Enzyme-treated mushroom composition with enhanced contents and Anti-inflammatory activity of betaglucan, seasoned salt containing the same, and method for manufacturing the same

Abstract

The present invention relates to an enzyme-treated pine mushroom extract in which the extraction yield of functional components of natural pine mushrooms is improved and antioxidant and anti-inflammatory substances are enhanced by steaming and enzyme-treating natural pine mushrooms, seasoning salt containing the same, and a method for manufacturing the same.

Description

Technology Field

[0001] The present invention relates to an enzyme-treated pine mushroom extract in which the extraction yield of functional components of natural pine mushrooms is improved and antioxidant and anti-inflammatory substances are enhanced by steaming and enzyme-treating natural pine mushrooms, seasoning salt containing the same, and a method for manufacturing the same. Background Technology

[0002] As interest in health intensifies day by day, there is a surging demand for materials and products capable of boosting the body's immunity to strengthen resistance to disease and maintain health. In particular, as viral diseases such as avian influenza spread globally, they pose a significant threat to people seeking to consume food safely. To address this, various immune-boosting health functional foods are being launched and widely distributed, with ginseng, chitosan products, and aloe being representative examples.

[0003] Meanwhile, beta-glucan is a representative indicator substance of medicinal mushrooms and is well known as a non-specific immune-enhancing substance. Beta-glucans contained in mushrooms such as Phellinus linteus, Shiitake mushrooms, Pleurotus ostreatus, and Trametes versicolor are known for their anticancer properties and are used as medical treatments. Additionally, beta-glucan known as zymosan, derived from yeast cell walls, is another representative immune-enhancing substance. Furthermore, beta-glucan secreted extracellularly by microorganisms of the genus Agrobacterium has been reported to possess excellent immune-enhancing capabilities. Since beta-glucan produced by microorganisms can be mass-produced with high purity, it has the advantage of being widely used as a health functional food and food additive. Structurally, microorganism-produced beta-glucan is characterized by glucose units consisting solely of beta 1,3 bonds; it is linear and has a molecular weight of approximately 300,000 daltons. Consequently, there is a demand for technology capable of obtaining health-beneficial beta-glucan with high yield. The problem to be solved

[0004] The present invention was devised to solve the above-mentioned requirements and aims to provide an enzyme-treated pine mushroom extract in which the extraction yield of functional components of natural pine mushrooms is improved and antioxidant and anti-inflammatory substances are enhanced by steaming and enzyme-treating natural pine mushrooms.

[0005] In addition, the present invention aims to provide seasoning salt containing the above-mentioned pine mushroom extract as an active ingredient.

[0006] Furthermore, the present invention aims to provide a method for manufacturing the above-mentioned pine mushroom extract and a method for manufacturing the above-mentioned seasoning salt. Through this, the invention aims to provide seasoning salt capable of preventing health hazards caused by the consumption of excessive salt.

[0007] The objectives of the present invention are not limited to those mentioned above. The objectives of the present invention will become clearer from the following description and will be realized by the means and combinations thereof described in the claims. means of solving the problem

[0008] A method for preparing a functional pine mushroom extract according to one embodiment of the present invention may include the steps of: steaming a pine mushroom at 70°C to 80°C for 30 minutes to 1 hour; drying the steamed pine mushroom; grinding the dried material; and enzymatically reacting a mixture of the ground material and a cell wall degrading enzyme at 40°C to 60°C for 3 hours to 5 hours.

[0009] The above cell wall degrading enzyme may include at least one of Viscozyme L, Celluclast, Shearzyme, and Frontia Fiberwash K.

[0010] The above extract may be characterized by an increased beta-glucan content.

[0011] The above extract may be characterized by enhanced alpha-glucosidase (α-Glucosidase) inhibitory ability.

[0012] The above extract may be characterized by enhanced nitric oxide (NO) activity inhibitory ability.

[0013] The above extract may be characterized by enhanced interleukin-6 (IL-6) activity inhibitory ability.

[0014] A method for producing functional seasoning salt according to another embodiment of the present invention may include the steps of: steaming matsutake mushrooms at 70°C to 80°C for 30 minutes to 1 hour; drying the steamed matsutake mushrooms; grinding the dried material; preparing a matsutake mushroom extract by enzymatically reacting a mixture of the ground material with a cell wall degrading enzyme at 40°C to 60°C for 3 hours to 5 hours; and mixing the matsutake mushroom extract with salt.

[0015] The above salt may include at least one of refined salt, solar salt, and processed salt.

