Hib solid culture medium, preparation method thereof and fermentation culture method of haemophilus influenzae type b

Abstract

The invention discloses a Hib solid culture medium, a preparation method thereof and a fermentation culture method of haemophilus influenzae type b. The solid culture medium comprises 1 to 3g / L of yeast extract powder, 3 to 6g / L of tryptone, 3 to 6g / L of sodium chloride, 0.5 to 1.5 g / L of anhydrous disodium hydrogen phosphate, 25 to 35g / L of agar powder, 2 to 3g / L of glucose, 0.01 to 0.03 g / L of NAD (Nicotinamide Adenine Dinucleotide), 0.02 to 0.05 g / L of hemin and 5 to 10g / L of osmotic pressure protective agent. The osmotic pressure protective agent comprises taurine, polyhydric alcohols and derivatives of the polyhydric alcohols in a mass ratio of (1-2): (1-2). The culture medium disclosed by the invention does not contain animal-derived components, so that the negative effects of pollutants such as nucleic acid, cell protein and endotoxin in capsular polysaccharide on a product purification process and immunogenicity are reduced.

Description

Technical Field

[0001] The present application relates to the field of microbial culture, and in particular to a Hib solid culture medium and a preparation method thereof, and a fermentation culture method of Haemophilus influenzae type b. Background Art

[0002] Haemophilus influenzae type b (Hib) is a common pathogen that causes serious infections in children. Its main pathogenic factor is capsular polysaccharide (CPS). Hib vaccine is mainly formed by combining Hib capsular polysaccharide with protein carrier to form a conjugate vaccine to enhance the immune effect and provide long-term protection. Therefore, high-quality Hib capsular polysaccharide is crucial to the effectiveness of the vaccine, which makes the fermentation culture of Hib of great significance.

[0003] In industrial production, in order to obtain high yield and high purity of Hib capsular polysaccharides, the growth conditions of microorganisms must be optimized. At present, brain heart infusion (BHI) is usually added to the culture medium used for Hib culture. This is a complex nutrient solution made from animal tissues that can provide microorganisms with necessary amino acids, vitamins and other nutrients. However, as an animal-derived ingredient, BHI has certain limitations. Since the raw materials and processes used in its production process may fluctuate, the composition of the ingredients in the final product may be unstable, which may not only affect the growth efficiency of Hib, but also the subsequent extraction and purification process of capsular polysaccharides.

[0004] More importantly, in the process of large-scale production of capsular polysaccharides, components in BHI such as nucleic acids and cellular proteins may contaminate the product, especially the lipopolysaccharide (LPS) called endotoxin, which is highly variable (in size, charge, hydrophobicity, etc.), which poses great challenges to purification. Existing purification methods are often complicated, time-consuming and costly, and may damage precious CPS components during the purification process, reducing their yield. In addition, the use of animal-derived components also poses a potential risk of immunogenicity changes, which is an issue that cannot be ignored in vaccine manufacturing.

[0005] In view of the above reasons, the development of a new culture medium that does not contain animal-derived ingredients, has good stability and can effectively promote the growth of Hib is of great significance for improving the production efficiency and quality of Hib capsular polysaccharides. Summary of the invention

[0006] In order to solve the adverse effects of animal-derived components in current Hib culture medium on the purity, yield and immunogenicity of Hib capsular polysaccharide, the present application provides a Hib solid culture medium without brain heart infusion and a preparation method thereof, and a fermentation culture method of Haemophilus influenzae type b.

[0007] In a first aspect, the present application provides a Hib solid culture medium, comprising: 1-3 g / L yeast extract powder, 3-6 g / L tryptone, 3-6 g / L sodium chloride, 0.5-1.5 g / L anhydrous disodium hydrogen phosphate, 25-35 g / L agar powder, 2-3 g / L glucose, 0.01-0.03 g / L NAD, 0.02-0.05 g / L hemin, and 5-10 g / L osmotic pressure protective agent; the osmotic pressure protective agent comprises taurine and polyols and their derivatives in a mass ratio of 1-2:1-2.

[0008] Preferably, the polyol and its derivatives include propylene glycol, glycerol, butylene glycol, sugar alcohol or a combination of several thereof.

[0009] Preferably, the sugar alcohol includes one or a combination of sorbitol, mannitol, erythritol, maltitol, lactitol and xylitol.

