CN110669684A - Swine erysipelas vaccine enrichment medium - Google Patents
Swine erysipelas vaccine enrichment medium Download PDFInfo
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- CN110669684A CN110669684A CN201811100955.9A CN201811100955A CN110669684A CN 110669684 A CN110669684 A CN 110669684A CN 201811100955 A CN201811100955 A CN 201811100955A CN 110669684 A CN110669684 A CN 110669684A
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- glucose
- erysipelas
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Abstract
The invention provides a erysipelas swine erysipelas vaccine enrichment medium, which relates to the field of biology and is prepared from the following raw materials in parts by weight: 5.0-10.0g of peptone, 5.0-10.0g of yeast extract, 5.0-10.0g of beef liver extract, 3.0-5.0g of dipotassium phosphate, 5.0-8.0g of sodium dihydrogen phosphate, 5.0-10.0g of glucose, 1.0-1.0 g of tween-800.5 and 2.0-5.0g of gelatin, and purified water is added to 1000 ml; the preparation method of the erysipelas swine erysipelas vaccine enrichment medium comprises the following steps: adding all raw materials except glucose into purified water, fully dissolving, adjusting pH to 7.8-8.2 with sodium hydroxide solution, sterilizing at 116 deg.C for 30 min, adding 12ml of glucose solution with final concentration of 50% by aseptic technique before use, and mixing; preparing the 50% glucose solution: weighing 50g of glucose, adding purified water for dissolving, fixing the volume to 100ml, sterilizing at 116 ℃ for 20 minutes, and storing at 2-8 ℃. The invention relates to application of a synthetic dry powder culture medium in production of swine erysipelas vaccines, and has the beneficial effects of suitability for high-density culture of swine erysipelas vaccine thalli, selection of commercial raw materials and high number of cultured bacteria.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a porcine erysipelas vaccine enrichment medium.
Background
The swine erysipelas is one of three infectious diseases of swine, is distributed worldwide, is also a zoonosis, and has erysipelas suis as a disease source. The existing swine erysipelas live vaccines in China at present have single vaccine, double vaccine and triple vaccine, and play an important role in preventing swine erysipelas. But the gap between the culture of vaccine bacteria liquid and the developed countries is still large. First, the culture stability is poor. In the sixty-seven decades, the pork liver and stomach (membrane) digestion soup containing 2% -4% of cracked whole blood is used as a culture medium for producing the vaccine, and after 80 years, domestic animal biological product enterprises begin to produce the swine erysipelas vaccine by replacing the cracked blood cell whole blood with tween-80, so that the number of cultured live bacteria is averagely increased by 40%, although the dependence on animal blood is eliminated in the culture process, the quality of meat, liver and stomach immersion liquid which are main components in the pork liver and stomach membrane digestion soup is greatly influenced by the type, age, freshness and digestion degree of animals, the number of cultured bacteria is low, and the batch is unstable.
Plum red clouds and the like (experimental discussion of producing the live vaccine of erysipelothrix rhusiopathiae by using a culture medium prepared from mixed peptone to prepare dry powder, Jilin animal veterinarian [ J ]. 7 th year in 2012, 24-25), which indicates that the production of the live vaccine of erysipelothrix rhusiopathiae by using the culture medium prepared from mixed domestic and imported peptone is superior to that by using a culture medium of single domestic peptone, although the number of cultured bacteria is increased, the problem of high culture cost exists; the patent application with the publication number of CN104306963A provides a synthetic culture medium for culturing erysipelothrix rhusiopathiae, and the research direction is to prolong the storage time of the erysipelothrix rhusiopathiae live vaccine, so that the bacterium increasing effect cannot be judged from the application document.
Disclosure of Invention
The invention provides a erysipelas swine erysipelas vaccine enrichment medium, which solves the technical problems of high synthesis cost, low culture bacteria number and unstable batch-to-batch in the prior art.
The invention is realized by the following steps: the feed comprises the following raw materials in parts by weight: 5.0-10.0g of peptone, 5.0-10.0g of yeast extract, 5.0-10.0g of beef liver extract, 3.0-5.0g of dipotassium phosphate, 5.0-8.0g of sodium dihydrogen phosphate, 5.0-10.0g of glucose, 1.0-1.0 g of tween-800.5 and 2.0-5.0g of gelatin, and purified water is added to 1000 ml.