[0016] The weight ratio of the above matsutake mushroom extract and salt may be 8:1 to 10:1.

[0017] A food composition for anti-diabetic, anti-inflammatory, and antioxidant purposes according to another embodiment of the present invention comprises a matsutake mushroom extract as an active ingredient, and the matsutake mushroom extract may be extracted from steamed matsutake mushrooms using hot water at 40°C to 60°C with added cell wall degrading enzymes as a solvent.

[0018] A functional seasoning salt according to another embodiment of the present invention may include pine mushroom extract and salt. Effects of the invention

[0019] By utilizing the present invention, the extraction yield of functional components of wild pine mushrooms is improved, and a composition comprising an enzyme-treated pine mushroom extract with enhanced antioxidant and anti-inflammatory substances can be provided. The pine mushroom extract exhibits excellent alpha-glucosidase (α-Glucosidase) inhibitory activity and excellent inhibition of the activity of antioxidant and anti-inflammatory factors such as nitric oxide (NO) and IL-6.

[0020] Therefore, using the seasoned salt according to the present invention can prevent health hazards caused by excessive salt intake.

[0021] The effects of the present invention are not limited to those mentioned above. It should be understood that the effects of the present invention include all effects that can be inferred from the following description. Brief explanation of the drawing

[0022] FIG. 1 schematically illustrates the manufacturing process of a functional seasoning salt according to one embodiment of the present invention. Figure 2 is a graph confirming the NO production inhibitory activity of a pine mushroom extract according to one experimental example of the present invention. Figure 3 is a graph confirming the IL-6 production inhibitory activity of a pine mushroom extract according to one experimental example of the present invention. Specific details for implementing the invention

[0023] The above objects, other objects, features, and advantages of the present invention will be easily understood through the following preferred embodiments associated with the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to ensure that the disclosed content is thorough and complete and to ensure that the spirit of the invention is sufficiently conveyed to a person skilled in the art.

[0024] Terms such as "first," "second," etc., may be used to describe various components, but said components should not be limited by said terms. These terms are used solely for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, the first component may be named the second component, and similarly, the second component may be named the first component. A singular expression includes a plural expression unless the context clearly indicates otherwise.

[0025] In this specification, terms such as "comprising" or "having" are intended to specify the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof. Furthermore, when a part such as a layer, film, region, or plate is described as being "on" another part, this includes not only the case where it is "immediately above" the other part, but also the case where there is another part in between. Conversely, when a part such as a layer, film, region, or plate is described as being "below" another part, this includes not only the case where it is "immediately below" the other part, but also the case where there is another part in between.

[0026] Unless otherwise specified, all numbers, values, and / or expressions used herein to represent amounts of ingredients, reaction conditions, compositions, and formulations should be understood to be modified by the term “approximately” in all cases, as these numbers are essentially approximations reflecting the various uncertainties of measurement that occur in obtaining these values ​​among other things. Furthermore, where numerical ranges are disclosed herein, such ranges are continuous and, unless otherwise indicated, include all values ​​from the minimum value of such range to the maximum value including the maximum value. Moreover, where such ranges refer to integers, they include all integers from the minimum value to the maximum value including the maximum value, unless otherwise indicated.

[0027] In this specification, where a range is described for a variable, it will be understood that the variable includes all values ​​within the described range, including the described endpoints of the range. For example, the range “5 to 10” will be understood to include not only the values ​​5, 6, 7, 8, 9, and 10, but also any sub-ranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc., and any values ​​between integers valid for the category of the described range, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9. Also, for example, the range “10% to 30%” will be understood to include all integers including values ​​such as 10%, 11%, 12%, 13%, etc. and up to 30%, as well as any sub-range such as 10% to 15%, 12% to 18%, 20% to 30%, etc., and any value between valid integers within the stated range category such as 10.5%, 15.5%, 25.5%, etc.

[0029] One example of the present invention relates to a method for preparing a functional pine mushroom extract comprising the steps of: steaming pine mushrooms at 70°C to 80°C for 30 minutes to 1 hour; drying the steamed pine mushrooms; grinding the dried material; and enzymatically reacting the mixture of the ground material with a cell wall degrading enzyme at 40°C to 60°C for 3 hours to 5 hours.

[0030] In the present invention, the pine mushroom is a natural pine mushroom ( Tricholoma matsutake It can be.

