KR960014622B1 - Culture ground for streptcoccus pneumoniae - Google Patents

Abstract

The improved culture medium for Streptococcus pneumoniae is composed of 0.5-1.5w/v% casein hydrolysate, 0.1-1.0w/v% trypton, 0.1-1.5w/v% NaCl, 0.1-0.5w/v% yeast extract, 0.017N K2HPO4, and 0.8-2.0w/v% carbon source in a distilled water. The above culture medium provides much more slime layer polysaccharides by cultivating aerobically.

Description

Improved preparation of pneumococcal culture medium and pneumococcal capsular polysaccharide components

Figure 1 shows the growth curve in modified CAT medium according to the present invention of type 1 pneumococci with carbohydrate addition.

Figure 2 shows the growth curve in modified CAT medium according to the invention of type 23 pneumococci with carbohydrate addition.

Figure 3 shows the comparison of polysaccharide production (P.S) and growth rate (absorbance: O.D.) in modified CAT media according to the invention of pneumococci of types 1,4 and 23 according to the amount of glucose in addition to carbohydrates.

Figure 4 shows the growth curve in modified CAT medium according to the invention of type 1 pneumococci with metal ion addition.

Figure 5 shows the growth curve in modified CAT medium according to the invention of type 1 pneumococci with amino acid addition.

FIG. 6 is a graph comparing polysaccharide production (P.S.) and growth rate (absorbance: O.D) in a modified CAT medium according to the invention of pneumococci of types 1, 4 and 23 according to the change in aeration rate.

Figure 7 shows the sedimentation reaction of capsular polysaccharides and standard serum isolated by culturing pneumococci in BHI medium and modified CAT medium according to the present invention by double-immunophoresis.

The present invention relates to a medium optimized for culturing Streptococcus pneumoniae, especially a medium for culturing pneumococci containing casein hydrolysates. The present invention also relates to an improved method for culturing pneumococci, which produces pneumococcal polysaccharide vaccines in large amounts by culturing pneumococci under optimized conditions in the above optimized medium.

Streptococcus pneumoniae has a mucosal capsular structure composed of polysaccharides outside its cell wall. The capsular polysaccharides of pneumococci represent about 80 types of serotypes, and the types of pneumococci are classified according to their type. Small doses of these capsular polysaccharides to mice produce antibody-specific immunoglobulins. Thus, the capsular polysaccharide of pneumococci can act as an antigen that provides immunity to infection of pneumococci.

In order to produce such useful antigenic capsular polysaccharide components, the prior art generally refers to pneumococci in blood medium, brain heart infusion (BHI) medium, trypsin soy broth medium, Todd Hewitt juice. (Todd Hewitt Broth) The culture method in a medium and a defined medium (defined media) has been used.

However, these media were not only complex or too expensive, but also produced very low yields of capsular polysaccharides when cultured in these media. For example, when BHI medium is used, pneumococci are well cultured, but because of the use of expensive calf brain leachate and small cardiac leachate as a constituent of the medium, the production cost of the medium is too high, which is suitable for mass production. I can't. In addition, since blood of sheep or horse must be added to the blood medium, the production of the medium is very demanding and expensive, and the yield of capsular polysaccharides is significantly lower than that of the BHI medium. Certain types of pneumococci provide good culture results but in most cases are inferior to those cultured in BHI medium.

Accordingly, the present inventors have concentrated on a method for producing a large amount of useful antigenic capsular polysaccharides by efficiently culturing pneumococci using inexpensive media without exhibiting the disadvantages of culturing pneumococci using conventional media as described above. As a result, in the prior art, a medium obtained by partially modifying components of CAT medium, which has been used only for cultivation of pneumococci which do not form a capillary under anaerobic conditions, for easy genetic manipulation, is grown and ventilated under aeration. It was confirmed that it has an amazing effect on the production of polysaccharides. In other words, a modified CAT medium containing casein hydrolysate as a main ingredient and an increased carbohydrate content compared to conventional CAT medium (carbohydrate content: glucose 0.2w / v%) was used as a medium for pneumococcal culture. By culturing pneumococci, it was confirmed that the above object can be achieved and the present invention has been completed.

