JPS624701A - Lipopolysaccharide and its production - Google Patents

Abstract

PURPOSE:To obtain a lipopolysaccharide in good yield, by extracting bacterial cells, ground or not ground, obtained by cultivating actinomyces and/or their related bacteria with a surfactant solution and purifying the culture by a combination of enzymatic digestion, molecular weight fractionation and solvent fractionation. CONSTITUTION:The bacterial cells obtained by cultivating actiomyces and/or their related bacteria are killed by an appropriate method to obtain killed bacterial cells. These cells, ground or not ground, are extracted with a surfactant solution to obtain and extract. A suitable organic solvent is added to this extract to percipitate a crude polysaccharide. The obtained crude polysaccharide is purified by a combination of enzymatic digestion, MW fractionation and solvent fractionation to obtain a lipopolysaccharide. This lipopolysaccharide consists of 5-20wt% fatty acid and 80-95wt% polysaccharides of a composition of one D-arabinose unit and 1-3 D-mannose units. This lipopolysaccharide has activation such as antitumoral effect, cell-rejuvenating effect and immunopotentiation effect.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to lipopolysaccharide and a method for producing the same. More specifically, the present invention relates to a lipopolysaccharide having physiological activities such as antitumor activity, immunomodulatory activity, cell activation activity, and infection prevention activity, and a method for efficiently producing lipopolysaccharide from actinomycetes and/or related bacteria.

[Prior Art] Live Mycobacterium tuberculosis BCG cells, anaerobic Propionibacterium cells, etc. have been used as immune enhancers and are known to have antitumor activity, but on the other hand, they have strong side effects. It is also clear that this phenomenon occurs, which poses an obstacle to clinical application.

Therefore, various attempts have been made to extract active ingredients from these effective bacterial cells or related bacterial cells and to remove side-effect components.

In particular, research on the bacterial body components and their activities of bacteria such as Mycobacterium, Propionibacterium, and Nocardia has been actively conducted, and various physiologically active components have been isolated and purified, and further modifications have been made to these components. Attempts to increase the activity have also been abandoned. That is, cell wall skeleton components (Unexamined Japanese Patent Publication No. 5
No. 4-28813, etc.), muramyl dipeptide (Unexamined Japanese Patent Publication No. 5
No. 2-156812 (1!), modified products of muramyl dipeptide (Japanese Patent Publication No. 56-49396, etc.), adjuvant active substances (Japanese Patent Publication No. 58-409, etc.), hot water extracts (Japanese Patent Publication No. 48-43842, etc.) et al.), tumor immunotherapeutics containing lipopolysaccharide as an active ingredient (Japanese Patent Application Laid-Open No. 56-8320, etc.) have already been disclosed, and recently lipopolysaccharide and its production method (Japanese Patent Application Laid-open No. 59-161320) have been disclosed. Disclosed.

[Problems to be solved by the invention] However, all of these lipopolysaccharides are not necessarily sufficient in terms of activity and side effects, and their production methods do not have very high yields, so further improvements are needed. It was wanted.

[Means for Solving the Problems] The present inventors have discovered that the large tuberculosis bacterium Mycobacterium laberculosis, the viable tuberculosis bacterium Mycobacterium bovis, and the non-pathogenic anaerobe Propionibacterium acnes Research is being conducted to obtain ingredients with higher yields and purify them into forms with high specific activity, such as those that do not cause side effects and exhibit efficacy such as antitumor activity, immunomodulatory activity, and cell rejuvenation activity. I have continued to do so.

As a result, they discovered that the target product could be obtained in good yield by using nonionic or ionic surfactants and physically crushing these bacterial cells, and crude lipopolysaccharide The present invention was completed based on the discovery that the specific activity could be increased several times by fractionating the protein using a combination of organic solvents and then removing protein using an immobilized protease.

