JP2024042229A - Composition containing lipopolysaccharide - Google Patents

Abstract

[Problem] The present invention aims to provide a composition containing LPS of a gram-negative bacterium having a specific structure or specific activity. [Solution] A composition containing lipopolysaccharide of a gram-negative bacterium, said lipopolysaccharide having lipid A, said lipid A having a specific structure and having a specific limulus activity. [Selected Figure] None

Description

The present invention relates to compositions containing lipopolysaccharides of Gram-negative bacteria.

Lipopolysaccharide (hereinafter, also referred to as "LPS" or "lipopolysaccharide") is a component that constitutes the cell wall of gram-negative bacteria such as Escherichia coli. When gram-negative bacteria are killed or destroyed, LPS released from them can cause reactions such as fever in mammals, and can even be fatal if administered in large quantities; therefore, LPS is also called endotoxin.

The basic structure of LPS includes a lipid A portion, which is a lipid portion, and a polysaccharide portion. The polysaccharide moiety is further composed of a core and O-antigen polysaccharide side chains. Normally, LPS is incorporated into the cell wall with lipid A embedded in the outer membrane and O antigen polysaccharide side chains protruding outward, and is not easily released from the cell wall. When Gram-negative bacteria are killed and cells are lysed, LPS is liberated, and when it acts on, for example, animal cells, various physiological activities are expressed.

The released LPS exerts its action via TLR4 (toll like receptor 4) on the cell membrane of target cells. When LPS binds to TLR4, the production of inflammatory cytokines such as TNFα, IL-6, and IL-12 and type I interferon (IFN) is triggered via the intracellular signal transduction system (Non-Patent Document 1). In particular, LPS such as E. coli activates these signals simultaneously and strongly, causing strong inflammation.

In this way, LPS has the effect of promoting cytokine production via the TLR4 signal transduction system. Cytokine production generally leads to strong immunostimulatory activity and plays a certain role in biological defense reactions for protection against bacterial infection.

As described above, since LPS is known to have immunostimulatory activity, the use of the immunostimulatory activity of LPS in pharmaceuticals, cosmetics, foods, feeds, etc. is also being considered. However, when obtaining LPS from Gram-negative bacteria using the usual method, it is difficult to obtain LPS with high concentration or high activity.

Furthermore, although the basic structure of LPS is similar among Gram-negative bacteria, it is known that the structure of LPS differs depending on the type of Gram-negative bacteria from which it is derived. However, it is generally not easy to determine the structure of LPS of Gram-negative bacteria.

Biochemistry, 79, 769-776, 2007

An object of the present invention is to provide a composition containing LPS of a Gram-negative bacterium, in which the LPS has a predetermined structure or activity.

を有する、請求項１～３のいずれかに記載の組成物。
［５］
グラム陰性菌がパラコッカス属に属する細菌である、請求項１～４のいずれか１項に記載の組成物。 The present inventor conducted extensive research and discovered a composition containing LPS of a Gram-negative bacterium having a predetermined structure or a predetermined activity. That is, the present invention is as follows.
[1]
A composition comprising a lipopolysaccharide of a gram-negative bacterium, the lipopolysaccharide having lipid A;
Four 3-hydroxyacyl chains having 8 to 16 carbon atoms are bonded to the glucosamine skeleton of Lipid A, and a hydroxyl group having 8 to 13 carbon atoms is bonded to the 3-hydroxyl group of one or two of the 3-hydroxyacyl chains. The acyl chain is further bonded,
A composition having a Limulus activity of 30,000 EU/mg or more.
[2]
Four 3-hydroxyacyl chains having 10 to 14 carbon atoms are bonded to the glucosamine skeleton of lipid A, and the 3-hydroxyacyl chain having 10 to 14 carbon atoms is bonded to the 2-position of the glucosamine skeleton. The composition according to claim 1, wherein an acyl chain having 12 carbon atoms is further bonded to the hydroxyl group.
[3]
The acyl chain having 12 carbon atoms is bonded to the hydroxyl group at the 3-position of the 3-hydroxyacyl chain having 14 carbon atoms, which is bonded to the 2-position of glucosamine on the non-reducing end side, and is bonded to the 2-position of glucosamine on the reducing end side. The composition according to claim 2, which does not bond to the hydroxyl group at the 3-position of a 3-hydroxyacyl chain having 14 carbon atoms.
[4]
The lipid A has a structure of formula (I):

The composition according to any one of claims 1 to 3, having the following.
[5]
The composition according to any one of claims 1 to 4, wherein the Gram-negative bacterium is a bacterium belonging to the genus Paracoccus.

The present invention provides a composition comprising LPS of a gram-negative bacterium having lipid A having a defined structure and limulus activity.
The composition provided by the present invention has a specified structure and high limulus activity, and is therefore useful for utilizing the immunostimulatory activity of LPS in pharmaceuticals, cosmetics, foods, feeds, and the like.

