阅读说明：本技术 一种替代饲用抗生素的中草药复合物智能筛选方法 (Intelligent screening method of Chinese herbal medicine compound for replacing feed antibiotics ) 是由 吴信 于 2020-10-20 设计创作，主要内容包括：本发明公开了一种替代饲用抗生素的中草药复合物智能筛选方法,包括以下步骤：按一定比例称取所用中草药残枝,去除杂质后晒干混合粉碎,然后对发酵前中草药中残枝成份分析以及生物学功能性分析,再按中草药、水、营养盐一定比重混合,升温并浸泡一段时间,从相应的中草药残枝分离和筛选适用于发酵的菌株,通过16S rDNA鉴定其菌属,确立筛选菌株在以相应中草药残枝为底物的成长曲线以及pH和还原糖的变化,将筛选的菌种加入到发酵罐中,并向发酵罐中加入营养物,混匀后得到发酵培养基。本发明可以有针对性的对菌属进行筛选,减少对菌属培养的盲目性,可以较好的替代原有的抗生素制品,且可以对中草药中的废物进行利用,比较环保。(The invention discloses an intelligent screening method of a Chinese herbal medicine compound for replacing feed antibiotics, which comprises the following steps: weighing the used Chinese herbal medicine residual branches according to a certain proportion, removing impurities, drying, mixing, crushing, analyzing the components of the residual branches in the Chinese herbal medicine before fermentation and analyzing the biological functionality, mixing the Chinese herbal medicine, water and nutrient salt according to a certain proportion, heating and soaking for a period of time, separating and screening strains suitable for fermentation from the corresponding Chinese herbal medicine residual branches, identifying the strains through 16S rDNA, determining the growth curve of the screened strains by taking the corresponding Chinese herbal medicine residual branches as substrates and the change of pH and reducing sugar, adding the screened strains into a fermentation tank, adding nutrients into the fermentation tank, and uniformly mixing to obtain a fermentation culture medium. The invention can be used for screening the bacterial genus with pertinence, reduces the blindness of bacterial genus culture, can better replace the original antibiotic product, can utilize the waste in the Chinese herbal medicine, and is more environment-friendly.)

1. An intelligent screening method of a Chinese herbal medicine compound for replacing feed antibiotics is characterized by comprising the following steps:

(1): weighing residual branches of the Chinese herbal medicines according to a certain proportion, removing impurities, drying in the sun, mixing and crushing;

(2): analyzing the components of the residual branches in the Chinese herbal medicine before fermentation and analyzing the biological functionality;

(3): mixing the Chinese herbal medicines, water and nutrient salt according to the weight ratio of 2: 8: 6, heating to 95-100 ℃, soaking for 2-4 hours, and leaching and sterilizing the water-soluble active substances of the Chinese herbal medicines;

(4) separating and screening strains suitable for fermentation from corresponding Chinese herbal medicine residual branches, identifying the genus of the strains through 16S rDNA, determining the activities of cellulase, ligninase, xylanase, protease and amylase, detecting the hemolytic property, antibiotic resistance and the like of the separated strains, and determining the growth curve of the screened strains and the change of pH and reducing sugar by taking the corresponding Chinese herbal medicine residual branches as substrates;

(5): adding the strains screened in the step (4) into a fermentation tank, adding nutrients into the fermentation tank, and uniformly mixing to obtain a fermentation culture medium;

(6): filtering the fermentation liquor after fermentation is finished, adding the obtained filtrate into a concentrator, drying the filter residue and the concentrated solution to obtain a blocky antibiotic substitute, and crushing the blocky antibiotic substitute to obtain a product;

(7) analyzing the components of the residual branches in the fermented Chinese herbal medicines and analyzing the biological functionality.

