Composition for enhancing immunity, containing, fermented product of cucurbitaceae plant bacillus sp. strain as active ingredient

Abstract

The present invention relates to a composition for enhancing immunity, containing a fermented product of a Cucurbitaceae plant Bacillus sp. strain as an active ingredient. This method for preparing a composition for enhancing immunity comprises the steps of: (a) obtaining a substrate by mixing a crushed liquid of the Cucurbitaceae plant and water; (b) putting the substrate into a fermentation tank and sterilizing same; (c) inoculating a Bacillus sp strain into the fermentation tank; (d) performing fermentation at 25 to 35℃ for 12-96 hours to obtain a fermented product; and (e) freeze-drying the fermented product.

Description

A composition for enhancing immunity containing a fermented product of a Bacillus genus strain of a Cucurbitaceae plant as an active ingredient

[0001] The present invention relates to an immune-enhancing composition comprising a fermented strain of a Cucurbitaceae plant as an active ingredient, and can be used for various purposes such as medicine and food.

[0002] Immunity is a self-defense system that exists in the body and is the process of recognizing various substances or organisms that invade from the outside as foreign substances and eliminating and metabolizing them.

[0003] It protects itself from damage caused by external stimuli or invasion by pathogenic microorganisms, but it can also cause damage to its own tissues, such as through inflammatory reactions.

[0004] It can be divided into an immune function enhancement action that regulates changes in immune function and restores it to normal, and an immune function suppression action that reduces the range of changes in immune function.

[0005] Alleviation of hypersensitivity immune responses refers to suppressing undesirable increased immune responses, such as allergic reactions caused by adverse reactions to foreign substances, reactions to self-antigens, or reactions to altered self-antigens.

[0006] Recently, there have been reports that a decline in the body's immune system function can lower resistance to viral respiratory diseases such as the common cold, as well as shingles and thyroid disease. Therefore, various studies are being conducted to enhance the body's immune response through food intake, and in particular, research on health functional foods using natural materials is also actively underway.

[0007] Meanwhile, the immune response is one of the important biological defense mechanisms that eliminates and repairs the disease environment induced by the invasion of external infectious substances other than self-components, and is divided into innate immunity response and adaptive immunity response.

[0008] Macrophages, which are the front line of the innate immune system's defense mechanism, are distributed throughout all tissues in the body and have the ability to primarily remove infectious pathogens such as bacteria and viruses, as well as aged normal cells and cancer cells, through phagocytosis.

[0009] Activated macrophages act as important mediators that maximize the immune response by enhancing phagocytic capacity, nitric oxide (NO), interleukin family, and various cytokines such as TNF-α, as well as morphological changes in cells such as enhanced proliferation and proliferation ability.

[0010] Immunity is a self-defense system that exists in the body. It is a process in which the body recognizes various substances or living organisms that invade from the outside as foreign substances and eliminates and metabolizes them. It protects itself from damage caused by external stimuli or invasion by pathogenic microorganisms, but it can also cause damage to its own tissues, such as through inflammatory reactions.

[0011] It is categorized as immune function suppression or immune function enhancement based on the action of regulating changes in immune function to restore it to normal or reduce the extent of changes.

[0012] Health functional foods with immune function regulation functions are currently divided into three categories: functional foods that enhance reduced immune function, functional foods that regulate excessive immune function, and functional foods that regulate autoimmune function that regulates immune response to self-ingredients.

[0013] When immune function is weakened, hypersensitivity reactions may occur, and these hypersensitivity reactions can manifest in various ways, such as asthma, seasonal or annual rhinitis, allergic sinusitis, conjunctivitis, food and drug allergies, atopic dermatitis, hives, and bee sting allergies.

[0014] To overcome immune dysfunction caused by various reasons, various immune boosters are being used; however, due to issues with side effects and biosafety, there is a need for an immune boosting composition that can be taken for a long period of time and is also suitable for preventive purposes.

[0015] Meanwhile, Cucurbitaceae plants are mainly distributed in tropical and subtropical regions and are annual vine plants. In Korea, there are about 70 species that are wild or cultivated, including melons, watermelons, pumpkins, cucumbers, gourds, and water parsley.

[0016] Most cucurbits are rich in dietary fiber and are mostly consumed raw.

[0017] In addition, young leaves, stems, and unripe fruits are consumed as food, but the consumption of parts other than the fruit is minimal. A huge amount of agricultural by-products such as parts other than the edible part of the fruit (pericarp, seeds, etc.), unsellable defective fruits, and harvested stems, leaves, and roots are being collected, which is becoming a new cause of environmental pollution, and the development of new processing methods is required.

[0018] Accordingly, the inventors of the present invention have confirmed that the immune-enhancing activity can be maximized when a plant material of the Cucurbitaceae family is fermented under certain conditions, and have made efforts to devise a method for industrially utilizing the same, and as a result, have completed the present invention.

[0019] The present invention is intended to solve the problems of the prior art described above, and the purpose of the present invention is to provide a functional food and pharmaceutical product containing a natural material having excellent biosafety.

