Preparation method of traditional Chinese medicine compound microecological preparation for preventing and treating rabbit coccidiosis

Abstract

The invention relates to the technical field of veterinary traditional Chinese medicines, and discloses a preparation method of a traditional Chinese medicine compound microecological preparation for preventing and treating rabbit coccidiosis, which comprises the following steps: weighing antifebrile dichroa extract, corn flour, wheat bran, soybean meal and calcium carbonate in parts by mass, and mixing; seed solutions and fermentation solutions of bifidobacterium lactis, bacillus subtilis and enterococcus faecalis are prepared respectively; mixing the bifidobacterium lactis liquid with sterile water, inoculating the mixture of the antifebrile dichroa extract and the auxiliary materials with the mixture, and carrying out first-stage anaerobic fermentation; and cooling the liquid medicine after the first-stage fermentation to 40 DEG C or below, inoculating a mixed bacteria solution of bacillus subtilis and enterococcus faecalis, and carrying out second-stage aerobic fermentation with sterile water. Through a modern pharmaceutical preparation technology, a traditional Chinese medicine antifebrile dichroa extract is subjected to staged fermentation through bifidobacterium lactis, bacillus subtilis and enterococcus faecalis to prepare the traditional Chinese medicine compound microecological preparation, so that the malaria prevention efficacy of antifebrile dichroa is enhanced, the disadvantage of spitting of antifebrile dichroa is avoided, intestinal injury caused by coccidiosis is improved, and the immune function of the organism is enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese veterinary medicine, and specifically to a preparation method of a traditional Chinese medicine compound microecological preparation for preventing and treating rabbit coccidiosis. Background Art

[0002] Rabbit coccidiosis is a protozoal disease caused by coccidia of the genus Eimeria or Isospora parasitizing in the epithelial cells of the small intestine or bile duct of rabbits, and it is one of the main diseases seriously endangering the development of the rabbit industry. Every year, the economic losses caused by this disease to the rabbit industry are huge. In particular, young rabbits within 4 months after weaning are most susceptible to the disease, and the mortality rate of infected young rabbits is as high as over 60%, seriously affecting the healthy development of the rabbit industry and the quality and safety of rabbit products. At present, chemical synthetic drugs are mainly used at home and abroad to prevent and treat rabbit coccidiosis. Among them, preventive drugs include robenidine, rabbit coccidia clear, monensin, salinomycin, etc., and treatment drugs include diclazuril, sulfonamide drugs quinoxaline, dimethoxypyrimidine, compound sulfamethoxazole, etc.

[0003] The traditional Chinese medicine compound microecological preparation uses modern fermentation technology to compound traditional Chinese medicine with beneficial microorganisms or beneficial growth promoting factors into a new type of biological preparation that is green, safe, and pollution-free, belonging to the third generation of microecological preparations. The traditional Chinese medicine compound microecological preparation is rich in various nutrient elements and active ingredients, and has the effects of improving animal growth performance and feed utilization rate, enhancing immune function, antibacterial and antiviral, and can improve the host intestinal microecological balance, improve the body's immunity, etc. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a preparation method of a traditional Chinese medicine compound microecological preparation for preventing and treating rabbit coccidiosis. The provided traditional Chinese medicine compound microecological preparation has the functions of killing coccidia, regulating intestinal flora, and enhancing the body's immunity, is suitable for preventing and treating rabbit coccidiosis and mixed infections of coccidia and bacteria, improving intestinal damage caused by coccidia, and enhancing the body's disease resistance.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: A traditional Chinese medicine compound microecological preparation for preventing and treating rabbit coccidiosis includes dichroa febrifuga extract, Bifidobacterium lactis, Bacillus subtilis, Enterococcus faecalis, corn flour, wheat bran, soybean meal, and calcium carbonate. Among them, the mass fraction of dichroa febrifuga extract accounts for 30 - 60% of the total mass, the mass fraction of Bifidobacterium lactis, Bacillus subtilis, and Enterococcus faecalis accounts for 10 - 25% of the total mass, the mass fraction of corn flour, wheat bran, and soybean meal accounts for 15 - 35% of the total mass, and the mass fraction of calcium carbonate accounts for 0.1 - 1% of the total mass.

[0006] Preferably, the mass fraction ratio of Bifidobacterium lactis, Bacillus subtilis, and Enterococcus faecalis is: Bifidobacterium lactis: Bacillus subtilis: Enterococcus faecalis = 1:1:1.

[0007] A preparation method of a traditional Chinese medicine compound microecological preparation for preventing and treating rabbit coccidiosis, comprising the following steps:

[0008] S1. Weigh and mix dichroa febrifuga extract, corn flour, wheat bran, soybean meal, and calcium carbonate according to parts by mass;

[0009] S2. Prepare seed solutions and fermentation broths of Bifidobacterium lactis, Bacillus subtilis, and Enterococcus faecalis respectively;

[0010] S3. Mix the Bifidobacterium lactis broth with sterile water and then inoculate it into the mixture of dichroa febrifuga extract and auxiliary materials for the first-stage anaerobic fermentation. The fermentation duration is 24 - 72 h, the fermentation temperature is 35 - 41 °C, and the addition amount of the complex enzyme is 0.1 - 0.2%;

[0011] S4. Cool the medicinal liquid after the first-stage fermentation to below 40 °C, and then inoculate it with the mixed broth of Bacillus subtilis and Enterococcus faecalis and sterile water for the second-stage aerobic fermentation. The fermentation duration is 24 - 50 h, the fermentation temperature is 32 - 38 °C, and the addition amount of the complex enzyme is 0.05 - 0.15%;

[0012] S5. Subject the fermented traditional Chinese medicine compound microecological preparation to vacuum freeze-drying treatment. The freezing temperature is -50 - -60 °C, and the freeze-drying time is 20 - 26 h, finally obtaining the traditional Chinese medicine compound microecological preparation for preventing and treating rabbit coccidiosis.

[0013] Preferably, the preparation method of the dichroa febrifuga extract in step S1 is to crush the dichroa febrifuga raw material, perform ultrasonic extraction with a 70% ethanol solution, combine the extraction solutions after extracting twice, filter, extract with chloroform, and recover chloroform under reduced pressure and dry to obtain the dichroa febrifuga extract.

[0014] Preferably, the parts by mass of the dichroa febrifuga extract and the auxiliary materials in step S1 are: 200 - 260 g of corn flour, 100 - 200 g of wheat bran, 100 - 150 g of soybean meal, and 1.0 - 2.0 g of calcium carbonate are added to every 1 kg of dichroa febrifuga extract.

[0015] Preferably, the raw materials in the fermentation tank are sterilized before being inoculated for fermentation. The sterilization temperature is 121 °C, and the sterilization time is 30 min.

[0016] Preferably, the preparation methods of the seed solutions and fermentation broths of Bifidobacterium lactis, Bacillus subtilis, and Enterococcus faecalis in step S2 include strain activation, shake flask scale-up culture, seed solution culture, and fermentation broth culture. The specific steps are as follows:

[0017] The activation and culture of Bifidobacterium lactis, Bacillus subtilis, and Enterococcus faecalis are carried out according to conventional methods, and they are cultured using MRS medium and inoculated into the corresponding seed solution and fermentation broth media for culture.

