CN111388527A - Preparation for preventing and treating mycoplasma of laying hens and preparation method thereof - Google Patents
Preparation for preventing and treating mycoplasma of laying hens and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a preparation for preventing and treating mycoplasma of laying hens and a preparation method thereof, wherein the preparation method comprises the following steps: inoculating bacillus subtilis to a beef extract peptone liquid culture medium to obtain bacillus subtilis seed liquid, adding water to a solid culture medium to prepare a fermentation material, and inoculating the bacillus subtilis seed liquid to the fermentation material for fermentation, wherein the solid fermentation culture medium contains one or more of scutellaria baicalensis, ephedra and liquorice. The preparation for preventing and treating the mycoplasma of the laying hens, which is prepared by the invention, has better effect on treating the mycoplasma gallisepticum.
Description
Technical Field
The invention relates to a preparation for preventing and treating mycoplasma of laying hens and a preparation method thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Mycoplasma (mycoplasma), also known as a mycoplasma, is the smallest of the simplest prokaryotes currently found. The only organelle visible in mycoplasma cells is the ribosome. Most mycoplasma are lethal. The mycoplasma which is pathogenic to the laying hens at present comprises mycoplasma gallisepticum and mycoplasma synoviae. The mycoplasma gallisepticum can cause contact and slow respiratory tract infectious diseases of laying hens, and is also called mycoplasma gallisepticum, and clinically common symptoms are gong, cough, rhinorrhea and lacrimation. Under normal conditions, the laying rate of laying hens infected with the mycoplasma gallisepticum is reduced by about 10 percent, and the serious laying rate can reach 20-30 percent. At present, antibacterial drugs such as tylosin, enrofloxacin, florfenicol and the like have better treatment effect on mycoplasma gallisepticum. However, the antibacterial drug can only relieve clinical symptoms, has high toxicity, is difficult to completely eliminate, and causes increasingly serious drug resistance and drug residue if the antibacterial drug is used for a long time unreasonably, so that the curative effect of the antibacterial drug on laying hens is gradually deteriorated. In order to solve this problem, development of a novel pharmaceutical preparation for controlling mycoplasma gallisepticum is required.
The traditional Chinese medicine is a natural substance taken from the nature, completely retains the natural structure and the biological activity thereof, and has small toxic and side effects. The Chinese veterinary medicine self-forming system has the advantages that the traditional Chinese medicine is applied to treating the mycoplasma gallisepticum at present and has good effect. However, through the research of the inventor of the invention, the traditional Chinese medicine for treating the mycoplasma gallisepticum is more in medicinal flavor and needs to be improved in treatment effect at present.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a preparation for preventing and treating the mycoplasma from the laying hens and a preparation method thereof, which adopt fewer medicinal flavors and have better effect of treating the mycoplasma gallisepticum.
In order to achieve the purpose, the technical scheme of the invention is as follows:
on the one hand, the preparation method of the preparation for preventing and treating the mycoplasma of the laying hens comprises the steps of inoculating the bacillus subtilis into a beef extract peptone liquid culture medium to obtain a bacillus subtilis seed liquid, adding water into a solid culture medium to prepare a fermentation material, and inoculating the bacillus subtilis seed liquid into the fermentation material to ferment, wherein the solid fermentation culture medium contains one or more of scutellaria baicalensis, ephedra and liquorice.
According to the invention, the bacillus subtilis is utilized to ferment the scutellaria baicalensis, the ephedra herb or the liquorice, and the fermented product has the mycoplasma gallisepticum resistant activity, wherein when the bacillus subtilis is utilized to ferment the scutellaria baicalensis, the ephedra herb or the liquorice, the fermented product has more remarkable mycoplasma gallisepticum resistant activity, and the egg laying rate of the laying hens can be prevented from being reduced.
On the other hand, the preparation for preventing and treating the mycoplasma of the laying hens is obtained by the preparation method.
The invention has the beneficial effects that:
according to the invention, the bacillus subtilis is used for carrying out microbial fermentation treatment on the scutellaria baicalensis, the ephedra and/or the liquorice, and the fermented product is used as a preparation for preventing and treating the mycoplasma gallisepticum. Meanwhile, the traditional Chinese medicine is fermented and metabolized by the bacillus subtilis to generate a plurality of new active ingredients, so that the number of the traditional Chinese medicines is reduced, and the effective utilization rate of the traditional Chinese medicine is increased.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In view of the problems of more medicinal ingredients, pending improvement of treatment effect and the like of the traditional Chinese medicine for treating the mycoplasma gallisepticum, the invention provides a preparation for preventing and treating the mycoplasma gallisepticum and a preparation method thereof.
