Extraction and purification method suitable for swine-derived bacterium capsular polysaccharide

Abstract

The invention provides an extraction and purification method suitable for swine-origin bacteria capsular polysaccharide, and belongs to the technical field of vaccine preparation, and the extraction and purification method comprises the following steps: S1, bacterial fermentation; s2, centrifugally collecting bacteria; s3, thallus splitting decomposition and polysaccharide crude extraction; s4, removing nucleic acid: adding a branched polyethyleneimine flocculant, adjusting the pH value to 6-8, carrying out oscillating reaction at 25 DEG C for 0-50 minutes, centrifuging to remove precipitate, and collecting supernate; s5, protein removal: adding phenol with the final concentration of 5%-10% into the supernate, carrying out oscillation reaction at room temperature, centrifuging, and collecting the supernate; and S6, ultrafiltration and concentration: performing equal-volume ultrafiltration on the obtained supernate with pure water for 6-10 times, and washing to remove impurities. And freeze-drying and storing the obtained polysaccharide pure solution.

Description

Technical Field

[0001] The invention relates to a method for extracting and purifying porcine-derived bacterial capsular polysaccharide, and belongs to the technical field of vaccine preparation. Background Art

[0002] Porcine bacteria are common pathogenic microorganisms in swine infections, including multiple genera such as Streptococcus, Salmonella, and Pasteurella, and are mostly Gram-positive or Gram-negative. These porcine bacteria typically have spherical, oval, or rod-shaped structures, are generally devoid of flagella and spores, and may or may not have a capsule. They are aerobic or facultatively anaerobic, and often require the addition of blood or serum to the culture medium. Some strains exhibit hemolytic properties on plates containing 5% sheep blood. For example, porcine Streptococcus infections are most typically manifested by meningitis and sepsis. Affected pigs often exhibit clinical manifestations such as elevated temperature, skin hemorrhages, respiratory distress, neurological symptoms (such as convulsions and circling), and lameness. Without timely treatment, these infections can rapidly lead to death.

[0003] Capsular polysaccharide (CPS) is one of the most studied virulence factors in swine-derived bacterial research. It is widely present on the cell surface of pathogens and is crucial for their survival, pathogenicity, and evasion of the immune system. Capsular polysaccharides typically consist of long chains of 2-5 repeating monosaccharide units that aggregate on the bacterial surface to form a thick, stable protective capsule. Studies have shown that capsular polysaccharides exhibit excellent immunogenicity, making them ideal vaccine antigen candidates. They are highly safe and unlikely to induce adverse immune reactions in animals.

[0004] Capsular polysaccharide extraction and purification are key steps in the development of both capsular polysaccharide vaccines and polysaccharide-protein conjugate vaccines. During the preparation of bacterial capsular polysaccharide vaccines, it is crucial to remove impure proteins and endotoxins as much as possible to minimize vaccine side effects. During the extraction process, various bacterial components are released after cell lysis, complicating subsequent purification. Therefore, pretreatment is generally required before purification to remove cell fragments. Polysaccharide extraction processes primarily include cell lysis, continuous flow centrifugation, clarification filtration, ultrafiltration, and concentration. Currently, there are two main lysis methods: mechanical lysis and lysis with lytic agents. Mechanical lysis is more intense, but it can alter the important structural components and relative molecular weight of CPS, or it can be insufficient, leading to incomplete lysis. Therefore, lysis with lytic agents is currently the primary method. Commonly used lytic agents, such as formaldehyde, can affect vaccine safety, while lysozyme can increase production costs. The purification process for bacterial capsular polysaccharides has undergone years of development, with numerous patents and non-patent literature describing purification methods. These reported purification methods vary, and even within the same purification method, the process sequence can vary. These methods include cold benzene extraction, ethanol graded precipitation, chromatography, and fermentation broth acidification. The key processing steps involved include: removing solid impurities by centrifugation or microfiltration after bacterial lysis; removing impurities such as foreign proteins and nucleic acids by adjusting pH or using proteases, nucleases, etc.; further removing impurities using activated carbon; precipitating capsular polysaccharides with ethanol or CTAB, and then obtaining refined capsular polysaccharides by chromatography or phenol extraction. However, these purification methods currently have one or more of the following technical problems: (1) A large amount of phenol is used in the process of removing impurities such as proteins, which is toxic and corrosive. In addition to endangering the health of workers, it also causes serious harm to the environment. (2) When using ethanol to purify bacterial capsular polysaccharides, a large amount of ethanol is required, resulting in a sharp increase in production costs. At the same time, the factory and facilities need to be specially designed for fire and explosion prevention, and operators need to be subject to higher requirements and more frequent training, supervision, and management. (3) Other purification methods have high purification costs and complex and cumbersome processes.

[0005] Existing capsular polysaccharide purification processes mainly include cold benzene extraction, ethanol graded precipitation, chromatography, acidification, etc. These methods generally involve the following steps: centrifugation or microfiltration to remove bacterial fragments; pH adjustment or enzymatic hydrolysis to remove protein and nucleic acid impurities; activated carbon adsorption to remove other impurities; precipitation and chromatographic purification of capsular polysaccharides. However, the above methods generally have the following problems: (1) phenolic compounds are highly toxic, causing harm to the environment and the health of operators; (2) the use of large amounts of ethanol increases costs and places high safety requirements on equipment and facilities; (3) chromatographic purification equipment and consumables are expensive, and the process steps are complicated.