As a preferred embodiment, the feed comprises the following raw materials in parts by weight: 10.0g of peptone, 10.0g of yeast extract powder, 10.0g of beef liver extract powder, 4.0g of dipotassium phosphate, 10.0g of sodium dihydrogen phosphate, 10.0g of glucose, 801.0g of tween-801.0 g and 5.0g of gelatin, and purified water is added to 1000 ml.
The invention also provides a preparation method of the erysipelas swine erysipelas vaccine enrichment medium, which comprises the following steps:
raw materials of the swine erysipelas vaccine culture medium except glucose: adding peptone, yeast extract powder, beef liver extract powder, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, tween-80 and gelatin into purified water, fully dissolving, adjusting pH to 7.8-8.2 with sodium hydroxide solution, sterilizing at 116 deg.C for 30 min, adding 12ml of glucose solution with final concentration of 50% by aseptic operation before use, and mixing;
preparing the 50% glucose solution: weighing 50g of glucose, adding purified water for dissolving, fixing the volume to 100ml, sterilizing at 116 ℃ for 20 minutes, and storing at 2-8 ℃.
The invention has the beneficial effects that: peptone provides various nitrogen sources for the growth of microorganisms; the yeast extract powder, the beef extract powder and the beef liver extract powder contain rich free amino acids, polypeptides, vitamins, nucleotides, trace elements and the like, are rich in various nutrient components and coordinated in proportion, and can provide comprehensive nutrition for microbial fermentation culture; the dipotassium phosphate and the sodium dihydrogen phosphate play a buffering role; glucose provides a carbon source for the growth of microorganisms; tween-80 is used as emulsifier to reduce the surface tension of the culture medium, so that toxic metabolites of cells such as lactic acid can be smoothly discharged out of cells to stimulate the growth of microorganisms; function of gelatin: the erysipelothrix rhusiopathiae is microaerophilic bacterium which has better growth effect in the presence of a small amount of oxygen than in the presence of sufficient oxygen, and the addition of gelatin can increase the viscosity of a culture medium, create a microaerophilic environment for the thalli and enable the thalli to grow better. The invention has the advantages of promoting the proliferation of the thallus, ensuring high number of cultured bacteria, realizing stable quality among batches and being simple and convenient to operate.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below. It is to be understood that the described embodiments are merely exemplary of some, and not necessarily all, embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example one
A pig erysipelas vaccine enrichment medium comprises, by weight, 5.0g of peptone, 5.0g of yeast extract powder, 5.0g of beef liver extract powder, 3.0g of dipotassium hydrogen phosphate, 5.0g of sodium dihydrogen phosphate, 5.0g of glucose, 800.5g of tween-800.5 g and 2.0g of gelatin, and purified water is added to 1000 ml.
The preparation method of the swine erysipelas vaccine culture medium comprises the following steps:
the raw materials except glucose are weighed according to the formula, the components of the culture medium are sequentially added into purified water, the pH value is adjusted to 7.8-8.2 by using sodium hydroxide solution after the components are fully dissolved, and the raw materials are sterilized for 30 minutes at 116 ℃. For application, 12ml of 50% final concentration glucose solution is added by aseptic technique and mixed.
Preparing the 50% glucose solution: weighing 50g of glucose, adding purified water for dissolving, fixing the volume to 100ml, sterilizing at 116 ℃ for 20 minutes, and storing at 2-8 ℃.
Example two
A pig erysipelas vaccine enrichment medium comprises, by weight, 8.0g of peptone, 8.0g of yeast extract powder, 8.0g of beef liver extract powder, 4.0g of dipotassium hydrogen phosphate, 6.0g of sodium dihydrogen phosphate, 8.0g of glucose, 800.8g of tween-800, 4.0g of gelatin, and purified water added to 1000 ml.
The preparation method of the swine erysipelas vaccine culture medium comprises the following steps:
the raw materials except glucose are weighed according to the formula, the components of the culture medium are sequentially added into purified water, the pH value is adjusted to 7.8-8.2 by using sodium hydroxide solution after the components are fully dissolved, and the raw materials are sterilized for 30 minutes at 116 ℃. For application, 12ml of 50% final concentration glucose solution is added by aseptic technique and mixed.
