WO2024058253A1 - Composition comprising lipopolysaccharide - Google Patents

Composition comprising lipopolysaccharide Download PDF

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WO2024058253A1
WO2024058253A1 PCT/JP2023/033603 JP2023033603W WO2024058253A1 WO 2024058253 A1 WO2024058253 A1 WO 2024058253A1 JP 2023033603 W JP2023033603 W JP 2023033603W WO 2024058253 A1 WO2024058253 A1 WO 2024058253A1
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lps
bonded
glucosamine
carbon atoms
hydroxyacyl
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PCT/JP2023/033603
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French (fr)
Japanese (ja)
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大知 桑原
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Ｅｎｅｏｓ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C07H13/06—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a composition containing lipopolysaccharide 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. LPS is also called endotoxin because LPS released from Gram-negative bacteria by killing or destroying them causes reactions such as fever in mammals, and can even lead to death if given in large amounts. be done.
  • LPS 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.
  • LPS is incorporated into the cell wall with lipid A embedded in the outer membrane of Gram-negative bacteria and the O antigen polysaccharide side chain protruding outward, and is not easily released from the cell wall.
  • 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.
  • TLR4 toll-like receptor 4
  • LPS binds to TLR4
  • the production of inflammatory cytokines such as TNF ⁇ , IL-6, and IL-12 and type I interferon (IFN) is triggered via the intracellular signal transduction system (Non-Patent Document 1).
  • LPS such as Escherichia coli activates these signals simultaneously and strongly, thereby causing a strong inflammatory response.
  • 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.
  • LPS is known to have immunostimulatory activity
  • the use of LPS's immunostimulatory activity in pharmaceuticals, cosmetics, foods, feeds, etc. is also being considered.
  • LPS when obtaining LPS from Gram-negative bacteria using the usual method, it is difficult to obtain LPS with high concentration or high activity.
  • 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.
  • 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 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.
  • [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 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 lipid A has a structure of formula (I): The composition according to any one of [1] to [3], which has the following.
  • 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.
  • a GC-MS spectrum (retention time 9.440 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 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.
  • 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.
  • 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.
  • the Gram-negative bacterium is preferably a bacterium belonging to the genus Paracoccus.
  • bacteria belonging to the genus Paracoccus are not particularly limited.
  • 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.
  • 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.
  • Lipid A LPS of Gram-negative bacteria has lipid A.
  • lipid A has a skeleton of two glucosamine molecules in which two glucosamine molecules are bonded through a ⁇ (1 ⁇ 6) bond.
  • this lipid A skeleton is referred to as a "glucosamine skeleton.”
  • a phosphate group is bonded to the 1-position of glucosamine on the reducing end side of the glucosamine skeleton, and a phosphate group is bonded to the 4-position of glucosamine on the non-reducing end side.
  • a hydroxyl group may be used instead of a phosphoric acid 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.
  • an acyl chain having a hydroxyl group at the 3-position is also referred to as a 3-hydroxyacyl chain. That is, Lipid A has two glucosamine molecules bonded to the 2-position of glucosamine, with a 3-hydroxyacyl chain bonded through an amide bond, and the 3-hydroxyacyl chain bonded with an ester bond to 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.
  • 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.
  • the 3-hydroxyacyl chain bonded to the glucosamine skeleton is preferably 3-OH10:0 (3-hydroxyacyl chain having 10 carbon atoms and 0 double bonds) or 3-OH14: 0 (3-hydroxyacyl chain with 14 carbon atoms and 0 double bonds).
  • the 3-hydroxyacyl chain bonded to the 2-position of glucosamine is 3-OH14:0
  • the 3-hydroxyacyl chain bonded 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.
  • an acyl chain having 8 to 13 carbon atoms is further ester-bonded to the hydroxyl group at the 3-position 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.
  • 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.
  • 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.
  • the acyl chain bonded to the 3-hydroxyl group of the 3-hydroxyacyl chain directly bonded to the glucosamine skeleton is preferably C12:1 (3-hydroxyl chain having 12 carbon atoms and 1 double bond). hydroxyacyl chain), and more preferably C12:1 (C12:1 ( ⁇ 5 )) having a double bond (cis) at the fifth carbon (between C5 and C6) counting from the carbon of the carbonyl group. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Lipid A has four 3-hydroxyacyl chains each having 10 or 14 carbon atoms bonded to the glucosamine skeleton, and 3-hydroxyacyl chains having 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 3rd position of the 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.
  • 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.
  • Lipid A has the structure of formula (I).
  • composition of the Present Invention it has been found that a composition containing a treated product obtained by treating Gram-negative bacteria can obtain 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.
  • composition of the present invention may be in the form of an extract or dried bacterial cells of Gram-negative bacteria, as long as it contains LPS of Gram-negative bacteria.
  • the composition of the present invention may be an extract obtained by appropriately culturing Gram-negative bacteria and extracting the obtained culture, or dried bacterial cells obtained by drying the culture.
  • 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.
  • 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.
  • commercially available products may be used.
  • Panaferd-AX ENEOS Techno Material Co., Ltd.
  • Paracoccus Paracoccus carotinifaciens
  • 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.
  • 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)). can do.
  • PCP phenol-chloroform-petroleum ether extraction
  • 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.
  • the composition of the present invention may contain lecithin or lysolecithin.
  • 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.
  • 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.
  • 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.
  • 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.
  • the Limulus activity is determined by the turbidimetric method or the colorimetric method (kinetic colorimetric method). It is measured using Limulus activity can be measured using a commercially available endotoxin measurement kit.
  • 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.
  • 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.
  • PCP phenol/chloroform/petroleum ether
  • Step 1 Degreasing dried bacterial cells of Paracoccus bacteria Panaferd-AX (ENEOS Techno Material Co., Ltd.) 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 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.
  • a 20 mg/mL aqueous solution of proteinase K was added to the nuclease-treated solution to give a final concentration of 100 ⁇ g/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.
  • 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.
  • 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.
  • 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.
  • Test 2 Identification of structure of LPS derived from Paracoccus by fatty acid analysis method using GC-MS (1) Sample preparation As LPS derived from Paracoccus, the LPS derived from Paracoccus (highly purified product) prepared in Test 1 was used. 1 mg of purified LPS was placed in a test tube and 5% HCl methanol solution was added. It was covered and incubated at 100°C for 2 hours. After cooling, 1 mL of water and 1 mL of standard solution (10 ⁇ g/mL methyl palmitate-d31) were added and mixed with stirring. It was centrifuged (3,000 rpm, 5 min) and the supernatant was obtained as a sample. A fatty acid standard Mix (Supelco® 37 component FAME Mix) was also analyzed.
  • 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 estimated to be 3-OH-10:0 from the MS fragment peak. That is, 3-hydroxy fatty acid usually has three characteristic peaks, [M-18], [M-50] and [M-92], and 3-hydroxy fatty acid-1 has three characteristic peaks, [M-18], [M-50] and [M-92]. :0, these three peaks are observed at [M-18]+ 184, [M-50]+ 152 and [M-92]+ 110, respectively. 3-hydroxy fatty acid-1 was estimated to be 3-OH-10:0 from having 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).
  • 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
  • the Paracoccus-derived LPS prepared in Test 1 was used as Paracoccus-derived LPS.
  • 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.
  • MALDI-TOF MS AXIMA Performance (SHIMADZU CORPORATION) was used as a measurement apparatus, and measurements were carried out under the measurement conditions shown in Table 3.