[0020] According to one aspect of the present invention, an immune-enhancing composition is provided, which comprises a fermented product of a bacillus sp. strain of a Cucurbitaceae plant as an active ingredient.

[0021] In one embodiment, the cucurbit plant may be one or more selected from the group consisting of pumpkin, cucumber, melon, watermelon, gourd, and melon.

[0022] In one embodiment, the Bacillus genus strain may be Bacillus subtilis.

[0023] In one embodiment, the fermented product can be obtained by adding a Bacillus genus strain to a crushed liquid of the cucurbit plant, fermenting at 25 to 35°C for 12 to 96 hours, and then freeze-drying.

[0024] In one embodiment, the fermentation product is 1×10 7 May contain Bacillus spp. strains exceeding CFU.

[0025] According to another aspect of the present invention, a health functional food for enhancing immunity is provided, which comprises a fermented product of a bacillus spp. strain of a Cucurbitaceae plant as an active ingredient.

[0026] In one embodiment, the health functional food for enhancing immunity can be manufactured in any one formulation selected from the group consisting of tablets, granules, powder, capsules, liquid solutions, and pills.

[0027] According to another aspect of the present invention, a method for producing an immune-enhancing composition is provided, comprising the steps of: (a) mixing a crushed liquid of a Cucurbitaceae plant and water to obtain a substrate; (b) introducing the substrate into a fermentation tank and sterilizing it; (c) inoculating the fermentation tank with the Bacillus spp. strain; (d) fermenting at 25 to 35°C for 12 to 96 hours to obtain a fermented product; and (e) freeze-drying the fermented product.

[0028] The composition according to one aspect of the present invention not only has excellent biosafety but also has excellent immune-enhancing activity, and thus can be usefully utilized not only as a health functional food but also for the treatment of immune-related diseases.

[0029] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the composition of the invention described in the claims.

[0030] Figure 1 shows the results of evaluating the effect of a fermented product according to one embodiment of the present invention on NO production.

[0031] Figure 2 shows the results of evaluating the effect of a fermented product according to one embodiment of the present invention on the production of cytokines.

[0032] Figure 3 shows the results of evaluating the effect of a fermented product according to one embodiment of the present invention on the expression of iNOS and COX2.

[0033] Figure 4 shows the results of evaluating the effect of a fermented product according to one embodiment of the present invention on phosphorylation of MAPK.

[0034] Figure 5 shows the results of evaluating the effect of a fermented product according to one embodiment of the present invention on the phosphorylation of Iκ and NFκ.

[0035] The terms used in this specification are selected from the most widely used general terms possible while taking into account the functions of the present invention, but these may vary depending on the intention of a technician working in the field, precedents, the emergence of new technologies, etc.

[0036] Additionally, in certain cases, the applicant may arbitrarily select terms, in which case their meanings will be described in detail in the description of the relevant invention. Therefore, the terms used in this invention should be defined based on their meaning and the overall content of the invention, rather than simply their names.

[0037] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0038] Numerical ranges are inclusive of the numbers defined in the ranges above. Every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if that lower numerical limitation were explicitly stated. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if that higher numerical limitation were explicitly stated. Every numerical limitation given throughout this specification will include every better numerical range within that broader numerical range, as if that narrower numerical limitation were explicitly stated.

[0039] Hereinafter, examples of the present invention will be described in detail, but it is obvious that the present invention is not limited to the following examples.

[0040] The terms used in this specification are selected from the most widely used general terms possible while taking into account the functions of the present invention, but these may vary depending on the intention of a technician working in the field, precedents, the emergence of new technologies, etc.

[0041] Additionally, in certain cases, the applicant may arbitrarily select terms, in which case their meanings will be described in detail in the description of the relevant invention. Therefore, the terms used in this invention should be defined based on their meaning and the overall content of the invention, rather than simply their names.

[0042] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0043] Numerical ranges are inclusive of the numbers defined in the ranges above. Every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if that lower numerical limitation were explicitly stated. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if that higher numerical limitation were explicitly stated. Every numerical limitation given throughout this specification will include every better numerical range within that broader numerical range, as if that narrower numerical limitation were explicitly stated.

[0044] Hereinafter, examples of the present invention will be described in detail, but it is obvious that the present invention is not limited to the following examples.

[0045] One aspect of the present invention provides an immune-enhancing composition comprising a fermented product of a bacillus spp. strain of a Cucurbitaceae plant as an active ingredient.

[0046] The above “immunity” refers to a mechanism that protects a living organism from disease by detecting and neutralizing pathogens and tumor cells within the organism, and the above “immunostimulation” may include a series of therapeutic or preventive strategies that reinforce the body’s defense mechanisms against various diseases such as cancer and inflammation.

[0047] The above “comprising an active ingredient” means including an amount sufficient to provide a therapeutic or preventive effect to the subject to be administered, and since the fermented product has excellent biosafety, those skilled in the art can appropriately adjust the upper limit of the quantity.

