阅读说明：本技术 一种分段式发酵制备畜禽抗病毒中草药制剂的方法 (Method for preparing antiviral Chinese herbal medicine preparation for livestock and poultry by sectional fermentation ) 是由 李登云 柴保国 韩露 陈佩格 乔彬 崔俊亚 李小佳 申雅琼 于 2021-08-27 设计创作，主要内容包括：本发明属于畜禽抗病毒制剂技术领域,具体公开一种分段式发酵制备畜禽抗病毒中草药制剂的方法。将黄连20-25份、黄芩10-15份、黄柏15-20份和连翘10-15分别粉碎；将黄芩和连翘进行提取,得提取液；将提取液和凝结芽孢杆菌、地衣芽孢杆菌和植物乳杆菌三种益生菌的发酵培养液进行混合发酵；将黄芩、连翘提取后的药渣与黄连、黄柏粉混合,用复合发酵液进行拌料并发酵,即得。本发明制备的中草药制剂具有清热解毒、补气益气、巩固根本和增强食欲的功效,可增强提高养殖动物抗病能力,提高养殖动物机体免疫力。本发明制备的微生态制剂还具有绿色无残留的特点,有效提高畜禽养殖效益和养殖质量。(The invention belongs to the technical field of livestock and poultry antiviral preparations, and particularly discloses a method for preparing an livestock and poultry antiviral Chinese herbal medicine preparation by sectional fermentation. Respectively pulverizing 20-25 parts of Coptidis rhizoma, 10-15 parts of Scutellariae radix, 15-20 parts of cortex Phellodendri and 10-15 parts of fructus forsythiae; extracting Scutellariae radix and fructus forsythiae to obtain extractive solution; mixing the extracting solution with fermentation culture solutions of three probiotics, namely bacillus coagulans, bacillus licheniformis and lactobacillus plantarum; mixing the residues after extraction of Scutellariae radix and fructus forsythiae with Coptidis rhizoma and cortex Phellodendri powder, stirring with composite fermentation liquid, and fermenting. The Chinese herbal medicine preparation prepared by the invention has the effects of clearing away heat and toxic materials, tonifying qi and qi, consolidating the root and enhancing appetite, and can enhance and improve the disease resistance of cultured animals and the immunity of organisms of the cultured animals. The microecological preparation prepared by the method also has the characteristic of environmental protection and no residue, and effectively improves the livestock and poultry breeding benefit and the breeding quality.)

1. A method for preparing antiviral Chinese herbal medicine preparation for livestock and poultry by sectional fermentation is characterized by comprising the following preparation steps:

(1) weighing 20-25 parts of coptis chinensis, 10-15 parts of scutellaria baicalensis, 15-20 parts of phellodendron and 10-15 parts of fructus forsythiae in parts by weight, and respectively crushing for later use;

(2) and preparing fermentation liquor:

(2.1) respectively activating and then carrying out expanded culture on bacillus coagulans, bacillus licheniformis and lactobacillus plantarum to obtain corresponding bacterial liquids;

(2.2) mixing the bacillus coagulans liquid, the bacillus licheniformis liquid and the lactobacillus plantarum liquid according to the volume ratio of (8.5-9.5) to (1.8-2.2) to 1 to obtain a fermentation culture solution;

(3) and liquid state fermentation:

(3.1) an extraction process: soaking the scutellaria baicalensis powder and the forsythia suspense powder obtained in the step (1) in water for at least 30 min, boiling, stirring and extracting for at least 1 h, and then performing centrifugal filtration to obtain a traditional Chinese medicine extracting solution and dregs of a decoction;

(3.2) adding the fermentation culture solution obtained in the step (2.2) and the traditional Chinese medicine extracting solution obtained in the step (3.1) into a fermentation tank according to the volume ratio of 1: 3-5, adding a composite culture medium into the fermentation tank, wherein the volume ratio of the total amount of the fermentation culture solution and the traditional Chinese medicine extracting solution to the composite culture medium is (0.5-1.5) to 100, and fermenting for 48-72 hours under the conditions of a stirring speed of 50-80 rpm and an air flow of 1.5-2.5 vvm to obtain a liquid fermentation broth; the composition of the complex culture medium is as follows: 0.8-1.2 g/L of beef extract, 8-12 g/L of peptone, 4-6 g/L of yeast extract, 18-22 g/L, NaCl 4-4 g/L of glucose, 4-6 g/L of triammonium citrate, 1-3 g/L of potassium bicarbonate, 1-1.5 g/L of Tween-800.5, 0.1-0.3 g/L of magnesium sulfate heptahydrate, 0.04-0.06 g/L of manganese sulfate and the balance of water;

