A high-density fermentation medium for streptococcus suis and preparation and application thereof

Abstract

The present application relates to the technical field of fermentation medium, more specifically, it relates to a high-density fermentation medium for streptococcus suis and preparation and application thereof.The medium comprises beef infusion powder 1-5 parts, tryptone 1-10 parts, pancreatic casein peptone 1-10 parts, soybean peptone 1-5 parts, dipotassium hydrogen phosphate 1-5 parts, sodium chloride 1-10 parts, yeast extract 1-10 parts and glucose 1-5 parts.

Description

Technical Field

[0001] This invention relates to the field of fermentation culture medium technology, and more specifically, to a high-density fermentation culture medium for Streptococcus suis, its preparation and application. Background Technology

[0002] Streptococcus suis (S. suis), a significant zoonotic pathogen, has caused severe economic losses to the global pig industry. Infection in pigs can lead to various diseases, including meningitis, septicemia, and arthritis. Currently, whole-cell inactivated vaccines are widely used for the control of streptococcal disease in pigs. While live attenuated vaccines are theoretically effective, they pose a risk of shedding in practice, and attenuated strains may undergo virulence reversion or mutation during preparation and passage, potentially affecting vaccine safety. Furthermore, these vaccines typically have poor stability and require stringent storage and transportation conditions. Subunit vaccines face limitations such as high manufacturing costs, short duration of immunity, limited immunogenicity, and strong dependence on adjuvants. Their long development cycles also make them ill-equipped to effectively address the continuous mutation of pathogens, often failing to respond promptly to sudden or emerging outbreaks. While novel vaccine technologies such as DNA vaccines and live vector vaccines show great promise, they still face numerous challenges before practical application, including challenges in areas such as duration of immune response, breadth of immune spectrum, and production cost control.

[0003] Vaccination remains one of the most effective means of controlling streptococcal disease in pigs, but its large-scale application is significantly limited by fermentation levels. Currently used commercial culture media, such as TSB and Martin's broth, while easy to prepare, generally face problems such as poor batch stability and low viable cell counts in large-scale production. According to existing patent reports, the highest viable cell count can only reach 6.767 billion to 7.1 billion CFU / mL. Furthermore, related studies indicate that to achieve ideal culture results, the minimum addition of newborn calf serum needs to reach 5% or more when culturing Streptococcus suis, which undoubtedly increases production costs.

[0004] Given the limitations of existing culture media in large-scale production and the challenges faced by new vaccine technologies, developing a culture medium suitable for large-scale industrial production, with readily available raw materials, low cost, and the ability to significantly increase the number of viable bacteria is of great practical significance for improving the production capacity and quality stability of streptococcal vaccines and effectively controlling the spread of the disease. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, one aspect of the present invention is to provide a high-density fermentation medium for Streptococcus suis, the medium comprising 1-5 parts beef extract powder, 1-10 parts tryptone, 1-10 parts casein peptone, 1-5 parts soybean peptone, 1-5 parts dipotassium hydrogen phosphate, 1-10 parts sodium chloride, 1-10 parts yeast extract powder, and 1-5 parts glucose.

[0006] Preferably, the culture medium comprises 2.5 parts beef extract powder, 5 parts tryptone, 5 parts casein peptone, 2.5 parts soybean peptone, 2.5 parts dipotassium hydrogen phosphate, 5 parts sodium chloride, 5 parts yeast extract powder, and 2.5 parts glucose.

[0007] Another objective of this invention is to provide a method for preparing a high-density fermentation medium for Streptococcus suis, wherein the specific steps of the preparation method are as follows: Mix the high-density fermentation medium of Streptococcus suis according to the ratio, bring the volume to 1L with distilled water, adjust the pH to 7.4 with 2mol / L NaOH, and sterilize by steaming at 116℃ for 30min.

[0008] Preferably, the high-density fermentation medium for Streptococcus suis is formulated with the following proportions: 1-5 parts beef extract powder, 1-10 parts tryptone, 1-10 parts casein peptone, 1-5 parts soybean peptone, 1-5 parts dipotassium hydrogen phosphate, 1-10 parts sodium chloride, 1-10 parts yeast extract powder, and 1-5 parts glucose.

[0009] Preferably, when using the culture medium, 0.5% of the volume of 50% glucose solution, 2% v / v newborn calf serum, horse serum, and 0.1% v / v sheep lysed blood are added to the culture medium to culture the strain together.

[0010] Preferably, the glucose is prepared as a 50% glucose solution, i.e., 50g of glucose is weighed, diluted to 100mL with distilled water, sterilized by steam at 110℃ for 30min, and added at 0.5% v / v during inoculation.