[0006] Specifically for Streptococci from porcine bacteria, the extraction and purification of Streptococcus suis type 2 capsular polysaccharide has been extensively studied. Existing methods, such as lysozyme lysis combined with strong anion exchange chromatography, yield high-purity capsular polysaccharides, but they are complex and costly. Other methods, such as ultrafiltration, simplify the process but struggle to meet the requirements of large-scale production and have limited purity.

[0007] Therefore, the field of porcine bacterial capsular polysaccharide extraction and purification urgently needs to develop a safer, more environmentally friendly, more efficient and economical method for obtaining capsular polysaccharides to meet the needs of large-scale production and clinical vaccine development. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for extracting and purifying capsular polysaccharides from porcine bacteria.

[0009] A method for extracting and purifying capsular polysaccharides from porcine bacteria, comprising the following steps: S1 bacterial fermentation: culturing and harvesting porcine bacterial strains to obtain fermented bacterial liquid; S2: Centrifugal collection of bacteria: The bacterial liquid after fermentation is collected by centrifugation, washed 2 to 3 times with buffer, and concentrated to 1 / 5 to 1 / 10 of its volume with the buffer.

[0010] S3 bacterial cell lysis and polysaccharide crude extraction: after adding lysozyme to react, centrifuge and collect the supernatant; S4: Remove nucleic acids: Add branched polyethyleneimine flocculant, adjust the pH to 6-8, shake at 25°C for 0-50 minutes, centrifuge to remove the precipitate, and collect the supernatant; S5 protein removal: add phenol to a final concentration of 5% to 10% to the supernatant, shake at room temperature, then centrifuge and collect the supernatant; S6 Ultrafiltration Concentration: The obtained supernatant is ultrafiltered 6 to 10 times with an equal volume of pure water to remove impurities. The obtained pure polysaccharide solution is freeze-dried and stored.

[0011] Furthermore, the pig-derived bacteria is one of Streptococcus suis types 2, 4, 9, Haemophilus parasuis, and Actinobacillus pleuropneumoniae.

[0012] Furthermore, the porcine-derived bacterial strain was deposited in the China Center for Type Culture Collection on December 6, 2024, with a preservation number of CCTCC NO: M20242747 and a classification name of Streptococcus suis type 2 (Streptococcus suisS068).

[0013] Furthermore, the molecular weight of the branched polyethyleneimine flocculant is controlled within the range of 25-70 kDa.

[0014] Furthermore, in the nucleic acid removal step S4, the concentration of the branched polyethyleneimine flocculant is 350 mg / L.

[0015] Furthermore, in the nucleic acid removal step S4, the flocculant is a 350 mg / L branched polyethyleneimine aqueous solution, and the pH is adjusted to 8.0±0.2.

[0016] Furthermore, the buffer in the S2 centrifugation and bacteria collection step is 10 mM PBS.

[0017] Furthermore, in the nucleic acid removal step S4, the oscillation reaction time of the flocculant is controlled within 30 minutes.

[0018] Furthermore, in the S2 centrifugal bacteria collection step, the centrifugal speed is controlled to be 3000-5000 rpm, and the centrifugal time is controlled to be 20-40 min.

[0019] Furthermore, in the S2 centrifugal bacteria collection step, the centrifugal speed is controlled to be 3000-5000 rpm, and the centrifugal time is controlled to be 20-40 min.

[0020] Furthermore, in the S3 bacterial cell lysis and polysaccharide crude extraction step, the concentration of lysozyme is 1 mg / mL, and the reaction is carried out at 37°C and 120-180 rpm for 12-16 hours, and then the enzymatically hydrolyzed bacterial solution is collected by centrifugation at 10,000 rpm for 20-40 minutes.

[0021] The beneficial effects of the present invention are: The present invention optimizes the extraction and purification process of capsular polysaccharides, adopts branched polyethyleneimine (PEI) as a flocculant, preferably a high molecular weight polyethyleneimine flocculant to remove nucleic acids, and combines the phenol oscillation method to efficiently remove proteins. This significantly simplifies the complex operations in the traditional polysaccharide purification process, reduces the process difficulty and operation steps, and facilitates industrial large-scale production implementation.

[0022] Among them, polyethyleneimine (PEI) is a positively charged high molecular polymer that can quickly combine with strongly negatively charged nucleic acids (such as DNA or RNA) through electrostatic interaction to form large particle complexes that precipitate, thereby effectively removing nucleic acid impurities. In contrast, although capsular polysaccharides carry a negative charge, their charge density is relatively low, and their structure exists in the form of linear or branched chains, with low steric hindrance, making it difficult to form a complex with PEI, thereby ensuring that capsular polysaccharides will not be co-precipitated in large quantities by PEI during the purification process, effectively improving the recovery rate of capsular polysaccharides. At the same time, the present invention further reduces the risk of capsular polysaccharides being mistakenly precipitated by optimizing key parameters such as pH, ionic strength, and action time of PEI treatment, so that the recovery rate of capsular polysaccharides reaches more than 95%, greatly improving the production efficiency of polysaccharides.

