CN115887535A - Compound traditional Chinese medicine residue fermentation liquor for preventing and treating avian colibacillosis and preparation method thereof - Google Patents

Abstract

The invention provides a compound traditional Chinese medicine residue fermentation liquid for preventing and treating avian colibacillosis and a preparation method thereof, belonging to the technical field of traditional Chinese medicine residue fermentation liquids. The compound traditional Chinese medicine residue fermentation liquid is prepared by fermenting lactobacillus rhamnosus PC30-2-1, and can reduce the level of inflammatory cytokines of pathogenic escherichia coli of artificially infected birds, improve bursal index, improve appetite and disease resistance of livestock and poultry, promote weight gain, improve production benefit, reduce or replace the use of conventional antibacterial drugs, reduce drug residues, reduce the selection pressure of the antibacterial drugs, and inhibit or slow down the generation of drug resistance of clinical pathogenic bacteria.

Description

A compound Chinese medicine residue fermentation liquid for preventing and treating avian Escherichia coli disease and preparation method thereof

Technical Field

The invention belongs to the technical field of Chinese medicinal residue fermentation liquid, and particularly relates to a compound Chinese medicinal residue fermentation liquid for preventing and treating avian Escherichia coli disease and a preparation method thereof.

Background Art

Escherichia coli disease is a common infectious disease of various livestock and poultry, and its pathogenic Escherichia coli (E. coli) is an important zoonotic pathogen. It not only causes huge economic losses to the poultry farming industry and hinders its sustainable and healthy development, but also easily causes animal food safety problems, posing a potential threat to human health and living environment. At present, clinically, the disease is mainly prevented and treated by antimicrobial drugs, but the irrational use of antimicrobial drugs has led to the continuous emergence of multi-drug resistance, cross-resistance and even super-drug-resistant strains, which brings great difficulties to the prevention and treatment of the disease. For this reason, reducing or eliminating the drug resistance of pathogenic Escherichia coli is the key to effectively prevent and treat the disease. Traditional Chinese medicine has multiple biological activities such as antibacterial, antiviral, and immunomodulatory. Because of its diverse ingredients, it is not easy to develop drug resistance. It is of great significance for effectively preventing and treating bacterial diseases of livestock and poultry caused by pathogenic Escherichia coli and protecting human health and the ecological environment. With the release of relevant regulations such as the "National Action Plan for Reducing the Use of Veterinary Antimicrobial Drugs (2021-2025)" by the Ministry of Agriculture and Rural Affairs of my country, the use of veterinary Chinese medicine to reduce or replace conventional veterinary antimicrobial drugs has become a hot topic in industry research and a new direction for the green and healthy development of animal husbandry. Lactobacillus rhamnosus is a safe and widely used probiotic that can tolerate the animal digestive tract environment and can colonize in the intestines of humans and animals, playing a role in regulating intestinal flora, preventing and treating diarrhea, eliminating toxins, preventing dental caries, and improving the body's immunity. It is acid-resistant, oxygen-resistant, and has strong colonization ability, and is currently highly accepted.

In summary, based on the preparation of traditional Chinese medicine, the use of lactic acid bacteria fermentation technology can not only increase the content of active ingredients in traditional Chinese medicine, change or produce some active ingredients that are not originally contained in traditional Chinese medicine, but also transform the macromolecules of traditional Chinese medicine into small molecules that can be directly absorbed and utilized by the body, thereby improving the disease resistance and economic benefits of the animal body.

Summary of the invention

The purpose of the present invention is to provide a compound Chinese medicine residue fermentation liquid for preventing and treating avian Escherichia coli disease and a preparation method thereof. The compound Chinese medicine residue fermentation liquid of the present invention can reduce the level of inflammatory cytokines of artificially infected avian pathogenic Escherichia coli, reduce or replace the use of conventional antibacterial drugs, and reduce drug residues.