A GC chart for fatty acid analysis is shown. The upper figure shows the analysis results of fatty acids derived from Paracoccus LPS, and the lower figure shows the analysis results of fatty acid standard mix. The GC-MS spectrum (retention time 9.440 min) is shown. The upper figure shows the analysis results of fatty acids derived from Paracoccus LPS, and the lower figure shows the database data for C12:1 (C5-C6 cis). A GC-MS spectrum (retention time 14.417 minutes) is shown. The upper figure shows the analysis results of fatty acids derived from Paracoccus LPS, and the lower figure shows the data recorded in the database for 3-OH C10:0. A GC-MS spectrum (retention time 23.010 minutes) is shown. The upper figure shows the analysis results of fatty acids derived from Paracoccus LPS, and the lower figure shows the data recorded in the database for 3-OH C14:0. The results of MALDI-TOF MS measurement are shown when the lipid A moiety of LPS derived from a bacterium belonging to the genus Paracoccus was measured in negative ion mode.

The present invention will be explained in more detail below. The scope of the present invention is not limited to these descriptions, and other than the examples below may be modified and implemented as appropriate without departing from the spirit of the present invention.

1. Gram-negative bacteria LPS
The composition of the present invention contains LPS of Gram-negative bacteria. Gram-negative bacteria are known to have LPS in their cell walls. In the present invention, the Gram-negative bacteria may be any Gram-negative bacteria that has LPS, and examples thereof include, but are not limited to, Proteobacteria (E. coli, Salmonella, Pseudomonas, Paracoccus, etc.), Cyanobacteria, etc. Includes the phyla Spirochetes, Chlorobium, and Bacteroidetes. In the present invention, the Gram-negative bacterium is preferably a bacterium belonging to the genus Paracoccus.

In this specification, bacteria belonging to the genus Paracoccus are not particularly limited. As for the bacteria belonging to the genus Paracoccus, Paracoccus carotinifaciens, Paracoccus marcusii, Paracoccus haeundaensis, and Paracoccus zeaxanthinifaciens are preferably used, Paracoccus carotinifaciens or Paracoccus zeaxanthinifaciens are more preferably used, and Paracoccus carotinifaciens is particularly preferably used. Specific strains of bacteria belonging to the genus Paracoccus include, for example, Paracoccus carotinifaciens E-396 strain (FERM BP-4283), Paracoccus bacteria A-581-1 strain (FERM BP-4671), Paracoccus marcusii DSM 11574 strain, Paracoccus Bacteria of the genus N-81106 strain, Paracoccus haeundaensis BC 74171 strain, Paracoccus zeaxanthinifaciens ATCC 21588 strain, and Paracoccus sp. PC-1 strain are mentioned, and mutant strains of these are also preferably used in the present invention. Strains E-396 and A-581-1 are available at the National Institute of Technology and Evaluation (NITE) Patent Organism Depositary (NITE-IPOD) (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818) ) as the international depositary authority and has been internationally deposited as follows.
E-396 strain Identification indication: E-396, Accession number: FERM BP-4283, Original deposit date: April 27, 1993 A-581-1 strain Identification indication: A -581-1, Accession number: FERM BP-4671, Original deposit date: May 20, 1994

2. Structure of Lipid A LPS of Gram-negative bacteria has lipid A. In the present invention, lipid A has a skeleton of two glucosamine molecules in which two glucosamine molecules are bonded through a β (1→6) bond. In this specification, this lipid A skeleton is referred to as a "glucosamine skeleton."

In one embodiment of the present invention, a phosphate group is bound to position 1 of the glucosamine on the reducing end side of the glucosamine backbone, and a phosphate group is bound to position 4 of the glucosamine on the non-reducing end. In another embodiment of the present invention, the number of bound phosphates may be 1 to 3, and further, the phosphate may be modified. In another embodiment of the present invention, a hydroxyl group may be used instead of the phosphate group.

Lipid A in the present invention has four acyl chains having 8 to 16 carbon atoms directly bonded to the glucosamine skeleton, and the acyl chain has a hydroxyl group at the 3-position. In this specification, an acyl chain having a hydroxyl group at the 3-position is also referred to as a 3-hydroxyacyl chain.
That is, in lipid A, a 3-hydroxyacyl chain has an amide bond at the 2-position of glucosamine, and two molecules of glucosamine in which the 3-hydroxyacyl chain has an ester bond are bonded at the 3-position. The two molecules of glucosamine may be two molecules of the same glucosamine, or two molecules of glucosamine in which different 3-hydroxyacyl chains are bonded between the two molecules.

In the present invention, the number of carbon atoms in the 3-hydroxyacyl chain bonded to the 2nd and 3rd positions of glucosamine is 8 to 16, preferably 10 to 14, more preferably 10, 11, 12, 13 or 14. , more preferably 10 or 14.

The number of double bonds in the 3-hydroxyacyl chain directly bonded to the glucosamine skeleton is 0, 1 or 2, preferably 0.