[0023] Compared with the existing technology, the present invention avoids the safety hazards and environmental pollution problems caused by the large-scale use of ethanol and organic solvents in traditional methods, effectively reduces the use of toxic and hazardous chemicals, and improves the environmental friendliness and production safety of the entire extraction and purification process.

[0024] By optimizing the pH conditions and reaction time during the key nucleic acid removal step, the present invention reduces the residual nucleic acid content to 0.12% and the residual protein content to 0.65% during the capsular polysaccharide purification process, achieving a polysaccharide purity of 81.4%, significantly surpassing the extraction and purification results of existing technologies. Furthermore, this method significantly improves the polysaccharide recovery rate, reaching 95%, while maintaining the integrity of the capsular polysaccharide molecular weight (KD value of 0.51), ensuring the high molecular weight structural stability of the polysaccharide antigen. This facilitates the subsequent preparation of polysaccharide-protein conjugate vaccines and enhances the vaccine's immune protection.

[0025] Furthermore, the method provided by the present invention has strong universal applicability and promotional value. It is not only suitable for the extraction and purification of capsular polysaccharides from Streptococcus suis type 2, but is also applicable to the extraction of capsular polysaccharides from other porcine bacteria (such as Streptococcus suis types 4, 7, and 9, Haemophilus parasuis, and Actinobacillus pleuropneumoniae). The obtained capsular polysaccharides have a purity exceeding 90%, and the impurity protein and nucleic acid contents are controlled below 2%, demonstrating excellent versatility and process stability. Compared with existing polysaccharide extraction processes, the present invention offers significant advantages in terms of ease of operation, short production cycle, high purification efficiency, and low production cost. This significantly improves the economic and practicality of vaccine production and reduces the overall costs for enterprises in terms of production facilities, safety management, and operator training.

[0026] In summary, the capsular polysaccharide extraction and purification method of the present invention is efficient, safe, and environmentally friendly. The obtained capsular polysaccharide product has high purity, high yield, and stable molecular weight, which can provide a solid technical guarantee for the development of capsular polysaccharide vaccines and related diagnostic products for Streptococcus suis type 2 and other various porcine bacterial capsular polysaccharide vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a standard curve of protein concentration.

[0028] Figure 2 This is the standard curve of polysaccharide concentration. DETAILED DESCRIPTION

[0029] In order to make the technical means, creative features, objectives and effects of the present invention easier to understand, the present invention is further explained below with reference to specific illustrations.

[0030] Example 1

[0031] This embodiment provides a method for extracting and purifying capsular polysaccharides from porcine bacteria, and uses fermentation culture of Streptococcus suis type 2 as raw material to prepare capsular polysaccharides. The specific method is as follows: S1 Bacterial Fermentation: A high-capsular polysaccharide-producing strain of Streptococcus suis type 2 was cultured in tryptone soy broth (TSB) supplemented with 5% newborn calf serum. During the logarithmic growth phase, a 500 g / L glucose solution was continuously fed to maintain a sugar concentration of 7 g / L in the fermentation broth. The rotational speed was adjusted to 130 rpm, and the pH was controlled between 7.2 and 7.4. The culture broth was harvested when the OD value reached 1.5 or above.

[0032] S2: Centrifugation to collect bacteria: The fermented bacterial liquid was centrifuged at 4000 rpm for 30 min, washed three times with 10 mM PBS, and concentrated to 1 / 5 of its volume with 10 mM PBS.

[0033] S3 cells were lysed and polysaccharide was crudely extracted. Lysozyme was added to a final concentration of 1 mg / mL and reacted at 37°C and 180 rpm for 12 h. The mixture was then centrifuged at 10,000 rpm for 20 min and the supernatant was collected. S4: Remove nucleic acids: Add a branched polyethyleneimine (PEI) aqueous solution with a final concentration of 350 mg / L to the supernatant, adjust the pH to 8.0 ± 0.2, shake at 25°C for 30 min, centrifuge at 10,000 rpm for 20 min, remove the precipitate, and collect the supernatant. S5 protein removal: add phenol to a final concentration of 5% to the supernatant, shake at room temperature for 4 hours, centrifuge at 10,000 rpm for 30 minutes, and collect the supernatant; S6 Ultrafiltration Concentration: The obtained supernatant was ultrafiltered 8 times with an equal volume of pure water to remove impurities. The obtained pure polysaccharide solution was freeze-dried for storage.

[0034] Detection method The polysaccharide yield and impurity content of the prepared product were tested as follows: 1. Nucleic acid content determination method: The nucleic acid content in polysaccharides is determined by ultraviolet absorption. First, the sample to be tested is diluted so that its absorbance value is between 0.3 and 0.7. Then, according to the Lambert-Beer law: A=kbc, k is the absorption coefficient, b is the absorption layer thickness (unit: cm), and c is the solution concentration (unit: mol / L). When the absorbance value is detected with a 1 cm cuvette, the absorption coefficient of nucleic acid at a wavelength of 260 nm is 200. Therefore, the nucleic acid content is estimated based on the absorbance.