[0031] In the present invention, the pine mushroom is an edible mushroom of the genus Trichosanthes in the family Trichosanthesaceae. It grows mainly in places where pine needles have accumulated and is distributed evenly across the Eurasian and North American continents, including northeastern and southern China, Taiwan, the Korean Peninsula, and Japan. The diameter of the cap is about 8 to 10 cm, and the surface may have grayish-brown or fibrous dark brown scales. The stem is cylindrical and may be white, and it is generally harvested from nature.

[0032] In the present invention, since pine mushrooms are generally distributed in their natural state with soil attached to the root portion, the pine mushrooms used in the present invention may be washed to remove foreign substances such as soil.

[0033] In the present invention, the steaming step may involve cooking the pine mushrooms. Steaming generally refers to the process of cooking ingredients, and may mean cooking the pine mushrooms until they become soft and edible.

[0034] In the present invention, the steaming step may be performed at 70°C to 80°C for 30 minutes to 1 hour to remove undesirable components, microorganisms, scents, etc. while minimizing changes in the components of the pine mushroom.

[0035] In the present invention, the drying step may be a step of preparing a dried product by drying steamed pine mushrooms, and the drying time and temperature are not particularly limited and may be performed according to known methods at room temperature or in a dryer to the extent that the moisture of the steamed pine mushrooms is almost completely removed.

[0036] In the present invention, the grinding step may be a step of grinding dried matsutake mushrooms, and the size of the ground particles is sufficient as long as it can react smoothly with cell wall degrading enzymes and is not particularly limited.

[0037] In the present invention, the cell wall degrading enzyme may include at least one of Viscozyme L, Celluclast, Shearzyme, and Frontia Fiberwash K, and may be, for example, Viscozyme L.

[0038] In the present invention, Viscozyme L may be an enzyme comprising arabanase, cellulase, beta-glucanase, hemicellulase, and xylanase.

[0039] In the present invention, the concentration of the cell wall degrading enzyme may be 0.1% (v / v) to 3% (v / v), for example, 0.5% (v / v) to 1.5% (v / v), but is not limited thereto.

[0040] In the present invention, the step of enzymatic reaction may involve reacting a mixture of the crushed material and a cell wall degrading enzyme at 40°C to 60°C for 3 to 5 hours to enzymatically react the dried matsutake mushroom contained in the crushed material.

[0041] In the present invention, the step of enzymatic reaction may involve mixing a cell wall degrading enzyme at a ratio of 1% by weight relative to the dry weight of the ground material, but is not limited thereto.

[0042] In the present invention, the method for preparing a matsutake mushroom extract may include the step of filtering and vacuum concentrating the extract according to known methods. That is, extracts that have undergone a conventional purification process after extraction may also be included in the extract of the present invention without limitation. For example, fractions obtained through various additional purification methods, such as separation using an ultrafiltration membrane having a specific molecular weight cut-off value or separation by various chromatographs (designed for separation based on size, charge, hydrophobicity, or affinity), are also included in the extract of the present invention. Furthermore, the extract of the present invention may also be prepared in a powder state by additional processes such as vacuum distillation, freeze-drying, or spray drying.

[0043] In the present invention, the pine mushroom extract may include an enzymatic reaction product generated from natural pine mushrooms steamed with a cell wall degrading enzyme.

[0044] In the present invention, the pine mushroom extract is characterized by an increased content of beta-glucan.

[0045] In the present invention, the pine mushroom extract may be characterized by having enhanced alpha-glucosidase (α-Glucosidase) inhibitory ability.

[0046] In the present invention, the pine mushroom extract may be characterized by having an enhanced ability to inhibit nitric oxide (NO) activity.

[0047] In the present invention, the pine mushroom extract may be characterized by having enhanced IL-6 activity inhibitory ability.

[0048] In the present invention, since the pine mushroom extract has excellent inhibitory ability against anti-diabetic, anti-inflammatory, and antioxidant factors as described above, the pine mushroom extract can be used as a food composition for anti-diabetic, anti-inflammatory, and antioxidant purposes.

[0049] Another example of the present invention relates to a method for producing functional seasoning salt, comprising the steps of: steaming matsutake mushrooms at 70°C to 80°C for 30 minutes to 1 hour; drying the steamed matsutake mushrooms; grinding the dried material; preparing a matsutake mushroom extract by enzymatically reacting the mixture of the ground material with a cell wall degrading enzyme at 40°C to 60°C for 3 hours to 5 hours; and mixing the matsutake mushroom extract with salt.

[0050] In the present invention, the step of preparing the pine mushroom extract may include substantially the same content as the method for preparing the pine mushroom extract described above, and a repeated description is omitted within the scope of overlap.

