Method for producing lipopolysaccharide, lipopolysaccharide and its formulation

Abstract

To solve problems for example, as a method for isolating LMM-LPS from the Pantoea bacteria cultured object, there is developed a method for performing gel filtration using deoxycholate, after purifying LPS, then after separating LMM-LPS and HMM-LPS, then removing the deoxycholate, however, the same method is extremely complicated and production cost becomes greater, and marketing thereof as a food ingredient is difficult in an aspect of cost.SOLUTION: There is provided a culture method for, by setting a culture temperature and a pH condition being elements which affect culture of bacteria, a weight ratio of LMM / HMM is enhanced to 1.85-3.07 (in the method, a content of LMM-LPS is almost four times of that of prior arts).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for obtaining an immunostimulatory substance that is safe even when added to pharmaceuticals, veterinary drugs, quasi-drugs, cosmetics, foods, functional foods, feeds, bath agents, etc., which cover mammals including humans (specifically livestock, pets, etc.), birds (specifically poultry, pet birds, etc.), amphibians, insects, fish (specifically aquaculture fish, pet fish, etc.), and invertebrates, lipopolysaccharide, Lipopolysaccharide production and its formulation. and

Background Art

[0002] Regarding mammals including humans (specifically livestock, pets, etc.), birds (specifically poultry, pet birds, etc.), amphibians, insects, fish (specifically aquaculture fish, pet fish, etc.), and invertebrates, innate immunity plays an indispensable role in maintaining the health of the living body, such as defending against bacterial and viral infections, repairing wounds, and regulating metabolism. Macrophages, which are phagocytic cells that play a central role in innate immunity, are present in tissues throughout the body, and are known to enhance functions such as infection defense, wound healing, and metabolic regulation.

[0003] LPS is present in the cell membrane of Gram-negative bacteria and has a structure in which polysaccharide and lipid are bound. In Japanese, it is called "glycolipid" or "lipopolysaccharide" (in English, Lipopolysaccharide), and is abbreviated as LPS. LPS is a molecule with a very complex structure, but basically it is an amphiphilic substance composed of a lipid part called lipid A, a core polysaccharide bound to it, and an O antigen bound to the core polysaccharide (Non-Patent Document 1). LPS activates and controls macrophages, and as a result, enhances the innate immune function.

[0004] ​Incidentally, when LPS is injected, it induces systemic inflammation and has a historical background of being called endotoxin due to effects such as fever, diarrhea, and hypotension. On the other hand, we have found that LPS, when administered transmucosally such as orally, transdermally, or nasally, acts through macrophages to control bacteria and viruses, repair wounds, maintain allergic balance, prevent diabetes, etc., and has a beneficial effect on maintaining health. Also, it has been clarified that the intake of LPS contained in foods such as brown rice, wheat, and buckwheat works to maintain health (Non-Patent Document 2). Furthermore, LPS of Gram-negative bacteria such as Pantoea bacteria, Xanthomonas bacteria, and acetic acid bacteria with food experience has been developed as a functional ingredient in foods by fermentation culture.

[0005] We found by electrophoresis (SDS-PAGE) that LPS extracted from Pantoea bacteria exists as a mixture of a low molecular weight type (about 5,000 Da) and a high molecular weight type (about 50,000 Da) (Patent Document 1, Non-Patent Document 3). When the low molecular weight type was named LMM (low molecular mass)-LPS and the high molecular weight type was named HMM (high molecular mass)-LPS and analyzed, it was found that LMM-LPS and HMM-LPS have different biological activities, and LMM-LPS has low toxicity and high macrophage activation ability (Patent Document 1). From this, it is considered that LPS rich in LMM is more useful for maintaining the health of the living body, and a method for producing LPS rich in LMM is required.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Summary of the Invention

Problems to be Solved by the Invention

[0008] Low-molecular-weight LMM-LPS manufacturing technology As a method for isolating LMM-LPS from Pantoea bacteria cultures, after purifying LPS, gel filtration using deoxycholic acid was performed to separate LMM-LPS and HMM-LPS, and then a method for removing deoxycholic acid was developed (Patent Document 1). However, this method is an extremely complicated purification method, the manufacturing cost becomes enormous, and it has been difficult to commercialize as a food ingredient in terms of cost. Also, a method of purifying by HPLC (high performance liquid chromatography) using a C4 column is known, but the problem is that it takes time and cost for large-scale purification.

