Group B streptococcus fermentation medium, preparation method and fermentation culture method

Abstract

The group B streptococcus fermentation culture medium comprises salt substances, a carbon source, an amino acid source, a vitamin source, a purine source, a pyrimidine source, a nitrogen source and solvent water, salt substances: 0.2 g / L to 0.5 g / L of dipotassium phosphate, 0.3 g / L to 0.5 g / L of monopotassium phosphate, 3 g / L to 5 g / L of disodium hydrogen phosphate, 2 g / L to 5 g / L of sodium dihydrogen phosphate monohydrate, 0.5 g / L to 0.8 g / L of ammonium sulfate, 0.2 g / L to 0.5 g / L of magnesium sulfate heptahydrate, 0.01 g / L to 0.03 g / L of manganous sulfate monohydrate and 0.03 g / L to 0.06 g / L of ferrous sulfate; an amino acid source: 0.2 g / L to 0.5 g / L of glycine and 0.1 g / L to 0.3 g / L of tryptophan; a nitrogen source: 3-6g / L of acid hydrolyzed casein; the feed also comprises 5-10g / L of yeast extract powder. The group B streptococcus is fermented and cultured by using the fermentation culture medium disclosed by the invention, sufficient nutrient substances can be provided for strains, and the bacterium concentration is effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a group B streptococcus fermentation medium, a preparation method and a fermentation culture method. Background Art

[0002] Group B Streptococcus, also known as GBS and Streptococcus agalactiae, is the main pathogen of neonatal infectious diseases. It can cause neonatal pneumonia, sepsis and meningitis. It is also one of the important factors leading to neonatal death. At the same time, Group B Streptococcus can also cause sepsis and skin infections in pregnant women, reduce the body's resistance, and cause infectious diseases in adults. The surface structure of Group B Streptococcus is complex, including capsular polysaccharides covering the surface of Group B Streptococcus, inner peptidoglycan and cell membrane. Among them, the pathogenicity of Group B Streptococcus is mainly related to the capsular polysaccharide antigen. The capsular polysaccharide has high immunogenicity and is an important protective antigen and virulence factor of cells. It is suitable as a target antigen for vaccines. Therefore, the development of Group B Streptococcus capsular polysaccharide conjugate vaccine is crucial to prevent Group B Streptococcus infection.

[0003] Traditional culture of Streptococcus agalactiae is carried out using Columbia medium, THB medium or NB medium, etc. The culture time of the strain is long, the growth concentration is low, and the cost is high, which is not suitable for large-scale production. At the same time, most of the current research is only carried out in the shake bottle stage, the culture scale is small, and the controllable culture parameters are relatively limited. The preparation of group B streptococcal vaccine requires large-scale culture of group B streptococci to obtain sufficient capsular polysaccharides. Therefore, in order to achieve this goal, it is necessary to expand the culture and control the culture of group B streptococci with multiple parameters at the fermentation tank level to obtain a large number of group B streptococci and harvest enough capsular polysaccharides for subsequent vaccine preparation.

[0004] At present, there have been studies on the use of restrictive chemical culture media for the cultivation of Group B Streptococcus. The culture media use a modified culture medium composed of glucose combined with multiple sulfates, phosphates, vitamins, and amino acids. Compared with liquid culture media such as THB or NB, Group B Streptococcus grows better in restrictive chemical culture media, and the bacterial concentration and capsular polysaccharide production are significantly higher than those cultivated in THB or NB culture media. At the same time, there are also culture medium formulas that are further improved on the basis of restrictive culture media. Replacing a large amount of amino acids in the restrictive chemical culture media with trypsin can further increase the bacterial concentration of Group B Streptococcus, but the effect of the increase is limited.

[0005] Therefore, it is necessary to further study and obtain culture conditions that are more suitable for the growth of Group B Streptococcus in order to increase the production of Group B Streptococcus capsular polysaccharides and provide a solid raw material foundation for the research and development of Group B Streptococcus conjugate vaccines.

[0006] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0007] The object of the present invention is to provide a group B streptococcus fermentation medium, a preparation method and a fermentation culture method, which can effectively increase the bacterial concentration and help obtain a high yield of group B streptococcus capsular polysaccharide.

