JP7479620B1 - Lipopolysaccharide, method for producing lipopolysaccharide and lipopolysaccharide formulation - Google Patents

Abstract

To clarify and provide novel substances/factors involved in the anti-inflammatory action of beets. This will enable the development of healthcare products such as medicines, foods, and cosmetics for suppressing chronic inflammation. Lipopolysaccharide obtained from bacteria having accession numbers NITE BP-03839, NITE BP-03840, NITE BP-03841, NITE BP-03842, or NITE BP-03843. Medicines, veterinary medicines, quasi-drugs, cosmetics, foods, functional foods, feed, fertilizers, or bath additives containing the lipopolysaccharide.

Description

NPMD NITE BP-03839 NPMD NITE BP-03840 NPMD NITE BP-03841 NPMD NITE BP-03842 NPMD NITE BP-03843

The present invention relates to lipopolysaccharides, methods for producing lipopolysaccharides and lipopolysaccharide formulations, and in particular to lipopolysaccharide (LPS) derived from bacteria that are uniquely symbiotic to beets.

Beets are considered to be a highly nutritious and functional vegetable. They are rich in minerals such as potassium, magnesium, and iron. In addition, the nitrite contained in beets is converted into nitric oxide in the body and has the effect of dilating blood vessels, betalain has the effect of preventing cell aging due to its antioxidant effect, and dietary fiber is said to be useful for improving the intestinal environment and improving rough skin, so they are highly functional (Non-Patent Document 1). It is known that polyphenols and vitamin C contained in common vegetables have the effect of maintaining the function of immune cells, and betacyanin, a pigment component of beets, is known to have an anti-inflammatory effect and has been reported to have the effect of reducing the expression of inflammatory cytokines (TNF-α, IL-6) (Non-Patent Document 2). However, it is not known that beets activate macrophages, which are natural immune cells, and increase the expression of anti-inflammatory cytokines (IL-10).

We have found that lipopolysaccharide (LPS) derived from symbiotic bacteria is attached to plants (Non-Patent Document 3), and that this LPS is taken up by the body and activates the human innate immune system (Non-Patent Document 4). Therefore, it is possible that part of the immunomodulatory effect of beets is related to LPS derived from bacteria that are unique to beets and symbiotic with them (hereinafter referred to as "beet LPS").

L. Chen et al., “Beetroot as a functional food with huge health benefits: antioxidant, antitumor, physical function, and chronic metabolic activity”, Food Sci Nutr., 2021.11, 9(11), p.6406-6420 S. Saito et al., “Metabolic engineering of betacyanin in vegetables for anti-inflammatory therapy”, Biotechnol Bioeng, 2023.05, 120(5), p.1357-1365 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. H. Inagawa et al., “Usefulness of Oral Administration of Lipopolysaccharide for Disease Prevention Through the Induction of Priming in Macrophages”, Anticancer Res., 2014.08, 34(8), p.4497-4501

The present invention clarifies and provides novel substances and factors involved in the anti-inflammatory action of beets, which will enable the development of healthcare products such as medicines, foods, and cosmetics to suppress chronic inflammation.

The present invention includes the following:

[1] A lipopolysaccharide characterized by being obtained from a bacterium having the accession number NITE BP-03839, NITE BP-03840, NITE BP-03841, NITE BP-03842, or NITE BP-03843.
[2] A method for producing lipopolysaccharide, comprising obtaining lipopolysaccharide from a bacterium having accession number NITE BP-03839, NITE BP-03840, NITE BP-03841, NITE BP-03842, or NITE BP-03843.
[3] A lipopolysaccharide formulation comprising the lipopolysaccharide described in [1] above.
[4] A lipopolysaccharide preparation according to [1] or [3] above, further comprising a substance of less than 0.5 kDa contained in beets.
[5] An edible plant processed product, characterized in that it contains the lipopolysaccharide described in any one of [1] and [3] to [4] above.