[0010] The solid culture medium in the present application can achieve a similar bacterial growth rate as that of the culture medium with brain heart infusion, and achieve a similar bacterial concentration within the same culture time. Specifically, the present application increases the amount of agar in the culture medium to reduce the free water content in the solid culture medium, increase the oxygen and carbon dioxide exchange rate of the bacteria, and compensate for the negative impact of the lack of brain heart infusion on the decrease in bacterial growth rate during the culture process by increasing its metabolic rate. At the same time, it promotes the increase in the yield of capsular polysaccharides.

[0011] It should be noted that the reduction of free water content in the solid culture medium may also lead to dehydration of microorganisms, thereby slowing down bacterial growth. For this reason, the culture medium of the present application is added with an osmotic pressure protectant to maintain the balance of bacterial osmotic pressure, maintain the integrity of bacterial cells, promote the exchange of nutrients and the transfer of metabolic substances, and ensure the growth rate of bacteria. In addition, the above-mentioned osmotic pressure protectants can provide rapidly available nitrogen and carbon sources for bacterial growth and reproduction, respectively, and compensate for the loss of nutrients caused by the removal of brain heart infusion.

[0012] Preferably, the polyol is panthenol and glycerol in a mass ratio of 1:0.9 to 1.5.

[0013] The polyols using sugar alcohols and glycerol have different molecular structures, which can better balance the osmotic pressure. The hydrophilicity of sugar alcohols makes them better at maintaining cell activity, while glycerol has a smaller molecular weight and higher bioavailability. It can be used as a carbon source that is more easily absorbed and utilized by the flora, supporting the rapid growth of the flora and the growth of the propagating bacteria. Specifically. It should be noted that the osmotic pressure protectant added in this application is sufficient to achieve the above-mentioned multiple effects.

[0014] In a second aspect, the present application provides a method for preparing a solid culture medium, which is prepared by using any of the above-mentioned solid culture medium raw material ratios and following the following steps: Yeast extract powder, tryptone, sodium chloride, and anhydrous disodium hydrogen phosphate are added into water according to the formula amount, stirred to dissolve, and then fixed to volume, and the pH value is adjusted to 7.2-7.4 to obtain a premixed solution; Add agar powder to the premixed liquid and boil to dissolve, and after fully dissolved, dispense into gram bottles for sterilization to obtain a sterilized culture medium; preferably, the sterilization conditions are a temperature of 115-120° C. and a time of 20-30 min; Weigh glucose, NAD, and hemin, dissolve glucose and NAD in water and then make up to volume, dissolve hemin with 5% ammonia water and then make up to volume with water; The solution of glucose, NAD and hemin is sterilized by filtering with a sterile filter, then added to the sterilized culture medium and mixed evenly, and pre-incubated after solidification.

[0015] Preferably, the preincubation conditions are a temperature of 35 to 37° C. and a time of 70 to 75 hours.

[0016] Preferably, after the pre-incubation is completed, the culture medium is placed in a refrigerator at 4-8°C for storage.

[0017] In a third aspect, the present application provides a fermentation culture method of Haemophilus influenzae type b, comprising: First generation culture: dripping the bacterial liquid into Hib solid culture medium, placing it in a carbon dioxide incubator, maintaining the temperature at 35-37°C, the carbon dioxide concentration at 5±0.5%, and culturing for 16-24 hours; wherein the solid culture medium is any of the solid culture medium described above or the solid culture medium prepared by the above method; Second generation culture: Take liquid culture medium solution to rinse the bacterial lawn on Hib solid culture medium, scrape the colonies on the solid culture medium with a scraper, transfer them to liquid culture medium after aspiration and beating evenly, and then place them in a shaker for culture at a temperature of 35-37°C and a rotation speed of 100-300 rpm for 2-5 hours; Third generation culture: pump the liquid culture medium and the second generation liquid strain harvest liquid into a 20L fermenter for culture; during the culture process, increase the stirring speed and air flow rate according to the change of dissolved oxygen, and control the dissolved oxygen content at 20%±15%; culture for 2 to 6 hours, and end when the 20L fermenter culture grows to the logarithmic phase and the OD600nm is between 0.6 and 1.2; Fermentation culture: Transfer the third-generation liquid strain harvest liquid to a 200L fermenter filled with liquid culture medium for fermentation culture; during the culture process, increase the stirring speed and air flow rate according to the change of dissolved oxygen, and control the dissolved oxygen content at 20%±15%; culture for 3 to 10 hours until the OD600nm value of the culture slows down or decreases, and then terminate the fermentation culture.

