阅读说明：本技术 一种中药微生态制剂的制备方法及其应用 (Preparation method and application of traditional Chinese medicine microecological preparation ) 是由 王琳超 陈营 李晶 韩易航 梁玉 宋捷 赵志成 贾立华 张丽娜 孙利杰 付晓娟 于 2020-06-02 设计创作，主要内容包括：本发明提供了一种中药微生态制剂的制备方法及其应用,属于禽用中药微生态制剂技术领域。海带加水、辅料,灭菌后加入海带降解菌种子液培养发酵,随后以海带发酵液体积为基准,分别加入麦芽汁、红糖、黄芪粉灭菌备用；待发酵罐温度下降后加入5％混合菌种行二次发酵,发酵结束后过滤除渣,得液体制剂,或制成干粉,即得中药微生态制剂。本发明利用海带降解菌进行液态发酵,再加入黄芪粉等由混合菌种行二次液体发酵,有效解决了黄芪单味中药抗菌能力及其他药效低下的缺陷,最终达到显著提升抑菌效果和药物有效成分的效果。经试验证实,由本发明制得的中药微生态制剂相比传统抗生素对家禽禽源致病性大肠杆菌体外抑菌效果更佳,且不会产生耐药性。(The invention provides a preparation method and application of a traditional Chinese medicine microecological preparation, and belongs to the technical field of traditional Chinese medicine microecological preparations for poultry. Adding water and auxiliary materials into the kelp, sterilizing, adding kelp degradation strain seed liquid, culturing and fermenting, and then respectively adding wort, brown sugar and astragalus powder into the kelp fermentation liquid as a reference for sterilization for later use; adding 5% mixed strain for secondary fermentation after the temperature of the fermentation tank is reduced, filtering to remove residue after fermentation to obtain liquid preparation, or making into dry powder to obtain Chinese medicinal microecological preparation. The invention utilizes the kelp degrading bacteria to carry out liquid state fermentation, and then adds the astragalus powder and the like to carry out secondary liquid fermentation by the mixed bacteria, thereby effectively solving the defects of low antibacterial capability and other drug effects of single traditional Chinese medicine of astragalus and finally achieving the effect of obviously improving the antibacterial effect and the active ingredients of the medicine. Experiments prove that compared with the traditional antibiotics, the traditional Chinese medicine microecological preparation prepared by the invention has better in-vitro antibacterial effect on pathogenic escherichia coli of poultry and poultry sources, and does not generate drug resistance.)

1. A method for preparing Chinese medicinal microecological preparation comprises:

(1) material preparation and sterilization: slicing herba Zosterae Marinae, placing into a fermenter, adding clear water and adjuvants, and sterilizing;

(2) primary fermentation: when the temperature in the fermentation tank is reduced to below 40 ℃, adding 5 percent of kelp degradation strain seed liquid into the prepared materials for culture and fermentation to obtain kelp fermentation liquid;

(3) secondary material preparation and sterilization: centrifuging the kelp fermentation liquor to obtain a supernatant for later use; respectively adding 10% of wort (v/v), 10% of brown sugar (m/v) and 7% -10% of astragalus powder (m/v) based on the volume of the clear liquid for sterilization for later use;

(4) and (3) secondary fermentation: when the temperature in the fermentation tank is reduced to below 40 ℃, adding 5 percent of mixed strains for secondary fermentation;

(5) and (3) detection: detecting after the fermentation is finished, and stopping fermentation after the detection meets the conditions to obtain a fermentation product for later use;

(6) preparing a finished product: filtering the fermented product to remove residue, and packaging to obtain liquid preparation, or spray drying to obtain dry powder to obtain the final product.

2. The production method according to claim 1, wherein:

the kelp in the step (1) is selected from any one of dry kelp and fresh kelp;

the auxiliary materials include but are not limited to ammonium sulfate, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate and manganese sulfate.

The sterilization conditions are as follows: sterilizing at 100 deg.C for 30 min.

3. The production method according to claim 2, wherein:

when dried kelp is fermented, 50 times of water is added;

when fresh kelp is fermented, 6 times of water is added.

