Method for producing plant-based fermented powder of sword bean and Cordyceps militaris with enhanced functional component content using sword bean

Abstract

The present invention relates to a method for producing a fermented powder of Cordyceps militaris with enhanced active ingredients by culturing Cordyceps militaris mycelium using sword beans and brown rice as culture media, and extracting and pulverizing the mycelium. Specifically, the method comprises the steps of: (1) preparing a sterile culture medium by mixing pre-treated sword beans and brown rice in a certain ratio; (2) inoculating the Cordyceps militaris mycelium with accession number KCCM13296P into the culture medium and performing dark culture and photoculture; (3) preparing a complex fermented product by sterilizing, drying, and grinding the culture; and (4) preparing a final powder by adding ethanol to the fermented product to extract active ingredients, and concentrating and freeze-drying the extract. The fermented powder produced in this manner has increased content of beneficial components such as β-glucan, ergosterol, cordycepin, and adenosine, and exhibits excellent inhibitory effects on NO and inflammatory cytokines, making it useful as a raw material for health functional foods or pharmaceuticals.

Description

Technology Field

[0001] The present invention relates to a method for producing plant-based sword bean cordyceps fermented powder with improved active ingredient content and physiological activity by culturing cordyceps mycelium using pre-treated sword beans and brown rice as a culture medium, and extracting and pulverizing the resulting product. The plant-based sword bean cordyceps fermented powder of the present invention can be applied as a health functional food, a food additive, or a raw material for natural material-based pharmaceuticals.

[0003] This invention is the result of the research project 'Development and Industrialization of Bronchial Health Food Using Cordyceps Mycelium Fermented Sword Bean Extract' conducted with support from the 2024 Jeonnam Technopark Regional Demand-Tailored Research and Development Project (Fostering Regional Core Science and Technology). Background Technology

[0004] Sword bean, traditionally known as 'Dodu' in Korea, is a plant utilized in folk medicine and is known to be effective for respiratory diseases such as asthma, rhinitis, and cough. In particular, sword beans are rich in various bioactive substances, including isoflavones, drawing attention to their potential as a functional ingredient for improving immune function and promoting bronchial health.

[0005] Currently, Jeollanam-do is the largest producer of sword beans in Korea, recording an annual production of approximately 2,500 tons and accounting for more than half of the national total. However, most sword bean products remain in the form of simple dried and roasted infusion teas, resulting in low product turnover and limitations in industrial expansion into health functional foods. Consequently, farm incomes remain stagnant, and poor crop yields caused by abnormal weather conditions and farm abandonment continue to occur.

[0006] Meanwhile, due to the rise in immune-related diseases such as allergic conditions and respiratory infections, the demand for strengthening bronchial immunity is steadily increasing, leading to the expansion of the health functional food and pharmaceutical markets. Although the existing market is dominated by synthetic drugs and some traditional herbal medicines, consumer interest in naturally derived functional materials has recently been rising, making it necessary to adopt a technological approach to domestic resources such as sword beans.

[0007] Accordingly, there is a need for the development of technologies that can enhance the added value of sword bean as a functional food ingredient, moving beyond its role as a mere condiment; in particular, advanced fermentation-based processing technologies are required to scientifically prove its immune-boosting and anti-inflammatory effects.

[0008] Fermentation is well known as an effective strategy for increasing the content of bioactive substances in natural materials and alleviating irritation or side effects. In particular, solid medium fermentation using mushroom mycelium (e.g., Cordyceps) can not only efficiently enhance functional components within the material, but also maximize immune and anti-inflammatory functions by adding active ingredients such as beta-glucan, cordycepin, and adenosine produced during the growth process of the mycelium.

[0009] Therefore, there is a need for a functional-focused technology for utilizing sword bean that goes beyond the existing simple drying process by using sword bean as a main culture medium, inoculating it with Cordyceps mycelium for fermentation, and then extracting and drying it.

[0010] The present invention is significant in that it provides a method for manufacturing a composite fermented powder of sword bean and Cordyceps sinensis that enhances the content of functional active ingredients and increases anti-inflammatory activity by reflecting such technical requirements and by fermenting Cordyceps sinensis mycelium in a plant medium using sword bean and brown rice, followed by extraction, drying, and powdering.

[0011] In this regard, prior art includes Korean Registered Patent No. 1585159, which discloses a "composition for the prevention and treatment of inflammatory or allergic diseases containing fermented sword bean extract as an active ingredient," and Korean Published Patent No. 2023-0058291, which reports a "method for preparing a sword bean pod extract with increased rutin content and functionality." However, these technologies are limited to simple extracts, and do not disclose technologies for enhancing complex functionality through fermentation, particularly technologies for improving active ingredients and functional effects through complex fermentation with Cordyceps. The problem to be solved

[0012] This invention was conceived out of the need to develop technology capable of enhancing the utilization of sword bean and maximizing the content of active ingredients and physiological activity of Cordyceps sinensis. Existing sword bean products were mainly limited to simple dried or infused tea forms, which restricted their use as a functional, high-value-added material.

[0013] Furthermore, although Cordyceps contains various bioactive functional components such as immune modulators, antioxidants, and anti-inflammatory agents, the expression amount of active ingredients varies significantly depending on the culture substrate and conditions, requiring a manufacturing process capable of producing them stably and efficiently.

[0014] Accordingly, the present invention was conceived in response to the above requirements, and the objectives of the present invention are (1) to establish a culture medium composition and mixing ratio optimized for Cordyceps cultivation using sword bean and brown rice, and (2) deposited strain ( Cordyceps militaris The present invention provides a method for producing a fermented powder of plant-based Cordyceps sinensis with excellent functional ingredient content, physiological activity, and palatability by setting culture conditions suitable for the growth of (3) and optimizing extraction and pulverization processes that can increase the extraction efficiency of functional ingredients. means of solving the problem

[0015] To solve the above problem, the present invention comprises the steps of: (1) preparing a culture medium by placing sword bean and brown rice in water, soaking them, sterilizing them, and cooling them; (2) in the culture medium prepared in step (1), Cordyceps ( Cordyceps militaris The present invention provides a method for manufacturing fermented sword bean cordyceps powder, characterized by comprising the steps of: (3) inoculating and culturing the mycelium; (2) sterilizing and drying the fermented product cultured in step (2) and then grinding it to produce a complex fermented sword bean cordyceps product; and (4) adding ethanol to the complex fermented sword bean cordyceps product prepared in step (3) to extract, then concentrating and freeze-drying it and then grinding it.

[0016] In addition, the present invention provides a fermented sword bean cordyceps powder prepared by the above method.

[0017] In addition, the present invention provides a processed food product manufactured using the above-mentioned sword bean cordyceps fermented powder. Effects of the invention

[0018] The fermented sword bean cordyceps powder of the present invention contains high amounts of major immune-activating components derived from cordyceps, such as β-glucan, ergosterol, cordycepin, and adenosine, and also has increased content of functional substances such as free sugars and free amino acids, so it can be utilized as a health functional material effective for boosting immunity and improving metabolism.

[0019] Furthermore, the fermented sword bean cordyceps powder of the present invention exhibits anti-inflammatory activity that effectively inhibits the production of NO (nitric oxide) and inflammatory cytokines (TNF-α, IL-6, IL-1β), thereby enabling its application as a natural anti-inflammatory material useful for improving various inflammatory diseases.

