JPH0690745A - Lps-producing microorganism, lps and pharmaceutical and animal drug containing lps - Google Patents

Abstract

PURPOSE:To provide a new immune function activating agent which can be administered by oral administration, transcutaneous administration and injection, an analgesic, their animal drugs, new LPS acting as the active component of the agent and new bacterial strain capable of producing the LPS. CONSTITUTION:Three kinds of LPS compounds having the following physical properties. (1) LPS1, principal molecular weight, 5,000+ or -1,000 (SDS-PAGE process); number of phosphorus atoms, 2+ or -1/5,000 molecular weight; number of hexosamine groups, 9+ or -1/5,000 molecular weight; number of KDO groups, 2+ or -1/5,000 molecular weight: (2) LPS2, principal molecular weight, 6,500+ or -2,500 (SDS-PAGE process); number of phosphorus atoms, 1-2/5,000 molecular weight; number of hexosamine groups, 7+ or -1/5,000 molecular weight; number of KDO groups, 1-2/5,000 molecular weight: (3) LPS3, principal molecular weight, 6,500+ or -2,500 (SDS-PAGE process); number of phosphorus atoms, 2+ or -1/5,000 molecular weight; number of hexosamine groups, 5+ or -1/5,000 molecular weight; number of KDO groups, 2+ or -1/5,000 molecular weight.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel LPS-producing bacterium,
The present invention relates to a novel LPS, a drug containing the novel LPS, and an animal drug. More specifically, the present invention relates to three novel glucose-fermenting Gram-negative bacilli producing LPS, novel LPS derived therefrom, and novel immune function activators, analgesics containing these LPS, And a novel immune function activator and analgesic for animals.

[0002]

2. Description of the Related Art An organism has an immune function for preventing the internal environment of the organism from being disturbed by foreign and endogenous foreign substances and maintaining homeostasis of the organism. Therefore,
Deterioration of immune function causes deterioration of health, onset of various diseases, and promotion of aging, and its activation leads to improvement of health, prevention of onset of various diseases, healing, and prevention of aging. Therefore, there is a demand for the provision of substances that activate the immune function.
PSK [also known as Krestin (registered trademark of Kureha Chemical Co., Ltd.)], lentinan (registered trademark of Ajinomoto Co., Inc.), bestatin (registered trademark of Nippon Kayaku Co., Ltd.), Sonifiran (registered trademark of Kaken Pharmaceutical Co., Ltd.), OK- 432 [Cancer-chemotherapy report to part 1 (C
ancer Chemotherapy Report
s Part1), vol. 58, No. 1 and 10 (1972), also known as picibanil (registered trademark of Chugai Pharmaceutical Co., Ltd.)] and the like are known.

Analgesics are roughly classified into narcotic analgesics and non-narcotic analgesics. Since narcotic analgesics are narcotics, the utmost caution is required when administering them.
(Pages 70-74 of "Clinical Pain" published by Medical Friend Co., Ltd. in 1981) On the other hand, the analgesic action of non-narcotic analgesics is generally weaker than that of narcotics and is non-habitual. However, it is considered that the drug should be treated pharmacologically in exactly the same manner as narcotic analgesics, such as resistance and dependence in long-term use. (7 of “Clinical Pain” above)
(P. 4)

[0004]

Among the conventional immune function activators, PSK, lentinan, bestatin, and sonifiran do not have TNF-producing ability, and therefore their immune function-activating ability is low. On the other hand, although OK-432 has TNF-producing ability, fever, chills,
The occurrence of side effects such as lower blood pressure and thrombocytopenia cannot be avoided,
Therefore, the chemotherapy coefficient is small. Further, since simple oral administration or transdermal administration has no effect, convenience in administration is lacking.
Here, "TNF" refers to a tumor-damaging factor (Tumor Necrosis F) produced by macrophages.
generic name of the actor [The Journal of Biological Chemistry (The Journal of biological
Biol. Chem. ) 260, 2345-2354
Page (1985)], and the production amount increases as the activity of macrophages increases. "Macrofar
"Di" is a type of immunocompetent cell, and is a generic term for large ameboid cells that are distributed in almost all tissues in the animal body and prey on and digest particulate foreign substances and waste cells in the body. . “Chemotherapeutic coefficient” refers to the ratio of the maximum tolerated dose of the host to the drug and the minimum effective concentration of the drug to the pathogen, and the larger this value, the better the chemotherapeutic agent.

Further, the analgesics currently used have the above-mentioned drawbacks, and no satisfactory one has been provided yet. In particular, as an analgesic for chronic pain, development of a drug that is highly safe, has no side effects, is inexpensive and has a simple administration method is strongly desired.

The present invention provides a novel immune function activator, analgesic agent, and a novel immune function activator, analgesic agent for animals, in which the above-mentioned drawbacks of the prior art are solved, and It is a technical subject to provide a novel LPS that is an active ingredient, and to provide a novel bacterium that produces those LPS.

[0007]

[Means for Solving the Problems] The above technical problems have a high ability to activate immune functions and an analgesic effect, a high chemotherapeutic index, a long-term use, and any of oral, transdermal, and injection. This can be achieved by providing a novel LPS that can be administered by a route, has a low production cost, can be supplied in a large amount, and provides a novel bacterium as a source of these LPSs.

