Lipopolysaccharide, method for producing lipopolysaccharide, and lipopolysaccharide complex

Abstract

The purpose of the present invention is to identify a novel substance / factor relating to the anti-inflammatory activity of sugar beet, and to provide the substance / factor. This will make it possible to develop health products such as pharmaceuticals, foods and cosmetics for inhibiting chronic inflammation. Provided is a lipopolysaccharide characterized by being obtained from a bacterium that has been preserved at a preservation number of NITE BP-03839, NITE BP-03840, NITE BP-03841, NITE BP-03842, or NITE BP-03843, and a method for producing the same. The invention also provides a medicine, a medicine for animals, a quasi-medicine, a cosmetic, a food, a functional food, a feed, a fertilizer or a bath lotion additive in which the lipopolysaccharide is mixed.

Description

[Technical Field]

[0002] This invention relates to lipopolysaccharides, methods for manufacturing lipopolysaccharides, and lipopolysaccharide complexes, and particularly to lipopolysaccharides (LPS) derived from bacteria that have a specific symbiotic relationship with beets (beetroots). [Background Technology]

[0004] Beetroot is considered a highly nutritious and functional vegetable. It is rich in minerals such as potassium, magnesium, and iron. Furthermore, the nitrites in beetroot are converted into nitric oxide in the body and have a vasodilatory effect; beet pigments have antioxidant activity that prevents cellular aging; and dietary fiber regulates the intestinal environment and is said to help improve rough skin. Therefore, beetroot is considered to have high functional value (Non-Patent Literature 1). Regarding beetroot, it is known to maintain the activity of immune cells through polyphenols and vitamin C, which are commonly found in vegetables. The pigment component betalain is known to have anti-inflammatory activity and has been reported to reduce the expression of inflammatory cytokines (TNF-α, IL-6) (Non-Patent Literature 2). However, it is unclear whether beetroot activates innate immune cells (macrophages) and increases the expression of anti-inflammatory cytokines (IL-10).

[0005] We found that lipopolysaccharide (LPS) derived from symbiotic bacteria attaches to plants (Non-Patent Literature 3), and that when this LPS is ingested, it activates the human innate immune system (Non-Patent Literature 4). Therefore, it is conceivable that part of the immunomodulatory activity of beets involves LPS derived from bacteria that have a specific symbiotic relationship with beets (hereinafter referred to as "beet LPS").

[0006] [Existing Technical Documents]

[0007] [Non-patent literature]

[0008] [Non-patent document 1] L. Chen et al., "Beetroot as a functional food with hugehealth benefits: Antioxidant, antitumor, physical function, and chronicmetabolomics activity", Food Sci Nutr., 2021.11, 9(11), p.6406-6420

[0009] [Non-Patent Literature 2] S. Saito et al., “Metabolic engineering of βcyanin invegetables for anti-inflammatory therapy”, Biotechnol Bioeng, 2023.05, 120(5), p.1357-1365

[0010] [Non-patent document 3] H. Inagawa et al., "Homeostasis as regulated by activated macrophage. II. LPS of plant origin other than wheat flour and their concomitant bacteria", Chemical and Pharmaceutical Bulletin, 1992.04, 40(4), p.994-997.

[0011] [Non-patent document 4] H. Inagawa et al., "Usefulness of Oral Administration ofLipopolysaccharide for Disease Prevention Through the Induction of Priming in Macrophages", Anticancer Res., 2014.08, 34(8), p.4497-4501 [Summary of the Invention]

[0013] [Technical Issues]

[0014] This invention identifies and provides novel substances / factors involved in the anti-inflammatory activity of beets. This will enable the development of health products, such as pharmaceuticals, foods, and cosmetics, for the purpose of suppressing chronic inflammation.

[0015] [Solutions to the problem]

[0016] This invention includes the following:

[0017] [1] Lipopolysaccharide, which was obtained from bacteria with accession numbers NITE BP-03839, NITE BP-03840, NITE BP-03841, NITE BP-03842 or NITE BP-03843.

[0018] [2] A method for manufacturing lipopolysaccharide, comprising obtaining the lipopolysaccharide from bacteria with accession numbers NITE BP-03839, NITE BP-03840, NITE BP-03841, NITE BP-03842 or NITE BP-03843.

