JP2022160938A - Fermentation and culture method, lipoprotein polysaccharide, fermentation extract, fermentation extract powder and compound thereof - Google Patents

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

The present invention is applicable to mammals including humans (specifically livestock, pet animals, etc.), birds (specifically, poultry, pet birds, etc.), amphibians, reptiles, fish (specifically, aquaculture fish, pet animals, etc.). fish, etc.), pharmaceuticals for invertebrates, veterinary drugs, quasi-drugs, cosmetics, foods, functional foods, feeds, bath additives, etc. Methods, lipopolysaccharides, fermented extracts, fermented extract powders and their formulations.

Mammals including humans (specifically livestock, pet animals, etc.), birds (specifically poultry, pet birds, etc.), amphibians, reptiles, fish (specifically, aquaculture fish, pet fish, etc.), For invertebrates, innate immunity plays an essential role in maintaining the health of living organisms, 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, exist in tissues throughout the body and are known to enhance functions such as defense against infection, wound healing, and metabolic regulation.

LPS exists in the cell membrane of Gram-negative bacteria and has a structure in which polysaccharides and lipids are bound together. is Although LPS is a molecule with an extremely complex structure, it is basically an amphipathic substance consisting of a lipid portion called lipid A, a core polysaccharide that binds to it, and an O-antigen that binds to the core polysaccharide. (Non-Patent Document 1). LPS activates and regulates macrophages, and as a result, enhances innate immune function.

By the way, LPS induces systemic inflammation when injected, and historically has been called endotoxin due to its effects such as fever, diarrhea, and hypotension. On the other hand, we have found that LPS, through transmucosal administration such as oral, transdermal, and nasal administration, works through macrophages to control bacteria and viruses, repair wounds, maintain allergy balance, prevent diabetes, etc., and maintain health. I have found it useful. It has also 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). In addition, we have developed LPS of Gram-negative bacteria such as Pantoea, Xanthomonas, and Acetic acid bacteria, which have been eaten, as functional ingredients for food by fermenting and culturing them.

We found by electrophoresis (SDS-PAGE) that LPS extracted from Pantoea bacteria exists as a mixture of low-molecular-weight (about 5,000 Da) and high-molecular-weight (about 50,000 Da) types (Patent Document 1). , Non-Patent Document 3). We named the low molecular mass (LMM)-LPS and the high molecular mass (HMM)-LPS, and analyzed them. It was found that it is toxic and has a high macrophage activation ability (Patent Document 1). Therefore, LPS rich in LMM is considered to be highly useful for maintaining the health of living bodies, and a method for producing LPS rich in LMM is required.

Japanese Patent No. 4043533 JP-A-08-245702

Wikipedia "lipopolysaccharide", [online], [searched on March 15, 2021], Internet C. Kohsaccharidechi et al., "Applications of lipopoly derived from Pantoea agglomerans (IP-PA1) for health care based on macrophage network theory", Journal of Bioscience and Bioengineering, 2006.12, 102(6), p.485-496 T. Kadowaki et al., “Induction of nitric oxide production in RAW264.7 cells under serum-free conditions by O-antigen polysaccharide of lipopolysaccharide”, ANTICANCER RESEARCH, 2013.07, 33(7), p.2875-9 H. Nagano et al., “p53-inducible DPYSL4 associates with mitochondrial supercomplexes and regulates energy metabolism in adipocytes and cancer cells”, Proceedings of the National Academy of Sciences, 2018.08, 115(33), p.8370-8375 Y. Lee et al., “Increased expression of glial cell line-derived neurotrophic factor (GDNF) in the brains of scrapie-infected mice”, Neuroscience Letters, 2006.12, 410(3), p.178-182 M. D. Wood et al., “Fibrin gels containing GDNF microspheres increase axonal regeneration after delayed peripheral nerve repair”, REGENERATIVE MEDICINE, 2013.01, 8(1), p.27-37 E. Joo et al., “Inhibition of Gastric Inhibitory Polypeptide Receptor Signaling in Adipose Tissue Reduces Insulin Resistance and Hepatic Steatosis in High-Fat Diet-Fed Mice”, Diabetes, 2017.04, 66(4), p.868-879 T. Tanaka et al., "IL-6 in Inflammation, Immunity, and Disease", Cold Spring Harbor Perspectives in Biology, 2014.09, 6(10), a016295 T. Tanaka et al., "Regulation of IL-6 in Immunity and Diseases", Advances in Experimental Medicine and Biology, 2016, 941, p.79-88 Q. Liu et al., "IL-10 targets Th1/Th2 balance in vascular dementia", European Review for Medical and Pharmacological Sciences, 2018.09, 22(17), p5614-5619 H. Mollazadeh et al., “Immune modulation by curcumin: The role of interleukin-10”, Critical Reviews in Food Science and Nutrition, 2019, 59(1), p.89-101 S. Sharba et al., “Formyl peptide receptor 2 orchestrates mucosal protection against Citrobacter rodentium infection”, Virulence, 2019.12, 10(1), p.610-624 T. Nishizawa et al., "Homeostasis as Regulated by Activated Macrophage. I. Lipopolysaccharide (LPS) from Wheat Flour: Isolation, Purification and Some Biological Activities", Chemical and Pharmaceutical Bulletin, 1992, 40(2), p.479- 483