In the present invention, the 3-hydroxyacyl chain attached to the glucosamine backbone is preferably 3-OH10:0 or 3-OH14:0.

In one aspect of the invention, the 3-hydroxyacyl chain attached to the 2-position of glucosamine is 3-OH14:0 and the 3-hydroxyacyl chain attached to the 3-position of glucosamine is 3-OH10:0.

Lipid A in the present invention has an acyl group having 8 to 13 carbon atoms in the hydroxyl group at the 3-position of the 3-hydroxyacyl chain in one or two of the four 3-hydroxyacyl chains directly bonded to the glucosamine skeleton. The chains are further ester bonded. In one embodiment of the present invention, an acyl chain having 8 to 13 carbon atoms is further ester bonded to the 3-hydroxyl group of the 3-hydroxyacyl chain (one or two) bonded to the 2-position of the glucosamine skeleton. Due to the attachment of such additional acyl chains, lipid A has 5 or 6 acyl chains.

In the present invention, the number of carbon atoms in the acyl chain bonded to the 3-hydroxyl group of the 3-hydroxyacyl chain directly bonded to the glucosamine skeleton is 8 to 13, preferably 8 to 12, and more preferably 12.

In the present invention, the number of double bonds in the acyl chain bonded to the 3-hydroxyl group of the 3-hydroxyacyl chain directly bonded to the glucosamine skeleton is 0, 1 or 2, preferably 1.

In the present invention, the acyl chain bonded to the 3-hydroxyl group of the 3-hydroxyacyl chain directly bonded to the glucosamine skeleton is preferably C12:1, more preferably the 5th carbon (counting from the carbon of the carbonyl group) It is C12:1 (C12:1(Δ ⁵ )) having a double bond (cis) between C5 and C6).

In one aspect of the present invention, the acyl chain bonded to the 3-hydroxyl group of the 3-hydroxyacyl chain directly bonded to the glucosamine skeleton is the acyl chain bonded to the 3-hydroxyl group of the 3-hydroxyacyl chain bonded to the 2-position of the glucosamine on the non-reducing end side. However, it does not bond to the hydroxyl group at the 3-position of the 3-hydroxyacyl chain, which is bonded to the 2-position of glucosamine on the reducing end side.

Examples of embodiments of lipid A in the present invention are listed below.
In one embodiment of the present invention, lipid A has four 3-hydroxyacyl chains having 8 to 16 carbon atoms bonded to the glucosamine skeleton, and the hydroxyl group at the 3-position of one or two of the 3-hydroxyacyl chains is bonded to the glucosamine skeleton. It has a structure in which an acyl chain having 8 to 13 carbon atoms is further bonded.

In another embodiment of the present invention, lipid A has four 3-hydroxyacyl chains having 10 to 14 carbon atoms bonded to the glucosamine skeleton, and 3-hydroxyacyl chains having 10 to 14 carbon atoms bonded to the 2-position of the glucosamine skeleton. It has a structure in which an acyl chain having 8 to 13 carbon atoms is further bonded to the hydroxyl group at the 3rd position of one or two of the hydroxyacyl chains.

In another embodiment of the present invention, lipid A has four 3-hydroxyacyl chains having 10 to 14 carbon atoms bonded to the glucosamine skeleton, and 3-hydroxyacyl chains having 10 to 14 carbon atoms bonded to the 2-position of the glucosamine skeleton. It has a structure in which an acyl chain having 12 carbon atoms is further bonded to the hydroxyl group at the 3rd position of one or two of the hydroxyacyl chains.

In another embodiment of the present invention, lipid A has four 3-hydroxyacyl chains having 10 to 14 carbon atoms bonded to the glucosamine skeleton, and 3-hydroxyacyl chains having 10 to 14 carbon atoms bonded to the 2-position of the glucosamine skeleton. An acyl chain having 12 carbon atoms is further bonded to the hydroxyl group at the 3-position of the hydroxyacyl chain,
The acyl chain having 12 carbon atoms is bonded to the hydroxyl group at the 3-position of the 3-hydroxyacyl chain having 14 carbon atoms, which is bonded to the 2-position of glucosamine on the non-reducing end side, and is bonded to the 2-position of glucosamine on the reducing end side. It has a structure that does not bond to the hydroxyl group at the 3-position of a 3-hydroxyacyl chain having 14 carbon atoms.

In another embodiment of the present invention, lipid A has four 3-hydroxyacyl chains having 10 or 14 carbon atoms bound to a glucosamine backbone, and an acyl chain having 12 carbon atoms further bound to the hydroxyl group at the 3-position of the 3-hydroxyacyl chain having 14 carbon atoms bound to the 2-position of the glucosamine backbone;
The structure has such that the acyl chain having 12 carbon atoms is bonded to the hydroxyl group at the 3-position of a 3-hydroxyacyl chain having 14 carbon atoms bonded to the 2-position of glucosamine on the non-reducing end side, but is not bonded to the hydroxyl group at the 3-position of the 3-hydroxyacyl chain having 14 carbon atoms bonded to the 2-position of glucosamine on the reducing end side.

