CN113881607A - Research and development method for preventing and treating avian clostridium perfringens - Google Patents
Research and development method for preventing and treating avian clostridium perfringens Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 25
- 241000271566 Aves Species 0.000 title claims abstract description 21
- 241000193468 Clostridium perfringens Species 0.000 title claims abstract description 18
- 238000012827 research and development Methods 0.000 title abstract description 7
- 239000001963 growth medium Substances 0.000 claims abstract description 54
- 239000006041 probiotic Substances 0.000 claims abstract description 31
- 235000018291 probiotics Nutrition 0.000 claims abstract description 31
- 241001515965 unidentified phage Species 0.000 claims abstract description 21
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 16
- 238000012258 culturing Methods 0.000 claims abstract description 16
- 238000009630 liquid culture Methods 0.000 claims abstract description 14
- 230000004913 activation Effects 0.000 claims abstract description 10
- 238000004321 preservation Methods 0.000 claims abstract description 10
- 230000002265 prevention Effects 0.000 claims abstract description 10
- 238000005119 centrifugation Methods 0.000 claims abstract description 9
- 235000016709 nutrition Nutrition 0.000 claims abstract description 8
- 230000035764 nutrition Effects 0.000 claims abstract description 8
- 230000003213 activating effect Effects 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims description 20
- 241001052560 Thallis Species 0.000 claims description 17
- 238000007710 freezing Methods 0.000 claims description 16
- 230000008014 freezing Effects 0.000 claims description 16
- 230000001717 pathogenic effect Effects 0.000 claims description 16
- 230000000529 probiotic effect Effects 0.000 claims description 16
- 239000000725 suspension Substances 0.000 claims description 16
- 238000011161 development Methods 0.000 claims description 12
- 238000004108 freeze drying Methods 0.000 claims description 8
- 244000144977 poultry Species 0.000 claims description 8
- 238000010171 animal model Methods 0.000 claims description 7
- 230000036541 health Effects 0.000 claims description 7
- 239000003513 alkali Substances 0.000 claims description 6
- 238000010972 statistical evaluation Methods 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 4
- 229910021641 deionized water Inorganic materials 0.000 claims description 4
- 239000000839 emulsion Substances 0.000 claims description 4
- 238000011156 evaluation Methods 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 238000004806 packaging method and process Methods 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 239000003223 protective agent Substances 0.000 claims description 4
- 230000001681 protective effect Effects 0.000 claims description 4
- 238000004537 pulping Methods 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000009374 poultry farming Methods 0.000 claims description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims 1
- 235000015097 nutrients Nutrition 0.000 claims 1
- 239000003242 anti bacterial agent Substances 0.000 abstract description 15
- 241001465754 Metazoa Species 0.000 abstract description 14
- 229940088710 antibiotic agent Drugs 0.000 abstract description 10
- 241000894006 Bacteria Species 0.000 abstract description 8
- 229940079593 drug Drugs 0.000 abstract description 7
- 239000003814 drug Substances 0.000 abstract description 7
- 238000006243 chemical reaction Methods 0.000 abstract description 4
- 238000009395 breeding Methods 0.000 abstract 1
- 230000001488 breeding effect Effects 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 238000005728 strengthening Methods 0.000 description 5
- 230000009471 action Effects 0.000 description 3
- 230000004888 barrier function Effects 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 230000002238 attenuated effect Effects 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
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Abstract
The invention relates to the technical field of biological antibacterial agents, and discloses a research and development method for preventing and treating avian clostridium perfringens, which comprises the following steps: s1: activating strains, collecting and selecting excellent strains, and periodically putting the strains into an activation barrel for activation according to the preservation periods of different preservation temperatures; s2: culturing in a culture medium, namely culturing the activated strains in a liquid culture medium, preferably selecting a basic culture medium when culturing in the liquid culture medium, and then performing nutrition enrichment on the basic culture medium for use; s3: the cells were collected by centrifugation. The biological antibacterial agent can effectively prevent and treat NE and improve the feed conversion rate, provides a new way for the ecological healthy breeding of animals by combining the biological antibacterial agent with the bacteriophage cocktail, can establish a biological prevention and control system based on the bacteriophage and probiotics, formulate a reasonable antibacterial combination scheme, reduce the use of antibiotics, prevent the generation of drug-resistant bacteria and establish sustainable animal welfare.
