Composition comprising lipopolysaccharide

Abstract

To provide a composition comprising an LPS of a Gram-negative bacterium having a predetermined structure or predetermined activity.SOLUTION: According to the present invention, there is provided 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 predetermined limulus activity.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to compositions comprising lipopolysaccharides of Gram-negative bacteria. [Background technology]

[0002] 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 lead to death if given in large quantities. Therefore, LPS is also called endotoxin.

[0003] The basic structure of LPS contains a lipid A portion, which is a lipid moiety, and a polysaccharide portion. The polysaccharide portion is further composed of a core and an O-antigen polysaccharide side chain. Normally, LPS is incorporated into the cell wall with the lipid A embedded in the outer membrane and the O-antigen polysaccharide side chain protruding outward, and is not easily released from the cell wall. When gram-negative bacteria die and the cells are lysed, LPS is released and acts on animal cells, for example, to express various physiological activities.

[0004] Released LPS exerts its effect via TLR4 (toll-like receptor 4) on the cell membrane of target cells. When LPS binds to TLR4, it induces the production of inflammatory cytokines such as TNFα, IL-6, and IL-12, as well as type I interferon (IFN), via the intracellular signal transduction system (Non-Patent Document 1). In particular, LPS from E. coli and other bacteria simultaneously and strongly activates these signals, inducing strong inflammation.

[0005] Thus, LPS promotes the production of cytokines through the TLR4 signaling pathway. Cytokine production generally leads to strong immunostimulatory activity and plays a certain role in the host defense response against bacterial infection.

[0006] As described above, since LPS is known to have immunostimulatory activity, the use of the immunostimulatory activity of LPS in medicines, cosmetics, foods, feed, etc. is being considered. However, when extracting LPS from Gram-negative bacteria by conventional methods, it is difficult to obtain LPS with high concentration or activity.

[0007] Although the basic structure of LPS is similar among gram-negative bacteria, it is known that the structure of LPS varies depending on the species of gram-negative bacteria from which it is derived. However, it is generally not easy to determine the structure of LPS from gram-negative bacteria. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Biochemistry, 79, 769-776, 2007 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a composition containing LPS from a Gram-negative bacterium, wherein the LPS has a given structure or activity. [Means for solving the problem]

[0010] The present inventors have conducted intensive research and found a composition containing LPS of a Gram-negative bacterium having a specific structure or specific activity. [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 bound to the glucosamine backbone of the lipid A, and an acyl chain having 8 to 13 carbon atoms is further bound to the hydroxyl group at the 3-position of one or two of the 3-hydroxyacyl chains; A composition having limulus activity of 30,000 EU / mg or more. [2] The composition according to claim 1, wherein four 3-hydroxyacyl chains having 10 to 14 carbon atoms are bonded to the glucosamine backbone of the lipid A, and an acyl chain having 12 carbon atoms is further bonded to the hydroxyl group at the 3-position of each of the 3-hydroxyacyl chains having 10 to 14 carbon atoms bonded to the 2-position of the glucosamine backbone. [3] The composition of claim 2, wherein the 12-carbon acyl chain is bonded to the 3-hydroxyl group of a 14-carbon 3-hydroxyacyl chain bonded to the 2-position of glucosamine on the non-reducing end side, and is not bonded to the 3-hydroxyl group of a 14-carbon 3-hydroxyacyl chain bonded to the 2-position of glucosamine on the reducing end side. [4] The lipid A has the structure of formula (I): [ka] The composition according to any one of claims 1 to 3, comprising: [5] 5. The composition according to claim 1, wherein the gram-negative bacterium is a bacterium belonging to the genus Paracoccus. Effect of the Invention

[0011] The present invention provides a composition comprising an LPS of a Gram-negative bacterium having lipid A with a defined structure and limulus activity. The composition provided by the present invention has a specified structure and has high Limulus activity, making it useful for utilizing the immunostimulatory activity of LPS in pharmaceuticals, cosmetics, foods, feeds, and the like.

