CN110787247B

CN110787247B - Traditional Chinese medicine microecological preparation for preventing and treating livestock and poultry diarrhea and preparation method thereof

Info

Publication number: CN110787247B
Application number: CN201911136334.0A
Authority: CN
Inventors: 江国托; 刘艳; 顾艳丽; 单春乔; 刘秋晨; 王效禹; 林洋; 曹艳子
Original assignee: Jiangsu Sanyi Bioengineering Co ltd; Dalian Sanyi Animal Drug Co ltd
Current assignee: Jiangsu Sanyi Bioengineering Co ltd; Dalian Sanyi Animal Drug Co ltd
Priority date: 2019-11-19
Filing date: 2019-11-19
Publication date: 2021-06-01
Anticipated expiration: 2039-11-19
Also published as: CN110787247A

Abstract

The invention provides a preparation method of a traditional Chinese medicine microecological preparation for preventing and treating livestock and poultry diarrhea, which comprises the following steps: inoculating seed liquid into the culture medium for fermentation; the culture medium contains a compound traditional Chinese medicine extracting solution and a matrix culture medium, and the seed solution contains a traditional Chinese medicine fermentation strain; the invention also provides a traditional Chinese medicine microecological preparation for preventing and treating livestock and poultry diarrhea and a bacillus coagulans CCTCC NO: m2019722. The traditional Chinese medicine microecological preparation is mainly used for preventing and treating livestock and poultry diarrhea, has an exact curative effect, not only greatly reduces the use amount of traditional Chinese medicines and saves the management cost of farmers through the fermentation of microorganisms, but also has good prevention and treatment effects.

Description

Traditional Chinese medicine microecological preparation for preventing and treating livestock and poultry diarrhea and preparation method thereof

Technical Field

The application relates to the field of animal husbandry and biotechnology, in particular to a traditional Chinese medicine microecological preparation for preventing and treating livestock and poultry diarrhea and a preparation method thereof.

Background

The national ministry of agriculture publishes that the drug feed additive will be completely withdrawn in 2020 in the notification about the development of veterinary antibacterial use reduction action test point work in 2018 and 2021, which means that the feed is 'nonreactive in the whole course', and the importance of provenance purification and livestock immunity improvement is further highlighted. Meanwhile, due to the ubiquitous existence of immune diseases, the humoral immunity and cellular immunity of the immunization using the vaccine are seriously damaged, so that the protective effect of the vaccine is reduced.

The main pathogenic bacteria of the livestock and poultry diarrhea are escherichia coli and salmonella. The colibacillosis of pigs, pullorum disease, avian colibacillosis and the like caused by the colibacillosis are serious diseases which harm the breeding industry at present, cause serious economic loss for the intensive chicken raising production, and effectively prevent and treat the colibacillosis becomes one of the important problems which are urgently needed to be solved by the intensive chicken raising industry. Pullorum disease and fowl typhoid disease caused by salmonella seriously affect chicken flocks in poultry industry, and the whole chicken raising period is damaged due to intensive feeding.

At present, the treatment of colibacillosis by antibiotics leads to the continuous expansion of the drug resistance spectrum of colibacillosis and the reduction of the prevention effect, and meanwhile, the residue of antibacterial drugs in organisms is easy to cause flora imbalance, thereby not only reducing the quality of meat and egg products, but also influencing the health of human beings and the public health and safety. The traditional Chinese medicine compound has an unobvious effect on improving immunity and a non-timely effect on treating emergencies due to slow absorption of the medicine effect. Therefore, for the prevention and treatment of livestock and poultry diarrhea, a preparation capable of effectively inhibiting pathogenic bacteria and replacing antibiotics is urgently needed to be found.

Disclosure of Invention

The invention aims to provide a preparation which can effectively inhibit pathogenic bacteria of livestock and poultry diarrhea and can replace antibiotics and a preparation method thereof.

In order to achieve the above object, the present disclosure provides a preparation method of a traditional Chinese medicine microecological preparation for preventing and treating livestock and poultry diarrhea, the preparation method comprising: inoculating seed liquid into the culture medium for fermentation; the culture medium contains a compound traditional Chinese medicine extracting solution and a matrix culture medium, and the seed solution contains a traditional Chinese medicine fermentation strain;

wherein the compound traditional Chinese medicine comprises herba Andrographitis, radix Pulsatillae, Coptidis rhizoma, cortex Phellodendri, radix aucklandiae, herba Patriniae and Glycyrrhrizae radix; the traditional Chinese medicine fermentation strain comprises the following components with the preservation number of CCTCC NO: m2019722 Bacillus coagulans (Bacillus coagulans), the preservation number is CGMCC NO: 2196 Enterococcus faecalis (Enterococcus faecalis), having a commercial product number of CICC NO: 10020 Bacillus subtilis (Bacillus subtilis) with a commercial product number of CICC NO: 31170 Candida utilis (Candida utilis), having a commercial product number of ACCC NO: 20039 Lactobacillus plantarum (Lactobacillus plantarum), having a commercial product number CICC NO: 24498 Clostridium butyricum (Clostridium butyricum) and having the commercial product number ACCC NO: 10649 of Rhodopseudomonas palustris (Rhodopseudomonas palustris).