[0020] Thus, the present invention overcomes the limitations of existing simply processed products through a manufacturing process that maximizes the beneficial components of sword bean and stably secures the physiologically active substances of Cordyceps, and has very high potential for utilization as a high-value-added health functional food material equipped with a combination of immune function improvement and anti-inflammatory functions. Brief explanation of the drawing

[0021] Figure 1 is a schematic diagram of the manufacturing process of the plant-based sword bean cordyceps complex fermented product of Manufacturing Example 3. Specific details for implementing the invention

[0022] To achieve the objective of the present invention, the present invention

[0023] (1) A step of preparing a culture medium by placing sword beans and brown rice in water, soaking them, sterilizing them, and cooling them;

[0024] (2) Cordyceps ( Cordyceps militaris ) A step of inoculating and culturing mycelium;

[0025] (3) A step of sterilizing and drying the cultured fermented product from step (2) above, and then grinding it to produce a composite fermented product of sword bean cordyceps; and

[0026] (4) A method for manufacturing a fermented powder of Cordyceps sinensis is provided, characterized by including the step of adding ethanol to the fermented Cordyceps sinensis complex product prepared in step (3) above, extracting, concentrating, freeze-drying, and then grinding.

[0027] In the method for preparing the fermented sword bean cordyceps powder of the present invention, the culture medium of step (1) can preferably be prepared by adding 5.5 to 6.5 parts by weight of sword beans and 3.5 to 4.5 parts by weight of brown rice to 25 to 35 parts by weight of water, immersing for 50 to 70 minutes at 20 to 30°C, sterilizing for 30 to 40 minutes at 110 to 130°C, and cooling to 20 to 30°C; more preferably, it can be prepared by adding 5.5 to 6.5 kg of sword beans and 3.5 to 4.5 kg of brown rice to 25 to 35 L of water, immersing for 50 to 70 minutes at 20 to 25°C, sterilizing for 30 to 40 minutes at 110 to 130°C, and cooling to 20 to 25°C; and most preferably, by adding 6 kg of sword beans and 4 kg of brown rice to 30 L of water and immersing for 60 minutes at 20 to 25°C It can be prepared by immersing, sterilizing at 121°C for 35 minutes, and cooling to 20–25°C. When a culture medium is prepared under the above conditions, nutrients from sword beans and brown rice are supplied in a balanced manner, allowing for active growth of Cordyceps mycelium; furthermore, the sterilization and immersion processes are effective in softening the substrate and preventing contamination, thereby enabling stable fermentation. As a result, the production of functional components such as cordycepin, adenosine, and β-glucan is maximized, and a fermented powder with excellent quality and reproducibility as a functional food material can be produced.

[0028] The above sword bean may preferably be pretreated by soaking the sword bean at 20–30°C for 4–6 hours, removing the moisture, roasting the sword bean at 110–130°C for 3–7 minutes, drying it at 45–55°C for 6–10 hours, cooling it to 20–30°C, and then freeze-drying it at -40–-50°C for 24–72 hours; more preferably, the sword bean may be pretreated by soaking it at 20–25°C for 5 hours, removing the moisture, roasting it at 120°C for 5 minutes, drying it at 50°C for 8 hours, cooling it to 20–25°C, and then freeze-drying it at -40–-50°C for 48 hours. The above pretreatment conditions allowed for the removal of the unpleasant odor of the sword bean and the minimization of the loss of active ingredients, while pretreating it to a state suitable for the growth of Cordyceps mycelium. In particular, hygiene, functionality, and fermentation efficiency could be simultaneously secured through processing steps of roasting, low-temperature drying, and freeze-drying.

[0029] In addition, the brown rice may preferably be pretreated by soaking at 20-30°C for 1-3 hours, removing the moisture, roasting at 110-130°C for 1-3 minutes, grinding, steaming at 75-85°C for 15-25 minutes, cooling to 20-30°C, and drying at 70-90°C for 4-6 hours; more preferably, the brown rice may be pretreated by soaking at 20-25°C for 2 hours, removing the moisture, roasting at 121°C for 2 minutes, grinding, steaming at 80°C for 20 minutes, cooling to 20-25°C, and drying at 80°C for 5 hours. The above pretreatment process is an optimal treatment condition that can increase the fermentation suitability of the brown rice, improve mycelial absorption efficiency, suppress contamination by unwanted bacteria, and form a substrate favorable for preserving functional components.

[0030] In addition, in the method for preparing the fermented sword bean cordyceps powder of the present invention, the culture in step (2) can preferably be dark cultured at 20 to 30°C for 2 to 4 days, followed by light cultured at 18 to 20°C for 50 to 70 days, and more preferably dark cultured at 25°C for 3 days, followed by light cultured at 18 to 20°C for 60 days. Culturing under such conditions can stably induce the growth of cordyceps mycelia while maximizing the production of active ingredients such as cordycepin and adenosine, thereby improving the functionality and quality of the final product.

[0031] In addition, in the method for preparing the sword bean cordyceps fermented powder of the present invention, the cordyceps of step (2) ( Cordyceps militaris ) The mycelium is Cordyceps ( Cordyceps militaris ) JMIM00002 mycelium was assigned the international accession number KCCM13296P by the Korean Culture Collection of Microorganisms on December 2, 2022 (Accession number: KCCM13296P).

[0032] When a fermented powder is prepared using the specific Cordyceps mycelium deposited above, it was possible to produce a fermented powder with excellent anti-inflammatory activity, which has excellent growth efficiency in the medium of the present invention, high content of functional components such as beta-glucan, ergosterol, cordycepin, and adenosine, and enhanced inhibitory activity of inflammation-inducing NO, IL-1β, TNF-α, and IL-6.

[0033] In addition, in the method for manufacturing the fermented sword bean cordyceps powder of the present invention, the composite fermented sword bean cordyceps of step (3) can preferably be manufactured by sterilizing the photo-cultured fermented product at 70 to 90°C for 10 to 20 minutes, drying it at 60 to 80°C for 10 to 14 hours, and then grinding it; more preferably, the fermented product can be manufactured by sterilizing the photo-cultured fermented product at 80°C for 15 minutes, drying it at 70°C for 12 hours, and then grinding it. Sterilizing and drying under the above conditions is the optimal processing condition for stably manufacturing high-quality and high-functionality fermented sword bean cordyceps powder by minimizing the loss of active ingredients while increasing hygiene and shelf life.

[0034] In addition, in the method for preparing the fermented powder of the sword bean cordyceps according to the present invention, step (4) can preferably be performed by adding 8 to 12 times (v / w) of 40 to 60% (v / v) ethanol to the sword bean cordyceps complex fermented product, extracting at 70 to 90°C for 6 to 10 hours, concentrating, freeze-drying at -40 to -50°C for 24 to 72 hours, and then grinding; more preferably, by adding 10 times (v / w) of 50% (v / v) ethanol to the sword bean cordyceps complex fermented product, extracting at 80°C for 8 hours, concentrating, freeze-drying at -40 to -50°C for 48 hours, and then grinding. Extracting and freeze-drying under the above conditions is the optimal extraction and drying condition that maximizes the extraction efficiency of functional components derived from cordyceps and simultaneously achieves stable preservation of heat-sensitive components and high-quality powdering.