[0019] [3] Lipopolysaccharide complexes comprising the lipopolysaccharide according to [1].

[0020] [4] According to the lipopolysaccharide complex of [1] or [3], it also contains substances from beets with a molecular weight of less than 0.5 kDa.

[0021] [5] Edible plant processed products containing lipopolysaccharides according to any one of [1], [3] and [4].

[0022] [6] Edible plant processed products according to any one of [1] and [3] to [5] also include substances from sugar beets with a molecular weight of less than 0.5 kDa.

[0023] [7] Edible plant products according to [5] or [6], wherein the edible plant products are beet products.

[0024] [8] An edible plant product according to any one of [5] to [7], wherein the edible plant product is dried vegetables or dried edible plant powder.

[0025] [9] Foods that include processed edible plant products according to any one of [5] to [8].

[0026]

[10] The lipopolysaccharide complex according to [3] or [4], wherein the lipopolysaccharide complex is a pharmaceutical, veterinary drug, quasi-pharmaceutical, cosmetic, food, functional food, feed, fertilizer or bath additive.

[0027] [Beneficial effects of the invention]

[0028] Beetroot LPS, derived from bacterial cells isolated from beets, activates macrophages, but is associated with known clumping pantothenia (…). Pantoea agglomeran Compared to LPS, beet LPS exhibits unique macrophage activation, such as a higher ability to induce the anti-inflammatory cytokine IL-10, and its activity has been shown to be due to the activity of polymyxin B in LPS. Furthermore, macrophage activation has been shown not to be caused solely by beet LPS, but rather through interaction with low-molecular-weight substances in beets. This will enable the development of health products for suppressing chronic inflammation, such as pharmaceuticals, foods, and cosmetics.

Detailed Implementation Methods

[0030] In this instruction manual, the term "beetroot" refers to the beetroot described in the 2020 edition (8th edition) of the Japanese Standard Food Ingredients Table.

[0031] The term "edible plant" in this specification refers to a plant that is consumed or drunk by humans. There are no limitations on the definition of edible plant in this specification, as long as it can be consumed or drunk by humans, and examples include vegetables, grains, tuber crops, legumes, seeds, fruits, mushrooms, algae, etc., with vegetables being preferred, and beets being particularly preferred. A single type of edible plant can be used alone, or two or more types can be used in any combination. Edible plants can be used as is, or after various treatments (e.g., drying, heating, de-bittering, peeling, deseeding, ripening, salting, skin processing, etc.). For components whose edible parts (e.g., edamame, green peas) are considered vegetables, their legume status can be determined by observing the whole plant (e.g., soybeans and peas) combined with the inedible parts (e.g., pods). Furthermore, the classification of edible plants can be determined from the whole plant including the inedible parts. Specifically, for example, one can understand which foods correspond to the edible plants of the present invention by referring to the “Japanese Food Standard Composition Table 2015 Edition (7th Edition) Supplement 2018” (a food composition table established by the Ministry of Health, Labour and Welfare, see Table 1 on page 236 in particular).

[0032] The properties of the edible plant processed products in this invention are not limited, but are preferably selected from one or more of edible plant powders, edible plant pastes, or aqueous extracts of edible plants. For example, it is preferred to prepare the processed products by applying heat treatment (e.g., above 80°C) such as drying, baking, or hot water extraction to the edible plants. The edible plant processed products of this invention are preferably dried vegetables or dried edible plant powders.

[0033] The following describes embodiments of the present invention, but the present invention is not limited to the following embodiments.

[0034] [Example 1: Evaluation of the effect of hot water extract of beet on macrophage activation]

[0035] [1] Method

[0036] (1) Preparation of beet extract

[0037] Wash the beets with the skin on using water and cut them into 1cm cubes. Heat the cut beets in a steamer at a temperature above 90°C for 15 minutes. Then, spread the heated beets on a perforated tray without overlapping, place the tray in a hot air dryer set to 70°C, and dry until the moisture content is below 6% (7–8 hours) to make beet flakes. Subsequently, grind the flakes using a grinder (Osaka Chemical) and pass them through a 355µm sieve to make dried beet powder.