That is, the present invention provides a polysaccharide 8O-95 (Kan 1) having a composition of 1 D-arabinose and 1 to 3 D-mannose.
A lipopolysaccharide consisting of 5% to 20% (w/su) fatty acids, and a surfactant with or without crushing the bacterial cells obtained by culturing actinomycetes and/or related bacteria. The present invention relates to a method for producing lipopolysaccharides, which is characterized by extraction with a solution and purification by a combination of enzymatic digestion, molecular weight fractionation, and solvent fractionation.

The method for producing lipopolysaccharide of the present invention will be explained.

Bacterial cells obtained by culturing aerobically or anaerobically in an appropriate medium are collected by sterile filtration or centrifugation and sterilized by an appropriate method, or directly sterilized and killed in the culture medium by an appropriate method. Obtain bacterial cells. After thoroughly washing the bacterial cells, they are crushed by physical means such as freeze-thawing, ultrasonication, or French press. The crushing may be carried out in an aqueous suspension or in a suspended state in an aqueous solution of a surfactant. If the crushed bacterial cells are in an aqueous suspension, add a surfactant to the suspension, or if they are in the presence of a surfactant, stir at room temperature, remove the bacterial layer by centrifugation, and prepare the extract. obtain. still,
If the dead bacteria are not crushed, the dead bacteria are stirred in an aqueous surfactant solution and then filtered or centrifuged to obtain an extract.

A suitable organic solvent is added to the thus obtained extract to precipitate the crude polysaccharide. The obtained crude polysaccharide is emulsified in an appropriate buffered salt solution and treated with amylase and amyloglycosidase. After the enzymes are precipitated and removed by heat treatment, the crude polysaccharide expressed as a polymer is purified by ultrafiltration or a molecular sieve column. The popolysaccharide fraction is separated and freeze-dried.

The obtained crude lipopolysaccharide is fractionated using a suitable combination of organic solvents such as pyridine-methanol, chloroform-methanol, etc. to remove mixed phospholipids and the like. If proteins are present, the purified lipopolysaccharide is obtained by dissolving in an appropriate salt solution, treating with an immobilized protease such as actinase E, and freeze-drying.

The bacteria used are Actinomycetes and related bacteria [Berge
y'S Manual New Edition (1974) Bacterial Classification System], the bacterial cells used may have been degreased in advance with a combination of appropriate organic solvents, or It may be treated with a protein-digesting enzyme.

The lipopolysaccharide of the present invention has a white to slightly off-white powder appearance and is slightly cloudy and soluble in distilled water or physiological saline, but is insoluble in organic solvents. The composition of the substance of the present invention is a polysaccharide whose main constituent sugars are D-arabinose and D-mannose, to which C14 to C10 fatty acids are ester-bonded, and the polysaccharide has 80 to 95
(corrected buttocks)% and fatty acids 5-20 (W/W)%. In addition, this substance is thought to form a hydrophobic micelle structure in an aqueous solvent, and in various molecular weight measurement methods,
It exhibits behavior similar to that of a polymer.

The ribopolysaccharide of the present invention can also be extracted with an alkaline aqueous solution or an alkaline buffer, and can be obtained by similar purification operations. However, with strong alkali or weak alkali heating, arabinomannan with a molecular weight of about 12,000 is saponified.
Provides mannan with a molecular weight of about 7000 and alkali salts of fatty acids.

[Effect] Next, regarding the physiological activities of the ribopolysaccharide obtained by the above procedure, we will discuss in detail the test results of antitumor activity, cell rejuvenation activity, polyclonal B cell activation ability, and phagocytic activation ability. explain. In addition, the test substances include Examples 1.2 and 2.
The ribopolysaccharide prepared in step 3 was used, and a corresponding amount of physiological saline was used as a control as appropriate.

First, the test results of antitumor activity using tumor growth suppressive effect and tumor regression effect as indicators will be explained.

Syngeneic methylcholanthrene induced subcutaneous injection in the upper abdomen of 8-week-old C57BL/611tlt mice! II tumor MC-1 was inoculated at 1 x 10''/mouse, and 10 mice were transplanted as 1 u10 mice.
On the second day, a sample dissolved in 0.2 ml of physiological saline is intraperitoneally administered, and on the 24th day after transplantation, the tumor is excised and weighed. The T/C value was calculated using the following formula to determine the tumor growth inhibitory effect. The control group received 0.2 d of physiological saline on the same schedule, and the results are shown in Table 1.