Bacterial Separation Source The three kinds of bacteria of the present invention have been isolated from wheat examined by the present inventors regardless of their origin and type. Therefore, it is presumed that it is separated from wheat and wheat products of all production areas and types. The origins and types of flours that the present inventors have confirmed to be able to separate these three types of bacteria are as follows. Name-producing areas da of wheat flour - click, Northern Springs US 1 Canadian Hui - Doo Canada ha - de Red wintering - - Semiha - de the United States - Stora Rian Sutanda - De Hui - The reserve − Stralia Horosiri Japan

Separation of LPS In order to separate the LPS of the present invention from the above-mentioned bacteria, Westphal et al. Described “Methods in Carbohydrate Chemistry”.
n Carbohydrate Chemistry)
Vol. V [published by Academic Press, Inc., New York, USA, 1965], page 83, using the thermal phenol method and further purified with an anion exchange resin. That is, after suspending the cells in distilled water, the cells are stirred together with distilled water and an equal volume of hot phenol, and then the aqueous layer is recovered by centrifugation, and the aqueous layer is dialyzed to obtain phenol. Was removed and concentrated by ultrafiltration to obtain a crude LPS fraction. This fraction was subjected to a conventional method, for example, using a Pharmacia FPLC system to produce a Pharmacia Mono Q-Sepharose (Sephar).
ose) and Q-Sepharose, followed by purification by anion exchange chromatography and further desalting according to a conventional method. By the above operation, a purified sample with a purity of 96% or more can be obtained.

Physical Properties of LPS As will be described in detail in Examples below, three types of LPS of the present invention
The physical properties of (96% or more pure sample) were as follows.
(SDS-PAGE method is defined in Example 1) LPS1 Main molecular weight: 5,000 ± 1,000 (S
DS-PAGE method) Phosphorus number: 2 ± 1 / molecular weight 5,000 Hexosamine number: 9 ± 1 / molecular weight 5,000 KDO number: 2 ± 1 / molecular weight 5,000 LPS2 Main molecular weight: 6,500 ± 2,500 ( S
DS-PAGE method) Phosphorus number: 1-2 / molecular weight 5,000 Hexosamine number: 7 ± 1 / molecular weight 5,000 KDO number: 1-2 / molecular weight 5,000 LPS3 Main molecular weight: 6,500 ± 2,500 ( S
DS-PAGE method) Phosphorus number: 2 ± 1 / molecular weight 5,000 Hexosamine number: 5 ± 1 / molecular weight 5,000 KDO number: 2 ± 1 / molecular weight 5,000

Form of Provision The LPS of the present invention can be provided as it is or in a concentrated form to an arbitrary degree. Further, in order to improve the storage stability, it can be provided as a dry powder by any means such as freeze drying and spray drying. Any of these can be produced by a conventional method.

Measurement of immunostimulatory ability The immunostimulatory ability of the LPS of the present invention was confirmed by the ability to produce endogenous TNF through macrophage activity.

In order to produce TNF in the animal body,
The requirement of a production precursor (priming) stage and a production initiation (triggering) stage is due to the fact that Carswell (Car
Swell) et al., Proceeding of National Academic Science of USA
(Proc. Natl. Acad. Sci. US
A. ), 72, 3666-3670 (1975), after which investigations on drugs that can be used at each stage are also being promoted. The drug administered for the initiation of the priming phase is "primer" (endogenous TNF production promoter), and the drug administered for the initiation of the triggering phase is "trigger" (endogenous TNF production agent) is there.

[0014] TNF activity is determined by L-929 cells [procedure
Ding of National Academic Science of USA 72, 3666-3670]
It is measured as follows based on the cytotoxicity against. L929 cells were grown in an Igrum minimum essential medium (hereinafter, referred to as MEM medium) supplemented with 5% fetal bovine serum so that 8 × 10 ⁴ cells were contained in 100 μl of the same medium, and 96 wells were prepared. Breed on a flat bottom plate. Breeding conditions are 37 ⁰ C, 2 hours, 5% CO ₂ , 100%
H ₂ O, which may be a method used in ordinary cell culture. After that, actinomycin D was added to the medium at a final concentration of 1 μm.
g / ml and add 150 μl of culture solution
And Immediately, add 50 μl of the sample diluted appropriately with MEM medium (at this time, adjust the dilution ratio appropriately, and add ED
You can ask for ₅₀ ). Further, L929 cells having a final volume of 200 μl are cultured under the above conditions for 18 hours.

To measure the cytotoxic activity, first, the whole medium is removed, and then a 1% methyl alcohol solution containing 0.1% crystal violet is added to perform fixed staining. Crystal violet stains all nucleated cells, but dead cells are removed by washing with water from the bottom of the plate after staining.
The cytotoxic activity can be measured directly from the results of viable cells. This staining degree is measured using the absorbance at OD (590 nm) as an index, and the cytotoxic activity is measured by comparing with the staining degree for the control group. The definition of activity is as follows. L929
The dilution ratio (N) of the specimen in which the cells can survive 50% is determined.
As a control, rabbit TNS [tumor-damaged serum (Tumor
Necrosis Serum)], and the activity n (unit / ml) of this rabbit TNS is determined using 2.4 × 10 ⁶ units / mg / ml TNF-α. The dilution ratio (C) giving the ED _{50 of} this rabbit TNS is determined. The sample activity (unit / ml) is calculated by N / C × n.

Measurement of Analgesic Effect The analgesic effect of the LPS of the present invention has been established as one of non-narcotic analgesic assay methods, “acetic acid-rising (Wr.
Iting method ”(page 415 of“ Inflammation and anti-inflammatory therapy ”published by Ito Denryaku Shuppan Co., Ltd. in 1982).

The LPSs of the present invention can be used separately, and as long as the intended use thereof is not impaired, they can be used in any combination of two or more kinds thereof, or in combination with any other substance. it can.
Also, as immune function test agents, animal immune function test agents, quasi-drugs, cosmetics, foods, functional foods, beverages, feeds, etc.,
Alternatively, it can be used as one component thereof.