(4) and solid state fermentation:

(4.1) mixing the dregs obtained in the step (3.1) with the coptis powder and the phellodendron bark powder obtained in the step (1);

(4.2) mixing the liquid fermentation liquor obtained in the step (3.2) and the solid mixture obtained in the step (4.1) according to the mass ratio of 1: 3-5, uniformly mixing, subpackaging the mixture into a fermentation bag with a one-way exhaust valve, sealing, and fermenting at 30-32 ℃ for 48-72 hours to obtain the target product, namely the livestock and poultry antiviral Chinese herbal medicine preparation.

2. The method for preparing the antiviral Chinese herbal medicine preparation for livestock and poultry by sectional fermentation according to claim 1, which is characterized in that: in the step (1), the powder is crushed to 40-80 meshes.

3. The method for preparing the antiviral Chinese herbal medicine preparation for livestock and poultry by sectional fermentation according to claim 1, which is characterized in that: in the step (2.1), the bacillus coagulans liquid, the bacillus licheniformis liquid and the lactobacillus plantarum liquid are respectively prepared according to the following processes:

preparing a bacillus coagulans liquid and a bacillus licheniformis liquid: inoculating the preserved Bacillus coagulans/Bacillus licheniformis into a seed slant culture medium, and performing activated culture at 35-37 deg.C for 24-48 h; then inoculating the activated culture solution into a basic fermentation culture medium according to the volume ratio of (0.5-1.5) to 100, and performing shaking amplification culture at 35-37 ℃ for 24-48 h at the speed of 150-; the seed slant culture medium comprises the following components: 8-12 g/L of peptone, 14-16 g/L of beef extract, 14-16 g/L of sodium chloride, 16-20 g/L of agar and the balance of water; the basic fermentation medium consists of: 8-12 g/L of peptone, 14-16 g/L of beef extract, 14-16 g/L of sodium chloride and the balance of water;

preparing lactobacillus plantarum bacterial liquid: inoculating the preserved lactobacillus plantarum into an MRS culture medium, and performing shaking activation culture at 35-37 ℃ and 200 r/min for 24-48 h; then inoculating the activated culture solution into an MRS culture medium according to the volume ratio of 0.5-1.5: 100, and carrying out shake amplification culture at 35-37 ℃ and 200 r/min for 24-48 h to obtain the lactobacillus plantarum bacterial solution.

Technical Field

The invention belongs to the technical field of livestock and poultry antiviral preparations, and particularly relates to a method for preparing livestock and poultry antiviral Chinese herbal medicine preparations through sectional fermentation.

Background

The fermented traditional Chinese medicine is a green feed additive, the probiotics is utilized to ferment the traditional Chinese medicine, macromolecular substances in the traditional Chinese medicine are decomposed and converted into micromolecular substances, and the micromolecular substances can be directly absorbed and utilized by the digestive tract of animals, so that the drug effect of the traditional Chinese medicine is improved, the action effect of the traditional Chinese medicine is enhanced, and the traditional Chinese medicine is a healthy and safe antibiotic substitute with great application potential.

After the effective components in the Chinese herbal medicine are purified, the effective components also contain a large amount of components which are not easy to degrade, and if the effective components are not utilized, the pollution to the ecological environment is influenced to a certain extent. Therefore, the Chinese herbal medicine is reasonably and effectively utilized, so that the resource utilization rate can be improved, the resources are saved, the waste is reduced, the pollution to the ecological environment can be reduced, and the combination of the Chinese herbal medicine and the probiotics can provide a new method for solving the problem of environmental pollution and recycling resources.

Disclosure of Invention

Aiming at the defects and shortcomings of the prior art, the invention aims to provide a method for preparing an antiviral Chinese herbal medicine preparation for livestock and poultry by sectional fermentation.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a method for preparing antiviral Chinese herbal medicine preparation for livestock and poultry by sectional fermentation comprises the following steps:

(1) weighing 20-25 parts of coptis chinensis, 10-15 parts of scutellaria baicalensis, 15-20 parts of phellodendron and 10-15 parts of fructus forsythiae in parts by weight, and respectively crushing for later use;

(2) and preparing fermentation liquor:

(2.1) respectively activating and then carrying out expanded culture on bacillus coagulans, bacillus licheniformis and lactobacillus plantarum to obtain corresponding bacterial liquids;