Accordingly, the present invention relates to a medium optimized for culturing pneumococci to produce antigenic capsular polysaccharides of pneumococci. In particular, the present invention is a casein hydrolyzate to 0.5 to 1.5 w / v%, tryptone 0.1 to 1 w / v%, NaCl 0.1 to 1.5 w / v%, yeast extract 0.1 to 0.5 w / v%, potassium phosphate (K ₂ HPO ₄ ) relates to a pneumococcal culture medium containing 0.017 N and a carbon source of 0.8 to 2.0 w / v%.

Carbon sources which can be added to the medium of the present invention include, for example, carbohydrates of monosaccharides or disaccharides such as glucose, sucrose and maltose, most preferably glucose. The amount of carbon source added is 0.8 to 2.0 w / v% of the medium composition, and the optimum amount is 0.8 w / v%. When cultivating pneumococci using a medium containing such a carbon source, the maximum growth point of pneumococci is 1.5 times higher than when a medium containing a carbon source is added in a trace amount of less than 0.8 w / v%, or when a medium without a carbon source is used. It increases more than twice, and the time to reach the maximum growth point is more than doubled.

The medium of the present invention may also contain metal ions and amino acid components as necessary.

Suitable metal ions that can be added to the medium according to the invention are calcium, magnesium and copper. These metal ions are preferably added in the form of each of CaCl ₂ , MgSO ₄ and CuSO ₄ , and the addition concentration thereof is preferably 1 mM to 10 mM, in particular 1 mM. When culturing pneumococci in the medium according to the present invention to which such metal ions are added, the time for reaching the maximum growth point of pneumococci is shortened by two or more times compared to when culturing in a medium containing no metal ions.

In addition, the yield of the desired capsular polysaccharide can be increased by two or more times by adding the amino acid component to the medium of the present invention. Suitable amino acids for this purpose may be any amino acid that does not adversely affect the culture of pneumococci, such as asparagine, phenylalanine, threonine, cysteine, isoleucine or methionine, but asparagine, phenylalanine or Most preferred is the use of threonine. At the time of addition of imino acid, the amount thereof is preferably 0.1 to 1000 ppm.

In view of the maximum growth point of pneumococci and the yield of capsular polysaccharide components, the most preferred pneumococcal culture medium according to the present invention is 1w / v% casein hydrolyzate, 0.5w / v% tryptone, 0.5w / v% yeast, yeast Extract 0.1w / v%, potassium phosphate (K ₂ HPO ₄ ) 0.017N, glucose 0.8w / v%, magnesium sulfate (MgSO ₄ ) copper sulfate (CuSO) or calcium chloride (CaCl ₂ ) 1 mM, and asparagine, phenylalanine or threonine 3 mg Medium containing / L.

When culturing pneumococci in the medium according to the present invention, the culture can be carried out by agitation by shaking at pH 5 to 8 in order to maximize the desired effect.

Under these conditions, the maximum growth point of pneumococcus did not change much compared to the case of culture without aeration, but the production cost was reduced to about 35% or less while doubling the yield of the desired capsular polysaccharide component. Can be. Aeration for this purpose can be achieved by gently shaking the culture, usually in the atmosphere, preferably below 50 rpm.

The medium according to the present invention can be prepared by the following simple method. That is, the medium of the present invention is a case of dissolving casein hydrolyzate, tryptone, sodium chloride and yeast extract in distilled water and autoclaving, and then separately sterilized potassium phosphate (K ₂ HPO ₄ ) and carbon source, and optionally a metal ion source or It can manufacture by adding an amino acid component.

As described above, according to the present invention, unlike the BHI medium, blood medium, and limited medium, which have been used in the prior art, the production cost of the antigenic capsular polysaccharide of pneumococcus of the target pneumoniae can be reduced because it uses a simple and inexpensive medium. In addition, the cultivation is carried out under aeration rather than anaerobic conditions, thus providing the advantage of simple culture operation. In addition, culturing pneumococci under the culture conditions according to the present invention provides a better or at least equivalent yield of capsular polysaccharide components than when cultured in BHI medium, which is recognized to provide the best yields in the prior art. This excellent yield demonstrates a clear advance in the art in that it is an effect obtained by simple manipulation using inexpensive media.

The invention is illustrated in more detail by the following examples.