[0009] On the other hand, until now, in the culture stage of Pantoea bacteria, the mixing ratio of LMM-LPS and HMM-LPS has not been controlled and the content has remained unknown. Therefore, when examining the weight ratio of LMM-LPS and HMM-LPS of Pantoea bacteria cultured under normal conditions, the ratio of the LMM-LPS content to the HMM-LPS content (LMM / HMM weight ratio) was 0.72.

[0010] From the above, in order to increase the content of low-molecular-weight LPS, it is difficult to provide products on an industrial scale with conventional physicochemical methods. Therefore, we have found and optimized the culture conditions of bacteria that biologically contain high LMM-LPS, and have conceived and earnestly studied the establishment of a manufacturing technology for LPS products containing high LMM-LPS that can also cope with industrial scale.

Means for Solving the Problem

[0011] It has not been known until now to change the LMM-LPS content by changing the culture conditions of bacteria. There are conditions such as temperature, nutrients, oxygen concentration, pH, and culture time that affect the culture of bacteria. Therefore, after earnestly studying these conditions, a culture method was found that increases the LMM / HMM weight ratio to 1.85 to 3.07 (about 4 times the LMM-LPS content of the conventional method) by setting the culture temperature and pH conditions, and the present invention was completed.

[0012] There was a need to establish a method for simply evaluating the weight ratio of LMM-LPS and HMM-LPS, which had not been evaluated so far. This was established by combining the Limulus reaction that can detect all LPS and ELISA that can detect only HMM. The reference culture conditions are 30.0 °C and pH 7.0.

[0013] Nutrition and oxygen concentration were examined, but since they had little effect on the LMM / HMM weight ratio, the culture temperature was examined, and it was found that the LMM / HMM weight ratio increased during culture at a predetermined temperature. Therefore, next, when the temperature was changed to 37.0 °C and the pH was varied, a higher LMM / HMM weight ratio than the reference was obtained at pH 8.0 or higher and 9.0 or lower.

[0014] To find the detailed temperature, the culture temperature was changed from 30.0 °C to 40.0 °C at pH 8.8, and it became clear that a high LMM / HMM weight ratio was shown at 37.0 °C or higher and 38.0 °C or lower.

Advantages of the Invention

[0015] According to the present invention, in the conventional LPS purification method, high LMM-LPS-containing LPS can be produced simply by changing the pH and and temperature, so cost reduction can be achieved. Furthermore, the LPS produced by this method had the effect of inducing Dpysl4, GDNF, and GIPR, which were not induced by the conventional method (from 2.55 times to 14.42 times), and thus was different from the conventional LPS. · Dpysl4 (Dihydropyrimidinase-related protein 4): Suppresses tumor growth and metastasis (Non-Patent Document 4) · GDNF (Glial cell line-derived neurotrophic factor): Involved in protection against neuronal loss and atrophy (Non-Patent Document 5), promotes motor axon regeneration (Non-Patent Document 6) · GIPR (Gastric inhibitory polypeptide receptor): Directly induces energy accumulation in adipose tissue (Non-Patent Document 7)

[0016] From this, it was found that the new culture has unprecedented functionality. In addition, it was found that the LPS extract produced by this method has a significantly high effect on IL-6, IL-10, and FPR2, which cannot be explained by the increase in LMM, being 15.51 to 271.54 times higher than that induced by the conventional method. ·IL-6 (Interleukin 6): Contributes to host defense through stimulation of hematopoiesis and immune response (Non-Patent Document 8), and contributes to the recovery of damaged tissues (Non-Patent Document 9) ·IL-10 (Interleukin 10): Suppresses apoptosis of nerve cells (Non-Patent Document 10), and is an anti-inflammatory and immunosuppressive cytokine (Non-Patent Document 11) ·FPR2 (Formyl Peptide Receptor 2): Reduces the inflammatory response (Non-Patent Document 12)

[0017] From the above, it is possible to provide formulations such as foods, cosmetics, feeds, and drugs, which are LMM-LPS high-content products with excellent biological activity without the need for high costs and complicated procedures.

