicine induced liver enzymes such as CYP450, accelerating the metabolism of enrofloxacin in the liver and shortening its residence time in the body. Most of the components in traditional Chinese medicine are contained in plant cells. The cell walls of plants are composed of cellulose, hemicellulose, pectin, etc. The physical barrier of the cell walls prevents fish from utilizing the effective components of traditional Chinese medicine. Bacillus subtilis, a fermentation strain, can secrete a variety of enzyme systems such as cellulase, hemicellulase, and pectinase when growing on a traditional Chinese medicine substrate. It has a significant cell wall breaking effect. Then, it is enzymatically hydrolyzed by Saccharomyces boulardii and Lactobacillus plantarum, which transforms the large nutritional molecules such as proteins and polysaccharides into easily absorbed small molecules. This makes the nutrients and beneficial components in the cells of traditional Chinese medicine easily available to fish, promotes the absorption, transport, and metabolism of the effective components of traditional Chinese medicine in the fish, improves the efficacy of traditional Chinese medicine, avoids the loss of activity or large loss of some components after high-temperature decoction, and has great potential to transform and modify the active components of traditional Chinese medicine and generate new active substances.

[0106] <The Effects of Fermented Traditional Chinese Medicine Additives on Enrofloxacin Residues in Common Farmed Species such as Carp and Crucian Carp>

[0107] (1) Select carp weighing (100±10) grams and crucian carp weighing (50±5) grams and stock them in aquariums with temperature control and circulating water control systems. The basic feed was the basic feed described in (1) of the experiment on the effect of fermented Chinese medicine additive on the growth of catfish. The control group was fed enrofloxacin daily at a dose of 50 mg / (kg·d) for 5 consecutive days. After 5 days, the basic feed was fed until the end of the experiment. The enrofloxacin feeding method for the fermented Chinese medicine group was the same as that for the control group. On the 5th day, 2.5% of fermented Chinese medicine was added to the feed. After 4 consecutive days, the basic feed was fed until the end of the experiment. The fermented Chinese medicine group used the microbial fermented Chinese medicine additive prepared by the method in Example 2, replacing an equal amount of soybean meal in the basic feed with 2.5% of the microbial fermented Chinese medicine additive.

[0108] (2) Timing began after the last administration of enrofloxacin-medicated feed. Samples were taken at 1, 3, 5, 10, 20, 60, 120, 180, 240, and 300 hours. Three fish were taken as one sample from each group. Blood samples were collected from the tail vein and placed in centrifuge tubes containing heparin sodium anticoagulant. The blood was centrifuged, and the supernatant was collected. After the fish were euthanized, liver and muscle tissue samples were taken for testing.

[0109] Table 5. Effects of fermented traditional Chinese medicine additives on enrofloxacin residues in carp liver, muscle, and blood.

[0110]

[0111] Note: Different shoulder notes in the same row indicate significant differences (P<0.05), and any identical shoulder note number indicates no significant difference (P>0.05). The data in the table are (mean ± standard error).

[0112] Table 6. Effects of fermented traditional Chinese medicine additives on enrofloxacin residues in the liver, muscle, and blood of crucian carp.

[0113]

[0114]

[0115] Note: Different shoulder notes in the same row indicate significant differences (P<0.05), and any identical shoulder note number indicates no significant difference (P>0.05). The data in the table are (mean ± standard error).

[0116] As shown in Tables 5 and 6, the peak enrofloxacin content in the liver, muscle, and blood of carp and crucian carp was higher in the fermented herbal medicine group than in the control group, indicating that the fermented herbal medicine can promote the absorption of enrofloxacin. After reaching the peak, similar to the enrofloxacin metabolism in catfish, the enrofloxacin content in each experimental group gradually decreased over time. However, unlike the enrofloxacin metabolism in catfish, after reaching the peak, the enrofloxacin content in the fermented herbal medicine group with oral fermented herbal medicine additive was higher than that in the control group at all sampling points, indicating that the fermented herbal medicine did not significantly affect the metabolism of enrofloxacin in the liver, muscle, and blood. Similar results were obtained using common farmed species such as grass carp and Wuchang bream, indicating that the fermented herbal medicine preparation obtained in this invention only accelerates the metabolism of enrofloxacin in catfish, removes enrofloxacin drug residues, and shortens the withdrawal period, demonstrating its specificity.