In one embodiment of the present invention, lipid A has 3-OH14:0 bonded to the 2-position of glucosamine, 3-OH10:0 bonded to the 3-position of glucosamine, and a double bond (cis) between C5-6. It has a structure in which C12:1 contained in is bound to 3-OH14:0, which is bound to the 2-position of glucosamine on the non-reducing end side.

In one aspect of the invention, Lipid A has the structure of formula (I).

3. Composition of the Invention The present invention has found that a composition containing a treated Gram-negative bacterium can provide higher Limulus activity than conventional bacterial cells. The composition of the present invention can be produced by subjecting Gram-negative bacteria to treatments such as purification and extraction.

The composition of the present invention may be in the form of an extract or dried bacterial cells of a gram-negative bacterium, so long as it contains LPS of the gram-negative bacterium. For example, the composition of the present invention can be an extract obtained by appropriately culturing a gram-negative bacterium and extracting the culture obtained, or a dried bacterial cell obtained by drying the culture.

Further, before or after the above-mentioned extraction treatment, treatments such as concentration, drying, dilution, crushing, pulverization, and heating may be performed alone or in combination. Drying processes include spray drying, freeze drying, vacuum drying, and drum drying. The crushing process includes crushing by means such as a homogenizer, French press, mortar, glass beads, etc. It is expected that by crushing or crushing the bacterial cells, the surface area will increase and more LPS will be exposed at the interface. Those skilled in the art can culture Gram-negative bacteria and carry out treatments such as extraction according to known methods.

As mentioned above, dried bacterial cells of Gram-negative bacteria after cultivation or a processed product thereof can also be used as the composition of the present invention. Alternatively, commercially available products may be used. For example, Panaferd-AX (ENEOS Co., Ltd.), which is dried bacterial cells after culturing of bacteria belonging to the genus Paracoccus (Paracoccus carotinifaciens), can be used.

Those skilled in the art can further purify the extracted LPS derived from bacteria belonging to the genus Paracoccus. The purification method can follow a conventionally known method.

For example, Gram-negative bacteria can be treated using the hydrothermal phenol method (O. Westphal, K. Jann, "Methods in Carbohydrate Chemistry", ed. by R. Whistler, Vol. 5, p. 83, Academic Press, New York (1965)). , LPS is extracted by processing with phenol-chloroform-petroleum ether extraction (PCP) method (C. Galanos, O. Luderitz, O. Westphal, Eur. J. Biochem., 9, 245 (1969)), etc. can do.

The composition obtained in each purification step of the LPS or PCP method can be used as the composition of the present invention. Purification can improve the purity of LPS. For details of the PCP method, reference can be made to the description of Examples. The LPS extract of Gram-negative bacteria obtained by these methods can be used as the composition of the present invention. Furthermore, in the present invention, the structure of LPS could be specified using LPS highly purified by the PCP method.

In another aspect of the invention, the composition of the invention may contain lecithin or lysolecithin. One skilled in the art can incorporate lecithin or lysolecithin into the composition using conventional techniques. For example, an aqueous solution of lecithin or lysolecithin, an LPS extract of Gram-negative bacteria, dried bacterial cells after cultivation of Gram-negative bacteria, or a processed product thereof are mixed using a mixer or the like. By treating Gram-negative bacteria with lecithin or lysolecithin, which are amphipathic molecules, it is expected that the water dispersibility of LPS (particularly LPS of bacteria belonging to the genus Paracoccus) will be improved and the absorption thereof in animals will be improved.

LPS is known to have immunostimulatory activity. The composition of the present invention exhibits a limulus activity of 30,000 EU/mg or more and is therefore useful as a feed composition, a pharmaceutical composition, a food composition, and a cosmetic composition. When used as feed, medicine, food, or cosmetics, it may further contain a carrier acceptable for each use.

4. Limulus Activity It has been found that the composition of the present invention exhibits higher Limulus activity than before. Limulus activity is the coagulation-promoting activity of horseshoe crab blood cell extract and is the biological activity of endotoxin at the molecular level. Limulus activity is measured by the Limulus test, which detects or quantifies endotoxin using a lysate reagent prepared from horseshoe crab blood cell extract. The Limulus test includes a gelation method that uses gel formation of a lysate reagent as an indicator, and an optical quantitative method (nephelometry, colorimetry) that uses optical changes as an indicator. Nephelometry is a method for measuring endotoxin concentration by measuring changes in turbidity (absorbance or transmittance) accompanying gelation of a lysate reagent. In addition, the colorimetric method is a method of measuring the amount of chromogenic group released from a chromogenic synthetic substrate by a coagulating enzyme produced by a reaction between endotoxin and a lysate reagent using absorbance or transmittance. Colorimetric methods include kinetic colorimetry and endpoint colorimetry. Since the turbidimetric method and the colorimetric method show substantially equivalent Limulus activity values, in the present invention, the Limulus activity is measured using the turbidimetric method or the colorimetric method (kinetic colorimetric method). Limulus activity can be measured using a commercially available endotoxin measurement kit.