Brief Description of the Drawings

[0018]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0019] The "Pantoea bacteria LPS" of the present invention, unless otherwise specified, refers to lipopolysaccharide obtained by culturing Pantoea agglomerans, a Gram-negative bacterium symbiotic with wheat, in wheat flour according to the procedure described in Patent Document 1, extracting lipopolysaccharide from the bacterial cells by hot water extraction, and removing the solid content.

[0020] The Pantoea bacteria LPS of the present invention can be applied to humans, non-human mammals (livestock such as pigs, cows, sheep, horses, dogs, and cats), birds (poultry such as chickens, turkeys, and ducks), and fish (cultured fish such as eels, sea breams, tuna, and yellowtails, and ornamental fish such as koi carps).

[0021] Examples of the administration routes of the present invention include oral administration, transdermal administration, buccal administration, subcutaneous injection, intradermal injection, intraperitoneal injection, and intramuscular injection. Preferably, they are oral administration, transdermal administration, and buccal administration. Examples of the dosage form include powders, granules, liquids, capsules, fine granules, pills, syrups, and emulsions. This pharmaceutical composition can be orally administered and is effective. In addition to the Pantoea bacteria LPS, these preparations can contain various pharmaceutically acceptable additives, such as stabilizers, fillers, emulsifiers, extenders, excipients, binders, humectants, disintegrants, surfactants, suspending agents, coating agents, coloring agents, fragrances, flavoring agents, sweetening agents, preservatives, and antioxidants.

[0022] The food composition of the present invention can be used as it is with the Pantoea bacteria LPS or can be used according to conventional methods in food compositions, such as mixing it with other foods or food ingredients. Also, its form is not particularly limited and can be any of the commonly used food states, such as solid (powder, granular, etc.), paste, liquid, or suspension. The food composition of the present invention can be a nutrient-functional food, a food for specified health use, a food with functional claims, a health food, a dietary supplement, a drink, a soft drink, an alcoholic beverage, a supplement, a feed, and a feed additive, etc.

[0023] The method for culturing Pantoea bacteria of the present invention will be described in detail below as an example. The present invention uses Pantoea agglomerans as the microorganism described in the examples, and as the medium, in addition to the medium generally used for culturing bacteria, grains (including wheat flour, rice flour, wheat bran powder, rice bran, or sake lees, etc., which are materials derived from grains), seaweeds (including wakame powder, kelp powder, or kombu powder, etc., which are materials derived from seaweeds), and beans (including okara, etc., which are materials derived from beans) are also applicable. It is well known that these plants contain proteins and saccharides and are suitable for fermentation and culture using Pantoea agglomerans. Also, it is widely known that bacteria that are indigenous to these plants, such as the genus Serratia and the genus Enterobacter, coexist (Non-Patent Document 13). Needless to say, the microorganisms used for fermentation are also applicable to facultative anaerobic Gram-negative bacteria that coexist with these plants.

Example

[0024] Establishment of the evaluation method for the Pantoea bacteria culture test 1. Preparation of LB (Luria broth) medium Commercially available LB medium powder (Lennox, Nacalai Tesque) was dissolved in distilled water at a concentration of 2% (w / v) and sterilized by high-pressure steam using an autoclave (MLS-3750, Sanyo Electric). The pH of the LB medium was adjusted (pH 7.0, pH 8.0, pH 8.5, pH 8.8, pH 8.9, pH 9.0) with sodium hydroxide (FUJIFILM Wako Pure Chemical Industries).

[0025] 2. Culture of Pantoea bacteria A single colony of Pantoea agglomerans was inoculated into 5 mL of LB medium and cultured in an osmotic incubator (Bio Shaker BR-21UM, Taitec) at a temperature of 37.0 °C and a shaking speed of 140 rpm for 18 hours to obtain a bacterial solution. 2 mL of the cultured bacterial solution was inoculated into 100 mL of pH-adjusted LB medium, and the mixture was reciprocally shaken and cultured in a 500 mL Sakaguchi flask (AGC Techno Glass) using an osmotic incubator (Incubator Shaker RMS-20R, Sanki Seiki) at a temperature of 37.0 °C and a speed of 160 rpm for 24 hours to obtain a Pantoea agglomerans culture solution. The absorbance (optical density: OD) at 660 nm of the Pantoea agglomerans culture solution was measured using a spectrophotometer (UV mini 1240, Shimadzu Corporation) to confirm the growth of Pantoea agglomerans.

