KR101045881B1 - Capsular Polysaccharide Production Medium, Production Method and Purification Method from Streptococcus - Google Patents

Abstract

The present invention is a medium for producing capsular polysaccharide from Streptococcus sp., Particularly Streptococcus pneumoniae , which produces capsular polysaccharide, a method for producing the capsular polysaccharide from pneumococci, and a method for purifying the same. In the present invention, by increasing the productivity of the capsular polysaccharide, and by simplifying the separation and purification of the produced capsular polysaccharide, it is possible to economically produce the capsular polysaccharide in high yield. Capsular polysaccharide produced according to the present invention can be used directly or as a carrier (carrier) as a raw material for vaccine production.

Capsular polysaccharide, capsular polysaccharide, pneumococcal, synthetic media, glucose, yeast extract, sodium chloride, potassium phosphate buffer solution, economics, productivity, vaccine

Description

Culture medium for production of capsular polysaccharide from Streptococcus sp., Process for production according, and process for purification

The present invention relates to the production of capsular polysaccharides from Streptococcus sp. More specifically, a medium for producing capsular polysaccharides from streptococci, in particular pneumococci, which produce the capsular polysaccharide, pneumonia The present invention relates to a method for producing capsular polysaccharide from cocci and a method for purifying the same.

Streptococcus pneumoniae is a Gram-positive bacterium that causes pneumonia, meningitis, rheumatic heart disease, otitis media, and sepsis. More than 90% of all pneumonia is caused by these bacteria. In particular, pneumococcal pneumonia is known as a major cause of meningitis in children and adults over 15 years of age.

Among the pneumococcal components, substances that act as antigens are capsular polysaccharides, C-substances, F-antigens, and M proteins. Capsular polysaccharide, the main antigen, is a complex polysaccharide that forms a hydrophilic gel and is composed of a complex of various monosaccharides, disaccharides, and compounds. The pathogenic bacterium is S type that produces capsular polysaccharide. Although pneumococci exist in 94 serotypes depending on the antigenicity of the capsular polysaccharide, the number of serotypes is expected to increase further due to the high transformation efficiency. The serotypes of pneumococcal capillaries were identified from 5,800 pneumococcal strains isolated from US patients from 1967 to 1984. , 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F), accounting for more than 90% of patients with pneumonia. Therefore, toxoids are currently used to obtain specific immunity by separating and purifying capsular polysaccharides from these 23 types of pneumococci and directly using them as vaccines, or by promoting the production of antibodies with detoxified toxins by chemical or physical treatment. It is used in combination with the vaccine. Currently, the main vaccine against otitis media, pneumonia, and meningitis used in Korea is a mixture of seven serotypes (4, 6B, 9V, 14, 18C, 19F, 23F). However, in most of these cases, the efficient induction of the immune response as a vaccine is difficult or weak, so many improvements are necessary.

To date, culture media for pneumococcus include defined media (Adams and Roe, 1945), synthetic medium (Sicard, 1964), casein hydrolysate (CAT) medium (Porter and Guild, 1976; Kim S.-N. et al., 1996), Brain Heart Infusion (BHI) medium (Min K.-K. et al., 1999), tryptic soy broth medium, and the like. This is known. However, little is known about which medium is suitable for the production of capsular polysaccharides, and the amount of capsular polysaccharides obtained from the culture medium (1 L) is very small, such as 100 mg, although it varies slightly depending on the serotype of the strain.

Relatively expensive BHI media (Difco, USA) has been used for capsular polysaccharide production, and there are very few studies on the media components that completely replace these media components. Therefore, in the production of capsular polysaccharides used to prepare pneumococcal vaccines, it is very important to develop alternative culture media having high cell growth and capsular polysaccharide production efficiency, easy supply of raw materials, and low cost. In particular, like most other vaccines, carbohydrate vaccines do not currently develop and produce vaccines in Korea, and depend on imports. In addition, the development of a vaccine for Korean pneumococcal requires the development of efficient production and purification methods of capsular polysaccharide, which occurs mainly in Korea.