[0051] In the present invention, salt may include at least one of refined salt, solar salt, and processed salt, and may include, for example, solar salt, but is not limited thereto.

[0052] In the present invention, the step of mixing the pine mushroom extract with salt may involve mixing the pine mushroom extract and salt in a weight ratio of 8:1 to 10:1, and, for example, may involve mixing in a ratio of 9:1.

[0053] In one embodiment, the matsutake mushroom extract may be in the form of a concentrated liquid. The functional seasoning salt may contain 10% (w / w) to 30% (w / w) of the concentrated liquid matsutake mushroom extract based on the total weight, which may be substantially the same as containing 10% (w / w) to 40% (w / w) of the solid powder form (based on Brix) matsutake mushroom extract.

[0054] Another example of the present invention relates to a food composition for anti-diabetic, anti-inflammatory, and antioxidant purposes, comprising a matsutake mushroom extract as an active ingredient, wherein the matsutake mushroom extract is extracted from steamed matsutake mushrooms using hot water at 40°C to 60°C to which a cell wall degrading enzyme is added as a solvent.

[0055] In the present invention, the food composition can be used as an anti-diabetic food composition due to the enhanced alpha-glucosidase inhibitory ability of the pine mushroom extract.

[0056] In the present invention, the food composition can be used as an anti-inflammatory and antioxidant food composition due to the enhanced inhibitory activity of nitric oxide (NO) and IL-6 possessed by the pine mushroom extract.

[0057] In the present invention, in addition to containing the active ingredient of the present invention, the food composition may contain various flavoring agents or natural carbohydrates, etc., as additional ingredients without limitation, as in conventional food compositions.

[0058] Examples of natural carbohydrates include monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; and polysaccharides, such as conventional sugars like dextrin, cyclodextrin, etc.; and sugar alcohols such as xylitol, sorbitol, erythritol, etc. The above-mentioned flavoring agents may advantageously use natural flavoring agents (taumatin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention may be formulated in the same manner as the pharmaceutical composition above and used as a functional food or added to various foods. Foods to which the composition of the present invention may be added include, for example, beverages, meat, chocolate, food products, confectionery, pizza, ramen, other noodles, chewing gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0059] In addition, the above food composition may contain, in addition to the extract which is an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, etc.

[0060] Another example of the present invention relates to a functional seasoning salt comprising matsutake mushroom extract and salt.

[0061] In the present invention, the functional seasoning salt is prepared according to the method of preparing seasoning salt using the aforementioned pine mushroom extract, and may include substantially the same content as the preparation method; therefore, a repeated description is omitted within the scope of overlap.

[0062] In the present invention, functional seasoning salt refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body in accordance with the Act on Health Functional Foods, and means consuming it for the purpose of obtaining effects useful for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body. The functional seasoning salt of the present invention may include conventional food additives, and unless otherwise stipulated, suitability as a food additive may be determined in accordance with the specifications and standards for the relevant item based on food industry compendiums and general test methods approved by the Ministry of Food and Drug Safety.

[0063] Other forms of the present invention will be described in more detail below through manufacturing examples and experimental examples. The following manufacturing examples and experimental examples are merely illustrative to aid in understanding the present invention and do not limit the scope of the present invention.

[0065] Preparation Example 1. Preparation of Matsutake mushroom extract

[0066] Washed wild matsutake mushrooms were steamed in a steamer at 70–80°C for 1 hour, dried, and then ground into an appropriate size. 20 ml of distilled water was added to 1 g of the ground matsutake mushroom powder, and four commercially available enzymes (Viscozyme L, Celluclast, Shearzyme, Frontia Fiberwash K) were added at 1% by weight relative to the powder (0.01–1% by weight of enzyme relative to the powder weight of the matsutake mushroom extract), and the mixture was reacted at 50°C for 3 hours. The enzyme reaction product was filtered and concentrated to complete the preparation of the extract.

[0067] division Beta-glucan content (%) Extraction yield (%) Comparative Example 1 Control 14.6 8.2 Comparative Example 2 Steaming O + Enzyme X 31.7 9.8 Example 1 Viscozyme L 45.7 22.3 Example 2 Celluclast 28.9 18.0 Example 3 Shearzyme 21.1 17.4 Example 4 Frontia Fiberwash K 34.2 19.2

[0068] As shown in Table 1, Viscozyme L had the highest beta-glucan content compared to other enzymes, and also showed the best results in terms of extraction yield at 22.3%, which represents the dry weight of the total extract containing beta-glucan relative to the dry weight of the matsutake mushroom. This is an improved result compared to the hot water extraction control group without added enzymes.