[0026] 3. Extraction of Lipopolysaccharide (LPS) from Pantoea agglomerans Culture Solution 1 mL of the Pantoea agglomerans culture solution was heated in a thermostat (Thermo Aluminum Bath ALB-101, Asahi Techno Glass) at 90.0 °C for 20 minutes, sonicated for 20 minutes using an ultrasonic cleaner (UT-305HS, Sharp), and then vigorously stirred for 2 minutes using a stirrer (Microtube Mixer MT-400, Tommy Medical). Thereafter, the supernatant was collected by centrifugation at 830 g for 15 minutes using a centrifuge (Micro Cooling Centrifuge 3740, Kubota Corporation) and used as the LPS extract.

[0027] 4. Measurement of LPS Quantity by Limulus Test of LPS Extract The Limulus test was performed by turbidimetry using Limulus ES-II Single Test Wako (FUJIFILM Wako Pure Chemical Corporation) and a toximeter (ET-6000, FUJIFILM Wako Pure Chemical Corporation). 200 μL of the LPS extract diluted with distilled water was added to a glass test tube attached to the kit, vigorously stirred for 5 seconds using a Digital Vortex Jenny 2 (SI-A286, Scientific Industries), and the turbidity was measured using a toximeter. The LPS quantity by the Limulus reaction was determined using LPS (Funakoshi, mac0001) as a standard substance.

[0028] 5. Measurement of LPS Quantity by Enzyme-Linked Immunosorbent Assay (ELISA) of LPS Extract (1) Pretreatment The ELISA for Pantoea bacteria detection is a sandwich ELISA method using antibodies that specifically detect two types of Pantoea bacteria LPS. 50 μL of the primary antibody (34-G2 antibody (Innate Immunity Applied Technology Research)) against Pantoea bacteria LPS diluted 2,000-fold with phosphate buffered saline (PBS(-)) (Sigma) was added to a 96-well plate (Immunoplate Maxisorp, Cat. 442404, Thermo Fisher), sealed with parafilm (PM-996, Bemis), and treated at 4.0 °C for 1 hour or more to immobilize the primary antibody. Next, 200 μL of 3% (w / v) BSA-PBS(-) (BSA (bovine serum albumin fraction V (Sigma)) dissolved in PBS(-) at a concentration of 3% (w / v)) was added to the primary antibody-immobilized plate, and blocking treatment was performed at 25.0 °C for 30 minutes to 2 hours. Thereafter, this was washed 3 times with 200 μL of the washing solution (10 mM Tris-HCl pH 7.5 (Tris (hydroxymethyl) aminomethane (Nacalai Tesque) dissolved in distilled water and adjusted to pH 7.5 with hydrochloric acid (FUJIFILM Wako Pure Chemical Corporation)), 150 mM sodium chloride (FUJIFILM Wako Pure Chemical Corporation), 0.05% (v / v) polyoxyethylene sorbitan monolaurate (Nacalai Tesque)) to obtain a blocked primary antibody-immobilized plate.

[0029] (2) Sample addition and secondary antibody treatment 50 μL of the LPS extract diluted with a solution of BSA (Sigma) dissolved in PBS(-) at a concentration of 1% (w / v) (1% (w / v) BSA-PBS(-)) was added to each well of the blocked primary antibody-immobilized plate, and after antibody reaction at 25.0 °C for 1 hour, the wells were washed 3 times with 200 μL of the washing solution. 50 μL of the secondary antibody (4-E11 antibody (Innate Immunity Applied Technology Research)) against Pantoea bacteria LPS diluted 1,000-fold with 1% (w / v) BSA-PBS(-) was added to each well, and after antibody reaction at 25.0 °C for 1 hour, each well was washed 3 times with 200 μL of the washing solution.

