阅读说明：本技术 一种猪链球菌9型高密度发酵培养基及其制备方法、应用 (Streptococcus suis 9 type high-density fermentation medium and preparation method and application thereof ) 是由 余姣 肖澄 周琳 车巧林 沈晓红 董彦鹏 缪芬芳 谢洁 张志华 于 2021-06-24 设计创作，主要内容包括：本发明公开了一种猪链球菌9型高密度发酵培养基及其制备方法、应用,包括基础培养基和培养基添加剂,基础培养基中包括以下组分：胰蛋白胨、酵母粉、牛肉粉、硫酸铵、葡萄糖、七水合硫酸镁、三水合磷酸氢二钾、磷酸二氢钾、剩余组分为纯水；培养基添加剂为马血清或牛血清；培养基添加剂按照所述基础培养基体积的2％～15％,与所基础培养基混合,得到猪链球菌9型高密度发酵培养基。使用本发明的猪链球菌9型高密度发酵培养基培养猪链球菌9型,有利于细菌的生长繁殖,培养的活菌滴度高。(The invention discloses a streptococcus suis 9 type high-density fermentation culture medium, a preparation method and application thereof, wherein the culture medium comprises a basic culture medium and a culture medium additive, and the basic culture medium comprises the following components: tryptone, yeast powder, beef powder, ammonium sulfate, glucose, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate and the balance of pure water; the culture medium additive is horse serum or bovine serum; the culture medium additive is mixed with the basic culture medium according to the volume of 2-15% of the basic culture medium to obtain the streptococcus suis 9 type high-density fermentation culture medium. The streptococcus suis 9 type high-density fermentation medium is used for culturing the streptococcus suis 9 type, so that the growth and the propagation of bacteria are facilitated, and the titer of the cultured viable bacteria is high.)

1. The streptococcus suis 9 type high-density fermentation culture medium is characterized by comprising a basic culture medium and a culture medium additive, wherein every 1000ml of the basic culture medium comprises the following components:

every 1000ml of the basic culture medium contains pure water as the residual component;

the culture medium additive is horse serum or bovine serum;

and mixing the culture medium additive with the basic culture medium according to the volume of 2-15% of the basic culture medium to obtain the streptococcus suis 9 type high-density fermentation culture medium.

2. The method for preparing a Streptococcus suis type 9 high-density fermentation medium according to claim 1, comprising the steps of:

s1: preparing an aqueous solution as a basic culture medium according to the following mixture ratio:

s2: adjusting the pH value of the basic culture medium to 7.4-7.8 by using 1mol/L sodium hydroxide solution;

s3: sterilizing and cooling;

s4: and adding horse serum or bovine serum according to 2-15% of the volume of the basic culture medium to obtain the streptococcus suis 9 type high-density fermentation culture medium.

3. The method for preparing a Streptococcus suis type 9 high-density fermentation medium according to claim 2, wherein in S3, the sterilization conditions are: autoclaving at 115 deg.C for 20 min.

4. The use of a Streptococcus suis type 9 high-density fermentation medium according to claim 1, wherein Streptococcus suis type 9 is cultured using a Streptococcus suis type 9 high-density fermentation medium.

5. The basic culture medium of the streptococcus suis 9 type high-density fermentation culture medium is characterized by comprising the following components in each 1000 ml:

6. a composition comprising the following components:

Technical Field

The invention relates to the technical field of veterinarians, in particular to a streptococcus suis 9 type high-density fermentation medium and a preparation method and application thereof.

Background

The streptococcus suis is divided into 33 serotypes, wherein the streptococcus suis type 2 (SS2) is most popular, has the highest morbidity, can infect people to die, is an important zoonosis pathogen, is usually sporadic or endemic, has high morbidity and mortality and seriously jeopardizes the development of the pig industry, and is caused by SS 2; until now, research on streptococcus suis has been a hotspot of swine diseases in all countries, particularly research on SS2 has never been stopped, although the streptococcus suis is distributed most widely in type 2 and has the strongest toxicity, in recent years, the separation rate and the pathogenic rate of SS9 in China tend to rise obviously, and new type 9 strains are separated continuously; as more and more type 9 strains are isolated in sick pigs, research on type 9 is also necessary; research finds that different types 9 strains have different pathogenicity, and a porcine cerebrospinal fluid isolate has stronger pathogenicity on mice, so that corresponding vaccine research is in the forefront with the strength of national resistance reduction.