2. The intelligent screening method of a Chinese herbal medicine compound for replacing feed antibiotics as claimed in claim 1, wherein the Chinese herbal medicine twig comprises 15-29 parts of eucommia ulmoides, 10-20 parts of astragalus membranaceus, 25-35 parts of ampelopsis grossedentata, 16-31 parts of mulberry and 10-21 parts of gardenia.

3. The intelligent screening method of Chinese herbal medicine compounds for replacing feed antibiotics as claimed in claim 2, wherein the Chinese herbal medicine twig comprises the following components in parts by weight: 16 parts of eucommia bark, 14 parts of astragalus, 26 parts of vine tea, 24 parts of mulberry and 20 parts of cape jasmine.

4. The intelligent screening method of Chinese herbal medicine compounds for replacing feed antibiotics as claimed in claim 2, wherein the Chinese herbal medicine twig comprises the following components in parts by weight: 18 parts of eucommia bark, 20 parts of astragalus, 27 parts of vine tea, 25 parts of mulberry and 10 parts of cape jasmine.

5. The intelligent screening method of Chinese herbal medicine compounds for replacing feed antibiotics as claimed in claim 2, wherein the Chinese herbal medicine twig comprises the following components in parts by weight: 17 parts of eucommia bark, 18 parts of astragalus, 25 parts of vine tea, 27 parts of mulberry and 13 parts of cape jasmine.

6. The intelligent screening method of Chinese herbal medicine compound for replacing feed antibiotics of claim 1, wherein the nutrients in the step (4) comprise sugar, peptone, growth factors, beef extract and inorganic salts, and the weight ratio of the nutrients to the nutrients is 1: 0.35-0.5: 0.6-0.8: 0.3-0.45: 0.2-0.3, said saccharides comprising: glucose and sucrose, the growth factors comprise vitamins, amino acids, purine and pyrimidine, and the inorganic salts comprise potassium, sodium, iron, magnesium, calcium, phosphorus and sulfur.

7. The intelligent screening method of Chinese herbal medicine compound for replacing feed antibiotics of claim 1, wherein the pH value of the fermentation in the step (5) is 7.0-7.3, and the fermentation temperature is 29-31 ℃.

8. The intelligent screening method of Chinese herbal medicine compound for replacing feed antibiotics of claim 1, wherein the fermentation in the step (5) is performed by repeated semi-batch type operation, and the repeated semi-batch type method comprises the following steps: after the feeding operation is finished, taking out part of the reaction system, adding a certain amount of substrate again into the rest part, and then carrying out the feeding operation according to the feeding operation.

9. The intelligent screening method of Chinese herbal medicine compound for replacing feed antibiotics according to claim 1, characterized in that the method for identifying 16S rDNA in the step (4) comprises the following steps: nucleic acid extraction → gene amplification → product purification → sequencing reaction → sequence alignment.

Technical Field

The invention relates to the field of feed additive production, in particular to an intelligent screening method of a Chinese herbal medicine compound for replacing feed antibiotics.

Background

The antibiotic plays an important role in the treatment of human and animal diseases, however, the long-term use of the antibiotic not only causes pathogenic bacteria to generate drug resistance, but also causes the problem of drug residue in animal products and ecological environment, thereby causing great threat to human health and safety, and the efficient, environment-friendly and non-toxic and side effects green feed additive is gradually replacing the antibiotic in livestock and poultry breeding industry, thus having wide application prospect.