[0048] The above cucurbitaceae plants are known worldwide with about 860 species in 100 genera, and in Korea, mainly watermelons, melons, cucumbers, melons, and pumpkins are cultivated.

[0049] Many exotic species, such as watermelon, which is native to Africa, and cucumber, which is native to West Asia, are widely cultivated for food or ornamental purposes, and their fruit, gourd, is widely used for food and other purposes.

[0050] The above cucurbit plant may refer to the fruit of the cucurbit plant, and may include both the pericarp and seeds of the fruit.

[0051] The fruit of the above cucurbit plant is characterized by having a thick skin and containing many seeds, and is rich in moisture and dietary fiber and contains large amounts of carbohydrates, minerals, calcium, phosphorus, potassium, etc. In particular, the inventors of the present invention confirmed that the immune-related activity of a fermented product obtained by inoculating and fermenting the above cucurbit plant with a Bacillus genus strain can be significantly enhanced.

[0052] In particular, the fermented product can exhibit excellent immune-enhancing activity as is after being dried after fermentation, but the immune-enhancing activity may decrease when the fermented product undergoes an extraction process, etc., so the immune-enhancing activity can be maintained and preserved through freeze-drying of the fermented product.

[0053] In one embodiment, the cucurbit plant may be one or more selected from the group consisting of, but not limited to, pumpkin, cucumber, melon, watermelon, gourd, and melon, and although there may be some differences depending on the characteristics of the cucurbit plant, the immune-enhancing activity may be commonly implemented or maximized by fermentation of a Bacillus genus strain.

[0054] The above Bacillus genus strains are also called bacilli, and may be a general term for rod-shaped gram-positive bacteria belonging to the genus Bacillus.

[0055] The above Bacillus strain is an aerobic or facultative anaerobic bacterium widely distributed in nature, which may be involved in denitrification, iron reduction, manganese oxidation, etc., and may form spores by undergoing structural changes within the bacterium depending on environmental conditions.

[0056] For example, the Bacillus strain may include, but is not limited to, Bacillus subtilis, B. coagulans, B. amyloliquefaciens, etc., and may preferably be Bacillus subtilis.

[0057] The above Bacillus subtilis is a gram-positive rod-shaped bacterium that is mainly found in soybean paste and is safe to ingest. It is known to survive high temperatures and have the vitality to reach the large intestine.

[0058] When the above Bacillus subtilis is fermented using a cucurbit plant as a substrate under predetermined conditions and the fermented product is freeze-dried to obtain a high level of immune-enhancing activity, it can be realized and maintained.

[0059] The above fermented product can be obtained by adding a Bacillus genus strain to the crushed liquid of the above cucurbit plant, fermenting at 25 to 35°C for 12 to 96 hours, and then freeze-drying.

[0060] Specifically, the fermented product can be manufactured through the steps of (a) obtaining a substrate by mixing a crushed liquid of a Cucurbitaceae plant and water; (b) introducing the substrate into a fermentation tank and sterilizing it; (c) inoculating the Bacillus spp. strain into the fermentation tank; (d) fermenting at 25 to 35°C for 12 to 96 hours to obtain a fermented product; and (e) freeze-drying the fermented product.

[0061] In the above step (a), a substrate can be obtained by mixing a crushed liquid of a Cucurbitaceae plant and water, and the crushed liquid obtained by washing and crushing the Cucurbitaceae plant and water can be mixed in a weight ratio of 100:10 to 100.

[0062] In the step (b), the substrate can be introduced into a fermentation tank, sterilized, and heated at 100 to 130°C for 30 to 90 minutes to kill or remove vegetative cells and spores of mold, bacteria, viruses, and protozoa present in the lysate, thereby allowing the subsequent fermentation process to proceed in a sterile state.

[0063] In the above step (c), the Bacillus spp. strain can be introduced into the fermentation tank, and the fermentation process can be carried out by inoculating the Bacillus spp. strain at 3 to 15 wt% of the total weight of the substrate.

[0064] In the above step (d), a fermented product can be obtained by fermenting at 25 to 35°C for 12 to 96 hours, and within the above conditions, the growth of the Bacillus genus strain is promoted, and the effective ingredient of the cucurbit plant is efficiently changed, so that the intended immune-enhancing activity can be realized.

[0065] In the above step (e), the fermented product can be freeze-dried, and by freezing the fermented product sample obtained in the solution state in the above step (d) and leaving it under reduced pressure in its original state, moisture in the sample can be removed by sublimation, and the fermented product can be preserved at room temperature for a long period of time while maintaining its efficacy, and can be easily consumed due to its excellent re-dissolution property in water.

[0066] In one embodiment, the fermentation product is 1×10 7 May contain Bacillus spp. strains exceeding CFU.

[0067] The above-mentioned immune-enhancing composition may include a fermented cucurbit plant material together with a large amount of Bacillus spp. strain, and may exhibit excellent immune-enhancing activity by including a large amount of the Bacillus spp. strain.