  • lipid A 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.
  • lipid A of Paracoccus carotinifaciens has 3-OH-14:0 bonded to the 2-position amino group of glucosamine via an amide bond, 3-OH-10:0 is bonded to the 3-position hydroxyl group of glucosamine via an ester bond, and a C12:1 fatty acid having a cis-type double bond at C5-C6 is bonded to the 3-position hydroxyl group of 3-OH-14:0. It was estimated that it has a structure in which it is bonded to a hydroxyl group via an ester bond (the structure is shown below).
  • lipid A of Paracoccus carotinifaciens has the above structure is consistent with the findings regarding 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, and 3-OH-14:0 and 3-oxo-14:0 (test 3 and not detected in Test 4) exists in lipid A in the form of an amide bond (FEMS Microbiology Letters 37 (1986) 63-67).
  • Test 4 Measurement of Limulus Activity
  • Limulus activity of LPS derived from Bacillus Paracoccus was measured.
  • Panaferd-AX Panaferd-AX (untreated)
  • Panaferd-AX finely ground product Panaferd-AX finely ground product + lecithin
  • Panaferd-AX finely ground product + lecithin Surfactant
  • Panaferd-AX pulverized product + lysolecithin Surfactant
  • “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".
  • lysate reagent 200 ⁇ L 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°C. 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.
  • 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.
  • LAL LONZA Limulus Amebocyte Lysate
  • 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 ⁇ L of the supernatant was placed in a 96-well plate and pre-incubated at 37° C. for 10 minutes using a plate reader.
  • 100 ⁇ L 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.
  • composition of the present invention was 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.
  • 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.

Abstract

The objective of the present invention is to provide a composition comprising an LPS of a Gram-negative bacterium having a predetermined structure or a predetermined activity. According to the present invention, provided is a composition comprising a lipopolysaccharide of a Gram-negative bacterium, wherein the lipopolysaccharide has lipid A, and the lipid A has a predetermined structure and a predetermined limulus activity.

Description

リポ多糖を含む組成物Composition containing lipopolysaccharide
　本発明は、グラム陰性菌のリポ多糖を含む組成物に関する。 The present invention relates to a composition containing lipopolysaccharide of Gram-negative bacteria.
　リポ多糖（以下、「ＬＰＳ」又は「リポポリサッカライド」ともいう）は、大腸菌等のグラム陰性菌の細胞壁を構成する成分である。グラム陰性菌の死滅や破壊によりグラム陰性菌から遊離したＬＰＳによって哺乳動物の発熱反応等の反応が引き起こされ、多量に与えられると死に至ることもあることから、ＬＰＳは内毒素（エンドトキシン）とも呼称される。 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. LPS is also called endotoxin because LPS released from Gram-negative bacteria by killing or destroying them causes reactions such as fever in mammals, and can even lead to death if given in large amounts. be done.
　ＬＰＳの基本構造は、脂質部分であるリピドＡ部分と多糖部分を含む。多糖部分はさらにコアとＯ抗原多糖側鎖で構成されている。通常、ＬＰＳは、リピドＡがグラム陰性菌の外膜に埋め込まれ、Ｏ抗原多糖側鎖が外に突き出た形で細胞壁に組み込まれており、細胞壁からは容易には遊離しない。グラム陰性菌が死滅して細胞が溶菌すると、ＬＰＳは遊離し、それが例えば動物細胞に作用することで様々な生理活性が発現する。 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 of Gram-negative bacteria and the O antigen polysaccharide side chain 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.
　遊離したＬＰＳは標的細胞の細胞膜上のＴＬＲ４（toll like receptor 4）を介して作用を発現する。ＴＬＲ４にＬＰＳが結合すると細胞内シグナル伝達系を介してＴＮＦα、ＩＬ－６、ＩＬ－１２といった炎症性サイトカインの産生やＩ型インターフェロン（ＩＦＮ）の産生が引き起こされる（非特許文献１）。特に、大腸菌等のＬＰＳは、これらのシグナルを同時にかつ強力に活性化するために強い炎症反応が惹起される。 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 Escherichia coli activates these signals simultaneously and strongly, thereby causing a strong inflammatory response.
　このようにＬＰＳはＴＬＲ４のシグナル伝達系を介してサイトカインの産生を促進する作用を有する。サイトカインの産生は、一般に、強い免疫賦活活性に繋がり細菌感染の防御のための生体防御反応に一定の役割を果たす。 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.
　上記のように、ＬＰＳは免疫賦活活性を有することが知られていることから、ＬＰＳの免疫賦活活性を医薬品、化粧品、食品、飼料等に利用することも検討されている。しかしながら、通常の方法でグラム陰性菌からＬＰＳを取得する場合には、高濃度又は高活性のＬＰＳを取得することが難しい。 As mentioned above, since LPS is known to have immunostimulatory activity, the use of LPS's immunostimulatory activity 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.
　また、ＬＰＳの基本構造は、グラム陰性菌で似通っているものの、由来するグラム陰性菌の種類によりＬＰＳの構造は異なることが知られている。しかし、一般に、グラム陰性菌のＬＰＳの構造を決定することは容易ではない。 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.
　本発明は、グラム陰性菌のＬＰＳを含有する組成物であって、前記ＬＰＳが所定の構造又は活性を有する前記組成物の提供を目的とする。 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.
　本発明者は、鋭意研究を行い、所定の構造又は所定の活性を有するグラム陰性菌のＬＰＳを含む組成物を見出した。すなわち、本発明は以下のとおりである。
［１］
　グラム陰性菌のリポ多糖を含む組成物であって、前記リポ多糖がリピドＡを有し、
　前記リピドＡのグルコサミン骨格に、炭素数８～１６の３－ヒドロキシアシル鎖が４本結合し、かつ、１又は２本の前記３－ヒドロキシアシル鎖の３位の水酸基に炭素数８～１３のアシル鎖がさらに結合し、
　リムルス活性が３００００ＥＵ／ｍｇ以上である、組成物。
［２］
　前記リピドＡのグルコサミン骨格に、炭素数１０～１４の３－ヒドロキシアシル鎖が４本結合し、かつ、グルコサミン骨格の２位に結合する炭素数１０～１４の３－ヒドロキシアシル鎖の３位の水酸基に炭素数１２のアシル鎖がさらに結合する、［１］に記載の組成物。
［３］
　前記炭素数１２のアシル鎖が、非還元末端側のグルコサミンの２位に結合する炭素数１４の３－ヒドロキシアシル鎖の３位の水酸基に結合し、還元末端側のグルコサミンの２位に結合する炭素数１４の３－ヒドロキシアシル鎖の３位の水酸基に結合しない、［２］に記載の組成物。
［４］
　前記リピドＡが、式（Ｉ）の構造：
を有する、［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、［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 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.
[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 [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 [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 [1] to [3], which has the following.
[5]
The composition according to any one of [1] to [4], wherein the Gram-negative bacterium is a bacterium belonging to the genus Paracoccus.
　本発明により、所定の構造及びリムルス活性を有するリピドＡを有する、グラム陰性菌のＬＰＳを含む組成物が提供される。
　本発明により提供される組成物は、構造が特定されており、また、高いリムルス活性を有する点で、ＬＰＳの免疫賦活活性を医薬品、化粧品、食品、飼料等に利用する上で有用である。 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.