The present invention first provides a method for preparing a compound Chinese medicine residue fermentation liquid for preventing and treating avian Escherichia coli disease, comprising:

Step 1: Lactobacillus rhamnosus PC30-2-1 was isolated from commercially available kimchi, and the strain was identified by 16S rDNA to obtain Lactobacillus rhamnosus PC30-2-1 (Lactobacillus rhamnosus PC30-2-1) with a preservation number of CCTCC NO: M 2022083, the preservation unit is: China Center for Type Culture Collection, the preservation address is: Wuhan University, China, and the preservation date is: January 17, 2022. Lactobacillus rhamnosus PC30-2-1 was inoculated into the culture medium, and cultured at 37° C., 150 r/min, and shaking for 12-18 hours to obtain a seed culture solution;

Step 2: Slice asarum, licorice, cinnamon, angelica, red peony root, jujube and akebia, add water and boil, and combine the filtrate;

Step 3: drying the drug residue obtained in step 2, adding distilled water to the obtained dry drug residue, sterilizing it, and then cooling it to room temperature to obtain a drug residue culture solution;

Step 4: inoculate the seed culture solution of step 2 into the medicinal residue culture solution of step 3, culture at 37° C., 150 r/min, with shaking for 12-18 h, collect the fermentation supernatant by centrifugation, and obtain the compound Chinese medicinal residue fermentation solution.

Preferably, the culture medium in step 1 is MRS liquid culture medium.

Preferably, the ratio of the Chinese medicinal raw materials in step 2 is: 12-15 parts of asarum, 12-15 parts of licorice, 12-15 parts of cinnamon, 12-15 parts of angelica, 12-15 parts of red peony root, 12-15 parts of jujube, and 8-10 parts of akebia.

Preferably, the ratio of Chinese medicinal materials in step 2 is: 12 parts of asarum, 12 parts of liquorice, 12 parts of cinnamon, 12 parts of angelica, 12 parts of red peony root, 12 parts of jujube, and 8 parts of akebia. Preferably, the number of decoctions in step 2 is 2 times, the first decoction time is 1 hour, and the second decoction time is 40-45 minutes.

Preferably, the drying temperature in step three is room temperature and the drying time is 48 hours.

Preferably, the solid-liquid ratio of the Chinese medicinal residue to distilled water in step three is 1:10.

Preferably, in step three, the sterilization temperature is 121° C. and the pressure is 103.4 kPa.

Preferably, the centrifugal collection in step 4 comprises: centrifuging the fermented liquor of the medicinal residue at 4°C, 8000r/min for 10min, vacuum filtering the supernatant obtained after centrifugation through a 0.45μm filter membrane, and then concentrating the filtrate in a rotary evaporator (45°C, 75r/min) to a volume ratio of 1:3.

The present invention also provides a compound Chinese medicinal residue fermentation liquid for preventing and treating avian Escherichia coli disease obtained by the preparation method.

The pharmacology and medicinal properties of each raw material in the medicinal residue of the present invention are as follows:

Asarum: warm in nature, pungent in taste, slightly toxic. It enters the heart, lung, and kidney meridians. It dispels wind, dispels cold, relieves pain, warms the lungs and removes phlegm. It is a pungent and warm antipyretic drug under the category of antipyretic drugs. 1. Local anesthetic effect; 2. Inhibits arthritis; 3. Antibacterial effect; 4. It has the effect of enhancing lipid metabolism and increasing blood sugar, and regulating the body's smooth muscle function. Volatile oil can reduce the blood pressure of anesthetized animals, while the decoction can increase blood pressure; 5. Anti-inflammatory and anticonvulsant effects; 6. Analgesic, sedative, fever suppression, antipyretic; 7. Antihistamine and antiallergic; 8. Excitatory respiratory effect.