[0117] <The Effect of Fermented Traditional Chinese Medicine Additives on Drug Residues in the Muscles of Catfish After Oral Administration of Other Antibiotics>

[0118] (1) Select catfish weighing (70±5) grams and raise them in aquariums with temperature and circulating water control systems. The basic feed was the basic feed described in (1) of the experiment on the effect of fermented Chinese medicine additives on the growth of catfish. Vancomycin control group was fed vancomycin daily at a dose of 1 mg / (kg·d); thiamphenicol control group was fed thiamphenicol daily at a dose of 30 mg / (kg·d); florfenicol control group was fed florfenicol daily at a dose of 10 mg / (kg·d). Each control group was fed medicated feed for 5 consecutive days, and then fed basic feed until the end of the experiment. The method of feeding vancomycin, thiamphenicol and florfenicol medicated feed in the fermentation group was the same as that in the corresponding control group. On the 5th day, 2.5% fermented Chinese medicine was added to the feed, and the feed was fed continuously for 4 days, and then fed basic feed until the end of the experiment. The fermentation group consists of microbial fermented traditional Chinese medicine additives prepared by the method in Example 2, with 2.5% of the microbial fermented traditional Chinese medicine additives replacing an equal amount of soybean meal in the basic feed of the catfish.

[0119] (2) Start timing after the last feeding of medicated bait. Take samples at 1, 3, 5, 10, 20, 60, 120, 180, 240 and 300 hours respectively. Take 3 fish in each group as a sample. After killing the fish, take muscle tissue samples for testing.

[0120] from Figure 1-3It can be seen that after oral administration of vancomycin, thiamphenicol, and florfenicol to catfish, the feeding of fermented traditional Chinese medicine additives did not show a significant effect in accelerating antibiotic metabolism. Similar results were obtained with antibiotics such as oxytetracycline and furazolidone. This indicates that the fermented traditional Chinese medicine preparation obtained in this invention only shows the effect of accelerating the in vivo metabolism of enrofloxacin, removing enrofloxacin drug residues, and shortening the withdrawal period for catfish after oral administration of enrofloxacin. It has specificity, and no significant effect was observed on catfish after oral administration of other antibiotics.

[0121] <The Effect of Compound Microbial Active Additives on Enrofloxacin Residue in Catfish>

[0122] (1) Select catfish weighing (70±5) grams and stock them in aquariums with temperature and circulating water control systems. The basic feed was the same as described in (1) of the experiment on the effect of fermented Chinese medicine additives on the growth of catfish. The control group was fed enrofloxacin daily at a dose of 50 mg / (kg·d) for 5 consecutive days. After 5 days, the basic feed was fed until the end of the experiment. The enrofloxacin feeding method for the compound microbial active additive group (i.e., Comparative Example 2) was the same as that for the control group. On the 5th day, 2.5% of the compound microbial active additive was added to the feed. After 4 consecutive days, the basic feed was fed until the end of the experiment. The compound microbial active additive group was prepared by the method of Comparative Example 2. 2.5% of the compound microbial active additive replaced an equal amount of soybean meal in the basic feed of catfish.

[0123] (2) Timing was started after the last feeding of enrofloxacin-treated food. Samples were taken at 1, 3, 5, 10, 20, 60, 120, 180, 240 and 300 hours. Three fish were taken as a sample in each group. After the fish were killed, muscle tissue samples were taken for testing to check for enrofloxacin residue in the muscle.

[0124] from Figure 4 It can be seen that the additive prepared by the method of the patented compound microbial active additive and its application in Comparative Example 2 (application number: 201410015670.0) did not show any significant effect in accelerating the metabolism of enrofloxacin or shortening the withdrawal period when fed to catfish.

