Method for obtaining LPS-removed plant fermentation extract, said LPS-removed plant fermentation extract, and formulation thereof

Abstract

The present invention is predicated on success at producing a substance that results from removing LPS from a plant fermentation extract and the discovery that said substance demonstrates a biological activity that cannot be explained by conventional scientific common knowledge. The present invention is a method for obtaining an LPS-removed plant fermentation extract, an LPS-removed plant fermentation extract that is obtained using said method, and a formulation in which said LPS-removed plant fermentation extract has been compounded, said method being characterized by comprising a step in which LPS is removed from a plant fermentation extract that is obtained by fermenting a material derived from an edible plant, using a facultative anaerobic gram-negative bacterium that is exclusively symbiotic with the plant, and simultaneously cultivating said facultative anaerobic gram-negative bacterium.

Description

Method for obtaining LPS-removed plant fermentation extract, the LPS-removed plant fermentation extract, and its formulation

[0001] The present invention relates to a method for obtaining a plant fermentation extract from which LPS has been removed, the LPS-removed plant fermentation extract, and its formulation.

[0002] Patent Document 1 describes a technique for producing a plant fermentation extract by fermenting and culturing wheat with Pantoea agglomerans. This plant fermentation extract has the ability to activate macrophages and exhibits an effect as an immunostimulant. In Patent Document 1, a method for producing an inexpensive and safe immunostimulant is provided compared to the prior art, and this plant fermentation extract is said to be usable in a wide range of fields such as pharmaceuticals, cosmetics, foods, and feeds, and it is characterized in that new biological activities can be obtained by the synergistic effect of plant components and microorganisms in the process of fermentation and culture.

[0003] On the other hand, the fact that the plant fermentation extract of Patent Document 1 has the ability to activate macrophages even in the presence of polymyxin B, which inhibits lipopolysaccharide (LPS) derived from Gram-negative bacteria, a limulus-positive glycolipid, is considered to suggest the following three points. <晓

[0004] (1) Polymyxin B usually binds to LPS and inhibits its biological activity. The fact that this plant fermentation extract can activate macrophages even in the presence of polymyxin B indicates that there are immunostimulatory factors other than LPS in this plant fermentation extract. (2) Components other than LPS in this plant fermentation extract also have various mechanisms for activating the immune system. (3) A new mechanism is involved, which is different from the conventional immunostimulatory effect of LPS. Thus, it is considered that this plant fermentation extract can exhibit a wider range of immunostimulatory effects.

[0005] The details of these mechanisms are unknown. Therefore, if it is possible to produce a plant fermentation extract from which LPS has been removed, it will be possible to produce a new immunostimulatory material that is materially different from the conventional plant fermentation extract and has new biological activities.

[0006] Japanese Patent No. 4026722

[0007] D. Li et al., "Pattern recognition receptors in health and diseases", Signal Transduction and Targeted Therapy, 2021, 6, 291https: / / doi.org / 10.1038 / s41392-021-00687-0Z. Wang et al., "KCTD12 is a prognostic marker of breast cancer and correlates with tumor immune cell infiltration", Translational Cancer Research, 2021, 10(1), p.261-272https: / / doi.org / 10.21037 / tcr-20-2099R. Witzel et al., "PACAP regulates VPAC1 expression, inflammatory processes and lipid homeostasis in M1- and M2-macrophages", Frontiers in Cardiovascular Medicine, 2023, 10https: / / doi.org / 10.3389 / fcvm.2023.1264901J. Leceta et al., "Receptors and transcriptional factors involved in the anti-inflammatory activity of VIP and PACAP", Annals of the New York Academy of Sciences, 2000, 921(1), p.92-102https: / / doi.org / 10.1111 / j.1749-6632.2000.tb06954.xD. Toth et al., "Protective Effects of PACAP in Peripheral Organs", Frontiers in Endocrinology, 2020, 11https: / / doi.org / 10.3389 / fendo.2020.00377A. Ciesielska et al., "Lysophosphatidic acid up-regulates IL-10 production to inhibit TNF-α synthesis in Mφs stimulated with LPS", Journal of Leukocyte Biology, 2019, 106(6), p.1285-1301https: / / doi.org / 10.1002 / JLB.2A0918-368RR.

