A method for the preparation of capsular polysaccharides using staphylococcus aureus

Abstract

The application discloses a method for preparing a capsular polysaccharide by using Staphylococcus aureus. The extraction method comprises the step of treating Staphylococcus aureus fermentation liquor by using acetic acid and sodium deoxycholate, and the method does not comprise the step of using enzyme treatment. Compared with the prior art, the application has the advantages that the fermentation culture system is free of any addition of animal-derived substances and has high cell density, polysaccharide extraction is free of the addition of organic solvents, lysozyme or staphylolytic enzyme and nuclease and the like, and column chromatography purification, the process is simple and easy to scale up, the yield is high, and the quality is good.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing capsular polysaccharides using Staphylococcus aureus. Background Technology

[0002] Staphylococcus aureus ( Staphylococcus aureus Staphylococcus aureus (SAA) is a group of Gram-positive opportunistic pathogens widely distributed in the natural environment and on the mucous membranes of humans and animals. It is one of the leading pathogens in clinical nosocomial infections and community-acquired infections, and a key pathogen inducing subclinical mastitis in dairy cows, purulent infections in livestock and poultry, and foodborne poisoning. The pathogenicity and immune evasion ability of SSA are highly dependent on the array of virulence factors on its surface. Among these, capsular polysaccharide (CPS), as a core extracellular protective antigen, can significantly enhance the bacteria's colonization, invasion, and transmission capabilities by shielding opsonin binding sites on the bacterial surface and inhibiting phagocytic adhesion and killing. It is a key molecule mediating the immune evasion of pathogens. Currently, more than 10 capsular serotypes of Staphylococcus aureus have been identified globally. Among them, serotypes 5, 8, and 336 account for more than 90% of clinical isolates and pathogenic livestock and poultry strains. They are the core target antigens for the development of Staphylococcus aureus vaccines, serological diagnostic kits, and passive immunization agents. Their efficient preparation and purity control directly determine the immunogenicity, safety, and batch-to-batch consistency of downstream biological products, and are the core technological bottlenecks restricting the industrialization of related products.

[0003] Regarding the fermentation process for preparing Staphylococcus aureus capsular polysaccharides, current technologies still commonly employ composite culture media containing animal-derived components (e.g., patents CN112646845A and CN107058421B both use Columbia medium). These media often contain animal-derived raw materials such as bovine serum, animal tissue digests, and plasma protein hydrolysates. While these can meet the nutritional requirements for bacterial proliferation and capsular polysaccharide synthesis, the sources of these animal-derived components are complex and their composition is not limited. They are prone to carrying potential contaminants such as exogenous viruses, mycoplasma, prions, and endotoxins, failing to meet the animal-free quality control standards of the Pharmacopoeia of the People's Republic of China and the WHO and EMA for upstream processes of biopharmaceuticals, posing significant safety risks for clinical applications. Furthermore, some technologies still use solid culture media (e.g., patent CN109134677A) to enrich bacterial cells for polysaccharide extraction; however, this method is difficult to scale up for commercial applications.

[0004] Traditional processes for extracting polysaccharides from Staphylococcus aureus capsularis involve lysing the bacterial cells with lysostaphin, removing nucleic acids with nucleases, and removing proteins with organic reagents such as ethanol. While these processes are simple to operate, they suffer from several key drawbacks: organic reagents are difficult to completely remove, posing safety risks and contradicting green pharmaceutical principles; nucleases and lysostaphin are expensive, hindering commercial production. Furthermore, existing technologies also suffer from poor product quality (high levels of impurities such as nucleic acids and proteins) and cumbersome process routes (most processes require column chromatography purification), resulting in significant process losses and low product yields.

[0005] The master's thesis, "Preparation of Monoclonal Antibody for Capsular Polysaccharide of Staphylococcus aureus Type 5 and Establishment of ELISA Method," shows that using Columbia liquid culture medium, 120 L of strain 5 yielded 157.35 mg of purified polysaccharide, with a yield of 1.35 mg / L fermentation broth. The thesis, "Preparation of Capsular Polysaccharide from Staphylococcus aureus by Liquid Fermentation" (Yang Shengyong, Master's Thesis, Dalian University of Technology), studied the fermentation process of Staphylococcus aureus type 5, using a 5 L fermenter (feed-feed process), achieving a maximum OD... 600 The yield reached 9.43, and the crude sugar yield in the fermentation broth was 101.49 mg / L. CN107058421A discloses that the extraction yield of type 336 capsular polysaccharide is 50 mg / L in the fermentation broth.

[0006] To address the aforementioned pain points, there is an urgent need to develop a culture medium for culturing Staphylococcus aureus that does not use animal-derived raw materials such as bovine serum, animal tissue digestion peptone, and plasma protein hydrolysates; and a process for preparing Staphylococcus aureus capsular polysaccharides that does not use enzymes (lysostaphin or nuclease) to purify capsular polysaccharides, in order to accelerate the industrialization and clinical application of Staphylococcus aureus vaccines and related capsular polysaccharide products. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing Staphylococcus aureus capsular polysaccharide and its application, so as to solve the technical problems of unfriendly process environment, low yield, poor purity and safety of Staphylococcus aureus capsular polysaccharide in the prior art.

[0008] This invention provides a method using Staphylococcus aureus (Staphylococcus aureus) Staphylococcus aureus A method for preparing capsular polysaccharides, the method comprising the following steps: (1) Add acetic acid to the Staphylococcus aureus fermentation broth to lyse it and obtain the lysate; (2) Adjust the pH of the lysis buffer to neutral using an alkaline solution, and add sodium deoxycholate while stirring and reacting; (3) Adjust the pH of the system obtained in step (2) to acidic using an acidic solution, and the reaction causes the impurity protein and sodium deoxycholate to precipitate out. (4) Solid-liquid separation: The system obtained in step (3) is subjected to solid-liquid separation, and the liquid phase containing the capsular polysaccharide is collected; (5) Drying: The liquid phase obtained in step (4) is dried to obtain the capsular polysaccharide; Furthermore, no additional enzyme treatment is added during the preparation process.

[0009] The enzymes referred to are exogenous enzymes such as lysozymes (e.g., lysostaphin, cytokinase, bacteriophage lyase), nucleases (e.g., Benzonase), or proteases (e.g., proteinase K).

[0010] In this invention, "no additional enzyme treatment" means that during the preparation of capsular polysaccharides, in the entire process of clarification and purification after cell disruption, no exogenous enzymes such as lysozyme (e.g., lysostaphin, cytokinase, phage lysin), nuclease (e.g., Benzonase), or protease (e.g., proteinase K) are artificially added. Sample processing is completed solely by chemical and physical means (centrifugation, filtration, chromatography) or endogenous enzyme systems, thus avoiding contamination from extragenous enzymes.

[0011] In some embodiments, in step (1) of the method, acetic acid is added to the Staphylococcus aureus fermentation broth to adjust the pH to 3.5-4.5, and the reaction is carried out at a temperature not lower than 60°C to obtain a lysate.

[0012] In some implementations, step (1) satisfies one or more of the following: 1) Adjust the pH value to 3.8-4.2, for example, 3.9-4.1; 2) The reaction occurs at 90-130℃, for example, 95-110℃; and, 3) The reaction time is 1-6 hours, for example, 1.5-2.5 hours.

[0013] The cell wall of Staphylococcus aureus has a thick peptidoglycan layer, and lysostaphin is commonly used for cell disruption. This enzyme specifically cleaves the glycine pentapeptide cross-links in the peptidoglycan, thereby specifically lysing the cell wall and releasing the capsular polysaccharide cross-linked with the peptidoglycan into the fermentation broth. Simultaneously, cell rupture releases large amounts of host genomic DNA and RNA, leading to a significant increase in the viscosity of the fermentation solution and increasing the risk of subsequent membrane clogging. Therefore, the addition of nucleases can degrade long-chain nucleic acids into short-chain oligonucleotides, effectively reducing the system viscosity and providing favorable flux conditions for subsequent chromatography or membrane filtration operations.

[0014] However, capsular polysaccharides obtained after treatment with lysostaphin and nucleases carry the risk of residual protein and nucleic acid impurities, and the process is costly.

[0015] In some implementations, a clarification step is also included between step (1) and step (2).

[0016] In some implementations, membrane clarification is used, preferably by solid-liquid separation via microfiltration membrane, and residual products are recovered by washing the membrane with purified water and combined to obtain a primary clarified liquid.

[0017] In some embodiments, the microfiltration membrane pack used in the clarification step is a microfiltration membrane pack with a pore size of 0.45 μm or 0.2 μm.

[0018] In some implementations, during the clarification step, purified water is added to wash the membrane twice.

[0019] In some implementations, step (2) uses a sodium hydroxide solution to adjust the pH to neutral.

[0020] In some embodiments, the sodium hydroxide solution is a 20% to 60% (w / v) sodium hydroxide solution, preferably 50% (w / v).

[0021] In some embodiments, sodium deoxycholate is added in step (2) to a final concentration of 0.05-0.5% (w / v) in the system, preferably 0.1-0.2% (w / v), for example 0.15% (w / v).

[0022] In some implementations, the stirring speed in step (2) is 600-1200 rpm, preferably 800-1000 rpm, for example 900 rpm.