[6] An edible plant processed product according to any one of [1] and [3] to [5], further comprising a substance of less than 0.5 kDa contained in beets.
[7] The edible plant processed product according to [5] or [6] above, characterized in that the edible plant processed product is a beetroot processed product.
[8] The edible plant processed product according to any one of [5] to [7] above, characterized in that the edible plant processed product is a dried vegetable or a dried edible plant powder.
[9] A food product comprising the edible plant processed product according to any one of [5] to [8] above.
[10] The lipopolysaccharide formulation described in [3] or [4] above, characterized in that the lipopolysaccharide formulation is a pharmaceutical, an animal drug, a quasi-drug, a cosmetic, a food, a functional food, a feed, a fertilizer, or a bath additive.

Beet LPS, derived from bacteria isolated from beets, activates macrophages, but compared to the known Pantoea agglomerans LPS, it exhibits special macrophage activation, such as a high ability to induce the anti-inflammatory cytokine IL-10, and this action was demonstrated to be due to LPS through the action of polymyxin B. It was also demonstrated that the macrophage activation is not the action of beet LPS alone, but an interaction with low molecular weight substances in beets. This will enable the development of healthcare products such as medicines, foods, and cosmetics to suppress chronic inflammation.

Beets in this specification refer to beets listed in the 2020 edition (8th revision) of the Standard Tables of Food Composition in Japan.

In this specification, the term "edible plant" refers to a plant that is consumed by humans. As used herein, the term "edible plant" is not limited as long as it is consumed by humans, but examples include vegetables, grains, potatoes, beans, nuts, fruits, mushrooms, algae, etc., with vegetables being preferred, and beets being particularly preferred. One type of edible plant may be used, or two or more types may be used in any combination. Edible plants may be used as is, or may be used after various treatments (e.g. drying, heating, removing lye, peeling, removing nuts and seeds, ripening, salting, peel processing, etc.). In addition, even if a foodstuff has a part of its edible parts (such as green soybeans and green peas) that is treated as a vegetable, it can be determined whether it is a legume or not based on the state of the whole plant (soybeans, peas, etc.) combined with the inedible parts (pods, etc.). In addition, the classification of edible plants can be determined based on the whole plant combined with the inedible parts. Specifically, for example, by referring to the classifications listed in the "Standard Tables of Food Composition in Japan, 2015 Edition (7th Revised Edition) Supplement 2018" (a food composition table established by the Ministry of Health, Labour and Welfare, see especially Table 1 on page 236), such as vegetables, grains, potatoes, beans, nuts and seeds, fruits, mushrooms, and algae, it is possible to understand what foods correspond to edible plants in the present invention.

The properties of the edible plant processed product of the present invention are not limited, but are preferably one or more selected from edible plant powder, edible plant paste, and aqueous extract of edible plant. For example, it is preferably a processed product obtained by subjecting an edible plant to a heat treatment (e.g., 80°C or higher) such as drying, roasting, or hot water extraction. The edible plant processed product of the present invention is preferably a dried vegetable or a dried edible plant powder.
Examples of the present invention will be described below, but the present invention is not limited to the following examples.

[Evaluation of macrophage activation by hot water extract of beets]
(1) Method 1) Preparation of beet extract

The beets with the skin on were washed with water and cut into 1 cm square cubes using a knife. The cut beets were then heated in a steamer at a temperature of 90°C or higher for 15 minutes. The heated beets were then spread out on a tray with holes so that they did not overlap, and the tray was placed in a hot air dryer set at 70°C. The beets were then dried (7-8 hours) until the moisture content was 6% or less, creating beet chips. The beets were then crushed using a grinder (Osaka Chemical) and passed through a 355 μm mesh to create dried beet powder.

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

2) LPS amount measurement method
The amount of LPS was measured using a toxinometer (Fujifilm Wako Pure Chemical Industries, Ltd.) as the Limulus value (value obtained by the Limulus test, converted value of LPS produced by the control standard endotoxin E. coli UKT-B).