[0018] Preferably, the liquid culture medium comprises: Acid hydrolyzed casein 5-10g / L, yeast extract powder 3-6g / L, sodium dihydrogen phosphate monohydrate 1-2g / L, anhydrous sodium dihydrogen phosphate 1-2g / L, sodium lactate aqueous solution 1-2ml / L, ammonium sulfate 0.5-1.5g / L, L-cystine 0.01-0.05g / L, L-tryptophan 0.01-0.05g / L, glucose 2-6g / L, NAD 0.01-0.1g / L, hemin chloride 0.01-0.05g / L, anhydrous calcium chloride 0.01-0.03g / L, magnesium sulfate 1-2g / L.

[0019] Glucose and L-cystine and L-cystine are added to the liquid culture medium to compensate for the carbon source and nitrogen source required for bacterial growth. Experiments have shown that the addition of phosphates can also help increase the yield of capsular polysaccharides, which may be due to the fact that the capsular polysaccharides contain more ribosyl phosphate.

[0020] Preferably, in the third generation culture, the ventilation flow rate is 2 to 10 L / min; the stirring speed is 100 to 300 rpm; the tank pressure is 0.01 to 0.03 MPa; the pH is 6.5 to 7.5; and the temperature is 36±1°C.

[0021] Preferably, in the fourth generation culture, the ventilation flow rate is 4-40 L / min; the stirring speed is 100-600 rpm; the tank pressure is 0.01-0.03 MPa; pH: 6.5-7.5; and the temperature is 36±1°C.

[0022] Preferably, when increasing the stirring speed and the air flow rate to control the dissolved oxygen content, the stirring speed is increased by 5 to 10 rpm or the air flow rate is increased by 1 to 2 L / min each time.

[0023] Increasing the dissolved oxygen in the culture medium can increase the growth rate of Hib bacteria and the yield of capsular polysaccharides. The purpose of increasing the stirring speed or air flow is to increase the dissolved oxygen content in the liquid culture medium. When the dissolved oxygen content decreases during the culture process, the dissolved oxygen content can be increased by increasing the stirring speed and / or increasing the air flow. However, within the allowable range of the stirring speed, increasing the stirring speed is preferred.

[0024] Preferably, the OD inspection frequency after the start of culture is once at 0 hour and once every 0 to 1 hour; the sampling time can be appropriately adjusted according to the actual fermentation situation.

[0025] The present application maintains dissolved oxygen by continuously rotating at a high speed during the operation phase, which helps to increase the growth rate and culture effect of bacteria.

[0026] In summary, this application has the following beneficial effects: The present application improves the stability of the quality of the capsular polysaccharide component, reduces the difficulty of purification and extraction, and reduces the risk of immunogenicity changes by removing the brain heart infusion from the traditional culture medium. At the same time, by adjusting the amount of agar in the solid culture medium for bacterial culture and using a specific osmotic pressure protective agent, the adverse effects of the absence of brain heart infusion on the bacterial culture rate and effect are effectively compensated. DETAILED DESCRIPTION

[0027] Preparation Example The raw materials selected in the preparation examples of this application are: the yeast extract powder is Angel Yeast Extract Powder FM802; the tryptone is LP0042 from Jiangsu Mag Biotechnology; the agar powder is purchased from Jiangsu Shunying Biotechnology (80-100 mesh); the brain heart infusion (BHI) is Oxoid CM1135B.

[0028] Preparation Example 1-1, a Hib solid culture medium, the preparation method is as follows: Weigh 2 g / L yeast extract powder, 5 g / L tryptone, 5 g / L sodium chloride, 0.99 g / L anhydrous disodium hydrogen phosphate, 4 g / L taurine, 1.8 g / L sorbitol, and 2.2 g / L glycerol, add appropriate amount of water and stir, make up to volume after fully dissolving, adjust the pH value to 7.2-7.4, and obtain solution 1.

[0029] Agar powder was weighed in an amount of 30.5 g / L and added to solution 1 for boiling and dissolution. After sufficient dissolution, it was dispensed into gram bottles for sterilization at 115° C. for 30 min to obtain a sterilized culture medium.

[0030] Weigh the amount of glucose 2g / L, NAD 0.01g / L, and hemin 0.05g / L, dissolve glucose and NAD in water and dilute to obtain glucose solution and NAD solution for later use. Dissolve hemin in an appropriate amount of 5% ammonia water, dilute to obtain hemin solution for later use.

[0031] The glucose solution, NAD solution, and hemin solution were sterilized by filtering with a 0.2 um sterile filter, and then added to the sterilized culture medium and mixed evenly, and pre-incubated after solidification. Pre-incubation conditions: temperature 35-37°C, time 72h.