[0023] In some implementations, the reaction time in step (2) is 20-40 min, for example 25-35 min, for example 30 min.

[0024] In some implementations, after adjusting the pH of the lysis buffer to neutral in step (2), a dilution step is also included before adding sodium deoxycholate.

[0025] In some embodiments, in step (2), the dilution step uses a sodium chloride solution for dilution, preferably a 0.8-0.9% (w / v) sodium chloride solution, for example a 0.85% (w / v) sodium chloride solution.

[0026] In some implementations, in step (2), the dilution step is a dilution of 1.5-3 times, preferably 1.8-2.2 times, for example, a dilution of 2 times.

[0027] In some implementations, in step (2), the dilution step is performed by stirring while diluting, with a stirring speed of 1500-2000 rpm, for example 1700 rpm.

[0028] In some implementations, the pH is adjusted to 3.5-4.5 in step (3), for example, 3.8-4.2 or 4.00 ± 0.1.

[0029] In some implementations, acetic acid is used to adjust the pH value in step (3).

[0030] In some implementations, the reaction time after adjusting the pH to acidity in step (3) is 0.5-2 hours, for example 0.5-1.5 hours, or for example 1 hour.

[0031] In some implementations, in step (4), the system obtained in step (3) is clarified and filtered using a microfiltration membrane, the flow-through is collected, and then the flow-through is concentrated using an ultrafiltration membrane with a molecular weight cutoff of 5 to 100 kDa, and replaced with a replacement solution. After replacement, the system is filtered using a sterile filter, and the liquid phase containing the capsular polysaccharide is collected.

[0032] In some implementations, the pore size of the microfiltration membrane pack in step (4) is 0.2 μm to 0.6 μm.

[0033] In some implementations, the pore size of the microfiltration membrane pack in step (4) is 0.45 μm or 0.2 μm.

[0034] In some implementations, the ultrafiltration membrane pack in step (4) is an ultrafiltration membrane pack with a molecular weight cutoff of 8-12 kDa.

[0035] In some implementations, the replacement in step (4) is an equal-volume replacement of 8-12 times the volume.

[0036] In some implementations, the replacement solution in step (4) is a salt solution and / or purified water; preferably, the solution is replaced 1-3 times with a salt solution and then replaced 2-6 times with purified water.

[0037] In some implementations, the replacement solution in step (4) is a sodium chloride solution with a concentration of 0.5-2 mol / L.

[0038] In some implementations, step (4) includes rinsing the ultrafiltration membrane pack with purified water and combining the rinsing solution with the retentate after the replacement is completed and before filtration.

[0039] In some implementations, the pore size of the sterilization filter in step (4) is 0.22 μm, and the material is polyethersulfone or polyvinylidene fluoride.

[0040] In some implementations, a 0.22 μm capsule filter is used for sterilization filtration.

[0041] In some embodiments, the protein content of the capsular polysaccharide prepared by the method is ≤1.0% (w / w).

[0042] In some embodiments, the protein content in the capsular polysaccharide prepared by the method is ≤0.5% (w / w).

[0043] In some embodiments, the protein content in the capsular polysaccharide is at a level that is undetectable. Undetectable level: Using the Lowry method, with a standard curve detection limit of 21.8 μg / mL, no protein was detected under these conditions.

[0044] In some embodiments, the nucleic acid content in the capsular polysaccharide prepared by the method is ≤0.8% (w / w), preferably ≤0.5% (w / w).

[0045] In some embodiments, the yield of capsular polysaccharide prepared by the method is not less than 200 mg / L.

[0046] In some embodiments, the capsular polysaccharide prepared by the method contains aminohexose, and the aminohexose content is ≥3.0% (w / w), preferably ≥8.0% (w / w).

[0047] In some embodiments, the capsular polysaccharide prepared by the method contains O-acetyl groups, and the O-acetyl group content is ≥0.8% (w / w), preferably ≥1.2% (w / w).

[0048] In some embodiments, the Staphylococcus aureus serotype is one or more of type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 336.

[0049] In some embodiments, the Staphylococcus aureus serotype is one or more of serotypes 5, 8, and 336.

[0050] In some implementations, the type designation of Staphylococcus aureus type 5 is ATCC 49521.

[0051] In some implementations, the type of Staphylococcus aureus serotype 336 is designated as ATCC 55804.

[0052] In some implementations, the type designation of Staphylococcus aureus type 8 is ATCC 49525.

[0053] In some embodiments, the method further includes the step of culturing Staphylococcus aureus using a culture medium combination to obtain a fermentation broth, and the step of culturing Staphylococcus aureus using a supplemental nutrient solution, wherein the culture medium combination comprises a basal culture medium, a glucose supplement solution, and a supplemental nutrient solution.

[0054] In some embodiments, the basal culture medium contains soybean peptone, yeast extract and glucose, and preferably also contains MgSO4·7H2O and / or K2HPO4.

[0055] In some implementations, the basal culture medium is further divided into seed culture medium and second-generation basal culture medium, depending on the content of soybean peptone, yeast powder and glucose.

[0056] In some embodiments, the glucose concentration in the glucose supplement solution is 20-500 g / L.

[0057] In some embodiments, the glucose concentration in the glucose supplement solution is 350-450 g / L, for example 400 g / L.

[0058] In some embodiments, the supplemental nutrient solution contains soybean peptone, yeast powder, uridine diphosphate (UDP), arginine, cysteine, glutamine, vitamin solution, T solution, and nicotinamide; the vitamin solution contains vitamin H, vitamin B9, vitamin B6, vitamin B1, vitamin B2, vitamin B3, vitamin B5, and vitamin B12; and the T solution contains MgSO4·7H2O, MnSO4·H2O, FeSO4·7H2O, CoCl·6H2O, CaCl2, ZnSO4·7H2O, CuSO4·5H2O, H3BO3, NaMoO4·2H2O, and NiSO4·6H2O.

[0059] In some embodiments, the concentration of soybean peptone in the seed culture medium is 20-30 g / L, for example 25 g / L; the concentration of yeast extract is 10-15 g / L, for example 12.5 g / L; and the concentration of glucose is 10-15 g / L, for example 12.5 g / L.

[0060] In some embodiments, the seed culture medium is formulated as follows: containing 20 g / L soybean peptone, 10 g / L yeast powder, and 10 g / L glucose.

[0061] In some embodiments, the concentration of soybean peptone in the second-generation culture medium is 15-25 g / L, for example 20 g / L; the concentration of yeast extract is 8-15 g / L, for example 10 g / L; the concentration of glucose is 8-15 g / L, for example 10 g / L; preferably the concentration of MgSO4·7H2O is 0.5-1.5 g / L, for example 1 g / L; and the concentration of K2HPO4 is 3-8 g / L, for example 5 g / L.

[0062] In some embodiments, the second-generation culture medium is formulated as follows: containing 20 g / L soybean peptone, 10 g / L yeast powder, 10 g / L glucose, 5 g / L K2HPO4, and 1 g / L MgSO4·7H2O.

[0063] In some embodiments, the amount of each component added to the supplemental nutrient solution is based on the volume of the second-generation basal culture medium, wherein, The amount of soybean peptone added is 50-70 g / L of second-generation basal medium, for example, 60 g / L of second-generation basal medium; The amount of yeast powder added is 25-35 g / L of second-generation basal medium, for example, 30 g / L of second-generation basal medium; The amount of uridine diphosphate (UDP) added is 0.05-0.15 g / L of second-generation basal medium, for example, 0.1 g / L of second-generation basal medium; Arginine was added at a rate of 0.05-0.15 g / L in the second-generation basal medium, for example, 0.1 g / L in the second-generation basal medium. The amount of cysteine ​​added is 0.05-0.15 g / L of second-generation basal medium, for example, 0.1 g / L of second-generation basal medium; The amount of glutamine added is 0.05-0.15 g / L of second-generation basal medium, for example, 0.1 g / L of second-generation basal medium; The amount of nicotinamide added is 0.8-6 mg / L in the second-generation culture medium, for example, 1.25-3 mg / L; The vitamin solution is added at a volume ratio of 2-10 mL / L of second-generation basal culture medium, such as 2.5 mL / L or 6 mL / L. The volumetric addition ratio of solution T is 2-20 mL / L, for example, 2.5 mL / L or 12 mL / L.

[0064] In some embodiments, the nicotinamide is added in solution form at a concentration of 0.4-0.6 g / L, for example, 0.5 g / L, and the volume ratio of the nicotinamide solution added to the secondary culture medium is 2-10 mL / L of the secondary basal culture medium, for example, 2.5 mL / L or 6 mL / L.

[0065] In some embodiments, the vitamins are added to the second-generation culture medium in the form of a mixed solution of different vitamins, and the T solution is added to the second-generation culture medium in solution form.

[0066] In some embodiments, the vitamin solution contains the following concentrations: vitamin H (2-6 mg / L, e.g., 4 mg / L); vitamin B9 (2-6 mg / L, e.g., 4 mg / L); vitamin B6 (20-60 mg / L, e.g., 40 mg / L); vitamin B1 (10-30 mg / L, e.g., 20 mg / L); vitamin B2 (10-30 mg / L, e.g., 20 mg / L); vitamin B3 (10-30 mg / L, e.g., 20 mg / L); vitamin B5 (10-30 mg / L, e.g., 20 mg / L); and vitamin B12 (0.2-0.6 mg / L, e.g., 0.4 mg / L).