[0038] Approximately 2g of the prepared dried beet powder was weighed into a 50mL tube, and distilled water was added to achieve a concentration of 100mg / mL. The mixture was then heated in an autoclave at 90°C for 20 minutes. After cooling, the mixture was stirred 10 times for 30 seconds each time using a vortex mixer (Vortex-genie2, Scientific Industries), and then sonicated at 37°C for 15 minutes. The supernatant collected after sonication was used as the beet extract.

[0039] (2) Methods for measuring LPS

[0040] Using Toxinometer (Fujifilm and Kojun Pharmaceutical Co., Ltd.) to measure the horseshoe crab test value (by horseshoe crab ( Limulus The values ​​obtained from the test were derived from the control standard endotoxin Escherichia coli (E. coli). Escherichia coli The LPS value generated by UKT-B is used to measure the LPS quantity.

[0041] (3) Preparation of macrophage activation evaluation samples using beet extract

[0042] RAW264.7 cells were passaged in RPMI 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 µg / mL streptomycin. Cultures were performed in T25 flasks and inoculated with 0.25 × 10⁻⁶ cells every 3 or 4 days. 5 Cells were passaged at 1000 cells / mL. Cells were cultured in a 37°C 5% CO2 incubator (hereinafter referred to as the incubator). All testing procedures were performed in a clean bench. Cells pre-cultured in T25 flasks were detached from the wall using a pipette, and the resulting cell suspension was transferred to a conical tube. The tube was centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant was discarded by decanting, and the cells were collected. After loosening the cells by tapping, culture broth was added, and the cells were resuspended evenly by pipetting. RAW264.7 cells were then passaged at 5 × 10⁻⁶ cells / mL. 5 0.5 mL / well of cells was seeded into a 24-well plate and incubated at 37°C for 3 hours. Then, 0.5 mL of culture broth, with or without double the concentration of each test substance, was added to each well. The culture broth derived from *Pantothia suspensa* (…) was then added to each well. Pantoea agglomerans LPS (LPSp: Funagoshi, mac0001) was used as a positive control. Samples other than the culture medium were diluted with the medium to a concentration of 10 ng / mL for LPS. After addition, cells were cultured in a 37°C 5% CO2 incubator, and the culture broth was collected after 4 hours.

[0043] (4) Methods for analyzing IL-10 gene expression

[0044] Total RNA extract (40 µL) was prepared from cells after the removal of culture broth using the RNeasy Mini Kit (QIAGEN) according to the manufacturer's instructions. Absorbance and RNA concentration were measured using a NanoVue Plus (GE HealthCare Japan) with 10 µL of the RNA extract. For the collected 2 µg of total RNA, chromosomal DNA was degraded by DNase treatment using the ReverTra Ace® qPCR RTMaster Mix with gDNA Remover (TOYOBO Co., Ltd.) according to the kit instructions, followed by cDNA synthesis via reverse transcription. Expression analysis of GAPDH (housekeeping gene) and IL-10 was performed by qPCR.

[0045] [2] Results

[0046] Table 1 shows the results of IL-10 gene expression in RAW264.7 cells for each sample. The average values ​​in the table represent the arithmetic mean of the fold change in IL-10 expression for each sample.

[0047] Culture medium: Culture medium only

[0048] LPSp: Culture medium supplemented with LPSp

[0049] Beetroot extract: Culture medium supplemented with beetroot extract

[0050] Compared to LPSp, beet extract showed a higher fold change in IL-10 expression.

[0051] Table 1: Fold change in IL-10 expression with addition of beet extract (as relative to LPSp=1)

[0052]

[0053] The LPS inhibitor polymyxin B (PB) was added to RAW264.7 cells, and IL-10 gene expression was measured for both LPS and beet extract. Furthermore, the inhibition rate by PB was calculated by comparing the fold change in IL-10 expression inhibited by PB with the fold change in IL-10 expression before PB addition. The results are shown in Table 2.

[0054] [Table 2: Fold change in IL-10 expression by addition of beet extract (as relative to LPSp=1) and inhibition rate by PB]

[0055]

[0056] PB (-): No PB added;

[0057] PB(+): PB has been added.

[0058] The results showed that both samples were inhibited by more than 90% with the addition of PB, indicating that the activity could be attributed to LPS.