[0035] 2. Protein content determination method The protein content in capsular polysaccharide was determined by Folin-phenol method.

[0036] Preparation of test solution: (1) 0.2 mol / L NaOH solution, (2) 4% Na2CO3 solution, (3) 2% potassium sodium tartrate solution, (4) 1% CuSO4 solution. The NaOH-Na2CO3 solution was prepared by mixing equal volumes of (1) and (2), and the potassium sodium tartrate-copper sulfate solution was prepared by mixing (3) and (4). Solution A was prepared by mixing these two solutions in a ratio of 50:1. Solution B was prepared by diluting the Folin-phenol reagent by half before use.

[0037] Reference solution: Accurately weigh 25.0 mg of standard bovine serum albumin, dry to constant weight, dissolve with a small amount of water, and then add water to a 50 mL volumetric flask to obtain a 0.5 mg / mL standard bovine serum albumin solution.

[0038] Draw a standard curve: Take 0, 50, 100, 150, 200, 250, and 300 μL of 0.5 mg / mL standard bovine serum albumin solution, add water to 500 μL, add 2.5 mL of reagent A, vortex and let it stand at room temperature for 10 minutes, then add 250 μL of reagent B, vortex again and let it stand for 1 hour, and measure its optical density at 750 nm. With the optical density of bovine serum albumin as the vertical axis and the concentration as the horizontal axis, the protein content standard curve equation is obtained (such as Figure 1 shown).

[0039] Quantitative determination: Accurately pipette an appropriate amount of the sample solution to be prepared into 5 mg / mL, operate in parallel with the bovine serum albumin standard, measure its optical density at 750 nm, and use the standard curve equation to obtain the protein content in the capsular polysaccharide.

[0040] 3. Polysaccharide content determination method The phenol-sulfuric acid method was used to determine the polysaccharide content in the sample.

[0041] Preparation of reference solution: Accurately weigh standard dextran and use a volumetric flask to make a 1.0 mg / mL standard solution.

[0042] To draw a standard curve: Take 0, 50, 100, 150, 200, 250, and 300 μL of a 1.0 mg / mL standard dextran solution, dilute to 500 μL, add 200 μL of 6% phenol and 1.0 mL of concentrated sulfuric acid, shake, and react in a boiling water bath for 10 minutes. Cool to room temperature and measure the optical density at 490 nm. Using the optical density of dextran as the ordinate and the concentration as the abscissa, the equation for the standard curve for sugar content is obtained. Figure 2 shown.

[0043] Quantitative determination: Accurately pipette an appropriate amount of sample solution to be prepared into 5 mg / mL, operate in parallel with the standard, measure its optical density at 490 nm, and use the standard curve equation to obtain the sugar content of capsular polysaccharide.

[0044] The freeze-dried capsular polysaccharide was tested for nucleic acid, protein and polysaccharide contents using the methods described in 1 to 3 above to investigate its purity and yield.

[0045] Polysaccharide assay of Example 1: The freeze-dried capsular polysaccharide was tested for nucleic acid, protein and polysaccharide content to investigate its purity and yield. The results are shown in Table 1.

[0046] Table 1 Polysaccharide assay results of Example 1 Example Nucleic acid content Protein content Polysaccharide content Polysaccharide yield Example 1 0.12% 0.65% 81.4% 353mg The difference between Example 2-3 and Example 1 is that the pH control after adding the flocculant is different.

[0047] By comparing Examples 2-3 with Example 1, it was verified that during the polysaccharide purification process, the flocculant PEI had an effect on the removal rate of nucleic acids and the purity of polysaccharides under different pH conditions.

[0048] Streptococcus suis type 2 was cultured, harvested by centrifugation, resuspended in 10 mM PBS, and lysed with lysozyme. A branched polyethyleneimine (PEI) aqueous solution was added to the harvested supernatant at a final concentration of 350 mg / L. The pH was adjusted to 6.0 ± 0.2 and 7.0 ± 0.2, respectively, as in Examples 2 and 3. The reaction was shaken at 25°C for 30 minutes, followed by centrifugation at 10,000 rpm for 20 minutes. The precipitate was removed and the supernatant collected. The protein precipitate was then removed with phenol. Finally, the supernatant was washed with pure water and the solution was replaced (see Example 1 for details) to obtain a pure polysaccharide solution. This solution was freeze-dried and stored, and the nucleic acid and protein impurity content and polysaccharide purity were measured. The results are shown in Table 2. At pH 8.0 ± 0.2, PEI significantly improved nucleic acid removal compared to the other two pH environments, with a polysaccharide purity of 84.7%. At pH 6.0 ± 0.2 and pH 7.0 ± 0.2, the polysaccharide purity was below 70%. Therefore, the optimal pH value for removing nucleic acids using flocculant PEI is pH 8.0±0.2.