[0067] In some embodiments, the concentration of MgSO4·7H2O in solution T is 1-5 g / L, for example, 3 g / L; the concentration of MnSO4·H2O is 0.2-0.8 g / L, for example, 0.5 g / L; the concentration of FeSO4·7H2O is 0.05-0.15 g / L, for example, 0.1 g / L; the concentration of CoCl·6H2O is 0.05-0.15 g / L, for example, 0.1 g / L; the concentration of CaCl2 is 0.05-0.15 g / L, for example, 0.1 g / L; the concentration of ZnSO4·7H2O is 0.005-0.02 g / L, for example, 0.1 g / L; the concentration of CuSO4·5H2O is 0.05-0.15 g / L, for example, 0.01 g / L; and the concentration of H3BO3 is 0.005-0.02 g / L, for example, 0.01 g / L. The concentration of NaoO4·2H2O is 0.005-0.02 g / L, for example 0.01 g / L; the concentration of NiSO4·6H2O is 0.05-0.15 g / L, for example 0.1 g / L.

[0068] In some embodiments, the formula of the supplemental compound nutrient solution is as follows: containing 240 g / L soybean peptone, 120 g / L yeast powder, 2 g / L UDP, 2 g / L arginine, 2 g / L cysteine, 2 g / L glutamine, 0.83% (v / v) vitamin solution, 1.7% (v / v) T solution, and 0.83% (v / v) 6% nicotinamide solution.

[0069] In some embodiments, the step of culturing Staphylococcus aureus to obtain fermentation broth includes: inoculating Staphylococcus aureus into a basal culture medium for cultivation, and adding supplemental glucose solution and supplemental compound nutrient solution during the cultivation process.

[0070] In some embodiments, the method includes culturing Staphylococcus aureus in a seed culture medium until the cells grow to the mid-logarithmic growth phase, obtaining a seed culture, and then using a fermentation broth culture medium, adding glucose supplementation solution and supplemental compound nutrient solution during the culture process.

[0071] In some embodiments, the OD of Staphylococcus aureus cultured by the method after 9 hours is... 600 The value is not less than 15.

[0072] In some implementations, the culture temperature is 30-38°C.

[0073] In some implementations, the pH of the culture is 6.5-8.0.

[0074] In some embodiments, the culture is stirred at a speed of 100-600 rpm.

[0075] In some implementations, the culture time is 8-15 hours.

[0076] In some implementation schemes, glucose solution and compound nutrient solution are added after 2-3 hours of cultivation.

[0077] In some implementations, the feeding method for the glucose solution is as follows: feed is added once per hour, each time at a rate of approximately 1 / 10 to 1 / 5 of the total volume.

[0078] In some implementations, the feeding method of the supplemental compound nutrient solution is as follows: feed is added once per hour, each time about 1 / 10 to 1 / 5 of the total volume.

[0079] In some implementations, the fermentation of Staphylococcus aureus is accomplished by the following steps: (1) Strain revival: The frozen strain was inoculated into the seed culture medium and cultured overnight at 32°C and 140 rpm to obtain the first generation seed liquid; (2) Batch culture: The first generation seed culture was inoculated into a fermenter containing the second generation basic culture medium at a ratio of 1:1 to 10 (v / v). The key culture parameters were: temperature 37℃, pH 7.2±0.1, stirring speed adjustable from 100 to 600 rpm, deep aeration rate 50 L / h, tank pressure 0.05 MPa, and dissolved oxygen level ≥15%. The feeding of glucose solution and compound nutrient solution was started at 2 to 3 hours of batch culture.

[0080] In some implementations, the inoculation volume ratio of the first-generation seed culture to the second-generation basal culture medium is 1:2.5; Termination of fermentation: Fermentation is stopped when the culture reaches the plateau phase.

[0081] In some implementations, the total batch culture time is 8 to 12 hours.

[0082] Another aspect of the present invention provides a culture medium combination for Staphylococcus aureus, the culture medium combination comprising the above-described culture medium combination.

[0083] Another aspect of the present invention provides a method for culturing Staphylococcus aureus to produce capsular polysaccharides, the method comprising culturing Staphylococcus aureus to produce capsular polysaccharides using the method provided by the present invention.

[0084] Another aspect of the present invention provides the application of capsular polysaccharides prepared by the method provided by the present invention in the preparation of vaccines, kits and diagnostic agents.

[0085] The reagents and raw materials used in this invention are all commercially available.

[0086] The positive and progressive effects of this invention are as follows: The method for preparing capsular polysaccharides provided by this invention does not use enzymes such as lysostaphylococcal enzymes or nucleases to purify capsular polysaccharides. Therefore, the process cost is low, the residual impurity proteins are reduced, the polysaccharide impurity content is extremely low, the yield is high, and the quality is good.

[0087] The culture medium provided by this invention does not contain animal-derived raw materials, reducing culture contaminants and yielding high capsular polysaccharide. Attached Figure Description

[0088] Figure 1 This is a bacterial growth curve diagram from Example 1.

[0089] Figure 2 This is a bacterial growth curve diagram from Example 2.

[0090] Figure 3 This is a chromatogram from the agarose immunodiffusion identification study in Example 4.

[0091] (Note: SA5, SA8, and SA336 are crude extracts of standard strains of Staphylococcus aureus types 5, 8, and 336, respectively; Newman and USA300 are crude extracts of clinical isolates of Staphylococcus aureus, respectively; CP5 is purified polysaccharide of Staphylococcus aureus type 5; pAb5 is hyperimmune serum of Staphylococcus aureus type 5; NC is physiological saline.)

[0092] Figure 4 A flowchart illustrating the extraction process of polysaccharides from the capsular region of Staphylococcus aureus. Detailed Implementation

[0093] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0094] The following embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the following embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0095] Example 1: Small-scale fermentation of Staphylococcus aureus

[0096] Example 1.1 Fermentation of Staphylococcus aureus type 5 (5L fermenter scale)

[0097] This embodiment discloses a high-efficiency fermentation culture process for Staphylococcus aureus type 5. The specific implementation steps are as follows: First, prepare a series of fermentation-specific culture media. The total volume of the seed culture medium is 0.2L. Each liter of basal culture medium contains 20g of soybean peptone, 10g of yeast powder, and 20g of sodium chloride. Accurately weigh 4.0g of soybean peptone, 2.0g of yeast powder, and 4.0g of sodium chloride. Stir thoroughly until all components are completely dissolved. Sterilize using 115℃ high-pressure steam for 20 minutes. After sterilization, cool to room temperature. The culture medium was sealed and stored in a sterile laminar flow hood for later use. The second-generation fermentation basal medium was prepared to a dissolving volume of 2.0L, with the concentrations of soybean peptone, yeast powder, sodium chloride, and glucose added at 20g / L, 10g / L, 20g / L, and 6g / L respectively, corresponding to weighed masses of 40g, 20g, 40g, and 12g. This second-generation fermentation basal medium was transferred to a 5L fermenter and autoclaved at 115℃ for 20 minutes. Separately, an inorganic salt stock solution was prepared by dissolving 10g of K₂HPO₄ in 50ml of pure water and 2g of MgSO₄·7H₂O in 50ml of pure water. Both inorganic salt solutions were separately autoclaved at 115℃ for 20 minutes to avoid co-sterilization with the basal medium and potential physicochemical reactions. 5.0ml of sterile defoaming agent was also prepared to suppress foam formation during fermentation.

[0098] The NaOH solution was prepared at a concentration of 500 g / L, with a dissolution volume of 0.5 L. It was then sterilized by high-pressure steam at 115 °C for 20 min and is specifically used for pH control in the fermentation process.

[0099] Preparation and sterilization of feed culture medium for fermentation tank: Glucose supplement solution 1 was prepared at a concentration of 500 g / L, with a dissolution volume of 0.5 L, and treated under the same sterilization conditions as a dedicated carbon source for fermentation sugar supplementation.

[0100] First-generation seed culture activation: Take one tube of Staphylococcus aureus glycerol bacillus type 5 (ATCC 49521) and inoculate it into an Erlenmeyer flask containing 0.2L of sterile seed culture medium at an inoculation ratio of 1:1000. Place the inoculated Erlenmeyer flask on a constant temperature shaking incubator, set the culture temperature to 32℃ and the shaking speed to 140rpm, and culture overnight with constant temperature shaking until the cells grow to the middle of the logarithmic growth phase to obtain mature first-generation seed culture for later use.

[0101] Batch fermentation was carried out: 0.2L of the first-generation seed culture was first added to a 5L fermenter containing 2.0L of sterile second-generation fermentation basal medium at an inoculation volume ratio of 1:10 (seed culture: basal medium), followed by the sequential addition of MgSO4·7H2O solution (1g / L) and K2HPO4 solution (5g / L). Throughout the fermentation process, core process parameters were precisely controlled: the culture temperature was kept constant at 37℃, the system pH was stably controlled at 7.2±0.1, the stirring speed was set to an adjustable range of 100~600rpm, the deep aeration rate was maintained at 50L / h, the fermenter pressure was maintained at 0.05MPa, and the dissolved oxygen level was maintained constant at 15%. During the culture, residual sugar levels were monitored hourly. When the residual sugar level in the medium fell below 4g / L, glucose feed solution 1 was used to supplement the sugar to 10g / L to ensure carbon source supply.