In the present invention, the Limulus activity measured by turbidimetry or colorimetry (kinetic colorimetry) is 30,000 EU/mg or more. Limulus activity in the present invention is preferably 4 x 10 ⁴ EU/mg or more, 1 x 10 ⁵ EU/mg or more, 1.2 x 10 ⁵ EU/mg or more, 1.9 x 10 ⁵ EU/mg or more, 1 ×10 ⁶ EU/mg or more, 2 × 10 ⁶ EU/mg or more, 3 × 10 ⁶ EU/mg or more, 5 × 10 ⁶ EU/mg or more, 1 × 10 ⁷ EU/mg or more, 2 × 10 ⁷ EU/mg mg or more, 3×10 ⁷ EU/mg or more, 4×10 ⁷ EU/mg or more, 4.5×10 ⁷ EU/mg or more, or 4.7×10 ⁷ EU/mg or more.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited only to these Examples.

Test 1. Purification of LPS Paracoccus carotinifaciens was used, and LPS derived from Paracoccus was purified by the PCP (phenol/chloroform/petroleum ether) method. Specifically, the purification of LPS derived from Bacterium Paracoccus was performed in the following four steps.

- Step 1: Degreasing dried paracoccus bacterial cells Panaferd-AX (ENEOS Corporation) was used as the dried bacterial cells. 60 g of dried bacterial cells were dispensed into 12 50 mL conical tubes (about 5 g/tube). Washing was performed in the order of ethanol (4 times), acetone (3 times), and diethyl ether (3 times) (40 mL/time for each tube). After washing, it was freeze-dried. Dry weight was 55.998g.

- Step 2: Extraction of LPS by PCP method Approximately 56 g of washed and degreased bacterial cells were added to a 500 mL wide-mouth glass reagent bottle. 300 mL of a PCP mixture consisting of liquid phenol (90 g + 11 mL of water), chloroform and petroleum ether (2:5:8) was added, and the mixture was stirred for 2 minutes with a Polytron under ice cooling. The suspension was transferred to 8 stainless steel centrifuge tubes and centrifuged at 5,000×g for 15 minutes using a high-speed refrigerated centrifuge model 7780, and the supernatant (150 mL) after centrifugation was collected into a 50 mL conical tube. The residue was further extracted twice with a PCP mixture, and the supernatant after centrifugation was collected. In the second time, 110 mL of supernatant was collected, and in the third time, 180 mL of supernatant was collected.

・Step 3: Precipitation purification of LPS (obtaining crudely purified LPS)
6 mL of each supernatant was dispensed into 16 glass test tubes, and the solvent (40°C) was distilled off under reduced pressure using a centrifugal evaporator. The supernatant was added to the glass tube in which the solvent had been reduced, and the solvent was distilled off from the entire amount of the supernatant. After removing the solvent, water was added to the supernatant (phenol solution) in an amount of 1/10 of the amount of the phenol solution. Centrifugation (3,500 rpm x 20 minutes) was performed, and the supernatant was decanted into another tube. A precipitate was observed at the bottom of the tube. The precipitate was washed twice with 80% phenol (3 mL each time) and 4 times with ether (about 3 mL each time), and after removing the ether, it was dried under reduced pressure. 447 mg of crudely purified LPS was obtained.