At present, a plurality of recommended culture mediums of streptococcus suis exist, but no special culture medium of streptococcus suis 9 is available, the streptococcus suis is cultured by using THB, TSB and Martin broth in research, and although the commercial THB and TSB culture mediums are convenient to use, in the culture process, bacteria grow slowly, the number of viable bacteria is low, and the highest number of viable bacteria in the fermentation process is 10-20 hundred million, so that the method is not suitable for large-scale production.

Disclosure of Invention

Therefore, the invention provides the streptococcus suis 9 type high-density fermentation medium, and the preparation method and the application thereof, which can lead bacteria to grow fast and have high viable count, are suitable for large-scale production, and solve the problems provided in the background technology.

In order to achieve the above object, the present invention provides a streptococcus suis type 9 high-density fermentation medium, comprising a basic medium and a medium additive, wherein the basic medium comprises the following components per 1000 ml:

tryptone 15-25 g;

3-5 g of yeast powder;

3 g-5 g of beef powder;

1-5 g of ammonium sulfate;

1 g-10 g of glucose;

0.2g to 1.0g of magnesium sulfate heptahydrate;

5-10 g of dipotassium hydrogen phosphate trihydrate;

0.5g to 1.0g of monopotassium phosphate;

every 1000ml of the basic culture medium contains pure water as the residual component;

the culture medium additive is horse serum or bovine serum;

and mixing the culture medium additive with the basic culture medium according to the volume of 2-15% of the basic culture medium to obtain the streptococcus suis 9 type high-density fermentation culture medium.

The preparation method of the streptococcus suis 9 type high-density fermentation medium comprises the following steps:

s1: preparing an aqueous solution as a basic culture medium according to the following mixture ratio:

tryptone 15g/1000 ml-25 g/1000 ml;

yeast powder 3g/1000 ml-5 g/1000 ml;

3g/1000 ml-5 g/1000ml of beef powder;

1g/1000 ml-5 g/1000ml of ammonium sulfate;

1g/1000 ml-10 g/1000ml of glucose;

magnesium sulfate heptahydrate 0.2g/1000 ml-1.0 g/1000 ml;

5g/1000 ml-10 g/1000ml of dipotassium phosphate trihydrate;

monopotassium phosphate is 0.5g/1000ml to 1.0g/1000 ml;

s2: adjusting the pH value of the basic culture medium to 7.4-7.8 by using 1mol/L sodium hydroxide solution;

s3: sterilizing and cooling;

s4: and adding horse serum or bovine serum according to 2-15% of the volume of the basic culture medium to obtain the streptococcus suis 9 type high-density fermentation culture medium.

Wherein, further, the sterilization conditions are as follows: autoclaving at 115 deg.C for 20 min.

The invention also provides application of the streptococcus suis 9 type high-density fermentation culture medium, and the streptococcus suis 9 type high-density fermentation culture medium is used for culturing the streptococcus suis 9 type.

The invention also provides a basic culture medium of the streptococcus suis 9 type high-density fermentation culture medium, wherein each 1000ml of the basic culture medium comprises the following components:

tryptone 15-25 g;

3-5 g of yeast powder;

3 g-5 g of beef powder;

1-5 g of ammonium sulfate;

1 g-10 g of glucose;

0.2g to 1.0g of magnesium sulfate heptahydrate;

5-10 g of dipotassium hydrogen phosphate trihydrate;

0.5g to 1.0g of monopotassium phosphate;

the remaining component was pure water.

In another aspect of the invention, a composition is provided, comprising the following components:

tryptone 15-25 weight portions;

3-5 parts of yeast powder;

3-5 parts of beef powder;

1-5 parts of ammonium sulfate;

1-10 parts of glucose;

0.2 to 1.0 portion of magnesium sulfate heptahydrate;

5-10 parts of dipotassium hydrogen phosphate trihydrate;

0.5 to 1.0 portion of monopotassium phosphate,

the basic culture medium of the streptococcus suis 9 type high-density fermentation culture medium provided by the invention can be obtained by dissolving the composition in pure water according to a proper proportion.