[0059] LPS is a lipopolysaccharide found in the outer membrane of Gram-negative bacteria. Based on the above results, it can be inferred that the substance in beets with characteristic macrophage-activating activity originates from LPS of Gram-negative bacteria that live in symbiosis with beets.

[0060] [Example 2: Search for substances with properties of beet hot water extract]

[0061] Next, the effects of sugar beet-derived components other than LPS were investigated by molecular fractionation.

[0062] [1] Method

[0063] (1) Preparation of beet extract

[0064] As described in Example 1.

[0065] (2) Grading of extracts

[0066] The above-mentioned hot water extract of beet was fractionated and separated into a retentate with a molecular weight fraction of 10 kDa or greater (ultrafiltration retentate) (containing LPS) and a permeate with a molecular weight fraction less than 10 kDa (ultrafiltration permeate) (without LPS) using a centrifugal 10 kDa ultrafiltration membrane (Merck Millipore Ltd.). Distilled water was added to the ultrafiltration retentate, and the centrifugation (washing) was repeated five times. Each fraction, including the distilled water used for washing, was freeze-dried, and then distilled water was added.

[0067] In addition, the ultrafiltration permeate (less than 10 kDa) was dialyzed against distilled water using a 0.5 kDa dialysis membrane (Funagoshi). Dialysis was repeated five times, and the dialysis membrane cutoff (0.5–10 kDa) and the dialysis membrane permeate (less than 10 kDa) were collected. The dialysis membrane permeate was freeze-dried and reconstituted with distilled water.

[0068] (3) IL-10 gene expression analysis

[0069] Additional samples were added as described in Example 1.

[0070] [2] Results

[0071] Each sample was added to RAW264.7 cells, and Table 3 shows the results of examining IL-10 gene expression. The mean values ​​in the table represent the arithmetic mean of the fold change in IL-10 expression for each sample.

[0072] LPSp: Culture medium supplemented with LPSp

[0073] Beetroot extract: Culture medium supplemented with beetroot extract

[0074] Ultrafiltration cut-off: Culture medium supplemented with ultrafiltration cut-off

[0075] Ultrafiltration permeate: Culture medium supplemented with ultrafiltration permeate

[0076] Ultrafiltration cut-off + ultrafiltration permeate: Culture medium supplemented with both ultrafiltration cut-off and ultrafiltration permeate.

[0077] Table 3: Fold change in IL-10 expression with addition of ultrafiltration permeate (relative to LPSp=1)

[0078]

[0079] The results showed that IL-10 gene expression in the ultrafiltration cut-off material was significantly lower than that in the beet extract. Furthermore, almost no IL-10 gene expression was observed in the ultrafiltration permeate. By reconstructing the ultrafiltration cut-off material and the ultrafiltration permeate, it was confirmed that some substances in the ultrafiltration permeate contribute to the properties of the beet extract. Therefore, it was found that the ultrafiltration permeate contains substances that induce the properties of the beet extract.

[0080] Here, the LPS inhibitor polymyxin B (PB) was added to RAW264.7 cells, and IL-10 gene expression in the ultrafiltration filtrate was examined. The results are shown in Table 4. The inhibition rate by PB was calculated in the same manner as in Example 1.

[0081] LPSp: Culture medium supplemented with LPSp

[0082] LPSp + Ultrafiltration Permeate: Culture medium supplemented with LPSp and ultrafiltration permeate

[0083] Beetroot extract: Culture medium supplemented with beetroot extract

[0084] Ultrafiltration cut-off: Culture medium supplemented with ultrafiltration cut-off.

[0085] Ultrafiltration cut-off + ultrafiltration permeate: Culture medium supplemented with ultrafiltration cut-off and ultrafiltration permeate.

[0086] [Table 4: Fold-up changes in IL-10 expression caused by the addition of each sample (as relative to LPSp=1) and inhibition rate by PB]

[0087]

[0088] PB (-): No PB added;

[0089] PB(+): PB has been added.

[0090] As described above, the fold change in IL-10 expression was inhibited by more than 90% with the addition of PB, and this activity was due to LPS.