[0068] The above immune enhancing composition is 1×10 7 When the number of Bacillus spp. strains exceeding CFU is included, the intended immune-enhancing activity can be exhibited, and if the number of Bacillus spp. strains is insufficient, the immune-enhancing activity may be reduced, so it is desirable to appropriately maintain the number of Bacillus spp. strains within a predetermined range.

[0069] Another aspect of the present invention provides a health functional food for enhancing immunity, which comprises a fermented product of a bacillus sp. strain of a Cucurbitaceae plant as an active ingredient.

[0070] The above composition for enhancing immunity can be used as a food composition, for example, a health functional food for enhancing immunity.

[0071] The above health functional food refers to a food manufactured and processed using raw materials or ingredients with functionality useful to the human body according to the Health Functional Food Act, and the functionality may mean consuming it for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological action.

[0072] The above composition may contain conventional food additives, and the suitability of the food additives may be determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.

[0073] The items listed in the above food additive code include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular-weight pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, and tar color preparations.

[0074] The above composition can be used in various ways in foods and beverages for enhancing immunity, and can be used in, for example, various foods, beverages, gum, tea, vitamin complexes, health functional foods, food additives, etc.

[0075] In addition, the above health functional food can be manufactured and processed into any one formulation selected from the group consisting of tablets, granules, powder, capsules, liquid solutions, and pills for the purpose of enhancing immunity.

[0076] Specifically, the above-mentioned health functional food in tablet form can be manufactured by granulating a mixture of the fermented product, excipients, binders, disintegrants, and other additives using a conventional method, then adding a lubricant, etc., and compressing and molding the mixture, or by directly compressing and molding the mixture. In addition, the above-mentioned health functional food in tablet form can contain a bittering agent, etc., if necessary, and can also be coated with an appropriate coating agent, if necessary.

[0077] Among the health functional foods in capsule form, hard capsules can be manufactured by filling a mixture of the fermented product and additives such as excipients, or a granular or coated granular material thereof, into a regular hard capsule, and soft capsules can be manufactured by filling a mixture of the fermented product and additives such as excipients into a capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as necessary.

[0078] The above-mentioned ring-shaped health functional food can be prepared by molding a mixture of the above-mentioned fermented product, excipient, binder, disintegrant, etc., in an appropriate manner, and, if necessary, can be coated with white sugar or another appropriate coating agent, or can be coated with starch, talc, or an appropriate substance.

[0079] The above granular health functional food can be manufactured into a granular form by a suitable method using a mixture of the above fermented product, excipient, binder, disintegrant, etc., and can contain a flavoring agent, a flavoring agent, etc., as needed.

[0080] In addition, the definitions of terms for the above excipients, binders, disintegrants, lubricants, maturing agents, flavoring agents, etc. are those described in documents known in the art and may include those having the same or similar functions.

[0081] According to another aspect of the present invention, a pharmaceutical composition for preventing or treating an immune disease is provided, which comprises a fermented product of a Bacillus genus strain of the Cucurbitaceae plant as an active ingredient.

[0082] The above composition for enhancing immunity can be usefully used for preventing, improving or treating diseases caused by immunodeficiency, immunosuppression or immune system damage by enhancing immune activity.

[0083] The above immune disease may be one or more selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, septic shock, allergic asthma, allergic rhinitis, atopic dermatitis, ulcerative colitis, dacryoadenitis, Alzheimer's disease, stroke, arteriosclerosis, vascular restenosis, type I diabetes, type II diabetes, urticaria, conjunctivitis, psoriasis, systemic inflammatory patch syndrome, polymyositis, dermatomyositis, nodular polyarthritis, mixed connective tissue disease, Sjogren's syndrome, gout, Parkinson's disease, amyotrophic lateral sclerosis, diabetic retinopathy, multiple sclerosis, Crohn's disease, chronic thyroiditis, celiac disease, myasthenia gravis, pemphigus vulgaris, viral disease, bacterial disease, radiation-induced damage, arteriosclerosis, hemangioma, angiofibroma, reperfusion injury, and cardiac hypertrophy, but is not limited thereto.

[0084] The term "prevention" above refers to any action that reduces the frequency or severity of a pathological phenomenon. Prevention may be complete or partial. In this case, it may refer to a reduction in symptoms caused by a weakened immune system within the subject compared to when the composition is not used.

[0085] The above “treatment” means any clinical intervention to alter the natural process of the subject or cell to be treated, and may be performed during the progression of a clinical pathological condition or to prevent it.

[0086] The desired therapeutic effect may include preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, alleviating or temporarily alleviating the disease state, or improving the prognosis.

[0087] The composition may be administered orally or parenterally. The composition may be administered systemically or topically, and administration may include oral or parenteral administration. The composition may be formulated with a suitable amount of a pharmaceutically acceptable vehicle or carrier to provide an appropriate dosage form.

[0088] The composition may further comprise carriers, excipients and diluents used in the manufacture of pharmaceutical compositions.

[0089] The carriers, excipients and diluents may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

[0090] In addition, the composition can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions.

[0091] Solid preparations for oral administration may be tablets, pills, powders, granules, capsules, etc., and the solid preparations may be prepared by mixing the fermented product and its fractions with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to the excipients, lubricants such as magnesium stearate and talc may be used.