[0032] Preparation Example 1-2, a Hib solid culture medium, the preparation method is as follows: Weigh 1 g / L yeast extract powder, 6 g / L tryptone, 3 g / L sodium chloride, 1.5 g / L disodium hydrogen phosphate anhydrous, 2 g / L taurine, 1.6 g / L mannitol, and 2.4 g / L glycerol, add appropriate amount of water and stir, make up to volume after fully dissolving, adjust the pH value to 7.2-7.4, and obtain a premix.

[0033] Agar powder was weighed in an amount of 26 g / L and added to the premixed solution for boiling and dissolution. After sufficient dissolution, it was dispensed into gram bottles for sterilization at 120° C. for 25 min to obtain a sterile culture medium.

[0034] Weigh the amount of glucose 3g / L, NAD 0.03g / L, and hemin 0.03g / L, dissolve glucose and NAD in water and dilute to obtain glucose solution and NAD solution for later use. Dissolve hemin in an appropriate amount of 5% ammonia water, dilute to obtain hemin solution for later use.

[0035] The glucose solution, NAD solution, and hemin solution were sterilized by filtering with a 0.2 um sterile filter, and then added to the sterilized culture medium and mixed evenly, and pre-incubated after solidification. Pre-incubation conditions: temperature 35-37°C, time 72h.

[0036] Preparation Example 1-3, a Hib solid culture medium, the preparation method is as follows: Weigh 3 g / L yeast extract powder, 3 g / L tryptone, 4 g / L sodium chloride, 0.58 g / L anhydrous disodium hydrogen phosphate, 4 g / L taurine, 3.1 g / L maltitol, and 2.9 g / L butanediol, add appropriate amount of water and stir, make up to volume after fully dissolving, adjust pH value to 7.2-7.4, and obtain solution 1.

[0037] Agar powder was weighed in an amount of 34.6 g / L and added to solution 1 for boiling and dissolution. After sufficient dissolution, it was dispensed into gram bottles for sterilization at 115° C. for 30 min to obtain a sterilized culture medium.

[0038] Weigh the amount of glucose 2g / L, NAD 0.02g / L, and hemin 0.05g / L, dissolve glucose and NAD in water and dilute to obtain glucose solution and NAD solution for later use. Dissolve hemin in an appropriate amount of 5% ammonia water, dilute to obtain hemin solution for later use.

[0039] The glucose solution, NAD solution, and hemin solution were sterilized by filtering with a 0.2 um sterile filter, and then added to the sterilized culture medium and mixed evenly, and pre-incubated after solidification. Pre-incubation conditions: temperature 35-37°C, time 72h.

[0040] Preparation Example 1-4, a Hib solid culture medium, differs from Preparation Example 1-1 in that glycerol is replaced by an equal amount of sorbitol.

[0041] Preparation Example 1-5, a Hib solid culture medium, differs from Preparation Example 1-1 in that sorbitol is replaced by an equal amount of glycerol.

[0042] Preparation Example 1-6, a Hib solid culture medium, differs from Preparation Example 1-1 in that taurine is replaced by an equal amount of sorbitol.

[0043] Preparation Example 1-7, a Hib solid culture medium, differs from Preparation Example 1-1 in that sorbitol and glycerol are replaced with equal amounts of taurine.

[0044] Preparation Example 1-8, a Hib solid culture medium, differs from Preparation Example 1-1 in that taurine, sorbitol and glycerol are replaced by equal amounts of glucose.

[0045] Preparation Example 1-9, a Hib solid culture medium, is different from Preparation Example 1-8 in that the agar concentration in the solid culture medium is 20 g / L.

[0046] Preparation Example 2-1, a Hib liquid culture medium, the preparation steps are as follows: Weigh 8 g / L of acid hydrolyzed casein, 4 g / L of yeast extract powder, 1 g / L of sodium dihydrogen phosphate monohydrate, 1 g / L of anhydrous sodium dihydrogen phosphate, 1.3 ml / L of sodium lactate aqueous solution, and 1 g / L of ammonium sulfate, add water and stir to fully dissolve to obtain Solution 1.

[0047] Weigh out 0.02 g / L L-cystine and 0.015 g / L L-tryptophan according to the formula, add 5% concentration of ammonia water and stir to dissolve, to obtain solution 2.

[0048] Pour solution 2 and solution 1 into the same container, dilute to the required volume with water and mix well, adjust the pH to 7.2-7.4, pour into a shaking bottle and sterilize with wet heat at 115°C for 30 min to obtain the basal culture medium.