Korean Patent Publication No. 1995-3439 (published on Feb. 16, 1995) discloses 0.5 to 1.5 w / v% of casein hydrolyzate, 0.1 to 1.0 w / v% of tryptone, 0.1 to 1.5 w / v% of sodium chloride, and yeast Aeration culture for pneumococcal culture and pneumococcal pneumococci, characterized in that the extract contains 0.1 to 0.5w / v%, potassium phosphate (K ₂ HPO ₄ ) 0.017 N and a carbon source 0.8 to 2.0w / v% Characterized in that, it has been disclosed a method for producing capsular polysaccharide of pneumococcal bacteria. This patent uses a modified CAT medium containing casein hydrolysate as a main component and having an increased carbohydrate content (0.8-2.0 w / v%) compared to conventional CAT medium (carbohydrate: glucose 0.2w / v%). Phenol was decomposed by adding 1w / v% of phenol to the culture medium, and 5w / v% of sodium acetate was added thereto to separate the capsular polysaccharides. The yield was not increased by using BHI medium or carbon source. It is reported to increase compared to the yield in the medium. However, this patent uses types 1, 4 and 23 of pneumococcus, and there is still room for improvement for more efficient capsular polysaccharide production and purification.

Of the 23 serotypes of pneumococci, the present inventors established optimal culture conditions using type 17 pneumococcal strains and capsular polysaccharides using 5 types (3, 15, 17, 22, and 9V) of pneumonia. Ongoing studies were conducted to simplify the method for the separation and purification. As a result, by developing a synthetic medium and fermentation conditions of a new composition that can replace the relatively expensive BHI medium to establish the optimum culture conditions of capsular polysaccharide production from pneumococcal, improve the productivity of the capsular polysaccharide, produced A method for separating and purifying polysaccharides more easily and efficiently has been devised, and the present invention has been completed.

An object of the present invention is to provide a medium for producing capsular polysaccharide from streptococci, in particular pneumococci, which produce capsular polysaccharide.

Another object of the present invention is to provide a method for producing capsular polysaccharide from streptococci, in particular pneumococci, which produce capsular polysaccharide.

Another object of the present invention is to provide a method for separating and purifying capsular polysaccharide from streptococci, in particular, pneumococcal medium, which produces capsular polysaccharide.

The first aspect of the present invention comprises at least one carbon source 5-30 g / l, yeast extract 5-30 g / l and 2-10 g / l sodium chloride selected from the group consisting of glucose, fructose, galactose and lactose It relates to a medium for producing capsular polysaccharide from streptococci producing capsular polysaccharide, having a pH adjusted to 5-9.

The second aspect of the present invention relates to a method for producing capsular polysaccharide from streptococci producing capsular polysaccharide comprising culturing the streptococci producing capsular polysaccharide in the medium.

The third aspect of the present invention

⑴ Sodium deoxycholate is added to the culture medium of streptococci producing capsular polysaccharide to lyse the cells,

(C) chloroform-butanol solution is added to the cell lysate to obtain a supernatant from which protein is removed;

에탄올 ethanol is added to the deproteinized supernatant to precipitate the capsular polysaccharide:

It relates to a method for separating and purifying capsular polysaccharide from streptococci producing capsular polysaccharide, comprising the step.

According to the present invention, by optimizing the media components and culture conditions for producing capsular polysaccharides from streptococci, particularly pneumococci, which produce capsular polysaccharides, the productivity of the capsular polysaccharides is increased, and the separation and purification of the produced capsular polysaccharides is simplified for a time. By shortening it, the capsular polysaccharide can be produced economically with high yield. Capsular polysaccharide produced according to the present invention can be used directly or as a carrier (carrier) as a raw material for vaccine production.

In the present invention, an economical culture medium capable of replacing expensive BHI medium in the production of capsular polysaccharides from streptococci, particularly pneumococci, which produces capsular polysaccharides has been sought. To this end, the effects of pH, type and concentration of carbon source, type and concentration of nitrogen source, concentration of potassium phosphate buffer, and metal ions on the capsular polysaccharide productivity from pneumococci were investigated. As a result, at least one carbon source selected from the group consisting of glucose, fructose, galactose and lactose, 5-30 g / l, yeast extract 5-30 g / l and sodium chloride 2-10 g / l, adjusted to 5-9 It was confirmed that a medium having a high pH can significantly improve the productivity of capsular polysaccharides from pneumococci. As a carbon source, glucose is the most preferable in consideration of economical efficiency, and the pH is most preferably adjusted to around 7. The most preferred concentration of sodium chloride is 5 g / l. In addition, it is preferable to adjust the pH by adding 25 ~ 200 mM potassium phosphate buffer solution, in particular it is preferred to add 50 mM potassium phosphate buffer solution. It is also preferred to add at least one of 0.5 to 2 mM, in particular 1 mM, of metal ions, for example magnesium, lithium, iron, cobalt, copper, calcium, manganese, in particular magnesium, lithium and iron ions. In the present invention, type 17 pneumonia strain was used, but the scope of the present invention is not particularly limited to this strain.

