CN110960623A

CN110960623A - Antiviral fermented traditional Chinese medicine for poultry and preparation method thereof

Info

Publication number: CN110960623A
Application number: CN201911413831.0A
Authority: CN
Inventors: 胡红伟; 李薇; 闫凌鹏; 麻啸涛; 杨亚奇; 杨京娥; 张婵娟; 党亚朋; 权豪强
Original assignee: Shanxi Dayu Bioengineering Co ltd
Current assignee: Shanxi Dayu Bioengineering Co ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2020-04-07

Abstract

The invention relates to an antiviral fermented traditional Chinese medicine for poultry, which comprises 30-50 parts of houttuynia cordata, 30-50 parts of gypsum, 20-30 parts of radix isatidis, 20-30 parts of honeysuckle, 10-20 parts of fructus forsythiae, 10-16 parts of rheum officinale, 10-16 parts of common andrographis herb, 10-16 parts of sweet wormwood herb, 10-16 parts of Chinese angelica, 10-16 parts of red paeony root, 10-16 parts of radix bupleuri, 10-16 parts of radix rehmanniae recen, 7-13 parts of scutellaria baicalensis, 7-13 parts of coptis chinensis, 7-13 parts of golden cypress, 5-10 parts of gardenia, 5-10 parts of purslane, 5-10 parts of radix ophiopogonis and 10-15 parts of liquorice, and further provides a preparation method of the antiviral fermented traditional Chinese medicine, wherein a complex enzyme and a complex bacterium are matched with each other in a fermentation process, the deeply fermented traditional Chinese medicine formula comprises bacillus subtilis, aspergillus niger, saccharomyces cerevisiae and clostridium butyricum, and the: cellulase, xylanase, glucanase, mannanase, mesophilic amylase, acid protease, neutral protease, etc. According to the invention, after mixed fermentation of bacteria and enzyme, the active ingredients of the traditional Chinese medicine are remarkably improved, and the traditional Chinese medicine has the effects of improving the immunity and the antiviral ability of animals.

Description

Antiviral fermented traditional Chinese medicine for poultry and preparation method thereof

Technical Field

The invention belongs to the technical field of fermented traditional Chinese medicines, and particularly relates to an antiviral fermented traditional Chinese medicine for poultry and a preparation method thereof.

Background

In recent years, with the rapid development of animal husbandry, in order to save breeding cost and improve breeding benefits, the density of poultry intensive breeding is higher and higher, poultry live in sub-health environment for a long time, and the introduction of new varieties, the spreading speed of viral diseases is faster and faster, and viruses are continuously mutated, so that the effects of western medicines and vaccines for preventing and treating viral diseases are poorer and poorer. Meanwhile, related laws and regulations prohibit the use of amantadine antiviral veterinary drugs, and the veterinary antiviral western drugs are less and less.

At present, the prevention of the virus is the main measure, the improvement of the poultry autoimmunity is the main measure, and the growth environment and the feeding management of the poultry are improved. China is a source of traditional Chinese medicine, the traditional Chinese medicine resources are rich, most of the traditional Chinese medicines have the characteristics of no toxicity, no side effect, no medicine residue and the like, and the traditional Chinese medicines contain rich active ingredients such as polysaccharides, alkaloids, glycosides, amino acids, volatile oil and the like, so that the traditional Chinese medicines have the characteristic of multi-target effect, are less influenced by virus variation, can activate immune cells, and improve the immunity and the antiviral ability of animals.

Because the Chinese herbal medicines are mostly natural plants, the cell walls of the plants limit the effective utilization of the traditional Chinese medicine components, in order to improve the utilization rate of the Chinese herbal medicines, the effective components are released by adopting a water extraction and alcohol precipitation mode, but in the high-temperature extraction process, the traditional Chinese medicine components are lost to different degrees, and the cost is higher. By using modern microbial fermentation technology, various enzyme preparations and various bacteria are matched with each other to thoroughly ferment and enzymolyze the cell walls of the traditional Chinese medicines, so that effective components are released to the maximum extent. The antiviral traditional Chinese medicine formula is fermented by the cooperation of the bacterial enzymes, antiviral traditional Chinese medicine components can be effectively released, the action condition is mild, the cost is low, the obtained fermented traditional Chinese medicine also contains various beneficial metabolites such as organic acid, vitamins and antibacterial peptide, and the animal immunity can be effectively improved.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide an antiviral fermented traditional Chinese medicine for poultry prepared by synergistic fermentation of bacteria and enzymes and a preparation method thereof, the fermented traditional Chinese medicine prepared by the invention has the functions of clearing away heat and toxic materials, enriching the blood, activating the blood and improving the immunity of poultry, is green and safe, has small toxic and side effects, and has the following specific scheme:

the invention provides an antiviral fermented traditional Chinese medicine for poultry, which comprises the following raw materials in parts by weight: 30-50 parts of houttuynia cordata, 30-50 parts of gypsum, 20-30 parts of isatis root, 20-30 parts of honeysuckle, 10-20 parts of fructus forsythiae, 10-16 parts of rheum officinale, 10-16 parts of common andrographis herb, 10-16 parts of sweet wormwood herb, 10-16 parts of Chinese angelica, 10-16 parts of red paeony root, 10-16 parts of radix bupleuri, 10-16 parts of radix rehmanniae recen, 7-13 parts of scutellaria baicalensis, 7-13 parts of coptis chinensis, 7-13 parts of golden cypress, 5-10 parts of cape jasmine, 5-10 parts of purslane, 5-10 parts of radix ophiopogonis, 10-15 parts of liquorice, 3-5 parts of a complex enzyme preparation, 20-30 parts of a microbial nutrient and 3-5 parts of a complex inorganic salt.

Preferably, the complex enzyme preparation consists of the following raw materials in parts by weight: 20-30 parts of cellulase, 10-15 parts of xylanase, 10-15 parts of glucanase, 5-10 parts of mannase, 10-15 parts of medium temperature amylase, 5-10 parts of acid protease and 5-10 parts of neutral protease.

In any scheme, preferably, in the complex enzyme preparation, the cellulase is more than or equal to 10000IU/g, the xylanase is more than or equal to 100000IU/g, the glucanase is more than or equal to 50000IU/g, the mannase is more than or equal to 100000IU/g, the medium-temperature amylase is more than or equal to 2000IU/g, the acid protease is more than or equal to 100000IU/g, and the neutral protease is more than or equal to 50000 IU/g.

In any of the above embodiments, preferably, the microbial nutrient is composed of the following components in parts by weight: 50-80 parts of corn flour, 20-30 parts of soybean meal, 5-10 parts of tryptone and 10-15 parts of yeast extract powder.

In any of the above schemes, preferably, the composite inorganic salt is composed of the following raw materials in parts by weight: 5-10 parts of sodium metabisulfite, 5-10 parts of ferric ammonium citrate, 5-10 parts of sodium chloride, 5-10 parts of light calcium carbonate, 3-8 parts of ammonium sulfate, 5-10 parts of sodium acetate and 5-10 parts of sodium citrate.

The invention also provides a preparation method of the poultry antiviral fermented traditional Chinese medicine, which comprises the following steps in sequence:

(1) crushing materials: pulverizing the medicinal components, and sieving with 40 mesh sieve;

(2) preparing bacterial liquid: respectively preparing bacillus subtilis liquid, aspergillus niger liquid, saccharomyces cerevisiae and clostridium butyricum mixed liquid;

(3) mixing: weighing the crushed traditional Chinese medicine components, the microbial nutrient and the composite components in sequence according to the formula proportion, starting a mixer to stir, putting the mixture into the mixer to stir for 2-3 minutes, adding the bacillus subtilis liquid, the aspergillus niger liquid, the composite enzyme preparation and the composite inorganic salt prepared in the step (2) into tap water to mix uniformly, controlling the ratio of the materials to the water to be 1: 0.5-1: 0.8, then pumping the mixed liquid into the mixer through a water pump, and continuing mixing for 3 minutes to obtain a mixed fermented material;

(4) solid state fermentation:

an aerobic stage: stacking the mixture fermentation materials into a stack with the height of 1.2 meters and the width of 1.5 meters, turning the stack once every 8 hours, controlling the temperature of a fermentation chamber to be 25-30 ℃, stopping turning the stack after fermenting for 5 days, and continuing fermenting for 12 hours;

an anaerobic stage: adding glucose oxidase into the mixed bacteria liquid of saccharomyces cerevisiae and clostridium butyricum, uniformly mixing, adding into the materials, subpackaging into one-way exhaust valve fermentation bags with the packaging amount of 50kg per bag, and carrying out anaerobic fermentation for 24 hours to obtain the poultry antiviral fermentation traditional Chinese medicine.