- Step 4: High-level purification of crudely purified LPS 440 mg of crudely purified LPS was suspended and dissolved in water for injection to a concentration of 10 mg/mL. 1M Tris-HCl pH 8.0 was added to 10mM. A 1M _MgCl2 aqueous solution was added to give a concentration of 2mM. 13.2 μL of benzonase solution was added to give a final concentration of 10 U/mL. Nucleolytic enzyme treatment was performed by incubating at 37°C for 6 hours. A 20 mg/mL aqueous solution of proteinase K was added to the nuclease-treated solution to give a final concentration of 100 μg/mL, and the mixture was incubated at 37° C. for 16 hours. The crude LPS solution treated with nucleolytic enzymes and proteolytic enzymes was divided into four tubes, and ultrafiltration was performed using four centrifugal ultrafiltration tubes with a molecular weight cutoff of 10 kDa. After ultrafiltration of the treated solution, ultrafiltration was performed four times using water for injection.
The inner solution of the ultrafiltration was collected and co-washed with water for injection. Water for injection was added to give a crude LPS concentration of 5 mg/mL to 88 mL. Triethylamine (TEA) was added to the solution to a final concentration of 0.2%, and sodium deoxycholate (DOC) was added to the solution to a final concentration of 0.5%, and the mixture was dissolved and suspended. The liquid was dispensed into eight 50 mL conical tubes in 11 mL portions. Equal amounts (11 mL) of water-saturated phenol were added, and the mixture was placed in ice water for 10 minutes, followed by centrifugation (3,500 rpm x 20 minutes), and the aqueous layer (upper phase) was collected into another 50 mL conical tube.
An aqueous solution of 0.2% TEA and 0.5% DOC in an amount equal to that of the collected aqueous layer was added to the phenol layer and mixed. After being left for 5 minutes, it was placed in ice water for 10 minutes, and then centrifuged (3,500 rpm x 20 minutes). The aqueous layer (upper phase) was collected into another 50 mL conical tube.
The collected aqueous layer was dispensed into eight 50 mL conical tubes, an equal amount of water-saturated phenol was added, and the liquids were mixed. After being left for 5 minutes, it was placed in ice water for 10 minutes, and then centrifuged (3,500 x 20 minutes). The aqueous layer (upper phase) was collected into another 50 mL conical tube.
The collected aqueous layer was divided into 8 tubes, 3M aqueous sodium acetate solution (pH 5.2) was added to the final concentration of 30 mM, and 33 mL of ethanol was added to each tube so that the final concentration was 75%. After mixing well, the mixture was stored at -20°C for 16 hours.
The liquid in the centrifuge tube was dispensed into eight centrifuge tubes and centrifuged (4°C, 10,000g x 30 minutes) using a high-speed refrigerated centrifuge. The supernatant was removed by decantation, and the resulting precipitate was washed four times with 20 mL of 100% ethanol each time. The obtained precipitate was freeze-dried and weighed to obtain 67.2 mg of highly purified LPS.

The purity of LPS was calculated using the following formula.
Formula: Amount of LPS = Amount of highly purified LPS - Amount of impurities LPS (purity) (%) = Amount of LPS / Amount of highly purified LPS x 100
Highly purified LPS contains nucleic acids and proteins as impurities. The amount of nucleic acid contained in highly purified LPS was measured by UV absorbance method, and the amount of protein was measured by Lowry method.

The purity of LPS after step 4 is shown below.
LPS (%) Nucleic acid (%) Protein (%) Total (%)
97.82 1.35 0.82 100.00

Test 2. Identification of the structure of Paracoccus LPS by fatty acid analysis using GC-MS (1) Sample preparation Paracoccus LPS (highly purified product) prepared in Test 1 was used as Paracoccus LPS. 1 mg of purified LPS was placed in a test tube and 5% HCl methanol solution was added. The tube was then capped and incubated at 100°C for 2 hours. After cooling, 1 mL of water and 1 mL of standard solution (10 μg/mL methyl palmitate-d31) were added and mixed by stirring. The mixture was centrifuged (3,000 rpm, 5 min) and the supernatant was collected as a sample. A fatty acid standard mix (Supelco® 37 component FAME Mix) was also analyzed.

(2) Equipment and measurement conditions
GC-MS: Measurement was performed under the measurement conditions shown in Table 1 using GCMS2010Ultra (SHIMADZU CORPORATION) as a measurement device.

(3) Results The measurement results are shown in Table 2 and FIG. 1.

From the component amounts shown in Table 2, it was determined that the top three fatty acids, ie, C12:1, 3-hydroxy fatty acid-1, and 3-hydroxy fatty acid-2, were included in the composition of LPS. Furthermore, since the content of C18:1 and C18:0 was small, they were judged to be impurities. In addition, in this test, it is possible that all ester and amide bonds were not decomposed, so the component ratio between C12:1, 3-hydroxy fatty acid-1 and 3-hydroxy fatty acid-2 was It was not considered in the structure specification.

(4) Structural identification of fatty acids Next, the structures of C12:1, 3-hydroxy fatty acid-1, and 3-hydroxy fatty acid-2 were identified based on MS fragment peaks.

The C12:1 fatty acid was identified as having a cis-type double bond at C5-C6 by comparing the GC retention time (9.440 min) with the MS fragment peak in the database (Fig. 2).

3-hydroxy fatty acid-1 was inferred to be 3-OH-10:0 based on the MS fragment peaks. That is, 3-hydroxy fatty acids usually have three characteristic peaks, [M-18], [M-50] and [M-92], and when 3-hydroxy fatty acid-1 is 3-OH-10:0, these three peaks are observed at [M-18] + 184, [M-50] + 152 and [M-92] + 110, respectively. 3-hydroxy fatty acid-1 was inferred to be 3-OH-10:0 based on the presence of these peaks (Figure 3).

3-hydroxy fatty acid-2 was also identified as 3-OH-14:0 using the same method as 3-hydroxy fatty acid-1 (Figure 4).

According to the fatty acid analysis method using GC-MS in this test example, LPS derived from Paracoccus bacteria is a C12:1 fatty acid having a cis-type double bond at C5-C6, 3-OH-10:0 and 3-OH-14: It turns out that it contains 0.