[0011] The 50% glucose solution consists of 50g of glucose, diluted to 1L (100mL) with distilled water, and sterilized by steam at 110℃ for 30min.

[0012] Another objective of the present invention is to provide an application of a high-density fermentation medium for Streptococcus suis, said high-density fermentation medium for inoculating high-density Streptococcus suis.

[0013] Preferably, the inoculation density of Streptococcus suis is 2% v / v.

[0014] The beneficial effects of this invention are as follows: Significantly increases viable cell yield: The high-density fermentation medium for Streptococcus suis provided by this invention can achieve a viable cell count of 1.79 × 10⁻⁶ for Streptococcus suis. 10 The CFU / mL count is significantly higher than the live bacteria count reported in existing technologies. This groundbreaking high-density fermentation process lays a solid foundation for preparing inactivated vaccines with high antigen content and enhancing vaccine efficacy.

[0015] Significantly reduced production costs: This invention selects inexpensive horse serum as a substitute raw material and reduces its addition amount from over 5% of the traditional culture medium volume to 2%. This optimization significantly reduces raw material costs while ensuring ultra-high viable cell counts, giving the vaccine product significant market competitiveness and economic benefits.

[0016] Enhancing fermentation process stability: The optimized culture medium has a clearly defined composition and stable ratio, effectively reducing batch-to-batch variations. This not only ensures the repeatability and stability of the fermentation process but also guarantees the consistency and controllability of vaccine antigen quality, making it more compliant with the requirements of large-scale industrial production and pharmaceutical manufacturing quality management standards.

[0017] This invention, through key optimization of the Streptococcus suis culture medium, successfully overcomes the technical challenges of low fermentation density and high production costs in existing technologies, providing crucial technical support and a new industrialization path for the efficient, stable, and economical production of high-quality Streptococcus suis vaccines.

[0018] Additional aspects and advantages of the invention will become apparent from the description which follows, or may be learned by practice of the invention. Detailed Implementation

[0019] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0020] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0021] Example 1: Optimization and screening of Streptococcus suis culture medium 1.1 Activation and propagation of Streptococcus suis strains The freeze-dried Streptococcus suis culture was reconstituted in 2 mL of Zhonghai Bio-Martin's Broth (55.5 g of Zhonghai Bio-Martin's Broth powder, 20 g of agar, diluted to 1 L with distilled water, natural pH, steam sterilized at 116℃ for 30 min; 5% v / v newborn calf serum and 0.1% v / v sheep lysed blood were added before use to culture the strain, and the temperature was stabilized at 48℃ in a water bath; 10% (v / v) sheep defibrinated blood was added during preparation). A small amount of the bacterial culture was dipped into Martin's Solid Medium (55.5 g of Martin's Broth powder, 16.0 g of agar, diluted to 1 L with distilled water, natural pH, steam sterilized at 116℃ for 30 min). Stir the medium at 37°C for 30 minutes, stabilize the temperature at 48°C using a water bath, and add 5% (v / v) newborn calf serum and 0.1% (v / v) sheep lysed blood during preparation. Incubate at 37°C inverted for 22 hours. Pick 10-12 single colonies into Martin's blood slant tubes (18×180) (55.5g of Zhonghai Biotech Martin's broth dry powder, 20g of agar, diluted to 1L with distilled water, natural pH, steam sterilized at 116°C for 30 minutes, stabilize the temperature at 48°C using a water bath, and add 10% (v / v) sheep defibrinated blood during preparation). Incubate at 37°C for 20 hours to form a uniform bacterial layer. Inoculate one Martin's blood slant into a 200 / 500 mL bottle of Zhonghai Biotech Martin's broth medium, incubate at 37°C and 120 rpm for 5 hours, and then inoculate with 2% (v / v) into 200 / 500 mL bottles of the medium. mL of homemade buffered broth (1L beef broth (or Martin's broth), 20.0g peptone, 2.0g sodium chloride, 1.0g disodium hydrogen phosphate, 2.0g sodium bicarbonate, and 2.0g glucose; add peptone and sodium chloride to the beef broth or Martin's broth according to the above ratio, heat to 80-90℃, adjust pH to 7.2 with sodium hydroxide solution, then add disodium hydrogen phosphate and sodium bicarbonate (add slowly), boil for 90 minutes, let stand for 2-8 hours (adjust according to the product), aspirate the supernatant, filter, dispense into large bottles or culture tanks, sterilize at 116℃ for 40 minutes, after sterilization the pH is 7.6-8.0, prepare a 50% glucose solution, sterilize intermittently 3 times, once a day, 30 minutes each time, or sterilize at 116℃ for 15-30 minutes, add at inoculation) and Zhonghai Bio Martin's broth medium, incubate at 37℃, 120r / min for 5h, then perform OD. 600 Measurement and CFU count.