[0008] As mentioned above, this plant fermentation extract has the characteristic of activating macrophages even in the presence of polymyxin B. We made diligent efforts to devise a method for producing a novel immune-activating material by removing LPS from this plant fermentation extract. As a result, we succeeded in producing a substance from which LPS has been removed from the plant fermentation extract, and found that this substance exhibits biological activity that cannot be explained by conventional scientific knowledge, thus completing the foundation of the present invention.

[0009] The present invention provides a method for obtaining an LPS-removed plant fermentation extract, characterized by comprising the step of fermenting a material derived from edible plants exclusively with facultative anaerobic gram-negative bacteria that coexist symbiotically with plants, and simultaneously culturing the facultative anaerobic gram-negative bacteria to obtain a plant fermentation extract from which LPS is removed.

[0010] Furthermore, the LPS-removing plant fermentation extract of the present invention is characterized by being obtained by the method described above. Furthermore, the formulation of the present invention is characterized by containing the above-described LPS-removing plant fermentation extract. The formulation is preferably a pharmaceutical product, a veterinary drug, a quasi-drug, a cosmetic product, a food product, a functional food product, a feed product, a fertilizer product, or a bath additive.

[0011] According to the present invention, it is possible to provide a method for obtaining an immunostimulant substance inexpensively and efficiently using safe materials, an LPS-removed plant fermentation extract obtained by this method, and a formulation containing the LPS-removed plant fermentation extract.

[0012] This figure shows the bioactivity-inducing ability of LPS-removed wheat fermentation extract in TLR-9-introduced HEK293 cells.

[0013] [Fermentation culture of Pantoea bacteria] [Production of wheat fermentation extract] In accordance with Patent Document 1, Pantoea bacteria, which are gram-negative bacteria, were fermented and cultured using wheat flour as a substrate to produce wheat fermentation extract.

[0014] (1) In a 2L Erlenmeyer flask, 64g of disodium phosphate-7 crystal water, 15g of potassium phosphate, 2.5g of sodium chloride, and 5g of ammonium chloride were taken and purified water was added to make a total volume of 1L (inorganic salt mixture solution). 13.1g of magnesium chloride-2 crystal water was taken and purified water was added to make a total volume of 100mL (magnesium chloride solution). 1.1g of calcium chloride-1 was taken and purified water was added to make a total volume of 100mL (calcium chloride solution). 4L of purified water was placed in a 5L Erlenmeyer flask (purified water). All of the above solutions and purified water were sterilized by autoclaving (TOMY BS-325 (Tomy Seikou Co., Ltd.), 120°C, 20 minutes).

[0015] (2) 24 g of wheat flour (Nisshin Flour Milling Co., Ltd.) was placed in a 1 L Erlenmeyer flask, and purified water was added to make a total volume of 600 mL. After autoclaving this in the same manner, 3 mg of α-amylase (SIGMA, Bacillus, enzyme activity of 1500 to 3000 units per 1 mg of protein) was added, and the mixture was heated in a 65°C water bath for 4 to 12 hours (wheat flour amylase treatment solution). (3) The inorganic salt mixture solution (200 mL) prepared in (1), purified water (550 mL), wheat flour amylase treatment solution (200 mL), magnesium chloride solution (2.0 mL), and calcium chloride solution (0.1 mL) were placed in a sterile 3 L Sakaguchi flask to prepare the wheat flour culture medium.

[0016] (4) A portion of the Pantoea agglomerans colonies isolated from wheat flour was added to 10 mL of the previously prepared (3), and incubated overnight in a 37°C constant temperature bath. (5) The entire amount of (4) was added to (3), and fermentation was carried out at 37°C with stirring for 20 to 30 hours. The pH of the fermentation liquid was measured, and ammonia water was added to adjust the pH to 7. (6) The wheat flour fermentation solution from (5) was centrifuged (Hitachi, high-speed refrigerated centrifuge SCR-20B, 5000 rpm, 20 minutes, 4°C), and the precipitate was collected.

[0017] (7) The precipitate from (6) was suspended in phosphate buffer, and the total volume was made 100 mL. 33 mL portions were transferred to 50 mL centrifuge tubes and heated in a boiling water bath for 30 minutes for extraction. After heating, the solution was cooled to room temperature and centrifuged (10,000 rpm, 20 minutes, 20°C). After centrifugation, 82 mL of the pale yellow supernatant was collected in a separate container by decanting.