[0035] The method for manufacturing the fermented sword bean cordyceps powder of the present invention is, more specifically,

[0036] (1) A step of preparing pretreated sword beans by soaking sword beans for 4 to 6 hours, removing the moisture, roasting them at 110 to 130°C for 3 to 7 minutes, drying them at 45 to 55°C for 6 to 10 hours, and then cooling and freeze-drying them;

[0037] (2) A step of preparing pretreated brown rice by soaking brown rice for 1 to 3 hours, removing excess water, roasting it at 110 to 130°C for 1 to 3 minutes, grinding it, steaming it at 75 to 85°C for 15 to 25 minutes, cooling it, and then drying it at 70 to 90°C for 4 to 6 hours;

[0038] (3) A step of preparing a culture medium by adding 5.5 to 6.5 parts by weight of sword beans pretreated in step (1) and 3.5 to 4.5 parts by weight of brown rice pretreated in step (2) to 25 to 35 parts by weight of water, soaking for 50 to 70 minutes, sterilizing at 110 to 130°C for 30 to 40 minutes, and cooling;

[0039] (4) Cordyceps with accession number KCCM13296P (in the medium prepared in step (3) above) Cordyceps militaris A step of inoculating JMIM00002 mycelium and culturing in the dark at 20–30℃ for 2–4 days, followed by photoculture at 18–20℃ for 50–70 days;

[0040] (5) A step of preparing a composite fermented product of sword bean cordyceps by sterilizing the photocultured fermented product of step (4) at 70~90℃ for 10~20 minutes, drying it at 60~80℃ for 10~14 hours, and then grinding it; and

[0041] (6) The method may include the step of adding 8 to 12 times (v / w) of 40 to 60% (v / v) ethanol to the fermented sword bean cordyceps complex prepared in step (5) above, extracting at 70 to 90°C for 6 to 10 hours, concentrating, freeze-drying, and then grinding.

[0042] More specifically,

[0043] (1) A step of preparing pretreated sword beans by soaking the sword beans for 5 hours, removing the moisture, roasting them at 120°C for 5 minutes, drying them at 50°C for 8 hours, and then cooling and freeze-drying them;

[0044] (2) A step of preparing pretreated brown rice by soaking brown rice for 2 hours, removing moisture, roasting it at 120°C for 2 minutes, grinding it, steaming it at 80°C for 20 minutes, cooling it, and then drying it at 80°C for 5 hours;

[0045] (3) A step of preparing a culture medium by adding 6 parts by weight of the sword beans pretreated in step (1) and 4 parts by weight of the brown rice pretreated in step (2) to 30 parts by weight of water, soaking for 60 minutes, sterilizing at 121°C for 35 minutes, and cooling;

[0046] (4) Cordyceps with accession number KCCM13296P (in the medium prepared in step (3) above) Cordyceps militaris A step of inoculating JMIM00002 mycelium and culturing in the dark at 25℃ for 3 days, followed by photoculture at 18~20℃ for 60 days;

[0047] (5) A step of preparing a composite fermented product of sword bean cordyceps by sterilizing the photocultured fermented product of step (4) at 80°C for 15 minutes, drying it at 70°C for 12 hours, and then grinding it; and

[0048] (6) The step of adding 10 times (v / w) of 50% (v / v) ethanol to the fermented sword bean cordyceps complex prepared in step (5) above, extracting at 80°C for 8 hours, concentrating, freeze-drying, and then grinding may be included.

[0049] The present invention also provides a fermented sword bean cordyceps powder prepared by the above method.

[0050] The present invention also provides a processed food product manufactured using the above-mentioned fermented sword bean cordyceps powder.

[0051] The fermented sword bean cordyceps powder according to the present invention has excellent functional substance content and anti-inflammatory function, so it can be usefully applied to various food products intended for maintaining health and improving functionality. The fermented sword bean cordyceps powder is not particularly limited to the type or formulation of processed food and can be added or blended according to a conventional food manufacturing process. Examples include health functional foods such as pills, tablets, powder sticks, and jellies, and it can be applied to various forms of food such as meat or fish products (e.g., sausage, ham, dumpling filling), confectionery (e.g., bread, cookies, chocolate, snacks, candy, gum), noodles (e.g., ramen, pasta, noodles), frozen foods, pizza, cereals, dairy products (e.g., yogurt, ice cream), beverages (e.g., health drinks, tea, energy drinks, alcoholic beverages), nutritional supplements, and vitamin complexes. Therefore, the fermented sword bean cordyceps powder of the present invention can be utilized as a high-value-added material for enhancing functionality throughout the food industry, and its applicable range includes not only health functional foods but also a wide range of food groups such as general foods, convenience foods, and functional snacks.

[0053] The present invention will be described in detail below with reference to manufacturing examples and embodiments. However, the following manufacturing examples and embodiments are merely illustrative of the present invention, and the content of the present invention is not limited to the following manufacturing examples and embodiments.

[0055] Preparation Example 1. Pretreatment for the preparation of a sword bean and brown rice culture medium composition

[0056] (1) The sword beans were soaked in water at 20–25°C for 5 hours, drained on a sieve, roasted in a roaster at 120°C for 5 minutes, and then dried in a hot air dryer at 50°C for 8 hours. Afterward, they were cooled to 20–25°C and then freeze-dried at -40–-50°C for 48 hours to obtain pre-treated sword beans.

[0057] (2) Brown rice was soaked in water at 20–25°C for 2 hours, drained using a sieve, roasted at 121°C for 2 minutes, and then ground. Afterwards, it was placed in a steamer and steamed at 80°C for 20 minutes, cooled to 20–25°C, and then dried in a hot air dryer at 80°C for 5 hours to produce pre-treated brown rice.

[0059] Preparation Example 2. Preparation of a plant-based sword bean and cordyceps complex fermented product using sword beans and brown rice

[0060] (1) 6 kg of sword beans and 4 kg of brown rice pretreated in Preparation Example 1 were placed in 30 L of water and soaked at 20–25°C for 1 hour to adjust the moisture content. Afterward, the mixture was divided into bags for mycelial culture and sterilized using an autoclave at 121°C for 35 minutes. After sterilization, the mixture was cooled to 20–25°C in a clean bench to prepare the culture medium.

[0061] (2) Cordyceps of deposit number KCCM13296P ( Cordyceps militaris JMIM00002 mycelium was aseptically collected and inoculated into the above medium. The inoculated medium was cultured in the dark for 3 days at 25°C and 65% humidity, and then cultured in the light for 60 days at 18-20°C and 65-70% humidity.

[0062] (3) After the fermented product from step (2) was completed, it was sterilized in an autoclave at 80°C for 15 minutes, then the culture bag was removed and it was dried in a drying oven at 70°C for 12 hours.

[0064] Preparation Example 3. Preparation of Plant-Based Sword Bean and Cordyceps Complex Fermented Powder

[0065] 10 kg of the plant-based sword bean cordyceps complex fermented product prepared in Preparation Example 2 was ground to 100 mesh using a blender. 100 L of 50% (v / v) ethanol was added to the ground sample, and extraction was performed at 80°C for 8 hours. The extract was filtered through a non-woven fabric, concentrated to 10 Brix based on solid content, freeze-dried at -40 to -50°C for 48 hours, and then passed through a 200 mesh sieve to obtain the final fermented powder.