Chinese herbal medicines as feed additives are recorded in China for a long time, are scientifically combined according to the theory of traditional Chinese medicines, are products of the special theory and practice of traditional Chinese medicines in China, generally consider that the effective components of the Chinese herbal medicine feed additives mainly comprise alkaloids, glycosides, volatile oil, tannins, saccharides, amino acids, proteins, enzymes, grease, inorganic components, pigments and the like, and the glycosides can obviously enhance the immunity of organisms; the tannin has inhibitory effect on Pseudomonas aeruginosa, Escherichia coli, cholera bacillus, diphtheria bacillus, etc.; the alkaloid can increase enterokinesia, promote digestive juice secretion, and stimulate appetite; some Chinese herbal medicines contain rich proteins, vitamins and mineral elements, and have dual functions of medicine effect and nutrition, in recent years, free organic acids in the Chinese herbal medicines are also found to have biological activity, for example, formic acid has anti-inflammatory and analgesic effects, chlorogenic acid has an antibacterial effect, phytochrome can also improve the color and luster of livestock and poultry products (meat, eggs), after Chinese herbal medicine dysentery stopping grass extracts are added into broiler feed, the feed reward of broiler chickens can be improved, the feed conversion ratio is reduced, the serum IgG level is increased, the coccidiosis of broiler chickens is reduced, at present, more Chinese herbal medicines such as pulsatilla chinensis, astragalus membranaceus, coptis chinensis, codonopsis pilosula, rheum officinale, honey-fried licorice roots and the like are used in chicken breeding industry, the types of plant extracts mainly comprise saponins, alkaloids, polysaccharides, saccharicterpenin, tea polyphenols, flavones, essential oils and the like, and the application research of plant essential oils in industrial production is further developed in recent years, such as clove oil, oregano oil, thymol, tea tree oil, cinnamon oil, rosemary essence oil and the like, all have better antibacterial, anti-inflammatory and antioxidant effects, and the Chinese herbal medicine preparation becomes an ideal feed additive due to the advantages of wide sources, low price, safety, convenience, small toxic and side effects, no residue, no drug resistance and the like, under the condition of ordinary feeding, the preparation is added into daily ration for feeding or drinking of animals so as to prevent animal diseases, accelerate growth, improve production performance and improve the quality of livestock and poultry products, however, the current research on the pharmacological action and action mechanism of Chinese herbs is weak, and a great deal of work is needed, such as whether toxic and side effects exist after long-term use, whether the feed has synergistic or resistant effect with other nutrient components or not needs to be further discussed and researched, which is beneficial to further development and utilization of the Chinese herbal medicine feed additive.

The existing screening method of the Chinese herbal medicine compound for replacing the feed antibiotics is not perfect enough, can not effectively screen and culture target products, and is not green enough, so that the intelligent screening method of the Chinese herbal medicine compound for replacing the feed antibiotics is provided.

Disclosure of Invention

The invention aims to provide an intelligent screening method of a Chinese herbal medicine compound for replacing a feed antibiotic, and solves the problems that the screening method of the Chinese herbal medicine compound for replacing the feed antibiotic is not perfect enough, a target product cannot be screened and cultured effectively, and the screening method is not green enough.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention provides an intelligent screening method of a Chinese herbal medicine compound for replacing feed antibiotics, which is prepared by the following steps:

(1): weighing residual branches of the Chinese herbal medicines according to a certain proportion, removing impurities, drying in the sun, mixing and crushing;

(2): analyzing the components of the residual branches in the Chinese herbal medicine before fermentation and analyzing the biological functionality;

(3): mixing the Chinese herbal medicines, water and nutrient salt according to the weight ratio of 2: 8: 6, heating to 95-100 ℃, soaking for 2-4 hours, and leaching and sterilizing the water-soluble active substances of the Chinese herbal medicines;

(4) separating and screening strains suitable for fermentation from corresponding Chinese herbal medicine residual branches, identifying the genus of the strains through 16SrDNA, determining the activities of cellulase, ligninase, xylanase, protease and amylase, detecting the hemolytic property, antibiotic resistance and the like of the separated strains, and establishing a growth curve of the screened strains and the changes of pH and reducing sugar by taking the corresponding Chinese herbal medicine residual branches as substrates;

(5): adding the strains screened in the step (4) into a fermentation tank, adding nutrients into the fermentation tank, and uniformly mixing to obtain a fermentation culture medium;

(6): filtering the fermentation liquor after fermentation is finished, adding the obtained filtrate into a concentrator, drying the filter residue and the concentrated solution to obtain a blocky antibiotic substitute, and crushing the blocky antibiotic substitute to obtain a product;

(7) analyzing the components of the residual branches in the fermented Chinese herbal medicines and analyzing the biological functionality.