脂肪酸分析のＧＣチャートを示す。上図がパラコッカスＬＰＳ由来の脂肪酸の分析結果であり、下図が脂肪酸標準Ｍｉｘの分析結果である。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. ＧＣ－ＭＳのスペクトル（リテンションタイム9.440分）を示す。上図がパラコッカスＬＰＳ由来の脂肪酸の分析結果であり、下図がC12:1（C5-C6 cis）のデータベース収録データである。A GC-MS spectrum (retention time 9.440 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 C12:1 (C5-C6 cis). ＧＣ－ＭＳのスペクトル（リテンションタイム14.417分）を示す。上図がパラコッカスＬＰＳ由来の脂肪酸の分析結果であり、下図が3-OH C10:0のデータベース収録データである。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. ＧＣ－ＭＳのスペクトル（リテンションタイム23.010分）を示す。上図がパラコッカスＬＰＳ由来の脂肪酸の分析結果であり、下図が3-OH C14:0のデータベース収録データである。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. パラコッカス属に属する細菌由来のＬＰＳのリピドＡ部分を陰イオンモードで測定した場合のMALDI-TOF MS 測定結果を示す。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.
　以下、本発明をさらに詳細に説明する。本発明の範囲はこれらの説明に限定されることはなく、以下の例示以外についても、本発明の趣旨を損なわない範囲で適宜変更し実施し得る。なお、本明細書で引用する出版物、特許出願および特許を含むすべての参考文献は、全体として、参照により本明細書に組み込まれる。 Hereinafter, the present invention will be explained in more detail. 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. All references, including publications, patent applications, and patents cited herein are incorporated by reference in their entirety.
１．グラム陰性菌のＬＰＳ
　本発明の組成物は、グラム陰性菌のＬＰＳを含有する。グラム陰性菌は、その細胞壁にＬＰＳを有することが知られている。本発明において、グラム陰性菌は、ＬＰＳを有するグラム陰性菌であればいずれでよく、限定されるわけではないが、例えば、プロテオバクテリア門（大腸菌、サルモネラ菌、シュードモナス菌、パラコッカス菌等）、シアノバクテリア門、スピロヘータ門、クロロビウム門、バクテロイデス門が含まれる。本発明において、グラム陰性菌は、好ましくはパラコッカス属に属する細菌である。 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.
　本明細書において、パラコッカス属に属する細菌は、特に限定されない。パラコッカス属に属する細菌は、Paracoccus carotinifaciens、Paracoccus marcusii、Paracoccus haeundaensis及びParacoccus zeaxanthinifaciensが好ましく用いられ、Paracoccus carotinifaciens又はParacoccus zeaxanthinifaciensがより好ましく用いられ、特にParacoccus carotinifaciensが好ましく用いられる。パラコッカス属に属する細菌の具体的な菌株として、例えば、Paracoccus carotinifaciens E-396株（ＦＥＲＭ ＢＰ－４２８３）、Paracoccus属細菌A-581-1株（ＦＥＲＭ ＢＰ－４６７１）、Paracoccus marcusii DSM 11574株、Paracoccus属細菌N-81106株、Paracoccus haeundaensis BC 74171株、Paracoccus zeaxanthinifaciens ATCC 21588株、及びParacoccus sp. PC-1株が挙げられ、これらの変異株も本発明に好ましく用いられる。Ｅ－３９６株及びＡ－５８１－１株は、独立行政法人製品評価技術基盤機構（NITE）特許生物寄託センター（NITE-IPOD）（〒292-0818 千葉県木更津市かずさ鎌足2-5-8）を国際寄託当局として以下のとおり国際寄託されている。
Ｅ－３９６株
　識別のための表示：E-396、受託番号：FERM BP-4283、原寄託日：平成５年（１９９３年）４月２７日
Ａ－５８１－１株
　識別のための表示：A-581-1、受託番号：FERM BP-4671、原寄託日：平成６年（１９９４年）５月２０日 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．リピドＡの構造
　グラム陰性菌のＬＰＳはリピドＡを有する。本発明において、リピドＡは、グルコサミン２分子がβ（１→６）結合で結合したグルコサミン２分子を骨格として有する。本明細書において、このリピドＡの骨格を「グルコサミン骨格」と称する。 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."
　本発明の一態様において、グルコサミン骨格の還元末端側のグルコサミンの１位にリン酸基が結合し、非還元末端のグルコサミンの４位にリン酸基が結合する。本発明の別の態様において、グルコサミン骨格に結合するリン酸は１～３であってもよく、さらに、リン酸は修飾されていてもよい。本発明の別の態様において、リン酸基の代わりに水酸基であってもよい。 In one embodiment of the present invention, a phosphate group is bonded to the 1-position of glucosamine on the reducing end side of the glucosamine skeleton, and a phosphate group is bonded to the 4-position of glucosamine on the non-reducing end side. In another embodiment of the invention, there may be 1 to 3 phosphoric acids attached to the glucosamine backbone, and the phosphoric acids may be modified. In another embodiment of the invention, a hydroxyl group may be used instead of a phosphoric acid group.
　本発明におけるリピドＡは、グルコサミン骨格に炭素数８～１６のアシル鎖が４本直接結合しており、当該アシル鎖は３位に水酸基を有する。本明細書において、３位に水酸基を有するアシル鎖を３－ヒドロキシアシル鎖とも称する。
　すなわち、リピドＡは、グルコサミンの２位に３－ヒドロキシアシル鎖がアミド結合し、３位に３－ヒドロキシアシル鎖がエステル結合したグルコサミン２分子が結合している。２分子のグルコサミンは、同一のグルコサミン２分子であってもよいし、２分子間で異なる３－ヒドロキシアシル鎖が結合したグルコサミン２分子でもよい。 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, Lipid A has two glucosamine molecules bonded to the 2-position of glucosamine, with a 3-hydroxyacyl chain bonded through an amide bond, and the 3-hydroxyacyl chain bonded with an ester bond to 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.
　本発明において、グルコサミンの２位及び３位に結合する３－ヒドロキシアシル鎖の炭素数は、８～１６であり、好ましくは１０～１４、より好ましくは１０、１１、１２、１３又は１４であり、さらに好ましくは１０または１４である。 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.
　グルコサミン骨格に直接結合する３－ヒドロキシアシル鎖の有する二重結合の数は、０、１または２であり、好ましくは０である。 The number of double bonds in the 3-hydroxyacyl chain directly bonded to the glucosamine skeleton is 0, 1 or 2, preferably 0.
　本発明において、グルコサミン骨格に結合する３－ヒドロキシアシル鎖は、好ましくは３－ＯＨ１０：０（炭素数が１０であり、二重結合の数が０の３－ヒドロキシアシル鎖）又は３－ＯＨ１４：０（炭素数が１４であり、二重結合の数が０の３－ヒドロキシアシル鎖）である。 In the present invention, the 3-hydroxyacyl chain bonded to the glucosamine skeleton is preferably 3-OH10:0 (3-hydroxyacyl chain having 10 carbon atoms and 0 double bonds) or 3-OH14: 0 (3-hydroxyacyl chain with 14 carbon atoms and 0 double bonds).
　本発明の一態様において、グルコサミンの２位に結合する３－ヒドロキシアシル鎖は３－ＯＨ１４：０であり、グルコサミンの３位に結合する３－ヒドロキシアシル鎖は３－ＯＨ１０：０である。 In one embodiment of the present invention, the 3-hydroxyacyl chain bonded to the 2-position of glucosamine is 3-OH14:0, and the 3-hydroxyacyl chain bonded to the 3-position of glucosamine is 3-OH10:0.