3) Preparation of samples for evaluating macrophage activation by beet extract
RAW264.7 cells were subcultured in RPMI1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were subcultured in a T25 culture flask at 0.25×10 ⁵ cells/mL every 3 or 4 days. The cells were cultured in a 5% CO2 incubator (hereinafter referred to as the incubator) at 37°C. All test procedures were performed in a clean bench. The cells precultured in the T25 culture flask were detached from the wall by pipetting, 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 decantation, and the cells were collected. The cells were loosened by tapping, and then culture medium was added and the cells were uniformly suspended by pipetting. The RAW264.7 cells were seeded in a 24-well plate at ^5x105 cells/0.5mL/well and cultured in a 37℃ incubator for 3 hours. Then, 0.5mL of culture medium containing or not containing 2x concentrations of each test substance was added to the wells. LPS derived from Pantoea agglomerans (LPSp: Funakoshi, mac0001) was used as a positive control. Samples other than the medium were diluted with medium to a LPS concentration of 10ng/mL. After addition, the cells were cultured in a 37℃ 5% CO2 incubator, and the culture medium was collected after 4 hours.

4) IL-10 gene expression analysis method Total RNA extract (40μL) was prepared from the cells after removing the culture medium using RNeasy Mini Kit (QIAGEN) according to the instructions. The absorbance and RNA concentration were measured using NanoVue Plus (GE Healthcare Japan, Ltd.) using 10μL of the RNA extract. Chromosomal DNA was degraded by DNase treatment using ReverTra Ace(R) qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd.) according to the kit's instructions, and then cDNA was synthesized by reverse transcription. Expression analysis of GAPDH (housekeeping gene) and IL-10 was performed by qPCR.

(2) Results The results of IL-10 gene expression in RAW264.7 cells for each sample are shown in Table 1. The average in the table represents the arithmetic mean value of the IL-10 expression fold for each sample.
Medium: Medium only
LPSp: LPSp was added to the medium. Beet extract: Beet extract was added to the medium. Beet extract showed a higher IL-10 expression fold than LPSp.

Polymyxin B (PB), an LPS inhibitor, was added to RAW264.7 cells, and IL-10 gene expression in LPSp and beet extract was measured. In addition, the inhibition rate by PB was calculated by comparing the IL-10 expression fold inhibition by PB addition with that before PB addition. The results are shown in Table 2.

*PB（-）：PB添加なし。PB（+）：PB添加あり。
両サンプルともにPB添加により90%以上阻害されることが示され、本活性はLPSによるものであることが示された。

*PB(-): No PB added. PB(+): PB added.
Both samples were shown to be inhibited by more than 90% by the addition of PB, indicating that this activity was due to LPS.

LPS is a lipopolysaccharide present in the outer cell membrane of gram-negative bacteria. From these results, it was speculated that the substance in beets that has the characteristic activation effect on macrophages is LPS derived from gram-negative bacteria that live symbiotically with beets.

[Searching for substances characteristic of hot water extract of beets]
Next, we used molecular fractionation to examine whether beet-derived components other than LPS had any effect.
(1) Method 1) Preparation of beet extract As described in Example 1.

2) Fractionation of the extract The above beet hot water extract was centrifuged using a centrifugal 10 kDa ultrafiltration filter (Merck Millipore Ltd) to separate it into an inner liquid (ultrafiltrate inner liquid) with a molecular weight of 10 kDa or more (containing LPS) and an outer liquid (ultrafiltrate outer liquid) with a molecular weight of less than 10 kDa (not containing LPS). Distilled water was added to the ultrafiltrate inner liquid, and the centrifugation (washing) procedure was repeated five times. Each fraction, including the distilled water used for washing, was freeze-dried, and distilled water was added.

Furthermore, the ultrafiltrate (less than 10 kDa) was dialyzed against distilled water using a 0.5 kDa dialysis membrane (Funakoshi). Dialysis was performed five times, and the fluid inside the dialysis membrane (0.5-10 kDa) and the fluid outside the dialysis membrane (less than 0.5 kDa) were collected. The fluid outside the dialysis membrane was freeze-dried and then prepared with distilled water.
3) IL-10 gene expression analysis Additional samples were added and the analysis was carried out as described in Example 1.