[0070] Preparation Example 2. Preparation of seasoning salt containing matsutake mushroom extract

[0071] Seasoning salt can be prepared by mixing the matsutake mushroom extract according to Example 1 of Preparation Example 1 with salt, sun-dried salt, or refined salt having a sodium chloride content of 70% or more. The salt and matsutake mushroom extract are mixed in a weight ratio of 9:1, and the matsutake mushroom extract is mixed according to a conventional method, but the ratio may vary slightly depending on the moisture content.

[0072] Specifically, a prepared matsutake mushroom extract can be mixed with salt at 50–60°C to prepare a mixed dough, and the mixed dough can be dried at 65–75°C and then ground to produce seasoned salt containing functional ingredients.

[0073] The above seasoning salt contains 1 to 20 weight percent of matsutake mushroom extract and is characterized by having a moisture content of less than 5% measured after being left for 24 hours under conditions of 20°C and 80% relative humidity.

[0074] The process for manufacturing seasoned salt according to Manufacturing Example 1 and Manufacturing Example 2 is schematically shown in FIG. 1.

[0076] Experimental Example 1. Establishment of Optimal Extraction Conditions

[0077] Experiments were conducted to establish optimal conditions for extracting pine mushrooms using the enzyme Viscozyme L. 20 ml of distilled water was added to 1 g of dried pine mushroom powder, and the antioxidant activity was evaluated according to the concentration of Viscozyme L. The results are shown in Table 2.

[0078] density(%) Beta-glucan content (%) Extraction yield (%) 0 14.6 8.2 0.1 15.1 10.1 0.2 16.8 11.6 0.5 21.7 12.9 1.0 45.7 22.3 1.5 45.5 22.2 2.0 45.8 22.4

[0079] Referring to Table 2, the extraction yield was 22.3% when the concentration of Viscozyme L enzyme was 1% (v / v), and it can be seen that the extraction yield did not increase at higher concentrations. Therefore, it can be confirmed that Viscozyme L at a concentration of 1% had the highest extraction yield of beta-glucan relative to the enzyme input.

[0081] Experimental Example 2. Comparison of Antioxidant Activities of Enzyme Extracts through Radical Scavenging Ability

[0082] 2-1. Measurement of DPPH Radical Scavenging Activity of Extracts

[0083] To confirm the antioxidant activity of the enzyme extract of the present invention, the DPPH radical scavenging activity of the control group (ascorbic acid), Comparative Example 1 (Matsutake mushroom hot water extract), Example 1 (steamed enzyme-treated Matsutake mushroom extract), and Comparative Example 2 (steamed Matsutake mushroom extract) was measured and compared.

[0084] The DPPH assay is one of the most common tests for measuring antioxidant efficacy. The test method evaluated the change in color of 1,1-diphenyl-2-picrylhydrazyl, which is dark purple, to pale yellow when reduced by an antioxidant. The radical activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH, Sigma-Aldrich, St. Louis, MO, USA) was measured as follows. 190 µl of DPPH dissolved in ethanol was added to 10 µl of each hot-water extract and reacted for 30 minutes in a dark room of a 37°C incubator, after which the absorbance was measured at 515 nm (VersaMax Microplate reader; Molecular Devices, Sunnyvale, CA, USA).

[0085] The DDPH radical scavenging activity was calculated by the following formula.

[0086] - DPPH radical scavenging activity (%) = [1 - ( Aa / Ab ) ] X 100

[0087] - Aa: Absorbance of the extract-added group, Ab: Absorbance of the untreated group

[0089] 2-2. Measurement of ABTS Radical Scavenging Activity of Extracts

[0090] In addition, the ABTS radical scavenging activity of the control group (ascorbic acid), Comparative Example 1 (matsutake mushroom hot water extract), Example 1 (steamed enzyme-treated matsutake mushroom extract), and Comparative Example 2 (steamed matsutake mushroom extract) was measured as an antioxidant activity using ABTS radicals by the ABTS+cation decolorization assay.

[0091] For the measurement of ABTS+ radical cation scavenging activity, 7.4 mM 2,2'azino-bis-3-rthylbenzothiaoline-6-sulfonic acid (ABTS, Sigma-Aldrich, St. Louis, MO, USA) and 2.5 mM potassium persulfate were dissolved in distilled water and mixed in a 1:1 ratio. The mixture was then reacted in a dark room at room temperature for more than 15 hours to form ABTS+ radicals. Before reacting with the extract at 732 nm (Versa Max microplate reader; Molecular Devices), the absorbance of the ABTS+ solution was 0.70 (±0.01), and it was diluted with distilled water.