Example 1

100 ml of medium prepared by dissolving casein hydrolyzate 1w / v%, tryptone 0.5w / v%, sodium chloride 0.5w / v%, yeast extract 0.1w / v% and potassium phosphate (K ₂ HPO ₄ ) 0.017N in distilled water Put in a 500ml Erlenmeyer flask and sterilize for 30 minutes at 121 ℃. Separately, each of the glucose, sucrose and maltose is sterilized using a carbon source, so that glucose is 0.8 w / v% (medium 1), sucrose is 2.0 w / v% (medium 2), and maltose is Each flask was introduced at a concentration of 2.0 w / v% (medium 3) to obtain three kinds of pneumococcal culture medium. The effect of the type of added carbon source on the growth of pneumococci was tested using this culture medium. As a control, a medium having the same composition as above and a BHI medium are used except that they do not contain a carbon source. Three kinds of pneumococci of type 1, type 4 and type 23 are inoculated in each medium at an amount of 10 ⁵ CFU / ml (CFU: Cell Forming Unit) and incubated at 37 ° C. Over time, the absorbance of the cultures in each flask was measured at 550 nm to determine the growth rate of pneumococci. (See FIGS. 1 and 2).

As a result, type 1 pneumococci showed a maximum absorbance of 0.831 after 5 hours of culture in medium containing glucose, and type 3 pneumococci showed a maximum absorbance after 11 hours of culture in medium 2 containing sucrose as a carbon source. 0.959. In contrast, the absorbance measured after 5 hours of type 1 pneumococci was 0.595 when cultured in BHI medium, and the maximum absorbance 0.735 after 7 hours of culture. In addition, the growth of pneumococci in the medium without the carbon source was minimal. That is, in the case of type 1 pneumococci, the absorbance was measured to be less than 0.1 after 9 hours of incubation, and even in the case of type 23 pneumococci, the absorbance was about 0.1. As seen above, the growth of pneumococci in medium containing glucose, sucrose or maltose as a carbon source is the time to reach the highest growth point compared to growth in medium without a carbon source or commonly used BHI medium. In terms of time, it provided an effect of reducing the time by about one third.

After incubation, 1 w / v% of phenol was added to each medium to decompose pneumoniae, and 5 w / v% of sodium acetate was added thereto to separate the generated capsular polysaccharides. As a result of measuring and comparing the yields of the polysaccharide components isolated from each medium, the production yields of pneumococci of types 1, 4 and 23 were about 1% compared to those of the medium containing no BHI medium or carbon source in the medium. It can be seen that more than 1.2 times increased.

In addition, the same composition as that of the medium 2, except that the culture was added in the same manner as described above using a medium added by changing the concentration of glucose to 0.2w / v%, 2w / v% and 5w / v%, The maximum growth rate of pneumococci and the yield of capsular polysaccharide components were measured according to glucose concentration (see FIG. 3). As a result, as shown in FIG. 3, satisfactory results were obtained at both the maximum growth rate and the yield of the capsular polysaccharide component when the glucose concentration added to the medium was 0.8 to 2.0 w / v%. However, when the glucose concentration is 0.2w / v%, the growth of pneumococci is not generally desirable, and in the case of type 23 pneumococci, polysaccharide production yield is markedly decreased when 5w / v% glucose is added. Therefore, the amount of carbon source added in the present invention was controlled from 0.8w / v% to 2.0w / v%.

Example 2

Casein hydrolyzate 1w / v%, tryptone 0.5w / v%, sodium chloride 0.5w / v%, yeast extract 0.1w / v% and potassium phosphate (K ₂ HPO ₄ ) 0.017N glucose 0.8w / v% 100 ml of the prepared medium is added to a 500 ml Erlenmeyer flask and sterilized at 121 ° C. for 30 minutes.

Separately, sterilization of magnesium sulfate, copper sulfate, calcium chloride, cobalt chloride, iron sulfate, and manganese chloride as metal ion sources, respectively, and each concentration of 1 mM was introduced into each flask containing 100 ml of the obtained medium. To obtain. The effect of the type of metal ion added on the growth medium and the yield of capsular polysaccharide component was tested using this medium. For control, a conventional CAT medium, a BHI medium containing no metal ions, and the medium containing EDTA 1 mM in place of the metal ion were used. Each medium is inoculated with three pneumococci of type 1, type 4 and type 23 in an amount of 10 ⁵ CFU / ml and incubated at 37 ° C. Over time, the absorbance of the cultures in each flask was measured at 550 nm to determine the growth rate of pneumococci (see Figure 4).