[0092] Liquid preparations for oral administration may include suspensions, solutions, emulsions, syrups, etc., and in addition to simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be used.

[0093] Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Examples of non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0094] The pharmaceutical composition may be administered to a subject in a pharmaceutically effective amount. The term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route of administration, and the excretion rate, the duration of treatment, concomitant medications, and other factors well known in the medical field.

[0095] The above pharmaceutical composition may be administered as an individual treatment or in combination with other treatments, sequentially or simultaneously with conventional treatments, or in single or multiple doses. Taking all of the above factors into account, it is desirable to administer an amount that achieves maximum effect with the minimum amount possible without causing side effects, a determination readily available to those skilled in the art.

[0096] The present invention is further described through the following examples, but it is obvious that the present invention is not limited to the following examples.

[0097] Manufacturing example: Manufacturing of fermented product of Bacillus spp. strain

[0098] Cleanly washed dong-a, pumpkin, and cucumber fruits were crushed to obtain a crushing solution.

[0099] Considering the properties of the crushed liquid, water was mixed in a predetermined ratio to obtain a substrate for fermentation.

[0100] After placing the substrate in the fermenter, it was sterilized by heating at 121℃ for 60 minutes.

[0101] After inoculating Bacillus subtilis in a sterilized substrate at 5 wt% of the total substrate weight, the fermentation process was carried out for 84 hours while maintaining a temperature of 31 to 33°C.

[0102] After the fermentation process was completed, the obtained fermented product was depressurized at -20°C to remove moisture and a freeze-dried product was obtained.

[0103] The fermented products of the Cucurbitaceae plants were named C-BH-DBS (dong-a), C-CM-DBS (pumpkin), and C-CS-DBS (cucumber), respectively.

[0104] Experimental Example 1: Cell Culture and Sample Preparation

[0105] Murine macrophage cell line RAW 264.7 was obtained from the Korea Cell Line Bank.

[0106] Dulbecco's Modified Eagles Medium (Hyclone Laboratories, Logan, UT, USA) was used with 10% fetal bovine serum (Gibco RBL Co., Grand Island, NY, USA) and 100 units / mL penicillin-streptomycin (Hyclone Laboratories, Logan, UT, USA) added, and cultured at 37°C, 5% CO2 incubator (311GP, Thermo Fisher Scientific, Waltham, MA, USA).

[0107] Experimental Example 2: Toxicity Evaluation (MTT Assay)

[0108] 5×10 RAW 264.7 cells 3The cells / 100 μL were ground into a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 24 hours, and then the samples were treated for 48 hours.

[0109] An MTT assay kit (Dozen, Seoul, Korea) was used, and the absorbance was measured at 450 nm using a microplate reader (Epoch, Biotek Instruments, Inc., Winooski, VT, USA).

[0110] Cell viability was measured by measuring the concentration of formazan produced by the conversion of tetrazolium salt (WST-1) to formazan by dehydrogenase in mitochondria of living cells using a spectrophotometer.

[0111] The MTT assay was performed according to the protocol using Ez-cytox (Dozen, Korea).

[0112] 1.5 × 10 in a 96-well plate 5 The cells were equally divided into 10 cells / well and cultured for 18 hours.

[0113] The existing medium was removed, and new medium containing various concentrations of fermented products and red ginseng (PC) was used, and then cultured for another 24 hours.

[0114] After incubation, 10 μL of WST solution (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium) was added to the medium and incubated for 2 hours.

[0115] The absorbance of each well was measured at 450 nm using a VerasaMax ELISA microplate reader.

[0116] Toxicity evaluation by cell viability for each sample was measured three times repeatedly, and the average value and standard deviation were calculated.

[0117] As a result of the evaluation, the fermented product of the present invention did not exhibit cytotoxicity in all concentration ranges.

[0118] Experimental Example 3: Measurement of NO production

[0119] 5×10 RAW 264.7 cells 4 The samples were dispensed into a 48-well plate at a density of 10 cells / mL and cultured for 24 hours at 37°C in a 5% CO2 incubator (311GP, Thermo Fisher Scientific, Waltham, MA, USA), and then treated for 48 hours.

[0120] As a positive control group, 100 ㎍ / mL of red ginseng (ginsenoside Rg1+Rb1+Rg3 5.5 mg / g) was used.

[0121] The supernatant was transferred to a 96-well plate, and NO in the supernatant was measured at 540 nm using a microplate reader (Epoch, Biotek Instruments, Inc., Winooski, VT, USA) using an NO assay kit (intron, Sungnam, Korea).

[0122] An appropriate amount of NO secreted from activated immune cells acts as an immune signal transmitter, activating immune cells to enhance toxicity against cancer cells and resistance to external pathogens.

[0123] Therefore, to confirm the effects of three types of fermented cucurbit products on NO production in macrophages by immune cell activation, NO production was measured in RAW 264.7 cells.