Any of the immunological activators including the LPS of the present invention can be administered as an oral drug, an intravenous drug, an intramuscular drug, a transdermal drug, by a conventional formulation technique or a conventional animal drug production method. As a single agent or as a mixture with other drugs, powders, granules, pills, tablets, troches, capsules, solutions, patches, ointments, liniments, lotions, suppositories, It can be provided in the form of an injection or the like. Especially for skin
Since it has a large amount of dice, it is more effective when administered as a skin application. Further, for animals, it can be further prepared as a feed additive, a premix preparation, and a drinking water additive. When used as a feed additive, it is preferably a powder or granules. The premix formulation refers to a product diluted with a feed component such as starch to facilitate mixing with a feed.

The feed additive containing the LPS of the present invention and the feed to which the premix preparation can be added may be any commercially available feed. It may also be a feed containing feed additives such as minerals, vitamins and amino acids.

If desired, additives such as excipients, preservatives and buffers may be added to these preparations according to a conventional method in order to maintain the storability and homogeneity. Further, a corrigent, a flavoring agent, and a coloring agent can be included. As the excipient, for example, lactose or starch can be used. Examples of preservatives that can be used include paraoxybenzoic acid esters such as methyl paraoxybenzoate, ethyl paraoxybenzoate and propyl paraoxybenzoate, sodium dehydroacetate, phenol, methylparaben, ethylparaben, propylparaben and the like. As the buffer, for example, citrate, acetate, phosphate and the like can be used.

Hereinafter, the present invention will be described in more detail with reference to Examples, Production Examples and Experimental Examples. The "Escherichia coli LPS" used in them is O128: B8 manufactured by Difco, USA.

Example 1 1.04 g of hard wheat flour (Canadian wheat from Canada) containing 1.09% ash was weighed and put into a 50 ml coning tube, and 20 ml of distilled water was added. Was added to prepare a 50 mg / ml flour solution.

[0023] The liquid and cultured with shaking in a water bath at 37 ⁰ C, at age 0, 1:00, 2:00, 3:00, 4:00, 6:00, 8
0 o'clock, 10 o'clock, 12 o'clock, 20 o'clock, 24 o'clock, 45 o'clock each.
5 ml was collected, diluted 10 ⁰ to 10 ⁵ times, and added to a standard agar medium (a medium manufactured by Nissui Pharmaceutical Co., Ltd., which has the following composition).
A 0 μl portion was sown, and the viable cell count was measured and the colony was observed.

Standard agar medium (Code No. 05618, Nissui Pharmaceutical Co., Ltd.) In 1 liter Yeast extract 2.5 g Peptone 5.0 g Glucose 1.0 g Agar 15.0 g pH 7.1 ± 0.1

Yellow to cream-colored opaque colonies (colony-1) and cream-colored opaque colonies (colony 2), yellow translucent colony (colony
3), milky white opaque colony (Coloni-4) and white opaque small colony (Coloni-5) are spread on another standard agar medium of the same kind as above, and subcultured, while colony-1 to
Gram stainability and limulus activity of 5 bacteria were examined.

The term "limulus activity" as used herein means to be positive in a limulus test, which is an endotoxin assay method using a horseshoe crab blood cell extract and a chromogenic synthetic substrate, which was invented by Levin in 1968. This limulus test is known as the LPS detection method,
For example, it can be carried out using a reagent set commercially available from Seikagaku Corporation under the name Toxicolor System.

Among the above colonies, the limus activity of Colony-4 and Colony-5 (both Gram stain +) was extremely lower than that of Colonies 1-3 (both Gram stain −). Except for the above, the morphology and biochemical properties of colonies 1-3 were observed using Nissui Pharmaceutical's medium and ID test EB-20. The following results were obtained.

Bacteria forming colony-1 (identification number: 9
(0081-1) (Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Microbial Technology)
Deposited in Japan as Microbiology Research Institute Contribution No. 11664 from August 17, 2013, and Microconservation Research Article Contribution No. 350 from August 12, 1991.
No. 9 was transferred to international deposit in accordance with the Budapest Treaty)

Based on the morphology and biochemical properties described below, the bacterium is presumed to belong to the genus Serratia of the Enterobacteriaceae family. (a) Morphology Short rod-like motility None Gram stainability :-( b) Growth state Standard agar medium: A round opaque colony of yellow to cream color is formed. SS agar: forms white, translucent colonies. [SS agar medium: Nissui Pharmaceutical Co., Ltd. code number: 05031] In 1 liter of composition Meat extract 5.0 g Bile salt 9.0 g Peptone 7.5 g Lactose 10.0 g Sodium citrate 8.5 g Sodium thiosulfate 5.5 g Ferric citrate 1.0 g Neutral red 0.025 g Brilliant gulin 0.033 g Agar 13.5 g pH: 7.1 ± 0.1 TSI agar medium: No change on slope, but higher layer The part turns yellow. Produces gas. [TSI agar medium: Nissui Pharmaceutical Co., Ltd. code number: 0510
3] Composition 1 liters Meat extract 5.0 g NaCl 5.0 g Peptone 15.0 g Lactose 10.0 g Sucrose 10.0 g Glucose 1.0 g Ferric citrate 0.2 g Sodium thiosulfate 0.2 g Feno -Ruled 0.02g Agar 15.0g pH: 7.6 ± 0.1 (c) Physiological properties Forges-Proscher's reaction: + Indole formation:-Hydrogen sulfide formation: -Use of citric acid : + Urease:-Oxidase:-OF test: + (d) Availability of carbon source Lactose: + Adnit:-Rhamnose: + Mannit: + Esculin: + Inosit:-Sorbit : + Arabinose: + Raffinose: + (10) Sucrose: + (e) Others Decarboxylation of lysine:-Utilization of malonic acid:-Decomposition of arginine:-Phenylalanine Deamination reaction of: -Decarboxylation of ornithine:-

Bacteria forming colony-2 (identification number: 9
(Headed to the Institute of Microbial Technology, Ministry of International Trade and Industry, Institute of Industrial Technology, Heisei 2)
Deposited in Japan as Microbiology Research Institute No. 11665 from August 17, 1996, and Microbiology Research Article No. 351 from August 12, 1991.
No. 0 was transferred to the international deposit in accordance with the Budapest Treaty)