The invention provides a preparation method of a preparation for preventing and treating mycoplasma gallisepticum, which comprises the steps of inoculating bacillus subtilis into a beef extract peptone liquid culture medium to obtain bacillus subtilis seed liquid, adding water into a solid culture medium to prepare a fermentation material, and inoculating the bacillus subtilis seed liquid into the fermentation material for fermentation, wherein the solid fermentation culture medium contains one or more of scutellaria baicalensis, ephedra and liquorice.
In traditional veterinary medicine, mycoplasma gallisepticum infection is reported to be caused by exogenous wind-cold or fire-heat, and is caused by heat transformation due to depression, so that the lung qi is abnormally dispersed and descended, and the healthy qi is weak, so that the traditional Chinese medicine is preferably treated by using formulas for clearing heat and removing toxicity, eliminating phlegm and relieving cough, and dispersing lung and relieving asthma. The traditional Chinese medicine for treating mycoplasma gallisepticum infection is prepared by compounding heat-clearing, detoxifying, pathogenic fire-reducing and dampness-drying medicines, exterior-syndrome-relieving medicines, phlegm-eliminating, cough-relieving and asthma-relieving medicines, yin-nourishing and qi-tonifying medicines, wind-dispelling and dampness-removing medicines, blood-activating and stasis-removing medicines, resuscitation-inducing medicines and the like. Wherein the heat-clearing, detoxifying, pathogenic fire-reducing and dampness-drying medicine comprises honeysuckle, weeping forsythia, isatis root, airpotato yam rhizome, stephanotis, common andrographis herb, heartleaf houttuynia herb, cape jasmine fruit, common anemarrhena rhizome, gypsum, cholic acid, golden thread, amur corktree bark, baical skullcap root and the like, the exterior-releasing medicine comprises ephedra herb, divaricate saposhnikovia root, mint, great burdock achene and the like, and the yin-nourishing and qi. According to the invention, one of the heat-clearing, detoxifying, fire-reducing and dampness-drying drugs, the exterior syndrome relieving drug and the wind-dispelling and dampness-removing drug is selected for fermentation research, and the fermentation product has the mycoplasma gallisepticum resisting activity by fermenting the scutellaria baicalensis, the ephedra or the liquorice with the bacillus subtilis.
In one or more embodiments of this embodiment, the solid fermentation medium comprises scutellaria baicalensis, ephedra herb, and liquorice. When the scutellaria baicalensis, the ephedra herb or the liquorice are fermented by utilizing the bacillus subtilis, the fermentation product has more remarkable anti-mycoplasma gallisepticum activity.
In the series of embodiments, the mass ratio of the scutellaria baicalensis to the ephedra to the liquorice is 2-3: 1-2. When the mass ratio of the scutellaria baicalensis to the ephedra to the liquorice is 3:2:2, the effect is better.
In one or more embodiments of this embodiment, the strain of bacillus subtilis is deposited under accession number cic 24713.
In one or more embodiments of this embodiment, the fermentation process is: aerobic fermentation is carried out at the temperature of 30-37 ℃.
In the series of embodiments, the aerobic fermentation time is 36-72 h.
In one or more embodiments of this embodiment, the fermented material is extracted under reflux with aqueous ethanol. The reflux extraction is carried out by heating to reflux and extracting under reflux condition.
In the series of embodiments, the ethanol aqueous solution is 94-96% by volume.
In the series of embodiments, the extraction is performed for 1-3 times, and the extraction time is 0.5-1.5 h each time.
In one or more embodiments of this embodiment, the fermentation material is sterilized prior to fermentation.
In another embodiment of the invention, the invention provides a preparation for preventing and treating the mycoplasma of the laying hens, which is obtained by the preparation method.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Bacillus subtilis in the following examples, purchased from china industrial microbial cultures collection management center, strain collection number: and CICC 24713.
The nutrient components of the beef extract peptone liquid medium comprise peptone 5.0 g/L, beef extract 3.0 g/L/L, agar 15.0 g/L and pH7.0.
Example 1
Removing impurities from Scutellariae radix, cleaning, oven drying, pulverizing, sieving with 40 mesh sieve to obtain Scutellariae radix powder, mixing 10g bran, 10g soybean meal and 30g Scutellariae radix powder, adding 2.5g potassium dihydrogen phosphate, 5g dipotassium hydrogen phosphate, 3g sodium chloride and 0.5g magnesium sulfate, mixing, and sterilizing at 115 deg.C for 30min to obtain solid fermentation culture medium.