[0018] (8) 80 mL of the supernatant from (7) was added to 8.9 mL of 5 M sodium chloride solution. When 178 mL of ethanol was added to this, turbidity was produced. This was left overnight in a freezer (-90°C), and then the solution was centrifuged (10,000 rpm, 20 minutes, 4°C). The supernatant was removed to obtain a precipitate. 10 mL of chilled 70% ethanol was added to this precipitate, and after suspension, the solution was centrifuged (10,000 rpm, 20 minutes, 20°C) to wash the precipitate. The precipitate was air-dried and dissolved in distilled water to obtain 11 mL of wheat fermentation extract.

[0019] [Confirmation of the inhibitory effect on polymyxin B (PXB)] Limulus-positive glycolipids have been identified as the component that activates macrophages in trace amounts within this wheat fermentation extract. This is LPS (LPSp), which is a component of the outer membrane of Pantoea agglomerans, a Gram-negative bacterium used in the fermentation culture, and shows a positive Limulus reaction. Highly purified LPS (purified LPSp) can be purchased from Funakoshi Co., Ltd.

[0020] First, we decided to confirm that adding polymyxin B, an LPS inhibitor, does not completely inhibit the biological activity of wheat fermentation extract. Polymyxin B is an antibiotic against Gram-negative bacteria and has a cyclic peptide structure consisting of polycationic peptides. By binding to the Lipid-A portion responsible for the biological activity of LPS, polymyxin B prevents LPS from binding to the LPS receptor on macrophages, thereby neutralizing the biological activity of LPS.

[0021] It is known that activating macrophages induces nitric oxide (NO), a physiologically active substance. NO is quickly converted to nitrite and nitrate. Since nitrite can be easily measured with Gries' reagent, NO induction evaluation is widely used as a simple method for measuring macrophage activation and for evaluating macrophage activators. Therefore, the inhibitory effect of polymyxin B on the biological activity of wheat fermentation extract was investigated using NO induction ability as the evaluation system, following the procedure below. In this evaluation, since culture medium was added to each sample, a control (culture medium only) was set up for comparison.

[0022] (1) RAW264.7 cells, a macrophage-based cultured cell line, were purchased from ATCC and subcultured in a carbon dioxide incubator (37°C, 5% CO2) using RPMI1640 supplemented with 10% fetal bovine serum (FBS) as the culture medium. (2) The number of cells from (1) was counted, diluted with the culture medium, and plated at 800,000 cells / 180 μL / well in a 96-well plate.

[0023] (3) 20 μL each of the samples diluted in culture medium (control, purified LPSp, wheat fermentation extract (10 ng / mL, 100 ng / mL)) was added to (2). Purified LPSp contains 100 ng / mL of LPSp. Wheat fermentation extract 10 ng / mL contains 1 μg / mL of wheat fermentation extract containing 1% LPSp, i.e., contains 10 ng / mL of LPSp. Wheat fermentation extract 100 ng / mL contains 10 μg / mL of wheat fermentation extract containing 1% LPSp, i.e., contains 100 ng / mL of LPSp. (4) The samples were incubated in a carbon dioxide incubator (37°C, 5% CO2) for 24 hours. (5) The culture supernatant was collected, Greiss reagent was added, and the nitrite (NO2) concentration was measured.

[0024] With 100 ng / mL of purified LPSp, the nitrite concentration, an indicator of nitric oxide (NO) induction ability from RAW264.7 macrophage culture cells, was 18.9 μmol / mL. However, when 10 μg / mL of polymyxin B was added, it became 0 μmol / mL, and the inhibition rate (1 - (LPSp + PXB) / LPS) × 100) was 100%. This confirmed that polymyxin B completely inhibits the biological activity of LPSp.

[0025] Similarly, 10 μg / mL of polymyxin B was added to 10 ng / mL and 100 ng / mL of wheat fermentation extract. In the unadded solution, the nitrite-inducing concentrations were 21.4 and 29.2 μmol / mL, respectively, while with the addition of polymyxin B, they were 7.3 and 26.1 μmol / mL, respectively, with inhibition rates of 65.9% and 10.6%, respectively.