Preparing the 50% glucose solution: weighing 50g of glucose, adding purified water for dissolving, fixing the volume to 100ml, sterilizing at 116 ℃ for 20 minutes, and storing at 2-8 ℃.
EXAMPLE III
A pig erysipelas vaccine enrichment medium comprises, by weight, 10.0g of peptone, 10.0g of yeast extract powder, 10.0g of beef liver extract powder, 4.0g of dipotassium hydrogen phosphate, 8.0g of sodium dihydrogen phosphate, 10.0g of glucose, 801.0g of tween-801.0 g and 5.0g of gelatin, and purified water is added to 1000 ml.
The preparation method of the swine erysipelas vaccine culture medium comprises the following steps:
the raw materials except glucose are weighed according to the formula, the components of the culture medium are sequentially added into purified water, the pH value is adjusted to 7.8-8.2 by using sodium hydroxide solution after the components are fully dissolved, and the raw materials are sterilized for 30 minutes at 116 ℃. For application, 12ml of 50% final concentration glucose solution is added by aseptic technique and mixed.
Preparing the 50% glucose solution: weighing 50g of glucose, adding purified water for dissolving, fixing the volume to 100ml, sterilizing at 116 ℃ for 20 minutes, and storing at 2-8 ℃.
Effect test
The synthetic dry powder culture medium is used for carrying out a bacterium increasing effect experiment on erysipelothrix rhusiopathiae as follows.
A bacterial strain for experiment
(1) The erysipelothrix rhusiopathiae strain CVCC1318, strain GC42, was identified, maintained and supplied by the harbin veterinary institute. The attenuated strain is prepared with virulent G370 strain and through subcutaneous injection of 500X 108CFU live bacteria non-lethal pig, 1X 107Live CFU immunization of mice resulted in 80% protection, serotype 1 a.
(2) The strain of erysipelothrix rhusiopathiae CVCC1319 strain, namely G4T10 strain, is separated by Jiangsu farm courtyard and is identified, stored and supplied by Chinese veterinary drug inspection. Attenuated strain, subcutaneous injection 50X 108～500×108CFU live pig 100% healthy and alive, 1X 107CFU live bacteria immunized mice can produce 81.8% protection, serotype 1 a.
B seed liquid preparation
1 each of the freeze-dried erysipelothrix rhusiopathiae CVCC1318 strains and 1 CVCC1319 strains is started, diluted by 1.0ml of Martin broth, inoculated on a gelatin plate, cultured at 15-18 ℃ for 3-5 days, 5-10 qualified bacterial colonies are selected respectively, mixed in a small amount of meat, liver and stomach membrane digestion soup, streaked and inoculated on a Martin agar plate, cultured at 36-37 ℃ for 24 hours, and taken as first-grade seeds after pure inspection is qualified, and stored at 2-8 ℃ for no more than 1 month. Inoculating the first-class seeds to meat liver and stomach membrane digestion soup, culturing at 36-37 ℃ for 20-22 h, and using the first-class seeds as seed liquid after pure inspection is qualified (Ministry of agriculture of the people's republic of China, second zero year edition of veterinary biological products regulation of the people's republic of China, chemical industry Press, 2001, hereinafter called regulation).
C bacterial liquid culture and determination
Inoculating 2% of the seed solution into 1 flask of 500ml containing 200ml of liquid culture medium, performing shake culture at 37 ℃ and 150r/min for 13-15 hours, and simultaneously setting 1 negative control without inoculation. Respectively sampling and counting viable bacteria according to the appendix of the current Chinese veterinary pharmacopoeia, and repeating the counting for three times. The number of bacteria should not be less than 140 × 108CFU/ml, and negative control should be grown aseptically. The enrichment effect is shown in Table 1.