(2.2) mixing the bacillus coagulans liquid, the bacillus licheniformis liquid and the lactobacillus plantarum liquid according to the volume ratio of (8.5-9.5) to (1.8-2.2) to 1 to obtain a fermentation culture solution;

(3) and liquid state fermentation:

(3.1) an extraction process: soaking the scutellaria baicalensis powder and the forsythia suspense powder obtained in the step (1) in water for at least 30 min, boiling, stirring and extracting for at least 1 h, and then performing centrifugal filtration to obtain a traditional Chinese medicine extracting solution and dregs of a decoction;

(3.2) adding the fermentation culture solution obtained in the step (2.2) and the traditional Chinese medicine extracting solution obtained in the step (3.1) into a fermentation tank according to the volume ratio of 1: 3-5, adding a composite culture medium into the fermentation tank, wherein the volume ratio of the total amount of the fermentation culture solution and the traditional Chinese medicine extracting solution to the composite culture medium is (0.5-1.5) to 100, and fermenting for 48-72 hours under the conditions of a stirring speed of 50-80 rpm and an air flow of 1.5-2.5 vvm to obtain a liquid fermentation broth; the composition of the complex culture medium is as follows: 0.8-1.2 g/L of beef extract, 8-12 g/L of peptone, 4-6 g/L of yeast extract, 18-22 g/L, NaCl 4-4 g/L of glucose, 4-6 g/L of triammonium citrate, 1-3 g/L of potassium bicarbonate, 1-1.5 g/L of Tween-800.5, 0.1-0.3 g/L of magnesium sulfate heptahydrate, 0.04-0.06 g/L of manganese sulfate and the balance of water;

(4) and solid state fermentation:

(4.1) mixing the dregs obtained in the step (3.1) with the coptis powder and the phellodendron bark powder obtained in the step (1);

(4.2) mixing the liquid fermentation liquor obtained in the step (3.2) and the solid mixture obtained in the step (4.1) according to the mass ratio of 1: 3-5, uniformly mixing, subpackaging the mixture into a fermentation bag with a one-way exhaust valve, sealing, and fermenting at 30-32 ℃ for 48-72 hours to obtain the target product, namely the livestock and poultry antiviral Chinese herbal medicine preparation.

Preferably, in step (1), the powder is ground to 40-80 mesh.

Preferably, in the step (2.1), the bacillus coagulans liquid, the bacillus licheniformis liquid and the lactobacillus plantarum liquid are respectively prepared according to the following processes:

preparing a bacillus coagulans liquid and a bacillus licheniformis liquid: inoculating the preserved Bacillus coagulans/Bacillus licheniformis into a seed slant culture medium, and performing activated culture at 35-37 deg.C for 24-48 h; then inoculating the activated culture solution into a basic fermentation culture medium according to the volume ratio of (0.5-1.5) to 100, and performing shaking amplification culture at 35-37 ℃ for 24-48 h at the speed of 150-; the seed slant culture medium comprises the following components: 8-12 g/L of peptone, 14-16 g/L of beef extract, 14-16 g/L of sodium chloride, 16-20 g/L of agar and the balance of water; the basic fermentation medium consists of: 8-12 g/L of peptone, 14-16 g/L of beef extract, 14-16 g/L of sodium chloride and the balance of water;

preparing lactobacillus plantarum bacterial liquid: inoculating the preserved lactobacillus plantarum into an MRS culture medium, and performing shaking activation culture at 35-37 ℃ and 200 r/min for 24-48 h; then inoculating the activated culture solution into an MRS culture medium according to the volume ratio of 0.5-1.5: 100, and carrying out shake amplification culture at 35-37 ℃ and 200 r/min for 24-48 h to obtain the lactobacillus plantarum bacterial solution.

Because culture mediums and culture conditions used in liquid fermentation and solid fermentation are different, the products of the liquid fermentation and the solid fermentation of the same traditional Chinese medicine are different, the pH yield is different, and some traditional Chinese medicine components are degraded and lose efficacy when the pH is changed or some metabolites are generated in the solid fermentation process, so that not all traditional Chinese medicines can be treated by the solid fermentation. The invention adopts sectional fermentation in the fermentation process, wherein scutellaria baicalensis and forsythia suspensa are extracted firstly, then the extracting solution and probiotics are subjected to mixed liquid fermentation, and the extracted dregs of a decoction are mixed with coptis chinensis and phellodendron, and then the mixture is mixed with liquid fermentation liquor for fermentation.