[0018] Preferably, in the step S3, the duration of the first-stage anaerobic fermentation is 48 - 60 h, the fermentation temperature is 38 - 40 °C; the addition amount of the complex enzyme is 0.12 - 0.16%, and the components and ratios of the complex enzyme are: protease: amylase: cellulase: dextranase = 1.0:0.6:0.8:0.6, and the water ratio of the fermentation material is controlled between 38 - 55%.

[0019] Preferably, in the step S4, the duration of the second-stage aerobic fermentation is 36 - 40 h, the fermentation temperature is 35 - 37 °C, the addition amount of the complex enzyme is 0.10 - 0.15%, and the components and ratios of the complex enzyme are: protease: amylase: glucoamylase: dextranase = 1.0:0.8:0.6:0.6; the water ratio of the fermentation material is controlled between 40 - 50%.

[0020] Preferably, the mixing ratio of the Dichroa febrifuga extract and the excipient and the fermentation conditions are adjusted according to specific requirements.

[0021] The present invention provides a preparation method of a traditional Chinese medicine compound microecological preparation for preventing and treating rabbit coccidiosis. It has the following beneficial effects:

[0022] 1. Based on the insecticidal, antimalarial and highly effective anti-coccidial activities of Dichroa febrifuga, through modern pharmaceutical preparation technology, the traditional Chinese medicine Dichroa febrifuga extract is prepared into a traditional Chinese medicine compound microecological preparation by segmented fermentation with Bifidobacterium lactis, Bacillus subtilis and Enterococcus faecalis, so as to enhance the antimalarial power of Dichroa febrifuga, eliminate the vomiting drawback of Dichroa febrifuga, improve the intestinal damage caused by coccidia and enhance the body's immune function.

[0023] 2. The three probiotics selected in the present invention can convert and decompose the drug active ingredients such as total alkaloids, crude polysaccharide starch, cellulose, etc. in traditional Chinese medicine Dichroa febrifuga more fully and thoroughly through fermentation in two different time periods, significantly improve the anti-coccidial activity of the drug, reduce the emetic and toxic side effects of the drug, and significantly improve the palatability of the drug.

[0024] 3. In the fermentation process of the present invention, corn flour, wheat bran and soybean meal are added to provide carbon source and nitrogen source for the probiotic fermentation process. At the same time, the polysaccharides released during the traditional Chinese medicine fermentation process provide nutrients for the growth of the bacterial strains and stimulate the proliferation of beneficial bacteria. After fermentation is completed, the fermentation product not only contains traditional Chinese medicine active ingredients, but also contains a large number of probiotics and their metabolites. The traditional Chinese medicine and probiotics synergistically enhance the immune system's defense ability of rabbits, reduce the production of toxic substances in the intestine, improve the cure rate, have no drug resistance, and effectively control the occurrence of rabbit coccidiosis. Description of the Drawings

[0025] Figure 1 It is a comparison diagram of the appearance characteristics of meat rabbits in Experimental Example 1 of the present invention;

[0026] Figure 2This is the intestinal anatomical change diagram of the meat rabbits in Experimental Example 1 of the present invention. Detailed implementation manners

[0027] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the specification of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0028] The present invention provides a traditional Chinese medicine compound microecological preparation for preventing and treating rabbit coccidiosis, which includes dichroa febrifuga extract, bifidobacterium lactis, bacillus subtilis, enterococcus faecalis, corn flour, wheat bran, soybean meal and calcium carbonate. Among them, the mass fraction of dichroa febrifuga extract accounts for 30-60% of the total mass, the mass fraction of bifidobacterium lactis, bacillus subtilis and enterococcus faecalis accounts for 10-25% of the total mass, the mass fraction of corn flour, wheat bran and soybean meal accounts for 15-35% of the total mass, and the mass fraction of calcium carbonate accounts for 0.1-1% of the total mass. Among them, the mass fraction ratio of bifidobacterium lactis, bacillus subtilis and enterococcus faecalis is: bifidobacterium lactis: bacillus subtilis: enterococcus faecalis = 1:1:1.

[0029] As part of this application, the present invention also provides a preparation method of a traditional Chinese medicine compound microecological preparation for preventing and treating rabbit coccidiosis, including the following steps:

[0030] S1. Weigh dichroa febrifuga extract, corn flour, wheat bran, soybean meal and calcium carbonate according to the mass fraction and mix them; the mass fraction of dichroa febrifuga extract and auxiliary materials is: add 200-260 g of corn flour, 100-200 g of wheat bran, 100-150 g of soybean meal and 1.0-2.0 g of calcium carbonate to every 1 kg of dichroa febrifuga extract. After mixing dichroa febrifuga extract with corn flour, wheat bran, soybean meal and calcium carbonate, sterilize them. The sterilization temperature is 121 °C and the sterilization time is 30 min.

[0031] S2. Prepare the seed liquid and fermentation liquid of bifidobacterium lactis, bacillus subtilis and enterococcus faecalis respectively;

[0032] The preparation sequence of the probiotic fermentation liquid includes the following steps:

[0033] S21. Strain activation: Respectively inoculate the freeze-dried strains of bifidobacterium lactis, bacillus subtilis and enterococcus faecalis into three MRS test tubes, culture them statically at 40 °C for 24 h, and use an inoculation needle to pick colonies for streak separation and purification.

[0034] S22. Shake flask scale-up culture: Respectively inoculate the activated single colonies of bifidobacterium lactis, bacillus subtilis and enterococcus faecalis into the corresponding MRS liquid medium above, culture them at 35-37 °C and 160 r / min for 20-24 h.

[0035] S23. Seed liquid culture: Inoculate the strains cultured in shake flasks into the seed liquid medium at a certain inoculation amount and perform constant-temperature culture.

[0036] ① Lactobacillus delbrueckii subsp. bulgaricus seed liquid culture: Inoculate the Lactobacillus delbrueckii subsp. bulgaricus liquid cultured in shake flasks into the seed liquid medium at a ratio of 1-2%, and culture at a temperature of 37.5 °C, a rotation speed of 180 r / min, and a time of 24 h to obtain the Lactobacillus delbrueckii subsp. bulgaricus seed culture solution.

[0037] The main components of the seed liquid medium include: glucose 3 g / L, peptone 5 g / L, beef extract 2 g / L, yeast extract 2 g / L, K 2 HPO 4 4.0 g / L, MgSO 4 2.0 g / L, MnSO 4 1.0 g / L, L-cysteine hydrochloride 0.5 g / L; Adjust the pH to 6.0-7.0 with NaOH and sterilize at 121 °C for 20 min.

[0038] ② Bacillus subtilis seed liquid culture: Inoculate the Bacillus subtilis liquid cultured in shake flasks into the seed liquid medium at a ratio of 1-2%, and culture at a temperature of 35 °C, a rotation speed of 220 r / min, and a time of 20 h to obtain its seed culture solution.

[0039] The main components of the seed liquid medium include: peptone 5 g / L, beef extract 8 g / L, sodium carboxymethyl cellulose 5 g / L, K 2 HPO 4 1.5%, NaNO 3 3 g / L, KCl 0.5 g / L, MgSO 4 0.5 g / L, Fe 2 (SO 4 ) 3 0.01 g / L; Adjust the pH to 6.0-7.0 with NaOH and sterilize at 121 °C for 20 min.