[0049] Table 2 Effect of different pH on PEI nucleic acid removal efficiency Example pH Polysaccharide yield Nucleic acid concentration Protein concentration Polysaccharide concentration Polysaccharide purity Example 2 6.0±0.2 1085mg 113mg / g 9.8mg / g 582mg / g 58.2% Example 3 7.0±0.2 1039mg 78.3mg / g 7.3mg / g 639mg / g 63.9% Example 1 8.0±0.2 1028mg 1.4mg / g 5.8mg / g 847mg / g 84.7% The difference between Example 4-5 and Example 1 is that different buffers are used instead of PBS in the S2 centrifugation bacteria collection step.

[0050] By comparing Examples 4-5 with Example 1, it can be verified that during the polysaccharide purification process, the effect of flocculant PEI on the removal rate of nucleic acids and the purity of polysaccharides in different buffers.

[0051] Streptococcus suis type 2 was cultured and harvested by centrifugation. The cells were then resuspended in 150 mM NaCl and 0.1 M glycine, respectively, and lysed with lysozyme. A branched polyethyleneimine (PEI) aqueous solution was added to the harvested supernatant at a final concentration of 350 mg / L, and the pH was adjusted to 8.0 ± 0.2. The reaction was shaken at 25°C for 30 minutes, followed by centrifugation at 10,000 rpm for 20 minutes. The precipitate was removed and the supernatant collected. The protein precipitate was then removed with phenol, and the supernatant was washed with pure water and replaced with the buffer (see Example 1 for details) to obtain a pure polysaccharide solution. This solution was lyophilized and stored, and the nucleic acid and protein impurity content and polysaccharide purity were measured. The results are shown in Table 3. In the 10 mM PBS buffer system, PEI achieved slightly better nucleic acid removal than the other two buffer systems, but significantly improved polysaccharide purity and content compared to the 150 mM NaCl and 0.1 M glycine buffer system. Therefore, the best buffer system when using flocculant PEI to remove nucleic acids is 10mM PBS.

[0052] Table 3 Effect of different buffers on PEI nucleic acid removal efficiency Example buffer Polysaccharide yield Nucleic acid concentration Protein concentration Polysaccharide concentration Polysaccharide purity Example 1 10mM PBS 1159mg 1.8mg / g 5.2mg / g 805mg / g 80.5% Example 4 150mM NaCl 914mg 36.4mg / g 7.3mg / g 629mg / g 62.9% Example 5 0.1 M glycine 948mg 57.4mg / g 6.5mg / g 597mg / g 59.7% The difference between Example 6-7 and Example 1 is that the action time of the flocculant is different.

[0053] By comparing Examples 6-7 with Example 1, it can be verified that during the polysaccharide purification process, the effects of different action times of flocculant PEI on the removal rate of nucleic acids and the purity of polysaccharides.

[0054] Streptococcus suis type 2 was cultured, harvested by centrifugation, and divided into three groups. The cells were resuspended in 10 mM PBS and lysed with lysozyme. A branched polyethyleneimine (PEI) aqueous solution at a final concentration of 350 mg / L was added to the harvested supernatant, and the pH was adjusted to 8.0 ± 0.2. The reaction was shaken at 25°C for 15 and 45 minutes, respectively, as in Examples 6 and 7. The reaction was then centrifuged at 10,000 rpm for 20 minutes, the precipitate removed, and the supernatant collected. The protein precipitate was then removed with phenol, and the supernatant was washed with pure water and replaced with the solution (see Example 1 for details) to obtain a pure polysaccharide solution. This solution was freeze-dried and stored, and the nucleic acid and protein impurity content and polysaccharide purity were measured. The results are shown in Table 4. After 15 minutes of PEI treatment, the final polysaccharide sample had high concentrations of residual nucleic acid and protein impurities, and the polysaccharide purity was less than 40%. However, after 30 and 45 minutes of treatment, the polysaccharide concentrations, as well as the concentrations of nucleic acid and protein impurities, were not significantly different. Therefore, the optimal treatment time for nucleic acid removal using the flocculant PEI is 30 minutes.

[0055] Table 4 Effect of different reaction times on PEI nucleic acid removal rate Example Action time Polysaccharide yield Nucleic acid concentration Protein concentration Polysaccharide concentration Polysaccharide purity Example 6 15min 1329mg 258mg / g 83.1mg / g 368mg / g 36.8% Example 1 30min 1057mg 1.6mg / g 4.7mg / g 829mg / g 82.9% Example 7 45min 1012mg 1.4mg / g 4.2mg / g 837mg / g 83.7% Example 8: Extraction of other porcine bacterial capsular polysaccharides Capsular polysaccharides from other porcine bacteria that share structural and functional similarities with the S. suis type 2 capsular polysaccharide, such as those synthesized by gene clusters regulated by glycosyltransferases and polymerases and that play a key role in bacterial immune evasion and pathogenicity, can also be extracted using the extraction and purification methods described in Example 1. Using the methods of Examples 1 to 7, we optimized the extraction and purification conditions for polysaccharides from other serotypes of S. suis (exemplified by types 4, 7, and 9), Haemophilus parasuis, and Actinobacillus pleuropneumoniae. The purities of these extracted capsular polysaccharides were all above 90%, with protein and nucleic acid impurity levels below 2%. This example demonstrates the feasibility of extending the S. suis type 2 capsular polysaccharide purification method to other porcine bacteria. This method, characterized by high efficiency and purity, is suitable for the extraction and purification of capsular polysaccharides from a variety of porcine bacteria, providing technical support for the development of related vaccines and diagnostic reagents.