The medium according to the present invention can be used without limitation for conventional capsular polysaccharide-producing microorganisms belonging to the Streptococci. The microorganism can be cultured using conventional batch, fed-batch, continuous culture method, depending on the biochemical characteristics of the microorganism to be cultured, the aerobic or anaerobic culture at a temperature of 30 ~ 50 ℃, pH 5 ~ 9 Can be. For example, capsular polysaccharides can be produced from pneumococci by shaking culture of pneumococci in the medium of the present invention. Pneumococci can be shaken at 100-250 rpm, especially 180 rpm for 12-24 hours, especially 12 hours, at a temperature of 30-37 ° C., especially 37 ° C. When the carbon source is converted to capsular polysaccharide, purified capsular polysaccharide can be prepared from the culture medium through a method capable of purely purifying the capsular polysaccharide.

In the present invention, to separate and purify the capsular polysaccharide produced from the culture medium, the following method can be used:

⑴ cell lysis process

Cells are lysed by adding 0.1% to 1%, especially 0.5%, of sodium deoxycholate to the cultured pneumococcal medium. Bacterial lysate is boiled after adding acetic acid to prevent gelation. After cooling and neutralizing the boiled sample, the precipitate is removed by centrifugation, and cold ethanol is added to the supernatant and centrifuged to obtain a precipitate.

⑵ Protein removal process

After dissolving the precipitate in distilled water, the chloroform-butanol solution is added in an amount of 0.1 to 0.5, in particular 1/5, extracted, and centrifuged to obtain a supernatant from which proteins are removed.

⑶ capsular polysaccharide precipitation process

To the supernatant from which the protein was removed, cold ethanol was added in a volume of 1 to 5 times, especially 2 times, and centrifuged to remove the supernatant to obtain a precipitate.

Purification method according to the present invention can be used to lyse cells more efficiently by using sodium deoxycholate, increased the protein removal efficiency by using a chloroform-butanol solution, and thus was previously performed two to three times Ethanol precipitation can be reduced to one time. According to the present invention, the purification time can be shortened from about one week to two days.

In the purification method of the present invention, pneumonia strains of type 3, 15, 17, 22, and 9V were used, but they are not particularly limited.

In the present invention, whether the purified content of each component is suitable for the vaccine reference content for each type is confirmed by measuring the total equivalent weight. For example, the purified capsular polysaccharide was lyophilized and then dissolved in distilled water, and quantitatively analyzed by phenol-sulfuric acid.

Furthermore, in the case of the type 17 strain, gel permeation chromatography (GPC) is further performed after ethanol precipitation, thereby obtaining a higher purity capsular polysaccharide. For example, gel permeation chromatography can be performed on a Sephadex ™ column (SigmaAldrich), and the Sephadex column can be one having a particle size in the range of 20 to 300 μm, in particular 20 to 120 μm, typically It may be a Sephadex G-100 column.

Hereinafter, the present invention will be described in more detail with reference to specific examples, but the scope of the present invention is not limited in any way by these examples.

Example 1 Culture of Pneumococcal Strains

⑴ strain

In this example, five strains of pneumococcal type 3 (KCCM41569, ATCC6303), type 15 (ATCC6315), type 17 (ATCC6317), type 22 (ATCC6322) and type 9V (ATCC700631) strains were used.

⑵ culture

Pneumococci were cultured in tryptic soy agar medium supplemented with 5% Defibrinated Sheep Blood. Colonies obtained here were inoculated into BHI liquid medium (Difco, USA) and incubated at 37 ° C. until absorbance reached the highest at 600 nm. At this time, type 3, type 15 and type 17 were cultured in an aerobic state, and type 22 and type 9V were cultured in an anaerobic state containing 5% carbon dioxide.

Example 2: Effect of Initial pH on the Production of Capsular Polysaccharides of Pneumococcal

Species Culture: A single colony of stored pneumococcal type 17 was inoculated into a 20 mm diameter tube containing 5 ml of growth medium and incubated for 12 hours at 180 rpm and 37 ° C. in a shaker.