Test 3. Measurement of LPS and Lipid A by MALDI-TOF MS (1) Preparation method of Lipid A Similar to Test 2, in Test 3, the Paracoccus-derived LPS prepared in Test 1 (highly purified product) was used as Paracoccus-derived LPS. Using. LPS was hydrolyzed by putting 4 mg of purified LPS into a test tube and incubating with a 2% acetic acid aqueous solution at 100°C for 2 hours. After cooling, the precipitate was collected by centrifugation (13,000×g, 2 minutes), washed several times with water, and used as a sample for MALDI-TOF MS.

(2) Equipment and measurement conditions
MALDI-TOF MS: Measurement was performed under the measurement conditions shown in Table 3 using AXIMA Performance (SHIMADZU CORPORATION) as a measurement device.

(3) Results
MALDI-TOF MS analysis (negative ion mode) is shown in Figure 5. From FIG. 5, the anion peak of Lipid A was considered to be 1472. Three fragment peaks 1392, 1302 and 1222 were identified as 1392( _-HPO3 ), 1302(-3-OH-10:0) and 1222( _-HPO3 and -3-OH-10:0), respectively. .
From this test example, it was found that LPS derived from Paracoccus bacteria has Lipid A phosphate ester and 3-OH-10:0 fatty acid.
Furthermore, it was revealed that the entire molecular weight of lipid A was 1473 (Figure 5). When fatty acids (C12:1 fatty acids with cis-type double bonds at C5-C6, 3-OH-10:0 and 3-OH-14:0) are combined to match this molecular weight, lipid A - It is understood that it has one C12:1 fatty acid with a cis-type double bond at C6, two 3-OH-10:0, and two 3-OH-14:0.

It is known that in lipid A of LPS, the fatty acid that is directly bound to two molecules of glucosamine is often a 3-hydroxyacyl chain with a hydroxyl group at the 3rd position. For this reason, it is presumed that C12:1 fatty acids that do not have a hydroxyl group at the 3rd position are not directly bound to glucosamine. In addition, 16S rRNA analysis shows that Paracoccus carotinifaciens has 93% identity with Rhodobacter sphaeroides, so they are related species and the structure of lipid A is presumed to be similar (FEMS Microbiology Reviews 4 (1988) 143-154). Considering the results of Tests 3 and 4 and previous findings on lipid A, it was estimated that the lipid A of Paracoccus carotinifaciens has a structure in which 3-OH-14:0 is bound to the 2-position amino group of glucosamine via an amide bond, 3-OH-10:0 is bound to the 3-position hydroxyl group of glucosamine via an ester bond, and a C12:1 fatty acid having a cis double bond at C5-C6 is bound to the 3-position hydroxyl group of 3-OH-14:0 via an ester bond. The fact that the lipid A of Paracoccus carotinifaciens has the above structure is consistent with the findings on the LPS structure of Paracoccus denitrificans, which has 97% identity with Paracoccus carotinifaciens in 16S rRNA analysis. That is, in the LPS of Paracoccus denitrificans, 3-OH-10:0 and C12:1 fatty acids are present in lipid A in the form of ester bonds, while 3-OH-14:0 and 3-oxo-14:0 (not detected in Tests 3 and 4) are present in lipid A in the form of amide bonds (FEMS Microbiology Letters 37 (1986) 63-67).

Test 4. Measurement of Limulus Activity Limulus activity of LPS derived from Bacillus Paracoccus was measured.

(1) Measurement sample In Test 4, Paracoccus-derived LPS purified by the PCP method in Test 1: "Highly purified product" (highly purified LPS obtained in Step 4), "Acetone defatted" (LPS obtained in step 1) and "crude purified LPS" (crude purified LPS obtained in step 3) were used.

In addition, as LPS derived from Paracoccus bacteria using a purification method other than the PCP method, Panaferd-AX ("Panaferd-AX (untreated)"), Panaferd-AX finely ground product ("Finely ground product"), Panaferd-AX finely ground product + lecithin (surfactant) ("finely pulverized product + lecithin") and Panaferd-AX pulverized product + lysolecithin (surfactant) ("finely pulverized product + lysolecithin") were also used as measurement samples.

These measurement samples were prepared as follows.
For "Panaferd-AX (untreated)", Panaferd-AX (ENEOS Co., Ltd.) was used.
"Finely pulverized product": 100 g of Panaferd-AX and 900 g of water were mixed and crushed by processing at 100 MPa 10 times using a high-pressure homogenizer Panda PLUS 2000. The crushed liquid was freeze-dried and used as a Panaferd-AX finely pulverized product.
"Finely ground product + lecithin": 300 mg of lecithin (SLP-PC70, manufactured by Tsuji Oil Co., Ltd.) was weighed in a 50 mL tube, 15 mL of ion exchange water was added, and the mixture was mixed with a direct mixer for 1 hour. Through this operation, a 2% aqueous solution of lecithin (uniform solvent) was obtained. 3 g of the finely pulverized product was weighed, put into an aqueous lecithin solution, and mixed for 1 hour with a direct mixer. The powder obtained by freeze-drying the mixed liquid was referred to as "finely ground product + lecithin".
"Finely pulverized product + lysolecithin": "Finely pulverized product + lysolecithin" was produced in the same manner as the "finely pulverized product + lecithin" except that lysolecithin (SLP-LPC70 manufactured by Tsuji Oil Co., Ltd.) was used instead of lecithin. Obtained.