4. The production method according to claim 1, wherein:

the kelp degrading bacteria seed liquid in the step (2) is prepared by the following method:

(1) adding a naturally-growing kelp sample into a culture solution prepared from kelp, ammonium sulfate, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate and the like according to 5% (m/v), and culturing at 37 ℃ and 150rpm until the kelp is decomposed by more than 90%;

(2) taking a certain amount of the kelp decomposition liquid, inoculating a new culture solution prepared from kelp, ammonium sulfate, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate and the like according to 5% (v/v), continuously culturing until the kelp is decomposed, and continuously repeating the step for 2-3 times;

(3) taking a certain amount of kelp decomposition liquid cultured for the last time, coating a beef extract peptone plate after a series of dilution, culturing at 37 ℃, selecting a single bacterial colony to continue to scribe on the beef extract peptone plate after bacterial colonies grow on the plate until the single bacterial colony appears, continuously selecting the single bacterial colony to repeat scribing on the beef extract peptone plate for 2 times, and obtaining a pure bacterial strain after the single bacterial colony grows;

(4) selecting the purified single colonies, respectively inoculating the single colonies on the surface of a culture medium containing sodium alginate and pectin as unique carbon sources, respectively, obtaining a strain for decomposing the sodium alginate or the pectin if the single colonies grow, and respectively selecting a strain for decomposing the sodium alginate and the pectin, wherein the strains with the best effect are combined together to be used as seed strains;

(5) inoculating the seed strain into a culture solution prepared from kelp, ammonium sulfate, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate and the like, and culturing at 37 ℃ and 150rpm until the kelp is decomposed by more than 90%, thus obtaining an original seed solution;

(6) inoculating 5% (v/v) of the original seed solution into a culture solution prepared from kelp, ammonium sulfate, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate and the like, and culturing at 37 ℃ and 150rpm for 1-2 days until 90% of the kelp is decomposed into liquid, namely the production seed solution.

5. The production method according to claim 1, wherein:

and (3) completely decomposing 90% of the kelp at the fermentation end point in the step (2).

6. The production method according to claim 1, wherein:

the sterilization conditions in the step (3) are as follows: sterilizing at 121 deg.C for 20 min.

7. The production method according to claim 1, wherein:

the mixed strain in the step (4) is prepared by mixing bacillus subtilis, bacillus coagulans and lactobacillus plantarum according to the ratio of bacteria number of 1:1: 2;

the bacillus subtilis is provided by Wuxi alpha Bauer bioengineering limited company;

bacillus coagulans is supplied by Luoyang Ogaku Biotech GmbH;

lactobacillus plantarum is supplied by Yunqi Biotechnology, Inc., Tianjin.

8. The production method according to claim 1, wherein:

the fermentation conditions in the step (5) are that the fermentation is carried out for 48 hours at 37 ℃, pH 6.5-6.8 and rotating speed of 100 rpm.

9. The production method according to claim 8, wherein:

the detection conformity conditions in the step (6) are any 3 or 4 of ① pH value not more than 5.0 and ② bacillus number not less than 1 × 10⁸The number of the CFU ② lactic acid bacteria is more than or equal to 1 × 10⁹④ the total polysaccharide content is increased by more than 30-50% compared with that before fermentation;

the total polysaccharide comprises astragalus polysaccharide, seaweed polysaccharide and seaweed oligosaccharide.

10. A Chinese medicinal microecological preparation prepared by the preparation method according to any one of claims 1 to 9.

Technical Field

The invention belongs to the technical field of traditional Chinese medicine microecologics for poultry, and particularly relates to a traditional Chinese medicine microecologics for improving the growth performance, disease resistance and survival rate of SPF (specific pathogen free) chickens, and a preparation method of the traditional Chinese medicine microecologics.

Background

Chinese medicine is profound and profound, and occupies an important position in Chinese disease prevention and treatment history. The Chinese herbal medicine is used as a natural organic matter, and has the characteristics of greenness, naturalness, no residue, no bacterial drug resistance and the like. After the traditional Chinese medicine is used in animals, the immunity of organisms can be obviously improved, the occurrence of diseases of the cultured animals can be effectively reduced, and the flavor and meat quality of animal products can be improved to different degrees. Not only has the efficacies of health care and disease treatment, but also can improve the ecological environment, and the function of the traditional Chinese veterinary medicine as a feed additive in the livestock and poultry breeding is increasingly important. However, because the raising cost of the cultured animals is limited and the price of the traditional Chinese medicinal materials is high, the raw materials of the traditional Chinese veterinary medicines which are often used in the breeding industry are all from leftovers or inferior-quality traditional Chinese medicinal materials, and the clinical curative effect cannot be well fed back. Therefore, how to improve the release of the active ingredients of the traditional Chinese medicinal materials and the exertion of the pharmacological effects at lower cost is of great importance in veterinary clinical.