Optionally, the extract of the compound traditional Chinese medicine is prepared by the following operations: drying and crushing the compound traditional Chinese medicine, extracting for 50-70 min by using 10-20 times of ethanol aqueous solution to obtain an ethanol extract of the compound traditional Chinese medicine, and then concentrating the extract to obtain a concentrated material; the ethanol aqueous solution contains 65-85 vol% of ethanol; the concentrated material is a liquid medicine containing 0.5-2g/ml crude drug.

Optionally, the fermentation is high density liquid submerged fermentation; the fermentation temperature is 30-37 ℃, and the fermentation time is 2-5 days.

Optionally, the compound traditional Chinese medicine contains the following components in parts by weight: 10-25 parts of common andrographis herb, 5-25 parts of Chinese pulsatilla root, 4-12 parts of coptis chinensis, 5-20 parts of amur corktree bark, 4-12 parts of costustoot, 4-20 parts of dahurian patrinia herb and 4-12 parts of liquorice.

Optionally, the compound traditional Chinese medicine contains the following components in parts by weight: 15-20 parts of common andrographis herb, 10-20 parts of Chinese pulsatilla root, 6-10 parts of coptis chinensis, 10-15 parts of amur corktree bark, 6-10 parts of costus root, 10-18 parts of dahurian patrinia herb and 5-10 parts of liquorice

Optionally, the number ratio of viable bacteria of the bacillus coagulans, the enterococcus faecalis, the bacillus subtilis, the candida utilis, the lactobacillus plantarum, the clostridium butyricum and the rhodopseudomonas palustris in the seed solution is (10-25): (5-15): (10-25): (10-25): (10-20): (5-15): (5-15); preferably (16-20): (10-13): (16-20): (16-20): (12-15): (10-13): (9-12); the seed solution is prepared by the following operation method: respectively culturing the bacillus coagulans, the enterococcus faecalis, the bacillus subtilis, the candida utilis, the lactobacillus plantarum, the clostridium butyricum and the rhodopseudomonas palustris to obtain culture solutions, and then mixing the culture solutions.

Optionally, the culture medium contains 5-20 volume (%) of extractive solution of FUFANGZHONGYAO and the rest of the matrix culture medium; the substrate medium contains: 10-50 g/L of brown sugar, 10-50 g/L of glucose, 10-50 g/L of honey, 1-5 g/L of peptone, 1-5 g/L of dipotassium phosphate, 1-5 g/L of sodium acetate, 0.1-1 g/L, L-cysteine, 0.1-1 g/L of magnesium sulfate, 0.05-0.3/L of manganese sulfate and 0.1-3 g/L of calcium carbonate; the pH value of the substrate culture medium is 6.0-8.0.

On the other hand, the present disclosure provides a traditional Chinese medicine microecological preparation for preventing and treating livestock and poultry diarrhea, which is prepared according to the preparation method; the dosage form of the traditional Chinese medicine microecological preparation is one of powder, granules, powder and solution.

Optionally, the total number of viable bacteria is (5-10) x 10 relative to 1g of the traditional Chinese medicine microecological preparation or 1mL of the traditional Chinese medicine microecological preparation⁸cfu。

In still another aspect, the Bacillus coagulans is classified and named Bacillus coagulans, and the Bacillus coagulans has a preservation number of CCTCC NO: m2019722.

According to the technical scheme, the traditional Chinese medicine microecological preparation is mainly used for preventing and treating livestock and poultry diarrhea, is definite in curative effect, not only greatly reduces the using amount of traditional Chinese medicines and saves the management cost of farmers through microbial fermentation, but also has the curative effect equivalent to that of antibiotics, can replace antibiotics to treat bacterial diarrhea, and has the capability of improving the immunity of animals. The invention also provides a bacillus coagulans strain, which has good stress resistance such as acid resistance, cholate resistance and the like, can also produce a large amount of lactic acid to assist and improve the inhibition effect of traditional Chinese medicines on pathogenic bacteria, grows well in a liquid fermentation culture medium with high medicine content, and provides a valuable strain for fermenting traditional Chinese medicines.

Additional features and advantages of the disclosure will be set forth in the detailed description which follows.

Biological material preservation information

The Bacillus coagulans is classified and named as Bacillus coagulans SAM-NJH-1Bacillus coagulans SAM-NJH-1 and is preserved in China center for type culture collection with the preservation addresses as follows: the preservation date of Wuhan university in Wuhan city of Hubei province is 2019, 9 and 16 days, and the strain preservation number is as follows: CCTCC NO: m2019722.

Drawings

FIG. 1 shows the colony morphology and microscopic examination of Bacillus coagulans (Bacillus coagulans).