[0125] The transformation and clearance of drugs in organisms are mainly carried out by drug-metabolizing enzymes. Cytochrome P450 enzymes (CYP450) are a superfamily of heme-containing isoenzymes and are the most important metabolic enzyme system in drug transformation. They participate in the metabolism of most endogenous and exogenous substances in the first-phase reaction of drugs. CYP450 is widely present in animals, plants, fish, and other organisms, and is expressed in tissues such as the liver, kidneys, and gastrointestinal tract, as well as in the endoplasmic reticulum, mitochondria, and nuclear membrane of cells within the same organism, but is mainly expressed in the liver. The first-phase reaction catalyzed by CYP450 enzymes is a key step in drug metabolism in vivo, and this step is often the rate-limiting step in drug elimination from the body. Therefore, the activity of CYP450 enzymes determines the rate of drug metabolism and is directly related to the drug clearance rate. Cytochrome P450 is a class of proteins composed of different numbers of amino acids. To date, more than 2,000 CYP450 enzymes have been discovered, and the subtypes and expression levels of CYP450 enzymes vary considerably among different organisms. The metabolic rate and residual time of drugs are often related to the induction and inhibition of CYP450. There is an interaction between CYP450 and drugs; CYP450 can catalyze drug metabolism, while drugs also act on drug-metabolizing enzymes, inducing or inhibiting their activity, thereby accelerating or slowing the metabolism of other drugs (substrate) or the drug itself. Different drugs activate or inhibit specific CYP450 enzymes. Different biological species often exhibit significant differences in the specific types and expression levels of CYP450 enzymes. Using different drugs on the same organism can activate completely different types of CYP450 enzymes, and the same drug used on different organisms can also activate or inhibit significantly different types of specific enzymes.

[0126] Enrofloxacin inhibits CYP450 in catfish. On the one hand, enrofloxacin has a long-lasting effect, providing good preventative and therapeutic benefits. On the other hand, the drug is slowly eliminated from the fish's body, leaving significant residues that require a considerable amount of time to reduce to below safe concentrations. The compound traditional Chinese medicine used in this invention, containing active ingredients such as baicalin and gallic acid, induces CYP450 in catfish, significantly accelerating the metabolism of enrofloxacin in the catfish. After fermentation, gallic acid, baicalin, and other active ingredients are more easily dissolved from the herbal cells, inducing efficient and rapid expression of genes related to the CYP450 enrofloxacin-degrading enzyme, resulting in a significant increase in enzyme activity and rapid degradation and excretion of enrofloxacin. Due to the differences in the types and expression levels of CYP450 enzymes among different fish species, the induction or inhibition effects of fermented traditional Chinese medicine feed additives on CYP450 enzymes in different fish species show significant differences. At the same time, the specific types and amounts of CYP450 enzymes activated or inhibited by enrofloxacin also vary considerably among different fish species. Among common farmed species, the compound fermented traditional Chinese medicine of this invention only has a significant effect on the degradation of enrofloxacin in catfish, while the effect on enrofloxacin in common farmed fish such as carp and crucian carp is not significant. As for other antibiotics, no significant degradation effect has been found so far.

[0127] <Preventive and adjunctive therapeutic effects of fermented traditional Chinese medicine additives on hemorrhagic septicemia in catfish>

[0128] (1) Preparation of infecting bacterial strains

[0129] Aeromonas hydrophila was inoculated into beef extract peptone broth and incubated at 28°C for 24 hours. The bacterial concentration was determined by turbidimetric assay, and the viable count was adjusted to 8 × 10⁻⁶ using sodium chloride injection. 7 per mL.

[0130] (2) Select catfish weighing (70±5) grams and temporarily house them in an indoor glass aquarium for 7 days, feeding them a basic diet. The water temperature during the experiment was 28-33℃. After the catfish adapted to the aquarium environment, 440 catfish of uniform size and without injury or disease were selected to conduct prevention and treatment experiments for hemorrhagic septicemia in catfish. In the prevention experiment, the catfish were fed a basic diet or medicated feed for 7 days, followed by intraperitoneal injection of Aeromonas hydrophila at a dose of 0.1 mL / fish. In the treatment experiment, the catfish were first injected intraperitoneally with Aeromonas hydrophila at a dose of 0.1 mL / fish, and then fed medicated feed or a basic diet 6 hours later. Both the prevention and treatment experiments were randomly assigned to groups, with one replicate in each group, and 20 fish in each group. The specific groupings were as follows: Negative control group: fed basal feed, no infection, injected with the same dose of sodium chloride injection as the experimental group; Positive control group: fed basal feed, infected; Traditional Chinese medicine group: fed basal feed + 2.5% traditional Chinese medicine (prepared in Comparative Example 1), infected; Fermented traditional Chinese medicine group: fed basal feed + 2.5% fermented traditional Chinese medicine (fermented traditional Chinese medicine obtained in Example 2), infected; Enrofloxacin group: fed basal feed + 0.25% enrofloxacin, infected; Traditional Chinese medicine enrofloxacin group: fed basal feed + 2.5% traditional Chinese medicine + 0.25% enrofloxacin, infected; Fermented traditional Chinese medicine enrofloxacin group: fed basal feed + 2.5% fermented traditional Chinese medicine + 0.25% enrofloxacin, infected. Feeding was done twice daily, until the animal was satiated with a slight surplus.