[0102] During fermentation, samples are taken every hour for monitoring. Each sample is tested for three core indicators. The cell morphology and growth activity are observed under an optical microscope, and the absorbance of the fermentation broth at an A600nm wavelength is measured. The cell growth morphology, A600nm value, and changes in residual sugar are used as the comprehensive criteria for judgment. Fermentation is terminated immediately when the cells grow to the late logarithmic growth phase or the early stationary phase.

[0103] Example 1.2 Fermentation of Staphylococcus aureus type 5 (5L fermenter scale)

[0104] This embodiment discloses an efficient fermentation culture process for Staphylococcus aureus type 5. Compared with Example 1.1, this embodiment changes the culture medium formula (including the seed culture medium formula and the second-generation basal culture medium formula), adds feed during fermentation, and changes the inoculation ratio. The specific implementation steps are as follows: First, prepare a series of fermentation-specific culture media. The total volume of the seed culture medium is 0.5L, with a fixed ratio of 20g soybean peptone, 10g yeast powder, and 10g glucose per liter of basal culture medium. Accurately weigh 10.0g soybean peptone, 5.0g yeast powder, and 5.0g glucose, and stir thoroughly until all components are completely dissolved. Sterilize at 115℃ for 20 minutes using autoclave. After sterilization, cool to room temperature and seal for later use in a sterile laminar flow hood. The second-generation fermentation basal culture medium is prepared with a dissolving volume of 1.25L, where the concentrations of soybean peptone, yeast powder, and glucose are 20g / L, 10g / L, and 10g / L, respectively, corresponding to weighed masses of 25.0g, 12.5g, and 12.5g. After mixing the above carbon and nitrogen source components, transfer to a 5L fermenter and autoclave at 115℃ for 20 minutes. Separately prepare an inorganic salt stock solution by weighing K2HPO4. 6.25g of MgSO4·7H2O was dissolved in 0.2L of pure water, and 1.25g of MgSO4·7H2O was dissolved in 50ml of pure water. Both inorganic salt solutions were separately autoclaved at 115℃ for 20min to avoid physicochemical reactions with the basal culture medium during sterilization. At the same time, 5.0ml of sterile defoaming agent was prepared to inhibit foam generation during fermentation.

[0105] NaOH solution: Prepared at a concentration of 400 g / L, with a dissolution volume of 0.2 L, and sterilized by high-pressure steam at 115℃ for 20 min, specifically for pH control in the fermentation process; The feed culture medium for the fermenter was divided into two groups, each prepared and sterilized independently: Glucose supplementation solution 2: Prepared at a concentration of 400 g / L, with a dissolution volume of 0.3 L, and treated under the same sterilization conditions, as a dedicated carbon source for fermentation sugar supplementation; Supplemental nutrient solution 1: Soybean peptone was added at 20 g / L of second-generation basal medium, and yeast powder was added at 10 g / L of second-generation basal medium, with corresponding weighing masses of 25.0 g and 12.5 g, respectively, and a dissolution volume of 0.3 L; 62.5 mL of amino acid mixture (containing 0.125 g each of uridine diphosphate (UDP), arginine, cysteine, and glutamine) was added to the supplemental nutrient solution; at the same time, 2.5 mL of vitamins, 5 mL of T solution, and 2.5 mL of 6% (g / 100 mL) nicotinamide were added to the supplemental nutrient solution, and all were sterilized by aseptic filtration. The specific components are shown in Table A.

[0106] Solution T: MgSO4·7H2O (3 g / L), MnSO4·H2O (0.5 g / L), FeSO4·7H2O (0.1 g / L), CoCl·6H2O (0.1 g / L), CaCl2 (0.1 g / L), ZnSO4·7H2O (0.1 g / L), CuSO4·5H2O (0.01 g / L), H3BO3 (0.01 g / L), NaMoO4·2H2O (0.01 g / L), and NiSO4·6H2O (0.1 g / L).

[0107] Vitamins: Biotin (vitamin H, 4 mg / L), Folic acid (vitamin B9, 4 mg / L), Vitamin B6 (40 mg / L), Vitamin B1 (20 mg / L), Riboflavin (vitamin B2, 20 mg / L), Nicotinic acid (vitamin B3, 20 mg / L), Calcium pantothenate (vitamin B5, 20 mg / L), Vitamin B12 (0.4 mg / L).

[0108] Table A: Components of Supplemental Compound Nutrient Solution

[0109] First-generation seed culture activation: Take one tube of Staphylococcus aureus glycerol bacillus type 5 (ATCC 49521) and inoculate it into a 1.0L Erlenmeyer flask containing 0.5L of sterile seed culture medium at an inoculation ratio of 1:1000. Place the inoculated Erlenmeyer flask on a constant temperature shaking incubator, set the culture temperature to 32℃ and the shaking speed to 140rpm, and culture overnight with constant temperature shaking until the cells grow to the middle of the logarithmic growth phase to obtain the first-generation seed culture for later use.

[0110] Batch fermentation was carried out: 0.5L of the mature first-generation seed culture was added to a 5L fermenter containing 1.25L of sterile second-generation fermentation basal medium at an inoculation volume ratio of 1:2.5 (seed culture: basal medium), followed by sequential addition of MgSO4·7H2O solution (1g / L) and K2HPO4 solution (5g / L). Throughout the fermentation process, core process parameters were precisely controlled: the culture temperature was kept constant at 37℃, the system pH was stably controlled at 7.2±0.1, the stirring speed was set to an adjustable range of 100~600rpm, the deep aeration rate was maintained at 50L / h, the fermenter pressure was maintained at 0.05MPa, and the dissolved oxygen level was maintained constant at 15%. Feeding and sugar addition operations were started simultaneously at the 1st hour of fermentation. Feeding (compound nutrient solution 1) was added in a segmented quantitative flow mode, with 50 ml added every hour. Sugar addition (glucose sugar solution 2) was added in a segmented quantitative flow mode, with 20 ml of glucose sugar solution added at the 1st hour of fermentation, 30 ml at the 2nd hour, 70 ml at the 3rd hour, 70 ml at the 4th hour, and 30 ml at the 5th hour, to avoid drastic fluctuations in carbon source concentration that could interfere with the normal growth and metabolism of the cells.

[0111] During fermentation, samples are taken every hour for monitoring. Each sample is tested for three core indicators. The cell morphology and growth activity are observed under an optical microscope, and the absorbance of the fermentation broth at an A600nm wavelength is measured. The cell growth morphology, A600nm value, and changes in residual sugar are used as the comprehensive criteria for judgment. Fermentation is terminated immediately when the cells grow to the late logarithmic growth phase or the early stationary phase.

[0112] Example 1.3 Fermentation of Staphylococcus aureus type 5 (5L fermenter scale)

[0113] This embodiment discloses an efficient fermentation culture process for Staphylococcus aureus type 5. Compared with Embodiment 1.2, this embodiment changes the seed liquid inoculation ratio and the feed culture medium formula. The specific implementation steps are as follows: First, prepare a series of fermentation-specific culture media. The total volume of the seed culture medium is 0.5L, with a fixed ratio of 20g soybean peptone, 10g yeast powder, and 10g glucose per liter of basal culture medium. Accurately weigh 10.0g soybean peptone, 5.0g yeast powder, and 5.0g glucose, and stir thoroughly until all components are completely dissolved. Sterilize at 115℃ for 20 minutes using autoclave. After sterilization, cool to room temperature and seal for later use in a sterile laminar flow hood. The second-generation fermentation basal culture medium is prepared with a dissolving volume of 1.25L, where the concentrations of soybean peptone, yeast powder, and glucose are 20g / L, 10g / L, and 10g / L, respectively, corresponding to weighed masses of 25.0g, 12.5g, and 12.5g. After mixing the above carbon and nitrogen source components, transfer to a 5L fermenter and autoclave at 115℃ for 20 minutes. Separately prepare an inorganic salt stock solution by weighing K2HPO4. 6.25g of MgSO4·7H2O was dissolved in 0.2L of pure water, and 1.25g of MgSO4·7H2O was dissolved in 50ml of pure water. Both inorganic salt solutions were separately autoclaved at 115℃ for 20min to avoid physicochemical reactions with the basal culture medium during sterilization. At the same time, 5.0ml of sterile defoaming agent was prepared to inhibit foam generation during fermentation.