[0026] These results show that purified LPS derived from Pantoea bacteria, at a concentration of 100 ng / mL, completely inhibited the NO-inducing ability of macrophages in the presence of polymyxin B. On the other hand, wheat fermentation extract showed inhibition rates of 65.9% at 10 ng / mL and 10.6% at 100 ng / mL, confirming that the addition of polymyxin B only partially inhibited the biological activity of wheat fermentation extract. (Table 1)

[0027]

[0028] [Preparation of LPS-Removed Wheat Fermentation Extract] To investigate the biological activity of this wheat fermentation extract after LPS removal, we attempted to produce an LPS-removed wheat fermentation extract using beads immobilized with polymyxin B (Affi-Prep Polymixin (Bio-Rad Laboratories, Inc.)). Polymyxin B is an antibiotic against Gram-negative bacteria, has a cyclic peptide structure composed of polycationic peptides, and has a high binding affinity to Lipid-A of LPS. Therefore, LPS can be removed by adding Affi-Prep Polymixin to a solution containing LPS and then separating the Affi-Prep Polymixin from the solution using a centrifuge or the like. Accordingly, we prepared an LPS-removed wheat fermentation extract using the following procedure.

[0029] (1) Wheat fermentation extract (LPS 12.5 mg / mL) was diluted 10,000 times with phosphate-buffered saline (PBS) to prepare diluted wheat fermentation extract (LPS 1.25 μg / mL). (2) The diluted wheat fermentation extract and the undiluted wheat fermentation extract (undiluted) were each dispensed into 2 mL Eppendorf tubes, and 100 mg of Affi-Prep Polymixin was added to each.

[0030] (3) Mixed in a rotator overnight at room temperature. (4) Centrifuged (3) at 3,000 rpm for 10 minutes. (5) 800 μL of the supernatant from (4) was collected and used as the LPS-removed diluted wheat fermentation extract and the LPS-removed stock wheat fermentation extract, respectively.

[0031] (6) The LPS content of the diluted wheat fermentation extract was 1.25 μg / mL, and the LPS content of the LPS-removed diluted wheat fermentation extract was 0.000246 μg / mL, so the LPS removal rate was 99.980%. The LPS content of the undiluted wheat fermentation extract was 12.5 mg / mL, and the LPS content of the LPS-removed undiluted wheat fermentation extract was 5.97 μg / mL, so the LPS removal rate was 99.952%.

[0032] [Measuring the Bioactivity of LPS-Removed Wheat Fermentation Extract] [HEK293 Cells with TLR-4, TLR-2, and TLR-9 Genes] To evaluate the bioactivity of LPS-removed wheat fermentation extract and investigate its mechanism, IL-8 expression was measured in HEK293 cells (TLR-4-introduced HEK293 cells, TLR-2-introduced HEK293 cells, and TLR-9-introduced HEK293 cells) into which the TLR-4, TLR-2, and TLR-9 genes were introduced and each gene expressed.

[0033] TLR-4 (Toll-like receptor 4) is the primary receptor in the innate immune system that recognizes LPS. LPS is a component of the outer membrane of Gram-negative bacteria and plays a role in maintaining the structural stability of bacteria, while also being known as a very potent immunostimulant. When LPS binds to TLR-4, it transmits signals to immune cells, activating the innate immune response by promoting cytokine production and antibacterial action. This allows the body to react rapidly to the invasion of pathogens and defend against infection. The responsiveness to LPS can be evaluated by examining the biological activity of TLR-4-introduced HEK293 cells.

[0034] TLR-2 (Toll-like receptor 2) is known as a receptor for peptidoglycan. Peptidoglycan is one of the main components of bacterial cell walls, and is particularly abundant in the cell walls of Gram-positive bacteria, but is also present in Gram-negative bacteria. Peptidoglycan has a structure in which sugar chains (N-acetylglucosamine and N-acetylmuramic acid) are alternately linked to form long chains, and these chains are cross-linked by peptides (short amino acid chains). Peptidoglycan is known as one of the important recognition molecules of the innate immune system. When peptidoglycan binds to TLR-2, it transmits signals to immune cells, activating the innate immune response by promoting cytokine production and antibacterial action. This allows the body to react rapidly to the invasion of pathogens and defend against infection. The responsiveness to peptidoglycan can be evaluated by examining the biological activity of TLR-2-introduced HEK293 cells.