[0030] (3) Color development treatment Alkaline phosphatase-labeled anti-mouse IgG specific goat immunoglobulin (Sigma) diluted 1,000-fold with 1% (w / v) BSA-PBS(-) was added to each well at 50 μL, and after allowing the antibody reaction to proceed at 25.0 °C for 1 hour, each well was washed 5 times with 200 μL of the washing solution. A chromogenic substrate (1 mg / mL disodium p-nitrophenyl phosphate hexahydrate (FUJIFILM Wako Pure Chemical Industries), 1 mM magnesium chloride (Nacalai Tesque), 50 mM sodium carbonate (Nacalai Tesque)) was added to each well at 50 μL, and after reacting at room temperature for 1 hour, the absorbance at 405 nm was measured using a microplate reader (iMark, Bio-Rad). Pantoea LPS standard (Funakoshi, mac0001) was measured simultaneously to obtain a calibration curve. The amount of LPS in the sample was measured from this calibration curve.

[0031] [Results] 1. Creation of an index for evaluating the LMM / HMM ratio A simple method for evaluating the ratio of LMM-LPS to HMM-LPS contained in a culture without purification was created. The limulus and ELISA values of pure LMM-LPS purified by the method described in Patent Document 1 and pure HMM-LPS purified by the method described in Patent Document 2 were measured. Since the limulus reaction occurs by binding to lipid A, both LMM-LPS and HMM-LPS are detected. On the other hand, since the ELISA used this time uses the 4-E11 antibody that discriminates O-antigen polysaccharide, LMM-LPS without O-antigen is not detected, and only HMM-LPS is detected. Also, in previous studies, it has been confirmed by Tricine SDS PAGE that the molecular weight of LMM-LPS is approximately 5,000 and that of HMM-LPS is approximately 50,000.

[0032] Each measured value was as follows. [Limulus value] For LMM-LPS, 3987 ± 1410 μg / mg (n = 9, mean plus or minus standard deviation) For HMM-LPS, 103 ± 55 μg / mg (n = 9, mean plus or minus standard deviation) [ELISA value] Below the detection limit (<26 μg / mg) with LMM-LPS (n = 6) 917 ± 225 μg / mg with HMM-LPS (n = 6, mean ± standard deviation) Note that the ELISA value of pure HMM-LPS was almost theoretically 1 mg / mg

[0033] [Conclusion] From the above, LMM-LPS does not contribute to the ELISA value of the mixture and can be considered to be derived from HMM-LPS From this, the respective weight concentrations of LMM-LPS and HMM-LPS in the mixture are represented by the following equations LMM-LPS = (Limulus value - ELISA value × 0.103) ÷ 3.987 × dilution factor HMM-LPS = ELISA measurement value × dilution factor ※ Limulus measurement value of pure LMM-LPS = 3.987 mg / mg (3987 μg / mg) Limulus value of pure HMM-LPS = 0.103 mg / mg (103 μg / mg) [Example]

[0034] Preliminary evaluation of the LMM / HMM weight ratio with respect to the Pantoea bacteria culture temperature Regarding the LMM / HMM weight ratio in the culture sample, a preliminary evaluation of the culture temperature was first performed. Usually, Pantoea bacteria are cultured at 30.0 °C and pH 7.0. Based on this, the bacteria were cultured at 25.0 °C and 37.0 °C, and the relative ratio considering the LMM / HMM weight ratio and growth was determined

[0035] [Results] Based on Example 1, the results of calculating the LMM / HMM weight ratio, as shown in Table 1, indicate that culturing at 37.0 °C and pH 7.0 results in a 1.74-fold increase in the LMM / HMM weight ratio compared to the conventional method, and the relative ratio of the increase in the weight of LMM-LPS considering growth (reference substance weight ratio × growth index (OD)) also improves from 2.22 to 3.06

[0036] [Table 1]

[0037] [Conclusion] From the above, it was first revealed that in order to increase the LMM / HMM weight ratio, the temperature during Pantoea bacteria culture should be higher than 25.0 °C and lower than 30.0 °C, and higher than 30.0 °C and lower than 37.0 °C.