[0040] ③ Enterococcus faecalis seed liquid culture: Inoculate the Enterococcus faecalis liquid cultured in shake flasks into the seed liquid medium at a ratio of 3-5%, and culture at a temperature of 37 °C, a rotation speed of 220 r / min, and a time of 24 h to obtain the seed culture solution.

[0041] The main components of the seed liquid medium include: peptone 10 g / L, yeast extract 15 g / L, brown sugar 20 g / L, KH 2 PO 4 2.0 g / L, MgSO 41.0 g / L, sodium acetate anhydrous 2 g / L, manganese sulfate 0.2 g / L, compound B vitamins 0.002 g / L; sterilize at 121 °C for 20 min.

[0042] S24. Fermentation broth culture: Inoculate the seed culture medium into the fermentation broth medium containing 1 mg / L dichroa febrifuga extract according to a certain inoculation amount for culture.

[0043] ① Fermentation broth culture of Bifidobacterium lactis: Inoculate the Bifidobacterium lactis seed culture medium into the fermentation broth medium containing 1 mg / L dichroa febrifuga extract at a ratio of 8 - 16%, culture at a temperature of 37.5 °C, rotation speed of 180 r / min, and culture time of 24 - 72 h.

[0044] The main components of the fermentation broth medium include: 1 mg / L dichroa febrifuga extract, glucose 5 g / L, peptone 10 g / L, beef extract 5 g / L, yeast extract 4 g / L, K 2 HPO 4 4.0 g / L, MgSO 4 2.0 g / L, MnSO 4 1.0 g / L, L-cysteine hydrochloride 0.5 g / L; adjust the pH to 6.5 - 7.0 with NaOH, sterilize at 121 °C for 20 min.

[0045] ② Fermentation broth culture of Bacillus subtilis - Enterococcus faecalis: First, mix the Bacillus subtilis and Enterococcus faecalis seed solutions according to a volume ratio of 1:1, and then inoculate the mixed seed solution into the fermentation broth medium containing 1 mg / L dichroa febrifuga extract at a ratio of 6 - 14%, culture at a temperature of 37 °C, rotation speed of 220 r / min, and culture for 24 h.

[0046] The main components of the mixed fermentation broth medium include: 1 mg / L dichroa febrifuga extract, peptone 10 g / L, sodium carboxymethylcellulose 5 g / L, yeast extract 15 g / L, brown sugar 20 g / L, K 2 HPO 4 1.5%, NaNO 3 3 g / L, KCl 0.5 g / L, MgSO 4 0.5 g / L, compound B vitamins 0.002 g / L, adjust the pH to 6.0 - 7.0 with NaOH, sterilize at 121 °C for 20 min.

[0047] S3. Mix the Bifidobacterium lactis bacterial liquid with sterile water, and then inoculate it into the mixture of Dichroa febrifuga extract and excipients for the first-stage anaerobic fermentation. The fermentation duration is 48 - 60 h, the fermentation temperature is 38 - 40 °C, the addition amount of the composite enzyme is 0.12 - 0.16%, and the components and ratios of the composite enzyme are: protease: amylase: cellulase: dextranase = 1.0:0.6:0.8:0.6. The water ratio of the fermentation material is controlled at 38 - 55%.

[0048] S4. Cool the medicinal liquid after the first-stage fermentation to below 40 °C, and then inoculate it with the mixed bacterial liquid of Bacillus subtilis and Enterococcus faecalis and sterile water for the second-stage fermentation. The fermentation duration is 36 - 40 h, the fermentation temperature is 35 - 37 °C, the addition amount of the composite enzyme is 0.10 - 0.15%, and the components and ratios of the composite enzyme are: protease: amylase: glucoamylase: dextranase = 1.0:0.8:0.6:0.6. The water ratio of the fermentation material is controlled at 40 - 50%.

[0049] S5. Subject the fermented traditional Chinese medicine compound microecological preparation to vacuum freeze-drying treatment. The freezing temperature is -50 - -60 °C, and the freeze-drying time is 20 - 26 h, finally obtaining the traditional Chinese medicine compound microecological preparation for preventing and treating rabbit coccidiosis. The specific steps of the vacuum freeze-drying of the traditional Chinese medicine compound microecological preparation are as follows: Put the material into a vacuum freeze-dryer, pre-freeze it at -50 - -60 °C for 2 - 3 h, then evacuate the air, the vacuum degree is 20 - 40 Pa, raise the temperature to -40 - -30 °C, and carry out freeze-drying for 20 - 26 h.

[0050] The present invention also provides the use of a traditional Chinese medicine compound microecological preparation for preventing and treating rabbit coccidiosis: After the traditional Chinese medicine compound microecological preparation is prepared, it is added by mixing with feed and used for preventing and treating rabbit coccidiosis. In the above use of the traditional Chinese medicine compound microecological preparation for preventing and treating rabbit coccidiosis, preventing and treating rabbit coccidiosis includes the following steps: When preventing rabbit coccidiosis, 25 mg is added by mixing with each kilogram of feed, once a day, for 5 consecutive days. When treating rabbit coccidiosis, 50 mg is added by mixing with each kilogram of feed, once a day, for 5 consecutive days.

[0051] Example 1: Preparation of the traditional Chinese medicine compound microecological preparation 1 for preventing and treating rabbit coccidiosis.

[0052] 1. Processing of Dichroa febrifuga extract

[0053] The specific extraction method is as follows: Crush the raw material of Dichroa febrifuga, accurately weigh 10 kg of Dichroa febrifuga powder, add 6 times the total weight of the raw material of 70% ethanol solution for ultrasonic extraction, extract for 60 minutes each time, repeat the extraction 2 times, combine the above extraction solutions, remove impurities with a small amount of chloroform, then adjust the pH value to 10 with concentrated ammonia water, extract with chloroform 3 times, combine the extraction solutions, recover chloroform under reduced pressure, and dry to obtain the Dichroa febrifuga extract.

[0054] 2. Preparation of probiotic fermentation liquid

[0055] (1) Preparation of culture medium

[0056] Step 1. MRS solid medium: peptone 1 g, beef powder 0.5 g, yeast powder 0.4 g, glucose 0.2 g, Tween-80 0.1 mL, dipotassium hydrogen phosphate 0.2 g, sodium acetate 0.5 g, ammonium citrate 0.2 g, magnesium sulfate 0.02 g, manganese sulfate 0.01 g, agar powder 1.5 g, distilled water 100 mL. Add the above components to distilled water, heat to dissolve, adjust the pH to 6.3 - 6.5, dispense, and sterilize at 121 °C under high pressure for 15 min.

[0057] Step 2. MRS liquid medium: peptone 1 g, beef extract 1 g, yeast extract 0.5 g, glucose 2 g, Tween-80 0.1 mL, ammonium citrate 0.2 g, manganese sulfate 0.025 g, dipotassium hydrogen phosphate 0.2 g, sodium acetate 0.5 g, agar 2 g, distilled water 300 mL, adjust the pH to 6.5, dispense, and sterilize at 121 °C under high pressure for 15 min.