[0124] Referring to Figure 1, the NO production increased 2.91-fold in C-CS-DBS (fermented cucumber), 3.58-fold in C-CS-DBS (fermented pumpkin), and 5.16-fold in C-BH-DBS (fermented Dong-A) compared to the control group at a concentration of 1000 ㎍ / mL.

[0125] The above results suggest that the fermented product of the Bacillus genus strain of the above-mentioned Cucurbitaceae plant can effectively activate immune cells and exhibit immune-enhancing activity.

[0126] Experimental Example 4: Measurement of Cytokine Production

[0127] Cytokines secreted from activated macrophages play an important role as cofactors in the immune response.

[0128] Therefore, the expression levels of TNFα, IL-6, and IL-1β secreted from RAW 264.7 cells were measured.

[0129] 5×10 RAW 264.7 cells 4 The cells / mL were dispensed into a 48-well plate and cultured for 24 hours at 37°C in a 5% CO2 incubator (311GP, Thermo Fisher Scientific, Waltham, MA, USA). The samples were then treated for 48 hours, and 100 μg / mL of red ginseng (ginsenoside Rg1+Rb1+Rg3 5.5 mg / g) was used as a positive control.

[0130] The supernatant was transferred to a 96-well plate, and TNF-α, IL-6, and IL-1β in the supernatant were measured using an Enzyme-Linked Immuosorbent Assay (ELISA) kit (R&D systems, Minneapolis, MN, USA) at an absorbance of 450 nm using a microplate reader (Epoch, Biotek Instruments, Inc., Winooski, VT, USA).

[0131] Referring to Figure 2, the TNF-α production increased about 16.4 times at 10 μg / mL, about 21.7 times at 100 μg / mL, and about 22.8 times at 1000 μg / mL compared to the control group in the case of C-CS-DBS, about 17.4 times at 10 μg / mL, about 21.9 times at 100 μg / mL, and about 21.9 times at 1000 μg / mL in the case of C-BH-DBS, and about 22.9 times at 10 μg / mL, about 26.3 times at 100 μg / mL, and about 28.2 times at 1000 μg / mL in the case of C-BH-DBS.

[0132] In the case of red ginseng, which was the positive control, there was an approximately 18-fold increase, and in LPS, there was an approximately 36.1-fold increase.

[0133] IL-6 production increased approximately 3.0 times at 10 μg / mL, approximately 8.7 times at 100 μg / mL, and approximately 142.3 times at 1000 μg / mL compared to the control group in the case of C-CS-DBS, approximately 4.3 times at 10 μg / mL, approximately 54.7 times at 100 μg / mL, and approximately 185.7 times at 1000 μg / mL in the case of C-CS-DBS, and approximately 9.0 times at 10 μg / mL, approximately 153.7 times at 100 μg / mL, and approximately 234.0 times at 1000 μg / mL in the case of C-BH-DBS.

[0134] In the case of red ginseng, which was the positive control, the increase was approximately 14.2 times, and in LPS, the increase was approximately 252.4 times.

[0135] IL-1β production increased approximately 12.7 times at 10 μg / mL, approximately 20.0 times at 100 μg / mL, and approximately 38.6 times at 1000 μg / mL compared to the control group in the case of C-CS-DBS, approximately 15.0 times at 10 μg / mL, approximately 22.7 times at 100 μg / mL, and approximately 62.7 times at 1000 μg / mL in the case of C-CS-DBS, and approximately 18.6 times at 10 μg / mL, approximately 101.8 times at 100 μg / mL, and approximately 105.0 times at 1000 μg / mL in the case of C-BH-DBS.

[0136] In the case of red ginseng, which was the positive control, the increase was approximately 18.8 times, and in LPS, the increase was approximately 105.1 times.

[0137] The above results suggest that the fermented product of the Bacillus spp. strain of the above Cucurbitaceae plant can effectively improve immune activity by enhancing cellular and humoral immune responses.

[0138] Experimental Example 5: Measurement of COX-2 and iNOS expression levels

[0139] iNOS is an enzyme involved in the biosynthesis of NO, and is produced in macrophages activated by inflammatory substances such as LPS or inflammatory cytokines.

[0140] COX2 is induced by activated macrophages to synthesize PGE2. Therefore, the signaling pathway activity of iNOS and COX2 was confirmed in RAW 264.7 cells using Western blotting analysis.

[0141] RAW 264.7 cells were seeded in a fixed amount on a 100 mm dish and cultured for 24 hours.

[0142] Samples were treated at different concentrations and cultured for differentiation in a CO2 incubator for 24 hours.

[0143] Cultured cells were harvested by centrifugation, washed with PBS, and then RIPA buffer (Sigma, St. Louis, USA) was added. The supernatant was centrifuged and the protein concentration was quantified using a BSA kit (Bio-Rad, California, USA).

[0144] The quantitative values ​​were separated by electrophoresis on a 10% sodium dodecyl sulfate (SDS)-polyacrylamaide gel at 80 to 90 V.

[0145] The separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane at 100 V for 1 hour, blocked in 5% skim milk for 1 hour at room temperature, and then washed three times with TBS-T for 15 minutes each.