Inoculating bacillus subtilis into a beef extract peptone liquid culture medium,culturing at 35 deg.C, and regulating effective viable bacteria number to be more than 5 × 108cfu/m L to obtain the seed liquid.
Adding 100m L distilled water into solid fermentation culture medium, mixing, sterilizing at 115 deg.C for 30min to obtain fermented material, inoculating seed solution into the fermented material, performing aerobic fermentation at 34 deg.C for 2 days, and oven drying at 50 deg.C.
And adding 95% ethanol water solution into the dried material according to the volume ratio of 1:10 for reflux extraction, and extracting twice for 1h each time. And (3) performing rotary evaporation on the extracted product to obtain a solid material, namely the preparation 1.
Example 2
Removing impurities from herba Ephedrae, cleaning, oven drying, pulverizing, sieving with 40 mesh sieve to obtain herba Ephedrae powder, mixing 10g of testa Tritici, 10g of soybean meal and 30g of herba Ephedrae powder, adding 2.5g of potassium dihydrogen phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride and 0.5g of magnesium sulfate, mixing, and sterilizing at 115 deg.C for 30min to obtain solid fermentation culture medium.
Inoculating Bacillus subtilis to beef extract peptone liquid culture medium, culturing at 35 deg.C, and regulating effective viable bacteria number to be more than 5 × 108cfu/m L to obtain the seed liquid.
Adding 100m L distilled water into solid fermentation culture medium, mixing, sterilizing at 115 deg.C for 30min to obtain fermented material, inoculating seed solution into the fermented material, performing aerobic fermentation at 34 deg.C for 2 days, and oven drying at 50 deg.C.
And adding 95% ethanol water solution into the dried material according to the volume ratio of 1:10 for reflux extraction, and extracting twice for 1h each time. And (3) performing rotary evaporation on the extracted product to obtain a solid material, namely the preparation 2.
Example 3
Removing impurities from Glycyrrhrizae radix, cleaning, oven drying, pulverizing, sieving with 40 mesh sieve to obtain Glycyrrhrizae radix powder, mixing 10g bran, 10g soybean meal and 30g Glycyrrhrizae radix powder, adding 2.5g potassium dihydrogen phosphate, 5g dipotassium hydrogen phosphate, 3g sodium chloride and 0.5g magnesium sulfate, mixing, and sterilizing at 115 deg.C for 30min to obtain solid fermentation culture medium.
Inoculating Bacillus subtilis to beef extract peptone liquid culture medium, culturing at 35 deg.C, and regulating effective viable bacteria number to be more than 5 × 108cfu/m L to obtain the seed liquid.
Adding 100m L distilled water into solid fermentation culture medium, mixing, sterilizing at 115 deg.C for 30min to obtain fermented material, inoculating seed solution into the fermented material, performing aerobic fermentation at 34 deg.C for 2 days, and oven drying at 50 deg.C.
And adding 95% ethanol water solution into the dried material according to the volume ratio of 1:10 for reflux extraction, and extracting twice for 1h each time. And (3) performing rotary evaporation on the extracted product to obtain a solid material, namely the preparation 3.
Example 4
Removing impurities from scutellaria baicalensis, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain scutellaria baicalensis powder, removing impurities from ephedra, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain ephedra powder, mixing 10g of bran, 10g of bean pulp, 15g of scutellaria baicalensis powder and 15g of ephedra powder, adding 2.5g of potassium dihydrogen phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride and 0.5g of magnesium sulfate, uniformly mixing, and sterilizing at 115 ℃ for 30min to obtain a solid fermentation culture medium.
Inoculating Bacillus subtilis to beef extract peptone liquid culture medium, culturing at 35 deg.C, and regulating effective viable bacteria number to be more than 5 × 108cfu/m L to obtain the seed liquid.
Adding 100m L distilled water into solid fermentation culture medium, mixing, sterilizing at 115 deg.C for 30min to obtain fermented material, inoculating seed solution into the fermented material, performing aerobic fermentation at 34 deg.C for 2 days, and oven drying at 50 deg.C.
And adding 95% ethanol water solution into the dried material according to the volume ratio of 1:10 for reflux extraction, and extracting twice for 1h each time. And (4) performing rotary evaporation on the extracted product to obtain a solid material, namely the preparation 4.