Preferably, the bacillus subtilis liquid is prepared by the following steps:

s1: strain activation and shake flask seed preparation

Marking the bacillus subtilis slant seeds on a flat plate respectively, performing static culture at 30-37 ℃ for 12-16 h to obtain single colonies, inoculating the single colonies to 500ml of liquid culture medium in a shake flask, and performing shake flask liquid filling of 100ml, 30-37 ℃ and 220r/min for culture for 16-24 h to obtain bacillus subtilis shake flask seed liquid. The plate culture medium comprises the following components: 3-5 g/L beef extract, 10-15 g/L peptone, 2-5 g/L sodium chloride, 1.5-3 g/L agar, pH 7.0-7.2, and sterilizing at 121 ℃ for 30 minutes; the shake flask culture medium comprises the following components: 3-5 g/L beef extract, 10-15 g/L peptone, 3-5 g/L yeast extract, 2-5 g/L sodium chloride, 5-8 g/L glucose, 7.0-7.2 pH, and sterilizing at 121 ℃ for 30 minutes;

s2: first order seed liquid preparation

Inoculating the shake flask seed solution into a seed tank according to the proportion of 1-3%, controlling the rotating speed at 200-250 rpm, controlling the ventilation ratio at 1:0.8, and culturing at 30-37 ℃ for 16-24 h to obtain a first-grade seed solution; the first-stage seeding tank culture medium comprises the following components: 20-25 g/L of bean cake powder, 10-15 g/L of corn flour, 5-10 g/L of glucose, 3-5 g/L of sodium chloride, 3-5 g/L of light calcium carbonate, 2-5 g/L of dipotassium phosphate, 2-5 g/L of manganese sulfate, pH 7.2-7.5, and sterilizing at 121 ℃ for 30-40 minutes;

s3: preparation of fermentation broth

Inoculating the primary seed liquid into a fermentation tank according to the proportion of 3-5%, controlling the rotating speed at 150-200 rpm for 0-3 h, and controlling the ventilation ratio at 1: 0.2-1: 0.3; controlling the ventilation ratio to be 1: 0.5-1: 0.7 within 3-6 h; after 6 hours, controlling the ventilation ratio to be 1: 0.8-1: 1; the fermentation tank culture medium comprises the following components: 30-40 g/L of corn flour, 5-10 g/L of glucose, 35-45 g/L of soybean meal, 3-5 g/L of corn steep liquor dry powder, 3-5 g/L of sodium chloride, 3-5 g/L of light calcium carbonate, 2-5 g/L of dipotassium hydrogen phosphate, 2-5 g/L of manganese sulfate, pH 7.2-7.5, and sterilizing for 30-40 minutes at 121 ℃.

In any of the above schemes, preferably, the aspergillus niger liquid is prepared by the following steps:

s1: strain activation and Aspergillus niger shake flask seed liquid preparation

Selecting an Aspergillus niger slant culture by using an inoculating needle, inoculating the Aspergillus niger slant culture into a liquid culture medium, carrying out shake culture at 25-28 ℃ and 200-250 rpm for 3-5 d, then inoculating the Aspergillus niger slant culture into a next shake flask according to 2-5%, repeating the culture once to complete strain activation, inoculating activated bacterium liquid into a shake flask expansion culture medium according to a proportion of 2-5%, carrying out shake culture at 25-28 ℃ and 200-250 rpm for 3-5 d to obtain shake flask seed liquid; the activating and shaking flask seed culture medium comprises the following components: 200g/L of potato, 20g/L of cane sugar, natural pH and sterilization at 121 ℃ for 30 minutes;

s2: preparation of aspergillus niger seeds by solid fermentation

And (4) preparing the shake flask seed liquid prepared in the step (S1) in a solid culture medium by adopting a tray fermentation method, wherein the thickness of the solid culture medium in a tray is controlled to be about 3-5 cm, the culture temperature is 25-28 ℃, and the culture time is 5-7 d. The solid medium comprises the following components: 80-90 parts of bran, 3-6 parts of soybean meal, 2-3 parts of ammonium sulfate, 1-2 parts of manganese sulfate, 0.5-1 part of dipotassium hydrogen phosphate, 0.5-1 part of magnesium sulfate, 1: 0.6-1: 0.8 of material-water ratio, and sterilizing at 121 ℃ for 40 minutes; before fermentation and inoculation, the Aspergillus niger culture is diluted with 0.1% Tween water according to a ratio of 1:10, stirred uniformly, and filtered by four layers of sterile gauze to obtain Aspergillus niger spore suspension for fermenting traditional Chinese medicines.

In any of the above schemes, preferably, the mixed bacterial liquid of saccharomyces cerevisiae and clostridium butyricum is prepared by the following steps:

s1: strain activation and shake flask seed liquid preparation

Activation of saccharomyces cerevisiae and preparation of shake flask seed liquid: a ring of saccharomyces cerevisiae slant seeds are selected and inoculated into a liquid activation culture medium, the shaking culture is carried out for 48-72 h at the temperature of 28 ℃ and the rpm of 200, then the activation seed liquid is inoculated into a shaking flask seed liquid according to the proportion of 1-5%, the shaking culture is carried out for 48-72 h at the temperature of 28 ℃ and the rpm of 200, and the activation and shaking flask seed culture medium comprises the following components: 10-15 g/L of yeast extract powder, 20-25 g/L of bovine bone peptone, 20-25 g/L of glucose, pH 5.8-6.0, and sterilizing at 121 ℃ for 30 minutes;

activating clostridium butyricum and preparing a shake flask seed solution: picking clostridium monobutyricum into an RCM liquid culture medium, culturing for 12-18 h in an anaerobic incubator at 37 ℃, and then carrying out one-time passage according to the proportion of 1-5% to obtain a shake flask seed solution, wherein the RCM culture medium comprises the following components: 3-5 g/L of yeast extract, 10-15 g/L of beef extract, 10-15 g/L of peptone, 1-3 g/L of soluble starch, 3-8 g/L of glucose, 0.5-1 g/L of cysteine hydrochloride, 3-5 g/L of sodium chloride, 3-5 g/L of sodium acetate, pH8.5, and sterilizing at 121 ℃ for 30 minutes;

s2: first order seed liquid preparation

Preparing first-grade seed liquid of saccharomyces cerevisiae: inoculating the shake flask seeds to a first-stage seed tank according to the proportion of 1-3%, wherein the culture temperature is 28-32 ℃, the rotating speed is 200-250 rpm, the ventilation ratio is 1:0.5, the tank pressure is 0.05-0.08 Mpa, and the culture time is 36-48 h; the first-stage seeding tank culture medium comprises the following components: 10-15 g/L of yeast extract powder, 20-25 g/L of bovine bone peptone, 20-25 g/L of glucose, 2-5 g/L of monopotassium phosphate and pH5.8-6.0;

preparing a clostridium butyricum primary seed solution: the inoculation amount is 1-2%, the temperature is 32-37 ℃, the rotating speed is 60-100 rpm, nitrogen is introduced in the culture process to form an anaerobic environment, the tank pressure is 0.05-0.08 Mpa, and the culture time is 12-16 h; the culture medium is an RCM culture medium;

s3: preparation of mixed bacterial liquid of saccharomyces cerevisiae and clostridium butyricum

Inoculating saccharomyces cerevisiae into a fermentation tank according to the proportion of 5-10%, wherein the culture temperature is 28-32 ℃, the rotating speed is 200-250 rpm, the ventilation ratio is 1:0.5, culturing is carried out for 30h, then ventilation is stopped, culturing is continued for 5-8 h, then inoculating clostridium butyricum primary seed liquid according to the proportion of 5-10%, stirring every half hour, controlling the temperature to be 30-35 ℃, and culturing for 8-12 h; the fermentation medium comprises the following components: 5-10 g/L of beef extract powder, 3-5 g/L of yeast extract powder, 40-50 g/L of glucose, 3-5 g/L of sodium chloride, 2-5 g/L of sodium acetate, 2-5 g/L of sodium citrate, 2-5 g/L of magnesium sulfate, 2-5 g/L of dipotassium hydrogen phosphate and 0.5-1 g/L of cysteine hydrochloride, and sterilizing for 30 minutes at 121 ℃.

In any of the above schemes, preferably, the bacillus subtilis inoculation amount is 3-5%; the aspergillus niger inoculation amount is 5-10%; the inoculation amount of the mixed bacterial liquid of the saccharomyces cerevisiae and the clostridium butyricum is 5-10%; the enzyme activity of the glucose oxidase is 3000IU/g, and the addition amount is 0.1%.

The invention has the following advantages and beneficial effects:

the invention provides an antiviral fermented traditional Chinese medicine for poultry, which comprises 19 Chinese herbal medicines such as houttuynia cordata, gypsum, isatis root, honeysuckle, forsythia, rhubarb, common andrographis herb, sweet wormwood herb, angelica, red peony root, radix bupleuri, radix rehmanniae, scutellaria baicalensis, coptis chinensis, phellodendron, gardenia, purslane, radix ophiopogonis, liquorice and the like, and the pharmacological actions of the traditional Chinese medicines are as follows:

houttuynia cordata: pungent taste and slightly cold nature; it enters lung, bladder and large intestine meridians. Has effects in clearing away heat and toxic materials, eliminating phlegm, expelling pus, eliminating carbuncle, promoting urination, relieving swelling, and treating stranguria.