Description
Technical Field
The invention relates to the technical field of biological antibacterial agents, in particular to a research and development method for preventing and treating avian clostridium perfringens.
Background
Bacterial drug resistance is a focus under the condition of great health, how to reduce antibiotics, and effective control of bacterial diseases are imperative, and bacteriophage is difficult to generate resistance due to the specificity of the bacteriophage and has the advantages of the biological antibacterial drug, and the like.
At present, no phage product is sold on the market, the phage cannot be effectively applied, and the traditional antibiotics are attenuated along with time, are not efficient and durable enough, and cannot meet the requirements of people, so that a research and development method for preventing and treating avian clostridium perfringens is provided.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a research and development method for preventing and treating avian clostridium perfringens, and solves the problems that bacteriophage cannot be effectively applied, and the traditional antibiotics are attenuated with time, are not efficient and durable enough and cannot meet the requirements of people.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme:
a development method for controlling avian perfringens clostridium comprises the following steps:
s1: activating strains, collecting and selecting excellent strains, and periodically putting the strains into an activation barrel for activation according to the preservation periods of different preservation temperatures;
s2: culturing in a culture medium, namely culturing the activated strains in a liquid culture medium, preferably selecting a basic culture medium when culturing in the liquid culture medium, and then performing nutrition enrichment on the basic culture medium for use;
s3: centrifugally collecting thalli, carrying out centrifugal treatment on the thalli after culture of a culture medium, and then collecting the thalli;
s4: preparing suspension, adding the centrifuged thallus into a mixing tank, and then adding a protective agent to prepare the suspension;
s5: subpackaging, namely subpackaging the prepared suspension, and performing ultraviolet sterilization treatment on packaging bottles during subpackaging;
s6: freeze drying, namely putting the subpackaged materials into a freezing chamber for freeze drying;
s7: establishing a model, establishing an animal model of the phage cocktail and the probiotic preparation, and performing clinical test and evaluation;
s8: in the test, the bacteriophage cocktail and the probiotic preparation are jointly applied to the prevention and treatment of NE in the large-scale poultry farm, and the antibacterial effect and the protective effect of preventing and treating NE are evaluated.
As a still further scheme of the present invention, in S1, Cp flora characteristics and pathogenic characteristics in poultry farming are analyzed by metagenome analysis of pathogenic flora data information of the avian NE.
Further, the basic culture medium in the S2 is NB liquid culture medium, 40ml of physiological saline with the mass fraction of 0.85% is added when the nutrition of the basic culture medium is strengthened, and the basal culture medium is cultured for 5 to 18 hours at the temperature of between 22 and 36 ℃.
On the basis of the scheme, the rotation speed during centrifugation in the S3 is 800-1800r/min, and the centrifugation time is 2-4 minutes.
Further, thalli are added into the S4, then the mixture is placed into an alkali solution with the temperature of 70-80 ℃ and the concentration of 2-6%, then the mixture is repeatedly washed by deionized water and an acid solution, and then the mixture is placed into a pulping machine to form emulsion; then placed in a centrifuge to obtain a suspension.
On the basis of the scheme, Cp specific bacteriophage and probiotic bacteria are obtained for pathogenic characteristics in S4, and a bacteriophage cocktail and probiotic health preparation capable of resisting the main pathogenic type in Cp is developed.
In a further aspect of the present invention, the temperature of the freezing step in S6 is-280-0 ℃, and the humidity in the freezing chamber is monitored in real time during the freezing step.
Further, in the step S8, data is collected during the application, and then statistical evaluation is performed, and the data is filtered during the statistical process to remove data that are seriously deviated from the exponential line, so as to improve the referential performance of the data.