Detailed Description

The following detailed description of specific embodiments of the present disclosure is provided in connection with the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

The invention discloses a preparation method of a traditional Chinese medicine microecological preparation for preventing and treating livestock and poultry diarrhea, which comprises the following steps: inoculating seed liquid into the culture medium for fermentation; the culture medium contains a compound traditional Chinese medicine extracting solution and a matrix culture medium, and the seed solution contains a traditional Chinese medicine fermentation strain;

Optionally, the preparation method of the traditional Chinese medicine microecological preparation specifically comprises the following steps:

the extracting solution of the compound traditional Chinese medicine is prepared by the following operations: drying and crushing the compound traditional Chinese medicine, extracting for 50-70 min by using 10-20 times of ethanol water solution to obtain an ethanol extract of the compound traditional Chinese medicine, and concentrating the extract to obtain a concentrated material. The ethanol aqueous solution contains 65-85 vol% of ethanol; the concentrated material is a liquid medicine containing 0.5-2g/ml crude drug.

According to the theory of Chinese veterinarian, livestock and poultry diarrhea mostly belongs to damp-heat diarrhea, and generally caused by exogenous summer-heat and dampness, pathogenic factors of epidemic toxin and dampness-heat invasion, gastrointestinal transport and transformation disorder, blood stasis and qi stagnation and damp-heat diarrhea. The invention discloses a compound traditional Chinese medicine prepared by screening several traditional Chinese medicines with strong bacteriostatic ability to pathogenic escherichia coli, salmonella and staphylococcus aureus from a single traditional Chinese medicine with bacteriostatic function.

The specific screening method of the compound traditional Chinese medicine comprises the following steps: preparing different compound traditional Chinese medicines from single traditional Chinese medicine with bacteriostatic function according to different combination proportions, extracting each composition, concentrating under reduced pressure to 1g/mL, and pouring 15mL nutrient agar solid culture medium (the components are 10g of peptone, 5g of beef extract, 5g of NaCl, 18g of agar, 1000mL of distilled water and pH7.0) into a sterilized plate. Mixing overnight-cultured pathogenic Escherichia coli, Salmonella, and Staphylococcus aureus solution with nutrient agar medium (containing 1.5% agar, w/v), and diluting to 2 × 10⁷About CFU/ml, 5ml was poured into prepared nutrient agar plates. Placing the Oxford cup on a flat plate with tweezers gently, adding 200 μ l of the medicinal liquid into the Oxford cup, standing for 30min, and culturing at 37 deg.CCulturing for 16h, and measuring the size of the zone of inhibition by using a vernier caliper.

By the screening method, the compound traditional Chinese medicine which is finally screened and has good inhibition effect on pathogenic escherichia coli, salmonella and staphylococcus aureus is prepared from the following components in parts by weight: 10-25 parts of common andrographis herb, 5-25 parts of Chinese pulsatilla root, 4-12 parts of coptis chinensis, 5-20 parts of amur corktree bark, 4-12 parts of costustoot, 4-20 parts of dahurian patrinia herb and 4-12 parts of liquorice; preferably 15-20 parts of common andrographis herb, 10-20 parts of Chinese pulsatilla root, 6-10 parts of coptis chinensis, 10-15 parts of amur corktree bark, 6-10 parts of costus root, 10-18 parts of dahurian patrinia herb and 5-10 parts of liquorice. The combination of the medicines has the effects of clearing away heat and toxic materials, eliminating dampness and stopping dysentery, and promoting qi circulation and removing food stagnation. In the formula, the common andrographis herb and the Chinese pulsatilla root are monarch drugs, and the common andrographis herb enters lung, stomach, large intestine and bladder channels, has the effects of clearing away heat and toxic materials, cooling blood and relieving swelling, and has an inhibiting effect on escherichia coli; the pulsatilla chinensis can clear away heat and toxic materials, cool blood and stop dysentery, and the active ingredients of pulsatilla chinensis saponin and coumarin have good antibacterial effects on pseudomonas aeruginosa, staphylococcus aureus, paratyphoid bacillus and escherichia coli; the composition takes the bitter of coptis root, clears away heat and toxic materials, eliminates dampness and thickens intestines, and the phellodendron bark purges the damp-heat, so as to play the effect of eliminating dampness and stopping dysentery together, the coptis root and the phellodendron bark are ministerial drugs, and the berberine which is the effective component of the coptis root and the phellodendron bark has good physiological activities of antibiosis, anti-inflammation, antivirus and the like; in the formula, the patrinia enters stomach, large intestine and liver channels, clears away heat and toxic materials, and has good effect on intestinal yellow dysentery; the compound Chinese medicine has the effects of promoting qi circulation, removing food stagnation, invigorating spleen and promoting digestion; the liquorice is used for harmonizing the effects of the other drugs, has the effect of detoxifying and can relieve the symptoms.

The inventor selects a plurality of microorganisms with strong enzyme production capacity, strong acid production capacity and certain bacteriostasis, such as lactic acid bacteria, cellulose production and saccharomycetes and the like as target strains to be respectively inoculated in a liquid culture medium added with 5-20% of compound traditional Chinese medicine extract for culture, performs primary screening by taking the growth condition of the strains in the liquid fermentation culture medium containing traditional Chinese medicine extract as an index, and selects 3 strains of bacillus, 3 strains of lactic acid bacteria, 2 strains of candida utilis and 1 strain of photosynthetic bacteria as target strains.