[0114] NaOH solution: Prepared at a concentration of 400 g / L, with a dissolution volume of 0.2 L, and sterilized by high-pressure steam at 115℃ for 20 min, specifically for pH control in the fermentation process; The feed culture medium for the fermenter was divided into two groups, each prepared and sterilized independently: Glucose supplementation solution 2: Prepared at a concentration of 400 g / L, with a dissolution volume of 0.3 L, and treated under the same sterilization conditions, as a dedicated carbon source for fermentation sugar supplementation; Supplemental nutrient solution 2: Soybean peptone was added at 60 g / L of second-generation basal culture medium, and yeast powder was added at 30 g / L of second-generation basal culture medium, with corresponding weighing masses of 75.0 g and 37.5 g, respectively, and a dissolution volume of 0.9 L; 62.5 mL of amino acid mixture (containing 0.125 g each of UDP, arginine, cysteine, and glutamine) was added to the supplemental nutrient solution; at the same time, 7.5 mL of vitamins, 15 mL of T solution, and 7.5 mL of 6% nicotinamide at the same concentration as in Example 1.2 were added to the supplemental nutrient solution, and all were sterilized by aseptic filtration.

[0115] First-generation seed culture activation: Take one tube of Staphylococcus aureus serotype 5 glycerol bacteria and inoculate it into a 1.0L Erlenmeyer flask containing 0.5L of sterile seed culture medium. The inoculation ratio is 1:2000. Place the inoculated Erlenmeyer flask on a constant temperature shaking incubator, set the culture temperature to 32℃ and the shaking speed to 140rpm, and culture overnight with constant temperature shaking until the bacteria grow to the middle of the logarithmic growth phase to obtain a mature first-generation seed culture for later use.

[0116] Batch fermentation culture was carried out: 0.5L of the above-mentioned mature first-generation seed culture was added to a 5L fermenter containing 1.25L of sterile second-generation fermentation basal medium at an inoculation volume ratio of 1:2.5 (seed culture: basal medium). The core process parameters were precisely controlled throughout the fermentation process: the culture temperature was kept constant at 37℃, the system pH was stably controlled at 7.2±0.1, the stirring speed was set to an adjustable range of 100~600rpm, the deep aeration rate was maintained at 50L / h, the fermenter pressure was maintained at 0.05MPa, and the dissolved oxygen level was kept constant at 15%. Feeding and sugar addition were initiated simultaneously at the 3rd hour of fermentation. Feeding (compound nutrient solution 2) was done in a segmented quantitative feeding mode: 100ml of feed was added at the 3rd hour of fermentation, 200ml at the 5th hour, 200ml at the 6th hour, 200ml at the 7th hour, and 200ml at the 8th hour. Sugar addition (glucose sugar solution 2) was done in a segmented quantitative feeding mode: 30ml of glucose solution was added at the 3rd hour of fermentation, 40ml at the 5th hour, 40ml at the 6th hour, 40ml at the 7th hour, and 40ml at the 8th hour. This avoided drastic fluctuations in carbon source concentration, which could interfere with the normal growth and metabolism of the cells.

[0117] During fermentation, samples are taken every hour for monitoring. Each sample is tested for three core indicators. The cell morphology and growth activity are observed under an optical microscope, and the absorbance of the fermentation broth at an A600nm wavelength is measured. The cell growth morphology, A600nm value, and changes in residual sugar are used as the comprehensive criteria for judgment. Fermentation is terminated immediately when the cells grow to the late logarithmic growth phase or the early stationary phase.

[0118] The results are as follows Figure 1 As shown in Table 1, under the process parameters of a seed liquid inoculation volume ratio of 1:2.5 and supplementation with glucose and high-concentration compound nutrient solution (Example 1.3), the final cell density A600nm can reach 30.64 after 9.5 hours of fermentation, achieving high-density fermentation.

[0119] Table 1 Study on small-scale fermentation process parameters of Staphylococcus aureus

[0120] Example 2: Pilot-scale fermentation of Staphylococcus aureus (30L fermenter)

[0121] First, prepare a series of fermentation-specific culture media. The total volume of the seed culture medium is 4.0L, with a fixed ratio of 20g soybean peptone, 10g yeast powder, and 10g glucose per liter of basal medium. Accurately weigh 80g soybean peptone, 40g yeast powder, and 40g glucose, and stir thoroughly until all components are completely dissolved. Dispense into four 2.0L Erlenmeyer flasks and autoclave at 115℃ for 20 minutes. After sterilization, cool to room temperature and seal for later use in a sterile laminar flow hood. The second-generation fermentation basal medium is prepared according to a 10L dissolution volume, with soybean peptone, yeast powder, and glucose added at concentrations of 20g / L, 10g / L, and 10g / L, respectively, corresponding to weighed masses of 200g, 100g, and 100g. After mixing the above carbon and nitrogen source components, transfer to a 30L fermenter and autoclave at 115℃ for 20 minutes. Separately prepare an inorganic salt stock solution by weighing K2HPO4. 50g of MgSO4·7H2O was dissolved in 0.2L of pure water, and 10g of MgSO4·7H2O was dissolved in 0.05L of pure water. Both inorganic salt solutions were separately autoclaved at 115℃ for 20min to avoid physicochemical reactions with the basal culture medium during sterilization. At the same time, 5.0ml of sterile defoaming agent was prepared to inhibit foam generation during fermentation.

[0122] The NaOH solution was prepared at a concentration of 400 g / L, with a dissolution volume of 2.0 L. It was then sterilized by high-pressure steam at 115 °C for 20 min and is specifically used for pH control in the fermentation process. The feed culture medium for the fermenter was divided into two groups, each prepared and sterilized independently: Glucose supplementation solution 2: Prepared at a concentration of 400 g / L, with a dissolution volume of 2.0 L, and treated under the same sterilization conditions, as a dedicated carbon source for fermentation sugar supplementation; Supplemental nutrient solution 3: Soybean peptone was added at 60g / L of second-generation basal culture medium, and yeast powder was added at 30g / L of second-generation basal culture medium, corresponding to weighing masses of 600g and 300g respectively, with a dissolving volume of 2.5L; 500mL of amino acid mixture (containing UDP, arginine, cysteine, and glutamine, 1.0g each) was added to the supplemental nutrient solution; at the same time, 25ml each of vitamins, T solution, and 6% nicotinamide were added to the supplemental nutrient solution, and each was sterilized by aseptic filtration.

[0123] First-generation seed culture activation: Take two vials of Staphylococcus aureus glycerol strain 5 (ATCC 49521) or 336 (ATCC 55804), each with a volume of 0.5 ml. Accurately transfer 0.25 ml of the glycerol suspension to a 2.0 L Erlenmeyer flask containing 1.0 L of sterile seed culture medium. Prepare a total of 4 seed culture systems according to this standard, with a total seed culture volume of 4.0 L. The inoculation ratio is 1:4000 or 1:10000. Place the inoculated Erlenmeyer flasks on a constant temperature shaking incubator, set the culture temperature to 30℃ and the shaking speed to 140 rpm, and culture at a constant temperature and shaking speed for 15 h until the cells grow to the middle of the logarithmic growth phase to obtain mature first-generation seed culture for later use.

[0124] Batch fermentation culture was carried out: 4.0L of the above-mentioned mature first-generation seed culture was added to a 30L fermenter containing 10.0L of sterile second-generation fermentation basal medium at an inoculation volume ratio of 1:2.5 (seed culture: basal medium). The core process parameters were precisely controlled throughout the fermentation process: the culture temperature was kept constant at 37℃, the system pH was stably controlled at 7.2±0.1, the stirring speed was set to an adjustable range of 100~600rpm, the deep aeration rate was maintained at 15L / min, the fermenter pressure was maintained at 0.05MPa, and the dissolved oxygen level was kept constant at 15%. Feeding and sugar addition operations were started simultaneously at the 2nd hour of fermentation. Feeding (compound nutrient solution 3) was added in a segmented quantitative feeding mode. 500ml of compound nutrient solution was added at the 2nd hour of fermentation, 1500ml at the 3rd hour, and 500ml at the 4th hour. The timing and dosage of feeding were strictly controlled. Sugar addition (glucose sugar solution 2) was added at a uniform rate of 5 rpm to complete the feeding operation of all glucose sugar solution. The feeding was kept stable throughout to avoid drastic fluctuations in carbon source concentration, which would interfere with the normal growth and metabolism of the cells.

[0125] During fermentation, samples are taken every hour for monitoring. Each sample is tested for three core indicators. The cell morphology and growth activity are observed under an optical microscope, and the absorbance of the fermentation broth at an A600nm wavelength is measured. The cell growth morphology, A600nm value, and changes in residual sugar are used as the comprehensive criteria for judgment. Fermentation is terminated immediately when the cells grow to the late logarithmic growth phase or the early stationary phase.

[0126] Based on the key process parameters determined in the above-mentioned small-scale process, pilot-scale fermentation was carried out, and the results are as follows: Figure 2As shown in Table 2, the A600nm of the final fermentation liquid for Staphylococcus aureus types 5 and 336, fermented for 8-9 hours according to the small-scale process, reached 22-23, indicating that the fermentation process of this invention has good applicability to different types of Staphylococcus aureus. Compared with the results of the 5L fermenter in the small-scale test, the A600nm decreased slightly, which is attributed to the decrease in the oxygen transfer coefficient after scale-up. Overall, this invention achieves a high cell density level under the conditions of controlling the fermentation system to be free of animal-derived components and fermentation time to 8-9 hours, laying a solid foundation for the extraction yield of capsular polysaccharides downstream.