[0092] Next, 10 µl of the control group, Comparative Example 1, Example 1, and Comparative Example 2 were added to 190 µl of ABTS+ solution and reacted for 5 minutes at room temperature in a dark room, and the absorbance was measured at 732 nm. The ABTS radical scavenging activity was calculated by the following formula.

[0093] - ABTS+ radical scavenging activity (%) = [1 - ( Aa / Ab ) ] X 100

[0094] - Aa: Absorbance of the extract-added group, Ab: Absorbance of the untreated group

[0096] 2-3. SOD-like activity test

[0097] Superoxide dismutase-like activity was tested. 20 mg of powder samples of the control group (ascorbic acid), Comparative Example 1 (Matsutake mushroom hot water extract), Example 1 (steamed enzyme-treated Matsutake mushroom extract), and Comparative Example 2 (steamed Matsutake mushroom extract) were placed in a 5 ml centrifuge tube, and 5 ml of distilled water was added to disperse them to prepare the samples.

[0098] 0.2 mL of sample, 3 mL of Tris-HCl buffer (50 mM Tris(hydroxymethyl) aminomethane + 10 mM EDTA, pH adjusted to 8.5), and 0.2 mL of 7.2 mM pyrogallol (Yakuri Pure Chemicals Co., Kyoto, Japan) were added and reacted at 25 ℃ for 10 minutes, and the reaction was stopped by adding 1 mL of 1 N HCl. The amount of oxidized pyrogallol in the reaction solution was measured by absorbance using a UV / VIS spectrophotometer at 420 nm, and SOD-like activity was expressed as the percentage difference in absorbance between the added group and the unadded group according to the following equation of the sample solution.

[0099] - SOD-like activity (%) = (1-(Aa / Ab))×100

[0100] - Aa: Absorbance of the sample-added group, Ab: Absorbance of the sample-free group

[0101] The results of measuring DPPH radical scavenging activity, ABTS radical scavenging activity, and SOD-like activity are shown in Table 3.

[0102] division DPPH radical scavenging activity (%) (Extract concentration: 500 ug / ml) ABTS radical scavenging activity (%) (Extract concentration: 500 ug / ml) SOD-like activity Control group (Ascorbic acid) 89.3 92.2 85.3 Example 1 (Steamed Enzyme-Treated Matsutake Mushroom Extract) 51.4 61.2 32.7 Comparative Example 1 (Matsutake mushroom hot water extract) 16.4 11.5 4.5 Comparative Example 2 (Steamed Matsutake Mushroom Extract) 31.1 35.6 21.5

[0103] Referring to Table 3, when the concentration of each extract is the same at 500 ug / ml, it can be confirmed that the DPPH radical scavenging activity of the steamed enzyme-treated matsutake mushroom extract of the present invention is higher than that of the hot water extract of Comparative Example 1.

[0104] In addition, when the concentration of each extract is the same at 500 ug / ml, it can be confirmed that the steamed enzyme-treated matsutake mushroom extract has an ABTS radical scavenging ability approximately 50% higher than Comparative Example 2.

[0106] Experimental Example 3. Comparison of Alpha-Glucosidase Inhibitory Activity of Enzyme Extracts

[0107] The alpha-glucosidase inhibitory activity of the control group [Acarbose], Comparative Example 1 (Matsutake mushroom hot water extract), Example 1 (steamed enzyme-treated extract), and Comparative Example 2 (steamed extract) was measured and compared.

[0108] Alpha-glucosidase inhibitory activity was measured by the following method. 120 µl of 0.19 U / ml alpha-glucosidase (α-Glucosidase) enzyme solution (in 0.1M PBS, pH 6.8) was mixed with 10 µl of the sample containing the dissolved extract and pre-incubated at 37°C for 10 minutes. Subsequently, 70 µl of 4 mM β-alpha-glucosidase (β-NPG, Sigma-Aldrich, St. Louis, MO, USA; in 0.1M PBS, pH 6.8) was added, and after reacting at 37°C for 10 minutes, the amount of β-nitrophenol produced was measured at 400 nm (VersaMax microplate reader; Molecular Devices), and the results are shown in Table 4.