　本発明におけるリピドＡは、グルコサミン骨格に直接結合する４本の３－ヒドロキシアシル鎖のうちの１本または２本について、３－ヒドロキシアシル鎖の３位の水酸基に、炭素数８～１３のアシル鎖がさらにエステル結合する。本発明の一態様において、グルコサミン骨格の２位に結合する３－ヒドロキシアシル鎖（１本又は２本）の３位の水酸基に、炭素数８～１３のアシル鎖がさらにエステル結合する。このようなさらなるアシル鎖の結合により、リピドＡは５本又は６本のアシル鎖を有する。 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 hydroxyl group at the 3-position 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.
　本発明において、グルコサミン骨格に直接結合する３－ヒドロキシアシル鎖の３位の水酸基に結合するアシル鎖の炭素数は、８～１３であり、好ましくは８～１２、より好ましくは１２である。 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.
　本発明において、グルコサミン骨格に直接結合する３－ヒドロキシアシル鎖の３位の水酸基に結合するアシル鎖の有する二重結合の数は、０、１または２であり、好ましくは１である。 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.
　本発明において、グルコサミン骨格に直接結合する３－ヒドロキシアシル鎖の３位の水酸基に結合するアシル鎖は、好ましくはＣ１２：１（炭素数が１２であり、二重結合の数が１の３－ヒドロキシアシル鎖）であり、より好ましくはカルボニル基の炭素から数えて５番目の炭素（Ｃ５－６間）に二重結合（シス）を有するＣ１２：１（Ｃ１２：１（Δ５））である。 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 (3-hydroxyl chain having 12 carbon atoms and 1 double bond). hydroxyacyl chain), and more preferably C12:1 (C12:1 (Δ 5 )) having a double bond (cis) at the fifth carbon (between C5 and C6) counting from the carbon of the carbonyl group. .
　本発明の一態様において、グルコサミン骨格に直接結合する３－ヒドロキシアシル鎖の３位の水酸基に結合するアシル鎖は、非還元末端側のグルコサミンの２位に結合する３－ヒドロキシアシル鎖の３位の水酸基に結合するが、還元末端側のグルコサミンの２位に結合する３－ヒドロキシアシル鎖の３位の水酸基に結合しない。 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.
　本発明におけるリピドＡの態様の例示を以下に挙げる。
　本発明の一態様において、リピドＡは、グルコサミン骨格に炭素数８～１６の３－ヒドロキシアシル鎖が４本結合し、かつ、1又は２本の前記３－ヒドロキシアシル鎖の３位の水酸基に炭素数８～１３のアシル鎖がさらに結合した構造を有する。 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.
　本発明の別の態様において、リピドＡは、グルコサミン骨格に炭素数１０～１４の３－ヒドロキシアシル鎖が４本結合し、かつ、グルコサミン骨格の２位に結合する炭素数１０～１４の３－ヒドロキシアシル鎖の１本又は２本について、その３位の水酸基に炭素数８～１３のアシル鎖がさらに結合した構造を有する。 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.
　本発明の別の態様において、リピドＡは、グルコサミン骨格に炭素数１０～１４の３－ヒドロキシアシル鎖が４本結合し、かつ、グルコサミン骨格の２位に結合する炭素数１０～１４の３－ヒドロキシアシル鎖の１本又は２本について、その３位の水酸基に炭素数１２のアシル鎖がさらに結合した構造を有する。 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.
　本発明の別の態様において、リピドＡは、グルコサミン骨格に炭素数１０～１４の３－ヒドロキシアシル鎖が４本結合し、かつ、グルコサミン骨格の２位に結合する炭素数１０～１４の３－ヒドロキシアシル鎖の３位の水酸基に炭素数１２のアシル鎖がさらに結合し、
　前記炭素数１２のアシル鎖が、非還元末端側のグルコサミンの２位に結合する炭素数１４の３－ヒドロキシアシル鎖の３位の水酸基に結合し、還元末端側のグルコサミンの２位に結合する炭素数１４の３－ヒドロキシアシル鎖の３位の水酸基に結合しない構造を有する。 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.
　本発明の別の態様において、リピドＡは、グルコサミン骨格に炭素数１０又は１４の３－ヒドロキシアシル鎖が４本結合し、かつ、グルコサミン骨格の２位に結合する炭素数１４の３－ヒドロキシアシル鎖の３位の水酸基に炭素数１２のアシル鎖がさらに結合し、
　前記炭素数１２のアシル鎖が、非還元末端側のグルコサミンの２位に結合する炭素数１４の３－ヒドロキシアシル鎖の３位の水酸基に結合し、還元末端側のグルコサミンの２位に結合する炭素数１４の３－ヒドロキシアシル鎖の３位の水酸基に結合しない構造を有する。 In another embodiment of the present invention, Lipid A has four 3-hydroxyacyl chains each having 10 or 14 carbon atoms bonded to the glucosamine skeleton, and 3-hydroxyacyl chains having 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 3rd position of the 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.
　本発明の一態様において、リピドＡは、グルコサミンの２位に３－ＯＨ１４：０が結合し、グルコサミンの３位に３－ＯＨ１０：０が結合し、二重結合（シス）がＣ５－６間に含まれるＣ１２：１が、非還元末端側のグルコサミンの２位に結合する３－ＯＨ１４：０に結合した構造を有する。 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.
　本発明の一態様において、リピドＡは式（Ｉ）の構造を有する。
 In one aspect of the invention, Lipid A has the structure of formula (I).
３．本発明の組成物
　本発明において、グラム陰性菌を処理した処理物を含む組成物が、従来の菌体等よりも高いリムルス活性を得られることが見出された。本発明の組成物は、グラム陰性菌を精製、抽出等の処理を経て製造することができる。 3. Composition of the Present Invention In the present invention, it has been found that a composition containing a treated product obtained by treating Gram-negative bacteria can obtain 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.
　本発明の組成物は、グラム陰性菌のＬＰＳを含む限り、グラム陰性菌の抽出物や乾燥菌体の態様であってもよい。例えば、グラム陰性菌を適宜培養し、得られた培養物を抽出処理した抽出物又は培養物を乾燥した乾燥菌体を、本発明の組成物とすることができる。 The composition of the present invention may be in the form of an extract or dried bacterial cells of Gram-negative bacteria, as long as it contains LPS of Gram-negative bacteria. For example, the composition of the present invention may be an extract obtained by appropriately culturing Gram-negative bacteria and extracting the obtained culture, or dried bacterial cells obtained by drying the culture.
　また、上記抽出処理の前又は後に、濃縮、乾燥、希釈、破砕、粉砕、加熱等の処理を単独又は組み合わせた処理に供してもよい。乾燥処理には噴霧乾燥、凍結乾燥、真空乾燥、ドラム乾燥が含まれる。破砕処理には、ホモジナイザー、フレンチプレス、乳鉢、ガラスビーズなどの手段による破砕が含まれる。菌体を破砕又は粉砕することにより、表面積が増え、より多くのＬＰＳが界面に露出することが期待される。当業者であれば、グラム陰性菌の培養や、抽出等の処理を公知の方法に従い実施することができる。 Furthermore, before or after the above 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.