And (3) carrying out a co-culture experiment on the preliminarily screened 9 strains, and investigating the interaction relationship. Selecting 7 strains of bacteria with reciprocal symbiosis or unrelated symbiotic relationship among strains as traditional Chinese medicine fermentation strains, wherein the strains are respectively as follows: the preservation number is CCTCC NO: bacillus coagulans (Bacillus coaguluns) of M2019722 (described herein), with a preservation number of CGMCC NO: 2196 Enterococcus faecalis (Enterococcus faecalis) (published by CN 101220342B), having a commercial product number of cic NO: 10020 Bacillus subtilis (from cic), with the commercial number cic NO: 31170 Candida utilis (available from CICC), having the commercial product ACCC NO: 20039 Lactobacillus plantarum (purchased from ACCC), accession number CICC NO: 24498 Clostridium butyricum (from CICC) and having the commercial product number ACCC NO: 10649 Rhodopseudomonas palustris (Rhodosedoudomoulus padustris) (available from ACCC).

Wherein the viable bacteria number ratio of the bacillus coagulans, the enterococcus faecalis, the bacillus subtilis, the candida utilis, the lactobacillus plantarum, the clostridium butyricum and the rhodopseudomonas palustris in the seed liquid is (10-25): (5-15): (10-25): (10-25): (10-20): (5-15): (5-15); preferably (16-20): (10-13): (16-20): (16-20): (12-15): (10-13): (9-12); the seed solution is prepared by the following operation method: respectively culturing the bacillus coagulans, the enterococcus faecalis, the bacillus subtilis, the candida utilis, the lactobacillus plantarum, the clostridium butyricum and the rhodopseudomonas palustris to obtain culture solutions, and then mixing the culture solutions.

Optionally, the culture medium contains 5-20 vol% of compound Chinese medicinal extractive solution and the rest of the matrix culture medium; the substrate medium contains: 10-50 g/L of brown sugar, 10-50 g/L of glucose, 10-50 g/L of honey, 1-5 g/L of peptone, 1-5 g/L of dipotassium phosphate, 1-5 g/L of sodium acetate, 0.1-1 g/L, L-cysteine, 0.1-1 g/L of magnesium sulfate, 0.05-0.3/L of manganese sulfate and 0.1-3 g/L of calcium carbonate; the pH value of the substrate culture medium is 6.0-8.0.

On the other hand, the disclosure also provides a traditional Chinese medicine microecological preparation for preventing and treating livestock and poultry diarrhea, and the dosage form of the traditional Chinese medicine microecological preparation is one of powder, granules, powder and solution.

The bacillus coagulans, the enterococcus faecalis, the lactobacillus plantarum and the clostridium butyricum have the functions of reducing the pH value of an intestinal tract and inhibiting the growth of putrefying bacteria, and the generated bacteriocin inhibits or kills pathogenic bacteria in the intestinal tract and maintains the flora balance; the enzymes generated by the bacillus and the microzyme in the feed additive can enhance the activity of animal digestive enzyme and improve the utilization rate of the feed, can antagonize pathogenic bacteria of animal organisms, enhances the immunity and disease resistance of the organisms, and plays a positive role in preventing and treating digestive system diseases. The Chinese herbal medicine compound extract is fermented by mixing various strains, the cell wall breaking of the Chinese herbal medicine is carried out by cellulose, hemicellulase, pectinase and other substances generated in the fermentation process to dissolve effective substances, meanwhile, the traditional Chinese medicine components are converted by microorganisms, macromolecular substances are converted into easily absorbed micromolecular substances, the effective components in blood quickly reach effective concentration, the fermented Chinese herbal medicine compound directly plays a role after entering an animal body, the decomposition and conversion process does not exist, and the slow-acting method of the Chinese herbal medicine is changed.

In still another aspect, the present disclosure further provides a Bacillus coagulans, the classification name of the Bacillus coagulans is Bacillus coagulans, and the preservation number of the Bacillus coagulans is CCTCC NO: m2019722. Lactic acid generated by the metabolism of the strain shows strong inhibition capability on escherichia coli, salmonella and staphylococcus aureus.

Bacillus coagulans CCTCC NO: the morphological characteristics of M2019722 (fig. 1) were: the bacterial colony is flat and yellowish, and the two ends of the bacterial colony are slightly stretched; gram-positive; the spore is grown and elliptic in microscopic examination, and the thallus is thin rod-shaped.

For bacillus coagulans CCTCC NO: 16S rDNA amplification and sequencing are carried out on the genome of M2019722, the homology of the strain and 10 kinds of Bacillus coagulans is found to be more than 99.65%, the strain is identified as Bacillus coagulans, and the Bacillus coagulans is preserved to China center for type culture Collection with the preservation number as follows: CCTCC NO: m2019722.

The present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited to the following examples.

The nutrient agar solid medium components used in the examples included: 10.0g/L of peptone, 5.0g/L, NaCl 5.0.0 g/L of beef extract, 18g/L of agar and pH 7.0.

The LB medium comprises the following components: tryptone 10.0g/L, yeast extract 5.0g/L, NaCl10.0g/L.