[0012] [Brief description of the drawings]

[0013] [Figure 1] The GC chart of fatty acid analysis is shown below. The upper chart shows the analysis results of fatty acids derived from Paracoccus LPS, and the lower chart shows the analysis results of the fatty acid standard mix. [Diagram 2]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). [Diagram 3] The GC-MS spectrum (retention time 14.417 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 3-OH C10:0. [Figure 4] The GC-MS spectrum (retention time 23.010 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 3-OH C14:0. [Diagram 5] 1 shows the results of MALDI-TOF MS measurement in negative ion mode for the lipid A portion of LPS derived from bacteria belonging to the genus Paracoccus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will be described in more detail below. The scope of the present invention is not limited to these descriptions, and other than the following examples, the present invention can be appropriately modified and implemented without departing from the spirit of the present invention.

[0015] 1. LPS from Gram-negative bacteria The composition of the present invention contains LPS of gram-negative bacteria. It is known that gram-negative bacteria have LPS in their cell walls. In the present invention, the gram-negative bacteria may be any gram-negative bacteria having LPS, and includes, but is not limited to, for example, Proteobacteria (Escherichia coli, Salmonella, Pseudomonas, Paracoccus, etc.), Cyanobacteria, Spirochaetes, Chlorobium, and Bacteroides. In the present invention, the gram-negative bacteria is preferably bacteria belonging to the genus Paracoccus.

[0016] In the present specification, the bacteria belonging to the genus Paracoccus are not particularly limited. As the bacteria belonging to the genus Paracoccus, Paracoccus carotinifaciens, Paracoccus marcusii, Paracoccus haeundaensis, and Paracoccus zeaxanthinifaciens are preferably used, Paracoccus carotinifaciens or Paracoccus zeaxanthinifaciens is more preferably used, and Paracoccus carotinifaciens is particularly preferably used. Specific examples of strains of bacteria belonging to the genus Paracoccus include Paracoccus carotinifaciens E-396 strain (FERM BP-4283), Paracoccus bacteria A-581-1 strain (FERM BP-4671), Paracoccus marcusii DSM 11574 strain, Paracoccus bacteria N-81106 strain, Paracoccus haeundaensis BC 74171 strain, Paracoccus zeaxanthinifaciens ATCC 21588 strain, and Paracoccus sp. PC-1 strain, and mutant strains of these are also preferably used in the present invention. The E-396 and A-581-1 strains have been internationally deposited with the National Institute of Technology and Evaluation (NITE) International Patent Organism Depositary (NITE-IPOD) (2-5-8 Kazusa Kamatari, Kisarazu, Chiba 292-0818) as the international depositary authority as follows: E-396 strain Identification: E-396, Accession number: FERM BP-4283, Original deposit date: April 27, 1993 A-581-1 strain Identification: A-581-1, Accession number: FERM BP-4671, Original deposit date: May 20, 1994

[0017] 2. Structure of Lipid A LPS of gram-negative bacteria has lipid A. In the present invention, lipid A has a backbone of two molecules of glucosamine, each molecule of glucosamine being linked by a β(1→6) bond. In this specification, this backbone of lipid A is referred to as the "glucosamine backbone."

[0018] In one embodiment of the present invention, a phosphate group is bound to the 1-position of the glucosamine on the reducing end side of the glucosamine backbone, and a phosphate group is bound to the 4-position 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.

[0019] In the lipid A of the present invention, four acyl chains having 8 to 16 carbon atoms are directly bonded to a glucosamine backbone, and the acyl chains have a hydroxyl group at position 3. In this specification, the acyl chain having a hydroxyl group at position 3 is also referred to as a 3-hydroxyacyl chain. That is, lipid A has two molecules of glucosamine bound to it, with a 3-hydroxyacyl chain at the 2nd position of glucosamine by an amide bond and a 3-hydroxyacyl chain at the 3rd position by an ester bond. The two molecules of glucosamine may be the same or may be two molecules of glucosamine bound to different 3-hydroxyacyl chains.