　上記のとおり、グラム陰性菌の培養後の乾燥菌体またはその処理物を本発明の組成物とすることもできる。また、市販品を用いてもよい。例えば、パラコッカス属に属する細菌（Paracoccus carotinifaciens）の培養後の乾燥菌体であるＰａｎａｆｅｒｄ－ＡＸ（ＥＮＥＯＳテクノマテリアル株式会社）を用いることができる。 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 Techno Material Co., Ltd.), which is dried bacterial cells after culturing of bacteria belonging to the genus Paracoccus (Paracoccus carotinifaciens), can be used.
　当業者であれば、抽出したパラコッカス属に属する細菌由来のＬＰＳをさらに精製することができる。精製方法は、従来公知の方法に従うことができる。 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.
　例えば、グラム陰性菌は、熱水フェノール法(O.Westphal, K. Jann, "Methods in Carbohydrate Chemistry", ed. by R. Whistler, Vol.5, p.83, Academic Press, New York (1965)、フェノール－クロロホルム－石油エーテル抽出法（ＰＣＰ）法（C.Galanos, O. Luderitz, O. Westphal, Eur. J. Biochem., 9, 245 (1969)）などで処理することにより、ＬＰＳを抽出することができる。 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)). can do.
　ＬＰＳやＰＣＰ法の各精製ステップで得られる組成物を、本発明の組成物とすることができる。精製することにより、ＬＰＳの純度を向上することができる。ＰＣＰ法の詳細は、実施例の記載を参照することができる。これらの方法で得られたグラム陰性菌のＬＰＳ抽出物を本発明の組成物することができる。また、本発明においては、ＰＣＰ法で高度に精製されたＬＰＳによって、ＬＰＳの構造の特定することができた。 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.
　本発明の別の態様において、本発明の組成物は、レシチンやリゾレシチンを含有してもよい。当業者は、通常の技術を用いてレシチン又はリゾレシチンを組成物に含有させることができる。例えば、レシチン又はリゾレシチンの水溶液と、グラム陰性菌のＬＰＳ抽出物、グラム陰性菌の培養後の乾燥菌体またはその処理物を、ミキサー等で混合する。両親媒性分子であるレシチンまたはリゾレシチンと一緒にグラム陰性菌を処理することにより、ＬＰＳ（特にパラコッカス属に属する細菌のＬＰＳ）の水分散性の改善や動物での吸収性の改善が見込まれる。 In another embodiment of the present invention, the composition of the present 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は免疫賦活活性を有することが知られている。本発明の組成物は、３００００ＥＵ／ｍｇ以上のリムルス活性を示すことから、飼料組成物、医薬組成物、食品組成物、化粧品組成物として有用である。飼料、医薬、食品、化粧品として用いる場合、それぞれの用途に許容可能な担体をさらに含有させてもよい。 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．リムルス活性
　本発明の組成物は、従来よりも高いリムルス活性を示すことが見出された。リムルス活性は、カブトガニ血球抽出物の凝固を促進する活性であり、分子レベルでのエンドトキシンの生物活性である。リムルス活性は、カブトガニの血球抽出物より調製されたライセート試薬を用いて、エンドトキシンを検出又は定量するリムルス試験により測定される。リムルス試験には、ライセート試薬のゲル形成を指標とするゲル化法と光学的変化を指標とする光学的定量法（比濁法、比色法）がある。比濁法は、ライセート試薬のゲル化に伴う濁度（吸光度又は透過率）の変化を測定することにより、エンドトキシン濃度を測定する方法である。また、比色法は、エンドトキシンとライセート試薬の反応により生じた凝固酵素によって発色合成基質から遊離される発色基の量を吸光度又は透過率で測定する方法である。比色法には、カイネティック比色法とエンドポイント比色法がある。比濁法と比色法（カイネティック比色法）は、ほぼ同等のリムルス活性値を示すことから、本発明においては、リムルス活性は比濁法又は比色法（カイネティック比色法）を用いて測定される。リムルス活性は、市販のエンドトキシン測定キットにより測定することが可能である。 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 (kinetic colorimetric method) show approximately the same Limulus activity value, in the present invention, the Limulus activity is determined by the turbidimetric method or the colorimetric method (kinetic colorimetric method). It is measured using Limulus activity can be measured using a commercially available endotoxin measurement kit.
　本発明において、比濁法又は比色法（カイネティック比色法）により測定したリムルス活性は、３００００ＥＵ／ｍｇ以上である。本発明におけるリムルス活性は、好ましくは４×１０４ＥＵ／ｍｇ以上、１×１０５ＥＵ／ｍｇ以上、１．２×１０５ＥＵ／ｍｇ以上、１．９×１０５ＥＵ／ｍｇ以上、１×１０６ＥＵ／ｍｇ以上、２×１０６ＥＵ／ｍｇ以上、３×１０６ＥＵ／ｍｇ以上、５×１０６ＥＵ／ｍｇ以上、１×１０７ＥＵ／ｍｇ以上、２×１０７ＥＵ／ｍｇ以上、３×１０７ＥＵ／ｍｇ以上、４×１０７ＥＵ／ｍｇ以上、４．５×１０７ＥＵ／ｍｇ以上、又は４．７×１０７ＥＵ／ｍｇ以上である。 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.
　以下、実施例により本発明をさらに詳しく説明するが、本発明はこれら実施例のみに限定されるものではない。 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.
試験１．ＬＰＳの精製
　パラコッカス菌は、Paracoccus carotinifaciensを用い、パラコッカス菌由来のＬＰＳはＰＣＰ（フェノール／クロロホルム／石油エーテル）法にて精製した。具体的には、パラコッカス菌由来のＬＰＳの精製は、以下に示す4ステップにて行った。 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.
・ステップ１：パラコッカス菌乾燥菌体の脱脂
　乾燥菌体として、Ｐａｎａｆｅｒｄ－ＡＸ（ＥＮＥＯＳテクノマテリアル株式会社）を用いた。乾燥菌体６０ｇを５０ｍＬのコニカルチューブ１２本（約５g／チューブ）に分注した。エタノール（４回）、アセトン（３回）およびジエチルエーテル（３回）の順で洗浄（各チューブ４０ｍＬ／回)を行った。洗浄後、凍結乾燥した。乾燥重量は５５．９９８gであった。 - Step 1: Degreasing dried bacterial cells of Paracoccus bacteria Panaferd-AX (ENEOS Techno Material Co., Ltd.) 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.
・ステップ２：ＰＣＰ法によるＬＰＳの抽出
　洗浄脱脂した菌体約５６ｇを５００ｍＬの広口のガラス試薬ビンに加えた。液体フェノール（９０ｇ＋水１１ｍＬの割合の混液）、クロロホルムおよび石油エーテル（２：５：８）から成るＰＣＰ混液を３００ｍＬ加え、氷冷下でポリトロンで２分間攪拌した。懸濁液をステンレス遠心管８本に移し高速冷却遠心機モデル７７８０にて５，０００×ｇ、１５分間遠心分離を行い、遠心分離後の上清（１５０ｍＬ）を５０ｍＬのコニカルチューブに回収した。残渣をさらに２回ＰＣＰ混液で抽出し、遠心分離した上清を回収した。２回目では上清１１０ｍＬ、３回目では上清１８０ｍＬが回収できた。 - 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.
