JPS6255075A - Cultivation of microorganism belonging to bordetella genus and medium therefor - Google Patents

Abstract

PURPOSE:To enable the stable and efficient culture of a microbial strain belonging to Bordetella genus, by using a medium containing specific amounts of casamino acid, ascorbic acid, etc. CONSTITUTION:A microbial strain belonging to Bordetella genus such as Bordetella pertussis, Bordetella bronchiseptica, etc., is detected and/or cultured by using a medium obtained by adding 0.1-20g/l of casamino acid, 0.01-1g/l of ascorbic acid, 0.1g/l of glutathione and 0.001-5g/l of cyclodextrin or its derivative to conventional medium such as SS medium, SSA medium, etc.

Description

[Detailed description of the invention] The present invention is directed to Borda bacterium, which is known as a pathogenic bacterium.

A method for cultivating microorganisms belonging to the genus B ordetella, the medium used for the cultivation, and the production of biologically active substances using this medium! ! Regarding the method of J-building.

Microorganisms belonging to the genus Bordetella include Bordetella pertussis, Bordetella pertussis, and Bordetella bronchiseptica.
It is known as the main pathogen of pertussis, which is an acute infection of the trachea, bronchi, and small bronchi that is accompanied by a characteristic cough that lasts for 100 days. Therefore, from a clinical perspective, rapid and reliable detection of Bordetella pertussis and the like is desired, and for this purpose a culture method or culture medium that promotes the growth of clinically isolated bacteria is required. In addition, pertussis vaccines (killed whole body vaccines or component vaccines) are used to prevent pertussis, but in order to efficiently produce vaccines, culture methods that promote the growth of pertussis bacteria are required. Alternatively, a medium is preferable. In addition, for example, a culture of Bordetella pertussis (culture medium and bacterial cells) can be used to produce an insulin secretion-enhancing active substance (I5uetact), which is expected to be developed as a diabetes treatment or prevention drug.
1) roll
otin(J ractor-hf311aQQHJ
t'1nrn (hereinafter abbreviated as PF-HA)
Yafi181entous-Mla([Iutinin
(hereinafter abbreviated as F-H8), medically effective 'biologically active substances can be obtained, but in order to efficiently produce such biologically active substances, it is necessary to efficiently cultivate B. pertussis. A method is needed.

[As a culture medium for the growth of bacteria belonging to Ordetella ffl, Toriwabanpu pertussis, Cohen et al.
American Journal of Public Health;
American Journal or Pu
blic l-1ealth36 371-37B
(1946) ) and Sutherland et al. (Journal of Biology and Bacteriology: Jou
rnal or P athology and
Bacteriology 82 431-438 (
(1961)). Culture media containing activated carbon, starch, or ion exchange resin are known. However, activated carbon and ion exchange resins form a heterogeneous system, making them unsuitable for tracking changes in bacterial growth over time.Also, because they have nonspecific adsorption properties, they do not absorb factors that inhibit bacterial growth, such as fatty acids. The removal effect cannot necessarily be said to be selective. The effect of starch on bacterial growth was not sufficient, and there was a desire to develop a new culture medium that would compensate for the above deficiencies. In addition, when clinically isolating B. pertussis as a single colony, a visual spray from a patient with pertussis is brought into direct contact with a solid agar medium containing blood, such as Poldey-Zhang medium (hereinafter abbreviated as BG medium). However, since these media contain fresh blood as an essential component, they have poor storage stability and have the disadvantage of poor reproducibility of colony forming ability. Therefore, from this point of view, it has been desired to develop a clinical separation medium that has a certain chemical composition and has a long shelf life.

In recent years, Stainer and Scholte (
A synthetic medium for the culture of Bordetella pertussis was developed by J. Scholte (Gen.

M 1crobiol) vol. 63, 211-22
p. 0, 1971). This Steady-Sholte medium (hereinafter abbreviated as SS medium) does not contain substances that may cause fluctuations in the temperature difference, such as blood and polypeptone derived from natural products, so the culture medium composition of bacteria can be strictly controlled. It has the characteristics that it can be cultured without causing any change in the bacterial properties, and that it can prevent unnecessary contamination with proteins from other species when separating and purifying biologically active substances such as IAP or LPF-HA mentioned above. In recent years, it has been widely used to produce pertussis vaccines and biologically active substances from Bordetella pertussis on an industrial scale. The production of IA is insufficient, and the inoculation size is 10' cells.
/d or less has the disadvantage that stable growth characteristics cannot be obtained.

In addition, in an agar medium (hereinafter abbreviated as SSA medium) obtained by adding 1 to 2% agar to SS medium and solidifying it,
It has a major defect in that colony formation is not observed when seeded at 10' cents or less.

