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Veterinary clostridium septicum toxin, preparation method thereof and special culture medium

A technology for Clostridium spoilage and toxin-producing culture medium, applied in biochemical equipment and methods, veterinary vaccines, microorganism-based methods, etc., can solve the problems of high cost, complicated preparation process, waste of vaccine production, etc. Reduced, simple to formulate, effective immune protection

Active Publication Date: 2018-07-31
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the vaccines currently on the market generally use the enzymatic digestion solution of beef and liver as raw materials to prepare the medium. The preparation process of the medium prepared by this method is cumbersome, time-consuming, and requires a lot of manpower. Uneven quality leads to unstable toxin production performance, which affects the quality of the vaccine, and also brings great waste and high cost to vaccine production
Moreover, among the 33 batches of products that failed the potency test results by serum neutralization method between 2006 and 2015, 15 batches of products failed to pass the potency of Clostridium putrefaction components

Method used

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  • Veterinary clostridium septicum toxin, preparation method thereof and special culture medium
  • Veterinary clostridium septicum toxin, preparation method thereof and special culture medium
  • Veterinary clostridium septicum toxin, preparation method thereof and special culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1, the screening of Clostridium putrefaction toxin-producing medium

[0078] 1. Three different toxin-producing medium formulations were designed:

[0079] Formula 1: Soybean peptone 15g, casein peptone 15g, yeast extract powder 5g, glucose 5g, purified water to 1000mL.

[0080] Formula 2: Show peptone 10g, casein peptone 10g, yeast extract powder 15g, sodium chloride 4g, sodium carbonate 0.6g, calcium chloride 0.1g, cystine 2g, dextrin 10g, add purified water to 1000mL.

[0081] Formula 3: peptone 15g, casein peptone 15g, yeast extract powder 5g, Na 2 HPO 4 12H 2 O 5g, KH 2 PO 4 0.3g, ZnSO 4 ·7H 2 O 1.4mg and glucose 10g, purified water was added to 1000mL.

[0082] 2. Prepare culture medium

[0083] Except for dextrin and glucose, weigh or measure each component according to the above content, add purified water, heat to fully dissolve, add purified water to make it to the final volume required for preparation, adjust with 10M sodium hydroxide pH...

Embodiment 2

[0104] Embodiment 2, Clostridium putrefaction toxin-producing medium preparation and optimization of using method

[0105] 1. Optimization of culture conditions in Erlenmeyer flasks

[0106] The culture temperature, initial pH value, and culture time were optimized by means of static culture in a triangular flask, and the optimal conditions were determined to be "the initial pH value of the medium was 7.5-8.0, and cultured at 37°C for 24h".

[0107] 2. Optimization of the culture conditions of the fermenter

[0108] The control of pH value, culture time, and sugar supplementation were optimized in the way of fermentation tank culture, and the optimal culture process was determined as "the initial glucose concentration of the medium was 1.0%, the initial pH value was 7.5-8.0, and the pH value was controlled throughout the fermentation process. 7.0, add 50% glucose aqueous solution according to 1% of the total volume of the medium when culturing for 16 hours, control the rotati...

Embodiment 3

[0126] Embodiment 3, preparation and effectiveness evaluation of Clostridium putrefaction toxoid vaccine

[0127] 1. Preparation of Clostridium putrefaction toxoid vaccine

[0128] 1) Detoxification of Clostridium putrefaction toxin

[0129] The fermented product obtained by fermenting and culturing at 37° C. for 24 h in the optimal fermenter fermentation condition and the optimal toxin-producing medium in Example 2 was added by volume with 0.7% formaldehyde aqueous solution (40%), and 10M sodium hydroxide was added to adjust the pH value to 6.8, placed at 35°C for inactivation and detoxification for 3 days to obtain the inactivated and detoxified bacterial liquid.

[0130] 2) Detection of Clostridium putrefactive toxin inactivation and detoxification effect

[0131] Take the above-mentioned inactivated and detoxified bacterial liquid to inoculate anaerobic meat liver soup, ordinary broth, and ordinary agar slant respectively, and observe the sterile growth for 5 days to sho...

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Abstract

The invention discloses a veterinary clostridium septicum toxin, a preparation method thereof and a special culture medium. Every 100 ml of culture medium is prepared from 1.5-2.0 g of proteose peptone, 1.5-2.0 g of casein peptone, 0.5-0.75 g of yeast extract powder, 0.14-0.28 mg of ZnSO4.7H2O, 0.5-0.75 g of Na2HPO4.12H2O, 0.03-0.045 g of KH2PO4, 1-1.5 g of glucose and the balance water; the pH value of the culture medium is 7.5-8.0. The veterinary clostridium septicum toxin is obtained by inoculating a production strain of clostridium septicum into the culture medium, collecting a culture object, conducting centrifugation and filtering a supernatant. By means of the method, the highest toxicity can be raised up to 10 times that of the vaccine-making standard of regulations for veterinarybiological products in China, and the output-input ratio can be increased to 20 times that of an original traditional process. Moreover, the neutralizing titers of toxoid vaccines prepared by using the veterinary clostridium septicum toxin to corresponding serums of rabbits and sheep are also increased to 8 times that of the regulation standard respectively.

Description

technical field [0001] The invention belongs to the field of veterinary biological products, and in particular relates to a veterinary Clostridium putrefactive toxin, a preparation method thereof and a special culture medium. Background technique [0002] Sheep rapid disease, sudden attack, lamb dysentery and enterotoxemia are common multiple infectious diseases of sheep caused by Clostridium putrefaction, Clostridium perfringens type C, Clostridium perfringens type B and Clostridium putrefaction respectively (Lu Chengping. Veterinary Microbiology [M]. Beijing: China Agricultural Press, 2013: 192-202.), they often occur in combination, the course of the disease is sharp, and the affected animals often die without showing symptoms, with high mortality and great harm. Therefore, immunization is the only effective way to control these diseases. Animal husbandry developed countries such as Europe, America and Australia all regard the four diseases of cattle and sheep as disease...

Claims

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Application Information

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IPC IPC(8): C12P21/00C12N1/20A61K39/08A61P31/04C12R1/145
CPCA61K39/08A61K2039/521A61K2039/552A61K2039/55505A61K2039/70C12N1/20C12P21/00
Inventor 蒋玉文彭小兵李旭妮彭国瑞
Owner CHINA INST OF VETERINARY DRUG CONTROL
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