The invention has the beneficial effects that:

1. the streptococcus suis 9 type high-density fermentation medium is used for culturing the streptococcus suis 9 type, so that the growth and the propagation of bacteria are facilitated, and the titer of the cultured viable bacteria is high.

2. The preparation method of the streptococcus suis 9 type high-density fermentation medium is simple.

3. Compared with the raw materials of the common brain heart infusion culture medium and the trypsin aged soybean culture medium, the raw materials used by the invention have lower cost.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.

Example one

Adding distilled water to 1000ml according to 25g of tryptone, 3g of yeast powder, 3g of beef powder, 2g of ammonium sulfate, 1g of glucose, 0.5g of magnesium sulfate heptahydrate, 10g of dipotassium phosphate trihydrate and 0.6g of potassium dihydrogen phosphate, adjusting the pH value to 7.45 by using 1mol/L sodium hydroxide solution, carrying out autoclaving at 115 ℃ for 20 minutes, cooling, and adding bovine serum according to the proportion of 10% (V/V) to obtain the streptococcus suis 9 type high-density fermentation culture medium.

Comparative example 1

TSB medium, the main component of which is tryptone, and which contains 0.25% glucose, 0.25% dipotassium hydrogen phosphate and 0.5% sodium chloride, was purchased from OXID, UK. 1000ml of TSB medium was prepared according to the instructions on the tip of the bottle, autoclaved at 115 ℃ for 20 minutes, cooled and then added with bovine serum at a ratio of 10% (V/V).

Culture and culture result comparison method

The high-density fermentation medium of Streptococcus suis type 9 prepared in example one was inoculated with Streptococcus suis type 9, the TSB medium purchased in comparative example was inoculated with Streptococcus suis type 9, both of the above media were simultaneously cultured in a shaker at 37 ℃ and 180RPM, and 1 sampling was performed every 1 hour, and the optical density value (OD6oo) of Streptococcus suis type 9 was measured using a spectrophotometer, and viable cell counting was performed for 8 hours continuously.

Usually, the OD value of the bacterial liquid in the logarithmic growth phase is proportional to the concentration, but the OD value and the peak value of the viable count of the bacteria do not appear simultaneously. Estimating the concentration interval of the bacteria liquid through the OD value, measuring the viable bacteria content by adopting a viable bacteria counting method (according to the appendix of the current Chinese animal pharmacopoeia), and recording and summarizing corresponding data as shown in the following table:

as can be seen from the above table, the OD value and the highest occurrence time of viable bacteria of Streptococcus suis type 9 in the TSB medium are later (7h), and the viable bacteria number is 19.1 hundred million, while the viable bacteria number of the medium used in the first embodiment of the present invention is 4h and 62.3 hundred million.

Therefore, the effect of the high-density fermentation medium for streptococcus suis 9 type prepared in the first embodiment of the invention when culturing streptococcus suis 9 type is obviously superior to that of the traditional TSB medium, the growth and the propagation of bacteria are facilitated, and the titer of the cultured viable bacteria is high.

Example two

According to 20g of tryptone, 4g of yeast powder, 4g of beef powder, 1g of ammonium sulfate, 5g of glucose, 0.2g of magnesium sulfate heptahydrate, 5g of dipotassium phosphate trihydrate and 0.5g of potassium dihydrogen phosphate, adding distilled water to 1000ml, adjusting the pH value to 7.45 by using 1mol/L sodium hydroxide solution, carrying out autoclaving at 115 ℃ for 20 minutes, cooling, and adding bovine serum according to the proportion of 2% (V/V) to obtain the streptococcus suis type 9 high-density fermentation culture medium.

Comparative example 2

TSB medium, the main component of which is tryptone, and which contains 0.25% glucose, 0.25% dipotassium hydrogen phosphate and 0.5% sodium chloride, was purchased from OXID, UK. 1000ml of TSB medium was prepared according to the instructions on the tip of the bottle, autoclaved at 115 ℃ for 20 minutes, cooled and then added with 2% (V/V) bovine serum.