Raw gypsum: sweet, pungent and cold in flavor; it enters lung and stomach meridians. Has effects of clearing heat-fire, relieving restlessness and quenching thirst.

Radix isatidis: bitter taste and cold nature; it enters heart, lung, liver and stomach meridians. Has effects of clearing away heat and toxic materials, cooling blood, and relieving sore throat.

Honeysuckle flower: sweet taste and cold nature; it enters lung and heart meridians. Has effects of clearing away heat and toxic materials, and dispelling pathogenic wind and heat. Can be used for treating carbuncle, furuncle, sore throat, erysipelas, toxic heat, dysentery, wind-heat type common cold, epidemic febrile disease, and fever.

Fructus forsythiae: bitter taste and mild nature; it enters heart, liver and gallbladder meridians. Has effects in clearing away heat and toxic materials, and dispersing pathogenic accumulation and swelling. Treating warm heat, erysipelas, macula, carbuncle, ulcer, scrofula, and urinary stranguria.

Rhubarb: bitter taste and cold nature; it enters spleen, stomach, large intestine, liver and pericardium meridians. Has the effects of purging, eliminating pathogenic accumulation, clearing away heat and fire, cooling blood, removing toxic substances, removing blood stasis, and dredging channels. Treating excess heat constipation, delirium, fever, food stagnation, abdominal fullness, dysentery, tenesmus, stasis, amenorrhea, abdominal mass, epidemic heat, acute conjunctivitis, hematemesis, epistaxis, yang jaundice, edema, stranguria with turbid urine, carbuncle, sore, furuncle, and injury due to fire and dampness.

Andrographis paniculata: bitter taste and cold nature; it enters heart, lung, large intestine and bladder meridians. Has effects in clearing away heat and toxic materials, cooling blood, relieving swelling, and eliminating dampness. Can be used for treating wind-heat common cold, early stage of epidemic febrile disease, cough and asthma due to lung heat, lung abscess with pus discharge, sore throat, damp-heat dysentery, pyretic stranguria with astringency, pruritus due to heat eruption, carbuncle, swelling, sore, and snake and insect bite.

Sweet wormwood herb: bitter and pungent taste and cold nature; it enters liver and gallbladder meridians. Has the effects of clearing deficiency heat, removing bone-steaming, relieving summer-heat, preventing malaria and eliminating jaundice, and can be used for treating yin deficiency, night fever, fever due to yin deficiency, bone-steaming fatigue fever, fever due to summer-heat, malaria fever due to cold and heat, damp-heat jaundice, etc.

Chinese angelica: sweet and pungent taste and warm nature; it enters liver, heart and spleen meridians. Has effects of replenishing blood, promoting blood circulation, regulating menstruation, relieving pain, and loosening bowel to relieve constipation. Can be used for treating blood deficiency, sallow complexion, giddiness, palpitation, menoxenia, amenorrhea, dysmenorrhea, asthenia cold, abdominal pain, constipation due to intestinal dryness, rheumatalgia, traumatic injury, superficial infection, pyocutaneous disease.

Red peony root: bitter taste and slightly cold nature; it enters liver meridian. Has effects of clearing heat, cooling blood, dispelling blood stasis and relieving pain. Can be used for treating heat syndrome of nutrient-blood, macula due to toxic heat, hematemesis and epistaxis, conjunctival congestion, swelling and pain, liver depression, hypochondriac pain, amenorrhea, dysmenorrhea, abdominal pain, traumatic injury, carbuncle, swelling, and sore.

Bupleurum root: pungent and bitter taste, slightly cold nature; it enters liver, gallbladder and lung meridians. Has the functions of harmonizing exterior and interior, soothing liver and invigorating yang. Can be used for treating common cold, fever, malaria, stagnation of qi due to depression of the liver, distending pain of chest and hypochondrium, rectocele, uterine prolapse, and menoxenia.

Dried rehmannia root: sweet and bitter taste and cool in nature; has effects in clearing away heat, promoting salivation, nourishing yin, and nourishing blood. Can be used for treating fever due to yin deficiency, diabetes, hematemesis, epistaxis, metrorrhagia, menoxenia, threatened abortion, and constipation due to yin deficiency.

Scutellaria baicalensis: bitter taste and cold nature; it enters lung, gallbladder, stomach and large intestine meridians. Has effects in clearing away heat, eliminating dampness, purging pathogenic fire, removing toxic materials, stopping bleeding, and preventing miscarriage. It can be used for treating epidemic febrile disease, upper respiratory infection, cough due to lung heat, yellow gallbladder due to damp-heat, pneumonia, dysentery, hemoptysis, conjunctival congestion, threatened abortion, hypertension, carbuncle, furuncle, and sore.

Coptis chinensis: bitter taste and slightly cold nature; it enters heart, stomach, liver and large intestine meridians. Has effects of clearing heat, eliminating dampness, purging pathogenic fire, and removing toxic substance. It can be used for treating damp-heat distention, fullness, emesis, acid regurgitation, dysentery, jaundice, hyperpyrexia, excessive heart-fire, vexation, insomnia, hematemesis, epistaxis, conjunctival congestion, toothache, diabetes, carbuncle, and furuncle.

Phellodendron bark: bitter taste and cold nature; it enters kidney and bladder meridians. Has the effects of clearing heat, eliminating dampness, purging fire, removing steam, removing toxic substance and treating sore. It can be used for treating dysentery due to damp-heat pathogen, jaundice, dark urine, leukorrhagia, pudendal pruritus, pyretic stranguria, pain, tinea pedis, atrophic debility cramped, hectic fever, night sweat, nocturnal emission, pyocutaneous disease, toxic swelling, and eczema.

Gardenia: bitter taste and cold nature; it enters heart, lung and triple energizer meridians. Has effects of purging pathogenic fire, relieving restlessness, clearing heat, promoting diuresis, cooling blood, and removing toxic substance. It can be used for treating vexation due to heat diseases, jaundice due to damp-heat, stranguria with blood, hematemesis, epistaxis, conjunctival congestion, swelling and pain, and pyocutaneous disease due to pathogenic fire.

Purslane: sour in taste, cold in nature; it enters liver and large intestine meridians. Has effects of clearing away heat and toxic materials, cooling blood, stopping bleeding, and relieving dysentery. It is mainly used for treating heat-toxin bloody dysentery, heat-toxin sores and ulcers, metrorrhagia and metrostaxis, and hematochezia.

Radix ophiopogonis: sweet, bitter and slightly cold in nature; it enters stomach, lung and heart meridians. Has effects in nourishing yin, moistening lung, benefiting stomach, promoting fluid production, clearing heart fire, and relieving restlessness, and can be used for treating dry cough due to lung dryness, tuberculosis cough due to yin deficiency, pharyngitis, pharyngalgia, body fluid injury, thirst, internal heat, diabetes, vexation, insomnia, and constipation due to intestinal dryness.

Licorice root: sweet in taste and neutral in nature; it enters spleen, stomach and lung meridians. Has effects in invigorating spleen, moistening lung, removing toxic materials, and regulating functions of the other medicines. It can be used for treating spleen deficiency, anorexia, gastric ulcer, duodenal ulcer, cough, and bronchitis.

The traditional Chinese medicines in the formula are compatible according to the principle of monarch, minister, assistant and guide, and the houttuynia cordata, the gypsum, the isatis root, the honeysuckle and the forsythia in the formula have the effects of clearing away heat and toxic materials, dispelling wind and heat, eliminating stagnation and reducing swelling, and clearing away heat and purging fire, and are monarch medicines together; the rhubarb, the common andrographis herb, the sweet wormwood herb, the Chinese angelica, the red paeony root, the Chinese thorowax root, the dried rehmannia root and the like have the effects of clearing away heat and toxic materials, cooling blood and reducing swelling, clearing away heat and purging the heat and the like, and can strengthen the drug effect of monarch drugs; scutellariae radix, Coptidis rhizoma, cortex Phellodendri, fructus Gardeniae, herba Portulacae, and radix Ophiopogonis as adjuvant drugs for clearing heat, eliminating dampness, purging pathogenic fire, and relieving restlessness; licorice root, radix Glycyrrhizae coordinates the effects of the other drugs in the recipe, and tonifies spleen and qi, acting as a guiding drug. The traditional Chinese medicines are matched with each other, and have the effects of clearing away heat and toxic materials, cooling blood and clearing heat, resisting bacteria and viruses, improving the organism immunity of poultry and enhancing the antiviral ability.