Preferably, the Chinese herbal medicine residual branches comprise 15-29 parts of eucommia ulmoides, 10-20 parts of astragalus membranaceus, 25-35 parts of vine tea, 16-31 parts of mulberry and 10-21 parts of cape jasmine.

Preferably, the Chinese herbal medicine twig comprises the following components in parts by weight: 16 parts of eucommia bark, 14 parts of astragalus, 26 parts of vine tea, 24 parts of mulberry and 20 parts of cape jasmine.

Preferably, the Chinese herbal medicine twig comprises the following components in parts by weight: 18 parts of eucommia bark, 20 parts of astragalus, 27 parts of vine tea, 25 parts of mulberry and 10 parts of cape jasmine.

Preferably, the Chinese herbal medicine twig comprises the following components in parts by weight: 17 parts of eucommia bark, 18 parts of astragalus, 25 parts of vine tea, 27 parts of mulberry and 13 parts of cape jasmine.

Preferably, the nutrient in step (4) is composed of sugar, peptone, growth factor, beef extract and inorganic salts, and the weight ratio is 1: 0.35-0.5: 0.6-0.8: 0.3-0.45: 0.2-0.3, said saccharides comprising: glucose and sucrose, the growth factors comprise vitamins, amino acids, purine and pyrimidine, and the inorganic salts comprise potassium, sodium, iron, magnesium, calcium, phosphorus and sulfur.

Preferably, the pH value of the fermentation in the step (5) is 7.0-7.3, and the fermentation temperature is 29-31 ℃.

Preferably, the fermentation in step (5) is carried out in a repeated semi-batch process, wherein the repeated semi-batch process comprises: after the feeding operation is finished, taking out part of the reaction system, adding a certain amount of substrate again into the rest part, and then carrying out the feeding operation according to the feeding operation.

Preferably, the method for identifying by using 16SrDNA in the step (4) comprises the following steps: nucleic acid extraction → gene amplification → product purification → sequencing reaction → sequence alignment.

The invention has the beneficial effects that: the method comprises the steps of firstly separating and screening strains suitable for fermentation from corresponding Chinese herbal medicine residual branches, identifying the strains through 16SrDNA, determining the activities of cellulase, ligninase, xylanase, protease and amylase, detecting the hemolytic property, antibiotic resistance and the like of the separated strains, determining the growth curve of the screened strains by taking the corresponding Chinese herbal medicine residual branches as substrates and the change of pH and reducing sugar, and being capable of screening the strains in a targeted manner, reducing the blindness of strain culture, better replacing the original antibiotic products, utilizing waste in Chinese herbal medicines and being more environment-friendly.

Detailed Description

The following further describes embodiments of the present invention with reference to examples. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.

Example 1

The preparation method comprises the following specific steps:

(1) preparing the following components in parts by weight: 16 parts of eucommia bark, 14 parts of astragalus, 26 parts of vine tea, 24 parts of mulberry and 20 parts of gardenia, removing impurities, drying in the sun, mixing and crushing;

(2): analyzing the components of the residual branches in the Chinese herbal medicine before fermentation and analyzing the biological functionality;

(3): mixing the Chinese herbal medicines, water and nutrient salt according to the weight ratio of 2: 8: 6, heating to 95-100 ℃, soaking for 2-4 hours, and leaching and sterilizing the water-soluble active substances of the Chinese herbal medicines;

(4) separating and screening strains suitable for fermentation from corresponding Chinese herbal medicine residual branches, identifying the genus of the strains through 16SrDNA, determining the activities of cellulase, ligninase, xylanase, protease and amylase, detecting the hemolytic property, antibiotic resistance and the like of the separated strains, and establishing a growth curve of the screened strains and the changes of pH and reducing sugar by taking the corresponding Chinese herbal medicine residual branches as substrates;