[0146] After reacting with primary antibodies diluted 1:200-1:1000, the cells were washed with TBS-T.

[0147] Next, the membrane was reacted with anti-rabbit IgG and anti-mouse IgG (Cell signaling, USA) conjugated with horseradish peroxidase (HRP) diluted 1:3,000 as secondary antibodies for 1 hour. The membrane was then washed with TBS-T.

[0148] The level of protein expression was confirmed by chemiluminescence by applying it to the membrane using an ECL kit (Bio-Rad, California, USA), and then the level of expression was quantified using an imaging densitometer (ImageJ bundled with 64-bit Java 1.8.0_112).

[0149] Referring to Figure 3, the protein expression of iNOS increased approximately 28.1-fold in C-BH-DBS 1000 μg / mL compared to the control group, and approximately 293.3-fold in LPS.

[0150] In addition, the protein expression of COX2 increased approximately 3.68-fold at 10 μg / mL of C-BH-DBS, approximately 3.38-fold at 100 μg / mL, and approximately 4.467-fold at 1000 μg / mL compared to the control group, and approximately 2.9-fold and 5.7-fold, respectively, in the positive control group, red ginseng, and LPS, respectively.

[0151] The above results suggest that the fermented product of the Bacillus spp. strain of the above Cucurbitaceae plant can effectively affect immune activity by increasing the expression level of enzymes involved in the biosynthesis of NO.

[0152] Experimental Example 6: Measurement of MAPK phosphorylation

[0153] The MAPK signaling pathway includes proteins such as p38, ERK1 / 2, and JNK, and is a signaling pathway that plays an important role in the activation of macrophages by promoting the production of cytokines such as TNF-α and IL-6 and NO production in macrophages.

[0154] Therefore, the signaling pathway activity of MAPKs in RAW 264.7 cells was confirmed using Western blotting analysis.

[0155] Referring to Figure 4, phosphorylation of p38 increased approximately 2-fold at 10 μg / mL of C-BH-DBS, approximately 3.1-fold at 100 μg / mL, and approximately 4.7-fold at 1000 μg / mL compared to the control group, and approximately 4.9-fold and 3.5-fold in the positive control group, red ginseng, respectively, and LPS.

[0156] Phosphorylation of ERK increased approximately 1.67-fold in 1000 μg / mL of C-BH-DBS compared to the control group, approximately 1.3-fold in the positive control group, red ginseng, and approximately 1.95-fold in LPS.

[0157] Phosphorylation of JNK increased approximately 1.23-fold at 100 μg / mL of C-BH-DBS and approximately 1.57-fold at 1000 μg / mL compared to the control group, and approximately 1.2-fold and 1.8-fold, respectively, in the positive control group, red ginseng, and LPS, respectively.

[0158] The above results suggest that the fermented product of the Bacillus spp. strain of the above Cucurbitaceae plant can effectively affect immune activity by increasing the expression level of enzymes involved in the biosynthesis of NO.

[0159] Experimental Example 7: Measurement of Iκ and NFκ phosphorylation

[0160] NF-κB exists in an inactive state by binding to IκB in a normal state, but in response to a specific signal, IκB is degraded by Iκκ, and the released NF-κB moves to the nucleus and promotes the gene expression of inflammatory response-related factors such as cytokines and adhesion molecules, thereby inducing the activation of macrophages.

[0161] Therefore, the signaling pathway activity of NF-κB and IκB in RAW 264.7 cells was confirmed using Western blotting analysis.

[0162] Referring to Figure 5, phosphorylation of IκB increased approximately 2.57-fold at 10 μg / mL of C-BH-DBS, approximately 2.76-fold at 100 μg / mL, and approximately 4.81-fold at 1000 μg / mL compared to the control group, and approximately 2.1-fold and 5.5-fold, respectively, in the positive control group, red ginseng, and LPS, respectively.

[0163] Phosphorylation of NFκB increased approximately 1.78-fold in 1000 μg / mL of C-BH-DBS compared to the control group, approximately 1.18-fold in the positive control group, red ginseng, and approximately 1.57-fold in LPS.

[0164] The above results suggest that the fermented product of the Bacillus spp. strain of the above Cucurbitaceae plant can effectively enhance immune activity by affecting the phosphorylation of IκB and NF-κB.

[0165] Experimental Example 8: Comparative Evaluation of Immune Activity According to Strain Changes

[0166] To evaluate the change in immune-enhancing activity according to strain change, the amount of NO production according to Experimental Example 3 was evaluated using different strain types.

[0167] Evaluation was conducted using Weissella koreensis and Aspergillus oryzae, which are not Bacillus strains, and Dong-a was used as a substrate.

[0168] The experimental method was maintained under the same conditions as Experimental Example 3, and the amount of NO production compared to the control group was measured at a concentration of 1000 ㎍ / mL (Table 1).