[0067] Experimental method

[0068] 1. Beta-glucan Analysis

[0069] The beta-glucan of the sample was measured using the Megazyme kit (Mushroom and Yeast β-glucan Assay Procedure K-YBGL, Megazyme, Ireland).

[0070] First, for total glucan, 100 mg of ground sample sieved through a 100 mesh sieve was placed in a tube, 1.5 mL of 37% HCl was added, and the tube was placed in a 30°C water bath for 45 minutes to decompose it. Afterward, 10 mL of distilled water was added, the mixture was vortexed, and incubated at 100°C for 2 hours. Subsequently, while cooling to room temperature, 10 mL of 2N KOH was added, and the volume was diluted to 100 mL with 200 mM sodium acetate buffer and thoroughly mixed. Then, the supernatant was obtained by centrifugation (1,500 g, 10 min), and 0.1 mL of exo-1,3-β-glucanase plus β-glucosidase dissolved in 200 mM sodium acetate buffer was added to 0.1 mL of the supernatant. For the reagent blank, 0.2 mL of acetate buffer was added, and for the D-glucose standard, 0.1 mL of D-glucose standard and 0.1 mL of acetate buffer were added and mixed, followed by incubation at 40°C for 60 minutes. Next, 3 mL of GOPOD (Glucose oxidase / peroxidase mixture) was added and incubated at 40°C for 20 minutes, after which the absorbance was measured at 510 nm.

[0071] For α-glucan, 100 mg of ground sample sieved through a 100 mesh sieve was placed in a tube, 2 mL of 2 M KOH was added, and the mixture was mixed for 20 minutes. After adding and mixing 8 mL of 1.2 M sodium acetate buffer, 0.2 mL of amyloglucosidase plus invertase was added and mixed well, followed by incubation in a 40°C water bath for 30 minutes. To 0.1 mL of the supernatant, 0.1 mL of 200 mM sodium acetate buffer and 3 mL of GOPOD were added, and after incubation at 40°C for 20 minutes, the absorbance was measured at 510 nm. The β-glucan content was quantified by subtracting the α-glucan content from the total glucan content.

[0073] 2. Ergosterol Analysis

[0074] For ergosterol analysis, 100 mL of ethanol was added to 5 g of the sample and refluxed at 80°C for 1 hour. The supernatant was collected, and 100 mL of ethanol was added to the residue and refluxed again at 80°C for 1 hour. To the ethanol extract, 20 mL of ethanol and 10 g of potassium hydroxide were added and saponified at 85°C for 1 hour. 50 mL of distilled water was added to the saponified solution, and the solution was fractionated three times with hexane in 50 mL aliquots. The hexane layer was then completely concentrated, dissolved in 5 mL of methanol, and analyzed by HPLC.

[0075] HPLC conditions for ergosterol analysis item Analysis conditions device Agilent Technologies 1200 Series column Agilent XDB-C 18 (Method Development Kit)(4.6×150 mm, 5 ㎛) menstruum 98% methanol column temperature 28.8℃ wavelength UV 280 nm flow velocity 1.0 mL / min Injection volume 20 ㎕

[0077] 3. Analysis of Cordycepin and Adenosine

[0078] 20 mL of distilled water was added to 1 g of the sample and extracted for 90 minutes, after which the obtained extract was centrifuged for 15 minutes, and the supernatant was filtered through a 0.45 μm filter and used for HPLC analysis.

[0079] Cordycepin and Adenosine Analysis Conditions item Analysis conditions equipment Agilent Technologies 1200 Series column Agilent ZORBAX Eclipse XDB-C18 column temperature 30℃ buffer solution A: Methanol B: DW hour A(%) B(%) 0 0 100 3 10 90 4 10 90 7 20 80 8 20 80 11 30 70 12 30 70 15 98 2 16 98 2 17 0 100 Absorbance (nm) UV 260 nm flow velocity 0.8 mL / min Injection volume 10 ㎕

[0081] 4. Analysis of Sugars

[0082] For the free sugar component, distilled water was added to 5 g of the sample, ground using a homogenizer, stirred, and leached to 100 mL, then centrifuged (3,000 rpm, 30 minutes) to obtain Sep-pak C 18 After purification, the filtrate filtered through a 0.45 µm membrane filter (Millipore Co., USA) was analyzed using HPLC (High Performance Liquid Chromatography). The analysis conditions are as shown in Table 3 below, and the content was calculated using the external standard method.

[0083] HPLC conditions for sugar analysis item Analysis conditions Instrument Agilent Technologies 1200 SeriesELSD detector Column ZORBAX Carbohydrate (4.6×150 mm) Solvent 85% Acetonitrile Column temp. 30℃ Flow rate 1.4 ml / min Injection volume 20 ㎕

[0085] 5. Analysis of free amino acids

[0086] For the analysis of free amino acids, 25 mg of sulfosalicylic acid was added to 10 mL of the filtrate obtained by the same method as for the quantification of free sugars, and the mixture was left at 4°C for 4 hours. Afterward, the mixture was centrifuged (50,000 rpm, 30 min) to remove proteins, etc., and the supernatant was filtered through a 0.45 μm membrane filter. A certain amount of the filtrate obtained was taken, derivatized with an Agilent amino kit reagent, and analyzed by HPLC. The analysis conditions are as shown in Table 4 below.

[0087] Amino acid analysis conditions item Analysis conditions equipment Agilent Technologies 1200 Series detector Agilent Technologies 1200 Series DAD column Poroshell HPH C 18 (2.1×150 mm, 4 ㎛) column temperature 40℃ buffer solution A: 10mM Sodium phosphate Di-basic : 10mM Sodium tetraborate ' 7H2O = 1:1 pH 8.2(adjusted with phsporic acid),B: Acetonitrile : Methanol : Water = 45 : 45 : 10 hour A(%) B(%) 0 98 2 5 84 16 9 72 28 13 60 40 15 40 60 15.1 10 90 17 10 90 Absorbance (nm) UV 338 flow velocity 0.35 mL / min Injection volume 5 ㎕

[0089] 6. Cell Culture

[0090] RAW 264.7 macrophages were cultured in a humid CO2 incubator (5% CO2 / 95% air) at 37°C using a cell culture medium prepared by adding 10% FBS (fetal bovine serum), 100 U / mL penicillin, and 100 µg / mL streptomycin to DMEM (Dulbecco Modified Eagle Medium, Welgene, Gyeongsan, Korea). When the cells filled about 80% of the culture dish, the cell monolayer was washed away with PBS (phosphate-buffered saline, pH 7.4), and the cells were subcultured with 0.25% trypsin-2.65 mM EDTA; the medium was changed every 2 days.

[0092] 7. Check cell viability

[0093] Cytotoxicity was measured using the MTT assay. Each cell was placed in a 96-well plate at a ratio of 1 × 10⁶ 5 The samples were cultured at a concentration of cell / mL and used in the experiment. After treating the samples at concentrations of 10, 50, 100, and 500 μg / mL for 24 hours, the medium was removed, and 10 μl of MTS was added to fresh medium and reacted for 4 hours, after which the absorbance was measured at 540 nm. The cell viability of each sample was calculated relatively, with the untreated group set as 100%.