[0058] Step 3. Seed medium for Bifidobacterium lactis: glucose 3 g / L, peptone 5 g / L, beef extract 2 g / L, yeast extract 2 g / L, KH 2 HPO 4 4.0 g / L, MgSO 4 2.0 g / L, MnSO 4 1.0 g / L, L-cysteine hydrochloride 0.5 g / L, sterilize at 121 °C under high pressure for 30 min.

[0059] Step 4. Seed medium for Bacillus subtilis: peptone 5 g / L, beef extract 8 g / L, sodium carboxymethyl cellulose 5 g / L, K 2 HPO 4 1.5%, NaNO 3 3 g / L, KCl 0.5 g / L, MgSO 4 0.5 g / L, Fe 2 (SO 4 ) 3 0.01 g / L, adjust the pH to 6.5 with NaOH, sterilize at 121 °C for 20 min.

[0060] Step 5. Seed medium for Enterococcus faecalis: peptone 10 g / L, yeast extract 15 g / L, brown sugar 20 g / L, KH 2 PO 4 2.0 g / L, MgSO 4 1.0 g / L, anhydrous sodium acetate 2 g / L, manganese sulfate 0.2 g / L, compound B vitamins 0.002 g / L, sterilize at 121 °C under high pressure for 30 min.

[0061] Step 6. Fermentation medium for Bifidobacterium lactis: 1 mg / L dichroa febrifuga extract, 5 g / L glucose, 10 g / L peptone, 5 g / L beef extract, 4 g / L yeast extract, K 2 HPO 4 4.0 g / L, MgSO 4 2.0 g / L, MnSO 4 1.0 g / L, L-cysteine hydrochloride 0.5 g / L; adjust the pH to 6.6 and sterilize at 121 °C for 20 min.

[0062] Step 7. Fermentation medium for mixed fermentation of Bacillus subtilis and Enterococcus faecalis: 1 mg / L dichroa febrifuga extract, 10 g / L peptone, 5 g / L sodium carboxymethyl cellulose, 15 g / L yeast extract, 20 g / L brown sugar, K 2 HPO 4 1.5%, NaNO 3 3 g / L, KCl 0.5 g / L, MgSO 4 0.5 g / L, compound B vitamins 0.002 g / L, adjust the pH to 6.8 with NaOH and sterilize at 121 °C for 20 min.

[0063] (2) Activation of probiotic strains

[0064] Respectively dissolve the freeze-dried strains of Bifidobacterium lactis, Bacillus subtilis and Enterococcus faecalis stored at low temperature in a 37 °C water bath. Use a sterile micropipette to respectively aspirate 1 - 2 drops of the bacterial liquid and inoculate it into Petri dishes containing MRS solid medium numbered 1 - 3. Incubate statically at 40 °C for 24 h, and use an inoculation needle to pick the bacterial liquid and streak for separation and purification.

[0065] (3) Shake flask scale-up culture

[0066] Respectively inoculate the single colonies of activated Bifidobacterium lactis, Bacillus subtilis and Enterococcus faecalis into test tubes containing MRS liquid medium numbered 1 - 3, and culture at 36 °C and 160 r / min for 22 h.

[0067] (4) Seed liquid culture

[0068] Inoculate the strains cultured in the shake flask into the seed liquid medium according to a certain inoculation amount and conduct constant temperature culture.

[0069] ① Seed liquid culture of Bifidobacterium lactis: Inoculate the Bifidobacterium lactis bacterial liquid cultured in the shake flask into the seed liquid medium at a ratio of 1 - 2%, and culture at a temperature of 37.5 °C, a rotation speed of 180 r / min, and a time of 24 h to obtain the Bifidobacterium lactis seed culture solution.

[0070] ② Culturing of Bacillus subtilis seed liquid: Inoculate the Bacillus subtilis bacterial liquid cultured in a shake flask into the seed liquid medium at a ratio of 1-2%, and culture it at a temperature of 35°C, a rotation speed of 220 r / min, and a time of 20 h to obtain its seed culture solution.

[0071] ③ Culturing of Enterococcus faecalis seed liquid: Inoculate the Enterococcus faecalis bacterial liquid cultured in a shake flask into the seed liquid medium at a ratio of 3-5%, and culture it at a culture temperature of 37°C, a rotation speed of 220 r / min, and a time of 24 h to obtain the seed culture solution.

[0072] (5) Culturing of fermentation broth

[0073] Inoculate the seed culture solution into the fermentation broth medium containing 1 mg / L dichroa root extract according to a certain inoculation amount for culturing.

[0074] ① Culturing of Bifidobacterium lactis fermentation broth: Inoculate the Bifidobacterium lactis seed culture solution into the fermentation broth medium containing 1 mg / L dichroa root extract at a ratio of 6-10%, with a culture temperature of 37.5°C, a rotation speed of 180 r / min, and a culture time of 18-20 h.

[0075] ② Culturing of Bacillus subtilis-Enterococcus faecalis mixed fermentation broth: First, mix the Bacillus subtilis and Enterococcus faecalis seed liquids according to a volume ratio of 1:1, and then inoculate the mixed seed liquid into the fermentation broth medium containing 1 mg / L dichroa root extract at a ratio of 8-12%, with a culture temperature of 37°C, a rotation speed of 220 r / min, and culture for 24 h.

[0076] 3. Preparation of traditional Chinese medicine compound microecological preparation

[0077] (1) Mixing: Weigh a certain amount of dichroa root extract, corn flour, wheat bran, soybean meal, and calcium carbonate by mass. Add 260 g of corn flour, 175 g of wheat bran, 150 g of soybean meal, and 1.5 g of calcium carbonate to 1 kg of traditional Chinese medicine dichroa root extract, and mix well for backup.

[0078] (2) Sterilization: After mixing the above raw materials, transfer them to a fermentation tank for sterilization at a sterilization temperature of 121°C and a sterilization time of 30 min.

[0079] (3) First-stage fermentation: After the raw materials are cooled to below 40°C, mix 14% Bifidobacterium lactis bacterial liquid, 0.15% of the compound enzyme with sterile water and then inoculate them into the traditional Chinese medicine mixture, and carry out first-stage anaerobic fermentation at 36°C for a fermentation duration of 50 h, and control the water ratio of the fermentation materials at 45%.

[0080] (4) Secondary fermentation: Cool the medicinal liquid after the first-stage fermentation to below 40°C, and then add 10% of the mixed bacterial liquid of Bacillus subtilis and Enterococcus faecalis, 0.10% of the complex enzyme, and sterile water to the traditional Chinese medicine mixture. Conduct secondary fermentation at 35°C for 40 hours, and control the water ratio of the fermentation materials at 40%.

[0081] (5) Freeze-drying: Put the fermented materials into a vacuum freeze-dryer, pre-freeze at -50°C for 2 hours, then evacuate the air, with the vacuum degree at 20 - 40 Pa, raise the temperature to -40°C, and conduct freeze-drying for 24 hours. The above is tentatively designated as Traditional Chinese Medicine Compound Microecological Preparation No. 1.

[0082] Example 2: Preparation of Traditional Chinese Medicine Compound Microecological Preparation 2 for Preventing and Treating Rabbit Coccidiosis

[0083] Processing of Dichroa febrifuga extract: Prepared according to the method in Example 1.