TABLE 1 enrichment Effect of synthetic Dry powder Medium on erysipelothrix rhusiopathiae
As can be seen from Table 1, the viable bacteria of examples one to three all satisfied the number of bacteria of not less than 140X 108CFU/ml can meet the requirement, and the average viable count of three batches in the enrichment medium prepared in the first example is 173 multiplied by 108CFU/ml, three batches in the enrichment medium prepared in the example, the average viable count is 195X 108CFU/ml. Wherein the viable count of three batches in the enrichment medium prepared in the third embodiment respectively reaches 240 multiplied by 108CFU/ml、248×108CFU/ml、248×108CFU/ml, three replicates over 200X 108CFU/ml, it can be seen that the enrichment effect is obvious.
Safety experiment
The culture medium obtained in the third embodiment is selected as a group A, the meat liver and stomach membrane digestion soup is selected as a group B, the CVCC1318 strain and the CVCC1319 strain of the same batch are cultured, and the strain obtained by culturing the culture medium and the digestion soup is subjected to safety experiments.
The method comprises the following steps: the three synthetic culture mediums of the example are used for culturing the same batch of CVCC1318 bacterial liquid and the same batch of CVCC1319 bacterial liquid, the CVCC1318 bacterial liquid is centrifuged for 20 minutes at 3500r/min, the supernatant is discarded, the precipitated bacterial cells are suspended by a proper amount of 20 percent of alumina gel physiological saline, and then viable bacteria are counted.
Selecting 120 mice with the weight of 20-22 g, and respectively injecting 10 subcutaneous injection doses of 10 bacterial liquids of two batches obtained by culturing the culture medium synthesized in the third embodiment and the meat liver and stomach membrane digestion soup with the injection amount of 0.2 ml/mouse, repeating each batch for 3 times, wherein the 120 mice are used as an immune group, and the CVCC1318 batch contains 14 multiplied by 10 mice8CFU viable bacteria/0.2 ml, CVCC1319 lot containing 10X 108CFU viable bacteria/0.2 ml, observed for 14 days, survival was recorded and the results are shown in Table 2.
TABLE 2 safety test results of two culture media for culturing thallus
As can be seen from Table 2, the cultured cells obtained in the medium obtained in example three were safe for mice, and the survival rate of 10/10 was achieved for each repetition.
Efficacy test
The culture medium and the meat liver and stomach membrane digestion soup obtained in the third embodiment are selected to culture the CVCC1318 strain and the CVCC1319 strain of the same batch respectively, and the strains cultured by the culture medium and the digestion soup are subjected to efficacy experiments.
The method comprises the following steps: the three synthetic culture mediums of the example are used for culturing the same batch of CVCC1318 bacterial liquid and the same batch of CVCC1319 bacterial liquid, the CVCC1318 bacterial liquid is centrifuged for 20 minutes at 3500r/min, the supernatant is discarded, the precipitated bacterial cells are suspended by a proper amount of 20 percent of alumina gel physiological saline, and then viable bacteria are counted.
Example three synthetic Medium culture two batches of bacterial liquid respectively subcutaneous injection mice 10, weight 16 ~ 18g, 0.2 ml/mouse, CVCC1318 batch contains 1.4 x 107CFU viable bacteria/0.2 ml, CVCC1319 batchContains 1.0X 107CFU viable bacteria/0.2 ml, and is used as group A.
10 mice are respectively injected subcutaneously with two batches of bacterial liquid cultured by the meat liver gastric-membrane digestion soup, the weight of each mouse is 16-18 g, and each mouse is 0.2ml, wherein the CVCC1318 batch contains 1.4 multiplied by 107CFU viable bacteria/0.2 ml, CVCC1319 lot containing 1.0 × 107CFU viable bacteria/0.2 ml, as group B, group A and group B constitute the immune group. After 14 days, 6 mice of 16-18 g were selected as non-immunized groups without immunization. Selecting 3 mice in an unimmunized group and all the mice in the immunized group, injecting the 1 type erysipelothrix rhusiopathiae and the 1 type erysipelothrix rhusiopathiae of 1000MLD subcutaneously respectively to prepare a virulent strain mixed bacterial solution, taking the 3 mice as a group C, injecting the 1MLD virulent strain mixed bacterial solution into the other 3 control mice in the unimmunized group as a group D, observing for 10 days, recording survival conditions, and obtaining results shown in Table 3.