Example

[0038] Evaluation of the Optimal pH for Culturing Pantoea Bacteria with a High LMM / HMM Weight Ratio Regarding the LMM / HMM weight ratio in the culture sample, the evaluation was carried out by changing the initial pH setting of the culture conditions from 7 to 10. In Example 2, Pantoea bacteria were cultured at 37.0 °C while changing the culture pH.

[0039] Normally, Pantoea bacteria are cultured at 30.0 °C and pH 7.0. Based on this, at a temperature of 37.0 °C, the initial culture pH was changed from 7 to 10 for culturing, and the relative ratio considering the LMM / HMM weight ratio and growth was determined.

[0040] [Results] Based on Example 1, as shown in Table 2, the results of calculating the LMM / HMM weight ratio showed that compared with the conventional method of culturing at 30.0 °C and pH 7.0, at pH 8.0 to 9.0, the reference substance weight ratio was 1.90 to 2.90 times, and the relative ratio of the LMM-LPS weight increase considering growth (reference substance weight ratio × growth index (OD)) was also improved from 3.53 to 6.00 times.

[0041]

Table 2

[0042] [Conclusion] From the above, it was first revealed that in order to increase the LMM / HMM weight ratio, the pH during Pantoea bacteria culture should be 8.0 or higher and 9.0 or lower.

Example

[0043] Optimal Temperature Evaluation of Pantoea Bacteria Culture at Optimal pH 8.8 with High LMM / HMM Weight Ratio To clarify the optimal temperature at an optimal pH of 8.8, the temperature was varied from 30 °C to 39 °C at pH 8.8 for culturing. As a result, it was confirmed that 37.7 °C gave the best LMM / HMM weight ratio. The results are shown in Table 3.

[0044]

Table 3

[0045] [Conclusion] By culturing Pantoea bacteria at 37.0 °C or higher and 38.0 °C or lower at pH 8.8, a method for culturing Pantoea bacteria with a higher LMM / HMM weight ratio (higher LMM-LPS content) compared to the conventional culturing at 30.0 °C and pH 7.0 could be achieved. In particular, by culturing at 37.7 °C and pH 8.8, the LMM / HMM weight ratio could be increased by more than four times.

Example

[0046] Biological Activity Evaluation Test of Pantoea Bacteria Culture (1) LPS Stimulation RAW264.7 cells (RIKEN) were diluted to 5x10 5 cells / mL with DMEM medium (Dulbecco's Modified Eagle Medium (Sigma), 10% FBS (Fetal Bovine Serum (Sigma), 100 U / mL Penicillin (Sigma), 100 μg / mL Streptomycin (Sigma)), and 1 mL of the diluted cells was added to each well of a 24-well cell culture plate (TPP Technoplate Products). LPS with a low LMM / HMM ratio and LPS with a high LMM / HMM ratio were each diluted with the medium to a final concentration of 1 ng / mL and added to the wells containing the cells. The 24-well cell culture plate containing the cells was subjected to LPS stimulation in a CO2 incubator (MCO-18AIC, Sanyo Electric) at 37.0 °C and 5% CO2 (Takamatsu Teisan) for 4 hours.

[0047] (2) RNA Extraction RNA extraction was performed using the RNeasy Mini kit (Qiagen). The culture supernatant in each well of a 24-well cell culture plate containing cells was removed, 350 μL of RLT was added, and the cells were lysed by pipetting. The cell lysate was collected in a 1.5 mL tube, 350 μL of ethanol (FUJIFILM Wako Pure Chemical Corporation) diluted to 70% with sterile water was added, and after mixing by pipetting, it was transferred to a spin column (attached to the RNeasy Mini kit) set in a collection tube (attached to the RNeasy Mini kit). It was centrifuged at 8,000 g for 15 seconds using a micro cooling centrifuge (3740, Kubota Corporation), and the filtrate was discarded. 700 μL of buffer RW1 was added to the spin column, and it was centrifuged at 8,000 g for 15 seconds using a micro cooling centrifuge, and the filtrate was discarded. 500 μL of buffer RPE was added to the spin column, and it was centrifuged at 8,000 g for 15 seconds using a micro cooling centrifuge, and the filtrate was discarded. 500 μL of buffer RPE was added to the spin column, and it was centrifuged at 8,000 g for 2 minutes using a micro cooling centrifuge. The spin column with the discarded filtrate was set in a new collection tube (attached to the RNeasy Mini kit), 40 μL of RNA-free water (attached to the RNeasy Mini kit) was added to the spin column, and it was centrifuged at 8,000 g for 1 minute using a micro cooling centrifuge, and the filtrate was collected. The purity and concentration of the collected sample were measured using NanoVue (GE Healthcare).