Low-molecular-weight LMM-LPS production technology As a method for isolating LMM-LPS from Pantoea culture, LPS was purified and then gel filtration using deoxycholic acid was performed to separate LMM-LPS and HMM-LPS. Later, a method for removing deoxycholic acid was developed (Patent Document 1). However, this method is an extremely complicated refining method, and the manufacturing cost is enormous, making it difficult to market it as a food ingredient. A method of purifying by HPLC (high performance liquid chromatography) using a C4 column is also known, but the problem is that large-scale purification requires time and effort and costs.

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

From the above, it is difficult to provide products on an industrial scale by conventional physicochemical methods to increase the content of low-molecular-weight LPS. By discovering and optimizing bacterial culture conditions, we invented the possibility of establishing a production technology for LPS products with a high LMM-LPS content that can be used on an industrial scale.

It has not been known to alter LMM-LPS content by altering bacterial culture conditions. Conditions such as temperature, nutrients, oxygen concentration, pH, and culture time are factors that affect the culture of bacteria. As a result of intensive investigation of these conditions, we found a culture method that increases the LMM/HMM weight ratio to 1.85 to 3.07 (4 times more LMM-LPS content than the conventional method) by setting the culture temperature and pH conditions. We have completed the present invention.

In the present invention, it was necessary to establish a method for easily evaluating the weight ratio of LMM-LPS and HMM-LPS, which has not been evaluated so far. This was established by combining the Limulus reaction, which can detect all LPS, and the ELISA, which detects only HMM.
Standard culture conditions are 30.0°C and pH 7.0.

We examined nutrients and oxygen concentration, but found that there was almost no effect on the LMM/HMM weight ratio. discovered. Then, when the temperature was changed to 37.0°C and the pH was changed, a LMM/HMM weight ratio higher than the standard was obtained at pH 8.0 or more and 9.0 or less.

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

According to the present invention, LPS with a high LMM-LPS content can be produced by simply changing the pH and/or temperature in the conventional LPS purification method, so cost reduction can be realized. Furthermore, the LPS produced by this method had the effect of inducing Dpysl4, GDNF and GIPR (2.55-fold to 14.42-fold), which were not induced by the conventional method, and thus differs from 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 cell loss and atrophy (Non-Patent Document 5), promotes motor axonal regeneration (Non-Patent Document 6)
・ GIPR (gastric inhibitory polypeptide receptor): directly induces energy accumulation in adipose tissue (Non-Patent Document 7)

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 had a remarkably high effect of IL-6, IL-10 and FPR2 from 15.51 to 271.54 times compared to the induction by the conventional method, which cannot be explained by the increase in LMM. was
・IL-6 (interleukin 6): Contributes to host defense through stimulation of hematopoiesis and immune response (Non-Patent Document 8), contributes to recovery of damaged tissues (Non-Patent Document 9)
・ IL-10 (interleukin 10): suppresses neuronal apoptosis (Non-Patent Document 10), anti-inflammatory and immunosuppressive cytokine (Non-Patent Document 11)
・FPR2 (formyl peptide receptor 2): decrease in inflammatory response (Non-Patent Document 12)

From the above, it is possible to provide formulations such as foods, cosmetics, feeds and medicines, which are LMM-LPS-rich products, which have excellent bioactivity without high cost and complicated labor.

FIG. 4 is a diagram showing relative ratios at each culture pH. FIG. 4 is a diagram showing relative ratios at each culture temperature.

Unless otherwise specified, the "Pantoea bacterium LPS" of the present invention is obtained by culturing Pantoea agglomerans, a gram-negative bacterium that lives symbiotically with wheat, in wheat flour according to the procedure described in Patent Document 1. It refers to lipopolysaccharide obtained by extracting lipopolysaccharide with hot water and removing the solid content.