[0095] 8. Measurement of NO (Nitric Oxide) Production Amount

[0096] 1 × 10⁶ Raw 264.7 cells per well in a 96-well plate 5 Cells were dispensed to a concentration of 1 / mL and cultured for 24 hours, after which the samples and LPS (Lipopolysaccharide) were added. LPS was applied at a concentration of 1 µg / mL, and each sample was diluted to concentrations of 100, 150, and 200 µg / mL and added to the cells. After 24 hours, the cell culture medium was collected, and the NO production inhibitory ability was measured using a NO assay with the Griess reagent system.

[0098] 9. Measurement of inflammatory cytokine (IL-6, TNF-α, IL-1β) production

[0099] The production of inflammatory cytokines in the cell culture medium was measured using an ELISA (mouse enzyme-linked immunosorbent assay) kit. Raw 264.7 cells were cultured in DMEM medium at a rate of 5×10⁻⁶ 5 After adjusting to cells / ml, the cells were seeded into 6-well plates and cultured in a 5% CO2 incubator for 24 hours. Cells were treated with 1 µg / mL LPS, and one hour later, each sample was added at different concentrations and cultured for 24 hours. The pro-inflammatory cytokine content of the supernatant obtained after culture was measured; quantification was performed using an ELISA kit, and the standard curve R for the standard was 2 The value was 0.99 or higher.

[0101] Example 1. Selection of Optimal Cordyceps Mycelium

[0102] Experiments were conducted to select the optimal strain by comparing and evaluating the growth efficiency of Cordyceps mycelia in sword bean-based media. To evaluate mycelial growth ability, various mushroom mycelia were inoculated into each medium and cultured for 28 days, and the degree of growth was quantified by measuring the length of the mycelia growing downward from the inoculation surface. The media conditions included a 100% mealworm medium, a mealworm:brown rice mixed medium, and media mixed with sword bean and brown rice in ratios of 60:40, 50:50, 40:60, and 30:70, respectively.

[0103] As a result, Cordyceps 2 mycelia exhibited excellent growth under all culture medium conditions, and showed the most active mycelial growth in a medium mixed with sword bean and brown rice in a ratio of 60:40 or 50:50. On the other hand, other mycelia, such as shiitake, oyster mushroom, Phellinus linteus, and Hericium erinaceus, showed generally poor growth or grew only under limited conditions.

[0104] Fermentation efficiency by mushroom mycelium badge Types of mushroom mycelium Mycelial growth (28 days) shiitake oyster mushrooms situation Cordyceps 1 Cordyceps 2 deer buttocks Control group 1 (100% mealworms) - - - ++ ++ - Control Group 2 (Mealworm 50%: Brown Rice 50%) - - - + ++ + Sword bean 60%:brown rice 40% - + + ++ +++ + Sword bean 50%:brown rice 50% - + ++ ++ +++ + Sword bean 40%:brown rice 60% - + ++ ++ ++ + Sword bean 30%:brown rice 70% + + + ++ ++ +

[0105] -: No mycelial growth (less than 1 cm), +: Weak mycelial growth (2–3 cm), ++: Good mycelial growth (4–6 cm), +++: Excellent mycelial growth (7 cm or more)

[0107] Example 2. Beta-glucan content

[0108] This study was conducted to evaluate the functional enhancement effect of sword bean-based media by comparing the β-glucan content in fermented products extracted from mushroom mycelia cultured according to various media compositions.

[0109] β-glucan is a type of polysaccharide, mainly found in grains in the (1→3)(1→4) linked form and in mushrooms and yeasts in the (1→3)(1→6) linked form, and the latter structure is known to be closely related to immune enhancement and anticancer activity.

[0110] In this experiment, various mushroom mycelia (shiitake, oyster mushroom, Phellinus linteus, Cordyceps 1 and 2, Hericium erinaceus) were inoculated into culture media prepared by using mealworms, brown rice, and sword beans, either individually or in combination, and then cultured. Afterward, hot water or ethanol extraction was performed on the fermented products to analyze the β-glucan content.

[0111] As a result, the highest β-glucan content of 77.4 mg / g was confirmed in the hot water extract of the fermented Cordyceps 2 mycelium cultured in a medium with a ratio of 60% sword bean to 40% brown rice, which was consistent with the conditions of high mycelial growth efficiency. In addition, the ethanol extract also showed a high content of 54.1 mg / g under the same conditions. This indicated that the sword bean-brown rice mixed medium is effective for the growth of Cordyceps 2 strains and the production of functional polysaccharides, and confirmed that the fermentation efficiency is high in terms of β-glucan content.

[0112] Beta-glucan content (mg / g) of mushroom mycelial fermentation Sample Beta-glucan content (mg / g) Types of mushroom mycelium shiitake oyster mushrooms situation Cordyceps 1 Cordyceps 2 deer buttocks Hot water extraction Control group 1 (100% mealworms) - - - 61.2 68.7 - Control Group 2 (Mealworm 50%: Brown Rice 50%) - - - 44.1 63.5 39.2 Sword bean 60%:brown rice 40% - 25.2 35.8 62.9 77.4 37.2 Sword bean 50%:brown rice 50% - 27.1 49.2 57.7 70.6 33.4 Sword bean 40%:brown rice 60% - 38.8 46.2 56.6 54.2 26.4 Sword bean 30%:brown rice 70% 28.1 22.4 26.7 56.4 64.6 25.8 Ethanol extraction Control group 1 (100% mealworms) - - - 44.8 50.8 - Control Group 2 (Mealworm 50%: Brown Rice 50%) - - - 37.5 48.2 21.5 Sword bean 60%:brown rice 40% - 22.8 14.1 50.6 54.1 20.1 Sword bean 50%:brown rice 50% - 20.5 22.9 51.7 48.9 19.8 Sword bean 40%:brown rice 60% - 19.7 21.1 43.5 47.3 17.2 Sword bean 30%:brown rice 70% 16 16.2 17.2 40.5 51.4 15.4

[0114] Example 3. Ergosterol content

[0115] The ergosterol content in fermented products extracted from mushroom mycelia cultured under various medium compositions was analyzed to evaluate the effect of sword bean-based media on enhancing bioactive substances. Ergosterol is a sterol compound specifically found in mushrooms, and its antioxidant, anti-inflammatory, and immunomodulatory effects have been reported, giving it high potential for use as a functional food and pharmaceutical material.

[0116] In the experiment, various mushroom mycelia (shiitake, oyster mushroom, Phellinus linteus, Cordyceps 1 and 2, Hericium erinaceus) were inoculated into a culture medium prepared by mixing mealworms, brown rice, and sword beans in various proportions, and after cultivation, the fermented product was extracted with hot water or ethanol to measure the ergosterol content.

[0117] As a result of the analysis, the ergosterol content was highest in Cordyceps 2 mycelium overall, and high content of 6.72 mg / g was observed in ethanol extraction and 6.42 mg / g in hot water extraction in a 60% sword bean:40% brown rice medium, which was consistent with conditions of high mycelial growth efficiency and β-glucan content.