[0008] In order to achieve the above object, a technical solution provided by a specific embodiment of the present invention is as follows:

[0009] A group B streptococcus fermentation medium comprising salt substances, a carbon source, an amino acid source, a vitamin source, a purine source, a pyrimidine source, a nitrogen source and solvent water;

[0010] The salt substances include: dipotassium hydrogen phosphate 0.2-0.5 g / L, potassium dihydrogen phosphate 0.3-0.5 g / L, disodium hydrogen phosphate 3-5 g / L, sodium dihydrogen phosphate monohydrate 2-5 g / L, ammonium sulfate 0.5-0.8 g / L, magnesium sulfate heptahydrate 0.2-0.5 g / L, manganous sulfate monohydrate 0.01-0.03 g / L, ferrous sulfate 0.03-0.06 g / L;

[0011] The amino acid source includes: 0.2-0.5 g / L of glycine and 0.1-0.3 g / L of tryptophan;

[0012] The nitrogen source includes 3-6 g / L of acid hydrolyzed casein;

[0013] It also includes 5-10 g / L of yeast extract powder.

[0014] In one or more embodiments of the present invention, the carbon source includes: 5-8 g / L sodium acetate trihydrate and 15-23 g / L anhydrous glucose.

[0015] In one or more embodiments of the present invention, the vitamin source includes: p-aminobenzoic acid 0.0002-0.0005 g / L, biotin 0.0002-0.0005 g / L, folic acid 0.0005-0.001 g / L, niacinamide 0.001-0.003 g / L, pantolactone 0.002-0.005 g / L, riboflavin 0.002-0.005 g / L, thiamine hydrochloride 0.001-0.003 g / L, and pyridoxine hydrochloride 0.0005-0.001 g / L.

[0016] In one or more embodiments of the present invention, the purine source includes: 0.002-0.005 g / L adenine and 0.002-0.005 g / L guanine.

[0017] In one or more embodiments of the present invention, the pyrimidine source includes 0.002-0.005 g / L of uracil.

[0018] Another specific embodiment of the present invention provides a technical solution as follows:

[0019] A method for preparing a group B streptococcus fermentation medium comprises the following steps:

[0020] Dissolve the salt substance and the carbon source in water to obtain solution Ⅰ;

[0021] Dissolve the amino acid source and the vitamin source in water to obtain solution II;

[0022] Dissolve the purine source and the pyrimidine source in water to obtain solution III;

[0023] Dissolve the nitrogen source and yeast extract powder in water to obtain solution IV;

[0024] The solution I, solution II, solution III and solution IV are mixed, the pH is adjusted to 7.0-7.5, and the volume is fixed to the target volume to obtain a group B streptococcus fermentation medium.

[0025] In one or more embodiments of the present invention, a certain proportion of a co-solvent is added when preparing solution II and / or solution III, and the co-solvent is selected from aqueous sodium hydroxide solution, aqueous potassium hydroxide solution, aqueous sodium bicarbonate solution, and aqueous sodium carbonate solution.

[0026] In one or more embodiments of the present invention, solution I, solution II and solution III are sterilized by membrane filtration before mixing, and solution IV is sterilized by wet heat before mixing.

[0027] Another specific embodiment of the present invention provides a technical solution as follows:

[0028] A fermentation and culture method for group B streptococci comprises the following steps:

[0029] Bacteria recovery;

[0030] Second generation liquid culture: transfer the revived strain to a seed tank for culture at a temperature of 35-37°C, a pH of 7.0-7.5, stirring at 100-200 rpm, aeration of 3-5 L / min, tank pressure of 0.005-0.01 MPa, and culture for 4-5 hours; the seed tank contains the above-mentioned Group B Streptococcus fermentation medium;

[0031] Third generation liquid culture: the strain is transferred to a fermentation tank for culture at a temperature of 35-37°C, a pH of 7.0-7.5, stirring at 100-200 rpm, aeration of 30-50 L / min, and a tank pressure of 0.005-0.01 MPa; the fermentation tank contains the above-mentioned group B streptococcus fermentation medium;

[0032] After the culture was completed, the bacterial culture was collected.

[0033] In one or more embodiments of the present invention, during the three-generation liquid culture process, 2 to 2.5 L of 40% glucose solution is added during the logarithmic growth phase for 1 to 2 hours.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The fermentation medium is composed of a variety of nutrient elements, which provides sufficient nutrients for the growth of the bacteria and effectively improves the growth concentration of the bacteria. At the same time, composite nutrients are used to replace a variety of amino acids, which simplifies the ingredients and improves the convenience of operation;

[0036] (2) The various nutrient elements are prepared into solutions according to their different types so that the nutrient elements can be fully dissolved. In addition, different solutions can be sterilized by membrane filtration or wet heat sterilization to avoid the decomposition of components with low thermal stability due to heat;