The Pantoea bacterium LPS of the present invention can be used in humans, mammals other than humans (livestock such as pigs, cattle, sheep, horses, dogs and cats), birds (poultry such as chickens, turkeys and ducks) and fish (eels, sea bream, tuna). , aquaculture fish such as yellowtail, and ornamental fish such as koi).

Examples of routes of administration of the present invention include oral administration, transdermal administration, buccal administration, subcutaneous injection, intradermal injection, intraperitoneal injection and intramuscular administration. Oral administration, transdermal administration and buccal administration are preferred. Dosage forms include powders, granules, liquids, capsules, fine granules, pills, syrups and emulsions. This pharmaceutical composition is orally administrable and effective. These formulations contain, in addition to Pantoea LPS, various pharmaceutically acceptable additives such as stabilizers, fillers, emulsifiers, bulking agents, excipients, binders, humectants, disintegrants, interfacial Active agents, suspending agents, coating agents, coloring agents, flavoring agents, flavoring agents, sweetening agents, preservatives and antioxidants may be included.

The food composition of the present invention can be used according to a conventional method for food compositions, such as using Pantoea LPS as it is or mixing it with other foods or food ingredients. Also, the form thereof is not particularly limited, and it may be in the form of a commonly used food such as solid (powder, granules, etc.), paste, liquid, or suspension. The food composition of the present invention can be a food with nutrient function claims, a food for specified health uses, a food with function claims, a health food, a dietary supplement, a drink, a soft drink, an alcoholic beverage, a supplement, a feed, a feed additive, or the like. can.

The method for cultivating the Pantoea bacterium of the present invention will be described in detail in the following examples. (Including wheat flour, rice flour, wheat bran flour, rice bran, or sake lees, which are materials derived from cereals), seaweed (including wakame powder, mekabu powder, or kelp powder, which are materials derived from seaweed), and beans (including bean curd refuse, which is a material derived from legumes). These plants are well known to contain proteins and sugars and are adaptable for fermentation and cultivation using Pantoea agglomerans. In addition, it is widely known that these plants have symbiotic bacteria such as the genus Serratia and the genus Enterobacter (Non-Patent Document 13). Needless to say, it can also be applied to anaerobic Gram-negative bacteria.

Establishment of Pantoea bacteria culture test evaluation method 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 autoclaved (MLS-3750, Sanyo Denki) with high-pressure steam sterilization. The pH of the LB medium was adjusted with sodium hydroxide (Fuji Film Wako Pure Chemical Industries, Ltd.) (pH 7.0, pH 8.0, pH 8.5, pH 8.8, pH 8.9, pH 9.0).

2. Cultivation of Pantoea agglomerans A single colony of Pantoea agglomerans was inoculated into 5 mL of LB medium and cultured for 18 hours in an infiltration culture machine (Bioshaker BR-21UM, Taitec) at a temperature of 37.0°C and a shaking speed of 140 rpm. A bacterial solution was obtained. 2 mL of the cultured bacterial solution was inoculated into 100 mL of pH-adjusted LB medium, and placed in a 500-mL Sakaguchi flask (AGC Techno Glass) using an infiltration culture machine (incubator shaker RMS-20R, Sanki Seiki) at a temperature of 37.0°C and 160°C. After culturing with reciprocating shaking at a speed of rpm for 24 hours, a pantoea bacterium culture solution was obtained. The optical density (OD) at 660 nm of the Pantoea culture solution was measured with an absorption photometer (UV mini 1240, Shimadzu Corporation) to confirm the growth of Pantoea bacteria.

3. Extraction of lipopolysaccharide (LPS) from Pantoea bacteria culture 1 mL of Pantoea bacteria culture was heated at 90.0°C for 20 minutes in a constant temperature vessel (Thermo aluminum bath ALB-101, Asahi Techno Glass), and then washed in an ultrasonic cleaner (UT- 305HS, Sharp) for 20 minutes, followed by vigorous stirring for 2 minutes with a stirrer (Microtube Mixer MT-400, Tommy Medico). After that, the mixture was centrifuged at 830 g for 15 minutes in a centrifuge (Micro Cooling Centrifuge 3740, Kubota Shoji), and the supernatant was collected and used as an LPS extract.