[0127] Table 2 Study on pilot-scale fermentation process parameters of Staphylococcus aureus

[0128] Example 3 Extraction of polysaccharides from the capsular region of Staphylococcus aureus type 5

[0129] Example 3.1 Preparation of Type 5 purified capsular polysaccharides by enzymatic hydrolysis-alcohol precipitation extraction

[0130] The fermentation broth of Staphylococcus aureus type 5 was centrifuged at 10,000 rpm for 25 min. The bacterial precipitate was collected and resuspended in 50 mmol / L phosphate buffer (PB buffer) at pH 7.5 to adjust the bacterial concentration to 0.5 g / L. The resuspended bacterial broth was thoroughly mixed to obtain a bacterial resuspension. Lysostaphin (Shanghai Gaoke United Biotechnology R&D Co., Ltd.) was added to the bacterial resuspension to adjust the final concentration of lysostaphin in the enzymatic hydrolysis system to 80 μg / mL of fermentation broth. The enzymatic hydrolysis system was then placed in a shaker and stirred overnight at 37°C and 180 rpm to obtain the bacterial enzymatic hydrolysate.

[0131] Add 50 mmol / L PB buffer (pH 7.5) containing 0.2 mol / L MgSO4 to the enzymatic hydrolysate to adjust the Mg content in the system. 2+ The final concentration was 2 mmol / L. Then, a totipotent nuclease (Xi'an Maiboteke Biotechnology Co., Ltd.) was added, with the enzyme dosage controlled at 25 U / g of bacterial cells. The enzymatic hydrolysis system was then placed in a shaker and hydrolyzed at 37°C and 180 rpm for 2 hours. Pre-cooled anhydrous ethanol (2-8°C) was added to the above acid hydrolysis product to adjust the final volume fraction of anhydrous ethanol in the system to 25%. After thorough mixing, the mixture was placed in a low-temperature environment of 2-8°C and allowed to stand for 4 hours to complete the first-stage ethanol precipitation. The first-stage ethanol precipitation mixture was transferred to a low-temperature high-speed centrifuge and centrifuged at 10,000 rpm and 4°C for 30 minutes. Impurities and precipitates were discarded, and the supernatant was collected for later use.

[0132] Add pre-cooled anhydrous ethanol (2-8°C) to the supernatant obtained from the first-stage ethanol precipitation centrifugation. Calculate the ethanol addition volume according to the dilution ratio to adjust the final volume fraction of anhydrous ethanol in the system to 75%. Mix thoroughly and let stand overnight at 2-8°C to complete the second-stage ethanol precipitation. Place the second-stage ethanol precipitation mixture in a low-temperature high-speed centrifuge and centrifuge at 10,000 rpm and 4°C for 30 min. Discard the supernatant and collect the target crude sugar precipitate. Resuspend the precipitate in 160 mL of pre-cooled anhydrous ethanol (2-8°C) and centrifuge again at 10,000 rpm and 4°C for 30 min. Collect the precipitate; this operation is recorded as a single wash. Repeat the above washing operation a total of 2 times to remove residual impurities and obtain the purified crude sugar precipitate.

[0133] Add 300 mL of purified water to the washed and purified crude sugar precipitate and stir continuously until the precipitate is completely dissolved and reconstituted. Transfer the reconstituted solution to a freeze dryer and start the freeze-drying program. After freeze-drying, the corresponding purified capsular polysaccharide sample is obtained. The purity and physicochemical properties of the purified capsular polysaccharide sample are characterized by protein content (Lowry method), nucleic acid content (ultraviolet spectrophotometry), O-acetyl content (Hestrin method), and aminohexose content (Elson Morgan method).

[0134] Example 3.2 Preparation of Type 5 purified capsular polysaccharides by enzymatic hydrolysis-DOC extraction method

[0135] Lysostaphin and totipotent nuclease (Xi'an Maibotai Biotechnology Co., Ltd.) were added to the fermentation broth of Staphylococcus aureus type 5. The lysostaphin (Shanghai Gaoke United Biotechnology R&D Co., Ltd.) was added at a concentration of 80 μg / mL of fermentation broth, and the totipotent nuclease was added at a concentration of 25 U / g wet cell weight. After mixing, the mixture was stirred at 37℃ for 10 h to complete enzymatic hydrolysis. After hydrolysis, the hydrolysate was diluted 20 times with 0.85% NaCl solution, stirred at 2200 rpm for 5 min, and the pH was adjusted to 7.00-7.30. Sodium deoxycholate (DOC) (Sigma, CAS: 302-95-4) solution was added to bring the final concentration to 0.15%, and the pH was adjusted again to 7.30. The mixture was mechanically stirred at 900 rpm for 1 h to complete DOC treatment. After DOC treatment, the pH was slowly adjusted to 4.00±0.05 with acetic acid, and mechanically stirred at 1500 rpm for 1 h to complete acetic acid adjustment. The acetic acid-adjusted feed solution was clarified using a 0.45 μm Millipore membrane (0.5 m² × 2), and the flow-through was collected. The retentate was washed with purified water, and the washing was repeated twice. All flow-through and washing solutions were combined. The combined clarified solution was diluted 2 times with a 0.85% NaCl solution, mechanically stirred at 1700 rpm, and the pH of the system was adjusted to 7.14 while stirring. Stirring was continued for 5 min. The diluted solution was concentrated and replaced by 10-fold volumetric ultrafiltration using a membrane with a molecular weight cutoff of 10 kDa (0.5 m² × 4). The membrane was first replaced twice with 1 mol / L NaCl solution, and then four times with purified water. After replacement, the retentate was collected into a clean collection bottle, and the membrane was rinsed twice with purified water. The rinsing and retentate solutions were combined and filtered through a 0.2 μm Capsule filter (Coplast). The resulting filtrate was a clear and transparent liquid. The filtrate was then lyophilized to obtain a purified capsular polysaccharide sample. The purity and physicochemical properties of the purified capsular polysaccharide sample were characterized by protein content (Lowry method), nucleic acid content (UV spectrophotometry), O-acetyl content (Hestrin method), and aminohexose content (Elson Morgan method).

[0136] Example 3.3 Preparation of Type 5 Refined Capsular Polysaccharides by High-Temperature Acid Cleavage-DOC Extraction Method

[0137] Acetic acid was added to the fermentation broth to adjust the pH to 4.00 ± 0.05, and the reaction was carried out at 100℃ for 2 hours to obtain a high-temperature acid lysis solution. The high-temperature acid lysis solution was then clarified by encapsulating it in a 0.45 μm Miebao membrane (0.5 m² × 4). The flow-through was collected, and the membrane was washed twice with purified water. The flow-through and washing solutions were combined to obtain a primary clarified solution. A 50% sodium hydroxide solution was added to the primary clarified solution to adjust the pH to neutral. The pH-adjusted primary clarified flow-through solution was diluted 2-fold with a 0.85% sodium chloride solution. During the dilution process, the solution was mechanically stirred at 1700 rpm. A 10% (w / v) sodium deoxycholate (DOC) solution was added to the diluted solution to bring the final DOC concentration to 0.15%. The system was kept at neutral pH and mechanically stirred at 900 rpm for 30 minutes at room temperature. After stirring, the system was mechanically stirred at 1500 rpm, and acetic acid was added to slowly adjust the pH to 4.00 ± 0.05. Stirring was continued for 1 hour. The acidified solution was clarified again using a 0.45 μm Millipore membrane (0.5 m² × 4), and the flow-through was collected. The clarified flow-through was then subjected to 10-fold volumetric ultrafiltration concentration and replacement using a 10 kDa molecular weight cutoff ultrafiltration membrane (0.5 m² × 4). The membrane was first washed twice with 1 mol / L sodium chloride solution, then washed four times with purified water. After replacement, the membrane retentate was collected, and the membrane was washed twice with purified water. The retentate and washing solutions were combined. The combined solution was filtered through a 0.2 μm Cornuaite Nenz filter, and the filtrate was freeze-dried to obtain a purified capsular polysaccharide sample. The purity and physicochemical properties of the purified capsular polysaccharide sample were characterized by protein content (Lowry method), nucleic acid content (UV spectrophotometry), O-acetyl content (Hestrin method), and aminohexose content (Elson Morgan method).

[0138] The results are shown in Table 3. The enzymatic hydrolysis-alcohol precipitation extraction method (Example 3.1) yielded 2.5469 g / L of capsular polysaccharide in the fermentation broth, but its nucleic acid content reached 14.86% and protein content reached 6.53%, indicating high impurity content, which could not meet the purity requirements for industrial production. The enzymatic hydrolysis-DOC extraction method (Example 3.2) and the high-temperature acid lysis-DOC extraction method (Example 3.3) yielded capsular polysaccharide and impurity content that were basically the same, both achieving the goals of high purity and high yield. However, the specific sugar content of the purified polysaccharide obtained by the enzymatic hydrolysis-DOC extraction method was significantly lower than that obtained by the high-temperature acid lysis-DOC extraction method, indicating that the enzymatic hydrolysis-DOC extraction method was more destructive to capsular polysaccharides than the high-temperature acid lysis-DOC extraction method. In addition, the high-temperature acid lysis-DOC extraction method uses an enzyme-free process, which, compared with the existing lysostaphin and nuclease processes, not only significantly reduces the cost of commercial production, but also has the technical advantages of simple process flow and easy industrial scale-up.