[0037] (3) Liquid culture is performed immediately after the strain is revived to ensure the vitality of the strain and help increase the bacterial concentration. At the same time, the number of culture generations is controlled at two times. When a higher concentration of bacterial solution is obtained, the possibility of strain mutation or even yield reduction can be reduced;

[0038] (4) During the fermentation process, the growth rate of bacteria that have not entered the logarithmic growth phase is slow, and the nutrients in the culture medium are sufficient to meet the growth of the bacteria. Supplementing nutrients before the logarithmic growth phase cannot effectively increase the bacterial concentration, and supplementing nutrients too late will cause the vitality of the bacteria to decrease or even die and cannot continue to grow, and the bacterial concentration cannot be further increased. Therefore, choosing to supplement glucose solution during the logarithmic growth phase can effectively increase the bacterial concentration and is conducive to obtaining a high yield of capsular polysaccharides;

[0039] (5) The fermentation culture process of the present invention is simple and fast, and the fermentation time is short, so that a high concentration of bacteria can be obtained in a short time, thereby obtaining large-scale capsular polysaccharides in a short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 The growth curves of type Ia bacteria in Example 3, Example 4 and Comparative Example 2 of the present invention are compared;

[0042] Figure 2 The growth curves of type Ⅰa bacteria in Example 5, Example 6 and Comparative Example 3 of the present invention are compared;

[0043] Figure 3 The growth curves of type II bacteria in Example 7, Example 8 and Comparative Example 4 of the present invention are compared;

[0044] Figure 4 The growth curves of type III bacteria in Example 9, Example 10 and Comparative Example 5 of the present invention are compared;

[0045] Figure 5 The growth curves of type IV bacteria in Example 11, Example 12 and Comparative Example 6 of the present invention are compared;

[0046] Figure 6 The growth curves of type V bacteria in Example 13, Example 14 and Comparative Example 7 of the present invention are compared;

[0047] Figure 7 This is the growth curve of the type Ⅰa strain in Example 15 of the present invention fermented in a 10L seed tank;

[0048] Figure 8 This is the growth curve of the type Ⅰa strain in Example 15 of the present invention fermented in a 100 L fermenter;

[0049] Fig. 9 This is the growth curve of the type II strain in comparative example 8 of the present invention fermented in a seed tank with a volume of 10 L;

[0050] Fig.10 This is the growth curve of the type II strain in Example 16 of the present invention fermented in a 10L seed tank;

[0051] Fig.11 This is the growth curve of the type II strain in Example 16 of the present invention fermented in a 100 L fermenter.

[0052] illustrate: Figure 1-6 In the figures, A means that no yeast extract powder is added to the fermentation medium; B means that 5 g / L yeast extract powder is added to the fermentation medium; and C means that 10 g / L yeast extract powder is added to the fermentation medium. DETAILED DESCRIPTION

[0053] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0054] The existing culture medium of group B streptococcus has complex components and many components. In addition to the carbohydrate substances that provide carbon sources, there are also a large number of salt substances, vitamin substances, amino acid substances, etc. that provide nutrients for the growth of strains. Moreover, many material components are unstable and cannot be sterilized at high temperatures or have low solubility. The culture medium preparation procedure is complicated and cumbersome, and it consumes a lot of costs during large-scale fermentation, the culture time is long, the harvested bacteria concentration is low, and the capsular polysaccharide yield is low. In view of these shortcomings, the present invention optimizes the culture medium formula and fermentation process, shortens the culture cycle, effectively improves the growth concentration of strains on the basis of cost saving, thereby increasing the yield of group B streptococcus capsular polysaccharides.

[0055] A specific embodiment of the present invention provides a group B streptococcus fermentation medium, salt substances, a carbon source, an amino acid source, a vitamin source, a purine source, a pyrimidine source, a nitrogen source and solvent water.

[0056] Specifically, the salt substances include dipotassium hydrogen phosphate 0.2-0.5g / L, potassium dihydrogen phosphate 0.3-0.5g / L, disodium hydrogen phosphate 3-5g / L, sodium dihydrogen phosphate monohydrate 2-5g / L, ammonium sulfate 0.5-0.8g / L, magnesium sulfate heptahydrate 0.2-0.5g / L, manganous sulfate monohydrate 0.01-0.03g / L, ferrous sulfate 0.03-0.06g / L; the carbon source includes: sodium acetate trihydrate 5-8g / L, anhydrous glucose 15-23g / L; the amino acid source includes: glycine 0.2-0.5g / L, tryptophan 0.1-0.3g / L; the vitamin source includes: p-aminobenzoic acid 0.0002-0.0005g / L , biotin 0.0002~0.0005g / L, folic acid 0.0005~0.001g / L, niacinamide 0.001~0.003g / L, pantolactone 0.002~0.005g / L, riboflavin 0.002~0.005g / L, thiamine hydrochloride 0.001~0.003g / L, pyridoxine hydrochloride 0.0005~0.001g / L; purine sources include adenine 0.002~0.005g / L, guanine 0.002~0.005g / L; pyrimidine sources include uracil 0.002~0.005g / L; nitrogen sources include acid hydrolyzed casein 3~6g / L; and yeast extract powder 5~10g / L.