[0131] Table 7. Preventive effect of fermented traditional Chinese medicine additives on hemorrhagic septicemia of catfish.

[0132]

[0133] Note: Different shoulder note letters in the same column indicate significant differences (P<0.05), and any identical shoulder note number indicates no significant difference (P>0.05). The data in the table are (mean ± standard error).

[0134] Feeding the catfish with medicated feed containing 2.5% compound traditional Chinese medicine (TCM) increased the survival rate by 42.5% compared to the positive control group fed with basal feed, indicating that this TCM formula has the effect of preventing Aeromonas hydrophila infection and improving the immunity of the catfish. TCM contains various active ingredients such as polysaccharides and flavonoids, which can enhance metabolic activity and improve immunity. The survival rate of the fermented TCM group increased by 65% ​​compared to the positive control group, and by 22.5% compared to the group with the same amount of TCM, indicating that the disease prevention effect of the TCM is improved after fermentation. After fermentation, the "cell wall breaking effect" of microorganisms makes the nutrients and beneficial components in the cells of traditional Chinese medicine (TCM) readily available to fish, promoting the absorption, transport, and metabolism of the active ingredients in the fish, thus enhancing the efficacy of the TCM. Some toxic substances are decomposed, and the taste of the TCM is also improved. Rhubarb and Scutellaria baicalensis in the compound have a direct killing effect on various intestinal bacteria and fish pathogens, such as Aeromonas hydrophila and Staphylococcus aureus, improving the intestinal environment and facilitating the colonization of lactic acid bacteria and Bacillus subtilis in the intestines. On the other hand, Bacillus subtilis and lactic acid bacteria inhibit the reproduction of harmful bacteria through "biological oxygen depletion," "spatial competition," and "food competition." Furthermore, the microbial fermentation process produces a large number of small peptides, organic acids, and various vitamins, which also promote the immune system. The synergistic effect of the TCM and microorganisms keeps the catfish in a good growth state, making it difficult for pathogens to invade.

[0135] Table 8. Therapeutic effects of fermented traditional Chinese medicine additives on hemorrhagic septicemia of catfish.

[0136]

[0137] Note: Different shoulder note letters in the same column indicate significant differences (P<0.05), and any identical shoulder note number indicates no significant difference (P>0.05). The data in the table are (mean ± standard error).

[0138] Approximately 6-8 hours after infection, most of the barbel catfish showed symptoms. Initially, mild congestion appeared in the mouth, upper and lower jaws, gill covers, and base of the pectoral fins, with punctate congestion on the abdomen and sides. As the disease progressed, congestion and swelling around the eyes and eye sockets occurred, the eyeballs bulged, bleeding on the body surface intensified, the abdomen swelled, and the anus became red and swollen. Upon dissection, congestion and hemorrhage were observed in multiple internal organs, including the gills, liver, kidneys, and intestines, which subsequently became lighter in color due to blood loss. The liver turned yellowish-brown, and the spleen and kidneys were significantly enlarged, losing their original elasticity and becoming easily broken. The intestinal wall was congested, and the abdominal cavity contained a large amount of bloody ascites, which was dark red in color and had low viscosity. The vast majority of deaths occurred within 4 days of the bacterial injection, indicating that hemorrhagic septicemia in barbel catfish is a rapidly developing and explosive disease.