4. Limulus ES-II Single Test Wako (Fujifilm Wako Pure Chemical) was used for the Limulus ES-II Single Test Wako (Fujifilm Wako Pure Chemical), and a turbidity method was used with a toxinometer (ET-6000, Fujifilm Wako Pure Chemical). gone. Add 200 μL of the LPS extract diluted with distilled water to the glass test tube provided with the kit, stir vigorously with a digital vortex Jenny 2 (SI-A286, Scientific Industries) for 5 seconds, and measure the turbidity with a toxinometer. did. LPS (Funakoshi, mac0001), a standard substance, was used for the amount of LPS determined by the Limulus reaction.

5. Determination of LPS content by enzyme-linked immunosorbent assay (ELISA) of LPS extract
(1) Pretreatment ELISA for detecting Pantoea is a sandwich ELISA method using antibodies that specifically detect two types of Pantoea LPS. 50 μL of the primary antibody (34-G2 antibody (Natural Immunology Applied Giken)) against Pantoea bacterium LPS diluted 2,000-fold with phosphate-buffered saline (PBS(-)) (Sigma) is placed in a 96-well (immunoplate) plate (immunoplate). Maxorp, 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, add 200 μL of 3% (w/v) BSA-PBS(-) (BSA (bovine serum albumin fraction V (Sigma))) to PBS(-) and add 3% (w/v) to the primary antibody-immobilized plate. v) dissolved at the concentration of v) was added, and blocking treatment was performed at 25.0° C. for 30 minutes to 2 hours. After that, 200 μL of a washing solution (10 mM Tris-HCl pH 7.5 (tris(hydroxymethyl)aminomethane (Nacalai Tesque) was dissolved in distilled water and adjusted to pH 7.5 with hydrochloric acid (Fuji Film Wako Pure Chemical Industries)), The plate was washed three times with 150 mM sodium chloride (Fuji Film Wako Pure Chemical) and 0.05% (v/v) polyoxyethylene sorbitan monolaurate (Nacalai Tesque) to obtain a blocked primary antibody-immobilized plate.

(2) Sample addition and secondary antibody treatment
LPS extract diluted with 1% (w/v) BSA (Sigma) in PBS(-) (1% (w/v) BSA-PBS(-)) After adding 50 μL to each well of the stratified plate and reacting with the antibody at 25.0° C. for 1 hour, the wells were washed 3 times with 200 μL of washing solution. Add 50 μL of a secondary antibody (4-E11 antibody (Natural Immunology Applied Giken)) against Pantoea bacterium LPS diluted 1,000 times with 1% (w/v) BSA-PBS(-) to each well and keep at 25.0°C for 1 hour. After antibody reaction, each well was washed 3 times with 200 µL of washing solution.

(3) Coloring treatment
Add 50 μL of alkaline phosphatase-labeled anti-mouse IgG-specific goat immunoglobulin (Sigma) diluted 1,000-fold with 1% (w/v) BSA-PBS(-) to each well and react with the antibody at 25.0°C for 1 hour. , each well was washed 5 times with 200 μL of washing solution. A chromogenic substrate (1 mg/mL p-nitrophenyl phosphate disodium hexahydrate (Fujifilm Wako Pure Chemical Industries, Ltd.), 1 mM magnesium chloride (Nacalai Tesque), 50 mM sodium carbonate (Nacalai Tesque)) was added to each well. After μL was added and allowed to react at room temperature for 1 hour, the absorbance at 405 nm was measured with a microplate reader (Imark, Biorad). Pantoair LPS standard (Funakoshi, mac0001) was measured at the same time to obtain a calibration curve. The amount of LPS in the sample was measured from this calibration curve.

[result]
1. Preparation of Index for Evaluating LMM/HMM Ratio A simple evaluation method was prepared without purifying the ratio of LMM-LPS and HMM-LPS contained in the culture. 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, the ELISA used this time uses the 4-E11 antibody that recognizes the O-antigen polysaccharide, so LMM-LPS without O-antigen is not detected, and only HMM-LPS is detected. Also, in a previous study, Tricine SDS PAGE confirmed that the molecular weight of LMM-LPS is approximately 5,000 and that of HMM-LPS is approximately 50,000.

Each measured value is as follows.
[Limulus value]
3987±1410 μg/mg for LMM-LPS (n=9, mean plus minus standard deviation)
103±55 μg/mg by HMM-LPS (n=9, mean plus minus standard deviation)
[ELISA value]
Below detection limit (<26 μg/mg) by LMM-LPS (n=6)
917±225 μg/mg by HMM-LPS (n=6, mean plus minus standard deviation)
The ELISA value of pure HMM-LPS was theoretically 1 mg/mg.