[0109] Alpha-glucosidase (α-Glucosidase) inhibitory activity was calculated using the following formula.

[0110] - Alpha-glucosidase (α-Glucosidase) inhibitory activity (%) = [1 - ( Aa / Ab ) ] X 100

[0111] - Aa: Absorbance of the extract-added group, Ab: Absorbance of the untreated group

[0112] division Alpha-glucosidase (α-Glucosidase) inhibitory activity (%) Control group (Acarbose, 100 mg / ml) 81.3 Example 1 (Steamed Enzyme-Treated Matsutake Mushroom Extract) 41.8 Comparative Example 1 (Matsutake mushroom hot water extract) 25.4 Comparative Example 2 (Steamed Matsutake Mushroom Extract) 29.5

[0113] Referring to Table 4, it can be confirmed that the steamed enzyme-treated pine mushroom extract of the present invention exhibits an alpha-glucosidase (α-Glucosidase) activity inhibitory effect improved by approximately 60% compared to the hot water extract. From the above results, it can be inferred that the steamed enzyme-treated pine mushroom extract of the present invention exhibits an improved anti-diabetic effect.

[0115] Experimental Example 4. Comparison of Anti-inflammatory Activity

[0116] 4-1. Cell Line Culture

[0117] The RAW 264.7 cell line, a murine macrophage cell line, was obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea) and used in the experiment. For cell growth, Dulbeco's modified Eagle's medium high glucose (DMEM) supplemented with 10% FBS (fetal bovine serum) and 1% antibiotics (penicillin / streptomycin) was used as the medium, and the cells were cultured in a 37°C incubator maintaining 5% CO2. The cultured cells were used in the experiment with the medium changed every 2-3 days, and when the cells had grown to over 80%, they were washed with phosphate buffered saline (PBS) and subcultured.

[0119] 4-2. Measurement of Cell Viability

[0120] Cell viability was measured using the MTT assay. RAW 264.7 cells were placed in a 96-well plate at a rate of 1 × 10⁶ 4Cells were dispensed at a concentration of cells / ml and cultured for 24 hours in a 37°C, 5% CO2 incubator. The cultured cells were treated with test samples at various concentrations (0, 100 µg / ml, 500 µg / ml, 1,000 mg / ml) and reacted for 24 hours. Subsequently, 100 µl of a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium (MTT) solution prepared at a concentration of 5 mg / mL was added to each sample, followed by 2 hours of incubation. After incubation, the MTT solution was removed, and 100 µl of dimethyl sulfoxide (DMSO) was added to completely dissolve the formazan. The absorbance was then measured at a wavelength of 540 nm using a microplate reader, and the viability was calculated as a percentage. At this time, the measured absorbance represents the amount of MTT reduced to formazan by the action of mitochondrial dehydrogenase in surviving cells, and is proportional to the number of surviving cells. The survival rate of cells pretreated with the test sample was expressed as a percentage relative to the average survival rate of the group without sample addition.

[0121] Concentration (ug / ml) 0 100 500 1,000 Comparative Example 1 100±1.2 98.4±0.6 99.4±2.7 82.3±0.4 Comparative Example 2 100±0.8 99.4±2.1 102.3±0.2 89.5±3.1 Example 1 100±0.6 101.2±1.0 98.7±0.5 92.4±1.6

[0122] Referring to Table 5, it can be seen that cell viability decreases slightly when the sample is treated at a concentration of 1,000 ug / ml. Accordingly, the antioxidant activity was measured by setting the sample concentration to 500 ug / ml.

[0124] 4-3. Measurement of NO production inhibitory activity

[0125] 1×10⁶ RAW 264.7 cells in a 24-well plate 5Cells were dispensed at a concentration of cells / ml and cultured for 24 hours in a 37°C, 5% CO2 incubator. The cultured cells were treated with Comparative Example 1, Comparative Example 2, and Example 1 at a concentration of 500 µg / ml and cultured for 2 hours. Afterward, lipopolysaccharides (LPS) were added to a final concentration of 1 µg / ml and stimulated for 24 hours. Subsequently, 100 µl of the culture supernatant was taken and transferred to a new 96-well plate. Then, 100 µl of Griess reagent [1% (w / v) sulfanilamide, 0.1% (w / v) naphylethylenediamine in 2.5% (v / v) phosphoric acid] was added to each well and reacted at room temperature for 15 minutes, after which the absorbance was measured at 540 nm. A standard calibration curve was constructed using sodium nitrite and used for content calculation.