(2) Results Each sample was added to RAW264.7 cells, and the results of examining IL-10 gene expression are shown in Table 3. The average in the table represents the arithmetic mean value of the IL-10 expression fold for each sample.
LPSp: LPSp added to the medium Beet extract: Beet extract added to the medium Ultrafiltrate inner solution: Ultrafiltrate inner solution added to the medium Ultrafiltrate outer solution: Ultrafiltrate outer solution added to the medium Ultrafiltrate inner solution + ultrafiltrate outer solution: Ultrafiltrate inner solution and ultrafiltrate outer solution added to the medium

These results show that IL-10 gene expression in the ultrafiltrate was significantly lower than in the beet extract. Furthermore, almost no IL-10 gene expression was observed in the ultrafiltrate. By reconstituting the ultrafiltrate and ultrafiltrate, it was confirmed that some substance in the ultrafiltrate gives rise to the characteristics of the beet extract. From this, it was found that the ultrafiltrate contains a substance that induces the characteristics of the beet extract.

Here, polymyxin B (PB), an LPS inhibitor, was added to RAW264.7 cells, and the IL-10 gene expression in the ultrafiltrate was measured, and the results are shown in Table 4. The inhibition rate by PB was calculated in the same manner as in Example 1.
LPSp: LPSp added to the medium
LPSp + UF: LPSp and UF added to the medium. Beet extract: Beet extract added to the medium. UF: UF added to the medium. UF + UF: UF and UF added to the medium.

*PB（-）：PB添加なし。PB（+）：PB添加あり。
上記のとおり、PB添加によりIL-10発現倍率が90%以上阻害されることが示され、本活性はLPSによるものであることが示された。

*PB(-): No PB added. PB(+): PB added.
As described above, it was shown that the addition of PB inhibited IL-10 expression by 90% or more, indicating that this activity was due to LPS.

Furthermore, in order to limit the molecular weight of low molecular weight substances contained in the 10 kDa ultrafiltrate, a 0.5 kDa dialysis membrane was used to prepare a dialysis membrane inner solution (0.5-10 kDa) and a dialysis membrane outer solution (less than 0.5 kDa), and their effects were examined. The results are shown in Table 5. The average in the table represents the arithmetic mean value of the IL-10 expression fold for each sample.
LPSp: LPSp added to the medium. Beet extract: Beet extract added to the medium. Ultrafiltrate inner fluid: Ultrafiltrate inner fluid added to the medium. Ultrafiltrate inner fluid + dialysis membrane outer fluid: Ultrafiltrate inner fluid and dialysis membrane outer fluid added to the medium.

The beet extract showed the characteristic of increasing the IL-10 expression ratio by reconstituting the ultrafiltrate inner fluid and the dialysis membrane outer fluid. From the above, it was narrowed down that any low molecular weight substances contained in the ultrafiltrate outer fluid that increase the IL-10 expression ratio are substances less than 0.5 kDa.

[Isolation of microorganisms with characteristics of hot water extract of beets]
(1) Method for isolating beet-resident bacteria

1) How to prepare beet agar medium (beet extract 25, 75%, yeast extract 0.5%, amphotericin 5μg/mL, vancomycin 10μg/mL, agar 1.5%)

(1): Hokkaido-grown beets were cut into 1 cm cubes and an amount of water for injection (Otsuka Pharmaceutical Co., Ltd.) equal to the weight of the beets was added. After crushing with a homogenizer, the mixture was centrifuged (3500 rpm, 10 min) and the supernatant was collected (50% beet extract). The 50% beet extract was heated at 68°C for 30 min and then freeze-dried. Distilled water was added to this freeze-dried product in an amount equal to the original 50% beet extract, to prepare beet extracts of the same concentration (50%) as the 50% beet extract and three times the concentration (150%). Amphotericin B (Fujifilm Wako Pure Chemical Corporation) was added to a final concentration of 10 μg/mL, and vancomycin (Fujifilm Wako Pure Chemical Corporation) was added to a final concentration of 20 μ/mL.

(2): In a separate container, agar (powder, Fujifilm Wako Pure Chemical Industries, Ltd.) and yeast extract (Nacalai Tesque, Inc.) were added to distilled water to final concentrations of 3% and 1%, respectively, and autoclaved at 121°C for 20 minutes.