[0048] (3) cDNA synthesis cDNA synthesis was performed using ReverTra Ace qPCR Master Mix with gDNA Remover (Toyobo) (kit). 500 ng of the extracted RNA, 1.96 μL of 4x DN Master Mix (supplied with the kit), 0.04 μL of gDNA Remover (supplied with the kit), and nuclease-free water (supplied with the kit) were prepared to a total volume of 8 μL and reacted at 37.0 °C for 5 minutes in a thermal cycler (Gene Atlas G02 (Astec)) to remove genomic DNA. 8 μL of the reaction solution with genomic DNA removed and 2 μL of 5x RT Master Mix II (supplied with the kit) were mixed and reacted in a thermal cycler under the temperature conditions of 37.0 °C for 15 minutes, 50.0 °C for 5 minutes, and 98.0 °C for 5 minutes.

[0049] (4) Real-time PCR 2 μL of 5 μM forward primer (Eurofins Genomics), 2 μL of 5 μM reverse primer (Eurofins Genomics), 5 μL of cDNA, and 10 μL of POWER SYBR(R) Green PCR Master Mix (Thermo Fisher) were mixed and subjected to PCR reaction in a Stratagene Mx3005p (Agilent Technologies) under the temperature conditions of 95.0 °C for 10 minutes, (95.0 °C for 15 seconds, 60.0 °C for 1 minute) x 40, 95.0 °C for 15 seconds, 60.0 °C for 30 seconds, and 95.0 °C for 15 seconds.

[0050] [Results] Two kinds of LPS with different LMM / HMM weight ratios (LMM-LPS 1 ng / mL fraction) were used to stimulate macrophage-like cells, and the macrophage activation function due to the high LMM-LPS content of LPS was evaluated. As a result, compared with the case of stimulation with LPS (LMM / HMM weight ratio 0.27) of the culture product by the conventional method, the gene expression level of cytokines increased when stimulated with LPS with a high LMM / HMM weight ratio (LMM / HMM weight ratio 6.5) by the developed method. The results are shown in Table 4 and Table 5. Table 4 shows that the induction of cytokine genes that were not induced in the conventional culture method LPS sample was induced in the developed culture method LPS sample. Table 5 shows that the cytokine induction in the developed culture method LPS sample was more than 15 times higher than that in the conventional culture LPS sample.

[0051]

Table 4

[0052]

Table 5

[0053] [Conclusion] It was clarified that the LPS extract with a high LMM / HMM weight ratio obtained by culturing Pantoea bacteria at 37.7 °C and pH 8.8 in the present invention exhibits biological activities different from those of LPS obtained by the conventional culture method. From the above, the newly developed LPS high-content product with a high LMM / HMM weight ratio in this patent can be manufactured as having extremely excellent biological activities compared with the conventional culture method.

Claims

1. A method for producing lipopolysaccharide, comprising culturing Pantoea agglomerans in a medium having an initial culture pH of 8.0 or higher and 9.0 or lower and a culture temperature of 37.0°C or higher and 38.0°C or lower, and obtaining lipopolysaccharide from the cultured Pantoea agglomerans.

2. 2. The method for producing lipopolysaccharide according to claim 1, wherein the medium contains a powder obtained from a plant.

3. 3. The method for producing lipopolysaccharide according to claim 2, wherein the powder is made of grains, seaweed or beans.

4. Lipopolysaccharide produced by the lipopolysaccharide production method described in any one of claims 1 to 3, characterized in that it has the effect of inducing Dpysl4, GDNF and GIPR.

5. A formulation characterized by containing the lipopolysaccharide described in claim 4.

6. 6. The composition according to claim 5, which is a pharmaceutical product, a veterinary drug, a quasi-drug, a cosmetic product, a food product, a functional food product, a feed product, a bath agent, or a daily necessities.

Images