Based on the morphology and biochemical properties described below, this bacterium is presumed to belong to the genus Enterobacter of the Enterobacteriaceae family. (a) Morphology, short rod shape, no mobility Gram stainability :-( b) Growth state Standard agar medium: A opaque colony with a cream color is formed. SS agar: A red, opaque colony is formed. TSI agar medium: There is no change on the slope, but the upper part turns yellow. Produces gas. (c) Physiological properties Forges-Proschauer reaction: + Indole formation:-Hydrogen sulfide formation:-Citric acid utilization: + Urease:-Oxidase:-OF test: + (d) Utilization of carbon source Lactose: + Adonite:-Rhamnose: + Mannitol: + Esculin: + Inosit:-Sorbite: + Arabinose: + Raffinose: + (10) Sucrose : + (E) Others Decarboxylation of lysine:-Utilization of malonic acid: + Decomposition of arginine: + Deamination of phenylalanine:-Decarboxylation of ornithine: +

Bacteria forming colony-3 (identification number: 9
(Heisei 2 in the Institute of Microbial Technology, Ministry of International Trade and Industry, Institute of Industrial Science and Technology)
Deposited in Japan as Microbiology Research Institute No. 11666 from August 17, 1996, and Microbiology Research Article No. 351 from August 12, 1991.
No. 1 was transferred to an international deposit in accordance with the Budapest Treaty)

Based on the morphology and biochemical properties described below, the bacterium is presumed to belong to the genus Pantoea of the Enterobacteriaceae family. (a) Morphology Short rod-like shape No mobility Gram stainability :-( b) Growth state Standard agar medium: A yellow, round, translucent colony is formed. SS agar medium: Does not form colonies. TSI agar medium: There is no change on the slope, but the upper part turns yellow. Does not generate gas. (c) Physiological properties Forges-Proschauer reaction: + Indole formation:-Hydrogen sulfide formation:-Citric acid utilization: + Urease:-Oxidase:-OF test: + (d) Utilization of carbon source Lactose: + Adonite:-Rhamnose: + Mannitol: + Esculin: + Inosit: -Sorbit: + Arabinose: + Raffinose:-(10) Sucrose : + (E) Others Decarboxylation of lysine:-Utilization of malonic acid: + Decomposition of arginine:-Deamination of phenylalanine:-Decarboxylation of ornithine:-

Colonies-1, 2 and 3 were each transferred to 1 liter of L-broth medium and shaken overnight at 37 ⁰ C,
5,000 G, cells were collected by centrifugation for 20 min at 4 ⁰ C. In addition, this L-broth medium was used for Difco (Difc).
o) Polypeptone 10g, yeast extract 5g,
Add Wako Pure Chemical Industries' special grade NaCl (5 g) to distilled water, and add N
It was adjusted to pH 7.5 with aOH, autoclaved, and separately prepared in advance with Wako Pure Chemical Industries' special grade glucose 40
% Solution was diluted 400 times and added.

Each of the cells was suspended in 50 ml of distilled water, and 50 ml of 90% hot phenol was added to the suspension to give 6 cells.
It stirred 5 to 70 ⁰ C for 20 minutes, after cooling, 10,00
The aqueous layer was collected by centrifugation at ⁰ G and 40 C for 20 minutes. The phenol layer was subjected to the same operation as above twice more. The three aqueous layers were combined, dialyzed overnight to remove the phenol, and the internal solution was advantech toyo (ADVA
It was subjected to ultrafiltration using UK-200 manufactured by NTEC TOYO) and concentrated by a molecular weight cut-off of 200,000 (N ₂ pressure: 2 atm).

This concentrated sample was used as Q-Sepharose Fast Flow (Q-Seph) manufactured by Pharmacia.
Arose Fast Flow) and subjected to anion exchange chromatography. That is, after applying the sample to the column with a buffer solution containing 10 mM Tris-HCl (pH 7.5) and 10 mM NaCl, 400 mM NaCl /
The limulus active fraction was eluted with 10 mM Tris-HCl (pH 7.5). This eluate is subjected to ultrafiltration under the same conditions as above to desalinize and concentrate to obtain LPS with a purity of 96% or more.
Got The nucleic acid is 1M NaCl / 10 mM Tris-
Elute with HCl (pH 7.5).

The results of each bacterial cell are shown in Tables 1 to 3 below. In addition, the amount of LPS is E. coli L
PS conversion value, sugar is phenol-sulfuric acid method [M. Authored by M. Dubois, Analytical
Chemistry (Analytical Chemistr
y), vol. 28, p. 350, 1956], the protein is the Lory method [O .. H. Rolly (OH Low
ry) et al., Journal of Biological Chemistry.
Chemistry)], vol. 193, page 65, 1
951]. In addition, the amount of nucleic acid is OD (260n
Based on the measured value in m) (1 OD = 50 μg), the purity (%) was calculated based on the following formula.

[0038]

[Equation 1]

[0039]

[Table 1]

[0040]

[Table 2]

[0041]

[Table 3]

Molecular weight LPS obtained from each microbial cell was dissolved in distilled water to obtain 2 m.
A g / ml solution was prepared and 10 μl thereof was placed in a 1.5 ml plastic tube. Separately, 180μ
l 10% (w / v) SDS, 45 μl 5% β-mercaptoethanol, 90 μl CBB dye solution, 11
2.5 μl of 0.5 M Tris-HCl (pH 6.8) and 2
SDS treatment solution 10 prepared by adding 2.5 μl of distilled water
μl was added and mixed well, then immersed in a boiling water bath for 5 minutes. Immediately after this heating, it was immersed in ice water and rapidly cooled.