[0057] The above types of nutrient elements are selected to form the fermentation medium, and the dosage of each nutrient element is controlled, so that the prepared fermentation medium can effectively promote the growth and metabolism of Group B Streptococcus, increase the growth concentration of the strain, and thus help to obtain more capsular polysaccharides.

[0058] Furthermore, the pH of the Group B Streptococcus fermentation medium is 7.0-7.5.

[0059] Specifically, by controlling the pH, it is possible to ensure that the bacteria grow in a suitable environment, which is conducive to better absorption of nutrients by the bacteria.

[0060] Another specific embodiment of the present invention provides a method for preparing a group B streptococcal fermentation medium, comprising steps S1-S2.

[0061] Step S1, solution preparation: dissolving salt substances and carbon sources in water to obtain solution I; dissolving amino acid sources and vitamin sources in water to obtain solution II; dissolving purine sources and pyrimidine sources in water to obtain solution III; dissolving nitrogen sources and yeast extract powder in water to obtain solution IV.

[0062] Specifically, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, anhydrous glucose, sodium acetate trihydrate, disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate, ammonium sulfate, magnesium sulfate heptahydrate, manganous sulfate monohydrate, and ferrous sulfate are fully dissolved in water for injection, and the volume is adjusted to 500 mL to obtain solution I. Para-aminobenzoic acid, biotin, folic acid, nicotinamide, pantolactone, riboflavin, ammonium sulfate hydrochloride, pyridoxine hydrochloride, glycine, and tryptophan are fully dissolved in water for injection, and the volume is adjusted to 50 mL to obtain solution II. Adenine, guanine, and uracil are fully dissolved in water for injection, and the volume is adjusted to 50 mL to obtain solution III. Acid hydrolyzed casein and yeast extract powder are fully dissolved in water for injection, and the volume is adjusted to 300 mL to obtain solution IV.

[0063] Furthermore, since the water solubility of the raw materials of Solution II and Solution III is low, a certain proportion of co-solvent is added during the preparation process to assist dissolution. The co-solvent is selected from a 30% concentration of sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, sodium bicarbonate aqueous solution, and sodium carbonate aqueous solution. When preparing 60L of fermentation medium, the amount of co-solvent added is 30-40mL.

[0064] Step S2, solution mixing: mixing solution I, solution II, solution III and solution IV, and fixing the volume to the target volume to obtain a group B streptococcus fermentation medium.

[0065] Specifically, since there are thermally unstable components in solution I, solution II and solution III, solution I, solution II and solution III are first sterilized and filtered using a 0.22um filter membrane, solution IV is sterilized by wet heat, and then the four solutions are mixed. After mixing, the pH of the solution is adjusted to 7.0-7.5, and then the volume is fixed to 1000 mL.

[0066] Another specific embodiment of the present invention provides a fermentation and culture method for Group B Streptococcus, comprising steps 1-4.

[0067] Step 1, strain recovery: open a working strain of group B streptococcus for fermentation, dissolve it completely and then spread it on TSA solid culture medium containing 5-10% sheep blood, place it in an incubator at 35-37°C and 5-10% CO2 for 18-24 hours.

[0068] Step 2, second-generation liquid culture: Use the above-mentioned Group B Streptococcus fermentation medium to wash and collect the revived strains, transfer them to a seed tank containing the above-mentioned Group B Streptococcus fermentation medium, control the culture temperature to 35-37°C, pH to 7.0-7.5, stir at 100-200rpm, ventilation volume to 3-5L / min, tank pressure to 0.005-0.01MPa, and culture for 4-5h.

[0069] Step 3, three-generation liquid culture: transfer the bacterial liquid in the seed tank to a fermentation tank containing the above-mentioned group B streptococcus fermentation medium for three-generation liquid culture, the culture temperature is 35-37°C, the pH is 7.0-7.5, the stirring is 100-200rpm, the ventilation volume is 30-50L / min, and the tank pressure is 0.005-0.01MPa.