In order to enhance the efficacy of the formula, the traditional Chinese medicine formula is subjected to deep fermentation enzymolysis by adopting a two-step fermentation method of bacterial enzyme synergistic fermentation, corn and soybean meal in the traditional Chinese medicine and the microbial nutrient can be subjected to enzymolysis under the action of a complex enzyme preparation, and cellulase, xylanase, glucanase and mannase are used for carrying out enzymolysis on plant cell walls to release the effective components of the traditional Chinese medicine; the corn starch is converted into dextrin by medium-temperature amylase enzymolysis, and the bean pulp is decomposed into micromolecular soybean peptide by protease, so that nutrition is provided for the quick start fermentation of microorganisms.

The saccharomyces cerevisiae and the clostridium butyricum are prepared in a co-culture mode, the toxic action of oxygen on the clostridium butyricum is solved, the saccharomyces cerevisiae is facultative anaerobe and can grow in the presence of oxygen, and meanwhile, multiple vitamins and amino acids are generated in the growth process to promote the growth of the clostridium butyricum.

In the aerobic fermentation stage, bacillus subtilis and aspergillus niger are adopted for fermentation, and the two aerobic bacteria generate a large amount of enzyme preparations such as amylase, protease, cellulase, pectinase and the like in the fermentation process to further crack the cell walls of the Chinese herbal medicines, and simultaneously consume oxygen in a fermentation substrate, thereby providing favorable conditions for anaerobic fermentation. The mixed bacteria liquid of saccharomyces cerevisiae and clostridium butyricum is added with glucose oxidase in the anaerobic fermentation stage, so that residual oxygen in the fermented material can be further consumed, a fermentation bag is adopted for creating an anaerobic environment to the maximum extent, clostridium butyricum can ferment saccharides in intestinal tracts to generate butyric acid, acetic acid and propionic acid, plant cell walls can be softened, the release of effective components can be promoted, the growth of pathogenic bacteria can be inhibited, and meanwhile, the butyric acid is an energy source of intestinal mucosa and can repair mucosal injury; the clostridium butyricum secreted and produced by clostridium butyricum has a killing effect on pathogenic bacteria.

Detailed Description

In order that the invention may be further understood, the invention will now be described in detail with reference to specific examples.

Example 1

The invention provides an antiviral fermented traditional Chinese medicine for poultry, which comprises the following components in parts by weight: 30 parts of heartleaf houttuynia herb, 30 parts of gypsum, 20 parts of indigowoad root, 20 parts of honeysuckle, 10 parts of weeping forsythia, 10 parts of rhubarb, 10 parts of common andrographis herb, 10 parts of sweet wormwood herb, 10 parts of Chinese angelica, 10 parts of red paeony root, 10 parts of Chinese thorowax root, 10 parts of raw rehmannia root, 7 parts of baical skullcap root, 7 parts of golden thread, 7 parts of amur corktree bark, 5 parts of cape jasmine fruit, 5 parts of purslane, 5 parts of dwarf lilyturf tuber, 10 parts of liquoric root, 3 parts of a complex enzyme preparation, 20 parts.

The compound enzyme preparation comprises the following raw materials in parts by weight: 20 parts of cellulase, 10 parts of xylanase, 10 parts of glucanase, 5 parts of mannase, 10 parts of medium temperature amylase, 5 parts of acid protease and 5 parts of neutral protease.

The microbial nutrient consists of the following components in parts by weight: 50 parts of corn flour, 20 parts of soybean meal, 5 parts of tryptone and 10 parts of yeast extract powder.

The composite inorganic salt is prepared from the following raw materials in parts by weight: 5 parts of sodium metabisulfite, 5 parts of ferric ammonium citrate, 5 parts of sodium chloride, 5 parts of light calcium carbonate, 3 parts of ammonium sulfate, 5 parts of sodium acetate and 5 parts of sodium citrate.

The invention also provides a preparation method of the antiviral fermented traditional Chinese medicine for poultry, which comprises the following steps:

(3) mixing:

a, weighing the crushed traditional Chinese medicine components, the microbial nutrient and the compound components in sequence according to a formula ratio, adding the components into a mixer, and stirring for 2-3 minutes;

b, adding the bacillus subtilis liquid, the aspergillus niger liquid, the complex enzyme preparation and the composite inorganic salt into tap water, uniformly mixing, controlling the material-water ratio to be 1: 0.5-1: 0.8, then pumping the mixed liquid into a mixer through a water pump, and continuously mixing for 3 minutes to obtain a fermented material;

(4) solid state fermentation:

a aerobic stage: stacking the fermented materials into a stack with the height of 1.2 meters and the width of 1.5 meters, turning the stack once every 8 hours, controlling the temperature of a fermentation chamber to be 25-30 ℃, stopping turning the stack after fermenting for 5 days, and continuing fermenting for 12 hours;

b, anaerobic phase: adding glucose oxidase into the mixed bacteria liquid of the saccharomyces cerevisiae and the clostridium butyricum, uniformly mixing, adding the mixture into the materials through a mixer, subpackaging the materials into one-way exhaust valve fermentation bags with the packaging amount of 50kg per bag, and carrying out anaerobic fermentation for 24 hours to obtain the product.

The bacillus subtilis liquid is prepared by the following steps:

s1: strain activation and shake flask seed preparation

Respectively streaking the bacillus subtilis slant seeds on a flat plate, standing and culturing for 12h at 30 ℃ to obtain single colonies, inoculating the single colonies to 500ml of liquid culture medium in a shake flask, filling the liquid in the shake flask with 100ml, and culturing for 16h at 30 ℃ and 220r/min to obtain bacillus subtilis shake flask seed liquid. The plate culture medium comprises the following components: 3g/L of beef extract, 10g/L of peptone, 2g/L of sodium chloride, 1.5g/L of agar, 7.0-7.2 of pH and 30-minute sterilization at 121 ℃; the shake flask culture medium comprises the following components: 3g/L of beef extract, 10g/L of peptone, 3g/L of yeast extract, 2g/L of sodium chloride, 5g/L of glucose, 7.0-7.2 of pH, and sterilizing for 30 minutes at 121 ℃;

s2: first order seed liquid preparation

Inoculating the shake flask seed solution into a seed tank according to the proportion of 1%, controlling the rotation speed at 200rpm and the ventilation ratio at 1:0.8, and culturing at 30 ℃ for 16h to obtain a first-stage seed solution; the first-stage seeding tank culture medium comprises the following components: 20g/L of bean cake powder, 10g/L of corn flour, 5g/L of glucose, 3g/L of sodium chloride, 3g/L of light calcium carbonate, 2g/L of dipotassium phosphate, 2g/L of manganese sulfate, pH 7.2-7.5, and sterilizing for 30-40 minutes at 121 ℃;

s3: preparation of fermentation broth

Inoculating the primary seed liquid into a fermentation tank according to the proportion of 3%, controlling the rotating speed at 150rpm for 0-3 h, and controlling the ventilation ratio to be 1: 0.2-1: 0.3; controlling the ventilation ratio to be 1: 0.5-1: 0.7 within 3-6 h; after 6 hours, controlling the ventilation ratio to be 1: 0.8-1: 1; the fermentation tank culture medium comprises the following components: 30g/L of corn flour, 5g/L of glucose, 35g/L of soybean meal, 3g/L of corn steep liquor dry powder, 3g/L of sodium chloride, 3g/L of light calcium carbonate, 2g/L of dipotassium phosphate, 2g/L of manganese sulfate, pH 7.2-7.5, and sterilizing for 30-40 minutes at 121 ℃;

the aspergillus niger liquid is prepared by the following steps:

s1 strain activation and Aspergillus niger shake flask seed liquid preparation

Selecting an Aspergillus niger slant culture by using an inoculating needle, inoculating the Aspergillus niger slant culture into a liquid culture medium, carrying out oscillation culture at 25 ℃ and 200rpm for 3d, then inoculating the Aspergillus niger slant culture into a next shake flask according to 2%, carrying out repeated culture once to complete strain activation, inoculating the activated bacterium liquid into a shake flask expansion culture medium according to 2% of the proportion, and carrying out oscillation culture at 25 ℃ and 200rpm for 3d to obtain a shake flask seed liquid; the activating and shaking flask seed culture medium comprises the following components: 200g/L of potato, 20g/L of cane sugar, natural pH and sterilization at 121 ℃ for 30 minutes;

preparation of Aspergillus niger seeds by solid fermentation of S2

And (4) preparing the shake flask seed liquid prepared in the step S1 in a solid culture medium by adopting a tray fermentation method, wherein the thickness of the solid culture medium in a tray is controlled to be about 3-5 cm, the culture temperature is 25 ℃, and the culture time is 5 d. The solid medium comprises the following components: 80 parts of bran, 3 parts of soybean meal, 2 parts of ammonium sulfate, 1 part of manganese sulfate, 0.5 part of dipotassium phosphate, 0.5 part of magnesium sulfate and 1:0.6 of feed-water ratio, and sterilizing for 40 minutes at 121 ℃; before fermentation and inoculation, the Aspergillus niger culture is diluted with 0.1% Tween water according to a ratio of 1:10, stirred uniformly, and filtered by four layers of sterile gauze to obtain Aspergillus niger spore suspension for fermenting traditional Chinese medicines.