Example 5
Removing impurities from scutellaria baicalensis, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain scutellaria baicalensis powder, removing impurities from liquorice, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain liquorice powder, mixing 10g of bran, 10g of bean pulp, 15g of scutellaria baicalensis powder and 15g of liquorice powder, adding 2.5g of potassium dihydrogen phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride and 0.5g of magnesium sulfate, uniformly mixing, and sterilizing at 115 ℃ for 30min to obtain a solid fermentation culture medium.
Inoculating Bacillus subtilis to beef extract peptone liquid culture medium, culturing at 35 deg.C, and regulating effective viable bacteria number to be more than 5 × 108cfu/m L to obtain the seed liquid.
Adding 100m L distilled water into solid fermentation culture medium, mixing, sterilizing at 115 deg.C for 30min to obtain fermented material, inoculating seed solution into the fermented material, performing aerobic fermentation at 34 deg.C for 2 days, and oven drying at 50 deg.C.
And adding 95% ethanol water solution into the dried material according to the volume ratio of 1:10 for reflux extraction, and extracting twice for 1h each time. And (3) performing rotary evaporation on the extracted product to obtain a solid material, namely the preparation 5.
Example 6
Removing impurities from herba Ephedrae, cleaning, oven drying, pulverizing, sieving with 40 mesh sieve to obtain herba Ephedrae powder, removing impurities from Glycyrrhrizae radix, cleaning, oven drying, pulverizing, sieving with 40 mesh sieve to obtain Glycyrrhrizae radix powder, mixing 10g of testa Tritici, 10g of bean cake, 15g of herba Ephedrae powder, and 15g of Glycyrrhrizae radix powder, adding 2.5g of potassium dihydrogen phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride and 0.5g of magnesium sulfate, mixing, and sterilizing at 115 deg.C for 30min to obtain solid fermentation culture medium.
Inoculating Bacillus subtilis to beef extract peptone liquid culture medium, culturing at 35 deg.C, and regulating effective viable bacteria number to be more than 5 × 108cfu/m L to obtain the seed liquid.
Adding 100m L distilled water into solid fermentation culture medium, mixing, sterilizing at 115 deg.C for 30min to obtain fermented material, inoculating seed solution into the fermented material, performing aerobic fermentation at 34 deg.C for 2 days, and oven drying at 50 deg.C.
And adding 95% ethanol water solution into the dried material according to the volume ratio of 1:10 for reflux extraction, and extracting twice for 1h each time. And (3) performing rotary evaporation on the extracted product to obtain a solid material, namely a preparation 6.
Example 7
Removing impurities from scutellaria baicalensis, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain scutellaria baicalensis powder, removing impurities from ephedra, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain ephedra powder, removing impurities from liquorice, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain liquorice powder, mixing 10g of bran, 10g of bean pulp, 10g of scutellaria baicalensis powder, 10g of ephedra powder and 10g of liquorice powder, adding 2.5g of monopotassium phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride and 0.5g of magnesium sulfate, uniformly mixing, and sterilizing at 115 ℃ for 30min to obtain a solid fermentation culture medium.
Inoculating Bacillus subtilis to beef extract peptone liquid culture medium, culturing at 35 deg.C, and regulating effective viable bacteria number to be more than 5 × 108cfu/m L to obtain the seed liquid.
Adding 100m L distilled water into solid fermentation culture medium, mixing, sterilizing at 115 deg.C for 30min to obtain fermented material, inoculating seed solution into the fermented material, performing aerobic fermentation at 34 deg.C for 2 days, and oven drying at 50 deg.C.
And adding 95% ethanol water solution into the dried material according to the volume ratio of 1:10 for reflux extraction, and extracting twice for 1h each time. And (3) performing rotary evaporation on the extracted product to obtain a solid material, namely the preparation 7.
Example 8
Removing impurities from scutellaria baicalensis, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain scutellaria baicalensis powder, removing impurities from ephedra, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain ephedra powder, removing impurities from liquorice, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain liquorice powder, mixing 10g of bran, 10g of bean pulp, 12g of scutellaria baicalensis powder, 9g of ephedra powder and 9g of liquorice powder, adding 2.5g of monopotassium phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride and 0.5g of magnesium sulfate, uniformly mixing, and sterilizing at 115 ℃ for 30min to obtain a solid fermentation culture medium.
Inoculating Bacillus subtilis to beef extract peptone liquid culture medium, culturing at 35 deg.C, and regulating effective viable bacteria number to be more than 5 × 108cfu/m L, namely the seedsAnd (4) liquid.