TABLE 3 results of the cell potency experiment
As can be seen from Table 3, the immune effect of the bacteria cultured in the medium obtained in the third example is better than that of the liver and stomach membrane digestion decoction, and most of the mice immunity of the CVCC1318 strain and the CVCC1319 strain can reach 10/10 protection, which is higher than that of the mice immunity protection rate of the two batches of bacteria liquid cultured in the liver and stomach membrane digestion decoction. As a result, the synthetic medium was found to be excellent in safety and immunogenicity.
The invention has the beneficial effects that: peptone provides various nitrogen sources for the growth of microorganisms; the yeast extract powder, the beef extract powder and the beef liver extract powder contain rich free amino acids, polypeptides, vitamins, nucleotides, trace elements and the like, are rich in various nutrient components and coordinated in proportion, and can provide comprehensive nutrition for microbial fermentation culture; the dipotassium phosphate and the sodium dihydrogen phosphate play a buffering role; glucose provides a carbon source for the growth of microorganisms; tween-80 is used as emulsifier to reduce the surface tension of the culture medium, so that toxic metabolites of cells such as lactic acid can be smoothly discharged out of cells to stimulate the growth of microorganisms; function of gelatin: the erysipelothrix rhusiopathiae is microaerophilic bacterium which has better growth effect in the presence of a small amount of oxygen than in the presence of sufficient oxygen, and the addition of gelatin can increase the viscosity of a culture medium, create a microaerophilic environment for the thalli and enable the thalli to grow better. The invention has the advantages of promoting the proliferation of the thallus, ensuring high number of cultured bacteria, realizing stable quality among batches and being simple and convenient to operate.
The foregoing is only a preferred embodiment of the present invention and is not intended to limit the present invention in any way, so that any person skilled in the art can change or modify the technical content disclosed above into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention will still fall within the protection scope of the technical solution of the present invention.
Claims (3)
1. The erysipelas of swine vaccine enrichment medium is characterized by comprising the following raw materials in parts by weight: 5.0-10.0g of peptone, 5.0-10.0g of yeast extract, 5.0-10.0g of beef liver extract, 3.0-5.0g of dipotassium phosphate, 5.0-8.0g of sodium dihydrogen phosphate, 5.0-10.0g of glucose, 1.0-1.0 g of tween-800.5 and 2.0-5.0g of gelatin, and purified water is added to 1000 ml.
2. The erysipelas of swine erysipelas vaccine enrichment medium of claim 1, which is characterized by comprising the following raw materials in parts by weight: 10.0g of peptone, 10.0g of yeast extract powder, 10.0g of beef liver extract powder, 4.0g of dipotassium phosphate, 10.0g of sodium dihydrogen phosphate, 10.0g of glucose, 801.0g of tween-801.0 g and 5.0g of gelatin, and purified water is added to 1000 ml.
3. The preparation method of the erysipelas of swine erysipelas vaccine enrichment medium, which is characterized by comprising the following steps:
raw materials of the swine erysipelas vaccine culture medium except glucose: adding peptone, yeast extract powder, beef liver extract powder, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, tween-80 and gelatin into purified water, fully dissolving, adjusting pH to 7.8-8.2 with sodium hydroxide solution, sterilizing at 116 deg.C for 30 min, adding 12ml of glucose solution with final concentration of 50% by aseptic operation before use, and mixing;
preparing the 50% glucose solution: weighing 50g of glucose, adding purified water for dissolving, fixing the volume to 100ml, sterilizing at 116 ℃ for 20 minutes, and storing at 2-8 ℃.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113046409A (en) * | 2021-03-22 | 2021-06-29 | 中海生物技术(枣庄)有限公司 | Selective chromogenic medium for colony of erysipelothrix suis and pasteurella multocida |
| CN114032199A (en) * | 2021-11-30 | 2022-02-11 | 国药集团扬州威克生物工程有限公司 | Enrichment medium and culture method of swine erysipelas weavins G4T10 and application of culture |
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| CN114032199A (en) * | 2021-11-30 | 2022-02-11 | 国药集团扬州威克生物工程有限公司 | Enrichment medium and culture method of swine erysipelas weavins G4T10 and application of culture |
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