[0020] In the present invention, the 3-hydroxyacyl chain bonded to the 2- and 3-positions of glucosamine has 8 to 16 carbon atoms, preferably 10 to 14 carbon atoms, more preferably 10, 11, 12, 13 or 14 carbon atoms, and further preferably 10 or 14 carbon atoms.

[0021] The number of double bonds in the 3-hydroxyacyl chain directly attached to the glucosamine backbone is 0, 1 or 2, preferably 0.

[0022] In the present invention, the 3-hydroxyacyl chain attached to the glucosamine backbone is preferably 3-OH10:0 or 3-OH14:0.

[0023] In one embodiment of the present 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.

[0024] In the lipid A of the present invention, one or two of the four 3-hydroxyacyl chains directly bonded to the glucosamine backbone are further ester-bonded to the hydroxyl group at the 3-position of the 3-hydroxyacyl chain with an acyl chain having 8 to 13 carbon atoms. 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 backbone. Due to the bond of such additional acyl chains, lipid A has five or six acyl chains.

[0025] In the present invention, the acyl chain bonded to the hydroxyl group at position 3 of the 3-hydroxyacyl chain directly bonded to the glucosamine backbone has 8 to 13 carbon atoms, preferably 8 to 12 carbon atoms, and more preferably 12 carbon atoms.

[0026] In the present invention, the number of double bonds in the acyl chain bonded to the hydroxyl group at position 3 of the 3-hydroxyacyl chain directly bonded to the glucosamine backbone is 0, 1 or 2, and preferably 1.

[0027] In the present invention, the acyl chain bonded to the hydroxyl group at the 3-position of the 3-hydroxyacyl chain directly bonded to the glucosamine backbone is preferably C12:1, more preferably C12:1 having a double bond (cis) at the 5th carbon (C5-6) counting from the carbonyl group (C12:1(Δ 5 )).

[0028] In one embodiment of the present invention, the acyl chain bonded to the hydroxyl group at position 3 of the 3-hydroxyacyl chain bonded directly to the glucosamine backbone is bonded to the hydroxyl group at position 3 of the 3-hydroxyacyl chain bonded to position 2 of the glucosamine on the non-reducing end side, but is not bonded to the hydroxyl group at position 3 of the 3-hydroxyacyl chain bonded to position 2 of the glucosamine on the reducing end side.

[0029] Examples of lipid A according to the present invention are given below. In one aspect of the present invention, lipid A has a structure in which four 3-hydroxyacyl chains having 8 to 16 carbon atoms are bound to a glucosamine backbone, and an acyl chain having 8 to 13 carbon atoms is further bound to the hydroxyl group at the 3-position of one or two of the 3-hydroxyacyl chains.

[0030] In another aspect of the present invention, lipid A has a structure in which four 3-hydroxyacyl chains having 10 to 14 carbon atoms are bonded to a glucosamine backbone, and an acyl chain having 8 to 13 carbon atoms is further bonded to the hydroxyl group at the 3-position of one or two of the 3-hydroxyacyl chains having 10 to 14 carbon atoms bonded to the 2-position of the glucosamine backbone.

[0031] In another aspect of the present invention, lipid A has a structure in which four 3-hydroxyacyl chains having 10 to 14 carbon atoms are bonded to a glucosamine backbone, and one or two of the 3-hydroxyacyl chains having 10 to 14 carbon atoms bonded to the hydroxyl group at the 3-position of the glucosamine backbone are further bonded to an acyl chain having a carbon number of 12.

[0032] In another embodiment of the present invention, lipid A has four 3-hydroxyacyl chains having 10 to 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 each of the 3-hydroxyacyl chains having 10 to 14 carbon atoms bound to the 2-position of the glucosamine backbone; The structure has such that the 12-carbon acyl chain is bonded to the 3-hydroxyl group of the 14-carbon 3-hydroxyacyl chain bonded to the 2-position of the glucosamine on the non-reducing end side, but is not bonded to the 3-hydroxyl group of the 14-carbon 3-hydroxyacyl chain bonded to the 2-position of the glucosamine on the reducing end side.