Adding 100m L distilled water into solid fermentation culture medium, mixing, sterilizing at 115 deg.C for 30min to obtain fermented material, inoculating seed solution into the fermented material, performing aerobic fermentation at 34 deg.C for 2 days, and oven drying at 50 deg.C.
And adding 95% ethanol water solution into the dried material according to the volume ratio of 1:10 for reflux extraction, and extracting twice for 1h each time. And (3) performing rotary evaporation on the extracted product to obtain a solid material, namely the preparation 8.
Example 9
Removing impurities from scutellaria baicalensis, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain scutellaria baicalensis powder, removing impurities from ephedra, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain ephedra powder, removing impurities from liquorice, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain liquorice powder, mixing 10g of bran, 10g of bean pulp, 9g of scutellaria baicalensis powder, 12g of ephedra powder and 9g of liquorice powder, adding 2.5g of monopotassium phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride and 0.5g of magnesium sulfate, uniformly mixing, and sterilizing at 115 ℃ for 30min to obtain a solid fermentation culture medium.
Inoculating Bacillus subtilis to beef extract peptone liquid culture medium, culturing at 35 deg.C, and regulating effective viable bacteria number to be more than 5 × 108cfu/m L to obtain the seed liquid.
Adding 100m L distilled water into solid fermentation culture medium, mixing, sterilizing at 115 deg.C for 30min to obtain fermented material, inoculating seed solution into the fermented material, performing aerobic fermentation at 34 deg.C for 2 days, and oven drying at 50 deg.C.
And adding 95% ethanol water solution into the dried material according to the volume ratio of 1:10 for reflux extraction, and extracting twice for 1h each time. And (3) performing rotary evaporation on the extracted product to obtain a solid material, namely the preparation 9.
Example 10
Removing impurities from scutellaria baicalensis, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain scutellaria baicalensis powder, removing impurities from ephedra, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain ephedra powder, removing impurities from liquorice, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain liquorice powder, mixing 10g of bran, 10g of bean pulp, 9g of scutellaria baicalensis powder, 9g of ephedra powder and 12g of liquorice powder, adding 2.5g of monopotassium phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride and 0.5g of magnesium sulfate, uniformly mixing, and sterilizing at 115 ℃ for 30min to obtain a solid fermentation culture medium.
Inoculating Bacillus subtilis to beef extract peptone liquid culture medium, culturing at 35 deg.C, and regulating effective viable bacteria number to be more than 5 × 108cfu/m L to obtain the seed liquid.
Adding 100m L distilled water into solid fermentation culture medium, mixing, sterilizing at 115 deg.C for 30min to obtain fermented material, inoculating seed solution into the fermented material, performing aerobic fermentation at 34 deg.C for 2 days, and oven drying at 50 deg.C.
And adding 95% ethanol water solution into the dried material according to the volume ratio of 1:10 for reflux extraction, and extracting twice for 1h each time. And (3) performing rotary evaporation on the extracted product to obtain a solid material serving as the preparation 10.
In order to verify whether the extract of the traditional Chinese medicine raw material subjected to 95% ethanol water solution has corresponding mycoplasma gallisepticum resistant activity, comparative examples 1-3 are set.
Comparative example 1:
removing impurities from Scutellariae radix, cleaning, oven drying, pulverizing, sieving with 40 mesh sieve to obtain Scutellariae radix powder, and extracting 30g Scutellariae radix powder with 95% ethanol water solution at a volume ratio of 1:10 for 1 hr twice. And (3) performing rotary evaporation on the extracted product to obtain a solid material serving as a preparation 11.
Comparative example 2
Removing impurities from herba Ephedrae, cleaning, oven drying, pulverizing, sieving with 40 mesh sieve to obtain herba Ephedrae powder, and extracting 30g herba Ephedrae powder with 95% ethanol water solution at a volume ratio of 1:10 for 1 hr twice. And (3) performing rotary evaporation on the extracted product to obtain a solid material, namely the preparation 12.
Comparative example 3
Removing impurities from Glycyrrhrizae radix, cleaning, oven drying, pulverizing, sieving with 40 mesh sieve to obtain Glycyrrhrizae radix powder, and extracting 30g Glycyrrhrizae radix powder with 95% ethanol water solution at a volume ratio of 1:10 for 1 hr twice. And (3) performing rotary evaporation on the extracted product to obtain a solid material serving as a preparation 13.
In order to verify whether the extract of the traditional Chinese medicine raw material subjected to 95% ethanol aqueous solution has corresponding anti-mycoplasma gallisepticum activity and simultaneously verify whether a new anti-mycoplasma gallisepticum activity substance is generated by reaction in the reflux of the 95% ethanol aqueous solution, a comparative example 4-10 is set.