Licorice: sweet in taste, neutral in nature, enters the heart, lung, spleen, and stomach meridians, and has the effects of tonifying the spleen and replenishing qi, clearing away heat and detoxifying, removing phlegm and relieving cough, relieving acute pain, and alleviating the properties of the medicine. It is mainly used to treat spleen and stomach weakness, fatigue, palpitations and shortness of breath, cough with sputum, acute pain in the abdomen and limbs, carbuncle and sore. Pharmacological effects include: 1. Adrenal cortex hormone-like effects (mineralocorticoid-like effects, glucocorticoid-like effects); 2. Enhance nonspecific immunity and cellular immunity, but have an inhibitory effect on humoral immunity; 3. Effects on the digestive system (anti-ulcer, liver protection, antispasmodic); 4. Detoxification; 5. Anti-inflammatory; 6. Antitussive and expectorant; 7. Anti-pathogenic microorganisms; 8. Anti-arrhythmia; 9. Anti-tumor; 10. Lowering blood lipids and anti-atherosclerosis.

Cinnamon bark: pungent, sweet, hot in nature, enters the spleen, kidney, heart, and liver meridians, and has the effects of replenishing fire and yang, guiding fire back to its source, dispelling cold and relieving pain, and promoting blood circulation and menstruation. It is mainly used to treat impotence, cold uterus, cold pain in the waist and knees, asthma due to kidney deficiency, red eyes and sore throat, cold pain in the heart and abdomen, vomiting and diarrhea due to deficiency and cold, cold hernia, amenorrhea and dysmenorrhea. Pharmacological effects include: 1. Effects on the cardiovascular system (cardiotonic, dilate blood vessels, lower blood pressure); 2. Effects on the digestive system (water decoction inhibits the peristalsis of the small intestine of mice, cinnamon oil promotes intestinal peristalsis, increases the secretion of digestive juices, and relieves gastrointestinal spasmodic pain); 3. Anti-ulcer; 4. Anti-platelet aggregation, anti-coagulation; 5. Excites the pituitary-adrenal cortex system; 6. Regulates the body's immunity.

Angelica sinensis: sweet and pungent, warm in nature, enters the liver, heart and spleen meridians, and has the effects of nourishing blood, activating blood circulation, regulating menstruation and relieving pain, and moistening the intestines and relieving constipation. It is mainly used to treat blood deficiency, sallow complexion, dizziness and palpitations, irregular menstruation, amenorrhea and dysmenorrhea, abdominal pain due to deficiency and cold, constipation due to dry intestines, rheumatic arthralgia, traumatic injury, carbuncle and ulcer, etc. Pharmacological effects include: 1. Effects on the blood system (promoting bone marrow hematopoietic function, inhibiting platelet aggregation, anti-thrombotic, lowering blood lipids, and anti-atherosclerosis); 2. Effects on the cardiovascular system (anti-myocardial ischemia, anti-fibrosis after myocardial infarction, anti-arrhythmia, dilating blood vessels, and lowering blood pressure); 3. Exciting or inhibiting uterine smooth muscle; 4. Enhancing the body's immune function; 5. Anti-radiation; 6. Anti-injury; 7. Protecting the liver; 8. Anti-tumor.

Red peony root: bitter, slightly cold. Enters the liver meridian. Has the effects of clearing heat and cooling blood; activating blood circulation and removing blood stasis. Mainly used for fever and toxicity, vomiting blood and epistaxis, intestinal wind and bleeding, red and swollen eyes, carbuncle, sore, amenorrhea, dysmenorrhea, metrorrhagia, stasis and pain in the ribs, hernia, and accumulation of masses; injuries from falls. Used for fever and toxicity, vomiting blood and epistaxis, red and swollen eyes, liver depression and pain in the ribs, amenorrhea, dysmenorrhea, abdominal pain due to symptoms, injuries from falls, carbuncle, sore. It is mostly used for fever and bleeding in the body caused by heat entering the blood, red and swollen eyes, carbuncle, sore and toxicity. Pharmacological effects include: 1. Anti-thrombotic effect; 2. Anti-platelet aggregation effect; 3. Hypolipidemic and anti-atherosclerotic effect; 4. Effect on the cardiovascular system; 5. Anti-tumor effect; 6. Liver protection effect.