As a result, in the medium containing Ca and Mg as metal ions, type 1 pneumococci exhibited an absorbance of 0.721 after about 5 to 6 hours of incubation, type 4 pneumococci 0.573, and type 23 pneumococci showed 0.728 absorbances. It was. Comparing these results with the growth of pneumococci in the control medium, the addition of metal ions can be judged not to significantly affect the growth rate of lung rum bacteria.

After incubation, 1w / v% of phenol was added to each medium to decompose pneumococci, and 5w / v% of sodium acetate was added thereto to separate the generated capsular polysaccharides. As a result of measuring and comparing the yields of the polysaccharide components isolated from each medium, the production yields of pneumococci of types 1, 4 and 23 were all BHI in the metal ion-containing medium, especially in the medium containing CuSO ₄ , MgSO ₄ or CaCl ₂ . It was found that the yield increased by about 1.4 times compared to the yield in the medium or the medium containing no metal ions.

Example 3

Casein hydrolyzate 1w / v%, tryptone 0.5w / v%, sodium chloride 0.5w / v%, yeast extract 0.1w / v% and potassium phosphate (K ₂ HPO ₄ ) 0.017N and glucose 0.8w / v% 100 ml of the prepared medium was dissolved in a 500 ml Erlenmeyer flask and sterilized at 121 ° C. for 30 minutes.

Separately sterilized each of cysteine, asparagine, phenylalanine, isoleucine, threonine and methionine as amino acid sources, and then introduced into each flask containing 100 ml of the obtained medium at a concentration of 3 mg / L, respectively, for six test culture media. To obtain. This culture medium was used to examine the effects of amino acid addition on the growth rate of pneumococci and the yield of capsular polysaccharide components. For control, conventional CAT medium and BHI medium containing no amino acid were used. Each medium is inoculated with three pneumococci of type 1, type 4 and type 23 in an amount of 10 ⁵ CFU / ml and incubated at 37 ° C. Over time, the absorbance of the culture in each flask was measured at 550 nm to determine the growth rate of pneumococci (see FIG. 5).

As a result, especially in the medium to which asparagine, phenylalanine or threonine was added as an amino acid, the maximum absorbance of type 1 pneumococcus was 0.763, type 4 pneumococci 0.587 and type 23 pneumococci 0.77. Comparing these results with the growth of pneumococci in the control medium, it can be seen that the addition of amino acids does not significantly affect the growth rate of pneumococci.

After incubation, 1w / v% of phenol was added to each medium to decompose pneumococci, and 5w / v% of sodium acetate was added thereto to separate the generated capsular polysaccharides. As a result of measuring and comparing the yields of the polysaccharide components isolated from each medium, the production yields of pneumococci of types 1, 4 and 23 were all free of BHI medium or amino acids in medium containing asparagine, phenylalanine or threonine. It was found that the yield increased more than about 2 times compared to the yield in the medium.

Example 4

Casein hydrolyzate 1w / v%, tryptone 0.5w / v%, sodium chloride 0.5w / v% yeast extract 0.1w / v% and potassium phosphate (K ₂ HPO ₄ ) 0.017N and glucose 0.8w / v% 100 ml of the medium prepared by dissolving at a concentration is added to a 500 ml Erlenmeyer flask and sterilized at 121 ° C. for 30 minutes. Inoculate pneumococci of type 1,4 and 23 into the sterilized culture medium in an amount of 10 ⁵ CFU / ml, and incubate with shaking at 37 ° C at 0, 50, 100 or 150 rpm to incubate the growth rate and capsular polysaccharides of pneumococci. The effect on the production yield of the components was tested (Figure 6).

Over time, the absorbance of the cultures in each flask was measured at 550 nm to determine the growth rate of pneumococci. As a result, as shown in FIG. 6, the effect of aeration on the growth rate of pneumococcus was hardly affected. The growth rate was slightly increased under mild agitation of about 50 rpm, but the growth rate was slightly decreased when the aeration by agitation above 100 rpm was performed. Could.

After incubation, 1w / v% of phenol was added to each medium to decompose pneumococci, and 5w / v% of sodium acetate was added thereto to separate the generated capsular polysaccharides. As a result of measuring and comparing the yields of the polysaccharide components isolated from each medium, as shown in FIG. 6, the yields of polysaccharide production of pneumococcus pneumoniae of type 1, 4, and 23 are not aeration when mildly ventilated at 50 rpm. It can be seen that an increase of about 1.2 to 2.0 times or more compared to the case of culturing with excessive aeration above 100 rpm.