[0169] Classification NO production amount (compared to the control group) Weissella koriensis 1.18 Lactobacillus plantarum 1.57

[0170] Referring to Table 1, the fermented products using Bacillus koreensis and Aspergillus oryzae, which are not Bacillus strains, were evaluated to have relatively low levels of NO production. The above results suggest a synergistic effect between cucurbits and Bacillus strains, in that the fermented products of Bacillus strains of cucurbits exhibit relatively high levels of immune-enhancing activity, whereas strains with different characteristics do not exhibit the same levels of immune-enhancing activity.

[0171] Experimental Example 9: Comparative Evaluation of Immune Activity According to the Form of the Final Product

[0172] In order to evaluate the change in immune-enhancing activity according to the form of the final harvest, the fermented product of the Bacillus spp. strain was prepared in the same manner as in the manufacturing example, but the form of the final harvest was changed to a fermented extract and the immune activity was evaluated.

[0173] Specifically, the fermented extract was obtained by obtaining a fermented product in the same manner as in the manufacturing example, but adding 5 times the amount of water to the fermented product, boiling it at 120°C for 4 hours, extracting it, filtering it, concentrating it to 10 to 20 brix, and pulverizing it using a freeze dryer.

[0174] The experimental method was maintained under the same conditions as Experimental Example 3, and the amount of NO production compared to the control group was measured at a concentration of 1000 ㎍ / mL (Table 2).

[0175] Classification NO Production Amount (compared to the control group) C-BH-DBS (Dong-a) 5.16 C-CM-DBS (Pumpkin) 3.58 C-CS-DBS (Cucumber) 2.91 Dong-a fermented extract 4.80 Pumpkin fermented extract 2.44 Cucumber fermented extract 2.29

[0176] Referring to Table 2, the sample of the manufacturing example obtained by freeze-drying according to the present invention was evaluated to have a significantly higher level of NO production compared to the fermented extract obtained by hot water extraction of the fermented product.

[0177] The above results suggest that the fermented product may not exhibit the same level of immune-enhancing activity as the manufacturing example due to reasons such as loss or deformation of useful components during the extraction process, and may have a significant impact on the immune activity depending on the final yield.

[0178] Experimental Example 10: Comparative Evaluation of Immune Activity According to Changes in Bacterial Count

[0179] To evaluate the change in immune-enhancing activity according to the number of bacteria, a fermented product of Bacillus spp. was prepared in the same manner as in the manufacturing example, but the number of bacteria in the fermented product was varied to evaluate the immune activity.

[0180] In Experimental Example 3, the immune activity was evaluated by varying the number of Bacillus strains in C-BH-DBS (Dong-A Fermentation).

[0181] The experimental method was maintained under the same conditions as Experimental Example 3, and the amount of NO production compared to the control group was measured at a concentration of 1000 ㎍ / mL (Table 3).

[0182] Classification NO production amount (compared to control group) 1×10 7 CFU5.021×10 8 CFU5.471×10 9 CFU5.51

[0183] Referring to Table 3, the immune-enhancing activity of the fermented Bacillus strain of the above-mentioned cucurbit plant can significantly change depending on the number of Bacillus strains, and 1×10 7 Below CFU, NO production was significantly reduced.

[0184] On the other hand, the number of bacteria in the above fermented product is 1×10 7 Even if the CFU was exceeded, the immune-boosting effect was not dramatically improved.

[0185] The above results show that the fermented product is 1×10 7 This suggests that if the Bacillus strains containing more than CFU are included, they may exhibit the intended high level of immune-enhancing activity.

[0186] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0187] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. An immune-enhancing composition comprising a fermented product of a bacillus sp. strain of a Cucurbitaceae plant as an active ingredient.

2. In paragraph 1, An immune-boosting composition, wherein the above-mentioned cucurbit plant is at least one selected from the group consisting of pumpkin, cucumber, melon, watermelon, gourd, and melon.

3. In paragraph 1, An immune-enhancing composition, wherein the above Bacillus strain is Bacillus subtilis.

4. In paragraph 1, The above fermented product is an immune-enhancing composition obtained by adding a Bacillus genus strain to a crushed liquid of the above cucurbit plant, fermenting it at 25 to 35°C for 12 to 96 hours, and then freeze-drying it.

5. In paragraph 4, The above fermented product is 1×10 7 An immune-enhancing composition comprising a Bacillus spp. strain having a CFU or greater.

6. A health functional food for enhancing immunity containing a fermented product of a bacillus sp. strain of a Cucurbitaceae plant as an active ingredient.

7. In paragraph 6, A health functional food for enhancing immunity, manufactured in any one formulation selected from the group consisting of tablets, granules, powder, capsules, liquid solutions, and pills. 8.(a) A step of obtaining a substrate by mixing a crushed liquid of a Cucurbitaceae plant and water; (b) a step of introducing the substrate into a fermentation tank and sterilizing it; (c) a step of inoculating the Bacillus genus strain into the fermentation tank; (d) a step of fermenting at 25 to 35°C for 12 to 96 hours to obtain a fermented product; (e) A method for producing an immune-enhancing composition, comprising the step of freeze-drying the fermented product.

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