The saccharomyces cerevisiae and clostridium butyricum mixed bacterial liquid is prepared by the following steps:

s1 bacterial activation and preparation of seed liquid by shaking bottle

Activation of saccharomyces cerevisiae and preparation of shake flask seed liquid: selecting a ring of saccharomyces cerevisiae slant seeds, inoculating the seeds into a liquid activation culture medium, performing shaking culture at 28 ℃ and 200rpm for 48 hours, then inoculating the activation seed liquid into a shaking flask seed liquid according to the proportion of 1%, and performing shaking culture at 28 ℃ and 200rpm for 48 hours, wherein the activation and shaking flask seed culture medium comprises the following components: 10g/L of yeast extract powder, 20g/L of bovine bone peptone, 20g/L of glucose, pH 5.8-6.0, and sterilizing at 121 ℃ for 30 minutes;

activating clostridium butyricum and preparing a shake flask seed solution: picking clostridium monobutyricum into an RCM liquid culture medium, culturing for 12h at 37 ℃ in an anaerobic incubator, and then carrying out passage once according to the proportion of 1% to obtain a shake flask seed liquid, wherein the RCM culture medium comprises the following components: 3g/L of yeast extract, 10g/L of beef extract, 10g/L of peptone, 1g/L of soluble starch, 3g/L of glucose, 0.5g/L of cysteine hydrochloride, 3g/L of sodium chloride, 3g/L of sodium acetate, pH8.5, and sterilizing for 30 minutes at 121 ℃;

s2 first order seed liquid preparation

Preparing first-grade seed liquid of saccharomyces cerevisiae: inoculating the shake flask seeds into a first-stage seeding tank according to the proportion of 1%, wherein the culture temperature is 28 ℃, the rotating speed is 200rpm, the ventilation ratio is 1:0.5, the tank pressure is 0.05Mpa, and the culture time is 36 h; the first-stage seeding tank culture medium comprises the following components: 10g/L of yeast extract powder, 20g/L of bovine bone peptone, 20g/L of glucose, 2g/L of potassium dihydrogen phosphate and pH5.8-6.0;

preparing a clostridium butyricum primary seed solution: inoculating at 1%, temperature 32 deg.C, rotation speed 60rpm, introducing nitrogen gas to form anaerobic environment, and culturing at 0.05Mpa for 12 hr; the culture medium is an RCM culture medium;

preparation of S3 mixed bacterial liquid of saccharomyces cerevisiae and clostridium butyricum

Inoculating saccharomyces cerevisiae into a fermentation tank according to the proportion of 5-10%, culturing at the temperature of 28 ℃, rotating at the speed of 200rpm, and ventilating at the ratio of 1:0.5, culturing for 30h, then stopping ventilation, continuing culturing for 5h, then inoculating clostridium butyricum primary seed liquid according to the proportion of 5%, stirring every half hour, controlling the temperature to be 30 ℃, and culturing for 8 h; the fermentation medium comprises the following components: 5g/L of beef extract powder, 3g/L of yeast extract powder, 40g/L of glucose, 3g/L of sodium chloride, 2g/L of sodium acetate, 2g/L of sodium citrate, 2g/L of magnesium sulfate, 2g/L of dipotassium hydrogen phosphate and 0.5g/L of cysteine hydrochloride, and sterilizing at 121 ℃ for 30 minutes.

The inoculation amount of the bacillus subtilis is 3 percent; the aspergillus niger inoculation amount is 5%;

the inoculation amount of the mixed bacteria liquid of the saccharomyces cerevisiae and the clostridium butyricum is 5 percent; the enzyme activity of the glucose oxidase is 3000IU/g, and the addition amount is 0.1%.

In the compound enzyme preparation, the cellulase is more than or equal to 10000IU/g, the xylanase is more than or equal to 100000IU/g, the glucanase is more than or equal to 50000IU/g, the mannase is more than or equal to 100000IU/g, the mesophilic amylase is more than or equal to 2000IU/g, the acidic protease is more than or equal to 100000IU/g, and the neutral protease is more than or equal to 50000 IU/g.

Example 2

A preparation method of an antiviral fermented traditional Chinese medicine for poultry comprises the following components in parts by weight: 50 parts of heartleaf houttuynia herb, 50 parts of gypsum, 30 parts of indigowoad root, 30 parts of honeysuckle, 20 parts of weeping forsythia, 16 parts of rhubarb, 16 parts of common andrographis herb, 16 parts of sweet wormwood herb, 16 parts of Chinese angelica, 16 parts of red paeony root, 16 parts of Chinese thorowax root, 16 parts of raw rehmannia root, 13 parts of baical skullcap root, 13 parts of golden thread, 13 parts of amur corktree bark, 10 parts of cape jasmine fruit, 10 parts of purslane, 10 parts of dwarf lilyturf tuber, 15 parts of liquoric root, 5 parts of a complex enzyme preparation, 30 parts of.

The compound enzyme preparation comprises the following raw materials in parts by weight: 30 parts of cellulase, 15 parts of xylanase, 15 parts of glucanase, 10 parts of mannase, 15 parts of medium temperature amylase, 10 parts of acid protease and 10 parts of neutral protease.

The microbial nutrient consists of the following components in parts by weight: 80 parts of corn flour, 30 parts of soybean meal, 10 parts of tryptone and 15 parts of yeast extract powder.

The composite inorganic salt is prepared from the following raw materials in parts by weight: 10 parts of sodium metabisulfite, 10 parts of ammonium ferric citrate, 10 parts of sodium chloride, 10 parts of light calcium carbonate, 8 parts of ammonium sulfate, 10 parts of sodium acetate and 10 parts of sodium citrate.

A preparation method of an antiviral fermented traditional Chinese medicine for poultry is characterized by comprising the following steps:

(1) crushing materials: pulverizing the above materials, and sieving with 40 mesh sieve.

(2) Preparing bacterial liquid: respectively preparing bacillus subtilis liquid, aspergillus niger liquid, saccharomyces cerevisiae and clostridium butyricum mixed liquid.

(3) Mixing:

a, weighing the crushed traditional Chinese medicine components, the microbial nutrient and the compound components in sequence according to the formula proportion, starting a mixer to stir, putting into the mixer, and stirring for 2-3 minutes.

b, adding the bacillus subtilis, the aspergillus niger, the complex enzyme preparation and the complex inorganic salt into tap water, uniformly mixing, controlling the material-water ratio to be 1: 0.5-1: 0.8, then pumping the mixed solution into a mixer through a water pump, and continuously mixing for 3 minutes.

(4) Solid state fermentation:

a aerobic stage: and (3) piling the fermented materials into a stack with the height of 1.2 meters and the width of 1.5 meters, turning the stack once every 8 hours, controlling the temperature of a fermentation chamber to be 25-30 ℃, stopping turning the stack after fermenting for 5 days, and continuing fermenting for 12 hours.

b, anaerobic phase: adding glucose oxidase into the mixed bacteria liquid of the saccharomyces cerevisiae and the clostridium butyricum, uniformly mixing, adding the mixture into the materials through a mixer, subpackaging the materials into one-way exhaust valve fermentation bags with the packaging amount of 50kg per bag, and carrying out anaerobic fermentation for 24 hours.