[0049] According to the formula of glucose 4g / L, NAD 0.015g / L, and hemin 0.02g / L, glucose and NAD were weighed separately, glucose and NAD were dissolved in water and then fixed to volume, hemin was dissolved in 5% ammonia water, and then fixed to volume with water. The dissolved glucose solution, NAD solution, and hemin solution were sterilized by filtering with a 0.2um sterile filter and then added to a sterilized container for storage.

[0050] According to the formula of 0.01g / L anhydrous calcium chloride and 1g / L magnesium sulfate, they were weighed separately, fixed to volume after fully dissolved, and sterilized by wet heat at 115℃ for 30min.

[0051] Preparation Example 2-2, a Hib liquid culture medium, the preparation steps are as follows: Weigh 5 g / L of acid hydrolyzed casein, 6 g / L of yeast extract powder, 1 g / L of sodium dihydrogen phosphate monohydrate, 1 g / L of anhydrous sodium dihydrogen phosphate, 1.8 ml / L of sodium lactate aqueous solution, and 0.5 g / L of ammonium sulfate, add water and stir to fully dissolve to obtain solution 1.

[0052] Weigh 0.01 g / L L-cystine and 0.03 g / L L-tryptophan according to the formula, add 5% concentration of ammonia water and stir to dissolve, to obtain solution 2.

[0053] Pour solution 2 and solution 1 into the same container, dilute to the required volume with water and mix well, adjust the pH to 7.2-7.4, pour into a shaking bottle and sterilize with wet heat at 115°C for 30 min to obtain the basal culture medium.

[0054] According to the formula of glucose 2g / L, NAD 0.03g / L, and hemin 0.01g / L, they were weighed separately, glucose and NAD were dissolved in water and then fixed to volume, hemin was dissolved in 5% ammonia water, and then fixed to volume with water. The dissolved glucose solution, NAD solution, and hemin solution were sterilized by filtering with a 0.2um sterile filter and then added to a sterilized container for storage.

[0055] According to the formula of 0.02g / L anhydrous calcium chloride and 2g / L magnesium sulfate, they were weighed separately, fixed to volume after fully dissolved, and sterilized by wet heat at 115℃ for 30min.

[0056] Preparation Example 2-3, a Hib liquid culture medium, the preparation steps are as follows: Weigh the following formula: 10 g / L acid hydrolyzed casein, 3 g / L yeast extract powder, 2 g / L sodium dihydrogen phosphate monohydrate, 1 g / L anhydrous sodium dihydrogen phosphate, 1.3 ml / L sodium lactate aqueous solution, and 1.5 g / L ammonium sulfate, add water, stir, and fully dissolve to obtain Solution 1.

[0057] Weigh 0.05 g / L L-cystine and 0.01 g / L L-tryptophan according to the formula, add 5% concentration of ammonia water and stir to dissolve, to obtain solution 2.

[0058] Pour solution 2 and solution 1 into the same container, dilute to the required volume with water and mix well, adjust the pH to 7.2-7.4, pour into a shaking bottle and sterilize with wet heat at 115°C for 30 min to obtain the basal culture medium.

[0059] According to the formula of glucose 6g / L, NAD 0.01g / L, and hemin 0.04g / L, glucose and NAD were weighed separately, glucose and NAD were dissolved in water and then fixed to volume, hemin was dissolved in 5% ammonia water, and then fixed to volume with water. The dissolved glucose solution, NAD solution, and hemin solution were sterilized by filtering with a 0.2um sterile filter and then added to a sterilized container for storage.

[0060] According to the formula of 0.03g / L anhydrous calcium chloride and 1g / L magnesium sulfate, they were weighed separately, fixed to volume after fully dissolved, and sterilized by wet heat at 120℃ for 30min.

[0061] Preparation Example 2-4, a Hib liquid culture medium, differs from Preparation Example 2-1 in that sodium dihydrogen phosphate monohydrate and anhydrous sodium dihydrogen phosphate are replaced with equal amounts of sodium bicarbonate.

[0062] Example 1, a fermentation culture method of Haemophilus influenzae type b, the steps are as follows: First generation culture: Place the Hib solid culture medium obtained in Preparation Example 1-1 in a 35-37°C incubator at least two hours in advance for rewarming, re-dissolve the freeze-dried Haemophilus influenzae type b (Hib strain is from the China Medical Bacteria Collection Center) with 1 ml of physiological saline, drop the re-dissolved bacterial solution into the rewarmed solid culture medium, and gently shake the bottle to evenly distribute the bacterial solution on the Hib solid culture medium. Place the Hib solid culture medium in a carbon dioxide incubator, maintain the temperature at 35-37°C and the carbon dioxide concentration at 5±0.5%, and culture for 20 hours to obtain the first generation of bacteria.