Example 5

Casein hydrolyzate 1w / v%, tryptone 0.5w / v%, sodium chloride 0.5w / v%, yeast extract 0.1w / v% potassium phosphate (K ₂ HPO ₄ ) 0.017N and glucose 0.8w / in distilled water 1.25% of the samples and distilled water diluted with 1: 1, 1, 2, 1: 4 and 1: 10 of the capsular polysaccharide components of pneumococcal bacteria isolated by culturing in a modified CAT medium containing v% in distilled water Agarose gel (1.25 g of agarose is dissolved in 25 ml of a solution consisting of 1.842 g / L of Veronal, 10.309 g / L of Veronal Sodium and 6.8 g / L of Sodium Acetate and 75 ml of distilled water and dispensed into an electrophoretic film 0.2 mm thick). (Manufactured), and then spread in a box saturated with moisture for 40 hours. The gel was prepared by adding 9000 ml of H ₂ O to 1.9 mM of nitrate buffered saline (NaH ₂ PO ₄ · 2H ₂ ), 8.1 mM Na ₂ HPO ₄ , and 154 mM NaCl, and adjusting the pH to 7.2 to 7.4) and distilled water. Wash 3 times for 15 minutes, put filter paper with gel side up to remove moisture, and dry. After dyeing with 0.5% Coomassie brilliant blue for 5 minutes, put into methyl alcohol: acetic acid: distilled water (4.5: 1: 4.5), remove the pigment by changing it with fresh liquid until the precipitation band is visible, and then remove the gel. Dry and observe. Polysaccharides obtained from BHI medium and polysaccharides obtained from modified CAT medium of the present invention react with standard serum to obtain the same settling line. The result is as shown in FIG. In FIG. 7, the central well contains the standard antiserum isolated from each pneumococcal type, peripheral well 1 is distilled water, peripheral well 2 is 1 mg / ml of purified polysaccharides, and peripheral well 3 is 0.5 mg / ml of purified polysaccharides. Peripheral well 4 contains 0.25 mg / ml of purified polysaccharide, and peripheral well 5 contains 1 mg / ml of purified polysaccharide. In addition, [A] shows the reaction of polysaccharides isolated from type 1 pneumococci cultured in BHI medium, [B] also shows the sedimentation reaction of polysaccharides isolated from type 4 pneumococci cultured in BHI medium, [C] shows the settling reaction of polysaccharides isolated from type 1 pneumococci cultured on the modified BHI medium, and [D] also shows the reaction of polysaccharides isolated from type 4 pneumococci cultured on modified BHI medium. It is shown.

As shown in the results of FIG. 7, it can be seen that the polysaccharide obtained from the modified BHI medium according to the present invention has no change in antigen specificity compared to the polysaccharide obtained in the BHI medium.

Claims (10)

Casein hydrolyzate in distilled water 0.5-1.5 w / v%, tryptone 0.1-1.0 w / v%, sodium chloride 0.1-1.5 w / v%, yeast extract 0.1-0.5 w / v% Carium phosphate (K ₂ HPO ₄ ) 0.017 N and a pneumococcal culture medium, characterized in that it contains 0.8 to 2.0 w / v% carbon source. The medium according to claim 1, which further contains 1 to 10 mM of metal ion source and 0.1 to 1000 ppm of amino acids. The medium of claim 1 wherein the carbon source is selected from glucose, sucrose and maltose. The medium of claim 3 wherein the carbon source is glucose. The medium of claim 2 wherein the metal ion source is selected from potassium chloride, magnesium sulfate and copper sulfate. The medium of claim 2 wherein the amino acid is selected from asparagine, phenylalanine and threonine. The distilled water hydrolyzate 1w / v%, tryptone 0.5w / v%, sodium chloride 0.5w / v%, yeast extract 0.1w / v% potassium phosphate (K ₂ HPO ₄ ) 0.017N, glucose 0.8w medium containing 1 mg / L of magnesium sulfate and 1 mg / L of asparagine. A method for producing a capsular polysaccharide component of pneumococcal bacteria, characterized by culturing the pneumococci under aeration in a medium according to any one of claims 1 to 7. The method according to claim 8, wherein the aeration is carried out by shaking at 50 rpm or less. The method of claim 8, wherein the culturing is performed at pH 5-8.