・ステップ３：ＬＰＳの沈殿精製（粗精製ＬＰＳの取得）
　ガラス試験管１６本に各６ｍＬ上清を分注し、遠心エバポレーターで減圧下溶媒（４０℃）を留去した。溶媒が減ったガラス管に上清を追加して上清の全量について溶媒を留去した。溶媒除去後の上清（フェノール液）に水をフェノール液の１／１０量加えた。遠心分離（３，５００ｒｐｍ×２０分）を行い、上清をデカントで別のチューブに移した。チューブの底に沈殿が認められた。沈殿を８０％フェノールで２回（１回につき３ｍＬ）、エーテルで４回順次洗浄（１回につき３ｍＬ程度）し、エーテル除去後減圧乾燥した。粗精製ＬＰＳを４４７ｍｇ取得した。 ・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.
・ステップ４：粗精製ＬＰＳの高度精製
　粗精製ＬＰＳ４４０ｍｇを１０ｍｇ／ｍＬになるように注射用水に懸濁・溶解した。１Ｍ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０を１０ｍＭとなるように加えた。１Ｍ　ＭｇＣｌ２水溶液を２ｍＭとなるように加えた。ベンゾナーゼ溶液を終濃度１０Ｕ／ｍＬになるように１３．２μＬ加えた。３７℃で６時間インキュベートすることにより核酸分解酵素処理をした。核酸分解酵素処理液にプロティナーゼＫを終濃度１００μｇ／ｍＬになるよう２０ｍｇ／ｍＬの水溶液を加え、３７℃で１６時間インキュベートした。核酸分解酵素処理とタンパク分解酵素処理を行った粗ＬＰＳ液を４本に分け、分画分子量１０ｋＤａの遠心式限外ろ過チューブ４本を用いて限外ろ過を行った。処理液を限外ろ過後に、注射用水で限外ろ過は４回行った。
　限外ろ過の内液を回収し、注射用水で共洗いした。粗ＬＰＳ濃度で５ｍｇ／ｍＬになるように注射用水を加えて８８ｍＬとした。トリエチルアミン（ＴＥＡ）を終濃度０．２％、デオキシコール酸Ｎａ（ＤＯＣ）を終濃度０．５％になるように加え溶解懸濁した。液を８本の５０ｍＬのコニカルチューブに１１ｍＬずつ分注した。水飽和フェノールを等量（１１ｍＬ）ずつ加え、氷水中で１０分間置き、その後遠心分離（３，５００ｒｐｍ×２０分間）を行い、水層（上相）を別の５０ｍＬコニカルチューブに回収した。
　フェノール層に、回収した水層と等量の０．２％ＴＥＡ、０．５％ＤＯＣ水溶液を加え混合した。５分間放置後、氷水中で１０分間置き、その後、遠心分離（３，５００ｒｐｍ×２０分間）を行った。水層（上相）を別の５０ｍＬコニカルチューブに回収した。
　回収した水層を８本の５０ｍＬコニカルチューブに分注し、等量の水飽和フェノールを加え、液を混ぜた。５分間放置後、氷水中で１０分間置き、その後、遠心分離（３，５００×２０分間）を行った。水層（上相）を別の５０ｍＬコニカルチューブに回収した。
　回収した水層を８本に分注し、３Ｍ酢酸ナトリウム水溶液（ｐＨ５．２）を終濃度３０ｍＭになるように加えて、終濃度が７５％になるようにチューブごとにエタノール３３ｍＬを加えた。よく混ぜた後、－２０℃で１６時間保存した。
　遠心チューブの液を遠心管８本に分注し高速冷却遠心機を用いて遠心分離（４℃、１０，０００ｇ×３０分）を行った。上清はデカントで除去し、得られた沈殿を、１００％エタノール２０ｍＬずつで計４回洗浄した。得た沈殿を凍結乾燥し、秤量し、６７．２ｍｇの高度精製ＬＰＳを得た。 - 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.
　ＬＰＳの純度は以下の式で算出した。
　　式：ＬＰＳ量＝高度精製ＬＰＳ量－不純物量
　　　　ＬＰＳ（純度）（％）＝ＬＰＳ量／高度精製ＬＰＳ量×１００
　高度精製ＬＰＳ中には、不純物として核酸およびタンパク質が含まれる。高度精製ＬＰＳに含まれる核酸の量はＵＶ吸光度法にて、タンパク質の量はＬｏｗｒｙ法にてそれぞれ測定した。 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.
　ステップ４実施後のＬＰＳの純度を以下に示す。
ＬＰＳ(％)　　　核酸（％）　　　タンパク質（％）　　　合計（％）　
９７．８２　　　１．３５　　　　０．８２　　　　　　１００．００ The purity of LPS after step 4 is shown below.
LPS (%) Nucleic acid (%) Protein (%) Total (%)
97.82 1.35 0.82 100.00
試験２．ＧＣ－ＭＳによる脂肪酸分析方法によるパラコッカス菌由来ＬＰＳの構造特定（１）サンプル調製
　パラコッカス菌由来ＬＰＳとして、試験１で調製したパラコッカス菌由来ＬＰＳ（高度精製品）を用いた。1 mgの精製ＬＰＳを試験管に入れ、5％HClメタノール溶液を加えた。蓋をして100℃で2時間インキュベートした。冷却後、1 mLの水と1 mLの標準液（10μg／mLパルミチン酸メチル-d31)を加え、攪拌混合した。遠心分離（3,000 rpm, 5 min）し、上清をサンプルとして取得した。脂肪酸標準Ｍｉｘ（Supelco（登録商標）37 component FAME Mix）も分析した。 Test 2. Identification of structure of LPS derived from Paracoccus by fatty acid analysis method using GC-MS (1) Sample preparation As LPS derived from Paracoccus, the LPS derived from Paracoccus (highly purified product) prepared in Test 1 was used. 1 mg of purified LPS was placed in a test tube and 5% HCl methanol solution was added. It was covered 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 with stirring. It was centrifuged (3,000 rpm, 5 min) and the supernatant was obtained as a sample. A fatty acid standard Mix (Supelco® 37 component FAME Mix) was also analyzed.
（２）装置及び測定条件
　GC-MS: GCMS2010Ultra (SHIMADZU CORPORATION)を測定装置として用い、表１の測定条件で測定した。 (2) Apparatus and measurement conditions GC-MS: Measurement was carried out under the measurement conditions shown in Table 1 using GCMS2010Ultra (SHIMADZU CORPORATION) as a measurement apparatus.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
（３）結果
　測定結果を表２及び図１に示す。 (3) Results The measurement results are shown in Table 2 and FIG. 1.
　表２に示す構成成分量から、上位の３つの脂肪酸、すなわち、C12:1、3-hydroxy fatty acid-1及び3-hydroxy fatty acid-2がLPSの構成に含まれていると判断された。また、C18:1とC18:0は含有量が少ないことから、不純物と判断した。なお、この試験では、すべのエステル、アミド結合が分解されなかった可能性もあるため、C12:1、3-hydroxy fatty acid-1及び3-hydroxy fatty acid-2間のcomponent ratioは、リピドＡの構造特定に考慮しなかった。 From the component amounts shown in Table 2, it was determined that the top three fatty acids, namely 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, there is a possibility 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 is based on lipid A. was not considered in the structure specification.
（４）脂肪酸の構造特定
　次に、C12:1, 3-hydroxy fatty acid-1, 3-hydroxy fatty acid-2のそれぞれについて、ＭＳのフラグメントピークに基づき構造を特定した。 (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.