The MRS culture medium comprises the following components: casein peptone 10.0g/L, beef extract 10.0g/L, yeast powder 5.0g/L, glucose 5.0g/L, sodium acetate 5.0g/L, diammonium citrate 2.0g/L, Tween 801.0 g/L, K₂HPO₄ 2.0g/L，MgSO₄.7H₂O 0.2g/L，MnSO₄.H₂O 0.05g/L，CaCO₃ 20.0g/L，pH6.8。

The wort medium comprises the following components: 5 ° Bee wort 1.0L, natural pH.

The TSA medium components include: tryptone 15.0g, soytone 5.0g, sodium chloride 5.0g, pH7.3 + -0.2.

Example 1

The components of the culture medium used in the embodiment comprise 20 volume percent of compound traditional Chinese medicine extracting solution and the balance of a substrate culture medium; wherein the matrix medium comprises: 30g/L of brown sugar, 30g/L of glucose, 30g/L of honey, 3g/L of peptone, 3g/L of dipotassium phosphate, 3g/L of sodium acetate, 0.5g/L, L g/L of magnesium sulfate-0.5 g/L of cysteine, 0.15 g/L of manganese sulfate, 0.20g/L of calcium carbonate and pH of 6.0-8.0.

The compound traditional Chinese medicine comprises: 18 parts of common andrographis herb, 15 parts of Chinese pulsatilla root, 8 parts of coptis root, 12 parts of amur corktree bark, 8 parts of costustoot, 15 parts of dahurian patrinia herb and 6 parts of liquoric root. Drying the compound traditional Chinese medicine at 55 deg.C for 2.5h, pulverizing, adding 75% ethanol water solution according to volume ratio of 1:15, extracting for 2 times, each time for 1h, standing the Chinese medicine raw material extract at 4 deg.C for 24h, and concentrating to obtain medicinal liquid containing crude drug amount of 1 g/ml. Obtaining the compound traditional Chinese medicine extracting solution.

The preservation number is CCTCC NO: m2019722 Bacillus coagulans (Bacillus coagulans), the preservation number is CGMCC NO: 2196 Enterococcus faecalis (Enterococcus faecalis), having a commercial product number of CICC NO: 10020 Bacillus subtilis with a commercial product number of CICC NO: 31170 Candida utilis (Saccharomyces cerevisiae), having a commercial product number of ACCC NO: 20039 Lactobacillus plantarum (Lactobacillus plantarum), having a commercial product number CICC NO: 24498 Clostridium butyricum (Clostridium butyricum) and having the commercial product number ACCC NO: 10649 enlarged culturing of Rhodopseudomonas palustris (Rhodopseudomonas palustris) in LB medium, MRS medium, Candida utilis medium, TSA medium, etc. to obtain the final product with viable count greater than 15 × 10⁸cfu/mL, and then according to the ratio of the number of viable bacteria of the bacillus coagulans, the enterococcus faecalis, the bacillus subtilis, the candida utilis, the lactobacillus plantarum, the clostridium butyricum and the rhodopseudomonas palustris being 2: 1: 2: 2: 1.5: 1:1 to obtain seed liquid, inoculating the seed liquid into a culture medium for fermentation, and culturing at 37 ℃ for 3 days to ensure that the effective viable count in the fermentation liquid reaches 5.0 multiplied by 10⁸cfu/mL to obtain the traditional Chinese medicine microecological preparation for preventing and treating livestock and poultry diarrhea, and the preparation form is solution.

Comparative example 1

The comparative example prepares the traditional Chinese medicine micro-ecological preparation according to the method of example 1, except that the preservation number is CCTCC NO: the Bacillus coagulans (Bacillus coaguluns) of M2019722 was replaced by Bacillus coagulans cic NO: 10069.

comparative example 2

The comparative example prepares the traditional Chinese medicine micro-ecological preparation according to the method of example 1, except that the preservation number is CCTCC NO: bacillus coagulans (Bacillus coagulons) from M2019722 was replaced by Bacillus coagulans powder (commercially available) purchased from Weifang Yihao biology.

Comparative example 3

Mixing 18 parts of common andrographis herb, 15 parts of Chinese pulsatilla root, 8 parts of coptis root, 12 parts of amur corktree bark, 8 parts of costustoot, 15 parts of dahurian patrinia herb and 6 parts of liquorice to obtain the compound traditional Chinese medicine. Drying the compound traditional Chinese medicine at 55 ℃ for 2.5h, crushing, adding 75% ethanol water solution according to the volume ratio of 1:15 for extraction, extracting for 2 times, 1h each time, standing the extraction product of the traditional Chinese medicine raw materials at 4 ℃ for 24h, concentrating, wherein the volume of the concentrated material is 1mL relative to 1g of the compound traditional Chinese medicine to obtain the extracting solution of the compound traditional Chinese medicine, and taking the prepared extracting solution of the compound traditional Chinese medicine as a traditional Chinese medicine preparation for preventing and treating livestock and poultry diarrhea.

Comparative example 4

The comparative example is a traditional Chinese medicine preparation prepared according to the method of the comparative example 3, except that the compound traditional Chinese medicine comprises 18 parts of common andrographis herb, 15 parts of Chinese pulsatilla root, 8 parts of coptis chinensis, 12 parts of amur corktree bark, 8 parts of costustoot and 6 parts of liquorice.