[0084] Preparation of probiotic fermentation broth: Prepared according to the method in Example 1.

[0085] Preparation of traditional Chinese medicine compound microecological preparation:

[0086] (1) Mixing: Weigh a certain amount of Dichroa febrifuga extract, corn flour, wheat bran, soybean meal, and calcium carbonate according to mass parts. Add 240 g of corn flour, 170 g of wheat bran, 140 g of soybean meal, and 1.4 g of calcium carbonate to 1 kg of traditional Chinese medicine Dichroa febrifuga extract, and mix well for backup.

[0087] (2) Sterilization: After mixing the above raw materials, transfer them to a fermentation tank for sterilization at a temperature of 121°C for 30 minutes.

[0088] (3) First-stage fermentation: After cooling the raw material liquid to below 40°C, mix 10% of the Bifidobacterium lactis bacterial liquid, 0.14% of the complex enzyme with sterile water, and then add them to the traditional Chinese medicine mixture. Conduct first-stage anaerobic fermentation at 39°C for 50 hours, and control the water ratio of the fermentation materials at 42%.

[0089] (4) Secondary fermentation: Cool the medicinal liquid after the first-stage fermentation to below 40°C, and then add 8% of the mixed bacterial liquid of Bacillus subtilis and Enterococcus faecalis, 0.12% of the complex enzyme, and sterile water to the traditional Chinese medicine mixture. Conduct secondary fermentation at 36°C for 38 hours, and control the water ratio of the fermentation materials at 43%.

[0090] (5) Freeze-drying: Put the fermented materials into a vacuum freeze-dryer, pre-freeze at -50°C for 2 hours, then evacuate the air, with the vacuum degree at 20 - 40 Pa, raise the temperature to -40°C, and conduct freeze-drying for 24 hours. The above is tentatively designated as Traditional Chinese Medicine Compound Microecological Preparation No. 2.

[0091] Example 3: Preparation of Traditional Chinese Medicine Compound Microecological Preparation 3 for Preventing and Treating Rabbit Coccidiosis

[0092] Processing of Dichroa febrifuga extract: Prepared with reference to the method of Example 1.

[0093] Preparation of probiotic fermentation broth: Prepared with reference to the method of Example 1.

[0094] Preparation of traditional Chinese medicine compound microecological preparation:

[0095] (1) Mixing: Weigh a certain amount of Dichroa febrifuga extract, corn flour, wheat bran, soybean meal, and calcium carbonate according to parts by mass. Among them, 210 g of corn flour, 180 g of wheat bran, 150 g of soybean meal, and 1.6 g of calcium carbonate are added to 1 kg of traditional Chinese medicine Dichroa febrifuga extract, and mix well for backup.

[0096] (2) Sterilization: After mixing the above raw materials, put them into a fermentation tank for sterilization. The sterilization temperature is 121 °C and the sterilization time is 30 minutes.

[0097] (3) First-stage fermentation: After the raw materials are cooled to below 40 °C, mix 12% of Bifidobacterium lactis bacteria solution, 0.16% of complex enzyme, and sterile water, and then add them to the traditional Chinese medicine mixture. Conduct first-stage anaerobic fermentation at 38 °C for 56 h, and control the water ratio of the fermentation materials at 46%.

[0098] (4) Second-stage fermentation: Cool the liquid medicine after the first-stage fermentation to below 40 °C, add 13% of the mixed bacteria solution of Bacillus subtilis and Enterococcus faecalis, 0.10% of complex enzyme, and sterile water to the traditional Chinese medicine mixture, and conduct second-stage fermentation at 37 °C for 39 h, and control the water ratio of the fermentation materials at 46%.

[0099] (5) Freeze-drying: Put the fermented materials into a vacuum freeze-dryer, pre-freeze at -50 °C for 2 h, then evacuate, the vacuum degree is 20 - 40 Pa, heat up to -40 °C, and freeze-dry for 24 h. The above is tentatively designated as traditional Chinese medicine compound microecological preparation No. 3.

[0100] Example 4: Preparation of traditional Chinese medicine compound microecological preparation 4 for preventing and treating rabbit coccidiosis

[0101] Processing of Dichroa febrifuga extract: Prepared with reference to the method of Example 1.

[0102] Preparation of probiotic fermentation broth: Prepared with reference to the method of Example 1.

[0103] Preparation of traditional Chinese medicine compound microecological preparation:

[0104] (1) Mixing: Weigh a certain amount of Dichroa febrifuga extract, corn flour, wheat bran, soybean meal, and calcium carbonate according to parts by mass. Among them, 250 g of corn flour, 160 g of wheat bran, 130 g of soybean meal, and 1.8 g of calcium carbonate are added to 1 kg of traditional Chinese medicine Dichroa febrifuga extract, and mix well for backup.

[0105] (2) Sterilization: The above - mixed raw materials are put into a fermentation tank for sterilization. The sterilization temperature is 121°C and the sterilization time is 30 min.

[0106] (3) First - stage fermentation: After the raw material liquid is cooled to below 40°C, a 15% Bifidobacterium lactis bacterial liquid, 0.12% of a complex enzyme, and sterile water are mixed and then added to the traditional Chinese medicine mixture. Anaerobic fermentation is carried out at 38°C for 40 h, and the water - to - material ratio of the fermentation material is controlled at 48%.

[0107] (4) Second - stage fermentation: The liquid medicine after the first - stage fermentation is cooled to below 40°C, and a 10% mixed bacterial liquid of Bacillus subtilis and Enterococcus faecalis, 0.13% of a complex enzyme, and sterile water are added to the traditional Chinese medicine mixture. Second - stage fermentation is carried out at 36°C for 36 h, and the water - to - material ratio of the fermentation material is controlled at 48%.

[0108] (5) Freeze - drying: The fermented material is put into a vacuum freeze - dryer and pre - frozen at - 50°C for 2 h, then evacuated. The vacuum degree is 20 - 40 Pa, the temperature is raised to - 40°C, and freeze - drying is carried out for 24 h. The above is tentatively designated as Traditional Chinese Medicine Compound Microecological Preparation No. 4.

[0109] To further verify the effect of the traditional Chinese medicine compound microecological preparation for preventing and treating rabbit coccidiosis, the following two test examples are provided. Through these tests, the curative effect of the traditional Chinese medicine compound microecological preparation is systematically evaluated and compared with the existing standard treatment plan.

[0110] Test Example 1: Efficacy Test of Traditional Chinese Medicine Compound Microecological Preparation for Treating Rabbit Coccidiosis

[0111] Test drugs:

[0112] Traditional Chinese Medicine Compound Microecological Preparation 3, prepared by the specific preparation method of Example 3. Usage method: Mix with feed and feed, once a day, and 5 days is one course of treatment.

[0113] The pilot - scale product of Dichroa febrifuga powder (100 g / bag, active ingredient content 0.35%). Feed at 0.1 g / kg by mixing with feed, once a day, and 5 days is one course of treatment.

[0114] Control drug: Diclazuril premix (50 g / bag, active ingredient content 0.2%), administered at a recommended dose of 0.5 g / kg. Usage method: Mix with feed and feed, once a day, and continuously feed for 5 days.