[0063] Second generation culture: Take 80ml of 0.01g / L calcium chloride solution, 80ml of 1g / L magnesium sulfate solution, 20ml of 4g / L glucose solution and 20ml of 0.02g / L hemin solution prepared in Preparation Example 2-1 and add them to a shake flask containing 380ml of basal culture solution (prepared in Preparation Example 2-1), shake well to obtain culture medium solution. Use a pipette to absorb about 10ml of culture medium solution in the shake flask, rinse the bacterial moss on the Hib solid culture medium after the first generation culture, then use a scraper to scrape off the colonies on the solid culture medium, then use a pipette to evenly suck them out into the shake flask containing the culture medium solution, and finally add 20ml of 0.015g / L NAD solution (prepared in Preparation Example 2-1). Put the inoculated shake flask into a shaker for culture, the culture conditions are temperature 35-37℃, rotation speed 100-300rpm, culture for 5 hours, and obtain the second generation liquid strain harvest liquid.

[0064] Third generation culture: check the second generation liquid strain harvest liquid, check the setting values ​​and control status of the parameters such as ventilation, stirring, tank pressure, pH, temperature, dissolved oxygen, etc. of the 20L fermenter, among which, the ventilation flow rate is 5-10L / min; stirring speed is 100-300rpm; tank pressure is 0.01-0.03MPa; pH is 6.5-7.5; temperature is 36±1℃; dissolved oxygen is 20%±15%. Pump the basic culture liquid, calcium chloride solution, magnesium sulfate solution, glucose solution and hemin chloride solution prepared in Preparation Example 2-1 into the 20L fermenter in sequence, then connect the second generation liquid strain harvest liquid straight-through head and the 20L fermenter inoculation tube under the flame, open the corresponding feed valve and pump it into the 20L fermenter with a peristaltic pump, and then pump the NAD solution after the inoculation. Click the culture start button on the screen at the beginning of the inoculation, and the inoculation is completed. Observe the foam changes at any time during the culture process, and add an appropriate amount of 20wt% defoaming agent to defoam. When dissolved oxygen approaches the lower limit of the control index, increase the stirring speed by 5 to 10 rpm each time. After the start of cultivation, the OD inspection frequency is: sampling once at 0 hours, sampling once every 1 hour, and cultivating for 2 to 6 hours. When the 20L fermentation tank culture grows to the logarithmic phase and the OD600nm is between 0.6 and 1.2, pressurize the 20L fermentation tank to 0.03 to 0.06MPa, start the transfer procedure, and transfer 15L culture to the 200L fermentation tank for cultivation.

[0065] Fermentation culture: Check the setting values ​​and control status of the ventilation, stirring, tank pressure, pH, temperature, dissolved oxygen and other parameters of the 200L fermentation tank, among which the ventilation flow rate is 30L / min; stirring speed is 350rpm; tank pressure is 0.01~0.03MPa; pH is 6.5~7.5; temperature is 36±1℃; dissolved oxygen is 20%±15%. Observe the foam changes at any time during the culture process, and add an appropriate amount of 20wt% defoaming agent to defoam. When the dissolved oxygen is close to the lower limit of the control index, increase the stirring speed, and increase the speed by 5~10rpm each time. The OD inspection frequency after the start of culture is: sampling once at 0 hours, sampling once every 1 hour, and culturing for 3~10 hours. The fermentation culture is terminated until the OD600nm value of the culture slows down or decreases.

[0066] At the end of fermentation, close the pH automatic control and feed valve of the fermenter, adjust the speed to 150rpm, adjust the air intake mode to shallow air intake, set the temperature to 20℃, add formaldehyde solution at 1%±0.3% (V / V) of the culture solution through the feed valve, and sterilize for 60±5min.

[0067] Example 2, a fermentation culture method for Haemophilus influenzae type b, is different from Example 1 in that the first-generation culture adopts the Hib solid culture medium prepared in Preparation Example 1-2, and the second-generation culture adopts the Hib liquid culture medium prepared in Preparation Example 2-2.

[0068] Example 3, a fermentation culture method for Haemophilus influenzae type b, is different from Example 1 in that the first-generation culture uses the Hib solid culture medium prepared in Preparation Examples 1-3, and the second-generation culture uses the Hib liquid culture medium prepared in Preparation Examples 2-3.