[0070] Specifically, in step 3, 2 to 2.5 L of 40% glucose solution is added during the logarithmic growth phase for 1 to 2 hours. Adding glucose solution during the logarithmic growth phase can supplement nutrients for bacterial growth to better increase bacterial concentration.

[0071] Step 4, bacterial liquid collection: when the growth curve tends to be flat or decreases, the culture is terminated, and ammonium glucose acetate solution is added to a final concentration of 3.7% (v / v). When the temperature drops below 30°C, sodium deoxycholate is added to a final concentration of 0.1‰ (m / v), and the mixture is stirred and sterilized for 30 minutes to obtain a fermented bacterial liquid.

[0072] The present invention is further described in detail below in conjunction with specific embodiments.

[0073] The original strain used in the present invention was purchased from the American Cell Culture Collection (ATCC), and other raw materials were obtained from commercial sources.

[0074] Preparation of fermentation medium

[0075] Example 1

[0076] The method for preparing a group B streptococcus fermentation medium comprises the following steps:

[0077] S1. Solution preparation

[0078] S1.1. Preparation of Solution I

[0079] Dissolve 0.30 g of dipotassium hydrogen phosphate, 0.44 g of potassium dihydrogen phosphate, 20 g of anhydrous glucose, 6 g of sodium acetate trihydrate, 3.15 g of disodium hydrogen phosphate, 2.05 g of sodium dihydrogen phosphate monohydrate, 0.60 g of ammonium sulfate, 0.20 g of magnesium sulfate heptahydrate, 0.01 g of manganous sulfate monohydrate, and 0.03 g of ferrous sulfate in water for injection, and dilute to 500 mL to obtain solution I;

[0080] S1.2. Preparation of Solution II

[0081] Fully dissolve 0.0002 g of p-aminobenzoic acid, 0.0002 g of biotin, 0.0008 g of folic acid, 0.0010 g of nicotinamide, 0.0020 g of pantolactone, 0.0020 g of riboflavin, 0.0010 g of ammonium sulfate hydrochloride, 0.0008 g of pyridoxine hydrochloride, 0.20 g of glycine, and 0.10 g of tryptophan in water for injection, add 0.5 mL of 30% sodium hydroxide aqueous solution to assist dissolution during the dissolution process, and then make the volume to 50 mL to obtain solution II;

[0082] S1.3. Preparation of Solution III

[0083] Dissolve 0.0020 g of adenine, 0.0020 g of guanine, and 0.0020 g of uracil fully with water for injection, add 0.5 mL of 30% sodium hydroxide aqueous solution to assist dissolution during the dissolution process, and then make the volume to 50 mL to obtain solution III;

[0084] S1.4. Preparation of Solution IV

[0085] Dissolve 5 g of acid hydrolyzed casein and 5 g of yeast extract powder in water for injection, and dilute to 300 mL to obtain solution IV.

[0086] S2, solution mixing

[0087] Solution I, solution II and solution III are sterilized and filtered respectively with a 0.22 μm filter membrane, and solution IV is sterilized by wet heat sterilization at 121°C for 30 min. After cooling, the sterilized and filtered solutions I, solution II and solution III are mixed with the solution, and the volume is made up to 1000 mL with water for injection. The pH is adjusted to 7.0 to obtain the fermentation medium. If the preparation amount needs to be expanded, it can be expanded proportionally.

[0088] Example 2

[0089] The method for preparing a group B streptococcus fermentation medium comprises the following steps:

[0090] S1. Solution preparation

[0091] S1.1. Preparation of Solution I

[0092] Dissolve 0.30 g of dipotassium hydrogen phosphate, 0.44 g of potassium dihydrogen phosphate, 20 g of anhydrous glucose, 6 g of sodium acetate trihydrate, 3.15 g of disodium hydrogen phosphate, 2.05 g of sodium dihydrogen phosphate monohydrate, 0.60 g of ammonium sulfate, 0.20 g of magnesium sulfate heptahydrate, 0.01 g of manganous sulfate monohydrate, and 0.03 g of ferrous sulfate in water for injection, and dilute to 500 mL to obtain solution I;

[0093] S1.2. Preparation of Solution II

[0094] Fully dissolve 0.0002 g of p-aminobenzoic acid, 0.0002 g of biotin, 0.0008 g of folic acid, 0.0010 g of nicotinamide, 0.0020 g of pantolactone, 0.0020 g of riboflavin, 0.0010 g of ammonium sulfate hydrochloride, 0.0008 g of pyridoxine hydrochloride, 0.20 g of glycine, and 0.10 g of tryptophan in water for injection, add 0.5 mL of 30% sodium hydroxide aqueous solution to assist dissolution during the dissolution process, and then make the volume to 50 mL to obtain solution II;