The Chinese herbal medicine fermentation technology is a new high-tech Chinese herbal medicine preparation technology formed by combining the research results of micro ecology with the modern microbial engineering on the basis of inheriting the preparation method of Chinese herbal medicines, and utilizes the biotransformation function of organisms to improve the content of active ingredients in the Chinese herbal medicines, change a certain ingredient or generate active ingredients which are not contained in some Chinese herbal medicines, thereby providing a new way for screening active compounds. The modern traditional Chinese medicine fermentation technology abandons the traditional natural fermentation process, takes probiotics derived from the alimentary canal of human or animals as fermentation strains, simulates an in vitro gastrointestinal fermentation system, completes the conversion of the effective components of the Chinese herbal medicine, and directly obtains absorbable micromolecular substances with stronger pharmacological activity metabolites and effective components. Greatly improves the content of active substances in the traditional Chinese medicine, improves the nature and taste of the traditional Chinese medicine, reduces the toxic and side effects of the traditional Chinese medicine, and removes macromolecular impurities in the traditional Chinese medicine. Meanwhile, some components in the Chinese herbal medicines can promote the growth of probiotics, and microorganisms generate a large amount of organic acids (lactic acid, propionic acid), peptides and the like in fermentation, thereby promoting the immune system of the organism.

With the attention of people on food safety and health problems, a green and environment-friendly healthy breeding mode is a necessary trend for the development of animal husbandry in future. Based on the characteristics, the research and development of a green and efficient veterinary preparation or feed additive which has no toxic or side effect, can prevent diseases and promote growth, and has a substitution resistance reducing type are significant.

Disclosure of Invention

According to the technical scheme, the kelp fermentation liquor is prepared by a biological technology, the astragalus powder and auxiliary nutrient substances are added, and then probiotics are inoculated for fermentation to prepare the traditional Chinese medicine microecological preparation containing algal polysaccharides, astragalus polysaccharides and algal oligosaccharides.

In order to realize the purpose, the invention adopts the following technical scheme that:

a method for preparing Chinese medicinal microecological preparation comprises:

(1) material preparation and sterilization: slicing herba Zosterae Marinae, placing into a fermenter, adding clear water and adjuvants, and sterilizing;

(2) primary fermentation: when the temperature in the fermentation tank is reduced to below 40 ℃, adding 5 percent of kelp degradation strain seed liquid into the prepared materials for culture and fermentation to obtain kelp fermentation liquid;

(3) secondary material preparation and sterilization: centrifuging the kelp fermentation liquor to obtain a supernatant for later use; respectively adding 10% of wort (v/v), 10% of brown sugar (m/v) and 7% -10% of astragalus powder (m/v) based on the volume of the clear liquid for sterilization for later use;

(4) and (3) secondary fermentation: when the temperature in the fermentation tank is reduced to below 40 ℃, adding 5 percent of mixed strains for secondary fermentation;

(5) and (3) detection: detecting after the fermentation is finished, and stopping fermentation after the detection meets the conditions to obtain a fermentation product for later use;

(6) preparing a finished product: filtering the fermented product to remove residue, and packaging to obtain liquid preparation, or spray drying to obtain dry powder to obtain the final product.

Further, the kelp in the step (1) is selected from any one of dry kelp and fresh kelp;

the auxiliary materials include but are not limited to ammonium sulfate, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate and manganese sulfate.

The sterilization conditions are as follows: sterilizing at 100 deg.C for 30 min.

Further, when the dried kelp is fermented, 50 times of water is added;

when fresh kelp is fermented, 6 times of water is added.