10 ml of 10% (w / v) SDS, 1
The migration buffer prepared by dissolving 7.9 g of tricine and 3.03 g of Tris in 1 liter of distilled water was placed in a slab gel electrophoresis tank manufactured by Marisol. A 20% polyacrylamide gel was fixed in an electrophoretic bath, a sample was put in a sample groove, the voltage was fixed at 50 v for 1 hour, and then fixed at 150 v, and the electrophoresis was continued until the dye was eluted from the gel (herein). This electrophoresis method is referred to as SDS-PAGE method). After electrophoresis, Bio-Rad's silver staining kit 161-04
Silver staining was carried out at room temperature using No. 43 to confirm the behavior.

Protein molecular weight marker simultaneously run [LMW kit E: Phosphorylase b manufactured by Pharmacia Co.]
(94k), albumin (67k), ovalbumin (43k), carbonic anhydrase (30k),
Trypsin inhibitor (20k), α-lactalbumin (14k)], peptide molecular weight marker [Pharmacia 1860-101 molecular weight marker: myoglobin (16.9k), myoglobin I & II (14.4).
k), myoglobin I (8.2k), myoglobin II
(6.0 k), myoglobin IV (2.5 k)] was used to calculate the molecular weight of the LPS of the present invention,
00 ± 1,000 (L derived from bacterial cell 900814-1
PS1), 6,500 ± 2,500 (Bacterial body 900814)
2) and LPS3) derived from bacterial cells 900814-3). E. coli LPS measured in the same manner (E. coli 0127: B8LP manufactured by Difco)
The molecular weight of S) was 30,000 ± 20,000.

LPS1, LPS2 in the above silver staining,
The stained band of LPS3 is shown in FIG. In FIG. 1, number 1
Is LPS1, number 2 is LPS2, number 3 is LPS3
It is a dyed belt. As shown in FIG. 1, LPS1 also showed a slightly aggregated staining band even at a molecular weight of around 30,000. LP
S2 has a dyeing zone between 30,000 and 43,000, but it is presumed that the number of macromolecules is extremely small in comparison with the dyeing degree of the dyeing zone of 14,000 or less.
LPS2 can be judged from the amount of sugar and hexosamine described below.
Has the lowest sugar content, followed by LPS3 and LPS1 in that order, which is considered to be in agreement with the pattern observed by electrophoresis. Also, the ratio of LPS amount / total dry yield is LPS.
2, LPS3, LPS1 in this order. From the above observation results, it is estimated that LPS2 has a relatively large amount of low-molecular-weight LPS, and then the ratio of LPS3 and LPS1 decreases in that order.

Phosphorus Content Chen-Toribara Method [Chen et al., "Analytical Chemistry (Ana
(Lytical Chemistry), vol. Two
8, 1756-1758 (1956).

LPS1, LPS2 and LPS3 were separately dissolved in distilled water to give 31.6 μg and 57.6 μg, respectively.
20 μl of a solution containing g, 103.6 μg of LPS was prepared and placed in a small test tube. 20 μl of 50 v / v% sulfuric acid was added and heated at 160 ⁰ C for 2 hours. Then 20
After adding 10 μl of 10 v / v% perchloric acid, the mixture was heated with a gas burner for 1 minute to incinerate. Then 0.5 ml distilled water, then 0.5 ml reaction reagent (1 ml 6N sulfuric acid, 2 ml distilled water, 2 ml 2.5 v / w% ammonium molybdate and 1 ml 10 v / w% ascorbic acid were mixed. Was prepared, and 0.5 ml thereof was added) and the mixture was allowed to stand at room temperature for 30 minutes, and then the absorbance OD at 820 nm (820 nm) was measured. As a sample for preparing the calibration curve, potassium dihydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) was diluted with distilled water to obtain phosphoric acid weights of 2.
0.5m including 5μg, 1μg, 0.25μg, 0μg
1 solution was prepared and used. In addition, 1 g of phosphorus corresponds to 4.39 g of potassium dihydrogen phosphate. The results are shown in Table 4 below. In the table, the numerical value indicating the absorbance is a value obtained by subtracting the data of the control which has not been heat-treated in order to avoid an error due to the incorporation of inorganic phosphorus (for example, originating from a phosphate buffer solution). The phosphorus amount (μg) is a value calculated from the absorbance. The phosphorus amount (% by weight) was calculated by the following formula. In addition,
In the formula, “0.67” refers to the OD value of 1 μg of standard phosphorus, and the sample concentration refers to the concentration (mg / ml) of each LPS dissolved in distilled water.

[0048]

[Equation 2]

The phosphorus number is a conversion number per 5,000 molecular weight calculated by the following formula.

[0050]

[Equation 3]

[0051]

[Table 4]

Hexosamine Content Elson-Morgan Method (Tokyo Kagaku Dojin Shuppan "Biochemistry Experiment Course" No. 4, 37)
7 to 379) and performed as follows.

1.58 mg of LPS dissolved in distilled water
(LPS1), 2.88 mg (LPS2), 5.18 m
A solution of g (LPS3) / ml was prepared and 100 μl thereof
Was placed in a Spitz with screw cap (manufactured by Iwaki Glass Co., Ltd.), 100 μl of 8N HCl was added thereto, and the mixture was heated at 110 ⁰ C for 16 hours. 4N NaOH about 20
The pH was adjusted to 7 by adding 0 μl. Aliquot 100 μl into another Spitz with screw cap,
After adding ⁰ μl of the following Reagent A, 1.
Heat for 5 hours, then cool with running water. Then 100
Aliquot μl, add 670 μl of 96% ethanol,
Further, 67 μl of the following reagent B was added, and the mixture was allowed to stand at room temperature for 1 hour, and the absorbance was measured at 535 nm. As a sample for preparing a calibration curve, 0.20 to 200 μg / ml N-acetylglucosamine (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

(Reagent A) 75 μl of acetylacetone and 2.5 ml of 1.25N sodium carbonate were mixed and prepared.