[0118] Ergosterol content (mg / g) of mushroom mycelial fermentation Sample Ergosterol content (mg / g) Types of mushroom mycelium shiitake oyster mushrooms situation Cordyceps 1 Cordyceps 2 deer buttocks Hot water extraction Control group (Mealworm 50%: Brown rice 50%) - - - 4.57 5.98 2.65 Sword bean 60%:brown rice 40% - 2.39 2.92 5.74 6.42 3.65 Sword bean 40%:brown rice 60% - 3.18 3.47 5.28 4.39 1.71 Sword bean 30%:brown rice 70% 0.87 1.54 1.28 4.48 5.58 1.24 Ethanol extraction Control group (Mealworm 50%: Brown rice 50%) - - - 4.38 6.02 2.73 Sword bean 60%:brown rice 40% - 2.82 3.12 5.82 6.72 4.07 Sword bean 40%:brown rice 60% - 2.69 3.34 5.49 4.89 1.81 Sword bean 30%:brown rice 70% 1.08 1.27 1.28 4.55 6.20 1.33

[0120] Example 4. Cordycepin content

[0121] The effects of applying sword bean-based media and Cordyceps strains were evaluated by analyzing the cordycepin content in mushroom mycelia cultured under various medium compositions. Cordycepin is Cordyceps ( Cordyceps militaris Cordycepin is a representative specific metabolite of Cordyceps and is a highly functional component with reported physiological activities such as anti-inflammatory, anticancer, immunomodulatory, and antiviral effects. Accordingly, the content of cordycepin is used as an important indicator to evaluate the quality of Cordyceps materials.

[0122] As a result of the experiment, cordycepin was detected only in the mycelia of Cordyceps 1 and 2, and was not detected in the mycelia of other mushrooms such as shiitake, oyster mushrooms, Phellinus linteus, and Hericium erinaceus. In particular, the mycelia of Cordyceps 2 showed a higher cordycepin content than Cordyceps 1 under all medium conditions, and the highest value was measured at 6.48 mg / g in a medium of 60% sword bean and 40% brown rice. This confirmed that a medium composition using sword bean and brown rice is the optimal condition to maximize cordycepin content when combined with the Cordyceps 2 strain.

[0123] Cordycepin content (mg / g) of Cordyceps Sample Cordycepin content (mg / g) Types of mushroom mycelium shiitake oyster mushrooms situation Cordyceps 1 Cordyceps 2 deer buttocks Control group 1 (100% mealworms) - - - 3.56 4.35 - Control Group 2 (Mealworm 50%: Brown Rice 50%) - - - 2.24 4.05 - Sword bean 60%:brown rice 40% - - - 4.39 6.48 - Sword bean 50%:brown rice 50% - - - 4.11 5.32 - Sword bean 40%:brown rice 60% - - - 1.69 3.15 - Sword bean 30%:brown rice 70% - - - 3.23 3.49 -

[0125] Example 5. Adenosine content

[0126] The adenosine content of mushroom mycelia cultured under various culture medium compositions was analyzed to confirm the effects of Cordyceps mycelia and culture medium components on the production of the relevant functional substance. Adenosine is a representative nucleic acid derivative with physiological activities such as immune modulation, anti-inflammatory, neuroprotective, and antioxidant properties; in particular, natural adenosine derived from Cordyceps has high potential for use as a material for health functional foods and pharmaceuticals.

[0127] As a result of the experiment, adenosine was detected at levels above a certain threshold in all mushroom mycelial cultures, but particularly high content was observed in the mycelia of Cordyceps 1 and 2. Among them, the mycelia of Cordyceps 2 showed an adenosine content in the range of 2.58–4.33 μg / g, recording the highest value under all medium conditions. The highest adenosine content was measured at 4.33 μg / g in the mycelia of Cordyceps 2 cultured in a medium of 60% sword bean and 40% brown rice.

[0128] Adenosine content of Cordyceps (µg / g) Sample Adenosine content (µg / g) Types of mushroom mycelium shiitake oyster mushrooms situation Cordyceps 1 Cordyceps 2 deer buttocks Control group 1 (100% mealworms) 1.66 1.15 1.63 2.67 3.58 1.37 Control Group 2 (Mealworm 50%: Brown Rice 50%) 1.38 1.48 1.93 2.21 3.24 1.31 Sword bean 60%:brown rice 40% 2.24 1.68 2.12 3.16 4.33 2.57 Sword bean 40%:brown rice 60% 0.59 0.81 1.11 1.55 2.58 0.29 Sword bean 30%:brown rice 70% 1.22 0.74 2.48 2.75 3.29 1.52

[0130] As a result of Examples 1 to 5 above, Cordyceps 2 mycelium showed excellent culture efficiency and high content of functional components such as β-glucan, ergosterol, cordycepin, and adenosine, and was selected as the optimal mycelium most suitable for the purpose of the present invention, and the said Cordyceps 2 mycelium is Cordyceps registered under accession number KCCM13296P ( Cordyceps militaris ) JMIM00002 was used as mycelium in subsequent experiments.

[0132] Example 6. Free sugar content

[0133] The free sugar content of fermented Cordyceps mycelia cultured under various media compositions was analyzed to evaluate the effect of sword bean and brown rice-based media compositions on sugar production. Free sugars are low-molecular-weight sugars that perform various functions, such as supplying energy within cells, protecting cell membranes, and enhancing physiological activity; they are receiving particular attention as functional ingredients that improve palatability and bioavailability in health functional foods.

[0134] In this experiment, Cordyceps 2 mycelia were cultured using a medium prepared by mixing mealworms, brown rice, and sword beans in various proportions, and the free sugar content, such as arabinose, fructose, glucose, sucrose, and maltose, was analyzed from the fermented mycelia.

[0135] As a result, free sugars were detected in all media, and in particular, the sample cultured in a 60% sword bean : 40% brown rice medium showed the highest free sugar content at a total of 44.07 mg / g. Conversely, the sample in a 30% sword bean : 70% brown rice medium showed the lowest free sugar content at a total of 34.92 mg / g.

[0136] The above results indicate that a medium with a high content of sword bean is advantageous for improving total free sugar concentration and, in particular, provides an effective basis for the accumulation of glucose and sucrose. Therefore, the medium composition and culture conditions of the present invention have value as a source of free sugars effective for improving flavor and enhancing functionality.

[0137] Free sugar content (mg / g) of fermented Cordyceps mycelium Sample Free sugar content (mg / g) Types of mushroom mycelium arabinose fructose glucose sucrose maltose total Control group (Mealworm 50%: Brown rice 50%) 6.89 0.65 25.69 2.5 0.93 36.66 Sword bean 60%:brown rice 40% 5.09 0.98 31.45 6.34 0.21 44.07 Sword bean 40%:brown rice 60% 4.64 0.62 25.33 7.1 0.11 37.80 Sword bean 30%:brown rice 70% 4.48 0.89 22.52 6.89 0.14 34.92

[0139] Example 7. Free amino acid content

[0140] The free amino acid content of fermented Cordyceps mycelia cultured in various media compositions was analyzed to evaluate the protein metabolite production efficiency of the sword bean-brown rice-based media. Free amino acids are key functional substances that regulate in vivo metabolic activity and contribute to immune responses, antioxidant activity, anti-fatigue, and improved palatability.

[0141] In this experiment, a total of 16 types of amino acids were quantitatively analyzed, and among them, glutamic acid, lysine, and arginine were detected as major components. As a result of the analysis, free amino acids were detected evenly in all samples; in particular, the sample cultured in a medium of 60% sword bean and 40% brown rice showed the highest content, with total free amino acids (TAA) at 51.29 mg / g and total essential amino acids (EAA) at 15.53 mg / g. This suggests that the composition of the medium provides a fermentation environment favorable for protein degradation activity and amino acid production.