[0022] Streptococcus suis bacterial cell OD 600 Measurement: Take 250 μL of the bacterial solution that has been thoroughly shaken, and measure its absorbance at a wavelength of 600 nm using a multi-functional microplate reader. This value is the bacterial concentration of the corresponding bacterial solution.

[0023] Determination of CFU count of Streptococcus suis: The collected bacterial suspension was thoroughly shaken and diluted to 10% of the original solution. -6Times (during fermentation, based on OD) 600 (Adjust the dilution factor according to the test results), take 100 μL of the diluted bacterial solution and spread it onto dry Martin solid medium. Incubate at 37℃ for 22 h, then observe and count the colonies on Martin solid medium with the naked eye.

[0024] The results are shown in Table 1 below. OD values ​​of Streptococcus suis cultured in buffered broth and Zhonghai Biotech Martin's broth medium... 600 The values ​​were 0.466 and 0.473, respectively, and the CFU counts were 0.99 × 10⁻⁶. 8 CFU / mL and 1.16×10 8 CFU / mL.

[0025] Table 1: Results of Streptococcus suis culture

[0026] 1.2 Screening and optimization of Streptococcus suis culture medium Table 1 shows that the culture effects of buffered broth and Zhonghai Biotech Martin's broth on Streptococcus suis were basically the same, with low viable cell counts in both. One Streptococcus suis blood slant was inoculated into a 200 / 500 mL bottle of Zhonghai Biotech Martin's broth and cultured at 37℃ and 120 rpm for 5 h. Then, it was inoculated at 2% (v / v) into both 200 / 500 mL of Zhonghai Biotech Martin's broth and the high-density fermentation medium of Streptococcus suis of this invention, and cultured at 37℃ and 120 rpm for 5 h before OD analysis. 600 Determination and CFU counting; results are shown in Table 2 below, OD of CNOOC Martin's broth medium. 600 The value was 0.494, and the CFU count result was 1.35 × 10⁻⁶. 8 CFU / mL, OD of the high-density fermentation medium for Streptococcus suis of this invention 600 The value was 0.983, and the CFU count result was 12.90 × 10⁻⁶. 8 CFU / mL.

[0027] Table 2. Results of culture medium screening and optimization

[0028] 1.3 Screening of inoculation ratio and culture time for Streptococcus suis One slant inoculum of Streptococcus suis was inoculated into a 200 / 500 mL bottle of Zhonghai Biotechnology Martin's Broth and cultured at 37°C and 120 rpm for 5 h. The inoculum was then inoculated at 2% (v / v) and 3% (v / v) into the high-density fermentation medium of Streptococcus suis of this invention (the medium consists of 2.5 g beef extract, 5 g tryptone, 5 g casein, 2.5 g soybean peptone, 2.5 g dipotassium hydrogen phosphate, 5 g sodium chloride, and 5 g yeast extract, diluted to 1 L with distilled water, and diluted with 2 mol / L...). Adjust the pH to 7.4 with NaOH, steam sterilize at 116℃ for 30 min, and before use, add 0.5% of the volume of 50% glucose solution, 5% (v / v) newborn calf serum, and 0.1% (v / v) sheep lysed blood to culture the strain together. Also add Zhonghai Bio Martin's broth medium (200 / 500 mL) and incubate at 37℃ and 120 rpm for 5 h, 5.5 h, and 6 h respectively to perform OD analysis. 600 Measurement and CFU counting; the results are shown in Table 3 below. The highest viable count of Streptococcus suis in the high-density fermentation medium of this invention was achieved when inoculated at 2% (v / v) and cultured for 5 hours, with OD... 600 The value was 0.887, and the CFU count result was 14.70 × 10⁻⁶. 8 CFU / mL; the highest viable count on Martin's broth medium from CNOOC Biotechnology was achieved when inoculated at 2% (v / v) and cultured for 5 hours, with OD... 600 The value was 0.486, and the CFU count result was 1.40 × 10⁻⁶. 8 CFU / mL; The viable cell count of the high-density fermentation medium for Streptococcus suis of this invention is 10.5 times that of the Martin's broth medium of Zhonghai Biotechnology.