Jujube: sweet, warm in nature. Enters the spleen and stomach meridians. Replenishes qi, nourishes blood and calms the mind. Used for spleen deficiency, poor appetite, fatigue, loose stools, and irritability in women. Pharmacological effects include: 1. Central nervous system inhibition; 2. Protects the liver; 3. Enhances muscle strength and resists fatigue; 4. Anti-allergic reactions; 5. Anti-tumor; 6. Calms and soothes the mind; 7. Increases leukocyte cAMP, enhances immunity and other functions.

Akebia: bitter, cold in nature. Enters the heart, small intestine, and bladder meridians. Clears away heat and promotes diuresis, activates blood circulation and unclogs meridians. Treats red urine, stranguria, edema, chest heat, throat pain, mouth sores, rheumatic pain, milk blockage, amenorrhea and dysmenorrhea. Pharmacological effects include: 1. Diuretic; 2. Antibacterial and antifungal.

Beneficial Effects of the Invention

The invention provides a compound Chinese medicine residue fermentation liquid for preventing and treating avian Escherichia coli disease and a preparation method thereof. The compound Chinese medicine residue fermentation liquid is fermented by utilizing rhamnosus lactobacillus PC30-2-1, and can reduce the level of inflammatory cytokines of artificially infected avian pathogenic Escherichia coli, improve the bursal index of Fabricius, improve the appetite and disease resistance of livestock and poultry, promote weight gain, improve production efficiency, reduce or replace the use of conventional antimicrobial drugs, reduce drug residues, reduce the selection pressure of antimicrobial drugs, and inhibit or slow down the generation of drug resistance of clinical pathogenic bacteria.

The seven Chinese medicines in the Chinese medicine residue prescription of the present invention are all common Chinese medicines, low in price, without any toxic side effects on the diseased livestock and poultry, no residue in the livestock and poultry, and no drug resistance. In vitro tests show that the drug has a good inhibitory effect on Escherichia coli; in vivo tests show that the compound Chinese medicine residue fermentation liquid has a good protective and therapeutic effect on artificially infected avian pathogenic Escherichia coli, and is expected to replace commonly used antibacterial drugs for the prevention and treatment of livestock and poultry Escherichia coli disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a bar graph showing the growth of mice gavaged with the fermented liquor of medicinal residues prepared in Example 2;

FIG2 is a bar graph showing the weekly weight gain of experimental chickens that were gavaged with the fermented medicinal residue liquid prepared in Example 2;

Fig. 3 is a diagram of lesions of autopsied organs;

FIG4 is a cytokine bar graph of IL-6 content;

FIG5 is a cytokine bar graph of IL-8 content;

FIG6 is a cytokine bar graph of serum IFN-γ levels.

DETAILED DESCRIPTION

Example 1 Isolation and Identification of Lactobacillus rhamnosus PC30-2-1

(1) Separation and purification

Place the commercially available pickle juice in a sterile clean bench, dilute the sample 10 times with physiological saline, and evenly spread 100 μL of the appropriate gradient dilution on the MRS medium containing 2% calcium carbonate. Place the coated medium in an anaerobic tank and culture at 37°C for 48 hours. Pick colonies with different morphologies with obvious calcium dissolving rings and inoculate them in the corresponding liquid culture medium for 24 hours, then streak them again on the MRS solid culture medium. After separation and purification three times, pick a single colony for Gram staining. The Gram-positive colonies are cultured for bacterial growth and stored at -80°C in MRS liquid culture medium (containing 20% glycerol). All strains need to be subcultured three times before use. After purification by MRS separation medium, round, milky white, neatly edged, smooth surface, 1-1.5 mm in diameter, and arranged in pairs or chains of colonies are obtained. Gram staining microscopy shows dispersed, short rod-shaped bacteria arranged in chains.

(2) 16S rDNA sequencing and identification

From the purified strain, a single colony was picked and placed in 5 mL of liquid culture medium for overnight culture, and the genomic DNA of the isolated strain was extracted using the Bacterial DNA Genome Extraction Kit of Sangon Biotech (Shanghai) Co., Ltd.

