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Serum-free high-density fermentation culture process for serum type 9 streptococcus suis

A high-density fermentation and Streptococcus suis technology, applied in the field of serum-free high-density fermentation and culture technology of serum type 9 Streptococcus suis, can solve the problems of lack of production and culture technology and inconvenience of serum type 9 Streptococcus suis, and achieve the prevention of serum type 9 Streptococcus suis Streptococcus suis, simple formula, fast bacterial production

Inactive Publication Date: 2021-03-09
山东滨州沃华生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the culture of Streptococcus suis mostly adopts high-density fermentation culture with serum or without serum, and the number of viable bacteria in culture is 5.0×10 9 ~9.0×10 9 Between CFU / ml, with the increase of prevalent serotypes of Streptococcus suis, the production of vaccine strains with multiple serotypes brings a lot of inconvenience to the production. Relieve the pressure on the production of multivalent vaccines, and there is currently a lack of research on the large-scale production and culture process of serotype 9 Streptococcus suis

Method used

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  • Serum-free high-density fermentation culture process for serum type 9 streptococcus suis
  • Serum-free high-density fermentation culture process for serum type 9 streptococcus suis
  • Serum-free high-density fermentation culture process for serum type 9 streptococcus suis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: 40L primary fermentation tank fermentation

[0036] Strain: SQ strain of Streptococcus suis serotype 9.

[0037] Seed culture: wash TSA (5% sheep blood) agar slant with 2ml TSB liquid medium, inoculate in TSB (5% adult bovine serum) liquid medium, culture at 37°C with shaking for 3.5h, OD 600nm The value reaches 0.4~0.8.

[0038] Fermentation culture: the fermentation medium is adjusted to pH 7.2 with ammonia water, sterilized at 121°C for 30 minutes, inoculated with 2% (V / V) inoculum in the fermenter, the stirring speed is controlled at 150rpm, the dissolved oxygen is controlled to 30%, and the tank body is kept positive. When the pressure is 0.04Mpa and the OD600nm of the fermented bacteria liquid is 1.0, stop the aeration culture. Add ammonia water to control the pH at 7.2. Add 400 ml each of 1% glucose solution and 1% skim milk solution when the concentration of the fermentation broth reaches OD600nm=0.8, 1.5, and 2.0, and the flow rate is controlle...

Embodiment 2

[0039] Embodiment 2: 500L secondary tank fermentation

[0040] Strain: SQ strain of Streptococcus suis serotype 9.

[0041] Primary seed culture: wash TSA (5% sheep blood) agar slant with 2ml TSB liquid medium, inoculate in TSB (5% adult bovine serum) liquid medium, culture at 37°C for 3.5 hours on a shaking table, OD 600nm The value reaches 0.6.

[0042]Secondary seed culture: the fermentation medium is adjusted to pH 7.2 with ammonia water, sterilized at 121°C for 30 minutes, inoculated with 2% (V / V) inoculum in the fermenter, the stirring speed is controlled at 100rpm, the dissolved oxygen is controlled to 45%, and the tank is maintained The positive pressure of the body is 0.05Mpa, and the pH is controlled at 7.2 by feeding ammonia water. When the culture OD600nm reaches 0.8, it is transplanted into a 500L fermenter.

[0043] Fermentation culture: the fermentation medium is adjusted to pH 7.4 with NaOH, sterilized at 121°C for 30 minutes, inoculated with 2% (V / V) inoculu...

Embodiment 3

[0044] Example 3: Immunogenicity assay

[0045] Immunogenicity in 28-35 day old piglets:

[0046] High-density fermented antigen prepared inactivated oil emulsion vaccine, 2ml / head, neck muscle immunization 5 healthy piglets aged 28-35 days, 28 days after immunization, together with 5 piglets of the control group, a minimum lethal dose (4.5 ×10 9 CFU / head) of the SQ strain viable bacterial liquid, observed 14 days after the challenge, and recorded the death of piglets, such as Figure 4 shown.

[0047] Immunogenicity on 18~22g clean grade Kunming mice

[0048] High-density fermented antigen prepared inactivated oil emulsion vaccine, 0.5ml / only, intraperitoneally immunized 10 Kunming rats of 18-22g clean grade, 21 days after immunization, together with 10 mice of the control group, intraperitoneally injected a minimum lethal dose (2.1×10 9 CFU / only) of the SQ strain live bacteria liquid, observed 7 days after the challenge, and recorded the death of the mice, such as Figu...

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Abstract

The invention relates to a serum-free high-density fermentation culture process for serum type 9 streptococcus suis. The process comprises the following steps of: harvesting secondary seeds by utilizing primary seeds, inoculating the secondary seeds into a fermentation tank according to an inoculation amount of 2 percent (V / V), fermenting until OD600nm =1.0 + / - 0.1, stopping ventilating, performing non-ventilating culture, supplementing 1-3 percent of glucose solution and 1-3 percent of skim milk powder solution in the fermentation process, harvesting when OD600nm of the culture bacterial solution is more than 2.5, thereby obtaining the serum type 9 streptococcus suis high-density antigen. According to the high-density fermentation culture medium and culture process for the serum type 9 streptococcus suis provided by the invention, the culture medium is simple in formula and low in cost. The fermentation parameters are easily controlled, the culture bacteria number is high, the immunogenicity is high, and large-scale high-density culture of the streptococcus type 9 antigen is easily realized. According to the process disclosed by the invention, the production cost can be saved, andthe streptococcus vaccine protection effect is guaranteed.

Description

technical field [0001] The invention relates to the technical field of microbial fermentation and cultivation, in particular to a serum-free high-density fermentation and cultivation process for serum type 9 Streptococcus suis. Background technique [0002] Streptococcus suis is an important and serious zoonotic infectious disease pathogen. According to the different antigenicity of capsular polysaccharides, it can be divided into 29 serotypes. It was originally thought that there were 35 serotypes, but it was verified later20, 22, 26, 32, 33, 34 are not Streptococcus suis. In my country, Streptococcus suis is mainly serotype 2, especially since 2003 or even earlier, the main pathogen of Streptococcus suis in my country has changed from zooepidemicus subspecies to Streptococcus suis. Serum types 2, 3, 7, and 9 have become common serotypes. Since 2016, serotype 9 has gradually become popular and has become another dominant serotype after type 2. It mainly causes meningitis a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12R1/46
CPCC12N1/20
Inventor 王楠杨灵芝朱杰耿兴良吴洪才杨平
Owner 山东滨州沃华生物工程有限公司
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