[0127] 4-4. Measurement of Inhibitory Activity on Inflammatory Cytokine Production

[0128] 1×10⁶ RAW 264.7 cells in a 24-well plate 5 Cells were dispensed at a concentration of cells / ml and cultured in a 37°C, 5% CO2 incubator for 24 hours. Then, LPS (1 µg / mL) and Comparative Example 1, Comparative Example 2, and Example 1 at a concentration of 500 µg / ml were simultaneously treated and cultured in a 37°C, 5% CO2 incubator for 24 hours. Afterward, the cell culture medium was centrifuged at 10,000 rpm for 3 minutes to remove the precipitate, and the supernatant was collected. The amount of pro-inflammatory cytokine produced was then measured using a Mouse IL-6 ELISA Kit.

[0129] The results of measuring NO production inhibition and IL-6 production inhibition activities are shown in Figures 2 and 3 and Table 6.

[0130] division NO (%) IL-6 (%) Control(LPS (-) ) 0 0 Control(LPS (+) ) 100 100 Comparative Example 1 (500 ug / ml) 92.1 89.7 Comparative Example 2 (500 ug / ml) 65.7 74.3 Example 1 (500 ug / ml) 56.4 61.2

[0131] Referring to Figures 2, 3 and Table 6, the enzyme-treated extract of steamed pine mushrooms according to Example 1 showed the best NO production inhibition and IL-6 production inhibition activities compared to the hot water extract of pine mushrooms of Comparative Example 1 and the hot water extract of steamed pine mushrooms of Comparative Example 2, based on the control group.

Claims

Claim 1 A method for preparing a functional pine mushroom extract comprising the steps of: steaming the pine mushrooms at 70°C to 80°C for 30 minutes to 1 hour; drying the steamed pine mushrooms; grinding the dried material; and enzymatically reacting the mixture of the ground material with a cell wall degrading enzyme at 40°C to 60°C for 3 hours to 5 hours, wherein the cell wall degrading enzyme is Viscozyme L. Claim 2 A method for preparing a functional pine mushroom extract according to claim 1, characterized in that the cell wall degrading enzyme is at a concentration of 1% (v / v) to 2% (v / v) relative to the total volume of the reaction solution obtained by mixing pine mushroom powder and distilled water. Claim 3 A method for preparing a functional pine mushroom extract according to claim 1, characterized in that the extract has an increased content of beta-glucan. Claim 4 A method for preparing a functional pine mushroom extract according to claim 1, characterized in that the extract has enhanced alpha-glucosidase inhibitory ability. Claim 5 A method for preparing a functional pine mushroom extract according to claim 1, characterized in that the extract has enhanced nitric oxide (NO) activity inhibitory ability. Claim 6 A method for preparing a functional pine mushroom extract according to claim 1, characterized in that the extract has enhanced interleukin-6 (IL-6) activity inhibitory ability. Claim 7 A method for producing functional seasoning salt comprising the steps of: steaming matsutake mushrooms at 70°C to 80°C for 30 minutes to 1 hour; drying the steamed matsutake mushrooms; grinding the dried material; preparing a matsutake mushroom extract by enzymatically reacting a mixture of the ground material with a cell wall degrading enzyme at 40°C to 60°C for 3 hours to 5 hours; and mixing the matsutake mushroom extract with salt, wherein the cell wall degrading enzyme is Viscozyme L. Claim 8 In claim 7, the method for manufacturing a functional seasoning salt comprises at least one of refined salt, solar salt, and processed salt. Claim 9 A method for manufacturing functional seasoning salt according to claim 7, wherein the weight ratio of the matsutake mushroom extract to the salt is 8:1 to 10:

1. Claim 10 A food composition for anti-diabetic, anti-inflammatory, and antioxidant purposes, comprising a pine mushroom extract as an active ingredient, wherein the pine mushroom extract is obtained by extracting steamed pine mushrooms using hot water at 40°C to 60°C to which a cell wall degrading enzyme is added as a solvent, and wherein the cell wall degrading enzyme is Viscozyme L. Claim 11 delete Claim 12 A functional seasoning salt comprising: a matsutake mushroom extract; and salt, wherein the matsutake mushroom extract is obtained by extracting steamed matsutake mushrooms using hot water at 40°C to 60°C to which a cell wall degrading enzyme is added as a solvent, and the cell wall degrading enzyme is Viscozyme L. Claim 13 delete Claim 14 In claim 12, the functional seasoning salt in which the weight ratio of the matsutake mushroom extract to the salt is 8:1 to 10:1.

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