[0091] In addition, to limit the molecular weight of low-molecular-weight substances contained in the 10 kDa ultrafiltration permeate, dialysis membranes with a capacity of 0.5 kDa were used to prepare dialysis membrane cut-offs (0.5–10 kDa) and dialysis membrane permeates (less than 0.5 kDa), and their effectiveness was examined. Table 5 shows the results. The average values ​​in the table represent the arithmetic mean of the fold change in IL-10 expression for each sample.

[0092] LPSp: Culture medium supplemented with LPSp

[0093] Beetroot extract: Culture medium supplemented with beetroot extract

[0094] Ultrafiltration cut-off: Culture medium supplemented with ultrafiltration cut-off.

[0095] Ultrafiltration cut-off + dialysis membrane permeate: Culture medium supplemented with ultrafiltration cut-off and dialysis membrane permeate.

[0096] Table 5: Fold change in IL-10 expression induced by addition of dialysis membrane permeate (relative to LPSp=1)

[0097]

[0098] By reconstructing the ultrafiltration retention and dialysis membrane permeate, the characteristics of beet extract with increased fold change in IL-10 expression were revealed. This indicates that the low-molecular-weight substance in the ultrafiltration permeate that causes the increased fold change in IL-10 expression is limited to substances smaller than 0.5 kDa.

[0099] [Example 3: Isolation of microorganisms with properties of beet hot water extract]

[0100] [1] Method for isolating bacteria resident in beets

[0101] (1) Method for preparing beet agar medium (beet extract 25.75%, yeast extract 0.5%, amphotericin B 5µg / mL, vancomycin 10µg / mL, agar 1.5%)

[0102] <1> Hokkaido beets were cut into cubes of approximately 1 cm and mixed with an equal weight of water for injection (Otsuka Pharmaceutical Co., Ltd.). The mixture was homogenized and centrifuged (3500 rpm, 10 minutes), and the supernatant (50% beet extract) was collected. The 50% beet extract was heated at 68°C for 30 minutes and then freeze-dried. Distilled water was added in an amount equal to that of the original 50% beet extract to prepare beet extracts of the same concentration (50%) and three times the concentration (150%) of the original 50% beet extract. Amphotericin B (Fujifilm and Koko Pure Chemicals Co., Ltd.) was added to a final concentration of 10 µg / mL, and vancomycin (Fujifilm and Koko Pure Chemicals Co., Ltd.) was added to a final concentration of 20 µg / mL.

[0103] <2> In addition, agar (powder, from Fujifilm and Kojun Pharmaceutical Co., Ltd.) and yeast extract (NACALAITESQUE, INC.) were added to distilled water to achieve final concentrations of 3% and 1%, respectively, and then autoclaved at 121°C for 20 minutes.

[0104] <3> When the solution in <2> reaches approximately 60°C, mix it with an equal volume of <1> to prepare beet culture media with final concentrations of 25% and 75%. Dispense each solution in 25 mL portions into 10 cm culture dishes.

[0105] (2) Methods for isolating bacteria

[0106] Perform the following procedures aseptically in a clean bench.

[0107] Wipe and wash the beet surface with Kimwipes soaked in water for injection. Collect the skin from the washed area and chop it with scissors. Combine the skins from three beets, add an equal weight of water for injection, and vortex mix (suspension). Dilute the suspension with physiological saline 5 × 10⁻⁶. 3 Multiplied by 5×10 4 Spread 100 µL of the diluted suspension onto beet agar (25% and 75% beet extract) and LB agar.

[0108] (3) Methods for culturing bacteria

[0109] Plates spread on each medium were cultured under both aerobic and anaerobic conditions. For aerobic culture, plates spread at 5 × 10⁶ m³ were cultured in an incubator set at 25°C. 4 Plate the diluted solution in 3-fold increments over 3-5 days. For anaerobic culture, spread the solution in 5×10⁻⁶ m² plates. 3 Plates containing the diluted solution were placed in anaerobic bags (AnaeroPouch, manufactured by Gen Sugiyama Co., Ltd.) and incubated for 3 days in an incubator set to 25°C. Two hours after the start of incubation, the anaerobic indicator was checked to confirm an anaerobic state (0.1% or less).

[0110] [2] Results

[0111] Aerobic culture for 5 days and anaerobic culture for 3 days were performed on beet culture medium (25, 75%). Table 6 shows the colony counts obtained.

[0112] 25% Beetroot Extract Medium: A medium containing 25% beetroot extract.