Main culture: The seeded strain was inoculated into a 200 ml flask containing 50 ml of the production medium and incubated at 180 rpm and 37 ° C. for 12 hours. As a culture medium, a synthetic medium consisting of BHI medium (Difco, USA) and 5 g of glucose per liter, 5 g of yeast extract, 5 g of sodium chloride, and 0.1725 M potassium phosphate was used. Because capsular polysaccharide production is closely related to cell growth, the growth curves of pneumococci were compared in BHI medium and synthetic media without pH control. Cell concentration was measured by diluting the sample taken from the culture solution every 20 hours by 20 times and measuring the suspension at 600 nm using a spectrophotometer (UV-160A, Shimadzu, Co., Japan). In conversion. Absorbance 1.0 unit corresponds to 0.90 g / l of dry cell weight.

The results are shown in FIG. As shown in FIG. 1, the growth of pneumococci in synthetic media without adjusting the pH was 0.65 g / l at 20 hours of cultivation, which was significantly lower than that of 2.61 g / l of BHI medium.

Example 3: Capsular Polysaccharide Production by pH Control

Species Culture: A single colony of stored pneumococcal type 17 was inoculated into a 20 mm diameter tube containing 5 ml of growth medium and incubated for 12 hours at 180 rpm and 37 ° C. in a shaker.

Main culture: The seeded strain was inoculated into a 200 ml flask containing 50 ml of the production medium and incubated at 180 rpm and 37 ° C. for 12 hours. As a culture medium, a synthetic medium containing BHI medium (Difco, USA) and 5 g of glucose per liter, 5 g of yeast extract, and 5 g of sodium chloride was used. The pH of the synthetic medium was adjusted using 50 mM potassium phosphate buffer, and the growth curves of pneumococci, which are closely related to capsular polysaccharide production, were compared.

The results are shown in FIG. As shown in Figure 2, when the pH was adjusted to 5.0 to 9.0 with 50 mM potassium phosphate buffer, the bacterial growth was very different, and the dry cell weight was 0.63 g / l (pH 5.0) at 22 hours after incubation. , 1.12 g / l (pH 6.0), 1.14 g / l (pH 7.0), 0.65 g / l (pH 8.0), 0.29 g / l (pH 9.0). Fungus growth. Therefore, in the subsequent experiments were fixed to pH 7.0 to investigate the components of each culture medium composition.

Example 4 Influence of Carbon Sources on Capsular Polysaccharide Production

Species Culture: A single colony of stored pneumococcal type 17 was inoculated into a 20 mm diameter tube containing 5 ml of growth medium and incubated for 12 hours at 180 rpm and 37 ° C. in a shaker.

Main culture: The seeded strain was inoculated into a 200 ml flask containing 50 ml of the production medium and incubated at 180 rpm and 37 ° C. for 12 hours. As a culture medium, a synthetic medium containing BHI medium (Difco, USA) and 10 g of various carbon sources per liter, 5 g of yeast extract, 5 g of sodium chloride, and 50 mM potassium phosphate buffer solution was used. Table 1 shows the results of investigating the capsular polysaccharides using various carbon sources. The total amount of capsular polysaccharide was calculated by the amount after primary alcohol precipitation.

Comparison of productivity of capsular polysaccharides of pneumococcal pneumoniae according to various carbon sources Carbon source Dry cell weight (g / l) Final pH Capsular Polysaccharide (mg / l) Glycerol 2.36 5.18    59.9 glucose 1.96 5.25 121 fruit sugar 2.18 5.56 109 Mannos 1.13 6.80     69.6 Galactose 1.69 7.02 109 Xylose 1.33 7.02     68.4 Maltose 2.41 5.38     84.3 Sugar 2.01 5.21     94.9 Lactose 1.51 7.23 131 Soluble starch 1.58 7.19 -

As shown in Table 1, glucose, fructose, galactose and lactose among the various carbon sources showed high bacterial growth and high capsular polysaccharide productivity. Among the most economical glucose, the effect of concentration was investigated specifically.

Example 5: Influence of Glucose Concentration

Species Culture: A single colony of stored pneumococcal type 17 was inoculated into a 20 mm diameter tube containing 5 ml of growth medium and incubated for 12 hours at 180 rpm and 37 ° C. in a shaker.