Comparative example 4
Removing impurities from scutellaria baicalensis, cleaning, drying, crushing, sieving by a 40-mesh sieve to obtain scutellaria baicalensis powder, removing impurities from ephedra, cleaning, drying, crushing, sieving by a 40-mesh sieve to obtain ephedra powder, mixing 15g of scutellaria baicalensis powder and 15g of ephedra powder, adding 95% ethanol water solution according to the volume ratio of 1:10, and extracting twice, wherein the extraction time is 1 hour each time. And (3) performing rotary evaporation on the extracted product to obtain a solid material, namely the preparation 14.
Comparative example 5
Removing impurities from scutellaria baicalensis, cleaning, drying, crushing, sieving by a 40-mesh sieve to obtain scutellaria baicalensis powder, removing impurities from liquorice, cleaning, drying, crushing, sieving by a 40-mesh sieve to obtain liquorice powder, mixing 15g of scutellaria baicalensis powder and 15g of liquorice powder, adding 95% ethanol aqueous solution according to the volume ratio of 1:10, and extracting twice, wherein the extraction time is 1 hour each time. And (3) performing rotary evaporation on the extracted product to obtain a solid material, namely the preparation 15.
Comparative example 6
Removing impurities from herba Ephedrae, cleaning, oven drying, pulverizing, sieving with 40 mesh sieve to obtain herba Ephedrae powder, removing impurities from Glycyrrhrizae radix, cleaning, oven drying, pulverizing, sieving with 40 mesh sieve to obtain Glycyrrhrizae radix powder, mixing 15g herba Ephedrae powder and 15g Glycyrrhrizae radix powder, adding 95% ethanol water solution at volume ratio of 1:10, extracting twice, each for 1 hr. And (3) performing rotary evaporation on the extracted product to obtain a solid material, namely the preparation 16.
Comparative example 7
Removing impurities from scutellaria baicalensis, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain scutellaria baicalensis powder, removing impurities from ephedra, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain ephedra powder, removing impurities from liquorice, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain liquorice powder, and adding 95% ethanol water solution into 10g of scutellaria baicalensis powder, 10g of ephedra powder and 10g of liquorice powder according to the volume ratio of 1:10 for extraction twice, wherein the extraction time is 1h each time. The extracted product is subjected to rotary evaporation to obtain a solid material, namely a preparation 17.
Comparative example 8
Removing impurities from scutellaria baicalensis, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain scutellaria baicalensis powder, removing impurities from ephedra, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain ephedra powder, removing impurities from liquorice, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain liquorice powder, and adding 95% ethanol water solution into 12g of scutellaria baicalensis powder, 9g of ephedra powder and 9g of liquorice powder according to the volume ratio of 1:10 for extraction twice, wherein the extraction time is 1h each time. And (3) performing rotary evaporation on the extracted product to obtain a solid material, namely the preparation 18.
Comparative example 9
Removing impurities from scutellaria baicalensis, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain scutellaria baicalensis powder, removing impurities from ephedra, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain ephedra powder, removing impurities from liquorice, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain liquorice powder, and adding 95% ethanol water solution into 9g of scutellaria baicalensis powder, 12g of ephedra powder and 9g of liquorice powder according to the volume ratio of 1:10 for extraction twice, wherein the extraction time is 1h each time. The extracted product is subjected to rotary evaporation to obtain a solid material, namely preparation 19.
Comparative example 10
Removing impurities from scutellaria baicalensis, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain scutellaria baicalensis powder, removing impurities from ephedra, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain ephedra powder, removing impurities from liquorice, cleaning, drying and crushing, sieving by a 40-mesh sieve to obtain liquorice powder, and adding 95% ethanol water solution into 9g of scutellaria baicalensis powder, 9g of ephedra powder and 12g of liquorice powder according to the volume ratio of 1:10 for extraction twice, wherein the extraction time is 1h each time. And (3) performing rotary evaporation on the extracted product to obtain a solid material, namely the preparation 20.
To verify whether the extract of the fermentation product produced after fermentation of Bacillus subtilis with 95% aqueous ethanol had the corresponding anti-Mycoplasma gallisepticum activity, comparative example 11 was set.
Comparative example 11
Mixing 25g of bran and 25g of soybean meal, adding 2.5g of monopotassium phosphate, 5g of dipotassium hydrogen phosphate, 3g of sodium chloride and 0.5g of magnesium sulfate, uniformly mixing, and sterilizing at 115 ℃ for 30min to obtain the solid fermentation culture medium.