　C12:1脂肪酸は、ＧＣのリテンションタイム（9.440 min）と、データベースのＭＳフラグメントピークとの照合により、Ｃ５－Ｃ６にcis型の二重結合を有するC12:1脂肪酸であると特定された（図２）。 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は、ＭＳフラグメントピークから3-OH-10:0であると推察された。すなわち、通常、3-ヒドロキシ脂肪酸は[M-18]、[M-50]及び[M-92]の３つの特徴的なピークを有し、3-hydroxy fatty acid-1が3-OH-10:0である場合は、これら３つのピークはそれぞれ、[M-18]+ 184、[M-50]+ 152及び[M-92]+ 110にて観測される。3-hydroxy fatty acid-1は、これらのピークを有することから3-OH-10:0であると推察された（図３）。 3-hydroxy fatty acid-1 was estimated to be 3-OH-10:0 from the MS fragment peak. That is, 3-hydroxy fatty acid usually has three characteristic peaks, [M-18], [M-50] and [M-92], and 3-hydroxy fatty acid-1 has three characteristic peaks, [M-18], [M-50] and [M-92]. :0, these three peaks are observed at [M-18]+ 184, [M-50]+ 152 and [M-92]+ 110, respectively. 3-hydroxy fatty acid-1 was estimated to be 3-OH-10:0 from having these peaks (Figure 3).
Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000006
　3-hydroxy fatty acid-2も3-hydroxy fatty acid-1と同様の手法で、3-OH-14:0と特定された（図４）。 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).
　本試験例におけるＧＣ－ＭＳによる脂肪酸分析方法により、パラコッカス菌由来ＬＰＳは、Ｃ５－Ｃ６にcis型の二重結合を有するC12:1脂肪酸、3-OH-10:0及び3-OH-14:0を含むことが明らかになった。 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.
試験３．ＭＡＬＤＩ－ＴＯＦ　ＭＳによるＬＰＳとリピドＡの測定
（１）Lipid Aの調製方法
　試験２と同様に、試験３では、パラコッカス菌由来ＬＰＳとして、試験１で調製したパラコッカス菌由来ＬＰＳ（高度精製品）を用いた。4 mgの精製ＬＰＳを試験管に入れ、2％酢酸水溶液で100℃、2時間インキュベートすることで、ＬＰＳを加水分解した。冷却後、沈殿を遠心分離(13,000×g, 2分)で集め、水で数回洗浄し、MALDI-TOF MS用サンプルとした。 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）装置と測定条件
　MALDI-TOF MS: AXIMA Performance（SHIMADZU CORPORATION）を測定装置として用い、表３の測定条件で測定した。 (2) Apparatus and measurement conditions MALDI-TOF MS: AXIMA Performance (SHIMADZU CORPORATION) was used as a measurement apparatus, and measurements were carried out under the measurement conditions shown in Table 3.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
（３）結果
　MALDI-TOF MS分析（陰イオンモード）を図５に示す。図５から、Lipid Aの陰イオンピークは1472であると考えられた。３つのフラグメントピーク1392、1302及び1222は、それぞれ1392（-HPO3）、1302（-3-OH-10:0）及び1222（-HPO3と-3-OH-10:0）と同定された。
　本試験例から、パラコッカス菌由来ＬＰＳは、Lipid Aリン酸エステルと3-OH-10:0脂肪酸を有することが分かった。
　また、リピドＡ全体の分子量は1473であることが明らかになった（図５）。この分子量に合うように脂肪酸（Ｃ５－Ｃ６にcis型の二重結合を有するC12:1脂肪酸、3-OH-10:0及び3-OH-14:0）を組み合わせると、リピドＡは、Ｃ５－Ｃ６にcis型の二重結合を有するC12:1脂肪酸を1本、3-OH-10:0を2本及び3-OH-14:0を2本有することが理解される。　 (3) Results MALDI-TOF MS analysis (negative ion mode) is shown in FIG. 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.
　ＬＰＳのリピドＡでは、グルコサミン2分子に直接結合する脂肪酸は、3位に水酸基を有する３－ヒドロキシアシル鎖であることが多いことが知られている。このため、3位に水酸基を有しないC12:1脂肪酸は、直接グルコサミンに結合していないと推定される。また、16S rRNA解析において、Paracoccus carotinifaciensはRhodobacter sphaeroidesと９３％の同一性を有することから、互いに類縁種であってリピドＡの構造も類似すると推定される（FEMS Microbiology Reviews 4 (1988) 143-154）。試験３及び試験４の結果と、リピドＡにおけるこれまでの知見を参酌すると、Paracoccus carotinifaciensのリピドＡは、3-OH-14:0がグルコサミンの2位アミノ基にアミド結合を介して結合し、3-OH-10:0がグルコサミンの３位水酸基にエステル結合を介して結合し、Ｃ５－Ｃ６にcis型の二重結合を有するC12:1脂肪酸が、3-OH-14:0の3位水酸基にエステル結合を介して結合する構造を有することが推定された（構造は下に示す）。Paracoccus carotinifaciensのリピドＡが上記構造を有することは、16S rRNA解析においてParacoccus carotinifaciensと９７％の同一性を有するParacoccus denitrificansのＬＰＳ構造に関する知見と一致する。すなわち、Paracoccus denitrificansのＬＰＳでは、3-OH-10:0及びC12:1脂肪酸はそれぞれエステル結合した形でリピドＡに存在し、3-OH-14:0と3-oxo-14:0（試験３と試験４では検出されず）はアミド結合した形でリピドＡに存在するとの知見（FEMS Microbiology Letters 37 (1986) 63-67）がある。 It is known that in lipid A of LPS, the fatty acid directly bonded to two glucosamine molecules is often a 3-hydroxyacyl chain having a hydroxyl group at the 3-position. Therefore, it is presumed that the C12:1 fatty acid, which does not have a hydroxyl group at the 3-position, is not directly bound to glucosamine. Furthermore, in 16S rRNA analysis, Paracoccus carotinifaciens has 93% identity with Rhodobacter sphaeroides, so it is assumed that they are related species and have similar lipid A structures (FEMS Microbiology Reviews 4 (1988) 143-154 ). Taking into account the results of Tests 3 and 4 and the previous findings regarding lipid A, lipid A of Paracoccus carotinifaciens has 3-OH-14:0 bonded to the 2-position amino group of glucosamine via an amide bond, 3-OH-10:0 is bonded to the 3-position hydroxyl group of glucosamine via an ester bond, and a C12:1 fatty acid having a cis-type double bond at C5-C6 is bonded to the 3-position hydroxyl group of 3-OH-14:0. It was estimated that it has a structure in which it is bonded to a hydroxyl group via an ester bond (the structure is shown below). The fact that lipid A of Paracoccus carotinifaciens has the above structure is consistent with the findings regarding 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, and 3-OH-14:0 and 3-oxo-14:0 (test 3 and not detected in Test 4) exists in lipid A in the form of an amide bond (FEMS Microbiology Letters 37 (1986) 63-67).
試験４．リムルス活性測定
　パラコッカス菌由来ＬＰＳのリムルス活性を測定した。 Test 4. Measurement of Limulus Activity Limulus activity of LPS derived from Bacillus Paracoccus was measured.
（１）測定サンプル
　試験４では、パラコッカス菌由来ＬＰＳとして、試験１でＰＣＰ法にて精製したパラコッカス菌由来ＬＰＳ：「高度精製品」（ステップ４で得られた高度精製ＬＰＳ）、「アセトン脱脂」（ステップ１で得られたＬＰＳ）、及び「粗精製ＬＰＳ」（ステップ３で得られた粗精製ＬＰＳ）を用いた。 (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.