An object of the present invention is to provide a stable and efficient method for culturing and separating microorganisms belonging to the genus Bordetella. Another object of the present invention is to provide a medium for use in such culture. Another object of the present invention is to provide a method for producing biologically active substances by culturing microorganisms belonging to the genus Bordetella using this medium.

The object of the present invention is to provide casamino acids of 0.1 to 20% when culturing microorganisms belonging to the genus Bordetella.
A method for culturing microorganisms belonging to the genus Bordetella, characterized by using a medium containing 9/Ma, ascorbic acid 0.01 to 19/1 and glutathione 0.1 to 59/1,
Biologically active substances are extracted from the culture (culture medium and bacterial cells) by culturing microorganisms belonging to the Bordetella genus in a medium containing gasamino acid, ascorbic acid, and geltadeone, and in a medium containing casamino acid, ascorbic acid, and glutathione. This is achieved by a method for producing biologically active substances, which is characterized by the collection of.

In the present invention, microorganisms belonging to the genus Bordetella include Bordetella pertussis, Bordetella pertussis, and Bordetella bronchiseptica. In the present invention, Bordetella pertussis is preferably used, and Pertussis I phase bacteria is particularly preferred.

Regarding the mycological properties and culture conditions of microorganisms belonging to the genus Bordetella, see 3 ergys Manusl.
orDeterminative Bacteria
logy, 8th edition, 1974, The Wi
lliams & willkins co.

Published by J, Exp Med, Volume 129, No. 523
-550 pages.

It is already publicly known, having been published in 1969 or Bacteriology Practical R Essentials, 3rd edition, pages 80 and below, published in 1971, round neck.

The casamino acids used as a component of the culture medium of the present invention are acid hydrolysates of casein and are available as a dry powder. Furthermore, ascorbic acid is known as vitamin C, and glutathione is a type of peptide that is widely distributed in yeast, animal liver, muscle, and the like.

Glutathione may be in an oxidized form, a reduced form, or a mixture thereof. The medium of the present invention contains casamino acids of 0.1 to 20S? /l, preferably 0.5~
10 g/l, preferably 0.1-0.49/l, glutathione 0.1-59/l, preferably 0.1-19/l
l Must be included. If it is outside the above range, the object of the present invention will not be fully achieved.

When the medium of the present invention contains cyclodextrin or a derivative thereof in an amount of 0.001 to 59/1°, preferably o, oos to 59/fL, in addition to the above-mentioned components, the objects of the present invention can be more advantageously achieved. . Cyclodextrin or its derivatives may be added to and mixed with the medium containing the above-mentioned components as they are, or may be added to the clathrate after forming a clathrate with glutathione in advance. In the present invention, cyclodextrin means α, β, and γ cyclodextrin, and cyclodextrin derivatives include, for example, aminated derivatives such as aminocyclodextrin and aminodeoxycyclodextrin, and esters such as acetylcyclodextrin and nitrocyclodextrin. It refers to etherified derivatives (etherified cyclodextrin) such as methylcyclodextrin, methylcyclodextrin, propylcyclodextrin, and carboxymethylcyclodextrin. Preferred in the present invention are etherified cyclodextrins, especially hexakis(2,6-0-dimethyl)α-cyclodextrin-(Meα-CD) and heptakis(2,6-0-dimethyl)α-cyclodextrin-(Meα-CD).
-0-dimethyl)β-cyclodextrin (Meβ-C
Methyldextrins such as D) are particularly preferred.

In the present invention, the medium means a conventionally known liquid medium such as bouillon or peptone water, or a conventionally known solid medium made by adding agar, gelatin, egg white, serum, etc. to a liquid medium, but preferred is SS medium (or SSB
These are an SS medium and an SSA medium obtained by adding about 1 to 2% (w/v) agar to the same and solidifying it. For example, SS medium usually contains 1 each of sodium glutamate, i-proline, potassium chloride, magnesium chloride, calcium chloride, and trishydroxymethylaminomethane per cross.
0,7. 0:24, 2.5. 0,5. 0.2
．． After adjusting the aqueous solution containing 1,525 g of 0.1° 0.02 to DH 7.6 with concentrated hydrochloric acid, it was sterilized in an autoclave at 121°C for 15 minutes.
Cystine.

f! Ferrous acid, ascorbic acid, niacin, glutathione per 11, 4. 1. 2. 0,
A solution containing 4°109 was filtered through a Millipore filter (0.45
Add the replacement fluid obtained by sterilization using 1.μ) to the basal medium.
It is obtained by adding at a ratio of 0% (V/V). In the present invention, the required amounts of cabmino acid, ascorbic acid, and glutathione are added to such a medium, and in some cases, the required amount of cyclodextrin or a derivative thereof is added.