[0056] Comparative Example 1 This comparative example uses fermentation culture of Streptococcus suis type 2 as raw material (the raw material is the same as that of Example 1, and the fermentation broth of Streptococcus suis type 2 cultured to the late logarithmic growth stage of fermentation is divided into three parts and used in Example 1, Comparative Example 1 and Comparative Example 2 respectively).

[0057] Referring to the method of Hu Qun et al., lysozyme was used to lyse the bacteria, calcium salt step-by-step precipitation was used to remove nucleic acid impurities, proteinase K enzymatic hydrolysis and Sevage method were used to remove protein impurities, and ethanol graded precipitation was used to precipitate pure polysaccharides (Preparation of Streptococcus suis capsular polysaccharide antiserum and its application in serotyping, "Jiangsu Agricultural Science", Vol. 51, No. 13, 2023, 189-196). The fermented Streptococcus suis type 2 bacterial liquid was extracted and purified to obtain capsular polysaccharide.

[0058] Bacterial fermentation: A high-capsular polysaccharide-producing strain of Streptococcus suis type 2 was cultured in tryptone soy broth (TSB) supplemented with 5% newborn calf serum. During the logarithmic growth phase, a 500 g / L glucose solution was continuously fed to maintain the sugar concentration in the fermentation broth between 5 g / L and 10 g / L. The rotational speed was adjusted to 120 rpm, and the pH was controlled between 7.2 and 7.4. The culture broth was harvested when the OD value reached 2.0.

[0059] 2. Harvest the bacteria by centrifugation: Centrifuge the fermented bacteria at 4000 rpm for 30 min, wash three times with 10 mM PBS, and resuspend in an equal volume of 10 mM PBS.

[0060] 3. Bacterial cell lysis and polysaccharide crude extraction: Add lysozyme to a final concentration of 1 mg / mL, shake at 37°C, 180 rpm for 12 hours, then centrifuge at 10,000 rpm for 20 minutes and collect the supernatant; 4. Remove the protein supernatant and add proteinase K at a final concentration of 100 μg / mL. Incubate at 55°C overnight, inactivate the enzyme at 80°C for 30 minutes, and cool to room temperature. 5. Remove nucleic acids: Add calcium chloride solution to a final concentration of 0.1 mol / L to the supernatant and stir for 1 hour. Add anhydrous ethanol to a final concentration of 25% by volume and mix thoroughly. Incubate at 4°C for 12 hours, then centrifuge at 10,000 rpm for 30 minutes to remove the nucleic acid precipitate and collect the supernatant. 6. Precipitation of Crude Capsular Polysaccharide: Add the above supernatant to anhydrous ethanol with a final concentration of 80%, mix thoroughly, let stand at 4°C for 12 hours, and then centrifuge at 10,000 rpm for 30 minutes. The resulting precipitate is the crude capsular polysaccharide.

[0061] 7. Polysaccharide Purification: Dissolve the crude capsular polysaccharide in distilled water and thoroughly mix with Sevage solution (n-butanol:chloroform = 1:4): polysaccharide = 1:4. Centrifuge at 10,000 rpm for 30 minutes at 4°C to separate the supernatant. Repeat 3-4 times until no protein is visible in the middle. Thoroughly mix the purified capsular polysaccharide with anhydrous ethanol at a final concentration of 80%, let it stand at 4°C for 12 hours, and then centrifuge at 10,000 rpm for 30 minutes. Collect the resulting polysaccharide precipitate and freeze-dry it for storage.

[0062] 8. Polysaccharide Assay The nucleic acid, protein and polysaccharide contents of freeze-dried capsular polysaccharide were tested to investigate its purity and yield. The results are shown in Table 5.

[0063] Comparative Example 2 This comparative example uses fermentation culture of Streptococcus suis type 2 as raw material (the raw material is the same as that of Example 1, and the fermentation broth of Streptococcus suis type 2 cultured to the late logarithmic growth stage of fermentation is divided into three parts and used in Example 1, Comparative Example 1 and Comparative Example 2 respectively).

[0064] Referring to Wu Chaoliang's method, lysozyme was used to lyse the bacteria, calcium salt step-by-step precipitation was used to remove nucleic acid impurities, sodium deoxycholate method was used to remove protein impurities, and gel permeation column chromatography was used to purify polysaccharides (Wu Chaoliang. Extraction, purification, identification and antigenicity detection of Streptococcus suis type 2 capsular polysaccharide [D]. Hunan Agricultural University, 2020). The fermented Streptococcus suis type 2 bacterial liquid was extracted and purified to obtain capsular polysaccharide.

[0065] Bacterial fermentation: A high-capsular polysaccharide-producing strain of Streptococcus suis type 2 was cultured in tryptone soy broth (TSB) supplemented with 5% newborn calf serum. During the logarithmic growth phase, a 500 g / L glucose solution was continuously fed to maintain the sugar concentration in the fermentation broth between 5 g / L and 10 g / L. The rotational speed was adjusted to 120 rpm, and the pH was controlled between 7.2 and 7.4. The culture broth was harvested when the OD value reached 2.0.