[0035] TLR-9 (Toll-like receptor 9) is an important immune receptor that primarily recognizes bacterial DNA and plays a role in activating innate immunity. TLR-9 is known to recognize unmethylated CpG motifs (sequences characteristically abundant in bacterial and viral DNA). While most nucleic acids containing CG sequences in eukaryotes are methylated, bacterial CG sequences are not. By examining the biological activity of TLR-9-introduced HEK293 cells, the responsiveness to bacterial nucleic acids can be evaluated.

[0036] [Measurement Method] IL-8 expression was measured in TLR-4, 2, and 9-introduced HEK293 cells as follows: (1) Each cell type (TLR2-introduced HEK293, TLR4-introduced HEK293, TLR9-introduced HEK293) was purchased from InvivoGen and subcultured in a carbon dioxide incubator (37°C, 5% CO2) using DMEM supplemented with 10% fetal bovine serum (FBS) as the culture medium.

[0037] (2) The number of cells was measured and diluted in culture medium, and 50,000 cells / 200 μL / well were plated in a 96-well plate. (3) The cells were incubated in a carbon dioxide incubator (37°C, 5% CO2) for 24 hours.

[0038] (4) When the medium was removed with a pipette, HEK cells remained adsorbed to the plate because they are adherent cells. To each cell remaining on the plate, 180 μL of medium and 20 μL of each sample diluted with water (negative control (water), purified LPSp, diluted wheat fermentation extract, LPS-removed diluted wheat fermentation extract) were added. (5) The cells were cultured in a carbon dioxide incubator (37 °C, 5% CO2) for 24 hours.

[0039] (6) Then, the culture supernatant was collected and stored at -20 °C. (7) The measurement of IL-8 was performed using ELISA MAXTM Deluxe Set Human IL-8 (BioLegend) according to the procedure described in the kit manual.

[0040] [Measurement Results] [TLR-4] The measurement results of TLR-4-introduced HEK293 cells are shown in Table 2.

[0041]

[0042] Reactivity was observed for 100 ng / mL of purified LPSp against TLR-4-introduced HEK293 cells. Reactivity was also observed for the diluted wheat fermentation extract. No reactivity was found for the LPS-removed diluted wheat fermentation extract. It was shown that LPS is responsible for the reactivity to TLR-4 in the wheat fermentation extract.

[0043] [TLR-2] The measurement results of TLR-2-introduced HEK293 cells are shown in Table 3.

[0044]

[0045] LPSp had no reactivity against TLR-2-introduced HEK293 cells and was equivalent to the negative control. On the other hand, surprisingly, reactivity was observed for the diluted wheat fermentation extract against TLR-2-introduced HEK293 cells, but no reactivity was found for the LPS-removed diluted wheat fermentation extract. It was suggested that LPS in the wheat fermentation extract is involved in the signal transduction to TLR-2. [TLR-9] The measurement results of TLR-9-introduced HEK293 cells are shown in Table 4 and Figure 1.

[0046]

[0047] While LPSp did not activate TLR-9-introduced HEK293 cells, slight activation was observed in the wheat fermentation extract. Even more surprisingly, the LPS-removed wheat fermentation extract showed 3.56 times higher biological activity than the original wheat fermentation extract.

[0048] LPS was shown to be a factor that controls the effects of peptidoglycan and nucleic acids within wheat fermentation extract. On the other hand, LPS-removed wheat fermentation extract was found to be a material with a strong effect on TLR-9.

[0049] TLR-9 is an important immune receptor that recognizes unmethylated CpG motifs, which are mainly found in bacterial and viral DNA, and its activation promotes innate immunity. Specifically, stimulation of TLR-9 induces the production of interferon (IFN) and antiviral cytokines, thereby strengthening early defense against viral infection. In addition, TLR-9 activates immune cells such as macrophages and neutrophils, enhancing their phagocytic and bactericidal capabilities against bacteria, thereby exerting an antibacterial effect. Furthermore, it also promotes the production of antimicrobial peptides, improving the body's defense against bacterial infection.

[0050] Activation of TLR-9 also induces a local inflammatory response through the production of inflammatory cytokines (e.g., TNF-α and IL-6). While this inflammatory response is important for eliminating pathogens at the site of infection, regulatory mechanisms are also in place to prevent excessive inflammation. In addition, some studies have shown that TLR-9 stimulation enhances tumor immunity and promotes the recognition and elimination of tumor cells, suggesting potential antitumor effects through innate immunity.