(Reagent B) 1.6 g of p-dimethylbenzaldehyde, 30 ml of concentrated hydrochloric acid and 30 ml of 96% ethanol were mixed and prepared. Result, LPS1, LPS
2. The hexosamine numbers of LPS3 were 9 ± 1 / molecular weight 5,000, 7 ± 1 / molecular weight 5,000, 5 ± 1 / molecular weight 5,000, respectively.

(10) KDO content The content of KDO (2-keto-3-deoxyoctaneate) was determined by the diphenylamine method [Shabby (Shabyal.
R. ) Et al., Analytical Biochem (Analy
mechanical Biochem. ), 58 (1), 123
~ 129 (1974)].

A KDO detection reagent was prepared by combining 500 mg of diphenylamine, 5 ml of ethanol, 45 ml of glacial acetic acid and 50 ml of concentrated hydrochloric acid (all manufactured by Wako Pure Chemical Industries, Ltd.). In 500 µl thereof, (1) 0.505 mg / ml of LPS1 was added in 250 µl distilled water solution; (2) 0.57
250 μl distilled aqueous solution containing 6 mg / ml LPS2;
(3) 250μ containing 0.518 mg / ml LPS3
l distilled water; combined either, 3 after heating in 100 ⁰ C boiling water bath for 33 minutes in cold water (24.5 ⁰ C)
Cool for 0 minutes, then use Hitachi spectrophotometer 320
UV absorption was measured at 0, 470, 630 and 650 nm (measured values were A420, A470, A630 and A, respectively).
650). As a standard sample, 0.5 μmol / m
1 KDO ammonium salt [US Sigma]
250 μl of distilled water containing] was used.

The value of the following equation was determined for each of the specimen sample and the standard sample. S = A420-A470 + A630-A650

The value (S _T ) of the sample specimen is 0.1 for LPS1.
09, LPS2 was 0.078, and LPS3 was 0.099. The value of the standard sample (S _S) is 0.246, the value of the distilled water alone was 0.005.

By comparing these values, LPS1 has a value of 2 ± 1.
/ Molecular weight 5,000, 1-2 for LPS2 / Molecular weight 5,
KD of 2 ± 1 / molecular weight of 5,000 for LPS3 and 5,000
It was estimated that O was included.

Note that these values are calculated as follows, taking LPS1 as an example. If the concentration of KDD contained in the solution is χ (μmol / ml),

[0062]

[Equation 4]

From the above equation, χ = 0.221. Therefore, letting y be the number of moles of KDD contained in 1 mole of LPS1 (assuming 5,000), y = 2.1
It becomes 9.

[0064]

[Equation 5]

The following is a formulation example of the preparation containing the LPS of the present invention. The LPS amount in Examples 2 to 5 is the E. coli LPS conversion amount by the limulus test.

Example 2 (tablet) LPS1 0.04 g 6% HPC lactose 178 g talc stearate 8 g potato starch 14 g The above ingredients were mixed and compressed to give 400 tablets of 0.5 g containing 0.1 mg of wheat LPS. Was prepared.

Example 3 (internal liquid formulation) LPS1 1 mg Purified water 100 ml

Example 4 (ointment)

Example 5 (injection)

Production Example 1 (Production of Bordetella pertussis LPS) A test Bordetella pertussis solution (2.
0 × 10 ¹⁰ cells / ml) were used as dead cells.

25 mg (dry weight) / ml of the dead cells
Was suspended in sterilized water. This equal volume of 90% heat phenol - added Le solution (68~70 ⁰ C), 1 in 68 ⁰ C
Extracted with shaking for hours. 8,000G, 2 at 4 ⁰ C
The aqueous layer was separated by centrifugation for 0 minutes. The same extraction was performed by adding the same amount of sterilized water as the above aqueous layer to the remaining phenol layer. The obtained aqueous layer was combined with the previous aqueous layer, dialyzed in running water overnight, and then concentrated to 1/10 with a rotary evaporator. This 8,000 G, was separated 20 minutes Centrifuge centered at 4 ⁰ C. Separate the supernatant, add a small amount of sodium acetate, and
4 ⁰ C cold ethanol - was left by adding 6 times Le overnight at -20 ⁰ C. 4,000 G, 4 ⁰ to C in the precipitate was collected by centrifugation for 30 min ethanol - 2 times Le, then once by centrifugation washed with acetone, aspirator - dried with data.

The residue was suspended in distilled water so as to have a concentration of 20 mg / ml, and subjected to ultrasonic treatment (output control 5, 15 minutes, room temperature) with a Sonifier 185 manufactured by Branson, USA. Then 2,500G, 4
^The mixture was centrifuged at ⁰ C for 10 minutes, and the supernatant was collected.

[0073] In this supernatant 4 ⁰ C, the United States sigma (Sig
ma) nucleic acid degrading enzymes DNaseI, RNase
Treated with A for 15 to 16 hours (finally 10 μg / m
l DNase I and 20 μg / ml RNase
A was used). Furthermore, the same concentration of nucleic acid degrading enzyme was added and the mixture was heated at 37 ⁰ C for 2 hours. Then 2,500G, 4 ⁰ C
The mixture was centrifuged at 10 minutes for 10 minutes, and the supernatant was collected.