Table 1 Antitumor Activity As a result of this test, it was found that the lipopolymer of the present invention had an extremely excellent growth-inhibiting effect and regression effect on mouse syngeneic tumors.

Next, the test results for cell rejuvenation activity will be explained.

Normal tile cells from an 8-week-old C57BL/6 female mouse were taken out and cultured with 10 ua of each ribopolymer obtained in each example, and the amount of 3H-demidine taken up by the cells was measured. The cell activation effect of the multi-body was determined. As a control, cells cultured with a corresponding amount of physiological saline were used. The test was carried out using cells from the same individual animal in each test group, and the test was repeated on five individual animals.

The results of the test are shown in Table 2.

As a result of this test, it was found that all of the ribopolysaccharides of the present invention significantly enhanced thymidine uptake in mouse tile cells and had excellent cell activation and cell rejuvenation effects.

Table 2 Cell rejuvenation effect Next, the results of the polyclonal B cell activation test will be explained.

Normal tile cells from a 100-week-old CDF+ female mouse were taken out and cultured with 10μ9 of the lipopolysaccharide volume obtained in each example, and the activity was evaluated by counting anti-trinitrophenylated horse red blood cell plaque-forming cells. As a control, cells cultured with a corresponding amount of physiological saline were used. The test was conducted using cells from the same individual animal for each test group.
The same test was repeated for two individual animals.

The test results are shown in Table 3.

Table 3 Results of polyclonal B cell activation ability test,
It was found that all of the lipopolysaccharides of the present invention have the ability to activate polyclonal B cells.

Next, the test results of phagocyte activation effect will be explained.

10 JJg of the lipopolysaccharide obtained in each example (dissolved in 0.2 ypl of physiological saline) was intraperitoneally administered to 8-week-old BALB/c male mice. After 2 days, the ascites was removed, and the peritoneal cells were collected in a plastic petri dish. 2X10 cells attached to
1×10 cells of syngeneic tumor (Meth) A cells were added to each cell, and after culturing for 24 hours in a medium supplemented with fetal bovine serum,
Day 3 - After adding thymidine and incubating for an additional 16 hours,
The effect of activated macrophages on the proliferation of tumor cells was determined based on the amount of H uptake, and the ability of the sample to activate macrophages was determined based on this. Table 4 shows the results of a test in which 1 group consisted of 10 animals and a corresponding amount of physiological saline was administered to the control group.

Table 4: Inhibitory effect on tumor cell growth by macrophages The results of this test confirmed that the lipopolysaccharide of the present invention is a substance that promotes the activation of macrophages in mice.

[Examples] The present invention will be explained in more detail by Examples, but the present invention is not limited in any way by these Examples.

Example 1 Mycobacterium laberculosis blue and white B()1yc
Obacterium tuberculosis 5
train AoyamaB) in two-tone medium.
After culturing at 15°C for 15 weeks and heat sterilizing at 100'C for 20 minutes, the dead bacteria obtained by filtration and washing with water was
/W)% Triton X-100 aqueous solution (approximately 5 of wet bacterial weight
After suspending and dispersing the cells by stirring and ultrasonication, the cells were further crushed using a French press (1500 kg/cm2). After centrifugation at 40,000xg for 30 minutes, 9 times the amount of ethyl alcohol was added to the supernatant obtained, stirred, and both precipitated fractions were collected by centrifugation at 5,000xg for 15 minutes, followed by ethyl alcohol. Wash with ethyl ether and dry. The resulting precipitate is treated with amylase and amyloglycosidase in an acetate buffer, heated and then centrifuged to remove the denatured enzyme. Both components with a molecular weight of 100,000 or more are concentrated by ultrafiltration and freeze-dried to obtain crude ribopolysaccharide.