[0115] Test strain: Eimeria magna.

[0116] Test reagents and preparation: Saturated saline: Heat 1000 mL of water to boiling, stir while adding salt until salt crystals appear in the water and no longer dissolve, then cool to room temperature and put into a container for standby.

[0117] 2.5% Potassium dichromate solution: 100 mL of distilled water, 2.5 g of potassium dichromate, fully dissolved, and stored in the dark for later use.

[0118] Experimental animals and grouping:

[0119] Experimental animals:

[0120] Forty-five-day-old healthy New Zealand meat rabbits were selected for the experiment. Fecal samples of the experimental meat rabbits were collected continuously for two days before the experiment, and the saturated saline flotation method was used to check for coccidian oocysts. If no oocysts were detected in the feces, the experiment could be carried out. The breeding environment, cages, and utensils used were all sterilized by flame, and free drinking water was provided during the breeding period.

[0121] Animal grouping:

[0122] Fifty 45-day-old meat rabbits with similar body weights and free of coccidia were selected, weighed one by one, and randomly divided into 5 groups, with 10 rabbits in each group. They included a healthy control group, an infected control group, a diclazuril group (chemical drug control), a traditional Chinese medicine compound microecological preparation group, and a dichroa febrifuga powder group (traditional Chinese medicine control). Except for the healthy control group, each meat rabbit in the other groups was orally inoculated with 1×10 5 sporulated oocysts of Eimeria maxima. After 24 hours of inoculation, administration was started respectively and continued for 5 days. The mental state, diet, drinking water, and bloody stools of the experimental meat rabbits in each group were observed every day and recorded. In case of death, autopsy was performed in a timely manner to observe the pathological changes of various organs. The experimental design and grouping are shown in Table 1.

[0123] Table 1: Experimental design and grouping

[0124] Group Number of animals (pcs) Treatment Health control 10 No challenge with parasites and no drug administration Infection control 10 Challenge with parasites and no drug administration Changshan powder 10 Add 0.1 g per 1 kg of feed and administer for 5 days Traditional Chinese medicine compound microecological preparation 10 Add 0.05 g per 1 kg of feed and administer for 5 days Diclazuril 10 Add 0.5 g per 1 kg of feed and administer for 5 days

[0125] Efficacy indicators and determination:

[0126] The therapeutic effect of the traditional Chinese medicine compound microecological preparation on artificially infected rabbit coccidiosis was evaluated based on clinical symptoms, body weight changes, oocyst excretion, mortality, and intestinal lesion scoring.

[0127] (1) Clinical symptoms: The appetite, mental state, shape and color of feces of rabbits in each group were observed every day and recorded.

[0128] (2) Body weight: The rabbits were weighed on an empty stomach on the first day of the experiment, and then on the 3rd, 7th, and 14th days after inoculation, and the average body weight of rabbits in each group was calculated.

[0129] (3) Survival rate: After the experiment, the number of surviving rabbits was counted, and the survival rate of the experimental meat rabbits was calculated. The formula is as follows:

[0130] Survival rate = (number of surviving rabbits / total number of rabbits) × 100%.

[0131] (4) Intestinal lesion scoring: Please refer to the appendix Figure 1 - Appendix Figure 2 , on the 7th and 14th days of the experiment, randomly select 1 experimental rabbit from each group and sacrifice it, take out the intestine, observe the intestinal lesion conditions and record them. The recording criteria for lesions are as follows: 0 points: no obvious visible abnormalities in the intestine; 1 point: hyperemia in the small intestine, slightly thickened mucosa, showing light yellow; 2 points: hyperemia in the small intestine, thickened mucosa, with white nodules in the mucosa, increased mucus, showing pink; 3 points: severe hyperemia in the small intestine, thickened mucosa, diffusely distributed with a large number of white nodules, thick and viscous mucus in the intestinal lumen, showing yellowish red; 4 points: swelling of the jejunum and ileum, the intestinal lumen is filled with bran-like substances or orange-yellow mucus masses, there are nodular patches or small nodules on the mucosa, and part of the mucosa falls off.

[0132] (5) Lesion value: The average intestinal lesion score of each group of rabbits × 10

[0133] (6) Oocyst count: When oocysts are excreted, collect the feces of the experimental rabbits in each group every day, and count the number of oocysts per gram of feces (OPG) by the McMaster's method.

[0134] Test results:

[0135] Clinical symptoms:

[0136] Healthy control group: Normal appetite, drinking water, and mental state, normal fecal form and color. Infected control group: On the 3rd day after coccidia inoculation, symptoms such as listlessness, ruffled hair, dull eyes, decreased body temperature, emaciation, and yellowish urine color appeared. The experimental meat rabbits had constipation first and then diarrhea, excreted soft and shapeless feces with blood in them, and the anus and hind limbs were contaminated with feces. Starting from the 5th day in the infected control group, some meat rabbits died, and a total of 3 died at the end of the experiment. Changshan powder group and probiotic group: Good mental state during the experiment, normal feeding and drinking water, no obvious clinical symptoms, and no deaths occurred. In the diclazuril control group, the experimental meat rabbits showed clinical symptoms from 3 days to 4 days and then gradually disappeared, and no deaths occurred.

[0137] Anatomical changes: Please refer to the appendix Figure 2 , the intestines of the rabbits in the healthy group were all normal, without swelling and bleeding. The intestines of the rabbits in the infected group were severely swollen and bleeding. After 5 days of administration in the Changshan powder group and the traditional Chinese medicine probiotic group, there was no obvious bleeding and swelling in the rabbits' intestines.

[0138] Average daily weight gain:

[0139] The average weight gain of the infection control group was the lowest, with a daily weight gain of only 22.01 g per rabbit. Compared with the healthy control group, there were no significant differences in the average weight gain among the Dichroa febrifuga powder group, the probiotic group, and the diclazuril group (P > 0.05); compared with the infection control group, the differences in all drug-administered groups were extremely significant (P < 0.01), and they were all much higher than the infection control group. Among them, the average weight gain of the probiotic group was the most obvious, reaching 66.41 g per rabbit. This indicates that the probiotic group has a better inhibitory effect on rabbit coccidia and has an obvious improvement effect on the weight loss of rabbits caused by coccidia. See Table 2 for details.

[0140] Table 2 Changes in body weight of rabbits in each group / g

[0141] Group Day 0 of drug administration Day 3 of drug administration Day 7 of drug administration Day 14 of drug administration Average daily weight gain Health control 848.2 1095.2 1308.7 1672.3 54.06±3.16** Infection control 873.6 894.5 912.6 1236.1 22.01±2.4 Changshan powder 864.7 1287.3 1476.5 1731.6 58.03±3.21** Traditional Chinese medicine microecology 897.1 1376.4 1519.8 1798.4 66.41±3.04** Diclazuril 856.4 1279.5 1476.4 1649.5 53.02±3.21**

[0142] ** indicates extremely significant difference (P < 0.01).