[0095] S1.3. Preparation of Solution III

[0096] Dissolve 0.0020 g of adenine, 0.0020 g of guanine, and 0.0020 g of uracil fully with water for injection, add 0.5 mL of 30% sodium hydroxide aqueous solution to assist dissolution during the dissolution process, and then make the volume to 50 mL to obtain solution III;

[0097] S1.4. Preparation of Solution IV

[0098] Dissolve 5 g of acid hydrolyzed casein and 10 g of yeast extract powder in water for injection, and dilute to 300 mL to obtain solution IV.

[0099] S2, solution mixing

[0100] Solution I, solution II and solution III are sterilized and filtered respectively with a 0.22 μm filter membrane, and solution IV is sterilized by wet heat sterilization at 121°C for 30 min. After cooling, the sterilized and filtered solutions I, solution II and solution III are mixed with the solution, and the volume is made up to 1000 mL with water for injection. The pH is adjusted to 7.0 to obtain the fermentation medium. If the preparation amount needs to be expanded, it can be expanded proportionally.

[0101] Comparative Example 1

[0102] The method for preparing a group B streptococcus fermentation medium comprises the following steps:

[0103] S1. Solution preparation

[0104] S1.1. Preparation of Solution I

[0105] Dissolve 0.30 g of dipotassium hydrogen phosphate, 0.44 g of potassium dihydrogen phosphate, 20 g of anhydrous glucose, 6 g of sodium acetate trihydrate, 3.15 g of disodium hydrogen phosphate, 2.05 g of sodium dihydrogen phosphate monohydrate, 0.60 g of ammonium sulfate, 0.20 g of magnesium sulfate heptahydrate, 0.01 g of manganous sulfate monohydrate, and 0.03 g of ferrous sulfate in water for injection, and dilute to 500 mL to obtain solution I;

[0106] S1.2. Preparation of Solution II

[0107] Fully dissolve 0.0002 g of p-aminobenzoic acid, 0.0002 g of biotin, 0.0008 g of folic acid, 0.0010 g of nicotinamide, 0.0020 g of pantolactone, 0.0020 g of riboflavin, 0.0010 g of ammonium sulfate hydrochloride, 0.0008 g of pyridoxine hydrochloride, 0.20 g of glycine, and 0.10 g of tryptophan in water for injection, add 0.5 mL of 30% sodium hydroxide aqueous solution to assist dissolution during the dissolution process, and then make the volume to 50 mL to obtain solution II;

[0108] S1.3. Preparation of Solution III

[0109] Dissolve 0.0020 g of adenine, 0.0020 g of guanine, and 0.0020 g of uracil fully with water for injection, add 0.5 mL of 30% sodium hydroxide aqueous solution to assist dissolution during the dissolution process, and then make the volume to 50 mL to obtain solution III;

[0110] S1.4. Preparation of Solution IV

[0111] After fully dissolving 5 g of acid hydrolyzed casein with water for injection, the volume was fixed to 300 mL to obtain solution IV;

[0112] S2, solution mixing

[0113] Solution I, solution II and solution III are sterilized and filtered using a 0.22 um filter membrane respectively, and solution IV is sterilized by wet heat sterilization at 121°C for 30 min. After cooling, the sterilized and filtered solutions I, solution II and solution III are mixed with the solution, and the volume is made up to 1000 mL with water for injection. The pH is adjusted to 7.0 to obtain the fermentation medium. If the preparation amount needs to be expanded, it can be expanded proportionally.

[0114] Shake flask culture of group B streptococci

[0115] Example 3

[0116] The fermentation and culturing method of group B streptococcus comprises the following steps:

[0117] Step 1: Open a strain of Type Ia Group B Streptococcus for fermentation, dissolve it completely and then spread it on TSA solid culture medium containing 5% sheep blood, and place it in an incubator at 37°C and 5% CO2 for 18 hours;

[0118] Step 2: Use the fermentation medium in Example 1 to wash and collect the revived strains, transfer them to a 1L shake flask containing 200 ml of the fermentation medium in Example 1, control the culture temperature to 37±1°C, stir at 200 rpm, and take samples every 2 hours to measure the OD value;

[0119] Step 3: When the growth curve tends to be flat or decreases, the culture is terminated, sodium deoxycholate is added to a final concentration of 0.1‰ (m / v), and the mixture is stirred and sterilized for 30 minutes to obtain a fermentation solution.