This supernatant was used in Gelman, USA
It was filtered with a pore size of 0.2 μm using an Acrodisc of the company. The filtrate was passed through a molecular sieve [resin: Sepharo manufactured by Pharmacia, USA].
Sepharose 6B, column size = inner diameter 5
cm × length 100 cm, buffer = 10 mM Tris-H
Cl, 10 mM NaCl (pH 7.5), flow rate = about 3
ml / cm ² / hour), the limus activity positive fractions were examined and combined using an LS-1 kit manufactured by Seikagaku Corporation, and filtered with the acrodisc manufactured by Gelman Co. at a pore size of 0.2 μm. The filtrate is subjected to ion exchange [apparatus: FPLC manufactured by Pharmacia, USA, resin: Mono Q HR10 / 10 manufactured by Pharmacia, USA, buffer = 1
0 mM Tris-HCl + 10 mM NaCl (pH
7.5) for 15 minutes, then adjust the amount of NaCl to 165
wash to 30 mM for 30 minutes, then for 20 minutes,
Washing while increasing the amount of NaCl so that the amount of NaCl becomes a concentration gradient from 165 mM to 1 M, and then washing with 1 M amount of NaCl for 30 times, flow rate = 2 ml / min], LS-1 kit manufactured by Seikagaku Corporation Fractions positive for limulus activity were examined by using and were combined. The combined fractions were desalted on a column [resin: Sephadex G-25 fine manufactured by Pharmacia, USA, column size = inner diameter 2 cm × length 25 cm, eluent = distilled water], and then freeze-dried. did.

The substance most likely contaminated with this freeze-dried preparation (4.50 mg) is nucleic acid. Therefore,
Take an ultraviolet absorption curve (200-400 nm), 260n
The absorbance at m was determined. Nucleic acid concentration is 5 when absorbance is 1.
It was 1% or less when the nucleic acid concentration was calculated from the above absorbance using 0 μg / ml. Also, SDS-PAG
No protein was clearly detected by method E. Therefore,
Considering the detection sensitivity, the protein mixed in the freeze-dried preparation is estimated to be 0 to 3% at most. Therefore, the purity of the freeze-dried preparation was estimated to be 96% or higher.

The physical properties of this B. pertussis LPS measured in the same manner as in Example 1 were as follows. Physical properties of B. pertussis LPS Main molecular weight = 6,000 ± 1,000 (SDS-PAG
E method) Phosphorus number = 4 / molecular weight 6,000 Hexosamine number = 12 / molecular weight 6,000 Fatty acid number = 4 / molecular weight 6,000 KDO number = 2 ± 1 / molecular weight 6,000

The physical properties of Escherichia coli LPS measured in the same manner were as follows. Physical properties of Escherichia coli LPS Main molecular weight = 40,000 ± 10,000 8,000 ± 4,000 (by SDS-PAGE method) Phosphorus number = 12 / molecular weight 30,000 Hexosamine number = 45 ± 6 / molecular weight 30,000 Fatty acid number = 18 / Molecular weight 30,000 Number of KDO = 5 ± 1 / Molecular weight 30,000

Experimental Example 1 (Immune function activating effect) Two or three mice in each group (7-week-old male C3H / H)
e. Average weight 25g. ) In the tail vein of 1, 10 or 100 μg of limulus activity per animal, LPS1, LP
0.2 ml of physiological saline containing S2 and LPS3 was injected, and 1 hour after that, serum was collected, and TNF activity was measured based on the toxicity to L929 cells. The results are shown in Table 5 below as the average of 2 or 3 animals in each group. In the table,
The number in () indicates the number of mice.

[0079]

[Table 5]

Experimental Example 2 (Analgesic Effect) Five C3H / He male mice (average body weight: about 28 g) of each group of 7 to 10 weeks of age were converted into LPS at 0, 1, 5, respectively.
200 μl of distilled water prepared to contain 25,400 μg / animal of each of LPS3 or E. coli LPS of the present invention was orally administered by a sonde. 1.5 hours after that, 500 μl of 0.7% acetic acid was intraperitoneally administered over 5 minutes, and the number of writhing of each mouse was counted over 30 minutes, and the results shown in Table 6 were obtained (5 for each group). Average of animals). "-" In the table
Indicates that the corresponding amount was not measured. In addition, "wrestling prevention rate (%)" was calculated by the following formula.

[0081]

[Equation 6]

[0082]

[Table 6]

FIG. 2 is a graph of the results shown in Table 6. From Figure 2, LPS3's writhing inhibition rate ED50
Was estimated to be 2.8 μg / animal and that of E. coli LPS was 17 μg / animal. Therefore, regarding the analgesic effect, LPS3
Is estimated to be about 6 times more effective than E. coli LPS.

Dose, Dosing Interval, Toxicity Value When the LPS of the present invention is administered as an immune function activator or analgesic, or as an animal immune function activator or analgesic, the amount and interval of administration are , Of course, under the strict control of the attending physician or veterinarian, the age and symptoms of the administration target,
Although it is determined individually in consideration of body weight and administration effect, it is an adult human (60 kg), and 1 μg to 100 mg by oral administration,
10 ng to 10 mg by intravenous administration, 100 ng by transdermal administration
A dose of ~ 1 mg is a rough guideline once a day. For animals, for large animals such as cows and horses, use 1/60 of the above amount as a guideline for the amount per 1 kg of body weight, and for medium-sized and small animals such as pigs, dogs and cats, double the amount. The dose per kg can be used as a standard, and for birds such as chickens, double the dose can be administered as a guide for the amount per kg of body weight.

Incidentally, Behrence Kelver (Behre
ns K

[Outer 1] rber) method, 7-week-old average body weight 22 g
, LPS1, LPS2 in C3H / He male mice
LDS of LPS3 is 150, 180, 180 respectively
μg / animal, and E. coli LPS value of 300 μg / animal 6
It was 0% or less. Also, E. coli LPS, B. pertussis LPS
Toxicity value LD ₅₀ of (Production Example 1) (2 male BALBs per group)
/ C mice, average body weight 45 g) were 3.4 and 11 mg / kg for intravenous administration and 16, 32 mg / kg for intradermal administration, respectively.