[0069] Example 4, a fermentation culture method for Haemophilus influenzae type b, is different from Example 1 in that the first-generation culture adopts the Hib solid culture medium prepared in Preparation Example 1-4.

[0070] Example 5, a fermentation culture method for Haemophilus influenzae type b, is different from Example 1 in that the first-stage culture adopts the Hib solid culture medium prepared in Preparation Example 1-5.

[0071] Example 6, a fermentation culture method for Haemophilus influenzae type b, is different from Example 1 in that the second generation culture adopts the Hib liquid culture medium prepared in Preparation Example 2-4.

[0072] Comparative Example Comparative Example 1 is a fermentation culture method for Haemophilus influenzae type b, which is different from Example 1 in that the Hib solid culture medium prepared in Preparation Example 1-6 is used in the first generation culture.

[0073] Comparative Example 2 is a fermentation culture method for Haemophilus influenzae type b, which is different from Example 1 in that the Hib solid culture medium prepared in Preparation Example 1-7 is used in the first generation culture.

[0074] Comparative Example 3, a fermentation culture method for Haemophilus influenzae type b, is different from Example 1 in that the first-stage culture adopts the Hib solid culture medium prepared in Preparation Examples 1-8.

[0075] Comparative Example 4 is a fermentation culture method for Haemophilus influenzae type b, which is different from Example 1 in that the first-generation culture adopts the Hib solid culture medium prepared in Preparation Examples 1-9.

[0076] Comparative Example 5, a fermentation culture method for Haemophilus influenzae type b, is different from Comparative Example 4 in that, in the preparation process of Hib solid culture medium and Hib liquid culture medium, an equal amount of brain heart infusion is used to replace yeast extract powder.

[0077] The following experiments illustrate the beneficial effects of this application: 1. Test results of physical and chemical properties of capsular polysaccharides The sterilized fermentation broth of the embodiment and the comparative example was used as a sample, centrifuged at 7500xg, the supernatant was collected, 0.1% hexadecyl trimethyl ammonium bromide was added, and the mixture was allowed to stand at 2-8°C for 5 hours, and the precipitate was collected by centrifugation to obtain crude polysaccharide. The conventional techniques in the art were used to remove nucleic acids by ethanol precipitation, remove proteins and bacterial endotoxins by phenol extraction, and then ultrafiltration was used to remove small molecules. Then, 70% ethanol was used for precipitation in the presence of 0.3 mol / L NaCl, and the polysaccharide was collected by centrifugation. The polysaccharide was washed three times with anhydrous ethanol and acetone. The polysaccharide was dried at 4°C to obtain refined capsule polysaccharide. The yield results are shown in Table 1.

[0078] The refined capsular polysaccharide was tested according to the method of "Pharmacopoeia of the People's Republic of China" (2010 edition, Part III).

[0079] Table 1. Test results of physical and chemical properties of refined capsule polysaccharide As shown in Table 1, the Hib capsular polysaccharide quality of the Hib flora fermented and cultured using the culture medium and method of the present application fully meets the requirements of the Chinese Pharmacopoeia standard. Compared with the capsular polysaccharide obtained using the culture medium containing heart and brain infusion, the content of ribose, nucleic acid, protein and endotoxin contained in it is significantly reduced, reducing the difficulty of product purification and the risk of immunogenicity changes.

[0080] 2. Comparison of growth rates of Haemophilus influenzae type b Table 2. OD value test results of first generation culture of Haemophilus influenzae type b Table 3. OD value test results of fermentation culture of Haemophilus influenzae type b Combining Examples 1 to 6 and Comparative Examples 1 to 5 and Table 2, it can be seen that in the first generation culture, using the culture medium of the present application without heart and brain infusion for bacterial culture can achieve a growth effect similar to that of using the culture medium containing heart and brain infusion.

[0081] 3. Capsular polysaccharide yield The yield of refined capsular polysaccharide extracted from the above-mentioned physical and chemical properties tests was recorded. The results are shown in Table 4.

[0082] Table 4. Capsular polysaccharide yield test results It can be seen from Example 1 and Example 6 and Table 4 that adding phosphate to the liquid culture medium helps to increase the yield of capsule.

[0083] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A Hib solid culture medium, characterized in that include: 1-3 g / L yeast extract powder, 3-6 g / L tryptone, 3-6 g / L sodium chloride, 0.5-1.5 g / L anhydrous disodium hydrogen phosphate, 25-35 g / L agar powder, 2-3 g / L glucose, 0.01-0.03 g / L NAD, 0.02-0.05 g / L hemin, and 5-10 g / L osmotic pressure protective agent; the osmotic pressure protective agent comprises taurine and polyols and their derivatives in a mass ratio of 1-2:1-2.