[Conclusion]
From the above, it can be considered that LMM-LPS does not contribute to the ELISA value of the mixture and that it is 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 x 0.103) ÷ 3.987 x dilution factor
HMM-LPS = ELISA measurement value x dilution ratio *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)

Preliminary Evaluation of LMM/HMM Weight Ratio Against Pantoea Bacteria Culture Temperature Regarding the LMM/HMM weight ratio in the culture sample, the culture temperature was first preliminarily evaluated. Pantoea bacteria are usually cultured at 30.0°C and pH 7.0. Using this as a reference, the cells were cultured at temperatures of 25.0°C and 37.0°C, and the LMM/HMM weight ratio and the relative ratio taking proliferativeness into consideration were determined.

[result]
Based on Example 1, the LMM/HMM weight ratio was calculated. It was also found that the relative ratio of LMM-LPS weight increase (reference material weight ratio×proliferation index (OD)) was improved from 2.22 to 3.06.

[Conclusion]
From the above, it became clear for the first time that the temperature during culture of Pantoea bacteria should be 30.0°C rather than 25.0°C and 37.0°C rather than 30.0°C in order to increase the LMM/HMM weight ratio.

Evaluation of Optimal pH for Pantoea Bacteria Culture with High LMM/HMM Weight Ratio The LMM/HMM weight ratio in the culture sample was evaluated by changing the initial pH setting of the culture conditions from 7 to 10.
In Example 2, the Pantoea bacterium was cultured at 37.0°C while changing the culture pH.

Normally, Pantoea is cultured at 30.0°C and pH 7.0, but based on this, it is cultured at a temperature of 37.0°C with the initial pH setting changed from 7 to 10, and the LMM/HMM weight ratio, And the relative ratio was determined taking into consideration the proliferative property.

[result]
As shown in Table 2, the results of calculating the LMM/HMM weight ratio based on Example 1 show that the reference material weight ratio 1.90 to 2.90 times in , and the relative ratio of LMM-LPS weight increase (reference material weight ratio × proliferation index (OD)) was also improved from 3.53 to 6.00 times.

[Conclusion]
From the above, it became clear for the first time that the pH during culture of Pantoea bacteria should be 8.0 or more and 9.0 or less in order to increase the LMM/HMM weight ratio.

Optimal temperature evaluation at optimal pH 8.8 for culture of Pantoea bacteria with a high LMM/HMM weight ratio To clarify the optimal temperature at pH 8.8, the temperature was varied from 30°C to 39°C at pH 8.8 and cultivated. As a result, it was confirmed that the LMM/HMM weight ratio was the best at 37.7°C.
Table 3 shows the results.

[Conclusion]
By culturing Pantoea bacteria at pH 8.8 between 37.0°C and 38.0°C, the LMM/HMM weight ratio is higher (more LMM-LPS content) than the conventional 30.0°C pH 7.0 culture method. I was able to In particular, by culturing at 37.7°C and pH 8.8, the LMM/HMM weight ratio could be increased by 4 times or more.

Pantoea bacteria culture biological activity evaluation test
(1) LPS stimulation
DMEM medium (Dulbecco's Modified Eagle's Medium (Sigma), 10% FBS (Fetal Bovine Serum ⁽ Sigma), 100 U/mL Penicillin (Sigma) , 100 μg/mL streptomycin (Sigma)) and added 1 mL each to a 24-well cell culture plate (TPP Techno Plastic Products) LPS with a low LMM/HMM ratio and LPS with a high LMM/HMM ratio were terminated. It was diluted with medium to a concentration of 1 ng/mL and added to the wells containing the cells.The 24-well cell culture plate containing the cells was placed in a CO2 incubator (MCO-18AIC, Sanyo Electric) at 37.0°C for 5 hours. LPS stimulation was performed for 4 hours under % CO2 (Takamatsu Teisan).