(III) advantageous effects
Compared with the prior art, the invention provides a research and development method for preventing and treating avian clostridium perfringens, which has the following beneficial effects:
1. the biological antibacterial agent which can effectively prevent and control NE and improve the feed conversion rate is obtained by establishing the chick intestinal microbial barrier by combining with probiotics, and a new way is provided for the ecological healthy culture of animals by combining with bacteriophage cocktail.
2. According to the invention, through clinical experiments of animal models and application of large-scale farms, the speed of developing commercial phage antibacterial preparations and probiotic health-care products aiming at Cp can be increased, a biological prevention and control system based on phage and probiotic is established, a reasonable antibacterial combination scheme is formulated, the use of antibiotics is reduced, the generation of drug-resistant bacteria is prevented, and sustainable animal welfare is established.
3. In the invention, the activity of the strains in the culture medium can be improved by strengthening the culture medium, and the culture effect is improved.
4. According to the invention, the bacteriophage cocktail and probiotics are gradually developed into a commercialized combined antibacterial preparation for preventing and treating NE, so that the use of antibiotics is reduced, the generation of drug-resistant bacteria is avoided, the welfare of animals is enhanced, the economic loss caused by NE is reduced, and the health of the animals is guaranteed.
Drawings
Fig. 1 is a schematic flow structure diagram of a development method for controlling avian clostridium perfringens provided by the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Referring to fig. 1, a development method for controlling clostridium perfringens avian comprises the following steps:
s1: activating strains, collecting and selecting excellent strains, and periodically putting the strains into an activation barrel for activation according to the preservation periods of different preservation temperatures;
s2: culturing in a culture medium, namely culturing the activated strains in a liquid culture medium, preferably selecting a basic culture medium when culturing in the liquid culture medium, and then performing nutrition enrichment on the basic culture medium for use;
s3: centrifugally collecting thalli, carrying out centrifugal treatment on the thalli after culture of a culture medium, and then collecting the thalli;
s4: preparing suspension, adding the centrifuged thallus into a mixing tank, and then adding a protective agent to prepare the suspension;
s5: subpackaging, namely subpackaging the prepared suspension, and performing ultraviolet sterilization treatment on packaging bottles during subpackaging;
s6: freeze drying, namely putting the subpackaged materials into a freezing chamber for freeze drying;
s7: establishing a model, establishing animal models of bacteriophage cocktail and probiotic preparations, carrying out clinical tests and evaluation, establishing a chick intestinal microbial barrier by combining with probiotics to obtain a biological antibacterial preparation capable of effectively preventing and treating NE and improving feed conversion rate, and providing a new way for ecological and healthy culture of animals by combining with the bacteriophage cocktail;
s8: the experiment shows that the bacteriophage cocktail and the probiotic preparation are jointly applied to prevention and treatment of NE in the large-scale poultry farm, the antibacterial effect and the protective effect for preventing and treating the NE are evaluated, the speed of developing commercial bacteriophage antibacterial preparations and probiotic health-care products aiming at Cp can be accelerated through animal model clinical experiments and application of the large-scale farm, a biological prevention and control system based on the bacteriophage and the probiotic is established, a reasonable antibacterial combination scheme is formulated, the use of antibiotics is reduced, the generation of drug-resistant bacteria is prevented, and sustainable animal welfare is established.
In the invention, in S1, pathogenic flora data information of the poultry NE is analyzed through metagenome, Cp flora characteristics and pathogenic characteristics in poultry cultivation are analyzed, a basic culture medium in S2 is an NB liquid culture medium, 40ml of physiological saline with the mass fraction of 0.85% is added when the basic culture medium is subjected to nutrition strengthening, the culture medium is placed at 26 ℃ for 8 hours of culture, the activity of strains in the culture medium can be improved through strengthening of the culture medium, the culture effect is improved, the rotating speed is 1800r/min during centrifugation in S3, the centrifugation time is 4 minutes, thalli are obviously added in S4, then the culture medium is placed in an aqueous alkali with the temperature of 80 ℃ and the concentration of 6%, then the culture medium is repeatedly washed by deionized water and an acid solution, and then the culture medium is placed in a pulping machine to form an emulsion; then placed in a centrifuge to obtain a suspension.