The bacillus subtilis liquid is prepared by the following steps:

s1: strain activation and shake flask seed preparation

Respectively streaking the bacillus subtilis slant seeds on a flat plate, statically culturing for 16h at 37 ℃ to obtain single colonies, inoculating the single colonies to 500ml of liquid culture medium in a shake flask, filling the liquid in the shake flask with 100ml, and culturing for 24h at 37 ℃ and 220r/min to obtain bacillus subtilis shake flask seed liquid. The plate culture medium comprises the following components: 5g/L of beef extract, 15g/L of peptone, 5g/L of sodium chloride, 3g/L of agar, 7.0-7.2 of pH, and sterilizing for 30 minutes at 121 ℃; the shake flask culture medium comprises the following components: 5g/L beef extract, 15g/L peptone, 5g/L yeast extract, 5g/L sodium chloride, 8g/L glucose, pH 7.0-7.2, and sterilizing at 121 ℃ for 30 minutes;

s2: first order seed liquid preparation

Inoculating the shake flask seed solution into a seed tank according to the proportion of 3%, controlling the rotation speed at 250rpm and the ventilation ratio at 1:0.8, and culturing at 37 ℃ for 24h to obtain a first-grade seed solution; the first-stage seeding tank culture medium comprises the following components: 25g/L of bean cake powder, 15g/L of corn flour, 10g/L of glucose, 5g/L of sodium chloride, 5g/L of light calcium carbonate, 5g/L of dipotassium phosphate, 5g/L of manganese sulfate, pH 7.2-7.5, and sterilizing at 121 ℃ for 30-40 minutes;

s3: preparation of fermentation broth

Inoculating the primary seed liquid into a fermentation tank according to the proportion of 5%, controlling the rotating speed at 200rpm for 0-3 h, and controlling the ventilation ratio at 1: 0.3; controlling the ventilation ratio at 1:0.7 within 3-6 h; after 6 hours, controlling the ventilation ratio at 1: 1; the fermentation tank culture medium comprises the following components: 40g/L of corn flour, 10g/L of glucose, 45g/L of soybean meal, 5g/L of corn steep liquor dry powder, 5g/L of sodium chloride, 5g/L of light calcium carbonate, 5g/L of dipotassium phosphate, 5g/L of manganese sulfate, pH 7.2-7.5, and sterilizing for 30-40 minutes at 121 ℃;

the aspergillus niger liquid is prepared by the following steps:

s1 strain activation and Aspergillus niger shake flask seed liquid preparation

Selecting an Aspergillus niger slant culture by using an inoculating needle, inoculating the Aspergillus niger slant culture into a liquid culture medium, carrying out oscillation culture at 28 ℃ and 250rpm for 5d, then inoculating the Aspergillus niger slant culture into a next shake flask according to 5%, carrying out repeated culture once to complete strain activation, inoculating activated bacterium liquid into a shake flask expansion culture medium according to 5% of proportion, carrying out oscillation culture at 28 ℃ and 200-250 rpm for 5d, and obtaining shake flask seed liquid; the activating and shaking flask seed culture medium comprises the following components: 200g/L of potato, 20g/L of cane sugar, natural pH and sterilization at 121 ℃ for 30 minutes;

preparation of Aspergillus niger seeds by solid fermentation of S2

And (4) preparing the shake flask seed liquid prepared in the step S1 in a solid culture medium by adopting a tray fermentation method, wherein the thickness of the solid culture medium in a tray is controlled to be about 3-5 cm, the culture temperature is 28 ℃, and the culture time is 7 d. The solid medium comprises the following components: 90 parts of bran, 6 parts of soybean meal, 3 parts of ammonium sulfate, 2 parts of manganese sulfate, 1 part of dipotassium phosphate and 1 part of magnesium sulfate, wherein the feed-water ratio is 1:0.8, and the mixture is sterilized at 121 ℃ for 40 minutes; before fermentation and inoculation, the Aspergillus niger culture is diluted with 0.1% Tween water according to a ratio of 1:10, stirred uniformly, and filtered by four layers of sterile gauze to obtain Aspergillus niger spore suspension for fermenting traditional Chinese medicines.

s1 bacterial activation and preparation of seed liquid by shaking bottle

Activation of saccharomyces cerevisiae and preparation of shake flask seed liquid: selecting a ring of saccharomyces cerevisiae slant seeds, inoculating the seeds into a liquid activation culture medium, performing shaking culture at 28 ℃ and 200rpm for 72 hours, then inoculating the activation seed liquid into a shaking flask seed liquid according to a proportion of 5%, performing shaking culture at 28 ℃ and 200rpm for 48-72 hours, wherein the activation and shaking flask seed culture medium comprises the following components: 15g/L of yeast extract powder, 25g/L of bovine bone peptone, 25g/L of glucose, pH 5.8-6.0, and sterilizing at 121 ℃ for 30 minutes;

activating clostridium butyricum and preparing a shake flask seed solution: picking clostridium monobutyricum into an RCM liquid culture medium, culturing for 18h at 37 ℃ in an anaerobic incubator, and then carrying out passage once according to a proportion of 5% to obtain a shake flask seed liquid, wherein the RCM culture medium comprises the following components: 5g/L of yeast extract, 15g/L of beef extract, 15g/L of peptone, 3g/L of soluble starch, 8g/L of glucose, 1g/L of cysteine hydrochloride, 5g/L of sodium chloride, 5g/L of sodium acetate, 8.5 of pH, and sterilizing for 30 minutes at 121 ℃;

s2 first order seed liquid preparation

Preparing first-grade seed liquid of saccharomyces cerevisiae: inoculating the shake flask seeds into a first-stage seed tank according to the proportion of 3 percent, wherein the culture temperature is 32 ℃, the rotating speed is 250rpm, the ventilation ratio is 1:0.5, the tank pressure is 0.05-0.08 Mpa, and the culture time is 48 hours; the first-stage seeding tank culture medium comprises the following components: 15g/L of yeast extract powder, 25g/L of bovine bone peptone, 25g/L of glucose, 5g/L of potassium dihydrogen phosphate and pH5.8-6.0;

preparing a clostridium butyricum primary seed solution: the inoculation amount is 2 percent, the temperature is 37 ℃, the rotating speed is 100rpm, nitrogen is introduced in the culture process to form an anaerobic environment, the tank pressure is 0.08Mpa, and the culture time is 16 h; the culture medium is an RCM culture medium;

Inoculating saccharomyces cerevisiae into a fermentation tank according to the proportion of 5-10%, culturing at the temperature of 32 ℃, rotating at the speed of 250rpm, and ventilating at the ratio of 1:0.5, culturing for 30h, then stopping ventilation, continuing culturing for 8h, then inoculating clostridium butyricum primary seed liquid according to the proportion of 10%, stirring every half hour, controlling the temperature to be 35 ℃, and culturing for 12 h; the fermentation medium comprises the following components: 10g/L of beef extract powder, 5g/L of yeast extract powder, 50g/L of glucose, 5g/L of sodium chloride, 5g/L of sodium acetate, 5g/L of sodium citrate, 5g/L of magnesium sulfate, 5g/L of dipotassium hydrogen phosphate and 1g/L of cysteine hydrochloride, and sterilizing for 30 minutes at 121 ℃.

The inoculation amount of the bacillus subtilis is 5 percent; the aspergillus niger inoculation amount is 10%;

the inoculation amount of the mixed bacteria liquid of the saccharomyces cerevisiae and the clostridium butyricum is 10 percent; the enzyme activity of the glucose oxidase is 3000IU/g, and the addition amount is 0.1%.

Example 3

A preparation method of an antiviral fermented traditional Chinese medicine for poultry comprises the following components in parts by weight: 40 parts of heartleaf houttuynia herb, 40 parts of gypsum, 25 parts of indigowoad root, 25 parts of honeysuckle, 15 parts of weeping forsythia, 13 parts of rhubarb, 13 parts of common andrographis herb, 13 parts of sweet wormwood herb, 13 parts of Chinese angelica, 13 parts of red paeony root, 136 parts of Chinese thorowax root, 13 parts of raw rehmannia root, 10 parts of baical skullcap root, 10 parts of golden thread, 10 parts of amur corktree bark, 7 parts of cape jasmine fruit, 7 parts of purslane, 7 parts of dwarf lilyturf tuber, 13 parts of liquoric root, 4 parts of a complex enzyme preparation, 25 parts of.

The compound enzyme preparation comprises the following raw materials in parts by weight: 250 parts of cellulase, 13 parts of xylanase, 13 parts of glucanase, 7 parts of mannase, 13 parts of medium temperature amylase, 7 parts of acid protease and 8 parts of neutral protease.

The microbial nutrient consists of the following components in parts by weight: 65 parts of corn flour, 25 parts of bean pulp, 7.5 parts of tryptone and 12.5 parts of yeast extract powder.

The composite inorganic salt is prepared from the following raw materials in parts by weight: 7.5 parts of sodium metabisulfite, 7.5 parts of ammonium ferric citrate, 7.5 parts of sodium chloride, 7.5 parts of light calcium carbonate, 5.5 parts of ammonium sulfate, 7.5 parts of sodium acetate and 7.5 parts of sodium citrate.

A preparation method of an antiviral fermented traditional Chinese medicine for poultry comprises the following steps:

(3) Mixing:

b, adding the bacillus subtilis, the aspergillus niger, the complex enzyme preparation and the complex inorganic salt into tap water, uniformly mixing, controlling the material-water ratio to be 1:0.7, then pumping the mixed solution into a mixer through a water pump, and continuously mixing for 3 minutes.