Further, the seed liquid of the kelp degrading bacteria in the step (2) is prepared by the following method:

(1) adding a naturally-growing kelp sample into a culture solution prepared from kelp, ammonium sulfate, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate and the like according to 5% (m/v), and culturing at 37 ℃ and 150rpm until the kelp is decomposed by more than 90%;

(2) taking a certain amount of the kelp decomposition liquid, inoculating a new culture solution prepared from kelp, ammonium sulfate, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate and the like according to 5% (v/v), continuously culturing until the kelp is decomposed, and continuously repeating the step for 2-3 times;

(3) taking a certain amount of kelp decomposition liquid cultured for the last time, coating a beef extract peptone plate after a series of dilution, culturing at 37 ℃, selecting a single bacterial colony to continue to scribe on the beef extract peptone plate after bacterial colonies grow on the plate until the single bacterial colony appears, continuously selecting the single bacterial colony to repeat scribing on the beef extract peptone plate for 2 times, and obtaining a pure bacterial strain after the single bacterial colony grows;

(4) selecting the purified single colonies, respectively inoculating the single colonies on the surface of a culture medium containing sodium alginate and pectin as unique carbon sources, respectively, obtaining a strain for decomposing the sodium alginate or the pectin if the single colonies grow, and respectively selecting a strain for decomposing the sodium alginate and the pectin, wherein the strains with the best effect are combined together to be used as seed strains;

(5) inoculating the seed strain into a culture solution prepared from kelp, ammonium sulfate, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate and the like, and culturing at 37 ℃ and 150rpm until the kelp is decomposed by more than 90%, thus obtaining an original seed solution;

(6) inoculating 5% (v/v) of original seed solution into culture solution prepared from herba Zosterae Marinae, ammonium sulfate, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, etc., culturing at 37 deg.C and 150rpm for 1-2 days to 90% of sea

The band is decomposed into liquid, namely the production seed liquid.

Further, the fermentation end point of the step (2) is 90% of the kelp is completely decomposed.

Further, the sterilization conditions in the step (3) are as follows: sterilizing at 121 deg.C for 20 min.

Further, the mixed strain in the step (4) is prepared from bacillus subtilis, bacillus coagulans and lactobacillus plantarum according to the number of bacteria

Mixing according to the proportion of 1:1: 2;

the bacillus subtilis is provided by Wuxi alpha Bauer bioengineering limited company;

bacillus coagulans is supplied by Luoyang Ogaku Biotech GmbH;

lactobacillus plantarum is supplied by Yunqi Biotechnology, Inc., Tianjin.

Further, the fermentation conditions in the step (5) are that the fermentation is carried out for 48 hours at 37 ℃, pH 6.5-6.8 and rotating speed of 100 rpm.

Further, the detection conformity conditions in the step (6) are any 3 or 4 of ① pH value being less than or equal to 5.0 and ② bacillus number being more than or equal to 1 × 10⁸The number of the CFU ② lactic acid bacteria is more than or equal to 1 × 10⁹④ the total polysaccharide content is increased by more than 30-50% compared with that before fermentation;

the total polysaccharide comprises astragalus polysaccharide, seaweed polysaccharide and seaweed oligosaccharide.

The invention also discloses a traditional Chinese medicine microecological preparation prepared by any one of the preparation methods.

Through the technical scheme, the invention has the beneficial effects that:

1. the invention adopts the kelp degrading bacteria seed liquid to ferment the kelp, solves the defect of poor degrading effect of a complex enzyme preparation for enzymolysis of terrestrial plants in the prior art, and ensures that the degradation rate of the algin contained in the kelp reaches more than 90 percent. Then adding the astragalus and mixed strains (probiotics) for secondary liquid fermentation, effectively overcoming the defects of low antibacterial ability and other drug effects of single traditional Chinese medicine of the astragalus, and finally achieving the effect of remarkably improving the antibacterial effect and the active ingredients of the medicine.

2. The invention adopts liquid fermentation for two times of fermentation, has high technical content and controllable effect, and the fermentation product is thorough and clear and can be monitored at any time.

3. The traditional Chinese medicine microecological preparation prepared by the invention can effectively improve the test of the in vitro bacteriostatic effect of the traditional Chinese medicine microecological preparation on poultry pathogenic escherichia coli brought by the traditional antibiotics.

4. It should be noted that the technical effect of the present invention is the result of the mutual cooperation and interaction of the process steps and parameters, and is not the superposition of simple processes, and the effect produced by the organic combination of the processes far exceeds the superposition of the functions and effects of each single process, so the present invention has better advancement and practicability.

Drawings

FIG. 1 is a comparison of the effect of mixed strains on the crude polysaccharide content of Astragalus membranaceus before and after fermentation;

FIG. 2 is a comparison of the effect of the mixed strains on the total flavone content of Astragalus membranaceus before and after fermentation;

FIG. 3 is a comparison of the effect of the mixed strains on the total saponins content of Astragalus before and after fermentation.

Detailed Description