(2) RNA extraction
RNeasy Mini kit (Qiagen) was used for RNA extraction. The culture supernatant in each well of the 24-well cell culture plate containing the cells was removed, and 350 µL of RLT was added and the cells were lysed by pipetting. Collect the cell lysate in a 1.5 mL tube, add 350 μL of ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) diluted to 70% with sterilized water, mix by pipetting, and place in a collection tube (provided with the RNeasy Mini kit). The mixture was transferred to a spin column (attached to the RNeasy Mini kit), centrifuged at 8,000 g for 15 seconds in a micro refrigerated centrifuge (3740, Kubota Shoji), and the filtrate was discarded. 700 μL of buffer RW1 was added to the spin column, centrifuged at 8,000 g for 15 seconds in a micro-refrigerated centrifuge, and the filtrate was discarded. 500 μL of buffer RPE was added to the spin column, centrifuged at 8,000 g for 15 seconds in a micro-refrigerated centrifuge, and the filtrate was discarded. Add 500 μL of buffer RPE to the spin column, centrifuge at 8,000 g for 2 minutes in a micro refrigerated centrifuge, discard the filtrate, set the spin column in a new collection tube (provided with the RNeasy Mini kit), and add RNA-free water to the spin column. (attached to the RNeasy Mini kit) was added, centrifuged at 8,000 g for 1 minute in a micro refrigerated centrifuge, and the filtrate was collected. The purity and concentration of the collected samples were measured with NanoVue (GE Healthcare).

(3) cDNA synthesis
ReverTra Ace qPCR Master Mix with gDNA Remover (Toyobo) (kit) was used for cDNA synthesis. Prepare 500 ng of the extracted RNA, 1.96 μL of 4x DN Master Mix (included in the kit), 0.04 μL of gDNA Remover (included in the kit), and nuclease-free water (included in the kit) to make a total volume of 8 μL. Astec)) was allowed to react at 37.0°C for 5 minutes to remove the genomic DNA. Mix 8 μL of the reaction solution from which genomic DNA has been removed and 2 μL of 5x RT Master Mix II (included in the kit), and react in a thermal cycler at 37.0°C for 15 minutes, 50.0°C for 5 minutes, and 98.0°C for 5 minutes. let me

(4) Real-time PCR
Mix 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), and add Stratagene Mx3005p. (Agilent Technologies) 95.0°C, 10 minutes, (95.0°C, 15 seconds, 60.0°C, 1 minute) x 40, 95.0°C, 15 seconds, 60.0°C, 30 seconds, 95.0°C, 15 seconds. rice field.

[result]
Macrophage-like cells were stimulated with two different LMM/HMM weight ratios of LPS (LMM-LPS 1 ng/mL), and the macrophage-activating function of LPS with high LMM-LPS content was evaluated. As a result, when stimulated with LPS (LMM/HMM weight ratio 0.27) of the culture product in the conventional method, when stimulated with LPS with a high LMM/HMM weight ratio (LMM/HMM weight ratio 6.5) by the developed method increased the gene expression levels of cytokines in The results are shown in Tables 4 and 5. Table 4 shows that induction of cytokine genes, which was not observed in conventional culture method LPS samples, was induced in development culture method LPS samples. Table 5 shows that the developed culture method LPS samples induced more than 15-fold higher cytokine induction than the conventionally cultured LPS samples.

[Conclusion]
It was clarified that the LPS extract of the present invention, which has a high LMM/HMM weight ratio obtained by culturing Pantoea at 37.7° C. and pH 8.8, exhibits biological activity different from that of LPS obtained by conventional culture methods.
As described above, the LPS-rich product with a high LMM/HMM weight ratio newly developed in this patent could be produced as a product with extremely superior biological activity compared to the conventional culture method.

Claims (10)

A fermentation and culture method comprising fermenting a medium having a pH of 8.0 to 9.0 with facultative anaerobic Gram-negative bacteria and simultaneously culturing the facultative anaerobic Gram-negative bacteria. 2. The medium according to claim 1, wherein said medium having a temperature of 37.0° C. or higher and 38.0° C. or lower is fermented with said facultative anaerobic Gram-negative bacteria, and said facultative anaerobic Gram-negative bacteria are cultured at the same time. Fermentation and culture methods. 3. The fermentation and culture method according to claim 1 or 2, wherein the facultative anaerobic Gram-negative bacterium is Pantoea agglomerans. 3. The fermentation and culture method according to claim 1 or 2, wherein the medium contains powder obtained from plants. 5. The fermentation and culture method according to claim 4, wherein the powder is grains, seaweeds or legumes. A lipopolysaccharide obtained from a facultative anaerobic Gram-negative bacterium fermented and cultured by the fermentation and culture method according to claim 1, 2, 4 or 5. A fermented extract obtained by the fermentation and culture method according to any one of claims 1 to 5. A fermented extract powder obtained from the fermented extract according to claim 7 . A formulation comprising the lipopolysaccharide, the fermented extract, or the fermented extract powder according to any one of claims 6 to 8. 10. The formulation according to claim 9, wherein the formulation is pharmaceuticals, veterinary drugs, quasi-drugs, cosmetics, foods, functional foods, feeds, bath additives or daily necessities.

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