[0029] Table 3. Screening results based on inoculation ratio and culture time

[0030] 1.4 Screening of serum strains and dosages used for Streptococcus suis culture One vial of *Streptococcus suis* blood was obliquely inoculated into a 200 / 500 mL bottle of Zhonghai Biotechnology Martin's broth medium and incubated at 37°C and 120 rpm for 5 h. The inoculum was then inoculated at 2% (v / v) into both Zhonghai Biotechnology Martin's broth medium and the *Streptococcus suis* high-density fermentation medium of this invention (200 / 500 mL). 5% (v / v) newborn calf serum was added to the Zhonghai Biotechnology Martin's broth medium. 5% (v / v) newborn calf serum, horse serum, and 2% (v / v) horse serum were added to the *Streptococcus suis* high-density fermentation medium of this invention, respectively. Ovine lysate was added at 0.1%. CFU counts were performed after incubation at 37°C and 120 rpm for 5 h. The results are shown in Table 4 below. The CFU count of *Streptococcus suis* in the high-density fermentation medium of this invention, with the addition of 2% (v / v) horse serum for 5 h, was 15.10 × 10⁻⁶.8 Although the number of viable bacteria in the medium was slightly lower than that in the medium containing 5% (v / v) newborn calf serum, it was still 11.1 times that of the medium containing Martin's broth from Zhonghai Biotechnology. At the same time, the change from 5% (v / v) newborn calf serum to 2% (v / v) horse serum greatly reduced the production cost.

[0031] Table 4. Results of serum variety and dosage screening

[0032] Example 2: Fermentation with Streptococcus suis 2.1 After rehydrating the freeze-dried Streptococcus suis type 27 culture medium with Martin's broth medium from Zhonghai Biotechnology, use a sterile inoculation loop to take a small amount of bacterial solution and streak it on Martin's solid medium for incubation. Incubate at 37°C with the medium upside down for 22 hours until colonies grow.

[0033] 2.2 Preparation of primary seed culture: Select 10-12 single colonies, inoculate them into one tube of Martin blood slant medium, stopper with a rubber stopper, and incubate at 37°C for 20 hours to form a uniform bacterial layer. Then store at 4°C.

[0034] 2.3 Secondary seed preparation: Inoculate one vial of blood into a bottle of 200 / 500mL of the high-density fermentation medium of Streptococcus suis of this invention, incubate at 37℃ and 120r / min for 5h, and store at 4℃.

[0035] 2.4. The high-density fermentation medium of *Streptococcus suis* of this invention is pumped into the fermenter using an air pump, with a liquid volume of 5L. An antifoaming agent is added at 1‰. The secondary seed liquid is then pumped into the fermenter at an inoculation rate of 2% of the fermentation medium. The internal temperature of the fermenter is set at 37℃, the stirring rate at 120 r / min, and the dissolved oxygen is controlled at 30%. Alkali is supplemented using 2M sodium hydroxide, and a carbon source is supplemented using 50% glucose.

[0036] 2.5. The results of the changes in OD value and viable cell count over time during fermentation are shown in Table 5 below. As can be seen from the table, the optimized high-density fermentation medium for *Streptococcus suis* of this invention, as described in Example 1, showed that at 7 hours, the OD value... 600 The pH reached 7.55, and the viable count reached 1.79 × 10⁻⁶. 10 CFU / mL.

[0037] Table 5. Fermentation results of Streptococcus suis

[0038] Example 3: Preparation of inactivated Streptococcus suis vaccine 3.1 Virulence determination of Streptococcus suis After reconstitution of the freeze-dried Streptococcus suis type 27 strain in the high-density fermentation medium of this invention, a small amount of bacterial solution was taken with a sterile inoculation loop and streaked onto Martin's solid medium. The culture was incubated at 37°C inverted for 22 hours until colonies appeared. Ten to twelve single colonies were picked and inoculated into a Martin's blood slant medium. The culture was incubated statically at 37°C for 20 hours. One Streptococcus suis blood slant was then inoculated into a 200 / 500 mL bottle of the high-density fermentation medium of this invention and incubated at 37°C and 120 rpm for 5 hours. The bacterial solution was serially diluted 2-fold with the high-density fermentation medium of this invention to 2 / 3, 4 / 3, 6 / 3, and 8 / 3 of the original solution. Mice (16-20 g KM mice) were intraperitoneally injected with 0.2 mL per mouse. The lethality of the mice was observed daily until day 7. The results are shown in Table 6 below. The mouse 1 MLD was 3.9 × 10⁻⁶. 7 CFU (Streptococcus suis)

[0039] Table 6. Results of virulence assay for Streptococcus suis

[0040] 3.2 Preparation of inactivated vaccine against Streptococcus suis Streptococcus suis type 27 was selected as the strain for vaccine preparation, and the inactivated Streptococcus suis vaccine was prepared using the following method: 3.2.1 Culture of Streptococcus suis Streptococcus suis was cultured using the high-density fermentation medium of the present invention at 37°C and 120 r / min for 5 h to obtain a Streptococcus suis culture broth.