[0120] Examples 4-8, Comparative Examples 2-7

[0121] The difference between Examples 4-8 and Comparative Examples 2-7 and Example 3 is only the different bacterial species or fermentation medium used, as shown in Table 1.

[0122] Table 1 Bacterial species and fermentation medium used in Examples 3-8 and Comparative Examples 2-7

[0123]

[0124]

[0125] Group B Streptococcus fermentation tank culture

[0126] Embodiment 15

[0127] The fermentation and culturing method of group B streptococcus comprises the following steps:

[0128] Step 1: Resuscitation of bacterial strains

[0129] Open a strain of Type Ia Group B Streptococcus for fermentation, dissolve it completely, and then spread it on TSA solid medium containing 5% sheep blood, and place it in an incubator at 37°C and 5% CO2 for 18 hours;

[0130] Step 2: Second generation liquid culture

[0131] The revived strains were washed and collected with the fermentation medium prepared in Example 1, and transferred to a 10 L seed tank containing 6 L of the fermentation medium prepared in Example 1. The culture temperature was controlled at 37 ± 1 °C, the pH was set to 7.0, the stirring was 200 rpm, the ventilation was 4.5 L / min, the tank pressure was 0.01 MPa, and the culture was carried out for 4 h.

[0132] Step 3: Third generation liquid culture

[0133] The bacterial liquid in the seed tank was transferred to a 100L fermenter for three-generation liquid culture. The fermenter contained 60L of the fermentation medium prepared in Example 1, and the temperature was controlled at 37±1°C, the pH was set to 7.0, the stirring was 200rpm, the ventilation was 30L / min, and the tank pressure was 0.01MPa. During the culture process, 2L of 40% glucose solution was added during the logarithmic growth phase and the mixture was replenished within 1 hour;

[0134] Step 4: Bacterial liquid collection

[0135] When the growth curve tends to be flat or decreases, the culture is terminated, and ammonium glucose acetate solution is added to a final concentration of 3.7% (v / v). When the temperature drops below 30°C, sodium deoxycholate is added to a final concentration of 0.1‰ (m / v), and the mixture is stirred and sterilized for 30 minutes to obtain a fermentation bacterial liquid.

[0136] Example 16

[0137] The fermentation and culturing method of group B streptococcus comprises the following steps:

[0138] Step 1: Resuscitation of bacterial strains

[0139] Open a strain of Type II Group B Streptococcus for fermentation, dissolve it completely, and then spread it on TSA solid medium containing 5% sheep blood, and place it in an incubator at 37°C and 5% CO2 for 18 hours;

[0140] Step 2: Second generation liquid culture

[0141] The revived strains were washed and collected with the fermentation medium prepared in Example 1, and transferred to a 10 L seed tank containing 6 L of the fermentation medium prepared in Example 1. The culture temperature was controlled at 37 ± 1 °C, the pH was set to 7.0, the stirring was 200 rpm, the ventilation was 4.5 L / min, the tank pressure was 0.01 MPa, and the culture was carried out for 4 h.

[0142] Step 3: Third generation liquid culture

[0143] The bacterial liquid in the seed tank was transferred to a 100L fermenter for three-generation liquid culture. The fermenter contained 60L of the fermentation medium prepared in Example 1, and the temperature was controlled at 37±1°C, the pH was set to 7.0, the stirring was 200rpm, the ventilation was 30L / min, and the tank pressure was 0.01MPa. During the culture process, 2L of 40% glucose solution was added during the logarithmic growth phase and the mixture was replenished within 1 hour;

[0144] Step 4: Bacterial liquid collection

[0145] When the growth curve tends to be flat or decreases, the culture is terminated, and ammonium glucose acetate solution is added to a final concentration of 3.7% (v / v). When the temperature drops below 30°C, sodium deoxycholate is added to a final concentration of 0.1‰ (m / v), and the mixture is stirred and sterilized for 30 minutes to obtain a fermentation bacterial liquid.

[0146] Comparative Example 8

[0147] The only difference between this comparative example and Example 16 is that the fermentation culture medium prepared in Comparative Example 1 is used for fermentation culture.

[0148] Depend on Figure 1-6 It can be seen that in the small-scale fermentation culture in shake flasks, compared with not adding yeast extract powder, the fermentation medium in the embodiment of the present invention can significantly increase the culture concentration of different types of group B streptococci.