[0139] Table 3. Study on extraction process parameters of Staphylococcus aureus capsular polysaccharides

[0140] Example 4: Identification Study Using Agar Double Amplification

[0141] Preparation of hyperimmune serum for various types of Staphylococcus aureus: Standard strains of Staphylococcus aureus type 5 (ATCC 49521), Staphylococcus aureus type 8 (ATCC 49525), and Staphylococcus aureus type 336 (ATCC 55804) were completely inactivated with formaldehyde solution to remove pathogenicity. SPF-grade New Zealand rabbits were then subjected to gradient immunization using a multi-needle, multi-point immunization method. The entire immunization process was completed according to the standard animal immunization program. After the immunization cycle was completed, rabbit venous blood was collected, centrifuged, and specific hyperimmune serum for each type of Staphylococcus aureus was obtained and stored at -80℃ for later use.

[0142] Preparation of crude extracts of Staphylococcus aureus of various serotypes: Standard strains of each serotype were inoculated into 30 mL of antibiotic-free LB medium and cultured overnight at 30 °C and 140 rpm to obtain fresh bacterial suspension; centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the bacterial precipitate was collected; the bacterial precipitate was resuspended in sterile 50 mmol / L Tris-HCl buffer (pH 7.0, containing 2 mmol / L MgCl2), lysostaphin and totipotent nuclease were added to the bacterial suspension, mixed well, and incubated in a constant temperature shaker at 37 °C and 180 rpm for 4 h, and the complete lysis of the bacterial cells was confirmed by microscopic examination; the bacterial lysis digest was filtered through a 0.2 μm sterile filter to remove bacteria, and the filtrate was the crude extract of each serotype strain, which was stored at 2–8 °C for later use.

[0143] Two-way immunodiffusion assay: Prepare 100 mL agarose gel solution, consisting of 1% agarose, 1×PBS buffer, and the remainder autoclaved deionized water. Heat the mixture in a microwave oven until the agarose is completely dissolved and the gel is homogeneous and transparent. Transfer the solution to a 50-60°C oven to prevent gel solidification. Place a sterile glass slide stably on a horizontal platform in a laminar flow hood. Pour 5 mL of the incubated agarose solution evenly onto a single slide and allow it to stand until the gel is completely cooled and solidified. Use a special punch to create 10 holes per slide, arranged in a diamond pattern. Add the test sample and serum to the gel wells according to the pre-defined sample loading protocol. After loading, place the gel slide in a humidified chamber and incubate at 37°C for diffusion. Observe and record the formation, location, morphology, and fusion state of the white precipitate lines in the gel.

[0144] The results are as follows Figure 3As shown, type 5 hyperimmune serum showed strong positive reactions only with type 5 purified polysaccharide, crude extract of type 5 Staphylococcus aureus standard strain, and Newman strain (a known type 5 capsular serotype); type 336 hyperimmune serum showed strong positive reactions only with type 336 purified polysaccharide and crude extract of type 336 Staphylococcus aureus standard strain; type 8 hyperimmune serum showed very weak positive reactions with type 8 purified polysaccharide and crude extract of type 8 Staphylococcus aureus standard strain, which is speculated to be due to the low titer of the hyperimmune serum; the overall results indicate that all types of capsular polysaccharides prepared in this invention can specifically react with the corresponding types of strains of hyperimmune serum.

[0145] Example 5 Preparation of polysaccharide-protein conjugate stock solution

[0146] Weigh 100 mg of type 5 or type 8 purified polysaccharide into a beaker, add 10 mL of 0.85% NaCl solution to dissolve it. After complete dissolution, adjust the pH of the polysaccharide solution to 8.45-9.05, add 0.3 mL of 100 mg / mL CDAP solution (already dissolved), and then add 3% triethylamine solution to maintain the pH at 8.30-9.20. React for 2 min. Immediately after 2 min, add 10 mL of 10 mg / mL recombinant pneumococcal protamine to the reaction beaker and react for 6-24 h. After the reaction, dilute to 10 L with 0.85% NaCl solution, concentrate and replace 4 times with a 300 kDa ultrafiltration membrane (Millipore), and finally collect the retentate. After filtration through a 0.22 μm syringe filter, the stock solution of each type of monovalent conjugate is obtained.

[0147] The results are shown in Table 4. The sugar content, glycoprotein ratio, and free sugar content of the type 5 and type 8 conjugate stock solutions all meet the requirements for downstream immunogenicity studies.

[0148] Table 4. Quality properties of the stock solutions of type 5 and type 8 conjugates

[0149] Example 6 Immunogenicity Study

[0150] The antigen immunization dose was calculated based on the sugar concentration of each type of conjugate stock solution, and then mixed with an equal volume of liposome adjuvant (containing QS-21 50 μg / ml, MPL 25 μg / ml, cholesterol 0.5 mg / ml, DOPC 2 mg / ml) to obtain a composition for immunization.

[0151] Six- to eight-week-old SPF-grade BALB / c mice, half male and half female, were used in each group. Each immunization group received a three-dose immunization program with a two-week interval. Each mouse received 0.1 ml of immunization. Blood samples were collected two weeks after the second and final immunizations. The geometric mean titer (IgG GMT) of specific IgG for each protective antigen was determined using an indirect ELISA method. Each type of purified polysaccharide antigen was diluted to 10 μg / mL, and 100 μL / well was coated onto an ELISA plate and incubated overnight at 4°C. Antisera from each experimental group were serially diluted, and 100 μL was added to each well, incubated at 37°C for 120 min. Then, 200 μL of washing buffer was added to each well for washing, for a total of 5 washes. After washing, 4000-fold diluted goat anti-mouse alkaline phosphatase-labeled secondary antibody (Southern Biotech) was added, and incubated at 37°C for 120 min. After incubation, 200 μL of washing buffer was added to each well for washing, for a total of 5 washes. Then, 100 μL of chromogenic solution (prepared from magnesium chloride hexahydrate (Sinopharm) and diethanolamine (Sigma) at a ratio of 1 (g): 1 (ml)) was added to each well, and incubated at 20–25°C for 60 min. Finally, 50 μL of stop solution (3 mol / L) was added. NaOH solution); the values ​​of the ELISA plate were read using an ELISA reader, and the Cutoff value was determined based on the negative serum.

[0152] The results are shown in Table 5. After three immunizations, the IgG GMT titer of the type 5 and type 8 conjugates reached 10. 4 ~10 5 It exhibits good immunogenicity, providing a solid foundation for the subsequent development of Staphylococcus aureus vaccines.

[0153] Table 5. Immunogenicity studies of stock solutions of polysaccharide-protein conjugates of types 5 and 8

[0154] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A method using Staphylococcus aureus ( Staphylococcus aureus A method for preparing capsular polysaccharides, characterized in that, The method includes the following steps: (1) Add acetic acid to the Staphylococcus aureus fermentation broth to lyse it and obtain the lysate; (2) Adjust the pH of the lysis buffer to neutral using an alkaline solution, and add sodium deoxycholate while stirring and reacting; (3) Adjust the pH of the system obtained in step (2) to acidic using an acidic solution, and the reaction causes the impurity protein and sodium deoxycholate to precipitate out. (4) Solid-liquid separation: The system obtained in step (3) is subjected to solid-liquid separation, and the liquid phase containing the capsular polysaccharide is collected; (5) Drying: The liquid phase obtained in step (4) is dried to obtain the capsular polysaccharide; Furthermore, no additional enzyme treatment is added during the preparation process.

2. The method as described in claim 1, characterized in that, In step (1) of the method, acetic acid is added to the Staphylococcus aureus fermentation broth to adjust the pH to 3.5-4.5, and the reaction is carried out at a temperature not lower than 60°C to obtain the lysate; Preferably, step (1) satisfies one or more of the following: 1) Adjust the pH value to 3.8-4.2, for example, 3.9-4.1; 2) The reaction occurs at 90-130℃, for example, 95-110℃; and, 3) The reaction time is 1-6 hours, for example, 1.5-2.5 hours.

3. The method as described in claim 1 or 2, characterized in that, There is also a clarification step between step (1) and step (2); Preferably, membrane clarification is used, and solid-liquid separation is preferably performed by microfiltration membrane clarification. The membrane is then washed with purified water to recover residual products, and the products are combined to obtain a primary clarified liquid.

4. The method according to any one of claims 1-3, characterized in that, Step (2) satisfies one or more of the following: 1) Adjust the pH to neutral using sodium hydroxide solution; 2) Add sodium deoxycholate to the system until the final concentration of sodium deoxycholate is 0.05-0.5% (w / v), preferably 0.1-0.2% (w / v). 3) The stirring speed is 600-1200 rpm, preferably 800-1000 rpm; 4) The reaction time is 20-40 minutes, for example, 25-35 minutes; and, 5) After adjusting the pH of the lysis buffer to neutral, a dilution step is also included before adding sodium deoxycholate; Preferably, the dilution step satisfies one or more of the following: 1) Dilute with sodium chloride solution, preferably a 0.8-0.9% (w / v) sodium chloride solution; 2) The dilution is 1.5-3 times, preferably 1.8-2.2 times; and, 3) Dilution should be carried out while stirring, with a stirring speed of 1500-2000 rpm.