Comparative example 5

The comparative example is a traditional Chinese medicine preparation prepared according to the method of the comparative example 3, except that the compound traditional Chinese medicine comprises 15 parts of Chinese pulsatilla root, 8 parts of coptis root, 12 parts of phellodendron, 8 parts of costus root and 6 parts of liquorice.

Comparative example 6

Pulsatilla chinensis powder purchased from Fujian Ningdele crude drug industry Co.

Comparative example 7

Neomycin sulfate purchased from Daliansan animal medicine, Inc.

Test example 1

Bacillus coagulans CCTCC NO in example 1: m2019722, bacillus coagulans cic NO in comparative example 1: 10069 and comparative example 2, respectively, until the effective viable count is more than 15 × 10⁸cfu/mL, then inoculating into a medium containing 20 vol% of compound traditional Chinese medicine extract and the balance matrix according to the inoculation amount of 3%, shaking-culturing at 50 ℃ for 48h at 180r/min, and counting viable bacteria.

An overnight cultured chicken colibacillus standard serotype (O)₇₈) Mixing the bacterial liquid and Staphylococcus aureus (Staphylococcus aureus) bacterial liquid with nutrient agar culture medium (containing 1.5% agar, w/v), and diluting to 2 × 10⁷About CFU/ml, 5ml was poured into prepared nutrient agar plates. The Oxford cup was gently placed on a plate inoculated with different strains with forceps, and 200. mu.l of the Bacillus coagulans CCTCC NO: m2019722. Bacillus coagulans cic NO: 10069 adding oxford cup into the compound traditional Chinese medicine fermentation liquid fermented by the bacterial liquid of the commercial bacterial powder, standing for 30min, culturing for 16h at 37 ℃, and measuring the size of the inhibition zone of the compound traditional Chinese medicine fermentation liquid on the standard serotype of chicken colibacillus and staphylococcus aureus by using a vernier caliper, wherein the size is shown in table 1.

TABLE 1

As can be seen from table 1, the bacillus coagulans in comparative example 1 and comparative example 2 were inhibited from growing in the culture medium of the present disclosure, whereas bacillus coagulans in example 1 has CCTCC NO: m2019722 can not only grow well in the culture medium disclosed by the invention, but also the effective viable count reaches 1.63 multiplied by 10⁸CFU/ml; can also enhance the inhibition ability of traditional Chinese medicine on Escherichia coli and Staphylococcus aureus.

Test example 2

The standard serotype (O) of chicken E.coli cultured overnight was separately₇₈) Chicken colibacillus wild strain (O)_{Chicken with egg yolk}) Standard serotype of porcine escherichia coli (O)₁₃₉) Escherichia coli wild strain (O) of pig_Pig) Pathogenic salmonella (salmonella), duck colibacillus wild strain (O)_Duck) Mixing with Staphylococcus aureus (Staphylococcus aureus) solution and nutrient agar culture medium (containing 1.5% agar, w/v), and diluting to 2 × 10⁷About CFU/ml, 5ml was poured into prepared nutrient agar plates.

Placing the Oxford cup on a plate inoculated with different strains by using tweezers, respectively adding 200 μ l of traditional Chinese medicine microecologics (1g/ml) and neomycin sulfate raw powder diluent (the neomycin sulfate raw powder is diluted into four times ten thousand) into the Oxford cup, standing for 30min, culturing at 37 ℃ for 16h, and measuring the size of the inhibition zone by using a vernier caliper.

The test solutions used in the test examples were the traditional Chinese medicine microecologics for preventing and treating diarrhea of livestock and poultry obtained in example 1 and the neomycin sulfate purchased in comparative example 7, each group of experiments was performed twice, the average value was taken, and the specific results are shown in table 2.

TABLE 2

The in vitro bacteriostatic test is used for detecting the bacteriostatic ability of the medicament to various pathogenic bacteria, is the premise and the basis for clinical treatment, provides very valuable materials and data for the bacteriostatic effect of pathogenic bacteria such as escherichia coli and the like, and reflects the therapeutic effect of the medicament from the side surface by the in vitro bacteriostatic ability. As can be seen from the data in Table 2, the in vitro bacteriostasis test of the traditional Chinese medicine microecological preparation provided by the disclosure is compared with the in vitro bacteriostasis test of neomycin sulfate, and the standard serotype (O) of the chicken colibacillus is treated₇₈) Chicken colibacillus wild strain (O)_{Chicken with egg yolk}) Standard serotype of porcine escherichia coli (O)₁₃₉) Escherichia coli wild strain (O) of pig_Pig) Pathogenic salmonella (salmonella), duck colibacillus wild strain (O)_Duck) And the bacteriostatic ability of staphylococcus aureus (staphyloccocus aureus) is obviously improved, which shows that the traditional Chinese medicine microecological preparation disclosed by the invention has very good bacteriostatic ability on pathogenic bacteria of livestock and poultry diarrhea.