2. The solid culture medium according to claim 1, characterized in that The polyol and its derivatives include propylene glycol, glycerol, butylene glycol, sugar alcohol or a combination of several thereof.

3. The solid culture medium according to claim 2, characterized in that The sugar alcohol includes one or a combination of sorbitol, mannitol, erythritol, maltitol, lactitol and xylitol.

4. The solid culture medium according to claim 2, characterized in that The polyol is made of sugar alcohol and glycerol in a mass ratio of 1:0.9-1.

5.

5. A method for preparing a solid culture medium, characterized in that: The mixture is prepared by using any of the ratios described in claims 1 to 4 and following the steps as follows: Yeast extract powder, tryptone, sodium chloride, and anhydrous disodium hydrogen phosphate are added into water according to the formula amount, stirred to dissolve, and then fixed to volume, and the pH value is adjusted to 7.2-7.4 to obtain a premixed solution; Add agar powder to the premixed liquid and boil to dissolve, and after fully dissolved, dispense into gram bottles for sterilization to obtain a sterilized culture medium; preferably, the sterilization conditions are a temperature of 115-120° C. and a time of 20-30 min; Weigh glucose, NAD, and hemin, dissolve glucose and NAD in water and then make up to volume, dissolve hemin with 5% ammonia water and then make up to volume with water; The solution of glucose, NAD and hemin is sterilized by filtering with a sterile filter, then added to the sterilized culture medium and mixed evenly, and pre-incubated after solidification.

6. A fermentation culture method for Haemophilus influenzae type b, characterized in that: include: First generation culture: drop the bacterial liquid into Hib solid culture medium, place it in a carbon dioxide incubator, maintain the temperature at 35-37°C, the carbon dioxide concentration at 5±0.5%, and culture for 16-24 hours; wherein the solid culture medium is the solid culture medium described in any one of claims 1 to 4 or the solid culture medium prepared by the method of claim 5; Second generation culture: Take liquid culture medium solution to rinse the bacterial lawn on Hib solid culture medium, scrape the colonies on the solid culture medium with a scraper, transfer them to liquid culture medium after aspiration and beating evenly, and then place them in a shaker for culture at a temperature of 35-37°C and a rotation speed of 100-300 rpm for 2-5 hours; Third generation culture: pump the liquid culture medium and the second generation liquid strain harvest liquid into a 20L fermenter for culture; during the culture process, increase the stirring speed and air flow rate according to the change of dissolved oxygen, and control the dissolved oxygen content at 20%±15%; culture for 2 to 6 hours, and end when the 20L fermenter culture grows to the logarithmic phase and the OD600nm is between 0.6 and 1.2; Fermentation culture: Transfer the third-generation liquid strain harvest liquid to a 200L fermenter filled with liquid culture medium for fermentation culture; during the culture process, increase the stirring speed and air flow rate according to the change of dissolved oxygen, and control the dissolved oxygen content at 20%±15%; culture for 3 to 10 hours until the OD600nm value of the culture slows down or decreases, and then terminate the fermentation culture.

7. The method according to claim 6, characterized in that The liquid culture medium comprises: Acid hydrolyzed casein 5-10g / L, yeast extract powder 3-6g / L, sodium dihydrogen phosphate monohydrate 1-2g / L, anhydrous sodium dihydrogen phosphate 1-2g / L, sodium lactate aqueous solution 1-2ml / L, ammonium sulfate 0.5-1.5g / L, L-cystine 0.01-0.05g / L, L-tryptophan 0.01-0.05g / L, glucose 2-6g / L, NAD 0.01-0.1g / L, hemin chloride 0.01-0.05g / L, anhydrous calcium chloride 0.01-0.03g / L, magnesium sulfate 1-2g / L.

8. The method according to claim 6, characterized in that In the third generation culture, the ventilation flow rate is 2-10 L / min; the stirring speed is 100-300 rpm; the tank pressure is 0.01-0.03 MPa; pH: 6.5~7.5; temperature: 36±1℃.

9. The method according to claim 6, characterized in that In the fourth generation culture, the ventilation flow rate is 4-40L / min; the stirring speed is 100-600rpm; the tank pressure is 0.01-0.03MPa; pH: 6.5~7.5; temperature: 36±1℃.

10. The method according to claim 6, characterized in that When increasing the stirring speed and air flow to control the dissolved oxygen content, increase the stirring speed by 5 to 10 rpm or increase the air flow by 1 to 2 L / min each time.

Images