[0142] Free amino acid content (mg / g) of fermented Cordyceps mycelium Free amino acids Control group (Mealworm 50%: Brown rice 50%) Sword bean 60%:brown rice 40% Sword bean 30%:brown rice 70% Aspartic acid 3.88 4.58 3.82 Serine 0.90 0.96 0.90 Glutamic acid 4.91 5.83 5.31 Glycine 3.48 4.24 3.73 Histidine 0.52 0.60 0.53 Arginine 9.16 10.41 9.13 Threonine 0.95 1.04 0.97 Alanine 3.13 3.59 3.13 Proline 2.60 3.23 2.63 Tyrosine 0.48 0.59 0.47 Valine 4.24 5.20 4.51 Methionine 0.61 0.72 0.64 Lysine 5.18 5.81 5.41 Isoleucine 0.91 1.02 0.92 Leucine 1.00 1.14 1.04 Phenylalanine 1.94 2.33 1.81 Total Amino Acids (TAA) 43.89 51.29 44.95 Total Essential Amino Acids (EAA) 13.41 15.53 14.02 EAA / TAA(%) 30.55 30.28 31.19

[0144] Example 8. Anti-inflammatory activity

[0145] (1) Confirmation of cell viability (MTT analysis)

[0146] The effects of fermented sword bean and Cordyceps sinensis extracts on macrophage survival were investigated to examine toxicity prior to evaluating anti-inflammatory activity. RAW 264.7 mouse-derived macrophages were placed in 96-well plates at a rate of 1 × 10⁶ 5 After inoculating at a concentration of cells / mL, fermented extracts (hot water and ethanol extracts) were treated at concentrations of 0, 10, 50, 100, and 500 μg / mL, respectively. Cell viability was measured via MTT analysis after 24 hours.

[0147] As a result, cell viability of over 90% was maintained in all extract treatment groups, and in particular, the hot water and ethanol extracts of the 60% sword bean : 40% brown rice medium showed viability of over 97% and 95%, respectively, even at high concentrations (500 μg / mL), confirming that they had low cytotoxicity.

[0148] Cell viability of Cordyceps hot water extract (RAW 264.7, unit: %) Sample Cell survival rate (%) Concentration (µg / mL) 0 10 50 100 500 Control group (Mealworm 50%: Brown rice 50%) 100±0.7 98±0.5 96±0.7 94±0.5 91±0.7 Sword bean 60%:brown rice 40% 99±0.5 97±0.3 95±0.7 97±0.6 Sword bean 40%:brown rice 60% 98±0.7 94±0.2 88±0.3 84±0.3 Sword bean 30%:brown rice 70% 99±0.6 98±0.3 96±0.3 92±0.5

[0150] Cell viability of Cordyceps militaris ethanol extract (RAW 264.7, unit: %) Sample Cell survival rate (%) Concentration (µg / mL) 0 10 50 100 500 Control group (Mealworm 50%: Brown rice 50%) 100±0.7 97±0.2 94±0.7 93±0.2 90±0.7 Sword bean 60%:brown rice 40% 98±0.7 96±0.5 96±0.4 95±0.5 Sword bean 40%:brown rice 60% 98±0.2 92±0.8 85±0.5 83±0.6 Sword bean 30%:brown rice 70% 98±0.5 98±0.3 95±0.7 90±0.5

[0152] (2) Measurement of NO (Nitric oxide) production amount

[0153] We investigated whether the complex fermented extract of sword bean and Cordyceps sinensis has an inhibitory effect on the production of nitric oxide (NO), a major marker of inflammatory responses. NO is a marker induced during the inflammatory response of macrophages, and its excessive production is known to cause cell damage, tissue inflammation, and various inflammatory diseases. Therefore, the inhibitory effect on NO production is utilized as an important evaluation indicator for anti-inflammatory functional food materials.

[0154] In the experiment, macrophages derived from RAW 264.7 mice were pretreated with hot water or ethanol extracts at different concentrations (0, 100, 150, 200 μg / mL) for 3 hours, followed by treatment with LPS (1 μg / mL) to induce an inflammatory response. Subsequently, the NO concentration in the culture medium was measured using Griess reagent.

[0155] As a result, the group treated with LPS alone exhibited a high NO production of 54 μM, whereas NO production decreased in a concentration-dependent manner in all extract treatment groups. In particular, the extract prepared from a 60% sword bean : 40% brown rice medium showed the strongest NO inhibitory effect under both hot water and ethanol conditions.

[0156] NO production amount of Cordyceps hot water extract (Unit: μM) Sample NO production amount Concentration (µg / mL) 0 LPS 100 150 200 Control group (Mealworm 50%: Brown rice 50%) 6.5±0.3 54±0.9 45±0.8 39±0.3 29±0.7 Sword bean 60%:brown rice 40% 40±0.4 32±0.3 24±0.5 Sword bean 40%:brown rice 60% 49±0.5 42±0.2 33±0.4 Sword bean 30%:brown rice 70% 43±0.6 38±0.2 27±0.5

[0158] NO production amount of ethanol extract of Cordyceps (Unit: μM) Sample NO production amount Concentration (µg / mL) 0 LPS 100 150 200 Control group (Mealworm 50%: Brown rice 50%) 6.5±0.3 54±0.9 43±0.8 35±0.8 25±0.8 Sword bean 60%:brown rice 40% 38±0.8 28±0.8 20±0.5 Sword bean 40%:brown rice 60% 45±0.2 38±0.8 30±0.4 Sword bean 30%:brown rice 70% 39±0.5 34±0.8 24±0.5

[0160] (3) Measurement of inflammatory cytokine production

[0161] This study was conducted to determine whether the fermented extract of sword bean and Cordyceps sinensis has an inhibitory effect on the production of major inflammatory cytokines induced during the inflammatory response of macrophages. To confirm the inhibitory effect of each extract on the production of inflammation-related cytokines, the extracts were pretreated at different concentrations (0, 100, 150, 200 μg / mL), LPS was added, and the amount of cytokine produced was analyzed using ELISA.

[0163] (a) Inhibitory effect on IL-1β production

[0164] IL-1β is a representative cytokine secreted during the early stages of the inflammatory response; it promotes the production of NO and PGE2 and is involved in the pathophysiological processes associated with various inflammatory diseases. Therefore, the inhibitory effect on IL-1β production is a key indicator for evaluating the anti-inflammatory activity of the extract.

[0165] As a result, the hot water and ethanol extracts prepared in a medium of 60% sword bean and 40% brown rice showed the best inhibition of IL-1β production, and when treated at a high concentration (200 μg / mL), the hot water extract showed an inhibition of about 40.3% (293→175 pg / mL), and the ethanol extract showed an inhibition of about 41.6% (293→171 pg / mL).