Particularly, in S4, specific Cp phages and probiotics are obtained aiming at pathogenic characteristics, phage cocktails and probiotics capable of resisting main pathogenic types in Cp and health-care preparations of probiotics are developed, the phage cocktails and the probiotics are gradually developed into commercial combined antibacterial preparations for NE prevention and treatment, the use of antibiotics is reduced, drug-resistant bacteria are avoided, the welfare of animals is enhanced, economic loss caused by NE is reduced, the health of the animals is guaranteed, the temperature is-280 ℃ when freezing is carried out in S6, the humidity in a freezing chamber is monitored in real time when freezing is carried out, data are collected when application is carried out in S8, then statistical evaluation is carried out, the data are filtered during statistical evaluation, data which are seriously deviated from an exponential line are removed, and the reference of the data is improved.
Example 2
Referring to fig. 1, a development method for controlling clostridium perfringens avian comprises the following steps:
s1: activating strains, collecting and selecting excellent strains, and periodically putting the strains into an activation barrel for activation according to the preservation periods of different preservation temperatures;
s2: culturing in a culture medium, namely culturing the activated strains in a liquid culture medium, preferably selecting a basic culture medium when culturing in the liquid culture medium, and then performing nutrition enrichment on the basic culture medium for use;
s3: centrifugally collecting thalli, carrying out centrifugal treatment on the thalli after culture of a culture medium, and then collecting the thalli;
s4: preparing suspension, adding the centrifuged thallus into a mixing tank, and then adding a protective agent to prepare the suspension;
s5: subpackaging, namely subpackaging the prepared suspension, and performing ultraviolet sterilization treatment on packaging bottles during subpackaging;
s6: freeze drying, namely putting the subpackaged materials into a freezing chamber for freeze drying;
s7: establishing a model, establishing animal models of bacteriophage cocktail and probiotic preparations, carrying out clinical tests and evaluation, establishing a chick intestinal microbial barrier by combining with probiotics to obtain a biological antibacterial preparation capable of effectively preventing and treating NE and improving feed conversion rate, and providing a new way for ecological and healthy culture of animals by combining with the bacteriophage cocktail;
s8: the experiment shows that the bacteriophage cocktail and the probiotic preparation are jointly applied to prevention and treatment of NE in the large-scale poultry farm, the antibacterial effect and the protective effect for preventing and treating the NE are evaluated, the speed of developing commercial bacteriophage antibacterial preparations and probiotic health-care products aiming at Cp can be accelerated through animal model clinical experiments and application of the large-scale farm, a biological prevention and control system based on the bacteriophage and the probiotic is established, a reasonable antibacterial combination scheme is formulated, the use of antibiotics is reduced, the generation of drug-resistant bacteria is prevented, and sustainable animal welfare is established.
In the invention, in S1, pathogenic flora data information of the poultry NE is analyzed through metagenome, Cp flora characteristics and pathogenic characteristics in poultry cultivation are analyzed, a basic culture medium in S2 is an NB liquid culture medium, 40ml of physiological saline with the mass fraction of 0.85% is added when the basic culture medium is subjected to nutrition strengthening, the culture medium is placed at 26 ℃ for culturing for 9 hours, the activity of strains in the culture medium can be improved through strengthening the culture medium, the culture effect is improved, the rotating speed is 1200r/min during centrifugation in S3, the centrifugation time is 3 minutes, thalli are added in S4 in number, then the culture medium is placed in an alkali solution with the temperature of 80 ℃ and the concentration of 6%, then deionized water and an acid solution are repeatedly washed, and then the mixture is placed in a pulping machine to form an emulsion; then placed in a centrifuge to obtain a suspension.