The Chinese herbal medicine preparation prepared by the invention has the effects of clearing away heat and toxic materials, tonifying qi and qi, consolidating the root and enhancing appetite, and can enhance and improve the disease resistance of cultured animals and the immunity of organisms of the cultured animals; the microecological preparation prepared by the method also has the characteristic of environmental protection and no residue, and effectively improves the livestock and poultry breeding benefit and the breeding quality.

Detailed Description

In order to make the invention clearer and clearer, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1

A method for preparing antiviral Chinese herbal medicine preparation for livestock and poultry by sectional fermentation comprises the following steps:

(1) weighing 20 parts of coptis chinensis, 15 parts of scutellaria baicalensis, 15 parts of golden cypress and 10 parts of fructus forsythiae by weight, and respectively crushing the materials to 60 meshes for later use;

(2) and preparing fermentation liquor:

(2.1) respectively preparing the bacillus coagulans liquid, the bacillus licheniformis liquid and the lactobacillus plantarum liquid according to the following processes:

preparing a bacillus coagulans liquid and a bacillus licheniformis liquid: inoculating the preserved bacillus coagulans/bacillus licheniformis into a seed slant culture medium, and performing activated culture at 37 ℃ for 24 hours; then inoculating the activated culture solution into a basic fermentation culture medium according to the volume ratio of 1: 100, and performing shaking amplification culture at 37 ℃ for 48 hours at 180 r/min to obtain bacillus coagulans liquid/bacillus licheniformis liquid; the seed slant culture medium comprises the following components: 10 g/L of peptone, 15 g/L of beef extract, 15 g/L of sodium chloride, 18 g/L of agar and the balance of water; the basic fermentation medium consists of: 10 g/L of peptone, 15 g/L of beef extract, 15 g/L of sodium chloride and the balance of water;

preparing lactobacillus plantarum bacterial liquid: inoculating the preserved lactobacillus plantarum into an MRS culture medium, and performing shaking activation culture at 37 ℃ and 180 r/min for 24 hours; then inoculating the activated culture solution into an MRS culture medium according to the volume ratio of 1: 100, and carrying out shake amplification culture at 37 ℃ for 48 h at 180 r/min to obtain lactobacillus plantarum liquid;

(2.2) mixing the bacillus coagulans liquid, the bacillus licheniformis liquid and the lactobacillus plantarum liquid according to the volume ratio of 9: 2: 1 to obtain a fermentation culture solution;

(3) and liquid state fermentation:

(3.1) an extraction process: soaking the scutellaria baicalensis powder and the forsythia suspense powder obtained in the step (1) in normal-temperature water for 30 min, boiling, stirring and extracting for 1 h, and then performing centrifugal filtration to obtain a traditional Chinese medicine extracting solution and dregs;

(3.2) adding the fermentation culture solution obtained in the step (2.2) and the traditional Chinese medicine extracting solution obtained in the step (3.1) into a fermentation tank according to the volume ratio of 1: 4, adding a composite culture medium into the fermentation tank, wherein the volume ratio of the total amount of the fermentation culture solution and the traditional Chinese medicine extracting solution to the composite culture medium is 1: 100, and fermenting for 48 hours under the conditions of stirring speed of 60 rpm and ventilation volume of 2.0 vvm to obtain liquid fermentation liquor; the composition of the complex culture medium is as follows: 1.0 g/L beef extract, 10 g/L peptone, 5 g/L yeast extract, 20 g/L, NaCl 5 g/L glucose, 5 g/L triammonium citrate, 2 g/L potassium bicarbonate, Tween-801.0 g/L, 0.2 g/L magnesium sulfate heptahydrate, 0.05 g/L manganese sulfate and the balance of water;

(4) and solid state fermentation:

(4.1) mixing the dregs obtained in the step (3.1) with the coptis powder and the phellodendron bark powder obtained in the step (1);

and (4.2) mixing the liquid fermentation liquor obtained in the step (3.2) and the solid mixture obtained in the step (4.1) according to the mass ratio of 1: 4, uniformly mixing, subpackaging the mixture into a fermentation bag with a one-way exhaust valve, sealing, and fermenting at 30 ℃ for 72 hours to obtain the target product, namely the livestock and poultry antiviral Chinese herbal medicine preparation.

Comparative example 1

The difference from example 1 is that: in the step (2.2), the bacillus coagulans liquid, the bacillus licheniformis liquid and the lactobacillus plantarum liquid are mixed according to the volume ratio of 9: 5: 1 to obtain a fermentation culture solution. Otherwise, the same procedure as in example 1 was repeated.