Using the above genomic DNA as a template, the bacterial 16S rDNA universal primers (forward sequence: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3', reverse sequence: 1492R: 5'-TACGGYTACCTTG TTACGACTT-3') synthesized by Sangon Biotechnology Co., Ltd. were used to perform PCR amplification of the 16S rDNA specific fragment of the isolated strain. The 50μL PCR reaction system is shown in Table 1-2. The PCR amplification program was set as follows: pre-denaturation: 94℃, 5min; denaturation: 94℃, 30s; annealing: 55℃, 30s; extension: 72℃, 90s; 30 cycles; end extension: 72℃, 10min.

Table 1 PCR reaction system

Tab.1 Reaction system of PCR

Take 5 μL of PCR amplification product, detect it by 1% agarose gel electrophoresis, and cut the sample with a clear band at 1600 bp for gel recovery. Connect the gel recovery product to the pMD18-T cloning vector and transform it into DH5α competent cells. Send the positive clone plasmid to Shanghai Biotech Co., Ltd. for sequencing analysis. The sequencing results were compared with the data in NCBI using BLAST to determine its species. The 16S rDNA sequence comparison results showed that the sequence identity of the bacteria and L. rhamnosus reached 99%.

Example 2 Preparation of compound Chinese medicine fermentation broth

Step 1: Inoculate 1% of the Lactobacillus rhamnosus PC30-2-1 stored in the laboratory into 5 mL of sterilized MRS liquid culture medium, and culture at 37°C, 150 r/min, with shaking for 12 hours to obtain a seed culture solution;

Step 2: Weigh 12g of asarum, 12g of licorice, 12g of cinnamon, 12g of angelica, 12g of red peony root, 12g of jujube, and 8g of akebia. Slice the above Chinese medicines, add 5L of water and boil for 60min until 2L is left. Filter the filtrate with gauze; add 3L of water again, boil the water until it boils, then simmer for 45min until 1L is left. Combine the filtrates;

Step 3: Dry the above-obtained drug residue, weigh a certain amount of the dried drug residue into a conical flask, add distilled water according to a solid-liquid ratio of 1:10, sterilize under high pressure at 121°C, and then cool to room temperature;

Step 4: Aseptically inoculate the seed culture solution of step 1 into the conical flask of step 3 at a volume ratio of 0.5%, and culture at 37°C, 150r/min, with shaking for 18h. Centrifuge the above-mentioned drug residue fermentation solution at 4°C, 8000r/min for 10min, and vacuum filter the supernatant obtained after centrifugation through a 0.45μm filter membrane. Then, concentrate the filtrate in a rotary evaporator (45°C, 75r/min) to a volume ratio of 1:3; obtain the compound Chinese medicine fermentation solution.

Example 3 Oxford cup method to test the antibacterial activity of compound Chinese medicine fermentation liquid against Escherichia coli standard strain ATCC25922

The ATCC25922 strain stored in the laboratory was inoculated into sterile LB liquid medium at a 1% inoculum, and cultured at 37°C, 150 r/min, with shaking until OD ₆₀₀ = 0.5 (about 10 ⁸ CFU/mL), and then serially diluted 100 times with LB liquid medium for drug sensitivity test;

100 μL of the cultured ATCC25922 strain was evenly spread on an LB solid culture medium plate and evenly placed in sterilized Oxford cups, and 200 μL of the concentrated fermentation liquid prepared in Example 2 was added to the Oxford cups, and the plates were placed in a 37°C constant temperature incubator for 12 h.

The plate was observed for inhibition zones, and the diameter of the inhibition zones was measured with a ruler. The Oxford cup test results showed that the diameter of the inhibition zone of the concentrated fermentation supernatant of the traditional Chinese medicine residues against Escherichia coli ATCC25922 strain was about 12 mm, and the supernatant had a certain antibacterial effect.

Example 4

Healthy Kunming mice (weight 20±2 g) were selected, half male and half female, 10 mice/group, and the fermentation liquid of the medicinal residue prepared in Example 2 was gavaged for 15 consecutive days, 200 ul/mouse/day, and the mice's food intake, water drinking, weight changes and health conditions were observed;

As shown in Figure 1, Figure 1 is a growth bar graph of mice gavaged with the fermented liquor of the medicinal residues prepared in Example 2. As can be seen from Figure 1, the experimental mice ate and drank better after gavage with the compound Chinese medicinal residues fermented liquor, their fur was smooth and shiny, and they were lively and had no abnormalities. Ten days before the experiment, the weight of mice in the group gavage with 1% compound Chinese medicinal residues fermented liquor showed a significant upward trend compared with the control group.