Particularly, in S4, specific Cp phages and probiotics are obtained aiming at pathogenic characteristics, phage cocktails and probiotics capable of resisting main pathogenic types in Cp and health care preparations of probiotics are developed, the phage cocktails and the probiotics are gradually developed into commercial combined antibacterial preparations for NE prevention and treatment, the use of antibiotics is reduced, drug-resistant bacteria are avoided, the welfare of animals is enhanced, economic loss caused by NE is reduced, the health of the animals is guaranteed, the temperature is-180 ℃ when freezing is carried out in S6, the humidity in a freezing chamber is monitored in real time when freezing is carried out, data are collected when application is carried out in S8, then statistical evaluation is carried out, the data are filtered during statistical evaluation, data which are seriously deviated from an exponential line are removed, and the reference of the data is improved.
In the description herein, it is noted that relational terms such as first and second, and the like, are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A development method for preventing and treating avian clostridium perfringens is characterized by comprising the following steps:
s1: activating strains, collecting and selecting excellent strains, and periodically putting the strains into an activation barrel for activation according to the preservation periods of different preservation temperatures;
s2: culturing in a culture medium, namely culturing the activated strains in a liquid culture medium, preferably selecting a basic culture medium when culturing in the liquid culture medium, and then performing nutrition enrichment on the basic culture medium for use;
s3: centrifugally collecting thalli, carrying out centrifugal treatment on the thalli after culture of a culture medium, and then collecting the thalli;
s4: preparing suspension, adding the centrifuged thallus into a mixing tank, and then adding a protective agent to prepare the suspension;
s5: subpackaging, namely subpackaging the prepared suspension, and performing ultraviolet sterilization treatment on packaging bottles during subpackaging;
s6: freeze drying, namely putting the subpackaged materials into a freezing chamber for freeze drying;
s7: establishing a model, establishing an animal model of the phage cocktail and the probiotic preparation, and performing clinical test and evaluation;
s8: in the test, the bacteriophage cocktail and the probiotic preparation are jointly applied to the prevention and treatment of NE in the large-scale poultry farm, and the antibacterial effect and the protective effect of preventing and treating NE are evaluated.
2. The development method for controlling clostridium perfringens of avian according to claim 1, wherein in the step S1, Cp flora characteristics and pathogenic characteristics in poultry farming are analyzed by metagenome analysis of pathogenic flora data information of avian NE.
3. The development method for controlling avian clostridium perfringens according to claim 1, wherein the basic culture medium in the S2 is NB liquid culture medium, 40ml of normal saline with the mass fraction of 0.85% is added during nutrient enrichment of the basic culture medium, and the mixture is cultured at 22-36 ℃ for 5-18 hours.
4. The development method for controlling clostridium perfringens of avian according to claim 3, wherein the rotation speed in the S3 centrifugation process is 800-1800r/min, and the centrifugation time is 2-4 minutes.
5. The development method for preventing and treating clostridium perfringens of avian according to claim 1, wherein thalli is obviously added in S4, then the thalli is placed in an alkali solution with the concentration of 2-6% at 70-80 ℃, then the alkali solution is repeatedly washed by deionized water and an acid solution, and then the alkali solution is placed in a pulping machine to form an emulsion; then placed in a centrifuge to obtain a suspension.
6. The development method for controlling clostridium perfringens of avian according to claim 5, wherein Cp-specific phages and probiotics for pathogenic characteristics are obtained in S4, and a health preparation capable of resisting the phages cocktail and probiotics in the main pathogenic type of Cp is developed.
7. The development method for controlling clostridium perfringens of avian according to claim 1, wherein the temperature in the S6 is-280-0 ℃ when freezing, and the humidity in the freezing chamber is monitored in real time when freezing.
8. The development method for controlling clostridium perfringens of avian according to claim 1, wherein data is collected in the application of S8, and then statistical evaluation is performed, and data is filtered in the statistical process, so that data which are seriously deviated from an exponential line are removed, and the referential performance of the data is improved.
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| CN115094008A (en) * | 2022-07-21 | 2022-09-23 | 宜兴市天石饲料有限公司 | Research and development method of biological antibacterial preparation for preventing and treating avian clostridium perfringens |
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