Test example 3

400 healthy AA broilers with similar weight and age of days in a certain chicken farm in Shandong province tobacco terrace city are selected, and the broilers are half male and half female. Performing mixed bacteria challenge of escherichia coli and salmonella, and performing clinical efficacy verification after the chickens are ill.

When the chicken flocks are attacked, the clinical symptoms are mainly manifested as lassitude, inappetence, loose and disorderly feathers, eye closure and lying down, and nest binding and lying; severe diarrhea, the feces being off-white, watery; the anus is adhered by feces and bloody stool.

The test method comprises the following steps: the chickens were randomly divided into 8 groups of 50: a blank control group (positive control), a toxicity counteracting non-treatment group (negative control), a traditional Chinese medicine microecological preparation group (example 2), a western medicine group (comparative example 7) and comparative examples 3-6. As the chickens can not eat freely, the stomach irrigation mode is adopted, the traditional Chinese medicine microecologics group (0.5g/ml diluted and irrigated by stomach irrigation for 0.5 ml/time and 2 times/day), the western medicine group (the usage amount of 1L of water is 0.77-1.15 g according to the specification), the comparative examples 3-6 are used for stomach irrigation treatment of the offensive chickens according to the usage amount of the test groups (0.5g/ml diluted and irrigated by stomach irrigation for 0.5 ml/time and 2 times/day), the positive control group normally takes food and drink water, the negative control group irrigates stomach physiological saline, the medicine is continuously administrated for 5 days, the clinical change of the sick chickens is observed every day, the cure, effective and ineffective numbers are counted, the cure rate and the total effective rate are calculated, and the bursa, thymus and spleen are weighed after the autopsy to calculate the immune organ index.

The judgment standard of the curative effect is as follows: and (3) curing: after the test is finished, the symptoms of the sick chicken disappear, the chicken turns to be good in spirit, the appetite is increased, the sick chicken completely recovers to be normal without loose stool, and no pathogenic bacteria are detected by performing a caesarean examination on heart, liver, spleen and intestines; the method has the following advantages: after the test is finished, the symptoms, spirit and appetite of the chicken diseases are obviously improved, and the chicken diseases are not watery; and (4) invalidation: symptoms appear during the test period, and no symptom disappears or even dies after the test is finished.

The specific therapeutic effect is shown in table 3.

TABLE 3

As can be seen from table 3, under a toxic dose of 56% lethal dose, the effective rate of the test group of the traditional Chinese medicine microecological preparation disclosed by the invention for diseases caused by escherichia coli and salmonella gallinarum is 88%, the cure rate is 82%, the ineffective rate is 12%, the effective rate is equivalent to the effect of the comparative example 7 (western medicine) using neomycin sulfate, but after the pathogenic bacteria are verified by plating after dissection, the cure rate of the test group of the traditional Chinese medicine microecological preparation is 5.12% higher than that of the western medicine group, the thymus index is increased by 17.80%, the spleen index is increased by 18.63%, and the bursal disease index is increased by 29.47%. The immune organ indexes (thymus index, spleen index and bursal index) of the chickens using the traditional Chinese medicine microecological preparation test group are higher than those of the western medicine group, so that the traditional Chinese medicine microecological preparation can replace antibiotics to treat bacterial diarrhea and has the capability of improving the immunity of the chickens.

It can also be seen from table 3 that example 2 is significantly higher than comparative examples in terms of number of surviving, effective rate, and cure rate of the chickens compared to comparative examples 3-6. Therefore, the effect of the traditional Chinese medicine microecological preparation is further proved from clinical data.

Test example 4

50 yellow and white diarrhea piglets with similar weight and age in days in a certain pig farm in Shandong province cigarette platform city are selected.

Sick pigs were randomly divided into 2 and 25 pigs per animal, and the animals were divided into a test group (example 2) and a control group (comparative example 7) under substantially identical feeding and management conditions. Administration mode and dosage: the syringe was drenched along the horns of piglets, and the medication was carried out at the same time, with the amount of the test group (example 2) being 0.2g/ml and 5 ml/time, and the amount of the control group (comparative example 7) being 2 times/day with reference to the instructions (0.75 g for 1kg body weight) and 5 days in succession. The average weight, the weight after healing, the healing condition, the weight at 60 days of age and the feed conversion ratio of the yellow-white dysentery piglets are recorded during treatment.

The judgment standard of the curative effect is as follows: and (3) curing: after the test is finished, the yellow-white dysentery phenomenon of the piglets disappears, the excrement is formed, and the mental state is good; the method has the following advantages: the frequency of treating the yellow and white scour of the piglets is reduced, and the stool character is recovered; and (4) invalidation: the frequency of treating the yellow and white scour of the piglets is unchanged or even dead before the treatment.

The specific therapeutic effect is shown in table 4.

As can be seen from table 4, the experimental group (example 2) has significant therapeutic effect on yellow-white scour of piglets, with an effective rate of 96% and a cure rate of 92%. Comparison with the control (comparative example 7): the effective rate is improved by 4.35%, the cure rate is improved by 9.52%, the average weight per 60 days is improved by 9.34%, and the feed conversion ratio is reduced by 0.07. The experimental result shows that the traditional Chinese medicine microecological preparation can effectively treat livestock and poultry diarrhea caused by bacteria such as escherichia coli and salmonella, the cure rate is higher than that of an antibiotic treatment group, indexes such as weight increment and feed conversion ratio reduction are obviously optimized, and the economic benefit can be obviously improved.