　また、ＰＣＰ法以外の精製法によるパラコッカス菌由来ＬＰＳとして、Ｐａｎａｆｅｒｄ－ＡＸ（「Panaferd-AX（未処理）」）、Ｐａｎａｆｅｒｄ－ＡＸ微粉砕品（「微粉砕品」）、Ｐａｎａｆｅｒｄ－ＡＸ微粉砕品＋レシチン（界面活性剤）（「微粉砕品＋レシチン」）、Ｐａｎａｆｅｒｄ－ＡＸ微粉砕品＋リゾレシチン（界面活性剤）（「微粉砕品＋リゾレシチン」）も測定サンプルとして用いた。 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.
　これらの測定サンプルは以下のように準備した。
「Panaferd-AX（未処理）」は、Panaferd-AX（ＥＮＥＯＳテクノマテリアル株式会社）を用いた。
　「微粉砕品」：１００ｇのＰａｎａｆｅｒｄ－ＡＸと９００ｇの水を混合し、高圧ホモジナイザーPanda PLUS 2000を用いて１００ＭＰａで１０回処理して破砕した。破砕液を凍結乾燥したものを、Ｐａｎａｆｅｒｄ－ＡＸ微粉砕品とした。
　「微粉砕品＋レシチン」：５０ｍＬチューブにレシチン（辻製油製ＳＬＰ－ＰＣ７０）を３００ｍｇ秤量し、１５ｍＬのイオン交換水を入れて、１時間ダイレクトミキサーで混合した。この操作により、レシチンの２％水溶液（均一溶媒）が得られた。微粉砕品を３ｇ秤量し、レシチン水溶液に入れ、１時間ダイレクトミキサーで混合した。混合液を凍結乾燥した粉末を「微粉砕品＋レシチン」とした。
　「微粉砕品＋リゾレシチン」：レシチンの代わりにリゾレシチン（辻製油製ＳＬＰ－ＬＰＣ７０）を用いた他は、「微粉砕品＋レシチン」の製造方法と同様の方法で「微粉砕品＋リゾレシチン」を得た。 These measurement samples were prepared as follows.
For "Panaferd-AX (untreated)", Panaferd-AX (ENEOS Techno Material 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）リムルス活性の測定方法１（比濁法）
　ＰＣＰ法にて精製したパラコッカス菌由来ＬＰＳのリムルス活性は、Limulus ES-II plus CS single test wako（富士フイルム和光純薬株式会社　Cat＃ 299-77201）を用いた比濁法により測定した。
　エッペンドルフチューブ（１．５ｍL）各サンプルを５ｍｇはかりとり、水を加えて５ｍｇ／ｍＬ懸濁液を作製した。懸濁液をＶｏｌｔｅｘにて５分撹拌した後、９０００ｒｐｍ、２０℃で１５分遠心し、その上清を測定に使用した。
　上清２００μＬをLimulus ES-II plus CS single test wakoのライセート試薬に入れ、Ｖｏｌｔｅｘにて１０秒撹拌した。ライセート試薬をトキシノメーター（富士フイルム和光純薬株式会社　Cat＃ ET-6000/J）にセットし、カイネティックモードで、４３０ｎｍの吸光度を経時測定した：測定間隔１５秒、総測定時間６０分、温度３７℃。なお、本測定は、ライセート試薬のゲル化にかかる時間を指標として定量しており、測定時間によって結果が変わるものではない。 (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）リムルス活性の測定方法２（比色法）
　ＰＣＰ法以外の精製法によるパラコッカス菌由来ＬＰＳのリムルス活性は、試験キット：LONZA Limulus Amebocyte Lysate (LAL) Kinetic-QCLを用い、Limulus測定（比色法）にて測定した。検量線作成にはキットに付属のE. coli O55:B5 Endotocin[E50-643]を５０ＥＵ／ｍＬとなるよう希釈し使用した。
　エッペンドルフチューブ（１．５ｍＬ）に各サンプルを５０ｍｇはかりとり、水を加えて１００ｍｇ／ｍＬ懸濁液を作製した。懸濁液をＶｏｌｔｅｘにて５分撹拌した後、９０００ｒｐｍ、２０℃で１５分遠心し、その上清を測定に使用した。
　上清１００μＬを９６ｗｅｌｌ ｐｌａｔｅに入れ、プレートリーダーで３７℃、１０分間プレインキュベートした。Ｌｉｍｕｌｕｓ試薬を１００μＬ入れ、プレートリーダーのｋｉｎｅｔｉｃモードで吸光度４０５ｎｍを経時測定した：測定間隔１分、測定総時間６０分、温度３７℃。なお、本測定は、吸光度が０．２上昇するのにかかった時間を指標として定量しており、測定時間によって結果が変わるものではない。 (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.
　既存のデータから、比濁法と比色法はほぼ同等のリムルス活性値が得られることが理解されるため、両方法によるリムルス活性値を比較することは可能である。 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）結果
　上記（２）及び（３）の方法で測定されたリムルス活性の値を表４に示す。 (4) Results Table 4 shows the values of Limulus activity measured by the methods (2) and (3) above.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　上記のように、本発明の組成物は、高いリムルス活性を示すことが示された。 As mentioned above, the composition of the present invention was shown to exhibit high Limulus activity.
　本発明により、所定の構造を有するリピドＡを有する、グラム陰性菌のＬＰＳを含む組成物が提供される。また、別の態様において、本発明により、所定のリムルス活性値を有するグラム陰性菌のＬＰＳを含む組成物が提供される。
　本発明により提供される組成物は、構造及び／又はリムルス活性の点でＬＰＳの免疫賦活活性を医薬品、化粧品、食品、飼料等に利用する上で有用である。 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.
[規則26に基づく補充 23.10.2023]
Figure WO-DOC-RO134
[Replenishment under Regulation 26 23.10.2023]
Figure WO-DOC-RO134

Claims (5)

  1. 　グラム陰性菌のリポ多糖を含む組成物であって、前記リポ多糖がリピドＡを有し、
    　前記リピドＡのグルコサミン骨格に、炭素数８～１６の３－ヒドロキシアシル鎖が４本結合し、かつ、１又は２本の前記３－ヒドロキシアシル鎖の３位の水酸基に炭素数８～１３のアシル鎖がさらに結合し、
    　リムルス活性が３００００ＥＵ／ｍｇ以上である、組成物。     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.
  2. 　前記リピドＡのグルコサミン骨格に、炭素数１０～１４の３－ヒドロキシアシル鎖が４本結合し、かつ、グルコサミン骨格の２位に結合する炭素数１０～１４の３－ヒドロキシアシル鎖の３位の水酸基に炭素数１２のアシル鎖がさらに結合する、請求項１に記載の組成物。 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. 　前記炭素数１２のアシル鎖が、非還元末端側のグルコサミンの２位に結合する炭素数１４の３－ヒドロキシアシル鎖の３位の水酸基に結合し、還元末端側のグルコサミンの２位に結合する炭素数１４の３－ヒドロキシアシル鎖の３位の水酸基に結合しない、請求項２に記載の組成物。 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. 　前記リピドＡが、式（Ｉ）の構造：
    を有する、請求項１～３のいずれかに記載の組成物。     The lipid A has a structure of formula (I):
    The composition according to any one of claims 1 to 3, having the following.
  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 composition according to any one of claims 1 to 4, wherein the Gram-negative bacterium is a bacterium belonging to the genus Paracoccus.
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