[0066] 2. Harvest the bacteria by centrifugation: Centrifuge the fermented bacteria at 4000 rpm for 30 min, wash three times with 10 mM PBS, and resuspend in an equal volume of 10 mM PBS.

[0067] 3. Bacterial cell lysis and polysaccharide crude extraction: Add lysozyme to a final concentration of 1 mg / mL, shake at 37°C, 180 rpm for 12 hours, then centrifuge at 10,000 rpm for 20 minutes and collect the supernatant; 4. Remove nucleic acids: Add calcium chloride solution to a final concentration of 0.1 mol / L to the supernatant and stir for 1 hour. Add anhydrous ethanol to a final concentration of 25% by volume and mix thoroughly. Incubate at 4°C for 12 hours, then centrifuge at 10,000 rpm for 30 minutes to remove the nucleic acid precipitate and collect the supernatant. 5. Precipitation of Crude Capsular Polysaccharide: Add the above supernatant to anhydrous ethanol with a final concentration of 80%, mix thoroughly, let stand at 4°C for 12 hours, and then centrifuge at 10,000 rpm for 30 minutes. The resulting precipitate is the crude capsular polysaccharide.

[0068] 6. Protein removal: Dissolve 1.5 g of crude polysaccharide in 150 mL of pyrogen-free water. Slowly add hydrochloric acid dropwise to adjust the pH to below 5.0. Then add sodium deoxycholate to a final concentration of 0.5%. Centrifuge at 3000 g for 5 min, remove the supernatant, and remove the protein precipitate.

[0069] 7. Further purification of polysaccharide: The supernatant after removing impurities was centrifuged at 10,000 g for 5 min to further remove impurities. The polysaccharide was then purified using a Sephacryl S-300 gel permeation chromatography column at a flow rate of 0.3 mL / min.

[0070] 8. Replace the solution and store it. Ultrafilter the supernatant eight times with an equal volume of pure water through a 100K ultrafiltration membrane pack to replace the solution. The resulting pure polysaccharide solution is freeze-dried and stored.

[0071] 9. Polysaccharide Assay The nucleic acid, protein and polysaccharide contents of freeze-dried capsular polysaccharide were tested to investigate its purity and yield. The results are shown in Table 1.

[0072] The results of three batches of polysaccharides purified by three different methods are shown in Table 5.

[0073] Table 5 Test results of polysaccharides purified by three methods batch Nucleic acid content Protein content Polysaccharide content Polysaccharide yield Example 1 0.12% 0.65% 81.4% 353mg Comparative Example 1 0.26% 0.48% 89.% 92mg Comparative Example 2 0.75% 1.23% 70.1% 105mg Polysaccharide molecular size distribution determination While some traditional purification methods can achieve high polysaccharide yields, the quality of the polysaccharide may not meet standards, and the molecular weight of the polysaccharide may be uneven. As a thymus-independent antigen, the molecular weight of the polysaccharide is closely related to its immunogenicity. The molecular weight of Streptococcus suis type 2 polysaccharide was determined according to General Chapter 3419 of Part III of the Chinese Pharmacopoeia.

[0074] 4.1 Reagents (1) Mobile phase: Weigh 11.7 g of sodium chloride and 0.1 g of sodium azide, dissolve in water to make 1000 mL, mix well, and adjust the pH to 7.0 with 0.1 mol / l sodium hydroxide.

[0075] (2) Blue Dextran 2000 solution: Weigh 20 mg of Dextran 2000 and dissolve it in mobile phase to make 10 mL.

[0076] (3) Vitamin B 12 Solution: Weigh 10 mg of vitamin B 12 , add mobile phase to dissolve into 10mL.

[0077] 4.2 Preparation of chromatographic columns Weigh 200 mL of agarose CL-4B gel, add 400 mL of mobile phase and stir thoroughly. Let it stand for 1 hour to allow it to settle. Pour off the supernatant suspension. Repeat this process 3 to 5 times. Add 200 mL of mobile phase and mix thoroughly. Remove the air from the gel, load the column, and elute and equilibrate with mobile phase.

[0078] 4.3 Column calibration Take 1 mL of blue dextran 2000 solution and vitamin B 12 The solution is 0.2 mL, the flow rate is 15-20 mL per hour, the detection wavelength is 206 nm, and the eluent is collected with a component collector and the chromatogram is recorded. The first peak is the blue dextran 2000 peak, and the volume of the eluent at the peak is the void volume V0; the second peak is vitamin B 12 Peak, the volume of the eluent at the peak is the column bed volume V i .

[0079] 4.4 Determination Take 1 mL of the test sample (polysaccharide concentration is 3-5 mg / mL) and add it to the calibrated chromatographic column. Add mobile phase for elution at a flow rate of 15-20 mL per hour and a detection wavelength of 206 nm. Collect the eluate with a fraction collector and record the chromatogram. The volume of the highest peak of the polysaccharide on the chromatographic column is V. e According to the formula K D =(V e - V0 ) / (V i - V0) Calculate KD value 4.5 K D Polysaccharide recovery rate K <0.7 D The chromatogram area <0.7 is A x , the total area of ​​the chromatogram is A t , according to the formula R x (%) = (A x / A t ) × 100, and calculate the polysaccharide recovery rate.