Main culture: The seeded strain was inoculated into a 200 ml flask containing 50 ml of the production medium and incubated at 180 rpm and 37 ° C. for 12 hours. As a culture medium, a synthetic medium containing BHI medium (Difco, USA) and 10 g of carbon source per liter, 5 g of yeast extract, 5 g of sodium chloride, and 50 mM potassium phosphate buffer was used. Table 2 shows the results of capsular polysaccharide production using various concentrations of glucose. The total amount of capsular polysaccharide was calculated by the amount after primary alcohol precipitation.

Productivity Comparison of Pneumococcal Capsular Polysaccharides at Different Concentrations of Glucose Glucose (g / ℓ) Dry cell weight (g / l) Final pH Capsular Polysaccharide (mg / l)  5 1.89 7.09 116 10 2.43 5.03 153 15 2.57 4.94 109 20 2.69 4.93 205 30 2.72 4.94 276

As shown in Table 2, it was confirmed that when the glucose concentration was changed to 5 to 30 g / L, cell growth was increased, the final pH was decreased, and the production of capsular polysaccharide was gradually improved.

Example 6 Influence of Nitrogen Sources

Species Culture: A single colony of stored pneumococcal type 17 was inoculated into a 20 mm diameter tube containing 5 ml of growth medium and incubated for 12 hours at 180 rpm and 37 ° C. in a shaker.

Main culture: The seeded strain was inoculated into a 200 ml flask containing 50 ml of the production medium and incubated at 180 rpm and 37 ° C. for 12 hours. As a culture medium, a synthetic medium containing BHI medium (Difco, USA), 10 g of carbon source per liter, 5 g of various nitrogen sources, 5 g of sodium chloride, and 50 mM potassium phosphate buffer solution was used. Table 3 shows the results of investigating the production of capsular polysaccharides using various nitrogen sources. The total amount of capsular polysaccharide was calculated by the amount after primary alcohol precipitation.

Comparison of Productivity of Capillary Polysaccharides of Pneumococcal by Different Nitrogen Sources Nitrogen source Dry cell weight (g / l) Final pH Capsular Polysaccharide (mg / l) Yeast Nitrogen Base 0.40 6.01    76.9 Casaic acid 0.59 5.31    67.1 Trypton 0.83 5.18    87.1 Casein hydrolyzate 0.97 5.35    76.3 H17 badge 1.24 5.38    56.3 Trypton Soy Sauce 1.35 5.23    47.1 Cassiton 1.53 5.38    86.6 peptone 1.93 5.24    69.1 Yeast extract 2.05 5.12 124 BHI 2.55 5.33 125

As shown in Table 3, the highest capsular polysaccharide productivity (124 mg / L) was obtained using yeast extract among various nitrogen sources, which was similar to that when using BHI medium (125 mg / L).

Example 7: Influence of Yeast Extract Concentration

Species Culture: A single colony of stored pneumococcal type 17 was inoculated into a 20 mm diameter tube containing 5 ml of growth medium and incubated for 12 hours at 180 rpm and 37 ° C. in a shaker.

Main culture: The seeded strain was inoculated into a 200 ml flask containing 50 ml of the production medium and incubated at 180 rpm and 37 ° C. for 12 hours. As a culture medium, a synthetic medium including BHI medium (Difco, USA) and 5 g of glucose per liter, yeast extract (Difco, USA) 5 to 30 g, sodium chloride 5 g and 50 mM potassium phosphate buffer solution was used. The results of investigating the production of capsular polysaccharides using various concentrations of yeast extract are shown in Table 4a. The total amount of capsular polysaccharide was calculated by the amount after primary alcohol precipitation.

Productivity Comparison of Pneumococcal Capsular Polysaccharides in Yeast Extracts at Different Concentrations Yeast Extract (g / ℓ) Dry cell weight (g / l) Final pH Capsular Polysaccharide (mg / l)  5 1.90 5.22 127 10 2.93 5.15 211 15 3.16 5.25 292 20 3.28 5.48 395 25 3.74 5.45 385 30 4.03 5.40 480

As shown in Table 4a, when the yeast extract concentration was changed from 5 to 30 g / l, the dry cell weight increased from 1.9 g / l to 4.03 g / l, and the pH change varied from 5.15 to 5.48. Polysaccharide productivity continued to increase from 127 mg / l to 480 mg / l. Industrial yeast extract (China) was used to reduce the price of nitrogen source, which accounts for the highest proportion of culture media.