Since the culture medium of the present invention allows bacteria to grow stably and efficiently, it can be preferably used for the growth and detection of clinical isolates of pertussis. In addition, IAP is expected to have medical effects as a diabetes treatment drug and q.

LP held as pertussis vaccine component
It is also an extremely advantageous medium for the production of active substances such as F-HA and bacterial vaccines.

The method and conditions for culturing microorganisms belonging to the genus Bordetella using such a medium are not particularly limited, and conventionally known methods and conditions can be adopted, but shaking culture is preferable to static culture, and culture temperature The temperature was 30-38°C, and the culture time was 10 to 100 hours.

The method 1 means for collecting the produced biologically active substance from the culture (culture medium and bacterial cells) is not particularly limited, and any known method 0 means can be used. For example, LPF
- To obtain HA, pertussis bacteria (Bordetella batatsis Higashihama strain) is cultured in the medium of the present invention at 35°C for 48 hours, and the resulting culture solution is centrifuged -Lm (1)l-18,3)
is passed through a hydroxyapatite column equilibrated with 0.01 M phosphate buffer at pi-18.0.

Then, the obtained permeate is 0.01 of pi-16,0.
The protein fraction is adsorbed onto a hydroxide xiapatite column equilibrated with M phosphate buffer, and eluted with a 0.1M phosphorus MW buffer (DH7.0) containing 0.5M sodium chloride to obtain a protein fraction. This protein fraction was converted into haptoglobin-cephalogram.
Adsorbed on affinity chromatography using 24 days as support, 0.5M NaCf and 3M thiosine.

0.0 containing Jjium anhydride. lMg4 acid buffer (rll
-17,0) to obtain LPF-1-IA.

In addition, the adsorbed material on a hydroxyl abatite 1-column with a pH of 0.0 and 0.1
Elute with M phosphoric acid** solution (1) I-17,0) to obtain a protein fraction. This protein fraction is passed through affinity chromatography using haptoglobin Sepharose 4B as a support, and F-1''A can be obtained from the passed-through liquid.

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 The 11 degrees of glutathione in normal SSA medium (containing ascorbic acid at 0.02 g/cross) was increased by 1.5 times, i.e. 0.
159/! 1, and further change the temperature of each casamino acid to 0.
．． 0,5. 1.0. 2,5. s, 0.10.
ogZ sentence, and the concentration of Meβ-CD is 0.
゜0.05, 0.10, 0.25, 0.5
G, improved S adjusted to be 1.09/Jl
Approximately 100 pertussis phase I Higashihama strains were spread on SA medium and cultured at 35°C for 3 days. The number of colonies formed was then compared with the number of colonies formed in BG medium under the same conditions. The results are shown in Table 1. Me
By adding 0.50 to 1.09/day of β-CD,
It can be seen that approximately the same number of colonies were formed as in the case of BG medium. 0.5-5.09/ of casamino acids
The presence of 41 made the morphology of the emergent colony more obvious.

Table 1 Example 2 Add 109 amino acids to the basal medium of normal SSB medium.
/ U, and glutathione is added to 1.
A replacement fluid adjusted to have 5 times ascorbic acid and 20 times ascorbic acid was added at a ratio of 1.0% (V/V).

The improved SSB medium thus obtained (with 109 casamino acids)
0.4 g/Jl of ascorbic acid, 0.159/41 of glutathione), and 0.0 g/Jl of Meβ-CD.
．． 0.05, 0.5. 2.0. 5. t) g/
After adding 200 d of each to a Sakaro flask, add 1.5 x 10 of pertussis 1 phase bacteria Higashihama strain.
The cells were inoculated at 9 cells/me and cultured at 35°C. The results are shown in Figures 1 and 2.

Figure 1 shows the relationship between bacterial concentration (IOU#) and culture time.
The figure shows the time course of LPF-HA activity. It can be seen that when the medium of the present invention is used, the proliferation of Bordetella pertussis and the production of LPF-HA are remarkable.

It can also be seen that this effect is promoted by the addition of Meβ-CD.

In addition, the bacterial concentration and LPF-1-(A activity) were determined by the following method.
*) was determined by measuring. l0tJ and I internat
It is an abbreviation for ional Opacity Ll nit, and corresponds to [l01J=109111i1/Ni].