[0139] Adding 2.5% compound traditional Chinese medicine (TCM) to the feed treated barbel catfish infected with Aeromonas hydrophila. Compared with the positive control group without TCM, the survival rate increased by 17.5%, indicating that the compound TCM has a certain therapeutic effect on hemorrhagic septicemia in barbel catfish caused by Aeromonas hydrophila. The ingredients in the formula, such as Scutellaria baicalensis, rhubarb, gallnut, and forsythia, have a bactericidal effect on Aeromonas hydrophila, inhibiting the damage of the pathogen to the fish. Compared with the positive control group, the survival rate of the fermented TCM group increased by 25%, slightly higher than that of the TCM group, indicating that the improvement in efficacy of TCM on hemorrhagic septicemia in barbel catfish after fermentation is limited. Compared with the prevention experiment, the survival rate after treatment in the TCM group was 30%, and in the fermented TCM group it was only 37.5%, indicating that the therapeutic effect of fermented compound TCM is lower than that of prevention. This may be related to the fact that the mechanism of action of TCM or fermented TCM is more indirect, through enhancing the fish's specific and non-specific immunity. The survival rate of the enrofloxacin group was significantly higher than that of the traditional Chinese medicine group and the fermented traditional Chinese medicine group (P<0.05), indicating that enrofloxacin has the characteristics of low dosage, good efficacy, and high efficiency in treating hemorrhagic septicemia in catfish. Although the survival rate improved after combining enrofloxacin with traditional Chinese medicine, there was no significant difference compared to the enrofloxacin group (P>0.05). However, the survival rate increased by 22.5% compared to the enrofloxacin group and by 15% compared to the enrofloxacin-traditional Chinese medicine group, with a significant difference (P>0.05). This indicates that the combined use of fermented traditional Chinese medicine and enrofloxacin can significantly improve the treatment effect on hemorrhagic septicemia in catfish.

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a fermented herbal feed additive for catfish, characterized in that: Includes the following steps: S1: Bacillus subtilis, Lactobacillus plantarum and Saccharomyces cerevisiae were activated and cultured and expanded to obtain Bacillus subtilis seed culture, Lactobacillus plantarum seed culture and Saccharomyces cerevisiae seed culture, respectively, for later use; S2: Take 70-90 parts by weight of peony leaves, 50-70 parts of scutellaria baicalensis, 30-50 parts of pigweed, 20-40 parts of gallnut, 20-40 parts of hawthorn, 20-40 parts of polygonum cuspidatum, 20-40 parts of ginkgo, 20-40 parts of forsythia, 20-40 parts of rhubarb, 15-25 parts of atractylodes macrocephala, and 15-25 parts of pomegranate peel, pulverize them, mix them evenly, and obtain Chinese medicine powder for later use. S3: Take 60-80 parts by weight of soybean meal powder, 60-80 parts of corn cob granules, 100-120 parts of wheat bran, 100 parts of mineral salt solution, and 450-490 parts of the Chinese medicine powder obtained in step S2. Add 340-400 parts of tap water or distilled water, mix evenly, and sterilize by steam to obtain a solid fermentation culture medium for later use. S4: Add 12-15 parts by weight of the above-mentioned *Bacillus subtilis* seed culture and 24-30 parts by weight of the above-mentioned *Bacillus subtilis* seed culture to the above-mentioned solid fermentation medium, mix well, control the temperature at 28-32 ℃, and ferment for 16-22 h by introducing sterile air or oxygen; stop introducing sterile air or oxygen, add 48-60 parts by weight of *Lactobacillus plantarum* seed culture, stir well, control the temperature at 30-36 ℃, and continue fermentation for 36 h-48 h to obtain fermented traditional Chinese medicine; S5: Pass hot air at 40-50 ℃ into the fermented Chinese medicine to dry it, then crush it to obtain the fermented Chinese medicine feed additive for catfish. The number of Bacillus subtilis in Bacillus subtilis seed culture, the number of Lactobacillus plantarum in Lactobacillus plantarum seed culture, and the number of Saccharomyces cerevisiae in Saccharomyces cerevisiae seed culture were all 3-8 × 10⁻⁶. 8 cells / mL; The mineral salt solution is prepared by weighing 30-40 parts of potassium dihydrogen phosphate, 60-70 parts of dipotassium hydrogen phosphate, 40-50 parts of sodium chloride and 6-8 parts of magnesium sulfate according to the weight ratio, placing them in a container, pouring in 100 parts of tap water or distilled water, and dissolving them completely to obtain the mineral salt solution.

2. The preparation method of the fermented Chinese herbal feed additive for catfish according to claim 1, characterized in that, The inoculation ratio of *Saccharomyces cerevisiae*, *Bacillus subtilis*, and *Lactobacillus plantarum* was 1:2:

4.

3. The method for preparing a fermented herbal feed additive for catfish according to claim 1, characterized in that, The method for preparing the corn cob granules is as follows: select clean, dry corn cobs free from mold contamination, and crush them into small pieces with a diameter of 5-10 mm in a pulverizer to obtain corn cob granules.