Inoculating Bacillus subtilis to beef extract peptone liquid culture medium, culturing at 35 deg.C, and regulating effective viable bacteria number to be more than 5 × 108cfu/m L to obtain the seed liquid.
Adding 100m L distilled water into solid fermentation culture medium, mixing, sterilizing at 115 deg.C for 30min to obtain fermented material, inoculating seed solution into the fermented material, performing aerobic fermentation at 34 deg.C for 2 days, and oven drying at 50 deg.C.
And adding 95% ethanol water solution into the dried material according to the volume ratio of 1:10 for reflux extraction, and extracting twice for 1h each time. The extracted product is subjected to rotary evaporation to obtain a solid material, namely the preparation 21.
In vitro bacteriostatic experiment:
mycoplasma gallisepticum strain S6 was purchased from the Chinese institute of veterinary medicine.
The Minimum Inhibitory Concentration (MIC) of the preparations obtained in the examples of the present invention was determined by agar double dilution. The experimental control medicament is selected from tylosin, and the tylosin is the veterinary antibiotic with better effect of resisting mycoplasma gallisepticum at present. Inoculating the bacterial liquid into culture dishes containing different concentrations of medicine by using a multi-point inoculator, wherein the inoculation bacterial amount is 106CFU/ml. The results were observed after 24 hours incubation at 37 ℃ with the concentration of compound in the non-growth dish as the Minimum Inhibitory Concentration (MIC) for the formulation.
The preparation prepared in examples 1 to 10 and comparative examples 1 to 11, which is 512mg, is precisely weighed, and is placed in a volumetric flask with the volume of 10m L, and is dissolved by a small amount of N, N-dimethylformamide, and then the volume of the preparation is determined to be 10m L by N, N-dimethylformamide, so that a stock solution with the volume of 51.2mg/m L is prepared, 25.6mg of tylosin is precisely weighed, and is placed in a volumetric flask with the volume of 10m L, and the volume of the preparation is determined to be 10m L by N, N-dimethylformamide, so that a control stock solution with the volume of 2560 mu g/m L is prepared.
The stock solution was diluted in Petri dishes by a double dilution method, each containing 1m of the drug solution, to 20m of molten MH agar, so that the final concentrations of the test agents 1 to 6 and 11 to 21 in the Petri dishes in the series were 25.6mg/m 0, 12.8mg/m 1, 6.4mg/m 2, 3.2mg/m 3, 1.6mg/m 4, 0.8mg/m 5, 0.4mg/m 6, 0.2mg/m 7, 0.1mg/m 8, 0.05mg/m 9 and 0.025mg/m, respectively, and so that the final concentrations of the test agents 7 to 10 in the Petri dishes in the series were 128. mu.g/m 0, 64. mu.g/m 1, 32. mu.g/m 2, 16. mu.g/m 3, 8. mu.g/m 4, 4. mu.g/m 5, 2. mu.g/m 6, 1. mu.g/m 7, 0.5. mu.0.25 g/m 2, 0310.g/m 2, 25. mu.g/m 3, 8. mu.g/m 4, 4 g/m 5, 25 g/m, 568 g/m, 25 g/m, respectively, 25 g/m, and 5 μm, respectively.
The results of the in vitro antibacterial experiments are shown in table 1.
TABLE 1 minimum inhibitory concentrations of the formulations of examples 1-10 and comparative examples 1-10
| MIC | |
| Preparation 1 | 6.4mg/mL |
| Preparation 2 | 12.8mg/mL |
| Preparation 3 | 25.6mg/mL |
| Preparation 4 | 1.6mg/mL |
| Preparation 5 | 1.6mg/mL |
| Preparation 6 | 6.4mg/mL |
| Preparation 7 | 64μg/mL |
| Preparation 8 | 0.5μg/mL |
| Preparation 9 | 4μg/mL |
| Preparation 10 | 16μg/mL |
| Preparation 11 | — |
| Preparation 12 | — |
| Preparation 13 | — |
| Preparation 14 | — |
| Formulation 15 | — |
| Preparation 16 | — |
| Preparation 17 | — |
| Preparation 18 | — |
| Preparation 19 | — |
| Preparation 20 | — |
| Preparation 21 | — |
| Tylosin | 0.0625μg/mL |
As can be seen from Table 1, the extracts obtained by directly extracting Scutellariae radix, herba Ephedrae, and Glycyrrhrizae radix did not detect anti-Mycoplasma gallisepticum activity in the detection range. After the bacillus subtilis is fermented, the obtained fermentation product is extracted, and the anti-mycoplasma gallisepticum activity is detected. The extract fermented by the single traditional Chinese medicine has lower anti-mycoplasma gallisepticum activity, while the extract fermented by the compound traditional Chinese medicine has higher anti-mycoplasma gallisepticum activity, wherein when the scutellaria baicalensis, the ephedra and the liquorice are fermented according to the proportion of 4:3:3, the anti-mycoplasma gallisepticum activity is better and is closer to that of tylosin.