Comparison of the Production of Pneumococcal Capsular Polysaccharides in Industrial Yeast Extracts at Various Concentrations Industrial Yeast Extract (g / ℓ) Dry cell weight (g / l) Final pH Capsular Polysaccharide (mg / l) 10 3.47 5.15 339 20 6.06 5.30 480 25 6.73 5.37 603 30 7.67 5.46 733

As shown in Table 4b, when the industrial yeast extract concentration was changed from 10 to 30 g / l, the capsular polysaccharide productivity increased significantly from 339 mg / l to 733 mg / l.

Example 8 Influence of Potassium Phosphate Buffer Concentration

Species Culture: A single colony of stored pneumococcal type 17 was inoculated into a 20 mm diameter tube containing 5 ml of growth medium and incubated for 12 hours at 180 rpm and 37 ° C. in a shaker.

Main culture: The seeded strain was inoculated into a 200 ml flask containing 50 ml of the production medium and incubated at 180 rpm and 37 ° C. for 12 hours. As a culture medium, a synthetic medium including BHI medium (Difco, USA) and 5 g of glucose per liter, 5 g of yeast extract, 5 g of sodium chloride, and 25 mM to 200 mM potassium phosphate buffer solution was used. Table 5 shows the results of investigating the production of capsular polysaccharides using various concentrations of potassium phosphate buffer. The total amount of capsular polysaccharide was calculated by the amount after primary alcohol precipitation.

Productivity Comparison of Pneumococcal Capsular Polysaccharides in Different Concentrations of Potassium Phosphate Buffer Buffer concentration (mM) Dry cell weight (g / l) Final pH Capsular Polysaccharide (mg / l)  25 1.80 5.25 121  50 2.40 5.65 106 100 1.60 5.64    87.9 150 1.44 6.43    64.3 200 1.34 7.05    18.9

As shown in Table 5, when the concentration of potassium phosphate buffer was changed from 25 mM to 200 mM, cell growth was increased up to 50 mM, but at higher concentrations, it was inhibited by high concentrations of salt, and the capsular polysaccharide productivity was the same. The trend was shown.

Example 9 Production of Capsular Polysaccharide by Addition of Metal Ions

Species culture: A single colony of stored pneumococcal type 17 was inoculated into a 20 mm diameter tube containing 5 ml of growth medium and incubated for 12 hours at 180 rpm and 37 ° C. in a shaker.

Main culture: The seeded strain was inoculated into a 200 ml flask containing 50 ml of the production medium and incubated at 180 rpm and 37 ° C. for 12 hours. As a culture medium, a synthetic medium containing 5 g of glucose per liter, 5 g of yeast extract, 5 g of sodium chloride, and 50 mM potassium phosphate buffer was used. Table 6 shows the results of investigating the production of capsular polysaccharides by adding various metal ions (1 mM). The total amount of capsular polysaccharide was calculated by the amount after primary alcohol precipitation.

Comparison of productivity of capsular polysaccharides of pneumococci with metal ion addition Metal ions Dry cell weight (g / l) Final pH Capsular Polysaccharide (mg / l) No additives 1.80 5.22 120.6 Mg ²⁺ 1.74 5.16 166.3 Li ⁺ 1.68 5.17 157.4 Fe ²⁺ 1.66 5.13 154.9 Co ²⁺ 1.47 6.00 136.9 Cu ²⁺ 1.73 5.31 135.7 Ca ²⁺ 1.82 5.14 134.3 Mn ²⁺ 1.73 5.30 130.9

As shown in Table 6, when various metal ions are added, magnesium, lithium and iron are about 20-40 mg per liter of culture, and cobalt, copper, calcium and manganese are less than 10-20 mg per liter. Appeared to increase.

Example 10 Capsular Polysaccharide Purification and Analysis

⑴ Purification of Capsular Polysaccharide

After inoculating BHI medium with pneumococcal incubation at 37 ° C. until the maximum absorbance was reached at 600 nm, sodium deoxycholate (Sigma Co., USA) was added at a concentration of 0.5% and at 37 ° C. for 30 minutes. The cells were lysed by standing. Since the cell lysate gelled, acetic acid was added thereto at a concentration of 0.5% and then boiled to a boiling point. The boiled sample was cooled, neutralized to pH 6.7 by adding NaOH, and then centrifuged at 10,000 × g for 10 minutes to remove the precipitate. Ethanol cooled to −70 ° C. was added to the supernatant at a double volume and left at 4 ° C. for 18 hours, followed by centrifugation at 10,000 × g for 30 minutes at 4 ° C. to remove the supernatant. The precipitate was completely dissolved in 1/10 volume of distilled water, the chloroform: butanol (5: 1, v / v) solution was added to 1/5 volume of the precipitate dissolved in distilled water, extracted, and centrifuged at 10,000 × g for 15 minutes. Supernatant with protein removed was taken. This process was repeated twice. Trimethylammonium bromide was added to 0.2% in the supernatant obtained therein, and ethanol cooled to -70 ° C. was added at a volume of 2 times. The precipitate was dissolved in 1/20 volume of distilled water. The dissolved solution was lyophilized to a powder. Figure 3 is a photograph of the capsular polysaccharide powder obtained. Total equivalent weight and weight were measured using the capsular polysaccharide powder obtained through the above procedure.