5. The method according to any one of claims 1-4, characterized in that, Step (3) satisfies one or more of the following: 1) Adjust the pH to 3.5-4.5, for example, 3.8-4.2; 2) Adjust the pH using acetic acid; and, 3) After adjusting the pH value to acidic, the reaction time is 0.5-2 hours, for example, 0.5-1.5 hours.

6. The method according to any one of claims 1-5, characterized in that, In step (4), the system obtained in step (3) is clarified and filtered using a microfiltration membrane, the flow-through is collected, and then the flow-through is concentrated using an ultrafiltration membrane with a molecular weight cutoff of 5~100 KDa, and replaced with a replacement solution. After replacement, it is filtered using a sterile filter, and the liquid phase containing the capsular polysaccharide is collected. Preferably, step (4) satisfies one or more of the following: 1) The pore size of the microfiltration membrane pack is 0.45 μm or 0.2 μm; 2) The ultrafiltration membrane pack is an ultrafiltration membrane pack with a molecular weight cutoff of 8-12 kDa; 3) The replacement is an equal-volume replacement of 8-12 times the volume; 4) The replacement solution is a salt solution and / or purified water; preferably, the solution is replaced 1-3 times with a salt solution first, and then replaced 2-6 times with purified water. 5) The replacement solution is a sodium chloride solution with a concentration of 0.5-2 mol / L; 6) After replacement and before filtration, the process also includes rinsing the ultrafiltration membrane pack with purified water and combining the rinsing solution with the retentate; and, 7) The pore size of the sterilization filter is 0.22 μm, and the material is polyethersulfone or polyvinylidene fluoride.

7. The method according to any one of claims 1-6, characterized in that, The capsular polysaccharide prepared by the method satisfies one or more of the following: 1) The protein content in the capsular polysaccharide is ≤1.0% (w / w), preferably ≤0.5% (w / w); 2) The nucleic acid content in the capsular polysaccharide is ≤0.8% (w / w), preferably ≤0.5% (w / w); 3) The yield of the capsular polysaccharide is not less than 200 mg / L; 4) The capsular polysaccharide contains aminohexose, and the aminohexose content is ≥3.0% (w / w), preferably ≥8.0% (w / w); and, 5) The capsular polysaccharide contains O-acetyl groups, and the O-acetyl group content is ≥0.8% (w / w), preferably ≥1.2% (w / w).

8. The method according to any one of claims 1-7, characterized in that, The Staphylococcus aureus serotype is one or more of type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 336; Preferably, the Staphylococcus aureus serotype is one or more of serotypes 5, 8, and 336.

9. The method according to any one of claims 1-8, characterized in that, The method further includes the step of culturing Staphylococcus aureus using a culture medium combination to obtain a fermentation broth, wherein the culture medium combination comprises a basal culture medium, a glucose supplementation solution, and a fed compound nutrient solution; the culture medium satisfies the following: 1) The basal culture medium contains soybean peptone, yeast powder and glucose, and preferably also contains MgSO4·7H2O and / or K2HPO4; 2) The basal culture medium is further divided into seed culture medium and second-generation basal culture medium according to the different contents of soybean peptone, yeast powder and glucose; 3) The glucose concentration in the glucose supplement solution is 20-500 g / L, preferably 350-450 g / L, for example 400 g / L; 4) The supplemental compound nutrient solution contains soybean peptone, yeast powder, uridine diphosphate, arginine, cysteine, glutamine, nicotinamide, vitamin solution, and T solution; the vitamin solution contains vitamin H, vitamin B9, vitamin B6, vitamin B1, vitamin B2, vitamin B3, vitamin B5, and vitamin B12; the T solution contains MgSO4·7H2O, MnSO4·H2O, FeSO4·7H2O, CoCl·6H2O, CaCl2, ZnSO4·7H2O, CuSO4·5H2O, H3BO3, NaMoO4·2H2O, and NiSO4·6H2O. Preferably, the culture medium combination satisfies: The seed culture medium contains 20-30 g / L soybean peptone, for example, 25 g / L; 10-15 g / L yeast extract, for example, 12.5 g / L; 10-15 g / L glucose, for example, 12.5 g / L; and / or, The second-generation basal culture medium contains soybean peptone at a concentration of 15-25 g / L, for example, 20 g / L; yeast extract at a concentration of 8-15 g / L, for example, 10 g / L; glucose at a concentration of 8-15 g / L, for example, 10 g / L; preferably MgSO4·7H2O at a concentration of 0.5-1.5 g / L, for example, 1 g / L; and K2HPO4 at a concentration of 3-8 g / L, for example, 5 g / L.

10. The method as described in claim 9, characterized in that, The addition amount of each component in the supplemental compound nutrient solution is based on the volume of the second-generation basal culture medium, wherein, The amount of soybean peptone added is 50-70 g / L of second-generation basal culture medium, for example, 60 g / L; The amount of yeast powder added is 25-35 g / L of second-generation basal culture medium, for example, 30 g / L; The amount of uridine diphosphate added is 0.05-0.15 g / L of the second-generation basal medium, for example, 0.1 g / L; Arginine was added at a rate of 0.05-0.15 g / L in the second-generation basal medium, for example, 0.1 g / L. The amount of cysteine ​​added is 0.05-0.15 g / L in the second-generation basal medium, for example, 0.1 g / L; The amount of glutamine added is 0.05-0.15 g / L of the second-generation basal culture medium, for example, 0.1 g / L; The amount of nicotinamide added is 0.8-6 mg / L in the second-generation culture medium, for example, 1.25-3 mg / L; The vitamin solution is added at a volume ratio of 2-10 mL / L of second-generation basal culture medium, such as 2.5 mL / L or 6 mL / L. The volume ratio of T solution added is 2-20 mL / L of second-generation basal culture medium, such as 2.5 mL / L or 12 mL / L; The vitamin solution contains the following concentrations: vitamin H (2-6 mg / L, e.g., 4 mg / L); vitamin B9 (2-6 mg / L, e.g., 4 mg / L); vitamin B6 (20-60 mg / L, e.g., 40 mg / L); vitamin B1 (10-30 mg / L, e.g., 20 mg / L); vitamin B2 (10-30 mg / L, e.g., 20 mg / L); vitamin B3 (10-30 mg / L, e.g., 20 mg / L); vitamin B5 (10-30 mg / L, e.g., 20 mg / L); and vitamin B12 (0.2-0.6 mg / L, e.g., 0.4 mg / L). In solution T, the concentration of MgSO4·7H2O is 1-5 g / L, for example, 3 g / L; the concentration of MnSO4·H2O is 0.2-0.8 g / L, for example, 0.5 g / L; the concentration of FeSO4·7H2O is 0.05-0.15 g / L, for example, 0.1 g / L; the concentration of CoCl·6H2O is 0.05-0.15 g / L, for example, 0.1 g / L; the concentration of CaCl2 is 0.05-0.15 g / L, for example, 0.1 g / L; the concentration of ZnSO4·7H2O is 0.005-0.02 g / L, for example, 0.1 g / L; the concentration of CuSO4·5H2O is 0.05-0.15 g / L, for example, 0.01 g / L; and the concentration of H3BO3 is 0.005-0.02 g / L, for example, 0.01 g / L. The concentration of NaMoO4·2H2O is 0.005-0.02 g / L, for example 0.01 g / L; the concentration of NiSO4·6H2O is 0.05-0.15 g / L, for example 0.1 g / L.

11. The method as described in claim 9 or 10, characterized in that, In the method, the step of culturing Staphylococcus aureus to obtain fermentation broth includes: inoculating Staphylococcus aureus into a basic culture medium for culture, and adding supplemental glucose solution and supplemental compound nutrient solution during the culture process; Preferably, the method satisfies one or more of the following: 1) The culture temperature is 35-38℃; 2) The pH of the culture is 6.5-8.0; 3) Stirring is performed during the cultivation process at a speed of 100~600 rpm; 4) The incubation time is 8-15 hours; 5) After culturing for 2-3 hours, add glucose solution and compound nutrient solution as supplementary feed; 6) The feeding method for the glucose solution is as follows: feed is added once per hour, each time about 1 / 10 to 1 / 5 of the total volume; 7) The feeding method of the compound nutrient solution is as follows: feed is added once per hour, each time about 1 / 10 to 1 / 5 of the total volume; 8) OD of Staphylococcus aureus cultured by the method for 9 hours 600 The value is not less than 15; and, 9) The method includes culturing Staphylococcus aureus in seed culture medium until the cells grow to the middle of the logarithmic growth phase, obtaining seed liquid, and then using second-generation basal culture medium, adding glucose supplementation solution and supplemental compound nutrient solution during the culture process.

12. A culture medium composition for Staphylococcus aureus, characterized in that, The culture medium combination is the culture medium combination defined in the method as described in claim 9 or 10.

13. A method for culturing Staphylococcus aureus to produce capsular polysaccharides, characterized in that, The method includes the method of claim 11 for culturing Staphylococcus aureus to produce capsular polysaccharides.

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