[0033] 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 12-carbon acyl chain is bonded to the 3-hydroxyl group of the 14-carbon 3-hydroxyacyl chain bonded to the 2-position of the glucosamine on the non-reducing end side, but is not bonded to the 3-hydroxyl group of the 14-carbon 3-hydroxyacyl chain bonded to the 2-position of the glucosamine on the reducing end side.

[0034] In one embodiment of the present invention, lipid A has a structure in which 3-OH14:0 is bound to the 2-position of glucosamine, 3-OH10:0 is bound to the 3-position of glucosamine, and C12:1, which contains a double bond (cis) between C5-6, is bound to 3-OH14:0 bound to the 2-position of glucosamine on the non-reducing end side.

[0035] In one embodiment of the invention, lipid A has the structure of formula (I): [ka]

[0036] 3. Composition of the Present Invention The present invention has discovered that a composition containing a treated product of Gram-negative bacteria can provide higher Limulus activity than conventional bacteria, etc. The composition of the present invention can be produced by subjecting Gram-negative bacteria to treatment such as purification and extraction.

[0037] 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 may 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.

[0038] In addition, before or after the above extraction treatment, the bacteria may be subjected to a single or combined treatment such as concentration, drying, dilution, crushing, pulverization, heating, etc. Drying treatments include spray drying, freeze drying, vacuum drying, and drum drying. Crushing treatments include crushing using a homogenizer, French press, mortar, glass beads, etc. It is expected that the surface area is increased by crushing or pulverizing the bacterial cells, and more LPS is exposed at the interface. Those skilled in the art can culture gram-negative bacteria and perform treatments such as extraction according to known methods.

[0039] As described above, the composition of the present invention can be made of dried cells or processed products thereof after culturing gram-negative bacteria. In addition, commercially available products can be used. For example, Panafert-AX (ENEOS Corporation), which is dried cells after culturing Paracoccus carotinifaciens, can be used.

[0040] Those skilled in the art can further purify the extracted LPS derived from bacteria belonging to the genus Paracoccus. The purification method can be according to a conventionally known method.

[0041] For example, LPS can be extracted from gram-negative bacteria by treating them with the hot water phenol method (O. Westphal, K. Jann, "Methods in Carbohydrate Chemistry", ed. by R. Whistler, Vol. 5, p. 83, Academic Press, New York (1965)), the phenol-chloroform-petroleum ether extraction method (PCP) (C. Galanos, O. Luderitz, O. Westphal, Eur. J. Biochem., 9, 245 (1969)), or the like.

[0042] The composition of the present invention can be LPS or a composition obtained in each purification step of the PCP method. The purity of LPS can be improved by purification. For details of the PCP method, see the description of the 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 identified by using LPS highly purified by the PCP method.

[0043] In another embodiment of the present invention, the composition of the present invention may contain lecithin or lysolecithin. Those skilled in the art can incorporate lecithin or lysolecithin into the composition using conventional techniques. For example, an aqueous solution of lecithin or lysolecithin is mixed with an LPS extract of a gram-negative bacterium, dried cells of a gram-negative bacterium after culture or a processed product thereof in 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 (especially LPS of bacteria belonging to the genus Paracoccus) and the absorbability in animals can be improved.

[0044] LPS is known to have immunostimulatory activity. The composition of the present invention has a limulus activity of 30,000 EU / mg or more, and is therefore useful as a feed composition, a pharmaceutical composition, a food composition, or a cosmetic composition. When used as a feed, a pharmaceutical composition, a food, or a cosmetic, it may further contain a carrier acceptable for each purpose.