⑵ total equivalence analysis

The lyophilized sample was dissolved at 1 mg / ml, diluted 10-fold, 100-fold and 1,000-fold, and then 25 μl and 25 μl of 5% phenol were added to the standard solution, mixed for 30 seconds, and then placed on ice for a while. 125 μl H ₂ SO ₄ was added thereto, mixed for 30 seconds, boiled at 100 ° C. for 10 minutes, cooled to room temperature, and absorbance was measured at 490 nm to calculate the equivalent weight. The standard solutions used were starch and Pneumococcal 3 polysaccharide (containing 2, 4, 6, 8, 16 and 32 μg). When the standard solution was used as starch, it was confirmed that Type 3 is 53.7 mg / L, Type 15 is 86.2 mg / L, Type 17 is 51.9 mg / L, Type 22 is 69.3 mg / L, and Type 9V is 48.6 mg / L. When using the two standard solutions, a slight numerical difference in total equivalents is regarded as the difference in the components of each unit.

Example 11: Further Purification of Capsular Polysaccharides

Type 17 capsular polysaccharide was further purified using Sephadex G-100 column (Sigma, USA) chromatography (1.6 × 60 cm) to obtain higher purity samples. After Sephadex G-100 column chromatography, 37.9 mg (containing 0.037 mg protein) of high-purity capsular polysaccharide (type 17) was obtained.

Comparison of Before and After Sephadex G-100 Column Chromatography of Capsular Polysaccharide (Type 17) Type 17 capsular polysaccharide Carbohydrate (mg) Protein (mg) Purification yield (%) Protein / Carbohydrate (%) Sephadex G-100 51.92 0.704 100 1.35 After Sephadex G-100 37.91 0.037  73 0.09

1 is a graph showing the change of dry cell weight of pneumococci for 22 hours according to the culture medium (BHI medium □, synthetic medium ○) of pneumococci;

Figure 2 is a graph showing the change in dry cell weight of pneumococcal according to the pH (5.0 ◇, 6.0 □, 7.0 ○, 8.0 ×, 9.0 △) of potassium phosphate buffer solution;

Figure 3 is a photograph showing the capsular polysaccharide powder obtained by separation and purification from each type of pneumonia strain after lyophilization.

Claims (7)

One or more carbon sources selected from the group consisting of glucose, fructose, galactose and lactose, 5-30 g / l yeast extract, 5-30 g / l yeast extract, 2-10 g / l sodium chloride and 25-50 mM potassium phosphate buffer And a medium for producing capsular polysaccharide from Streptococcus pneumoniae type 17, which does not contain casein hydrolysate and has a pH adjusted to 5-9. delete delete The medium for producing capsular polysaccharide from Streptococcus pneumoniae type 17 further comprising at least one metal ion selected from the group consisting of magnesium, lithium, iron, cobalt, copper, calcium and manganese. . A method for producing capsular polysaccharides from Streptococcus pneumoniae type 17, comprising culturing Streptococcus pneumoniae type 17 in a medium according to claim 1. The method of claim 5, 용 Sodium deoxycholate is added to the culture medium of Streptococcus pneumoniae type 17 to lyse the cells; ⑵ chloroform-butanol solution is added to the cell lysate to obtain the supernatant from which the protein is removed; 에탄올 ethanol is added to the deproteinized supernatant to precipitate the capsular polysaccharide: A method of producing capsular polysaccharide from Streptococcus pneumoniae type 17, further comprising the step. 7. The method of claim 6, a method of producing capsular polysaccharide from step ⑶, pneumococcus (Streptococcus pneumoniae) type 17 containing a capsular polysaccharide obtained in the further step of purification by gel permeation chromatography (Gel Permeation Chromatography, GPC) .

Images