(2) Limulus activity measurement method 1 (turbidimetry)
The Limulus activity of LPS derived from Paracoccus purified by the PCP method was measured by turbidimetry using Limulus ES-II plus CS single test wako (Fujifilm Wako Pure Chemical Industries, Ltd. Cat# 299-77201).
Weighed 5 mg of each sample into an Eppendorf tube (1.5 mL) and added water to prepare a 5 mg/mL suspension. The suspension was stirred with Vortex for 5 minutes, then centrifuged at 9000 rpm and 20°C for 15 minutes, and the supernatant was used for measurement.
200 μL of the supernatant was added to the lysate reagent of Limulus ES-II plus CS single test wako, and the mixture was stirred for 10 seconds using Vortex. The lysate reagent was set in a toxinometer (Fuji Film Wako Pure Chemical Industries, Ltd. Cat# ET-6000/J), and absorbance at 430 nm was measured over time in kinetic mode: measurement interval 15 seconds, total measurement time 60 minutes, Temperature 37℃. Note that this measurement is quantified using the time required for gelation of the lysate reagent as an index, and the results do not change depending on the measurement time.

(3) Limulus activity measurement method 2 (colorimetric method)
The Limulus activity of LPS derived from Paracoccus bacteria obtained by a purification method other than the PCP method was measured by Limulus measurement (colorimetric method) using a test kit: LONZA Limulus Amebocyte Lysate (LAL) Kinetic-QCL. To create a calibration curve, E. coli O55:B5 Endotocin [E50-643] included in the kit was diluted to 50 EU/mL.
50 mg of each sample was weighed into an Eppendorf tube (1.5 mL), and water was added to prepare a 100 mg/mL suspension. The suspension was stirred with Vortex for 5 minutes, then centrifuged at 9000 rpm and 20°C for 15 minutes, and the supernatant was used for measurement.
100 μL of the supernatant was placed in a 96-well plate and pre-incubated at 37° C. for 10 minutes using a plate reader. 100 μL of Limulus reagent was added, and absorbance at 405 nm was measured over time in the kinetic mode of a plate reader: measurement interval 1 minute, total measurement time 60 minutes, temperature 37°C. In this measurement, the time taken for the absorbance to increase by 0.2 is used as an index to quantify the amount, and the results do not change depending on the measurement time.

From existing data, it is understood that the turbidimetric method and the colorimetric method yield approximately equivalent Limulus activity values, so it is possible to compare the Limulus activity values obtained by both methods.

(4) Results Table 4 shows the values of Limulus activity measured by the methods (2) and (3) above.

As mentioned above, the compositions of the present invention were shown to exhibit high limulus activity.

The present invention provides a composition containing LPS of a Gram-negative bacterium having lipid A having a predetermined structure. In another aspect, the present invention provides a composition comprising LPS of a Gram-negative bacterium having a predetermined Limulus activity value.
The composition provided by the present invention is useful in utilizing the immunostimulatory activity of LPS in pharmaceuticals, cosmetics, foods, feeds, etc. in terms of structure and/or limulus activity.

Claims (5)

A composition comprising a lipopolysaccharide of a gram-negative bacterium, the lipopolysaccharide having lipid A;
Four 3-hydroxyacyl chains having 8 to 16 carbon atoms are bonded to the glucosamine skeleton of Lipid A, and a hydroxyl group having 8 to 13 carbon atoms is attached to the 3-hydroxyl group of one or two of the 3-hydroxyacyl chains. The acyl chain is further bonded,
A composition having a Limulus activity of 30,000 EU/mg or more. Four 3-hydroxyacyl chains having 10 to 14 carbon atoms are bonded to the glucosamine skeleton of lipid A, and the 3-hydroxyacyl chain having 10 to 14 carbon atoms is bonded to the 2-position of the glucosamine skeleton. The composition according to claim 1, wherein an acyl chain having 12 carbon atoms is further bonded to the hydroxyl group. The acyl chain having 12 carbon atoms is bonded to the hydroxyl group at the 3-position of the 3-hydroxyacyl chain having 14 carbon atoms, which is bonded to the 2-position of glucosamine on the non-reducing end side, and is bonded to the 2-position of glucosamine on the reducing end side. The composition according to claim 2, which does not bond to the hydroxyl group at the 3-position of a 3-hydroxyacyl chain having 14 carbon atoms. The lipid A has a structure of formula (I):

The composition according to any one of claims 1 to 3, having the following. The composition according to any one of claims 1 to 4, wherein the Gram-negative bacterium is a bacterium belonging to the genus Paracoccus.