[0143] Oocyst per gram of feces (OPG):

[0144] The oocyst excretion reached the highest level in each infected group on the 6th - 8th day, but the oocyst excretion in the drug-administered groups was much lower than that in the infection control group, and the peak oocyst excretion in the drug-administered groups was delayed compared with the infection control group, indicating that the Dichroa febrifuga powder group and the probiotic group can inhibit the development and reproduction of oocysts, thereby reducing the oocyst excretion. Among them, the oocyst excretion in the probiotic group was the least, indicating that the probiotic group has a good therapeutic effect on rabbit coccidia. See Table 3 for details.

[0145] Table 3 Average OPG value of rabbits in each group (1×10 5 )

[0146]

[0147] Survival rate and lesion value:

[0148] The experimental rabbits in the infection control group began to die on the 7th day, and a total of 3 rabbits died during the experimental period, with a survival rate of 70.0%. No deaths occurred in other groups. After the rabbits in the infected drug-administered groups (Dichroa febrifuga powder group, traditional Chinese medicine probiotic group, and diclazuril group) were infected with oocysts, all indicators were basically normal during the entire experimental period. The intestinal lesion value of the Dichroa febrifuga powder group was 9.0, the intestinal lesion value of the traditional Chinese medicine probiotic group was 2.0, and the intestinal lesion value of the diclazuril group was 12.0. The small intestine of the diseased rabbits in the infection control group was severely congested, the mucosa was thickened, and the intestine was severely damaged, with a lesion value of 30.0. See Table 4 for details.

[0149] Table 4 Comparison of the efficacy of rabbit coccidia in different experimental groups

[0150] Evaluation index Health control Infection control Changshan powder Traditional Chinese medicine compound microecological preparation Diclazuril Survival rate (%) 100 70 100 100 100 Average lesion score 0 3.0 0.9 0.2 1.2 Lesion value 0 30 9.0 2.0 12

[0151] Conclusion:

[0152] Rabbit coccidiosis can occur and prevail throughout the year. Especially in spring and summer when it is warm and there is more rain, the disease often shows endemic prevalence. When the temperature ranges from 20 to 28 °C, the relative humidity ranges from 55 to 75%, and there is sufficient oxygen, rabbit coccidia oocysts can develop and mature in 2 - 3 days. When rabbits eat the forage or drink water contaminated with infected oocysts, they will get sick. The traditional Chinese medicine compound microecological preparation is administered by mixing it with the feed at a dose of 0.05 g per 1 kg of feed, which can significantly increase the weight gain rate of the experimental rabbits and has a good therapeutic effect on the experimental rabbits artificially infected with Eimeria magna. The experimental results show that the traditional Chinese medicine compound microecological preparation can not only directly kill rabbit coccidia and inhibit the sporulation of coccidia oocysts, but also improve the intestinal damage caused by coccidia infection, significantly increase the average weight gain of the experimental rabbits, reduce the small intestine lesion score caused by coccidia infection, and thus greatly improve the survival rate of the experimental rabbits. The traditional Chinese medicine compound microecological preparation of the present invention has the advantages of being green and environmentally friendly, easy to use, having good anti-coccidial efficacy and improving intestinal damage, which is of great significance for effectively preventing and treating rabbit coccidiosis and ensuring food safety, and is expected to become a new type of anti-coccidial drug.

[0153] Experimental Example 2 Influence of Traditional Chinese Medicine Microecological Preparation on Sporulation of Rabbit Coccidia Oocysts

[0154] Test drugs:

[0155] Traditional Chinese medicine compound microecological preparation 4, prepared by the specific preparation method of Example 4. When in use, it is respectively prepared into 0.25, 0.5, 1.0 g / ml with 2.5% potassium dichromate solution for standby.

[0156] Rabbit coccidia oocysts: Eimeria magna is selected. Before the experiment, it is rejuvenated by artificially infecting coccidia-free meat rabbits to obtain fresh unsporulated coccidia oocysts for standby.

[0157] Test animals: 50-day-old coccidia-free meat rabbits are selected for coccidia rejuvenation experiment.

[0158] Test reagents and preparation:

[0159] Saturated saline: 1000 mL of water is heated and boiled, and salt is added to the boiling water while stirring until a large amount of salt crystals appear in the water and no longer dissolve, and then it is cooled and filled into a container for standby.

[0160] 2.5% potassium dichromate solution: 100 mL of distilled water and 2.5 g of potassium dichromate are fully dissolved and stored in the dark for standby.

[0161] Test method:

[0162] Collection of rabbit coccidia oocysts:

[0163] In the experiment, 5 healthy meat rabbits are selected, and each is orally inoculated with 1×10 5A large number of sporulated oocysts of Eimeria maxima were sacrificed on the 8th day after inoculation. The intestinal contents were collected, placed in a beaker, stirred with an appropriate amount of distilled water, filtered through a 240-mesh sieve, the filter residue was discarded, and the filtrate was taken. The filtrate was allowed to stand until sufficient precipitation occurred, the supernatant was discarded, and then 1 volume of saturated saline was added. After thorough mixing with a glass rod, it was centrifuged for 10 min (4000 r / min). After centrifugation, the supernatant was discarded. First, a small amount of saturated saline was poured in and stirred thoroughly with a glass rod, then a large amount of saturated saline was poured in and stirred evenly. After balancing, it was centrifuged at a speed of 4000 r / min for 8 min. After centrifugation, the supernatant was retained and the precipitate was discarded. 1 / 3 saturated saline and 2 / 3 distilled water were added and stirred thoroughly. After balancing, it was centrifuged at 4000 r / min for 12 min. After centrifugation, the supernatant was carefully poured off and the precipitate was collected. The precipitate was placed in a petri dish containing an appropriate amount of 2.5% potassium dichromate solution, and then added to 6-well cell culture plates respectively and labeled.

[0164] Effect of Chinese herbal compound microecological agents at different concentrations on the sporulation rate of rabbit coccidian oocysts:

[0165] The Chinese herbal compound microecological agents were prepared into three different mass concentrations of 0.25 g / mL, 0.5 g / mL, and 1.0 g / mL with 2.5% potassium dichromate solution for the experiment, and 2.5% potassium dichromate solution was used as the blank control. The Chinese herbal medicine and control solutions (2 mL / well) were added to the above-mentioned cell culture dish wells, shaken well, and then placed in an incubator at 27 °C for cultivation. The coccidian oocysts were counted at 24 h, 48 h, 72 h, 96 h, and 120 h respectively, and the oocyst sporulation rate of each group was calculated. The calculation formula is as follows: Oocyst sporulation rate (%) = number of sporulated oocysts in 100 oocysts / 100 × 100%.

[0166] Results and analysis:

[0167] The results showed that the traditional Chinese medicine compound microecological preparation had a good inhibitory effect on rabbit coccidia, and with the increase of the drug mass concentration, its inhibitory effect on the sporulation of coccidian oocysts gradually enhanced. At 24 h after drug treatment and culture, the inhibitory effect of the traditional Chinese medicine compound microecological preparation with a mass concentration of 1.0 g / mL was the best, and the sporulation rate was 28%. When the mass concentration was 0.5 g / mL, the inhibition rate was 36%. When the mass concentration was 0.25 g / mL, the inhibition rate was 45%. With the extension of the culture time, the sporulation rate of rabbit coccidian oocysts in the drug-administered group was significantly lower than that in the blank control group (P < 0.05). At 120 h after traditional Chinese medicine treatment and culture, the sporulation rate of the traditional Chinese medicine microecological preparation with a mass concentration of 0.25 g / mL was only 70%; the sporulation rate of the traditional Chinese medicine microecological preparation with a mass concentration of 0.50 g / mL was 68%; the sporulation rate of the traditional Chinese medicine microecological preparation with a mass concentration of 1.00 g / mL was 55%. The sporulation rate of coccidia in the blank control group reached 90% after 72 h of culture and was as high as 97% after 120 h of culture.