[0113] 75% Beetroot Extract Medium: A medium containing 75% beetroot extract.

[0114] Colonies were scraped off each plate using a cell scraper, and water for injection was added to obtain a wet bacterial cell weight of 10–100 mg / mL. The plates were then heated at 90°C for 20 minutes (hot water extract of the cultured bacterial groups). Table 6 shows the LPS content measurements of the hot water extracts for each cultured bacterial group. All cultured bacterial groups contained bacteria that could be absorbed by horseshoe crabs (Limulus amebocyte lysate). Limulus LPS detected by reagents.

[0115] Table 6: LPS content per 1 mg of bacterial cells collected under each culture condition (Limulus amebocyte lysate (LAL)) Limulus (Reagent method)

[0116]

[0117] Next, the expression of the IL-10 gene in the hot water extract of the cultured bacterial populations was examined with and without the addition of PB to confirm that the activity originated from LPS. As shown in Table 7, the fold change in IL-10 expression was significantly higher in anaerobic culture in 25% beet extract medium and in aerobic and anaerobic culture in 75% medium compared to LPSp (significance level P < 0.05 in the T-test). Furthermore, the inhibition rate by PB exceeded 90% in both aerobic and anaerobic cultures in 25% beet extract medium and in aerobic culture in 75% medium. Based on these results, anaerobic culture (colony count: 6) and aerobic culture (colony count: 13) in 25% beet extract medium were selected. The inhibition rate by PB was calculated in the same manner as in Example 1.

[0118] [Table 7: Fold change in IL-10 expression by addition to each sample (relative to LPSp=1) and inhibition rate by PB]

[0119]

[0120] PB (-): No PB added;

[0121] PB(+): PB has been added.

[0122] The significance level in the t-test is P < 0.05.

[0123] [Example 4: Identification of microorganisms exhibiting characteristics of beet hot water extract]

[0124] Strains were selected from the microbial communities that emerged when sugar beets were cultured anaerobically in 25% sugar beet extract medium and aerobically in 75% sugar beet extract medium, and the genus was identified by 16S rRNA method, and the strains were preserved.

[0125] [1] Identification methods

[0126] (1) Method for preparing LB agar medium

[0127] Add agar to LB medium to a concentration of 1.5% and autoclave at 121°C for 20 minutes. After cooling to approximately 60°C, add amphotericin B to a final concentration of 5 µg / mL and vancomycin to a final concentration of 10 µg / mL, and dispense 25 mL into each culture dish.

[0128] (2) Identification of bacteria

[0129] Nineteen colonies selected from replicas of 25% beetroot medium (anaerobic culture) and 75% beetroot medium (aerobic culture) (stored at 4°C) were streaked onto fresh LB agar plates. After aerobic incubation at 25°C for 2 days, single colonies were streaked onto fresh medium. After incubation at 25°C for 2 days and confirmation of colony presence, the plates were sent to Technosuruga Laboratory Co., Ltd. for 16S rDNA partial sequence analysis.

[0130] (3) Thermal phenolic water extraction

[0131] For the hot phenolic water extraction, phenol extraction was performed according to the method of Westphal et al. Water for injection was added to the wet cells to a concentration of 100 mg / mL, and the mixture was stirred using a vortex mixer. An equal volume of 90% phenol was added to the suspension and mixed using a vortex mixer. The mixture was heated in a water bath set to 68°C for 20 minutes. During heating, the mixture was stirred for approximately 10 seconds every 5 minutes. After cooling to room temperature, it was centrifuged (3500 rpm, 20 min, room temperature), and the aqueous layer was collected. An equal volume of water for injection was added to the collected aqueous layer, and phenol extraction was performed again.

[0132] Water for injection was added to the collected aqueous layer to dilute it 10-fold. To remove phenol from this solution, ultrafiltration was performed using a 10 kDa centrifugal ultrafiltration filter, and LPS was collected and passed through a horseshoe crab (LMS) filter. Limulus The amount of LPS in the solution was determined by the reagent method.

[0133] (4) Methods for analyzing IL-10 gene expression

[0134] As described in Example 1, IL-10 gene expression was analyzed for each sample. Here, the ultrafiltration permeate was used as a low molecular weight substance, but as mentioned above, any low molecular weight substance that actually increases the fold change in IL-10 expression is a substance less than 0.5 kDa.