[0045] 4. Limulus activity It has been found that the composition of the present invention exhibits higher limulus activity than conventional compositions. Limulus activity is the activity of promoting the coagulation of horseshoe crab hemocyte extracts, and is the biological activity of endotoxin at the molecular level. Limulus activity is measured by a limulus test that detects or quantifies endotoxin using a lysate reagent prepared from a horseshoe crab hemocyte extract. The limulus test includes a gelation method using the gel formation of the lysate reagent as an indicator, and an optical quantification method (turbidimetric method, colorimetric method) using optical changes as an indicator. The turbidimetric method is a method for measuring the endotoxin concentration by measuring the change in turbidity (absorbance or transmittance) associated with the gelation of the lysate reagent. The colorimetric method is a method for measuring the amount of chromogenic groups released from a color-forming synthetic substrate by a clotting enzyme generated by the reaction of endotoxin with the lysate reagent, using absorbance or transmittance. The colorimetric method includes a kinetic colorimetric method and an end-point colorimetric method. Since the turbidimetric method and the colorimetric method show almost the same limulus activity values, in the present invention, the limulus activity is measured using the turbidimetric method or the colorimetric method (kinetic colorimetric method). The limulus activity can be measured using a commercially available endotoxin measurement kit.

[0046] In the present invention, the Limulus activity measured by turbidimetry or colorimetry (kinetic colorimetry) is 30,000 EU / mg or more. The Limulus activity in the present invention is preferably 4×10 4 EU / mg or more, 1×10 5 EU / mg or more, 1.2×10 5 EU / mg or more, 1.9×10 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 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 x 10 7 EU / mg or more. EXAMPLES

[0047] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0048] Test 1. Purification of LPS Paracoccus carotinifaciens was used as the paracoccus bacterium, and the LPS derived from Paracoccus bacterium was purified by the PCP (phenol / chloroform / petroleum ether) method. Specifically, the purification of the LPS derived from Paracoccus bacterium was carried out in the following four steps.

[0049] Step 1: Defatting of dried Paracoccus cells Panafert-AX (ENEOS Corporation) was used as the dried cells. 60 g of dried cells were dispensed into 12 50 mL conical tubes (approximately 5 g / tube). The cells were washed in the order of ethanol (4 times), acetone (3 times), and diethyl ether (3 times) (40 mL / tube). After washing, the cells were freeze-dried. The dry weight was 55.998 g.

[0050] Step 2: Extraction of LPS by PCP method Approximately 56 g of washed and degreased cells were added to a 500 mL wide-mouth glass reagent bottle. 300 mL of PCP mixture consisting of liquid phenol (90 g + 11 mL water), chloroform, and petroleum ether (2:5:8) was added and stirred with a Polytron for 2 minutes under ice cooling. The suspension was transferred to eight stainless steel centrifuge tubes and centrifuged at 5,000 x g for 15 minutes in a high-speed refrigerated centrifuge model 7780, and the supernatant (150 mL) after centrifugation was collected in a 50 mL conical tube. The residue was extracted twice more with the PCP mixture, and the supernatant after centrifugation was collected. 110 mL of supernatant was collected the second time, and 180 mL of supernatant was collected the third time.

[0051] Step 3: Precipitation purification of LPS (obtaining crude LPS) 6 mL of the supernatant was dispensed into 16 glass test tubes, and the solvent was removed under reduced pressure (40°C) using a centrifugal evaporator. The supernatant was added to the glass tube with the reduced amount of solvent, and the solvent was removed from the entire amount of the supernatant. Water was added to the supernatant (phenol solution) after the solvent was removed in an amount of 1 / 10 the amount of phenol solution. Centrifugation (3,500 rpm x 20 minutes) was performed, and the supernatant was transferred to another tube by decantation. A precipitate was observed at the bottom of the tube. The precipitate was washed twice with 80% phenol (3 mL each time) and four times with ether (approximately 3 mL each time), and after removing the ether, it was dried under reduced pressure. 447 mg of crudely purified LPS was obtained.