[0166] Inhibitory effect of hot water extract on IL-1β production (Unit: pg / mL) Sample 1L-1β production amount Concentration (µg / mL) 0 LPS 100 150 200 Control group (Mealworm 50%: Brown rice 50%) 42±1.6 293±2.2 268±1.5 225±1.8 183±1.6 Sword bean 60%:brown rice 40% 260±2.1 218±1.5 175±1.5 Sword bean 40%:brown rice 60% 276±1.8 250±1.9 200±1.8 Sword bean 30%:brown rice 70% 266±1.7 223±1.7 181±1.9

[0168] Inhibitory effect of ethanol extract on IL-1β production (Unit: pg / mL) Sample 1L-1β production amount Concentration (µg / mL) 0 LPS 100 150 200 Control group (Mealworm 50%: Brown rice 50%) 42±1.6 293±2.2 264±1.9 221±1.7 180±1.6 Sword bean 60%:brown rice 40% 254±1.6 217±1.6 171±1.9 Sword bean 40%:brown rice 60% 271±2.0 246±2.1 196±2.2 Sword bean 30%:brown rice 70% 264±1.6 218±1.7 176±2.0

[0170] (b) Inhibitory effect on IL-6 production

[0171] IL-6 is known as a central inflammatory factor that induces the proliferation and differentiation of immune cells and mediates various physiological phenomena related to inflammatory responses. As a result of confirming the inhibition rate of IL-6 production, hot water and ethanol extracts prepared in a medium of 60% sword bean and 40% brown rice most effectively inhibited IL-6 production, and at a high concentration (200 μg / mL) compared to the LPS treatment group, they showed inhibition effects of approximately 59.7% (243→98 pg / mL) and 62.1% (243→92 pg / mL), respectively.

[0172] IL-6 production of hot water extract (Unit: pg / mL) Sample 1L-6 production volume Concentration (µg / mL) 0 LPS 100 150 200 Control group (Mealworm 50%: Brown rice 50%) 32±0.8 243±4.9 184±3.2 135±1.5 104±1.2 Sword bean 60%:brown rice 40% 175±2.1 131±3.2 98±2.6 Sword bean 40%:brown rice 60% 194±2.0 146±2.5 115±2.0 Sword bean 30%:brown rice 70% 183±1.2 135±2.6 103±2.2

[0174] IL-6 production of ethanol extract (Unit: pg / mL) Sample 1L-6 production volume Concentration (µg / mL) 0 LPS 100 150 200 Control group (Mealworm 50%: Brown rice 50%) 32±0.8 243±4.9 181±3.5 130±2.5 100±2.8 Sword bean 60%:brown rice 40% 172±3.3 124±3.4 92±2.6 Sword bean 40%:brown rice 60% 192±2.5 140±3.6 111±3.0 Sword bean 30%:brown rice 70% 180±3.5 130±3.5 100±2.9

[0176] (c) Inhibitory effect on TNF-α production

[0177] TNF-α is a representative pro-inflammatory cytokine that promotes inflammatory responses, activates immune cells, and induces tissue damage, and is one of the key indicators for evaluating the efficacy of anti-inflammatory materials. As a result of confirming the inhibition rate of TNF-α production, the extract prepared in a medium of 60% sword bean and 40% brown rice showed the most superior TNF-α inhibitory effect under both hot water and ethanol conditions. At high concentrations (200 μg / mL), inhibition was reduced to 355 pg / mL (hot water) and 344 pg / mL (ethanol), respectively, showing a reduction effect of approximately 48.0% to 49.6% compared to the LPS-treated group (683 pg / mL).

[0178] TNF-α production amount of hot water extract (Unit: pg / mL) Sample TNF-α production amount Concentration (µg / mL) 0 LPS 100 150 200 Control group (Mealworm 50%: Brown rice 50%) 71±2.1 683±4.3 526±3.5 430±3.0 380±2.9 Sword bean 60%:brown rice 40% 513±2.6 416±3.5 355±3.1 Sword bean 40%:brown rice 60% 546±2.9 462±3.1 416±3.4 Sword bean 30%:brown rice 70% 522±3.4 428±3.5 373±2.6

[0180] TNF-α production amount of ethanol extract (Unit: pg / mL) Sample TNF-α production amount Concentration (µg / mL) 0 LPS 100 150 200 Control group (Mealworm 50%: Brown rice 50%) 71±2.1 683±4.3 513±2.9 422±3.4 370±3.2 Sword bean 60%:brown rice 40% 505±3.1 410±3.1 344±2.6 Sword bean 40%:brown rice 60% 528±3.4 448±2.9 415±3.5 Sword bean 30%:brown rice 70% 514±2.6 415±3.5 359±4.3

[0182] Depository Name: Korean Culture Collection of Microorganisms (Overseas) Trustee Number: KCCM13296P Date of Deposit: 2022-12-02

Claims

Claim 1 delete Claim 2 delete Claim 3 (1) a step of preparing a culture medium by adding 5.5 to 6.5 parts by weight of sword bean and 3.5 to 4.5 parts by weight of brown rice to 25 to 35 parts by weight of water, soaking for 50 to 70 minutes, sterilizing at 110 to 130°C for 30 to 40 minutes, and cooling; (2) a Cordyceps sinensis with accession number KCCM13296P ( Cordyceps militaris A method for preparing a fermented sword bean cordyceps powder, characterized by comprising the steps of: (3) inoculating the mycelium of JMIM00002 and culturing it in the dark at 20~30℃ for 2~4 days, followed by culturing it in the light at 18~20℃ for 50~70 days; (2) sterilizing the photo-cultured fermented product from step (2) at 70~90℃ for 10~20 minutes, drying it at 60~80℃ for 10~14 hours, and then grinding it; and (4) adding 40~60% (v / v) ethanol in an amount of 8~12 times (v / w) to the fermented sword bean cordyceps product prepared in step (3), extracting it at 70~90℃ for 6~10 hours, concentrating it, freeze-drying it, and then grinding it. Claim 4 (1) A step of preparing pretreated sword beans by soaking sword beans for 4 to 6 hours, removing the moisture, roasting them at 110 to 130°C for 3 to 7 minutes, drying them at 45 to 55°C for 6 to 10 hours, and then cooling and freeze-drying them; (2) A step of preparing pretreated brown rice by soaking brown rice for 1 to 3 hours, removing the moisture, roasting it at 110 to 130°C for 1 to 3 minutes, grinding it, steaming it at 75 to 85°C for 15 to 25 minutes, cooling it, and then drying it at 70 to 90°C for 4 to 6 hours; (3) A step of preparing pretreated brown rice by adding 5.5 to 6.5 parts by weight of the sword beans pretreated in step (1) and 3.5 to 4.5 parts by weight of the brown rice pretreated in step (2) to 25 to 35 parts by weight of water and soaking them for 50 to 70 minutes Step of preparing a culture medium by sterilizing at 110~130℃ for 30~40 minutes and cooling; (4) Cordyceps sinensis with accession number KCCM13296P ( Cordyceps militaris A method for preparing a fermented sword bean cordyceps powder, characterized by comprising the steps of: inoculating the mycelium of JMIM00002 and culturing it in the dark at 20~30℃ for 2~4 days, followed by culturing it in the light at 18~20℃ for 50~70 days; (5) sterilizing the fermented product from step (4) at 70~90℃ for 10~20 minutes, drying it at 60~80℃ for 10~14 hours, and then grinding it to produce a complex fermented sword bean cordyceps; and (6) adding 40~60% (v / v) ethanol in an amount of 8~12 times (v / w) to the complex fermented sword bean cordyceps produced in step (5), extracting it at 70~90℃ for 6~10 hours, concentrating it, freeze-drying it, and then grinding it. Claim 5 Fermented sword bean cordyceps powder prepared by the method of paragraph 3 or 4. Claim 6 A processed food product manufactured using the fermented sword bean cordyceps powder of Paragraph 5.

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