Culture and culture result comparison method

The high-density fermentation medium of Streptococcus suis type 9 prepared in example two was inoculated with Streptococcus suis type 9, the TSB medium purchased in comparative example was inoculated with Streptococcus suis type 9, both of the above media were simultaneously cultured in a shaker at 37 ℃ and 180RPM, and 1 sampling was performed every 1 hour, and the optical density value (OD6oo) of Streptococcus suis type 9 was measured using a spectrophotometer, and viable cell counting was performed for 8 hours continuously.

Usually, the OD value of the bacterial liquid in the logarithmic growth phase is proportional to the concentration, but the OD value and the peak value of the viable count of the bacteria do not appear simultaneously. Estimating the bacterial liquid concentration interval through the OD value, calculating the bacterial liquid concentration by adopting a viable bacteria counting method (according to the appendix of the current Chinese veterinary pharmacopoeia), and recording and summarizing corresponding data as shown in the following table:

as can be seen from the above table, the OD value and the highest occurrence time of viable bacteria of Streptococcus suis type 9 in the TSB medium are later (7h), and the viable bacteria count is 15.9 hundred million, while the viable bacteria count of the medium used in the second embodiment of the present invention is 5h and 62.8 hundred million.

Therefore, the effect of the high-density fermentation medium for streptococcus suis 9 type prepared in the first embodiment of the invention when culturing streptococcus suis 9 type is obviously superior to that of the traditional TSB medium, the growth and the propagation of bacteria are facilitated, and the titer of the cultured viable bacteria is high.

EXAMPLE III

Adding distilled water to 1000ml according to 15g of tryptone, 5g of yeast powder, 5g of beef powder, 5g of ammonium sulfate, 10g of glucose, 1g of magnesium sulfate heptahydrate, 8g of dipotassium hydrogen phosphate trihydrate and 1g of potassium dihydrogen phosphate, adjusting the pH value to 7.45 by using 1mol/L sodium hydroxide solution, carrying out autoclaving at 115 ℃ for 20 minutes, cooling, and adding bovine serum according to the proportion of 15% (V/V) to obtain the streptococcus suis 9 type high-density fermentation culture medium.

Comparative example three

TSB medium, the main component of which is tryptone, and which contains 0.25% glucose, 0.25% dipotassium hydrogen phosphate and 0.5% sodium chloride, was purchased from OXID, UK. 1000ml of TSB medium was prepared according to the instructions on the tip of the bottle, autoclaved at 115 ℃ for 20 minutes, cooled and then added with bovine serum at 15% (V/V).

Culture and culture result comparison method

The high-density fermentation medium of Streptococcus suis type 9 prepared in example three was inoculated with Streptococcus suis type 9, the TSB medium purchased in comparative example was inoculated with Streptococcus suis type 9, both of the above media were simultaneously cultured in a shaker at 37 ℃ and 180RPM, and 1 sampling was performed every 1 hour, and the optical density value (OD6oo) of Streptococcus suis type 9 was measured using a spectrophotometer, and viable cell counting was performed for 10 hours in a row.

Usually, the OD value of the bacterial liquid in the logarithmic growth phase is proportional to the concentration, but the OD value and the peak value of the viable count of the bacteria do not appear simultaneously. Estimating the bacterial liquid concentration interval through the OD value, calculating the bacterial liquid concentration by adopting a viable bacteria counting method (according to the appendix of the current Chinese veterinary pharmacopoeia), and recording and summarizing corresponding data as shown in the following table:

as can be seen from the above table, the OD value and the highest occurrence time of viable bacteria of Streptococcus suis type 9 in the TSB medium are later (6h), and the number of viable bacteria is 18 hundred million, while the number of viable bacteria of the medium used in the first embodiment of the present invention is highest and appears at 5h, and the number of viable bacteria is 63.3 hundred million.

Therefore, the effect of the high-density fermentation medium for streptococcus suis 9 type prepared in the first embodiment of the invention when culturing streptococcus suis 9 type is obviously superior to that of the traditional TSB medium, the growth and the propagation of bacteria are facilitated, and the titer of the cultured viable bacteria is high.

The above description is only a preferred embodiment of the present invention, and any person skilled in the art may modify the present invention or modify it into an equivalent technical solution by using the technical solution described above. Therefore, any simple modifications or equivalent substitutions made in accordance with the technical solution of the present invention are within the scope of the claims of the present invention.