[0052] Step 4: High-purification of crude LPS 440 mg of crude 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 a concentration of 10 mM. 1M MgCl2 aqueous solution was added to a concentration of 2 mM. 13.2 μL of benzonase solution was added to a final concentration of 10 U / mL. Nuclease 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 treatment solution to a final concentration of 100 μg / mL, and incubated at 37°C for 16 hours. The crude LPS solution treated with nuclease and protease was divided into four tubes and ultrafiltered using four centrifugal ultrafiltration tubes with a molecular weight cutoff of 10 kDa. After ultrafiltration of the treated solution, ultrafiltration was performed four times with water for injection. The internal liquid of the ultrafiltration was collected and washed with water for injection. Water for injection was added to make the crude LPS concentration 5 mg / mL, making the total volume 88 mL. Triethylamine (TEA) was added to a final concentration of 0.2%, and sodium deoxycholate (DOC) was added to a final concentration of 0.5% to dissolve and suspend the LPS. The liquid was dispensed into eight 50 mL conical tubes, 11 mL each. An equal amount (11 mL) of water-saturated phenol was added to each tube, and the tube was left in ice water for 10 minutes, after which it was centrifuged (3,500 rpm x 20 minutes) and the aqueous layer (upper phase) was collected in another 50 mL conical tube. The phenol layer was mixed with an equal amount of 0.2% TEA and 0.5% DOC aqueous solution to the collected aqueous layer. After leaving it 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 in another 50 mL conical tube. The collected aqueous layer was dispensed into eight 50 mL conical tubes, and an equal amount of water-saturated phenol was added and mixed. After leaving it 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 in another 50 mL conical tube. The collected aqueous layer was dispensed into eight tubes, and a 3M aqueous sodium acetate solution (pH 5.2) was added to the tube to make the final concentration 30 mM. Then, 33 mL of ethanol was added to each tube to make the final concentration 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 min) using a high-speed refrigerated centrifuge. The supernatant was decanted and the resulting precipitate was washed a total of four times with 20 mL of 100% ethanol each time. The resulting precipitate was freeze-dried and weighed to obtain 67.2 mg of highly purified LPS.

[0053] The purity of LPS was calculated using the following formula. Formula: LPS amount = highly purified LPS amount - impurity amount LPS (purity) (%) = LPS amount / highly purified LPS amount x 100 Highly purified LPS contains nucleic acids and proteins as impurities. The amount of nucleic acids in the highly purified LPS was measured by UV absorbance and the amount of proteins was measured by the Lowry method.

[0054] The purity of LPS after step 4 is shown below. LPS (%) Nucleic acid (%) Protein (%) Total (%) 97.82 1.35 0.82 100.00

[0055] Test 2. Identification of the structure of Paracoccus LPS by fatty acid analysis using GC-MS (1) Sample preparation Paracoccus-derived LPS (highly purified product) prepared in Test 1 was used as Paracoccus-derived 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 (registered trademark) 37 component FAME Mix) was also analyzed.

[0056] (2) Apparatus and measurement conditions GC-MS: GCMS2010Ultra (SHIMADZU CORPORATION) was used as the measuring device, and measurements were performed under the measurement conditions shown in Table 1.

[0057] [Table 1]

[0058] (3) Results The measurement results are shown in Table 2 and FIG.

[0059] [Table 2]

[0060] 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. In addition, C18:1 and C18:0 were determined to be impurities because of their low content. 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 not taken into consideration in identifying the structure of lipid A.

[0061] (4) Identification of fatty acid structure Next, the structures of C12:1, 3-hydroxy fatty acid-1, and 3-hydroxy fatty acid-2 were identified based on MS fragment peaks.

[0062] The C12:1 fatty acid was identified as a C12:1 fatty acid having a cis double bond at C5-C6 by matching the GC retention time (9.440 min) with the MS fragment peak in the database (Figure 2).

[0063] 3-hydroxy fatty acid-1 was inferred to be 3-OH-10:0 from the MS fragment peak. That is, 3-hydroxy fatty acids usually have three characteristic peaks of [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 because it has these peaks (Figure 3).

[0064] [ka]

[0065] 3-hydroxy fatty acid-2 was also identified as 3-OH-14:0 using the same method as 3-hydroxy fatty acid-1 (FIG. 4).

[0066] [ka]

[0067] The fatty acid analysis method by GC-MS in this test example revealed that Paracoccus-derived LPS contains C12:1 fatty acid having a cis double bond at C5-C6, 3-OH-10:0, and 3-OH-14:0.