4. The preparation method of a fermented herbal feed additive for catfish according to claim 1, characterized in that, The activation and scale-up culture methods for Bacillus subtilis are as follows: Activation culture: Bacillus subtilis culture was placed in a 37 ℃ incubator for 4-6 hours, then inoculated into beef extract peptone medium and cultured at 37 ℃ for 24 hours on a shaker at 200 rpm to obtain activated Bacillus subtilis. The beef extract peptone medium contained 5 g beef extract, 10 g peptone, and 5 g sodium chloride per liter, with the remainder being water, and a pH of 7.2-7.

5. Expanded culture: Activated Bacillus subtilis cultured medium was inoculated into Bacillus subtilis expansion medium. The medium was shaken at 200 rpm and incubated at 37°C for 24 hours. Bacillus subtilis expansion medium was then added to adjust the viable count to 3-8 × 10⁻⁶ cells / day. 8 The seed culture of Bacillus subtilis is obtained by measuring 10 g / mL of the sample. The Bacillus subtilis expansion culture medium contains 10 g of corn flour, 5 g of glucose, 20 g of soybean meal, 5 g of calcium carbonate, 1 g of ammonium sulfate, 0.3 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.2 g of manganese sulfate hydrate, and the balance being water, with a pH of 7.0-7.

5.

5. The preparation method of a fermented herbal feed additive for catfish according to claim 1, characterized in that, The activation and scale-up culture methods for the aforementioned Lactobacillus plantarum are as follows: Activation culture: Lactobacillus plantarum was cultured at 34 ℃ for 4-6 hours, then inoculated into MRS medium and incubated statically at 34 ℃ for 18-24 hours; the MRS medium contained 10 g of peptone, 5 g of beef extract, 4 g of yeast powder, 20 g of glucose, 1.0 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 5 g of sodium acetate, 2 g of triammonium citrate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, and the remainder was water; Expanded culture: The activated Lactobacillus plantarum culture was inoculated into Lactobacillus plantarum expansion medium and incubated statically at 34 ℃ for 24 h. Lactobacillus plantarum expansion medium was then added to adjust the viable count to 3-8 × 10⁻⁶ cells / year. 8 The concentration of Lactobacillus plantarum in the culture medium is 5% bean sprout juice, 10% tomato juice, 5% potato, 2% sucrose, 4% salt, and the remainder is water.

6. The preparation method of a fermented herbal feed additive for catfish according to claim 1, characterized in that, The activation and scale-up culture methods for the capsule-coated yeast are as follows: Activation culture: The *Saccharomyces cerevisiae* was cultured at 30 ℃ for 4-6 hours, then inoculated into YPD liquid medium and cultured at 30 ℃ with a shaking speed of 180 rpm for 18-24 hours to obtain activated *Saccharomyces cerevisiae* culture. The YPD liquid medium contained 20 g of glucose, 20 g of peptone, 10 g of yeast extract, and the remainder was water per liter. Expanded culture: Inoculate the activated culture of *Saccharomyces cerevisiae* into expanded liquid culture medium, then incubate at 180 rpm and 30 ℃ for 18-24 h on a shaker. Add more expanded liquid culture medium to adjust the concentration to achieve a *Saccharomyces cerevisiae* count of 3-8 × 10⁻⁶. 8 The seed culture of *Saccharomyces cerevisiae* was obtained by measuring 1000 cells / mL. The expanded liquid culture medium for *Saccharomyces cerevisiae* consisted of 50 g glucose, 10 g ammonium sulfate, 5 g urea, 20 g yeast extract, 1 g dipotassium hydrogen phosphate, 0.5 g potassium dihydrogen phosphate, 1.2 g magnesium sulfate, and the remainder was water. The pH was adjusted to 5.5, and 1000 mL of distilled water was used.

7. The fermented Chinese herbal feed additive for catfish (Catfish spp.) obtained by the preparation method according to any one of claims 1-6.

8. The application of the fermented Chinese herbal feed additive for catfish as described in claim 7 in the preparation of feed for the prevention of hemorrhagic septicemia in catfish and the preparation of feed for the treatment of hemorrhagic septicemia in catfish, wherein the dosage in the feed is 2-3% of the feed mass.

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