Example 5

30 healthy Rock chicken chicks of 7 days old (basic immunity has been completed) were selected and adapted to the environment for 5 days, and set as the test group and the control group, 10 per group, half male and half female. The compound Chinese medicine fermentation broth of Example 2 of the present invention was continuously fed with drinking water at 1% and 5% addition for 21 days, twice a day, and the drinking water was calculated to ensure that each group of experimental chickens drank it up within 2 hours each time. On the 22nd day of the experiment, the addition of fermentation broth was stopped, and 0.5mL/chicken (about 10 ⁹ CFU/mL) of avian pathogenic Escherichia coli CC1 strain was dripped into the nose. 48h after the attack, the state and clinical symptoms of the experimental chickens were observed, venous blood was collected under the wings, and the levels of serum IL-2, IL-6, IL-8, IFN-γ and other cytokines were detected. Six experimental chickens were dissected in each group, and organ lesions were observed and organ indexes such as spleen and bursa of Fabricius were calculated.

As shown in Figure 2, Figure 2 is a bar graph of the weekly weight gain of the test chickens that were gavaged with the fermented liquor prepared from the medicinal residues of Example 2. It can be seen from Figure 2 that both 1% and 5% fermented liquors from the medicinal residues promoted the weight gain of the test chickens to a certain extent. Among them, the 5% addition group had the best weight gain effect in the second week, and the 1% addition group had the best weight gain effect in the third week, but the difference with the control group was not significant. The above results show that the compound fermented liquor from the medicinal residues is safe for the test animals, has no toxic side effects, can improve the appetite of the animals to a certain extent, promote weight gain, and reduce the feed-to-meat ratio.

Figure 3 is a diagram of organ lesions after autopsy, where a represents the control group (no medication), and b represents the pericardium and liver of the fermentation liquid addition group. Figure 3 shows that after the infection, the amount of food and water consumed by the experimental chickens in each group decreased to varying degrees compared with the usual time. Some chickens in the control group showed depression, increased eye and nasal secretions, and loose wings. The overall feeding and mental state of the compound Chinese medicine residue fermentation liquid addition group was slightly better than that of the control group. The autopsy of some chickens showed pathological changes such as thickened and turbid pericardium, fibrinous pericarditis, enlarged liver, hemorrhage and stasis, and fibrinous exudate on the liver surface. The compound Chinese medicine residue fermentation liquid addition group (Figure 3b) had fewer chickens with the above symptoms, and the overall symptoms were milder than those of the control group (Figure 3a,).

Figure 4 is a cytokine bar graph of IL-6 content, Figure 5 is a cytokine bar graph of IL-8 content, and Figure 6 is a cytokine bar graph of serum IFN-γ content. Figures 4-6 show that compared with the control group, 1% and 5% Chinese herbal medicine residue fermentation liquid can significantly reduce serum IL-6 levels; in addition, 1% Chinese herbal medicine residue fermentation liquid can also significantly reduce the secretion of serum IL-8, IFN-γ and other cytokines, indicating that it has a certain anti-inflammatory effect.

Table 2 shows the effects of the compound Chinese medicinal residue fermentation liquid of the present invention on the immune organ indexes of the test chickens. As shown in Table 2, 1% and 5% Chinese medicinal residue fermentation liquids have a promoting effect on the development of the bursa of Fabricius in the test chickens, and the bursa of Fabricius indexes are higher than those of the control group, but the differences are not significant; while 1% and 5% Chinese medicinal residue fermentation liquids reduce the spleen index of the test chickens, which is also not significant compared with the control group.