[0041] 3.2.2 Inactivate the bacterial suspension and perform sterility testing. The method for inactivating the cultured Streptococcus suis bacterial suspension was as follows: inactivation with 0.2% (V / V) formaldehyde solution, inactivation at 37℃ for 24 hours, and shaking 2-3 times daily.

[0042] Sterility testing method: Refer to the "Regulations for Veterinary Biological Products of the People's Republic of China, Version 2000" for operation.

[0043] The results showed that the sterility test of the inactivated bacterial solution obtained in this embodiment was qualified.

[0044] 3.2.3 Preparation of inactivated vaccines Streptococcus suis bacterial suspension and autoclaved aluminum hydroxide gel were mixed at a volume ratio of 5:1, thoroughly shaken, and aseptically dispensed. The prepared experimental vaccine was used to immunize mice (16-20g, KM mice) with a dose of 0.2 mL / mouse via subcutaneous immunization. A total of 10 mice were immunized, and a control group of 10 mice was also included. A second immunization was administered 21 days after the first immunization using the same method and dose. Fourteen days after the second immunization, mice were challenged intraperitoneally with a lethal dose of Streptococcus suis. The results are shown in Table 7. All 10 mice in the control group died, while 1 out of 10 mice in the immunized group died, leaving 9 survivors. Based on these experimental results, using the self-prepared high-density fermentation medium of this invention to culture Streptococcus suis type 27 yields a basic bacterial suspension with a high viable count, which can then be used to prepare a reliable inactivated vaccine with a 90% protection rate in immunized mice.

[0045] Table 7. Immunization Results of Experimental Vaccines

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A high-density fermentation medium for Streptococcus suis, characterized in that: The culture medium comprises 1-5 parts beef extract, 1-10 parts tryptone, 1-10 parts casein peptone, 1-5 parts soybean peptone, 1-5 parts dipotassium hydrogen phosphate, 1-10 parts sodium chloride, 1-10 parts yeast extract, and 1-5 parts glucose.

2. The high-density fermentation medium for Streptococcus suis according to claim 1, characterized in that: The culture medium comprises 2.5 parts beef extract powder, 5 parts tryptone, 5 parts casein peptone, 2.5 parts soybean peptone, 2.5 parts dipotassium hydrogen phosphate, 5 parts sodium chloride, 5 parts yeast extract powder, and 2.5 parts glucose.

3. A method for preparing a high-density fermentation medium for Streptococcus suis, characterized in that: The specific steps for preparing the culture medium are as follows: Mix the high-density fermentation medium of Streptococcus suis according to the ratio, bring the volume to 1L with distilled water, adjust the pH to 7.4 with 2mol / L NaOH, and sterilize by steaming at 116℃ for 30min.

4. The method for preparing a high-density fermentation medium for Streptococcus suis according to claim 3, characterized in that: The high-density fermentation medium for Streptococcus suis is formulated with the following proportions: 1-5 parts beef extract powder, 1-10 parts tryptone, 1-10 parts casein peptone, 1-5 parts soybean peptone, 1-5 parts dipotassium hydrogen phosphate, 1-10 parts sodium chloride, 1-10 parts yeast extract powder, and 1-5 parts glucose.

5. The method for preparing a high-density fermentation medium for Streptococcus suis according to claim 3, characterized in that: When using the culture medium, 0.5% of the volume of 50% glucose solution, 25% v / v newborn calf serum, horse serum, and 0.1% v / v sheep lysed blood are added to culture the strain together.

6. The method for preparing a high-density fermentation medium for Streptococcus suis according to claim 3, characterized in that: The 50% glucose solution is prepared by weighing 50g of glucose, making up to 1L (100mL) with distilled water, sterilizing with steam at 110℃ for 30min, and adding it at 0.5% v / v during inoculation.

7. The application of a high-density fermentation medium for Streptococcus suis, characterized in that: The high-density fermentation medium for Streptococcus suis is used for inoculating high-density Streptococcus suis.

8. The application of the high-density fermentation medium for Streptococcus suis according to claim 7, characterized in that: The inoculation density of Streptococcus suis was 2% v / v.

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