[0168] Table 5 Effects of traditional Chinese medicine microecological preparation on the sporulation of coccidian oocysts

[0169]

[0170] Conclusion:

[0171] Rabbit coccidiosis is a disease caused by coccidia of the genus Eimeria, which are obligate protozoa parasitizing in the digestive tract of rabbits. After coccidian oocysts are excreted from the feces of diseased rabbits, they need to carry out sporulation in a suitable external environment and develop into sporulated oocysts before they are pathogenic. Therefore, inhibiting the sporulation of coccidian oocysts is the key to preventing and treating coccidiosis. The test results showed that the traditional Chinese medicine microecological preparation could inhibit the sporulation of coccidian oocysts to varying degrees, and with the increase of the drug mass concentration, its inhibitory effect on the sporulation of coccidian oocysts enhanced. After 120 h of drug administration, the sporulation rate of the traditional Chinese medicine microecological preparation with a mass concentration of 1.00 g / mL was 55%; the sporulation rate of the blank control group was as high as 97%. It shows that the traditional Chinese medicine microecological preparation can significantly inhibit the sporulation of rabbit coccidian oocysts, thus achieving the effect of anti-coccidiosis.

[0172] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made therein without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A Chinese medicinal composite microecological preparation for preventing and treating rabbit coccidia, characterized in that: The invention comprises radix changshanii extract, bifidobacterium lactis, bacillus subtilis, enterococcus faecalis, corn flour, wheat bran, soybean meal and calcium carbonate, wherein the mass proportion of radix changshanii extract accounts for 30-60% of the total mass, the mass proportion of bifidobacterium lactis, bacillus subtilis and enterococcus faecalis accounts for 10-25% of the total mass, the mass proportion of corn flour, wheat bran and soybean meal accounts for 15-35% of the total mass, and the mass proportion of calcium carbonate accounts for 0.1-1% of the total mass.

2. A Chinese medicinal composite microecological preparation for preventing and treating rabbit coccidia according to claim 1, characterized in that: The mass ratio of the Bifidobacterium lactis, Bacillus subtilis and Enterococcus faecalis is: Bifidobacterium lactis: Bacillus subtilis: Enterococcus faecalis = 1:1:

1.

3. A method for preparing a traditional Chinese medicine composite microecological preparation for preventing and treating rabbit coccidia, characterized in that: A Chinese medicinal composite microecological preparation for preventing and treating rabbit coccidia as described in any one of claims 1-2 comprises the following steps: S1. Weigh and mix the Changshan extract, corn flour, wheat bran, soybean meal and calcium carbonate in parts by mass; S2. Prepare seed solution and fermentation broth of Bifidobacterium lactis, Bacillus subtilis and Enterococcus faecalis respectively; S3. The lactobacillus lactis liquid was mixed with sterile water and then connected to the mixture of Changshan extract and excipients for the first anaerobic fermentation, the fermentation time was 24 to 72h, the fermentation temperature was 35 to 41 ° C, and the amount of complex enzyme added was 0.1 to 0.2%; S4. The fermented liquid was cooled to below 40°C, and a mixed bacterial solution of Bacillus subtilis and Enterococcus faecalis was added with sterile water for a second aerobic fermentation, the fermentation time was 24 to 50 hours, the fermentation temperature was 32 to 38°C, and the amount of the complex enzyme added was 0.05 to 0.15%; S5. The fermented Chinese herbal compound probiotic preparation is subjected to vacuum freeze-drying treatment with a freezing temperature of -50 to -60°C and a freeze-drying time of 20 to 26 hours, thereby finally obtaining a Chinese herbal compound probiotic preparation for preventing and treating rabbit coccidia.

4. The method for preparing a traditional Chinese medicine composite microecological preparation for preventing and treating rabbit coccidia according to claim 3, characterized in that: The preparation method of the Dichroia extract in step S1 is: crush the Dichroia raw material, use 70% ethanol solution for ultrasonic extraction, combine the extracts after two extractions, filter and extract with chloroform, recover the chloroform under reduced pressure, and dry to obtain the Dichroia extract.

5. The method for preparing a traditional Chinese medicine composite microecological preparation for preventing and treating rabbit coccidia according to claim 3, characterized in that: The mass proportions of the Dichroa febrifuga extract and the auxiliary materials in step S1 are as follows: 200-260 g corn flour, 100-200 g wheat bran, 100-150 g soybean meal, and 1.0-2.0 g calcium carbonate are added to every 1 kg of Dichroa febrifuga extract.

6. The method for preparing a traditional Chinese medicine composite microecological preparation for preventing and treating rabbit coccidia according to claim 3, characterized in that: The raw materials in the fermentation tank were sterilized before being put into fermentation, the sterilization temperature was 121° C., and the sterilization time was 30 min.

7. The method for preparing a traditional Chinese medicine composite microecological preparation for preventing and treating rabbit coccidia according to claim 3, characterized in that: The method for preparing the seed solution and fermentation solution of Bifidobacterium lactis, Bacillus subtilis and Enterococcus faecalis in step S2 includes strain activation, shake flask expansion culture, seed solution culture and fermentation solution culture, and the specific steps are: The activation and cultivation of Bifidobacterium lactis, Bacillus subtilis and Enterococcus faecalis were carried out according to conventional methods, using MRS medium for cultivation and inoculating into corresponding seed liquid and fermentation liquid medium for cultivation.

8. The method for preparing a traditional Chinese medicine composite microecological preparation for preventing and treating rabbit coccidia according to claim 3, characterized in that: In step S3, after optimization, the first stage of anaerobic fermentation lasts for 48 to 60 hours, the fermentation temperature is 38 to 40° C., the amount of compound enzyme added is 0.12 to 0.16%, the components and proportions of the compound enzyme are: protease: amylase: cellulase: glucanase = 1.0:0.6:0.8:0.6, and the water ratio of the fermentation material is controlled between 38 and 55%.

9. The method for preparing a traditional Chinese medicine composite microecological preparation for preventing and treating rabbit coccidia according to claim 3, characterized in that: In step S4, after optimization, the second aerobic fermentation time is 36 to 40 hours, the fermentation temperature is 35 to 37° C., the amount of complex enzyme added is 0.10 to 0.15%, and the components and proportions of the complex enzyme are: protease: amylase: saccharifying enzyme: glucanase = 1.0:0.8:0.6:0.6; the water ratio of the fermentation material is controlled between 40 and 50%.

10. The method for preparing a traditional Chinese medicine composite microecological preparation for preventing and treating rabbit coccidia according to claim 3, characterized in that: The mixing ratio of the Dichroa febrifuga extract and auxiliary materials and the fermentation conditions are adjusted according to specific needs.

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