Table 2 Effect of compound Chinese medicine residue fermentation liquid on immune organ index of experimental chickens %

Claims (10)

1. A preparation method of compound traditional Chinese medicine residue fermentation liquor for preventing and treating avian colibacillosis is characterized by comprising the following steps:

the method comprises the following steps: lactobacillus rhamnosus PC30-2-1 is separated and obtained from the commercially available pickle, the strain identification is carried out through 16S rDNA, and the Lactobacillus rhamnosus PC30-2-1 (Lactobacillus rhamnous PC 30-2-1) with the preservation number of CCTCC NO: M2022083 is obtained, wherein the preservation unit is as follows: china center for type culture Collection, the collection addresses are: the preservation date of Wuhan university in China is as follows: inoculating Lactobacillus rhamnosus PC30-2-1 into culture medium at 37 deg.C and 150r/min for 12-18 hr in 2022 year, 1 month and 17 days to obtain seed culture solution;

step two: slicing herba asari, glycyrrhrizae radix, cortex Cinnamomi, radix Angelicae sinensis, radix Paeoniae Rubra, fructus Jujubae, and caulis Akebiae, decocting in water, and mixing filtrates;

step three: drying the dregs obtained in the step two, adding distilled water into the obtained dry dregs, sterilizing, and cooling to room temperature to obtain dregs culture solution;

step four: inoculating the seed culture solution in the second step into the residue culture solution in the third step, performing shaking culture at 37 ℃ for 12-18h at 150r/min, centrifuging, and collecting fermentation supernatant to obtain compound Chinese medicinal residue fermentation liquid.

2. The method for preparing the compound Chinese medicine residue fermentation liquid for preventing and treating the avian colibacillosis according to claim 1, wherein the culture medium in the first step is an MRS liquid culture medium.

3. The preparation method of the compound traditional Chinese medicine residue fermentation liquid for preventing and treating avian colibacillosis according to claim 1, wherein the mixture ratio of the traditional Chinese medicine raw materials in the second step is as follows: 12-15 parts of asarum, 12-15 parts of liquorice, 12-15 parts of cinnamon, 12-15 parts of angelica, 12-15 parts of red peony root, 12-15 parts of Chinese date and 8-10 parts of akebia stem.

4. The preparation method of the compound traditional Chinese medicine residue fermentation liquor for preventing and treating avian colibacillosis according to claim 3, wherein the traditional Chinese medicine raw materials in the step two are in the following proportion: 12 parts of asarum, 12 parts of liquorice, 12 parts of cinnamon, 12 parts of angelica, 12 parts of red peony root, 12 parts of Chinese date and 8 parts of akebia stem.

5. The preparation method of the compound traditional Chinese medicine residue fermentation liquor for preventing and treating avian colibacillosis according to claim 1, wherein the number of times of decoction in the second step is 2, the first decoction time is 1h, and the second decoction time is 40-45min.

6. The preparation method of the compound traditional Chinese medicine residue fermentation liquid for preventing and treating avian colibacillosis according to claim 1, wherein the drying temperature in the third step is room temperature, and the drying time is 48 hours.

7. The preparation method of the compound traditional Chinese medicine residue fermentation liquid for preventing and treating avian colibacillosis according to claim 1, wherein the material-liquid ratio of the traditional Chinese medicine residue to the distilled water in the third step is 1.

8. The preparation method of the compound traditional Chinese medicine residue fermentation liquid for preventing and treating avian colibacillosis according to claim 1, wherein the sterilization temperature in the third step is 121 ℃, and the pressure is 103.4kPa.

9. The preparation method of the compound traditional Chinese medicine residue fermentation liquid for preventing and treating avian colibacillosis according to claim 1, wherein the centrifugal collection in the fourth step comprises: centrifuging the residue fermentation liquid at 4 deg.C and 8000r/min for 10min, vacuum filtering the supernatant with 0.45 μm filter membrane, and rotary evaporating the filtrate in rotary evaporator (45 deg.C and 75 r/min) to volume ratio of 1:3.

10. The compound traditional Chinese medicine residue fermentation liquid for preventing and treating avian colibacillosis, which is obtained by the preparation method of claim 1.

Images