[0051] Furthermore, CpG oligonucleotides, which are TLR-9 ligands, are also used as vaccine adjuvants (agents that enhance the immune response), playing a role in strongly inducing acquired immunity after vaccination. Considering these diverse immune effects, LPS-removed wheat fermentation extract is considered a material that can be expected to be useful as an antiviral, antibacterial, antitumor, anti-inflammatory agent, and vaccine adjuvant via TLR-9.

[0052] [TNF-α Inducing Ability of TLR-4 Deficient Macrophages] Previous research using HEK293 cells into which TLR-2, 4, and 9 were introduced revealed that LPS-removed wheat fermentation extract possesses an unexpected new function: 3.56 times higher activation ability for TLR-9 compared to wheat fermentation extract (Table 4). However, specific information regarding the usefulness of LPS-removed wheat fermentation extract other than for TLR-9 has not yet been obtained.

[0053] Normal macrophages contain multiple TLRs (TLR-1, 2, 3, 4, 5, 6, 7, 8, 9, 10), as well as NLRs, RLRs, and CTRs that react with bacterial components (Non-Patent Literature 1). Components of wheat fermentation extract other than LPS express biological functions through various receptors. Therefore, to investigate the biological differences between LPS-removed wheat fermentation extract and wheat fermentation extract, we decided to compare wheat fermentation extract and LPS-removed wheat fermentation extract using thioglycolate-induced peritoneal macrophages (TLR-4 deficient macrophages) from C3H / HeJ mice. TNF-α, which exhibits biological diversity, was selected as the macrophage response to LPS.

[0054] [Measurement Method] TNF-α production by TLR-4-deficient macrophages using an LPS-removing agent was investigated. Five C3H / HeJJcl mice (6 weeks old, female, CREA Japan Co., Ltd.) were each administered 2 mL of 4.05% thioglycolate medium (Thioglycolate Medium Brewer Modified REF211716, Becton, Dickinson and Company) intraperitoneally.

[0055] Three days later, 5 mL of PBS(-) was administered into the peritoneal cavity of mice, and the abdomen was massaged. The fluid containing peritoneal cells was then collected. Peritoneal cells collected from five mice were pooled and suspended in RPMI-1640 medium (RPMI-1640 #189-02025, Fujifilm Wako Pure Chemical Corporation) containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cell counts were measured by trypan blue staining.

[0056] 1.5 x 10 ５Cells were seeded at a rate of 100 μL / well in 96-well plates (#353072, Corning). They were cultured for 2 hours in a 5% CO2 incubator (MCO-20A10, SANYO) at 37°C, and the culture medium containing non-adherent cells was removed. Then, 100 μL of RPMI-1640 medium was added to each well to the remaining adherent cells (peritoneal macrophages).

[0057] Purified LPSp was adjusted to 20 ng / mL using culture medium. Wheat fermentation extract was diluted with culture medium to achieve an LPSp concentration of 20 ng / mL (50,000-fold dilution). LPS-removed wheat fermentation extract was diluted 50,000-fold using culture medium to achieve the same dilution ratio as the wheat fermentation extract. RPMI-1640 medium, which does not contain LPS, was used as a control. 100 μL of each was added to peritoneal macrophages and incubated for 24 hours in a 5% CO2 incubator at 37°C. The supernatant was collected and used as the sample for measurement. The LPSp concentration of both LPSp and wheat fermentation extract was 10 ng / mL. TNF-α contained in each sample was measured by ELISA (ELISA MAX™ Deluxe Set Human TNF-α Cat#430204, Biolegend) (n=3).

[0058] [Measurement Results] The results are shown in Table 5. It was confirmed that LPSp (10 ng / mL) did not activate TLR-4 deficient macrophages compared to the control. Wheat fermentation extract containing the same LPSp produced 142 pg / mL of TNF-α, while the LPS-removed wheat fermentation extract produced a similar amount at 146 pg / mL. From this, it became clear that both wheat fermentation extract and LPS-removed wheat fermentation extract have macrophage activation capabilities that do not involve TLR-4, i.e., other components that do not involve LPS. Furthermore, no difference in TNF-α induction ability was observed between wheat fermentation extract and LPS-removed wheat fermentation extract.