[0086]

INDUSTRIAL APPLICABILITY The present invention provides a novel bacterium, a novel LPS derived therefrom, and a novel immune function activator, analgesic, animal immune function activator, and analgesic containing the same.

Further, the LPS of the present invention can be easily used as a main component or a single component of medicines, veterinary medicines, test agents, quasi drugs, cosmetics, foods, functional foods, beverages, feeds and the like by a conventional method. It can be blended.

[Brief description of drawings]

FIG. 1 is a diagram showing a pattern of LPS of the present invention in an SDS-PAGE method.

FIG. 2 is a graph showing the analgesic effect of LPS of the present invention in comparison with E. coli LPS.

[Explanation of symbols]

In FIG. 1, 1 is a pattern of LPS1, 2 is a pattern of LPS2, and 3 is a pattern of LPS3. In FIG. 2, □ indicates the data of LPS of the present invention, and ◯ indicates the data of Escherichia coli LPS.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location A61K 37/20 ADR AER C08B 37/00 P 7329-4C C12P 19/04 C 7432-4B // ( (C12N 1/20 C12R 1:01) (C12P 19/04 C12R 1:01) (72) Inventor Daisuke Tsukioka 1-2-21-17 Kasuga, Chiba City, Chiba Prefecture (72) Inventor Denichi Mizuno 18 Okamoto City, Kamakura City, Kanagawa Prefecture (72) Inventor Haruyuki Oshima 1097 Tatemachi Tachimachi, Hachioji City, Tokyo 2-1 −513

Claims (10)

[Claims] 1. An LPS-producing Gram-negative bacillus having the following properties: (a) Morphology Short rod-like motility None Gram stainability :-( b) Growth state Standard agar medium: A round opaque colony of yellow to cream color is formed. SS agar: forms white, translucent colonies. TSI agar medium: There is no change on the slope, but the upper part turns yellow. Produces gas. (c) Physiological properties Forges-Proschauer reaction: + Indole formation:-Hydrogen sulfide formation:-Citric acid utilization: + Urease:-Oxidase:-OF test: + (d) Utilization of carbon source Lactose: + Adonite:-Rhamnose: + Mannitol: + Esculin: + Inosit:-Sorbite: + Arabinose: + Raffinose: + (10) Sucrose : + (E) Others Decarboxylation reaction of lysine:-Utilization of malonic acid:-Decomposition of arginine:-Deamination reaction of phenylalanine:-Decarboxylation reaction of ornithine:- 2. An LPS-producing Gram-negative bacilli having the following properties. (a) Morphology, short rod shape, no mobility Gram stainability :-( b) Growth state Standard agar medium: A opaque colony with a cream color is formed. SS agar: A red, opaque colony is formed. TSI agar medium: There is no change on the slope, but the upper part turns yellow. Produces gas. (c) Physiological properties Forges-Proschauer reaction: + Indole formation:-Hydrogen sulfide formation:-Citric acid utilization: + Urease:-Oxidase:-OF test: + (d) Utilization of carbon source Lactose: + Adonite:-Rhamnose: + Mannitol: + Esculin: + Inosit:-Sorbite: + Arabinose: + Raffinose: + (10) Sucrose : + (E) Others Decarboxylation of lysine:-Utilization of malonic acid: + Decomposition of arginine: + Deamination of phenylalanine:-Decarboxylation of ornithine: + 3. An LPS-producing Gram-negative bacilli having the following properties. (a) Morphology Short rod-like shape No mobility Gram stainability :-( b) Growth state Standard agar medium: A yellow, round, translucent colony is formed. SS agar medium: Does not form colonies. TSI agar medium: There is no change on the slope, but the upper part turns yellow. Does not generate gas. (c) Physiological properties Forges-Proschauer reaction: + Indole formation:-Hydrogen sulfide formation:-Citric acid utilization: + Urease:-Oxidase:-OF test: + (d) Utilization of carbon source Lactose: + Adonite:-Rhamnose: + Mannitol: + Esculin: + Inosit: -Sorbit: + Arabinose: + Raffinose:-(10) Sucrose : + (E) Others Decarboxylation of lysine:-Utilization of malonic acid: + Decomposition of arginine:-Deamination of phenylalanine:-Decarboxylation of ornithine:- 4. The LPS derived from the bacterium according to claim 1, which exhibits the following physical properties. Main molecular weight: 5,000 ± 1,000 (SDS-PAG
E method) Phosphorus number: 2 ± 1 / molecular weight 5,000 Hexosamine number: 9 ± 1 / molecular weight 5,000 KDO number: 2 ± 1 / molecular weight 5,000 5. The LPS derived from the bacterium according to claim 2, which exhibits the following physical properties. Main molecular weight: 6,500 ± 2,500 (SDS-PAG
E method) Phosphorus number: 1-2 / molecular weight 5,000 Hexosamine number: 7 ± 1 / molecular weight 5,000 KDO number: 1-2 / molecular weight 5,000 6. The LPS derived from the bacterium according to claim 3, which exhibits the following physical properties. Main molecular weight: 6,500 ± 2,500 (SDS-PAG
E method) Phosphorus number: 2 ± 1 / molecular weight 5,000 Hexosamine number: 5 ± 1 / molecular weight 5,000 KDO number: 2 ± 1 / molecular weight 5,000 7. The L according to any one of claims 4 to 6.
LP selected from the group consisting of PS and mixtures thereof
An immune function activator containing S. 8. The L according to any one of claims 4 to 6.
LP selected from the group consisting of PS and mixtures thereof
An analgesic containing S. 9. The L according to any one of claims 4 to 6.
LP selected from the group consisting of PS and mixtures thereof
An animal immune function activator containing S. 10. L selected from the group consisting of LPS according to any one of claims 4 to 6 and mixtures thereof.
An analgesic for animals containing PS.