(5): adding the strains screened in the step (4) into a fermentation tank, adding nutrients into the fermentation tank, and uniformly mixing to obtain a fermentation culture medium;

(6): filtering the fermentation liquor after fermentation is finished, adding the obtained filtrate into a concentrator, drying the filter residue and the concentrated solution to obtain a blocky antibiotic substitute, and crushing the blocky antibiotic substitute to obtain a product;

(7) analyzing the components of the residual branches in the fermented Chinese herbal medicines and analyzing the biological functionality.

Example 2

The preparation method comprises the following specific steps:

(1) preparing according to the components and weight components: the Chinese herbal medicine residual branches comprise the following components in parts by weight: 18 parts of eucommia bark, 20 parts of astragalus, 27 parts of vine tea, 25 parts of mulberry and 10 parts of cape jasmine;

(2): analyzing the components of the residual branches in the Chinese herbal medicine before fermentation and analyzing the biological functionality;

(3): mixing the Chinese herbal medicines, water and nutrient salt according to the weight ratio of 2: 8: 6, heating to 95-100 ℃, soaking for 2-4 hours, and leaching and sterilizing the water-soluble active substances of the Chinese herbal medicines;

(4) separating and screening strains suitable for fermentation from corresponding Chinese herbal medicine residual branches, identifying the genus of the strains through 16SrDNA, determining the activities of cellulase, ligninase, xylanase, protease and amylase, detecting the hemolytic property, antibiotic resistance and the like of the separated strains, and establishing a growth curve of the screened strains and the changes of pH and reducing sugar by taking the corresponding Chinese herbal medicine residual branches as substrates;

(5): adding the strains screened in the step (4) into a fermentation tank, adding nutrients into the fermentation tank, and uniformly mixing to obtain a fermentation culture medium;

(6): filtering the fermentation liquor after fermentation is finished, adding the obtained filtrate into a concentrator, drying the filter residue and the concentrated solution to obtain a blocky antibiotic substitute, and crushing the blocky antibiotic substitute to obtain a product;

(7) analyzing the components of the residual branches in the fermented Chinese herbal medicines and analyzing the biological functionality.

Example 3

The preparation method comprises the following specific steps:

(1) preparing according to the components and weight components: the Chinese herbal medicine residual branches comprise the following components in parts by weight: 17 parts of eucommia bark, 18 parts of astragalus, 25 parts of vine tea, 27 parts of mulberry and 13 parts of gardenia;

(2): analyzing the components of the residual branches in the Chinese herbal medicine before fermentation and analyzing the biological functionality;

(3): mixing the Chinese herbal medicines, water and nutrient salt according to the weight ratio of 2: 8: 6, heating to 95-100 ℃, soaking for 2-4 hours, and leaching and sterilizing the water-soluble active substances of the Chinese herbal medicines;

(4) separating and screening strains suitable for fermentation from corresponding Chinese herbal medicine residual branches, identifying the genus of the strains through 16SrDNA, determining the activities of cellulase, ligninase, xylanase, protease and amylase, detecting the hemolytic property, antibiotic resistance and the like of the separated strains, and establishing a growth curve of the screened strains and the changes of pH and reducing sugar by taking the corresponding Chinese herbal medicine residual branches as substrates;

(5): adding the strains screened in the step (4) into a fermentation tank, adding nutrients into the fermentation tank, and uniformly mixing to obtain a fermentation culture medium;

(6): filtering the fermentation liquor after fermentation is finished, adding the obtained filtrate into a concentrator, drying the filter residue and the concentrated solution to obtain a blocky antibiotic substitute, and crushing the blocky antibiotic substitute to obtain a product;

(7) analyzing the components of the residual branches in the fermented Chinese herbal medicines and analyzing the biological functionality.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.