Comparative example 2

The difference from example 1 is that: in the step (2.2), the bacillus coagulans liquid, the bacillus licheniformis liquid and the lactobacillus plantarum liquid are mixed according to the volume ratio of 9: 1 to obtain a fermentation culture solution. Otherwise, the same procedure as in example 1 was repeated.

The number of bacillus coagulans, the lactic acid content, the small peptide content and the baicalin content as an effective ingredient in the target products obtained in example 1 and comparative examples 1-2 were detected, and the results are shown in table 1, which indicates that: the ratio of the species in example 1 has a very excellent fermentation effect.

Animal experiments

1.1 test drugs

The compound traditional Chinese medicine comprises the following components: example 1 the four traditional Chinese medicines of coptis chinensis, scutellaria baicalensis, phellodendron amurense and forsythia suspense in the step (1) are respectively ground according to the proportion and then uniformly mixed to obtain the traditional Chinese medicine.

Fermenting the traditional Chinese medicines: example 1 an antiviral Chinese herbal medicine preparation for livestock and poultry prepared by sectional type fermentation.

1.2 test animals

According to the diagnostic standard of chicken infected with Newcastle disease, 8200 clinically diseased chickens are collected.

1.2.1 diagnostic criteria for Newcastle disease in chickens

1.2.1.1 clinical symptoms

The chickens are not in spirits, the feathers are loose and disorderly, the feed intake and the water intake are reduced, the feathers are fluffy, and the feathers shrink and are piled up and are in a nodding pecking shape; the cockscomb and the meat beard are dark red or dark purple; dyspnea, stretching mouth and neck, frequent "grunt" and "purulent" sounds, mucus flows out from mouth and nose; the feces are green or yellow-green, thin and foul.

1.2.1.2 case Subsections

Bleeding of mucous membranes and serosa of all parts of the chickens died of diseases, particularly the glandular stomach papilla presents obvious needle-tip-like bleeding points; in the trachea of half of dead chickens, annular bleeding, cecal tonsil swelling bleeding, bleeding spots in the intestinal tract and bleeding spots in the cardiac fat are observed.

1.3 grouping method and dosing regimen for sick chickens

Infected chickens are randomly divided into 3 groups, 200 control groups, 4000 Chinese medicinal herbs in the compound Chinese medicinal herb and 4000 Chinese medicinal herbs in the fermented Chinese medicinal herb respectively. The dosing schedule for each group is shown in table 2. During the test period, all test chickens are uniformly fed with the complete feed of the conventional broiler chicken and subjected to daily conventional management.

1.4 Observation of therapeutic Effect and death

During the test period, the mental state, average feeding condition, fecal character and other indexes of the chickens in each test group are observed every day. Observing and recording the number of clinically cured chickens, the number of effective chickens and the number of dead chickens of each group in the test period according to a curative effect judgment standard in the test period; the criteria for therapeutic effect are as follows:

and (3) curing: various clinical symptoms disappear, the appetite and the mental state are good, and the feces are normal;

and (4) invalidation: various clinical symptoms are not effective or are not obvious;

death: die after administration.

1.5. Test results

The test period is seven days, and the specific test effects are as follows:

test groups: at the beginning of the trial, 200 chickens all had varying degrees of clinical symptoms, with 16 of the severe clinical symptoms; after the test is finished, 140 patients are cured clinically, and the cure rate is 70%; 40 invalid chickens are used, and the failure rate is 20%; only 20 dead chickens with a mortality rate of 10 percent;

the compound traditional Chinese medicine comprises: at the beginning of the trial, 4000 chickens all had varying degrees of clinical symptoms, with 830 severe clinical symptoms; after the test is finished, 3360 patients are cured clinically, and the cure rate is 84%; the number of ineffective chickens is 535, and the ineffective rate is 13.38%; 105 dead chickens with the death rate of 2.62%;

fermenting the traditional Chinese medicine: at the beginning of the trial, 4000 chickens all had varying degrees of clinical symptoms, with 835 severe clinical symptoms; after the test is finished, 3680 patients are cured clinically, and the cure rate is 92%; 275 ineffective chickens with 6.87% ineffective rate; 45 dead chickens were observed, and the mortality rate was 1.13%.

The comparative results are shown in Table 3.

1.6. Small knot

The test shows that: the fermented traditional Chinese medicine prepared by the invention can effectively reduce the death rate of infected chickens and improve the cure rate effect of infected chickens when treating newcastle diseases, and has higher clinical application value for preventing and treating viral diseases such as newcastle diseases and the like.