[0135] [2] Results

[0136] Technosuruga Laboratory Co., Ltd. was commissioned to determine the partial 16S rDNA sequences of the 19 strains obtained as described above, and to perform a BLAST search of the sequence data in international base sequence databases to identify the bacterial genera. Three strains from 25% beet culture medium (anaerobic culture) and two strains from 75% beet culture medium (aerobic culture) were deposited at the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary. Table 8 shows the genus and accession number for each bacterium.

[0137] Table 8: Genus and accession number for each strain

[0138]

[0139] IL-10 gene expression in preserved bacterial cells was measured, and the increase rate was calculated by comparing expression before and after the addition of low-molecular-weight substances to individual samples. The results are shown in Tables 9 and 10.

[0140] [Table 9: Fold change in IL-10 expression after adding each sample (relative to LPSp=1) and the increase rate after adding low molecular weight substances]

[0141]

[0142] [Table 10: Fold change in IL-10 expression with the addition of each sample (relative to LPSp=1) and the increase rate with the addition of low molecular weight substances]

[0143]

[0144] The results above show that when samples are used alone, LPS derived from bacteria isolated from beets has a higher fold change in IL-10 expression than LPSp (LPS derived from BR-4, BR-6, BR-15, and BR-19 strains). Alternatively, when low molecular weight substances are added (using ultrafiltration permeate less than 10 kDa, but in practice, substances less than 0.5 kDa are effective), the fold change in IL-10 expression increases by more than 20-fold compared to samples alone (LPS derived from BR-1 and BR-15 strains).

[0145] These findings have made it clear that sugar beets possess anti-inflammatory activity through the interaction between LPS derived from sugar beet-specific bacteria and low-molecular-weight substances that alter LPS activity. Therefore, new substances associated with the immunomodulatory functions of sugar beets have become available for the development of pharmaceuticals, veterinary drugs, quasi-pharmaceuticals, cosmetics, foods, functional foods, feeds, fertilizers, and bath additives for suppressing chronic inflammation, as well as health products such as foods blended with processed edible plants such as vegetables, fruits, and grains, particularly root vegetables.

[0146] All publications cited in this article are incorporated herein in their entirety.

[0147] [Collection Number]

[0148] Name of the depository: National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary (NITE)

[0149] Address of the depository: Room 122, 2-5-8 Kazusa-Kamazutari, Kisarazu City, Chiba Prefecture, Japan 292-0818

[0150] Date of preservation: March 2, 2023

[0151] Collection Number:

[0152] (1) NITE BP-03839

[0153] (2) NITE BP-03840

[0154] (3) NITE BP-03841

[0155] (4) NITE BP-03842

[0156] (5) NITE BP-03843 PCT / RO / 134 form

Claims

1. Lipopolysaccharide, which is obtained from bacteria with accession numbers NITE BP-03839, NITE BP-03840, NITE BP-03841, NITE BP-03842 or NITE BP-03843.

2. A method for producing lipopolysaccharide, comprising obtaining the lipopolysaccharide from bacteria with accession numbers NITE BP-03839, NITE BP-03840, NITE BP-03841, NITE BP-03842 or NITE BP-03843.

3. A lipopolysaccharide complex comprising the lipopolysaccharide according to claim 1.

4. The lipopolysaccharide complex according to claim 1 or 3, further comprising a substance of less than 0.5 kDa from beets.

5. Edible plant processed products comprising lipopolysaccharides according to any one of claims 1 and 3 to 4.

6. The edible plant processed product according to any one of claims 1 and 3 to 5, further comprising a substance of less than 0.5 kDa from sugar beets.

7. The edible plant product according to claim 5 or 6, wherein the edible plant product is a beet product.

8. The edible plant processed product according to any one of claims 5 to 7, wherein the edible plant processed product is dried vegetables or dried edible plant powder.

9. Food products comprising processed edible plant products according to any one of claims 5 to 8.

10. The lipopolysaccharide complex according to claim 3 or 4, wherein the lipopolysaccharide complex is a pharmaceutical, veterinary drug, quasi-pharmaceutical, cosmetic, food, functional food, feed, fertilizer, or bath additive.

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