[0059] TNF-α production for each sample is shown as Mean ± SD.

[0060] [Genetic Induction Ability in TLR-4 Deficient Macrophages] In order to identify the useful biological characteristics of LPS-removed wheat fermentation extract compared to wheat fermentation extract, we decided to comprehensively analyze the induced genes using DNA microarrays and examine the differences in the genes expressed by the two. DNA microarrays are a technique for simultaneously measuring the expression levels (amount of mRNA) of many genes within a cell.

[0061] [Measurement Method] Six C3H / HeJJcl mice (10 weeks old, female, CREA Japan Co., Ltd.) were each administered 2 mL of 4.05% thioglycolate medium intraperitoneally.

[0062] Three days later, 5 mL of PBS(-) (#166-23555, Fujifilm Wako Pure Chemical Corporation) was administered into the peritoneal cavity of mice, and the abdomen was massaged. The fluid containing peritoneal cells was then collected. Peritoneal cells collected from six mice were pooled and suspended in RPMI-1640 medium containing 10% FBS, penicillin 100 U / mL, and streptomycin 100 μg / mL. The cell count was measured by trypan blue staining.

[0063] 1.78 × 10 ６ Cells were seeded in 12 wells at a rate of 1187 μL / well. They were cultured for 2 hours in a 5% CO2 incubator at 37°C, and the culture medium containing non-adherent cells was removed. Then, 1.2 mL of RPMI-1640 medium was added to the remaining adherent cells (peritoneal macrophages).

[0064] The test involved a control (culture medium) group and a wheat fermentation extract group (10 ６ Dilution), LPS-removed wheat fermentation extract group (10 ６ The study was conducted in four groups: a diluted group and an LPSp group (12.5 ng / mL) (n=3 in each group).

[0065] Each group of peritoneal macrophages was given 1.2 mL of culture medium at twice the final concentration. The cells were incubated for 4 hours in a 5% CO2 incubator at 37°C. After incubation, the supernatant was collected and used as the sample for measurement. The sample was stored at -80°C. FastGene was used for RNA extraction. TMRNA Premium kit (FG-81050, Nippon Genetics Co., Ltd.) was used. RNA extraction was performed according to the kit's standard protocol. Specifically, 350 μL of lysis buffer (RL) containing 143 mM 2-mercaptoethanol was added to the cells after removing the supernatant, and the resulting cell suspension (lysate) was added to the FastGeneRNA filter column. The lysate was clarified, and the filtrate was collected. 350 μL of 70% ethanol was added to the filtrate, mixed by pipetting, and the entire volume was added to the FastGeneRNA binding column. After washing the membrane, 50 μL of buffer RE was added, and the eluate was collected by centrifugation (10,000 G, 30 seconds). 5 μL of 10× DNase I reaction buffer was added to the eluate. 1 μL of DNase I was mixed by pipetting and incubated at 25°C for 10 minutes. 250 μL of buffer RBD was mixed by pipetting and added entirely to the FastGeneRNA mini-elute column. After washing the membrane, 30 μL of buffer RE was added, and the eluate was collected by centrifugation (10,000 G, 60 seconds). The eluate was used as the purified RNA solution. RNA concentration, A260 / A280, and A260 / A230 were measured using nanoVue (GE electronics) with 5 μL of the purified RNA solution. The purified RNA solution was stored at -80°C.

[0066] Mouse DNA microarray analysis (Clariom™ S Assay, mouse, Applied Biosystems™) was outsourced to Philgen Co., Ltd. Total RNA samples of 1 μg or more were submitted. Overall gene expression patterns, pathways, and network interactions were analyzed using ScriptomeAnalysis Console (TAC) software.

[0067] [Measurement Results] The differences in the response (induced or suppressed mRNA gene groups) between TLR-4-deficient macrophages and LPS-removed wheat fermentation extract were comprehensively investigated using TAC, and the results are shown in Table 6. When comparing the genes of peritoneal macrophages stimulated with wheat fermentation extract to unstimulated peritoneal macrophages (control), 1074 genes showed a doubling or greater increase in gene induction, while 777 genes showed a decrease to half or less (Table 6, Comparison (1)). When comparing the genes of peritoneal macrophages stimulated with LPS-removed wheat fermentation extract to unstimulated peritoneal macrophages, 925 genes showed a doubling or greater increase in gene induction, while 752 genes showed a decrease to half or less (Table 6, Comparison (2)). A comparison of the genes of peritoneal macrophages stimulated with wheat fermentation extract and peritoneal macrophages stimulated with LPS-removed wheat fermentation extract revealed that 163 genes showed a more than twofold increase in gene induction, while 149 genes decreased to less than half (Table 6, Comparison (3)). This clearly indicates that LPS-removed wheat fermentation extract has a different macrophage activation ability than wheat fermentation extract.