(4) Solid state fermentation:

The bacillus subtilis liquid is prepared by the following steps:

s1: strain activation and shake flask seed preparation

Marking the slant seeds of the bacillus subtilis on a flat plate respectively, performing static culture at 33 ℃ for 14h to obtain single colonies, inoculating the single colonies into 500ml of liquid culture medium in a shake flask, filling the liquid in the shake flask with 100ml, and performing culture at 30-37 ℃ at 220r/min for 20h to obtain the shake flask seed liquid of the bacillus subtilis. The plate culture medium comprises the following components: 4g/L of beef extract, 12.5g/L of peptone, 3.5g/L of sodium chloride, 2g/L of agar, 7.0-7.2 of pH, and sterilizing at 121 ℃ for 30 minutes; the shake flask culture medium comprises the following components: 4g/L of beef extract, 12.5g/L of peptone, 4g/L of yeast extract, 3.5g/L of sodium chloride, 6.5g/L of glucose, 7.0-7.2 of pH and 30-minute sterilization at 121 ℃;

s2: first order seed liquid preparation

Inoculating the shake flask seed solution into a seed tank according to the proportion of 2%, controlling the rotation speed at 225rpm, the ventilation ratio at 1:0.8, and culturing at 33 ℃ for 20h to obtain a first-stage seed solution; the first-stage seeding tank culture medium comprises the following components: 22.5g/L of bean cake powder, 12.5g/L of corn flour, 7.5g/L of glucose, 4g/L of sodium chloride, 4g/L of light calcium carbonate, 3.5g/L of dipotassium phosphate, 3.5g/L of manganese sulfate, pH 7.2-7.5, and sterilizing for 30-40 minutes at 121 ℃;

s3: preparation of fermentation broth

The first-stage seed liquid is inoculated into a fermentation tank according to the proportion of 4%, the rotating speed is controlled at 175rpm for 0-3 h, and the ventilation ratio is 1: 0.25; controlling the ventilation ratio at 1:0.6 within 3-6 h; after 6 hours, controlling the ventilation ratio at 1: 0.9; the fermentation tank culture medium comprises the following components: 35g/L of corn flour, 7.5g/L of glucose, 40g/L of soybean meal, 4g/L of corn steep liquor dry powder, 4g/L of sodium chloride, 4g/L of light calcium carbonate, 3.5g/L of dipotassium phosphate, 3.5g/L of manganese sulfate, 7.2-7.5 of pH value, and sterilizing for 30-40 minutes at 121 ℃;

the aspergillus niger liquid is prepared by the following steps:

s1 strain activation and Aspergillus niger shake flask seed liquid preparation

Selecting an Aspergillus niger slant culture by using an inoculating needle, inoculating the Aspergillus niger slant culture into a liquid culture medium, carrying out shake culture at 26 ℃ and 225rpm for 4d, then inoculating the Aspergillus niger slant culture into a next shake flask according to 3.5%, carrying out repeated culture once to complete strain activation, inoculating activated bacterium liquid into a shake flask expansion culture medium according to a proportion of 3.5%, carrying out shake culture at 26 ℃ and 225rpm for 4d to obtain shake flask seed liquid; the activating and shaking flask seed culture medium comprises the following components: 200g/L of potato, 20g/L of cane sugar, natural pH and sterilization at 121 ℃ for 30 minutes;

preparation of Aspergillus niger seeds by solid fermentation of S2

And (4) preparing the shake flask seed liquid prepared in the step S1 in a solid culture medium by adopting a tray fermentation method, wherein the thickness of the solid culture medium in a tray is controlled to be about 3-5 cm, the culture temperature is 26 ℃, and the culture time is 6 d. The solid medium comprises the following components: 85 parts of bran, 4.5 parts of soybean meal, 2.5 parts of ammonium sulfate, 1.5 parts of manganese sulfate, 0.75 part of dipotassium hydrogen phosphate, 0.75 part of magnesium sulfate and a material-water ratio of 1:0.7, and sterilizing for 40 minutes at 121 ℃; before fermentation and inoculation, the Aspergillus niger culture is diluted with 0.1% Tween water according to a ratio of 1:10, stirred uniformly, and filtered by four layers of sterile gauze to obtain Aspergillus niger spore suspension for fermenting traditional Chinese medicines.

s1 bacterial activation and preparation of seed liquid by shaking bottle

Activation of saccharomyces cerevisiae and preparation of shake flask seed liquid: selecting a ring of saccharomyces cerevisiae slant seeds, inoculating the seeds into a liquid activation culture medium, performing shaking culture at 28 ℃ and 200rpm for 60 hours, then inoculating the activation seed liquid into a shaking flask seed liquid according to the proportion of 3%, performing shaking culture at 28 ℃ and 200rpm for 60 hours, wherein the activation and shaking flask seed culture medium comprises the following components: soaking yeast powder at a concentration of 12.5g/L, bovine bone peptone at a concentration of 22.5g/L, glucose at a concentration of 22.5g/L, pH of 5.8-6.0, and sterilizing at 121 ℃ for 30 minutes;

activating clostridium butyricum and preparing a shake flask seed solution: picking clostridium monobutyricum into an RCM liquid culture medium, culturing for 15h at 37 ℃ in an anaerobic incubator, and then carrying out passage once according to the proportion of 3% to obtain a shake flask seed liquid, wherein the RCM culture medium comprises the following components: 4g/L of yeast extract, 12.5g/L of beef extract, 12.5g/L of peptone, 2g/L of soluble starch, 5.5g/L of glucose, 0.75g/L of cysteine hydrochloride, 4g/L of sodium chloride, 4g/L of sodium acetate, 8.5 of pH value and 30-minute sterilization at 121 ℃;

s2 first order seed liquid preparation

Preparing first-grade seed liquid of saccharomyces cerevisiae: inoculating the shake flask seeds into a first-stage seed tank according to the proportion of 1-3%, wherein the culture temperature is 30 ℃, the rotating speed is 225rpm, the ventilation ratio is 1:0.5, the tank pressure is 0.06MPa, and the culture time is 42 h; the first-stage seeding tank culture medium comprises the following components: 12.5g/L of yeast extract powder, 22.5g/L of bovine bone peptone, 22.5g/L of glucose, 3.5g/L of monopotassium phosphate and pH5.8-6.0;

preparing a clostridium butyricum primary seed solution: the inoculation amount is 1.5 percent, the temperature is 35 ℃, the rotating speed is 80rpm, nitrogen is introduced in the culture process to form an anaerobic environment, the tank pressure is 0.06MPa, and the culture time is 14 hours; the culture medium is an RCM culture medium;

Inoculating saccharomyces cerevisiae into a fermentation tank according to the proportion of 5-10%, culturing at the temperature of 30 ℃, rotating at the speed of 225rpm, and ventilating at the ratio of 1:0.5, culturing for 30h, then stopping ventilation, continuing culturing for 6h, then inoculating clostridium butyricum primary seed liquid according to the proportion of 7.5%, stirring every half an hour, controlling the temperature to be 33 ℃, and culturing for 10 h; the fermentation medium comprises the following components: 7.5g/L of beef extract powder, 4g/L of yeast extract powder, 45g/L of glucose, 4g/L of sodium chloride, 3.5g/L of sodium acetate, 3.5g/L of sodium citrate, 3.5g/L of magnesium sulfate, 3.5g/L of dipotassium hydrogen phosphate, 0.75g/L of cysteine hydrochloride and sterilization at 121 ℃ for 30 minutes.

The inoculation amount of the bacillus subtilis is 4 percent; the aspergillus niger inoculation amount is 7.5%;

the inoculation amount of the mixed bacteria liquid of the saccharomyces cerevisiae and the clostridium butyricum is 7.5 percent; the enzyme activity of the glucose oxidase is 3000IU/g, and the addition amount is 0.1%.

Test examples

Research on influence of antiviral fermented traditional Chinese medicine on newcastle disease vaccine

1 materials and methods

1.1 materials

1.1.1 fermenting traditional Chinese medicines: the fermented antiviral traditional Chinese medicine is prepared by the embodiment 2, and the formula is as follows: 50 parts of heartleaf houttuynia herb, 50 parts of gypsum, 30 parts of indigowoad root, 30 parts of honeysuckle, 20 parts of weeping forsythia, 16 parts of rhubarb, 16 parts of common andrographis herb, 16 parts of sweet wormwood herb, 16 parts of Chinese angelica, 16 parts of red paeony root, 16 parts of Chinese thorowax root, 16 parts of raw rehmannia root, 13 parts of baical skullcap root, 13 parts of golden thread, 13 parts of amur corktree bark, 10 parts of cape jasmine fruit, 10 parts of purslane, 10 parts of dwarf lilyturf tuber.

1.1.2 drugs and reagents

The Newcastle disease Lasota live vaccine is produced by Boleway biotechnology, Inc. in Shandong Binszhou, and a Newcastle disease antibody (NDV-Ab) ElISA kit is purchased from Shanghai enzyme-linked biotechnology, Inc.; tetramethylazodicarbonyl blue (MTT), Phytohemagglutinin (PHA), available from sigma corporation; RPM11640 culture solution, Hank's solution, produced by Gibco corporation; lymphocyte separation medium, manufactured by Shanghai Hengxin chemical reagents, Inc.; lymphocyte lysate, dimethyl sulfoxide, produced by Suzhou chemical research institute.