The procedure of dissolving this substance in pyridine and adding methyl alcohol to precipitate it was repeated three times, and the solvent-soluble fraction was removed.
Next, the washing operation with chloroform-methyl alcohol is repeated to remove the solvent-soluble fraction. Further, the precipitate was dissolved in a phosphate buffer, and after shaking treatment with actinase immobilized on Sepharose 4B in the phosphate buffer, the immobilized enzyme was removed, and the solution was desalted by dialysis. Lyophilize to obtain lipopolysaccharide. The yield was 0.3% of the wet bacterial weight.

As a result of analysis, the composition of this substance is a polysaccharide 91 whose constituent sugars are D-7 rabinose: D-mannose (1:2.5).
(W/W)% and saturated fat n15m9 (1
4hl) It was a lipopolysaccharide consisting of χ.

Example 2 Mycobacterium bovis B CG ()lycoba
cte-rium bovis BCG) in a two-tone medium at 37°C for 15 weeks, filtered and sterilized, and the resulting bacterial cells were washed with water, washed several times with acetone, degreased, dried, and suspended in water. French press (1500ka/c
m2). Trines X-100 were added to give a final concentration of 1.5 (Δ/en)%, and stirring was continued for 24 hours at air temperature. Centrifugation @ (40,000X!II,
The supernatant obtained after 30 minutes) is treated in the same manner as in Example 1 to obtain lipopolysaccharide. The yield was about 1% of the dry bacterial cells.

As a result of analysis, D-arabinose: D-mannose (1:
88 (W/W)% polysaccharide having 3) as the main constituent sugar,
A lipopolysaccharide consisting of 12 saturated fatty acids (Δ/Old %), mainly valmitic acid and clostearic acid.

Example 3 Propionibacterium acnes CC-7 (Prop
ionibacteriu acnes C-7) was statically cultured under anaerobic conditions for 4 days in a modified thioglycolic acid liquid medium supplemented with meat extract, and the bacterial cells obtained by centrifugation were washed with water, and then concentrated at 1 (W/W)%. The cells were suspended in a solution of sodium lauryl sulfate and thulium, treated 6 times for 5 minutes with a 20KtlZ ultrasonic cell disrupter, and then centrifuged (40,000
xg, 30 minutes) is treated in the same manner as in Example 1 to obtain lipopolysaccharide.

The yield was 0.8% of the wet bacterial weight.

Analysis results D-arabinose: D-mannose (1:
Polysaccharide with 2.8> as the main constituent sugar 894/W)%
The composition was mainly 11% (en/kei) of fatty acids with CI5 to C+a.

[Effects of the Invention] The lipopolysaccharide according to the present invention is obtained from the order Actinomycetes and related bacteria, and is a polysaccharide whose main constituent sugars are D-arabinose and D-mannose, as well as valmitic acid and labeled clostearic acid. As explained in detail in the test examples, it is a lipopolysaccharide in which fatty acids are ester-linked, and it is an excellent substance that has anti-tumor effects, cell rejuvenation ability, immune-enhancing effects, etc., and is a tumor immunity enhancer. Alternatively, it is expected to be put to practical use as an immunomodulator.

Furthermore, the production method of the present invention allows lipopolysaccharide to be obtained from bacterial cells in a high yield through relatively simple operations, and is therefore advantageous for industrial production.

Claims (1)

[Claims] 1) D-arabinose is 1 to D-mannose 1 to 1
A lipopolysaccharide consisting of 80-95 (W/W)% of a polysaccharide having a composition of 3 and 5-20 (W/W)% of a fatty acid. 2) A lipopolysaccharide characterized by extracting bacterial cells obtained by culturing actinomycetes and/or related bacteria with a surfactant solution, and purifying them by a combination of enzymatic digestion, molecular weight fractionation, and solvent fractionation. manufacturing method. 3) The bacterial cells obtained by culturing actinomycetes and/or their related bacteria are crushed, extracted with a surfactant solution, and purified by a combination of enzymatic digestion, molecular weight fractionation, and solvent fractionation. A method for producing lipopolysaccharide.