(3): When the solution (2) reached about 60°C, it was mixed with an equal amount of (1) to prepare final concentrations of 25% and 75% beetroot medium. 25 mL of this was dispensed into 10 cm petri dishes.

2) Method for isolating bacteria The following operations were carried out aseptically in a clean bench.
The surface of the beet was scrubbed and washed with a Kimwipe soaked in water for injection. The skin of the washed area was collected and cut into small pieces with scissors. The skin of three beets was mixed, and an equal amount of water for injection was added by weight and stirred with a vortex mixer (suspension). This suspension was diluted 5 x 10 ³ times and 5 x 10 ⁴ times with physiological saline. 100 μL of the diluted suspension was spread on beet agar medium (beet extract 25, 75%) and LB agar medium.

3) Cultivation method of bacteria Plates on each medium were cultivated under aerobic and anaerobic conditions. For aerobic cultivation, plates were cultivated with 5x10 ⁴ -fold dilution in a thermostatic chamber set at 25°C for 3-5 days. For anaerobic cultivation, plates were cultivated with 5x10 ³ -fold dilution in an anaerobic pouch (Anelopouch Kenki: Sugiyamagen Co., Ltd.) and cultivated for 3 days in a thermostatic chamber set at 25°C. Two hours after the start of cultivation, it was confirmed that the anaerobic indicator showed no oxygen (0.1% or less).

(2) Results The bacteria were cultured aerobically for 5 days and anaerobicly for 3 days on beetroot medium (25, 75%). The number of colonies obtained is shown in Table 6.
Beet extract 25% medium: Beet extract medium with a concentration of 25% Beet extract 75% medium: Beet extract medium with a concentration of 75%

Colonies from each plate were scraped off with a cell scraper, water for injection was added to give a wet cell weight of 10-100 mg/mL, and the mixture was heated at 90°C for 20 minutes (hot water extract of cultured bacteria). The results of measuring the LPS content of the hot water extract of each cultured bacteria are shown in Table 6. Each cultured bacteria contained LPS that could be measured with Limulus.

Next, the IL-10 gene expression of the hot water extract of the cultured bacteria group was examined with and without the addition of PB, and it was confirmed that this activity was derived from LPS. As shown in Table 7, the IL-10 expression ratio was significantly higher in anaerobic culture in 25% beet extract medium, 75% aerobic culture, and anaerobic culture in 25% beet extract medium, compared to LPSp (P < 0.05 in the T-test). In addition, the inhibition rate by PB exceeded 90% in aerobic and anaerobic culture in 25% beet extract medium, and aerobic culture in 75% medium. Based on the above, anaerobic culture in 25% beet extract medium (number of colonies: 6) and aerobic culture in 75% beet extract medium (number of colonies: 13) were selected. The inhibition rate by PB was calculated using the same method as in Example 1.

*PB（-）：PB添加なし。PB（+）：PB添加あり。
*：T検定で危険率P<0.05

*PB(-): No PB added. PB(+): PB added.
*: P<0.05 in T-test

[Identification of microorganisms with characteristics of hot water extract of beets]
A strain was selected from the group of bacteria that emerged when beets were cultured anaerobically in a medium containing 25% beet extract and then aerobically in a medium containing 75% beet extract. The genus was identified by the 16S rRNA method and the strain was deposited.
(1) Identification method 1) Preparation method of LB agar medium

Agar was added to the LB medium to a concentration of 1.5%, and the medium was autoclaved at 121°C for 20 minutes. After cooling to approximately 60°C, reagents were added to make the final concentration of amphotericin B 5μg/mL and vancomycin 10μg/mL, and 25mL was dispensed into petri dishes.

2) Identification of bacteria
Nineteen colonies selected from replicas (stored at 4°C) of 25% beet medium (anaerobic culture) and 75% beet medium (aerobic culture) were streaked onto new LB medium. After aerobic culture at 25°C (2 days), a single colony was streaked onto new medium. After culturing at 25°C for 2 days and confirming the colonies, the plate was sent to Techno Suruga Lab, a subcontractor, to obtain partial 16s rDNA sequence analysis data.