[0068] Test 3. Measurement of LPS and lipid A by MALDI-TOF MS (1) Preparation of Lipid A As in Test 2, in Test 3, the Paracoccus-derived LPS (highly purified product) prepared in Test 1 was used as the Paracoccus-derived LPS. 4 mg of purified LPS was placed in a test tube and incubated in a 2% aqueous acetic acid solution at 100°C for 2 hours to hydrolyze the LPS. After cooling, the precipitate was collected by centrifugation (13,000 x g, 2 minutes), washed several times with water, and used as a sample for MALDI-TOF MS.

[0069] (2) Apparatus and measurement conditions MALDI-TOF MS: AXIMA Performance (SHIMADZU CORPORATION) was used as the measuring device, and measurements were performed under the measuring conditions shown in Table 3.

[0070] [Table 3]

[0071] (3) Results MALDI-TOF MS analysis (negative ion mode) is shown in Figure 5. From Figure 5, the negative ion 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 contains Lipid A phosphate ester and 3-OH-10:0 fatty acid. In addition, the molecular weight of the entire lipid A was found to be 1473 (Figure 5). By combining fatty acids (C12:1 fatty acid having a cis double bond at C5-C6, 3-OH-10:0, and 3-OH-14:0) to match this molecular weight, it is understood that lipid A has one C12:1 fatty acid having a cis double bond at C5-C6, two 3-OH-10:0, and two 3-OH-14:0.

[0072] 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. Therefore, it is presumed that C12:1 fatty acid, which does not have a hydroxyl group at the 3rd position, is not directly bound to glucosamine. In addition, in 16S rRNA analysis, Paracoccus carotinifaciens has 93% identity with Rhodobacter sphaeroides, so it is presumed that they are related species and that the structure of lipid A is similar to each other (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 with 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).

[0073] [ka]

[0074] Test 4. Limulus Activity Measurement The limulus activity of LPS derived from Paracoccus sp. was measured.

[0075] (1) Measurement sample In Test 4, the Paracoccus-derived LPS used was the Paracoccus-derived LPS purified by the PCP method in Test 1: "Highly purified product" (highly purified LPS obtained in step 4), "Acetone-defatted LPS" (LPS obtained in step 1), and "Crudely purified LPS" (crudely purified LPS obtained in step 3).

[0076] In addition, the following LPS derived from Paracoccus bacteria purified by methods other than the PCP method were also used as measurement samples: Panafert-AX ("Panaferd-AX (untreated)"), finely ground Panafert-AX ("finely ground product"), finely ground Panafert-AX + lecithin (surfactant) ("finely ground product + lecithin"), and finely ground Panafert-AX + lysolecithin (surfactant) ("finely ground product + lysolecithin").

[0077] These measurement samples were prepared as follows. "Panaferd-AX (untreated)" was Panaferd-AX (ENEOS Corporation). "Finely ground product": 100 g of Panaferd-AX was mixed with 900 g of water and crushed by processing 10 times at 100 MPa using a high-pressure homogenizer Panda PLUS 2000. The crushed liquid was freeze-dried to obtain the finely ground Panaferd-AX product. "Finely ground product + lecithin": 300 mg of lecithin (Tsuji Oil SLP-PC70) was weighed out into a 50 mL tube, 15 mL of ion-exchanged water was added, and the mixture was mixed for 1 hour using a direct mixer. This operation produced a 2% aqueous solution of lecithin (homogeneous solvent). 3 g of the finely ground product was weighed out, added to the aqueous lecithin solution, and mixed for 1 hour using a direct mixer. The mixture was freeze-dried to obtain a powder, which was designated "finely ground product + lecithin." "Finely ground product + lysolecithin": "Finely ground product + lysolecithin" was obtained in the same manner as "finely ground product + lecithin", except that lysolecithin (Tsuji Oil Mills, SLP-LPC70) was used instead of lecithin.