LPF-HA activity is determined by LP produced by the following method.
F-HA was collected and its activity was measured. The centrifuged supernatant (1) 88.6) of the culture solution was passed through a hydroxyapatite column equilibrated with 0.01 M phosphate buffer at l) 88.0, and the resulting effluent was passed through a hydroxyapatite column equilibrated with l) H6,0. After adjusting to 0.01 M phosphorus 8111% of DH6.0
Adsorption onto a hydroxyapatite column equilibrated with liquid6
1. This was eluted with OjM phosphate buffer (pH 7.0) containing 0.5M sodium chloride to obtain a protein fraction. This protein fraction was adsorbed on affinity chromatography using haptoglobin-Sepharose as a support.
P F -HA was desorbed with 0.1 M Tris buffer containing 5 M NaCl and 3 M potassium thiocyanide.
I got it. LPF-HA activity was determined using the enzyme antibody method (ELISA method) of Sahara et al., the 28th Toxin Symposium (198
January 23rd to 24th, 2015, Hataira, Iwate Prefecture) Collection of lecture abstracts,
(see pages 141-144), L P F
-1-IA The activity unit (tl) of A is OD 400m
It was calculated as the dilution factor for each sample giving 0.1 per unit volume (rd).

Example 3 Ascorbic acid, glutathione in the same manner as in Example 2 (but without adding Meβ-CD).

Example 2 A culture medium containing various concentrations of casamino acids was prepared.
Inoculate and culture Bordetella pertussis in the same manner as above, and inoculate with Shun's L for 48 hours.
PF-HA activity was measured.

The results were as shown in Table 2.3.4.

2nd surface power IJ''mino acid = 10 g/l Table 3 Table 4 Reduction C 1 long geltade A: 0.155 J/day Example 4 (1) Cultivation Add 109/l to the basal medium of normal SSB medium. Add casamino acids and 29/Jl of Meβ-CD, and add 1.0 g of replacement fluid adjusted to have 20 glutathionic acids.
% (V/V). The improvement S thus obtained
In SB medium 41, 1. Bordetella pertussis was inoculated at a concentration of 109 OX/-, and cultured using a 5.0 incubator. After 48 hours, the bacterial concentration was approximately 10″/ad, L.
PF-HA had an ELISA value of 1250 U/ae. The supernatant (3.6 Jl) obtained by centrifuging the finished culture solution at 6000 rpm x 3G was used for subsequent LPF-HAw.
It was made by I.

(2) 1 tM'l of Isl solution or 8 LPF-HA
J culture supernatant 3.61 (EIH8,3) was equilibrated with 0.01 molar phosphate buffer (EIH8) at around 4°C on a hytroxylapatite (B D l-1 Chemiforces) column (100 m). ) at a flow rate of 200 m/h. 0.01 M phosphate buffer (pl-18)
Wash the column with 4.0.0. Ofl
was adjusted to DH6.0 with concentrated hydrochloric acid. Add this solution to 0.0
A flow rate of 10 was applied to a Hyde O xylapatite column (140 m) equilibrated with 1 M phosphate buffer (IIH6,0).
0−/hr r″ flow. 0.01 M phosphate buffer (p
Wash the column with 0.5 mL (6.0), then 0.1 M in Xl buffer (1ll-17,0) 25
The sample was washed at 0 m, and further eluted and fractionated with 0.1 molar phosphate buffer (+)-17,0) containing 0.5 molar sodium chloride. The resulting protein fractions were collected (100 d) and passed through a haptoglobin-Sepharose 4Bi lum (30 ml!) equilibrated with 0.1 molar phosphate buffer containing 0.5 molar saline. The adsorbed material was eluted and fractionated into 9 fractions with a 0.1 molar Milli solution containing 3 molar thiocyanate and 0.5 molar common salt. The resulting protein fractions were collected and added to 0.0
．． Dialyzed against 1 molar phosphate buffer (OH7,Q) to
20.7 mg of PF-)-IA was obtained.

This product was prepared using polyacrylamide gel (polyacrylamide l [7.5%, INKOH-glacial acetic acid! III solution (p
H4,3>) shows a single band in disk electrophoresis,
The electrophoresis position matched that of existing LPF-1-IA. Also, the ELISA of this item is 111. It was OU/μ3.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the relationship between bacterial concentration and culture time when using the medium of the present invention, and FIG.
FIG. 3 is a diagram showing the relationship between activity and culture time. Figure 1: 4 gin hats hr) Figure 2, 1700 trowel, 11 gin (shi) ・ :O Mu: O, OS O? 0,5 11:2.0 Law 5.0 Procedural amendment (method) September 1985 Shinto date

Claims (1)

[Claims] 1. When culturing microorganisms belonging to the genus Bordetella, casamino acids are added at 0.1 to 20 g/
Bordetella spp., characterized by using a medium containing 0.01 to 1 g/l of ascorbic acid, 0.1 to 5 g/l of glutathione, and 0.001 to 5 g/l of cyclodextrin or its derivatives. Cultivation method of related microorganisms. 2. A medium for detecting and/or culturing microorganisms belonging to the genus Bordetella, containing 0.1 to 20 g of casamino acids.
1. A medium containing ascorbic acid, 0.01 to 1 g/l, glutathione, 0.1 to 5 g/l, and cyclodextrin or a derivative thereof, 0.001 to 5 g/l.