[0068]

[0069] Therefore, we analyzed the expressed genes that are useful for disease prevention and treatment based on comparative data of wheat fermentation extract and LPS-removed wheat fermentation extract, and the results are shown in Table 7. It was found that the expression of the Kctd12b (potassium channel tetramerization domain containing 12) gene was 3.0 times higher in LPS-removed wheat fermentation extract compared with wheat fermentation extract. Kctd12b has a positive correlation with markers such as B cells, CD8+ T cells, dendritic cells (DCs), and macrophages (TAMs), and it has been reported that high expression in cancer tissue is associated with a higher prognosis and a higher immune response (Non-Patent Literature 2). From this, it was shown that LPS-removed wheat fermentation extract is more useful as a cancer immunotherapy agent than wheat fermentation extract.

[0070] It was found that LPS-removed wheat fermentation extract induces 3.0 times more Adcyap1r1 (PAC1 receptor, Adenylate Cyclase Activating Polypeptide 1 Receptor 1) compared to wheat fermentation extract. Adcyap1r1 is an anti-inflammatory factor in macrophages, acting on the PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide) receptor and having a wide range of physiological effects, including neuroprotection, anti-inflammatory effects, and vasodilatory effects (Non-Patent Literature 3). Furthermore, when Adcyap1r1 expression is increased in macrophages, it promotes differentiation into anti-inflammatory macrophages (M2 type), suppresses neuroinflammation, cardiovascular inflammation, and metabolic inflammation, and shows suppression of excessive inflammation in innate immunity (Non-Patent Literature 4, Non-Patent Literature 5). Furthermore, these findings indicate that LPS-removed wheat fermentation extract is more useful than wheat fermentation extract for the prevention and treatment of chronic inflammatory diseases such as neuroinflammatory diseases (Alzheimer's disease, Parkinson's disease, multiple sclerosis), cardiovascular diseases (arteriosclerosis, heart failure), inflammatory bowel disease, rheumatoid arthritis, and post-stroke complications (inflammation suppression and neuroprotection).

[0071] It was found that LPS-removed wheat fermentation extract induces 2.7 times more Lpar5 (LPA5) than wheat fermentation extract. Lpar5 is a type of lysophosphatidic acid (LPA) receptor that is expressed in macrophages and microglia and has an anti-inflammatory effect that strongly suppresses the production of inflammatory cytokines (Non-patent Literature 6). These findings indicate that LPS-removed wheat fermentation extract is more useful than wheat fermentation extract for the prevention and treatment of inflammatory diseases.

[0072] Based on these findings, LPS-removed plant fermentation extract can be considered a novel immune-activating material that differs materially from conventional plant fermentation extracts and possesses new biological activity.

[0073] *: Gene expression increase ratio by LPS-removed wheat fermented extract, based on wheat fermented extract.

[0074] The disclosure of Japanese Patent Application No. 2024-169648, filed on 27 September 2024, including the specification, claims and drawings, is incorporated herein by reference. All publications, patents and patent applications referenced herein are incorporated herein by reference.

Claims

1. A method for obtaining an LPS-removed plant fermentation extract, characterized by comprising the step of fermenting a material derived from edible plants exclusively with facultative anaerobic gram-negative bacteria that coexist symbiotically with plants, and simultaneously culturing the facultative anaerobic gram-negative bacteria to obtain a plant fermentation extract from which LPS is removed.

2. An LPS-removing plant fermentation extract characterized by being obtained by the method described in claim 1.

3. A formulation characterized by containing the LPS-removing plant fermentation extract described in claim 2.

4. The formulation according to claim 3, characterized in that the formulation is a pharmaceutical product, a veterinary drug, a quasi-drug, a cosmetic product, a food product, a functional food product, a feed product, a fertilizer product, or a bath additive.

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