[0078] (2) Limulus activity measurement method 1 (turbidimetric method) The limulus activity of the Paracoccus LPS purified by the PCP method was measured by the turbidimetric method using Limulus ES-II plus CS single test wako (Fujifilm Wako Pure Chemical Corporation Cat# 299-77201). 5 mg of each sample was weighed out into an Eppendorf tube (1.5 mL) and water was added to prepare a 5 mg / mL suspension. The suspension was stirred in a Vortex for 5 minutes, then centrifuged at 9000 rpm and 20°C for 15 minutes, and the supernatant was used for the measurement. 200μL of the supernatant was placed in the lysate reagent of Limulus ES-II plus CS single test wako and stirred for 10 seconds with a Voltex. The lysate reagent was placed in a toxinometer (Fujifilm Wako Pure Chemical Corporation Cat# ET-6000 / J) and the absorbance at 430nm was measured over time in kinetic mode: measurement interval 15 seconds, total measurement time 60 minutes, temperature 37℃. Note that this measurement was quantified using the time it takes for the lysate reagent to gel as an index, and the results did not change depending on the measurement time.

[0079] (3) Limulus activity measurement method 2 (colorimetric method) The Limulus activity of Paracoccus LPS purified by a method other than the PCP method was measured by Limulus measurement (colorimetric method) using the 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 50EU / mL and used. 50 mg of each sample was weighed out into an Eppendorf tube (1.5 mL) and water was added to prepare a 100 mg / mL suspension. The suspension was stirred in a Vortex for 5 minutes, then centrifuged at 9000 rpm at 20°C for 15 minutes, and the supernatant was used for the measurement. 100 μL of the supernatant was placed in a 96-well plate and pre-incubated in a plate reader at 37°C for 10 minutes. 100 μL of Limulus reagent was added, and the absorbance at 405 nm was measured over time in the kinetic mode of the plate reader: measurement interval 1 minute, total measurement time 60 minutes, temperature 37°C. This measurement was quantified using the time it took for the absorbance to increase by 0.2 as an index, and the results did not change depending on the measurement time.

[0080] From existing data, it is understood that the turbidimetric method and the colorimetric method provide almost equivalent limulus activity values, so it is possible to compare the limulus activity values ​​obtained by both methods.

[0081] (4) Results The Limulus activity values ​​measured by the methods (2) and (3) above are shown in Table 4.

[0082] [Table 4]

[0083] As described above, the composition of the present invention was shown to exhibit high Limulus activity. [Industrial Applicability]

[0084] The present invention provides a composition comprising an LPS of a Gram-negative bacterium having a lipid A with a predetermined structure. In another aspect, the present invention provides a composition comprising an LPS of a Gram-negative bacterium having a predetermined Limulus activity value. The compositions provided by the present invention are useful in utilizing the immunostimulatory activity of LPS in pharmaceuticals, cosmetics, foods, feeds and the like in terms of their structure and / or Limulus activity.

Claims

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 bound to the glucosamine backbone of the lipid A, and one or two of the 3-hydroxyacyl chains are further bound to the hydroxyl group at the 3-position with an acyl chain having 8 to 13 carbon atoms; A composition having a limulus activity of 30,000 EU / mg or more.

2. The composition according to claim 1, wherein four 3-hydroxyacyl chains having 10 to 14 carbon atoms are bound to the glucosamine backbone of the lipid A, and an acyl chain having 12 carbon atoms is further bound to the hydroxyl group at the 3-position of each of the 3-hydroxyacyl chains having 10 to 14 carbon atoms bound to the 2-position of the glucosamine backbone.

3. The composition according to claim 2, wherein the 12-carbon acyl chain is bonded to the 3-hydroxyl group of a 14-carbon 3-hydroxyacyl chain bonded to the 2-position of glucosamine at the non-reducing end, but is not bonded to the 3-hydroxyl group of a 14-carbon 3-hydroxyacyl chain bonded to the 2-position of glucosamine at the reducing end.

4. The lipid A has the structure of formula (I): 【Chemistry 6】 10. The composition of claim 1, wherein

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.

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