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

The collected aqueous layer was diluted 10-fold with water for injection. To remove phenol from this solution, ultrafiltration was performed using a centrifugal 10 kDa ultrafiltration filter, and the LPS content of this solution was measured using the Limulus method.

4) IL-10 gene expression analysis method IL-10 gene expression analysis was performed for each sample as described in Example 1. Ultrafiltrate was used as the low molecular weight substance here, but as described above, any low molecular weight substance that actually increases the IL-10 expression fold is a substance of less than 0.5 kDa.

(2) Results The 16s rDNA partial base sequences of the 19 strains obtained above were determined by outsourcing to Techno Suruga Lab, and the sequence data was subjected to a BLAST search in the international base sequence database to identify the genus of the bacteria. Of these, three strains from the 25% beet medium (anaerobic culture) and two strains from the 75% beet medium (aerobic culture) were deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation. Table 8 shows the genus and accession number of each strain.

The IL-10 gene expression of the deposited bacteria was measured, and the increase rate when the low molecular weight substance was added to the sample alone was calculated compared to before the addition of the low molecular weight substance. The results are shown in Tables 9 and 10.

These results reveal that the IL-10 expression fold increase in the sample alone is higher than that of LPSp (LPS derived from BR-4, BR-6, BR-15, and BR-19 strains) or that the IL-10 expression fold increase is due to the addition of low molecular weight substances (ultrafiltrate of less than 10 kDa was used, but substances of less than 0.5 kDa were actually effective), with the increase in IL-10 expression increasing by more than 20-fold compared to the sample alone (LPS derived from BR-1 and BR-15 strains) in LPS derived from beet isolated bacteria.

From these findings, it has become clear that beets have an anti-inflammatory effect due to the interaction between LPS derived from bacteria specific to beets and low molecular weight substances that modify the action of LPS. The new substances related to the immune-regulating function of beets may enable the development of pharmaceuticals, veterinary medicines, quasi-drugs, cosmetics, food, functional foods, feed, fertilizers, and bath additives that suppress chronic inflammation, as well as healthcare products such as foods that incorporate edible plant products obtained by processing edible plants such as vegetables, fruits, and grains, in particular root vegetables.

All publications cited herein are incorporated herein by reference in their entirety.

Name of depository institution: National Institute of Technology and Evaluation, Patent Microorganism Depository Center Address of depository institution: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan Date of deposit: March 2, 2023 Accession number:
(1). NITE BP-03839
(2). NITE BP-03840
(3). NITE BP-03841
(4). NITE BP-03842
(5). NITE BP-03843

Claims (10)

A lipopolysaccharide characterized in that it is obtained from a bacterium having accession number NITE BP-03839, NITE BP-03840, NITE BP-03841, NITE BP-03842, or NITE BP-03843. A method for producing lipopolysaccharide, comprising obtaining lipopolysaccharide from bacteria having accession numbers NITE BP-03839, NITE BP-03840, NITE BP-03841, NITE BP-03842, or NITE BP-03843. A lipopolysaccharide formulation comprising the lipopolysaccharide according to claim 1. The lipopolysaccharide preparation according to claim 3, further comprising a substance less than 0.5 kDa in beets. 10. An edible plant processed product comprising the lipopolysaccharide according to claim 1 . 6. The edible plant processed product according to claim 5, further comprising a substance of less than 0.5 kDa contained in beets. 6. The edible plant processed product according to claim 5, characterized in that the edible plant processed product is a beetroot processed product. 6. The edible plant processed product according to claim 5 , wherein the edible plant processed product is a dried vegetable or a dried edible plant powder. A food comprising the edible plant processed product according to any one of claims 5 to 8. The lipopolysaccharide formulation according to claim 3 or 4, characterized in that the lipopolysaccharide formulation is a pharmaceutical product, an animal drug, a quasi-drug, a cosmetic product, a food product, a functional food product, a feed, a fertilizer, or a bath additive.