[0080] Table 6 Molecular weight determination results of polysaccharides purified by three methods Example <![CDATA[K D Value]]> Recovery rate Example 1 0.51 95% Comparative Example 1 0.48 94% Comparative Example 2 0.50 95% The results show that after treatment according to the steps of Example 1, the total amount of polysaccharide reaches about 3 times the total amount of polysaccharide after treatment in Comparative Example 1 and Comparative Example 2. Compared with the two comparative examples, the step of precipitating crude polysaccharides is reduced during the purification process, which greatly reduces the loss of capsular polysaccharides. At the same time, the impurity protein content in the refined polysaccharides obtained after treatment in Example 1 and Comparative Examples 1 and 2 is all below 2%, and the content of impurity nucleic acid is also below 2%. Among them, the nucleic acid content in Example 1 is the lowest among the three methods, and the protein content is slightly higher than that in Comparative Example 1. Its quality is significantly better than that of the refined polysaccharide obtained by treatment in Comparative Example 2. At the same time, Example 1 has simple steps, strong operability, few reagents used, and low cost. Therefore, high-quality purified polysaccharides can be obtained by treatment in Example 1, and a relatively high polysaccharide recovery rate can be obtained, while also having the advantages of simple steps and low cost.

[0081] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art will appreciate that the present invention is not limited to the foregoing embodiments and that various modifications and improvements may be made to the present invention without departing from the spirit and scope of the present invention. Such modifications and improvements are intended to fall within the scope of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for extracting and purifying porcine bacterial capsular polysaccharides, characterized in that: The following steps are involved: S1 bacterial fermentation: culturing and harvesting porcine bacterial strains to obtain fermented bacterial liquid; S2: centrifugation to collect the bacterial liquid after fermentation, washing it with a buffer solution, and concentrating it with the buffer solution; S3 bacterial cell lysis and polysaccharide crude extraction: After adding lysozyme to react, centrifuge at 10,000 rpm for 20-40 minutes to collect the supernatant; S4: Remove nucleic acids: Add branched polyethyleneimine flocculant, adjust the pH to 6-8, shake the reaction at 25°C, centrifuge at 10,000 rpm for 20-40 minutes to remove the precipitate, and collect the supernatant; S5 protein removal: add phenol to a final concentration of 5% to 10% to the supernatant, shake at room temperature, then centrifuge and collect the supernatant; S6 Ultrafiltration Concentration: The obtained supernatant is washed with an equal volume of pure water by ultrafiltration to remove impurities, and the obtained pure polysaccharide solution is freeze-dried for storage.

2. The method for extracting and purifying porcine bacterial capsular polysaccharides according to claim 1, characterized in that: The pig-derived bacteria is one of Streptococcus suis type 2, Streptococcus suis type 4, Streptococcus suis type 9, Haemophilus parasuis, and Actinobacillus pleuropneumoniae.

3. The method for extracting and purifying porcine bacterial capsular polysaccharides according to claim 2, characterized in that: The pig-derived bacterial strain was deposited in the China Center for Type Culture Collection on December 6, 2024, with a preservation number of CCTCC NO: M20242747 and a classification name of Streptococcus suis type 2.

4. The method for extracting and purifying porcine bacterial capsular polysaccharides according to claim 1, characterized in that: The molecular weight of the branched polyethyleneimine flocculant is controlled at 25-70 kDa.

5. The method for extracting and purifying porcine bacterial capsular polysaccharides according to claim 4, characterized in that: In the nucleic acid removal step S4, the concentration of the branched polyethyleneimine flocculant is 350 mg / L.

6. The method for extracting and purifying porcine bacterial capsular polysaccharides according to claim 1, characterized in that: In the S4 nucleic acid removal step, the pH is adjusted to 8.0±0.

2.

7. The method for extracting and purifying porcine bacterial capsular polysaccharides according to claim 1, characterized in that: In the S2 centrifugation and bacteria collection step, the buffer solution is 10 mM PBS solution.

8. The method for extracting and purifying porcine bacterial capsular polysaccharides according to claim 1, characterized in that: In the nucleic acid removal step S4, the oscillation reaction time of the flocculant is controlled at 30 minutes.

9. The method for extracting and purifying porcine bacterial capsular polysaccharides according to claim 1, characterized in that: In the S2 centrifugal bacteria collection step, the centrifugal speed is controlled to be 3000-5000 rpm, and the centrifugal time is controlled to be 20-40 min.

10. The method for extracting and purifying porcine bacterial capsular polysaccharides according to claim 1, characterized in that: In the S3 bacterial cell lysis and polysaccharide crude extraction step, the concentration of lysozyme is 1 mg / mL, and the reaction is carried out at 37° C. and 120-180 rpm for 12-16 hours, and then the enzymatically hydrolyzed bacterial solution is collected by centrifugation at 10,000 rpm for 20-40 minutes.

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