In vivo mycoplasma gallisepticum experiments:
200 laying hens aged 30 weeks in a laying hen farm in a chat city are selected, the laying hens are divided into 4 groups of 50, 1 group is used as a blank control group, the other 3 groups of laying hens are infected with mycoplasma gallisepticum S6, after 7 days of infection, the laying hens have clinical symptoms such as head swing, cough, neck stretching breathing, clear water sample nasal fluid flowing and the like, and the situation that eggs are not laid in one day, blood sampling is carried out by a serum plate agglutination test, and the result is positive, which indicates that the mycoplasma gallisepticum S6 is infected successfully.
Infection group 1 was used as a virus control group, infection group 2 was treated with tylosin mixed with water (test group 1) at a dose of 0.5g per chicken per day, and infection group 3 was treated with formulation 8 prepared in example 8 mixed with water (test group 2) at a dose of 0.5g per chicken per day. The treatment was administered for 5 days, then stopped and observed for 15 days, and the treatment effects are shown in table 2.
TABLE 2 therapeutic effect on Mycoplasma gallisepticum-infected laying hens
The standard of cure is as follows: the clinical symptoms completely disappeared, and each hen recovered to lay one egg a day (in the blank control group, each hen laid one egg a day, total egg production per day was 50).
As can be seen from Table 2, if the layer infected with Mycoplasma gallisepticum is not treated in time, the layer can be killed, and the fatality rate can reach 12%. The mortality rate can be obviously reduced by adopting both tylosin and the preparation 8, the cure rate of the test group 2 adopting the preparation 8 can reach 92 percent, and the obvious difference (P is more than 0.05) is avoided compared with the test group 1 adopting the tylosin.
In conclusion, the preparation prepared by the embodiment of the invention can be used for preventing and treating the mycoplasma gallisepticum, wherein the preparation prepared by the embodiment 8 has more excellent effect and can be used as a substitute of tylosin.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. 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 (10)
1. A preparation method of a preparation for preventing and treating mycoplasma of laying hens is characterized in that bacillus subtilis is inoculated into a beef extract peptone liquid culture medium to obtain bacillus subtilis seed liquid, water is added into a solid culture medium to prepare a fermentation material, the bacillus subtilis seed liquid is inoculated into the fermentation material to be fermented, and the solid fermentation culture medium contains one or more of scutellaria baicalensis, ephedra and liquorice.
2. The method for producing a preparation for controlling mycoplasma gallisepticum as claimed in claim 1, wherein the solid fermentation medium comprises scutellaria baicalensis, ephedra herb, and glycyrrhiza uralensis.
3. The method for preparing a preparation for preventing and treating mycoplasma gallisepticum as claimed in claim 2, wherein the mass ratio of scutellaria baicalensis, ephedra herb and liquorice is 2-3: 1-2.
4. The method for producing a preparation for controlling mycoplasma gallisepticum as claimed in claim 1, wherein the strain of bacillus subtilis has the accession number CICC 24713.
5. The method for preparing a preparation for controlling mycoplasma gallisepticum according to claim 1, wherein the fermentation process comprises: aerobic fermentation is carried out at the temperature of 30-37 ℃.
6. The method for preparing the preparation for preventing and treating the mycoplasma gallisepticum as recited in claim 5, wherein the aerobic fermentation time is 36-72 hours.
7. The method for preparing a preparation for controlling mycoplasma gallisepticum as recited in claim 1, wherein the fermented material is extracted under reflux with an aqueous solution of ethanol. The reflux extraction is carried out by heating to reflux and extracting under reflux condition.
8. The method for preparing a preparation for controlling mycoplasma gallisepticum according to claim 1, wherein the aqueous ethanol solution is 94-96% by volume.
9. The method for producing a preparation for controlling mycoplasma gallisepticum as claimed in claim 1, wherein the fermentation material is sterilized before fermentation.
10. A preparation for controlling mycoplasma in laying hens, which is obtained by the preparation method according to any one of claims 1 to 9.
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