The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. In order to avoid unnecessary repetition, various possible combinations will not be separately described in this disclosure.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Claims

1. A preparation method of a traditional Chinese medicine microecological preparation for preventing and treating livestock and poultry diarrhea is characterized by comprising the following steps: inoculating seed liquid into the culture medium for fermentation; the culture medium contains 5-20 vol% of compound traditional Chinese medicine extract and the balance of matrix culture medium, and the seed solution contains traditional Chinese medicine fermentation strain;

the compound traditional Chinese medicine extracting solution is prepared by the following operations: drying and crushing the compound traditional Chinese medicine, extracting the dried and crushed compound traditional Chinese medicine for 50-70 min at 60-70 ℃ by using 10-20 times of ethanol aqueous solution, and then concentrating the extract to obtain a concentrated material; the ethanol aqueous solution contains 65-85 vol% of ethanol; the concentrated material is a liquid medicine containing 0.5-2g/ml crude drug;

wherein the compound traditional Chinese medicine comprises, by weight, 10-25 parts of common andrographis herb, 5-25 parts of Chinese pulsatilla root, 4-12 parts of coptis chinensis, 5-20 parts of amur corktree bark, 4-12 parts of costus root, 4-20 parts of dahurian patrinia herb and 4-12 parts of liquorice;

the traditional Chinese medicine fermentation strain comprises:

the preservation number is CCTCC NO: m2019722 Bacillus coagulans,

The preservation number is CGMCC NO: 2196A preparation containing Enterococcus faecalis (Enterococcus faecalis),

The commodity number is CICC NO: 10020 Bacillus subtilis,

The commodity number is CICC NO: 31170 Candida utilis (Candida utilis),

The commodity number is ACCC NO: 20039 Lactobacillus plantarum (Lactobacillus plantarum),

The commodity number is CICC NO: 24498 Clostridium butyricum (Clostridium butyricum) and

the commodity number is ACCC NO: 10649 Rhodopseudomonas palustris (Rhodosedoudomoulous padustris);

the viable bacteria number ratio of the bacillus coagulans, the enterococcus faecalis, the bacillus subtilis, the candida utilis, the lactobacillus plantarum, the clostridium butyricum and the rhodopseudomonas palustris in the seed liquid is (10-25): (5-15): (10-25): (10-25): (10-20): (5-15): (5-15); relative to 1g of traditional Chinese medicine microecological preparation or 1mL of traditional Chinese medicine microecological preparation, the total number of viable bacteria is (5-10) multiplied by 10⁸cfu。

2. The method for preparing a microecological preparation according to claim 1, wherein the fermentation is high density liquid submerged fermentation; the fermentation temperature is 30-37 ℃, and the fermentation time is 2-5 days.

3. The method for preparing a microecological preparation according to claim 1, wherein the compound Chinese medicine comprises, by weight, 15 to 20 parts of andrographis paniculata, 10 to 20 parts of pulsatilla chinensis, 6 to 10 parts of coptis chinensis, 10 to 15 parts of phellodendron amurense, 6 to 10 parts of costus root, 10 to 18 parts of patrinia, and 5 to 10 parts of licorice.

4. The preparation method of the traditional Chinese medicine microecological preparation according to claim 1, wherein the viable bacteria number ratio of the bacillus coagulans, the enterococcus faecalis, the bacillus subtilis, the candida utilis, the lactobacillus plantarum, the clostridium butyricum and the rhodopseudomonas palustris in the seed solution is (16-20): (10-13): (16-20): (16-20): (12-15): (10-13): (9-12); the seed solution is prepared by the following operation method: respectively culturing the bacillus coagulans, the enterococcus faecalis, the bacillus subtilis, the candida utilis, the lactobacillus plantarum, the clostridium butyricum and the rhodopseudomonas palustris to obtain culture solutions, and then mixing the culture solutions.

5. The method for preparing a microecological preparation of a Chinese medicinal composition according to claim 1, wherein the substrate medium comprises brown sugar 10-50 g/L, glucose 10-50 g/L, honey 10-50 g/L, peptone 1-5 g/L, dipotassium hydrogen phosphate 1-5 g/L, sodium acetate 1-5 g/L, magnesium sulfate 0.1-1 g/L, L-cysteine 0.1-1 g/L, manganese sulfate 0.05-0.3/L, and calcium carbonate 0.1-3 g/L; the pH value of the substrate culture medium is 6.0-8.0.

6. A traditional Chinese medicine microecological preparation for preventing and treating livestock and poultry diarrhea is characterized by being prepared according to the preparation method of any one of claims 1 to 5; the dosage form of the traditional Chinese medicine microecological preparation is one of granules, powder and solution.

7. The bacillus coagulans is characterized in that the preservation number of the bacillus coagulans is CCTCC NO: m2019722, the preservation unit is China center for type culture Collection, and the preservation date is 2019, 9 and 16 days.