[0149] Combination Figure 7-11 , Type Ia group B streptococci were cultured for two generations in liquid fermentation, and the bacterial concentration (OD 650 ) can reach 10.62; in a 10L volume fermenter, when no yeast extract powder is added, the maximum concentration of type II group B streptococcus is only 1.73, and when yeast extract powder is added, the bacterial concentration can reach 9.20; in a 100L volume fermenter, the bacterial concentration can reach 12.38, indicating that in large-scale fermentation culture using a fermenter, using the fermentation medium in the embodiment of the present invention can also obtain a higher bacterial concentration, which is conducive to obtaining a high yield of capsular polysaccharides.

[0150] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0151] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A group B streptococcus fermentation medium, characterized in that: Including salt substances, carbon source, amino acid source, vitamin source, purine source, pyrimidine source, nitrogen source and solvent water; The salt substances include: dipotassium hydrogen phosphate 0.2-0.5 g / L, potassium dihydrogen phosphate 0.3-0.5 g / L, disodium hydrogen phosphate 3-5 g / L, sodium dihydrogen phosphate monohydrate 2-5 g / L, ammonium sulfate 0.5-0.8 g / L, magnesium sulfate heptahydrate 0.2-0.5 g / L, manganous sulfate monohydrate 0.01-0.03 g / L, ferrous sulfate 0.03-0.06 g / L; The amino acid source includes: 0.2-0.5 g / L of glycine and 0.1-0.3 g / L of tryptophan; The nitrogen source includes 3-6 g / L of acid hydrolyzed casein; It also includes 5-10 g / L of yeast extract powder.

2. The group B streptococcus fermentation medium according to claim 1, characterized in that The carbon source includes: 5-8 g / L sodium acetate trihydrate and 15-23 g / L anhydrous glucose.

3. The group B streptococcus fermentation medium according to claim 1, characterized in that The vitamin source comprises: p-aminobenzoic acid 0.0002-0.0005 g / L, biotin 0.0002-0.0005 g / L, folic acid 0.0005-0.001 g / L, nicotinamide 0.001-0.003 g / L, pantolactone 0.002-0.005 g / L, riboflavin 0.002-0.005 g / L, thiamine hydrochloride 0.001-0.003 g / L, and pyridoxine hydrochloride 0.0005-0.001 g / L.

4. The group B streptococcus fermentation medium according to claim 1, characterized in that The purine source includes: 0.002-0.005 g / L adenine and 0.002-0.005 g / L guanine.

5. The group B streptococcus fermentation medium according to claim 1, characterized in that The pyrimidine source includes 0.002-0.005 g / L of uracil.

6. A method for preparing a group B streptococcus fermentation medium according to any one of claims 1 to 5, characterized in that: The following steps are involved: Dissolve the salt substance and the carbon source in water to obtain solution Ⅰ; Dissolve the amino acid source and the vitamin source in water to obtain solution II; Dissolve the purine source and the pyrimidine source in water to obtain solution III; Dissolve the nitrogen source and yeast extract powder in water to obtain solution IV; The solution I, solution II, solution III and solution IV are mixed, the pH is adjusted to 7.0-7.5, and the volume is fixed to the target volume to obtain a group B streptococcus fermentation medium.

7. The method for preparing a group B streptococcus fermentation medium according to claim 6, characterized in that: When preparing solution II and / or solution III, a certain proportion of co-solvent is added, and the co-solvent is selected from sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, sodium bicarbonate aqueous solution, and sodium carbonate aqueous solution.

8. The method for preparing a group B streptococcus fermentation medium according to claim 6, characterized in that: Solution I, solution II and solution III were sterilized by membrane filtration before mixing, and solution IV was sterilized by wet heat before mixing.

9. A fermentation and culturing method for group B streptococci, characterized in that: The following steps are involved: Bacteria recovery; Second generation liquid culture: transfer the revived strain to a seed tank for culture at a temperature of 35-37°C, a pH of 7.0-7.5, stirring at 100-200 rpm, aeration of 3-5 L / min, tank pressure of 0.005-0.01 MPa, and culture for 4-5 hours; the seed tank contains the group B streptococcus fermentation medium according to any one of claims 1-5; Third generation liquid culture: the strain is transferred to a fermenter for culture at a temperature of 35-37°C, a pH of 7.0-7.5, stirring at 100-200 rpm, aeration of 30-50 L / min, and a tank pressure of 0.005-0.01 MPa; the fermenter contains the group B streptococcus fermentation medium according to any one of claims 1-5; After the culture was completed, the bacterial culture was collected.

10. The fermentation and culturing method of group B streptococcus according to claim 9, characterized in that: During the three-generation liquid culture, 2 to 2.5 L of 40% glucose solution was added during the logarithmic growth phase for 1 to 2 hours.

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