1.2 methods

500 feathers of healthy and uniformly-developed 1-day-old Hailan brown laying hens are randomly selected and divided into 5 groups, wherein the 1 st group is a blank control group, the 2 nd group is a vaccine control group, and the 3 rd to 5 th groups are fermented traditional Chinese medicines with different addition amounts respectively. The 3 test groups and the vaccine control group are subjected to primary immunization and secondary immunization at the 7 th day and the 28 th day respectively, Newcastle disease live vaccine eye-drop nose-drop immunization is adopted, the 3 test groups are fed with fermented traditional Chinese medicines in a material mixing mode, the addition amounts of the fermented traditional Chinese medicines are 0.5%, 1% and 1.5%, and the blank control group and the vaccine control group are not fed with the fermented traditional Chinese medicines respectively. Randomly selecting 8 chickens from each group at 21, 35, 42 and 56 days of age, collecting two parts of peripheral venous blood, separating serum from one part, adding sodium citrate anticoagulant into the other part, and storing at-20 deg.C.

1.2.1 determination of Newcastle disease antibody content

According to the specification of the ELISA kit for the Newcastle disease antibody, a standard curve is established by using a standard substance in the kit, the light absorption value of a sample is measured at 450nm, and the antibody content of each sample is calculated through a regression equation. 1.2.2 determination of Chicken T lymphocytes

Diluting anticoagulant blood sample with Hank's solution by 1 time by MTT method, carefully adding into upper layer of lymphocyte separation liquid, centrifuging at 2000r/min for 20min, collecting middle cloudy leucocyte layer, washing with serum-free RPM11640 nutrient solution for 2 times, and centrifuging at 1500 RPM for 15 min. Trypan blue staining, after the cell count is more than 90%, the cell concentration is adjusted to 2.5 × 10 by RPM11640 nutrient solution⁶One/ml of the suspension was added to a 96-well cell culture plate, 80. mu.L/well, followed by 20. mu.L/well of pHA, and each sample was plated in 4-well format at 39.5 ℃ with 5% CO₂Culturing for 44h in incubator, taking out, adding MTT solution 20 μ L/well, culturing for 4h, adding lysate 100 μ L/well, shaking for 5min, and measuring absorbance (A) at 570nm₅₇₀) As an index of proliferation of peripheral blood T lymphocytes.

1.3 data processing

Adopting SAS9.0 statistical software to measure the content of newcastle disease antibody and chicken T lymphocyte A₅₇₀The data were analyzed, the changes of the data at different ages of day were compared, the results were expressed as "mean ± standard deviation", and the significance analysis was performed using anova analysis of variance and Duncan multiple comparisons.

2 results and analysis

2.1 Effect of antiviral fermented traditional Chinese medicine on Newcastle disease antibody content

As can be seen from Table 1, at 21 days of age, the content of the Newcastle disease antibody in the group with 1% of the addition amount of the antiviral fermentation traditional Chinese medicine is significantly higher than that in other groups; at 35 and 42 days of age, the content of Newcastle disease antibody in the fermented traditional Chinese medicine addition group and the vaccine control group is obviously higher than that in a blank control (P is less than 0.05), and the content in the 1% addition group is obviously higher than that in the vaccine control group, 0.5% addition group and 1.5% addition group; at the age of 56 days, the content of the antibody in the 1% adding amount group is obviously higher than that in the other groups (P < 0.05); the results show that the newcastle disease antibody content and the duration of the newcastle disease group with 1% of the addition amount have better performances.

TABLE 1 Change in Newcastle disease antibody content (pg/ml)

Note: the letters shown after the same column of data indicate significant differences (P <0.05), and the letters shown are the same indicating insignificant differences (P > 0.05).

2.2 Effect of antiviral fermented traditional Chinese medicine on T lymphocyte proliferation

As can be seen from Table 2, the T lymphocyte content of the 1% antiviral fermentation traditional Chinese medicine adding amount group is significantly higher than that of the other groups (P < 0.05); at 35 days of age, 42 days of age and 56 days of age, the T lymphocyte content of the 1% adding amount group is obviously higher than that of the rest groups (P <0.05), the T lymphocyte content difference of the 0.5% adding amount group and the 1% adding amount group is not significant (P >0.05) but is obviously higher than that of the vaccine control group and the blank control group (P <0.05), and the T lymphocyte content of the vaccine control group is obviously higher than that of the blank control group (P < 0.05).

TABLE 2 Change in T lymphocyte proliferation (A)₅₇₀)

3 conclusion

The research result shows that the antiviral fermented traditional Chinese medicine added into the chicken daily ration can improve the newcastle disease antibody content of the chicken and promote the proliferation of T lymphocytes, and the antiviral effect is optimal when the addition amount is 1%.

It will be understood by those skilled in the art that the antiviral fermented traditional Chinese medicine for poultry and the preparation method thereof of the present invention include the summary of the invention and the detailed description of the present specification, which are limited to the space and are not described one by one for the sake of brevity of the specification. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An antiviral fermented traditional Chinese medicine for poultry is characterized by comprising the following raw materials in parts by weight: 30-50 parts of houttuynia cordata, 30-50 parts of gypsum, 20-30 parts of isatis root, 20-30 parts of honeysuckle, 10-20 parts of fructus forsythiae, 10-16 parts of rheum officinale, 10-16 parts of common andrographis herb, 10-16 parts of sweet wormwood herb, 10-16 parts of Chinese angelica, 10-16 parts of red paeony root, 10-16 parts of radix bupleuri, 10-16 parts of radix rehmanniae recen, 7-13 parts of scutellaria baicalensis, 7-13 parts of coptis chinensis, 7-13 parts of golden cypress, 5-10 parts of cape jasmine, 5-10 parts of purslane, 5-10 parts of radix ophiopogonis, 10-15 parts of liquorice, 3-5 parts of a complex enzyme preparation, 20-30 parts of a microbial nutrient and 3-5 parts of a complex inorganic salt.

2. The antiviral fermented traditional Chinese medicine for poultry according to claim 1, wherein the complex enzyme preparation is composed of the following raw materials in parts by weight: 20-30 parts of cellulase, 10-15 parts of xylanase, 10-15 parts of glucanase, 5-10 parts of mannase, 10-15 parts of medium temperature amylase, 5-10 parts of acid protease and 5-10 parts of neutral protease.

3. The antiviral fermented traditional Chinese medicine for poultry according to claim 2, characterized in that in the complex enzyme preparation, cellulase is more than or equal to 10000IU/g, xylanase is more than or equal to 100000IU/g, glucanase is more than or equal to 50000IU/g, mannanase is more than or equal to 100000IU/g, mesophilic amylase is more than or equal to 2000IU/g, acidic protease is more than or equal to 100000IU/g, and neutral protease is more than or equal to 50000 IU/g.

4. The antiviral fermented traditional Chinese medicine for poultry according to claim 1, wherein the microbial nutrient is composed of the following components in parts by weight: 50-80 parts of corn flour, 20-30 parts of soybean meal, 5-10 parts of tryptone and 10-15 parts of yeast extract powder.

5. The antiviral fermented traditional Chinese medicine for poultry according to claim 1, wherein the compound inorganic salt is composed of the following raw materials in parts by weight: 5-10 parts of sodium metabisulfite, 5-10 parts of ferric ammonium citrate, 5-10 parts of sodium chloride, 5-10 parts of light calcium carbonate, 3-8 parts of ammonium sulfate, 5-10 parts of sodium acetate and 5-10 parts of sodium citrate.

6. A preparation method of an antiviral fermented traditional Chinese medicine for poultry comprises the following steps in sequence:

(4) solid state fermentation:

7. The method for preparing an antiviral fermented traditional Chinese medicine for poultry according to claim 6, wherein the Bacillus subtilis liquid is prepared by the following steps:

s1: strain activation and shake flask seed preparation

s2: first order seed liquid preparation

s3: preparation of fermentation broth

8. The method for preparing an antiviral fermented traditional Chinese medicine for poultry according to claim 6, wherein the Aspergillus niger liquid is prepared by the following steps:

s1: strain activation and Aspergillus niger shake flask seed liquid preparation

s2: preparation of aspergillus niger seeds by solid fermentation

9. The method for preparing an antiviral fermented traditional Chinese medicine for poultry according to claim 6, wherein the mixed bacterial liquid of saccharomyces cerevisiae and clostridium butyricum is prepared by the following steps:

s1: strain activation and shake flask seed liquid preparation

s2: first order seed liquid preparation

10. The method for preparing an antiviral fermented traditional Chinese medicine for poultry according to claim 6, characterized in that the inoculation amount of Bacillus subtilis is 3-5%; the aspergillus niger inoculation amount is 5-10%; the inoculation amount of the mixed bacterial liquid of the saccharomyces cerevisiae and the clostridium butyricum is 5-10%; the enzyme activity of the glucose oxidase is 3000IU/g, and the addition amount is 0.1%.