KR101068692B1 - Immunological functions-improving health food containing ?-glucan derived from Sangwhang mushroom - Google Patents

Abstract

The present invention relates to a health supplement for improving immune function comprising beta glucan derived from Saccharomycetes fruiting body, and more particularly, dried, pulverized, hot water extraction, filtration, redrying, pulverization Β-glucan content is 87 ~ 162 mg / g to give the extract from the spores formed in the fruiting body, the appearance is brown powder, the daily dose (3.3 g / day) appeared to be suitable In addition to the excellent effect in the industry, it is used in the confectionery, bakery, noodle making, has an excellent effect in the health food industry.

Fruiting body, β-glucan, brown powder, dose, biopharmaceutical, health food industry

Description

Immunological functions-improving health food containing β-glucan derived from Sangwhang mushroom}

The present invention relates to a dietary supplement for improving immune function, characterized by containing β-glucan derived from Phellinus linteus as an active ingredient.

More specifically, after growing the fruiting bodies of the situation mushrooms, the fruiting bodies obtained by artificially forming a large amount of spores are obtained, and the brown powder containing a large amount of beta-glucan (β-glucan) component, which is accumulated from the fruiting bodies and the spores, is food. It relates to a health supplement for improving immune function added to.

Sangwhang mushroom is a Phellius linteus, a scientific name that has been used for stomach pain and knee joint pain since ancient times. It is very difficult to obtain the fruiting body in nature, and most spores of other poisonous mushrooms have been found in the wind or insects. In case of contact and eating it, it is often killed immediately.

Situation mushroom is a woody mildew fungus belonging to the Aphylloporales spores of the family Polyporaceae, and for this reason, its spores have been cultivated in liquid to produce polysaccharides, polysaccharides, to obtain anticancer immune substances. (Korean New Drug Registration No. 10-455906 and KIST Registration 174433)

In particular, all of the above patents are liquid culture of the mycelium, ethanol extraction of active ingredients such as α-heteroglycan, galactomannoglucan, or extracts and purification of polysaccharides consisting of glucose, galactose, mannose, gyros, arabinose Yes is disclosed.

In addition, as a situation mushroom mycelium cultivation method, Patent Publication No. 10-205-48748 is disclosed.

In contrast, the method of artificially cultivating the situation mushroom fruiting body for the first time at home and abroad was disclosed and disclosed in the first domestic patent No. 179725 by the present inventors. As a result, the beta glucan was inadequately produced and the alpha heteroglycan was mainly produced.

On the other hand, patents for cancer cell proliferation and metastasis inhibition to immune function effect of the ethanol extract derived from the situation mushroom fruiting body from nature has been disclosed in Korean Patent Publication No. 10-2005-72644.

Likewise, in the extraction method of the situation mushrooms, hot water extraction, ethanol extraction after hot water extraction, ethanol extraction, and hot water extraction after ethanol extraction each do not exist or are mixed with β-glucan or α-heteroglycan. Sometimes.

From the extract, the present inventors have already acquired the patent registration No. 10-543811 for β-glucan from the ethanol extract of fruiting bodies obtained by the unpublished situation mushroom mass production method.

Here, the hydrothermal extract of S. mushroom fruiting body did not have an antioxidant effect, but ethanol extract confirmed that it had an antioxidant effect and obtained a patent.

However, until now, there is no document that empirically proves that the hydrothermal extract derived from the situational fruiting body has an effect on improving immune function.

In addition, confectionery, bread and noodles added with the hot water extract of the situation mushroom fruiting body having such an immune function has not been disclosed so far.

In view of the above, the present invention is a mixture of various types of monosaccharides such as α-heteroglycan derived from the liquid culture of the situation mushroom mycelium, namely (1 → 3), (1 → 4) and (1 → 6). Unlike acid complex polysaccharides having an unusual composition, the present invention provides a method of producing fruiting bodies containing large amounts of situational mushroom spores containing only β-glucan, which is excellent in immuno-functionality derived from solid cultures of situational fruiting bodies, as an active ingredient. The purpose is to.

Another object of the present invention is to provide a confectionery, bread, and noodles to which the hot water extract containing β-glucan is added.

The above object of the present invention, after inoculating the mycelium of the liquid culture medium (KCTC0173BP) of the liquid culture medium sterilized by putting the solid medium in a plastic pot, and then cultivated in a plastic house for at least 24 months, and the spores obtained therein are formed in large quantities. The fruiting body was obtained, extracted with hot water, filtered, dried and pulverized to obtain a brown powder product, followed by visual inspection and quantitative test of total glucan, alpha-glucan and beta glucan. This was achieved by examining the sensory test after noodle making.

The present invention confirms that the brown powder as a result of the visual inspection by the problem solving means and glucan quantitative test results show that the β-glucan content of 87 ~ 162mg per unit gram has an excellent immune function effect and contains β-glucan It has an excellent effect on providing immune functional foods and has an excellent effect on providing immune functional foods containing β-glucan.

EMBODIMENT OF THE INVENTION Hereinafter, the content of this invention is described in detail, giving an Example.

Test strains of the situation mushroom used in the present invention was used as a test material for other known strains KCTC0173BP commonly used in liquid culture in the market.

In addition, K-41 (Kumsa-Sang mushroom) possessed by the present inventor was provided to the experiment as a test strain.

In order to maximize the spore formation in the artificial mass production of the situation mushroom fruiting body of the present invention, three-step operation treatment was performed over time in a low-temperature, high-humidity environment.

In order to obtain the preferred situation mushroom fruiting body of the present invention, liquid culture of the seed strains was performed in advance, and then inoculated in a plastic pot containing sterilized artificial solid medium and transferred to a plastic house, where it was cultivated in large quantities.

Preferred cultivation period is at least two years to maximize sporulation, preferably three to four years.

The fruiting body and spores obtained by mass cultivation of the situation mushroom of the present invention are shown in Fig. 1, coarsely pulverized them, and then extracted with hot water, dried, filtered and re-dried, and then pulverized to obtain brown powder.

Hereinafter, the specific configuration of the present invention has been described in detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited thereto.

Example 1.Liquid culture of mycelium of the present situation mushroom strain spawn

Situation of Mushroom Seedling Mycelium Liquid culture medium consisting of 0.3 wt% book, 0.2 wt% yeast extract, 0.02 wt% soybean oil, and remaining distilled water (H ₂ O) was prepared with KCTC0173BP. After adjusting for 90 minutes sterilization at 121 ℃ cooled to room temperature and inoculated in a clean room and then incubated for 3 days at 26 ℃ to obtain a starter starter.

Example 2 Cultivation for Mass Production of Situation Mushroom Fruiting Body

The water was added to a solid medium containing 73% by weight of mulberry whole rice and 27% of rice bran, and then purified by adding pure water (H ₂ O) to 80% water content, followed by mixing with calcium carbonate and 1 part by weight to pH 6.0.

The solid medium composition prepared as described above was added to an autoclave, sterilized at 121 ° C. for 90 minutes, cooled, and then placed in a plastic pot in a cleanbench. Liquid culture mycelia were inoculated as seed starters corresponding to 3% of the solid medium composition.

Transfer the inoculated plastic pot container to the plastic house for mass cultivation and cultivate the cultivation environment at room temperature of 17 ℃ and relative humidity of 65% for the first 3 ~ 4 weeks. After three months, three-step operation was performed at room temperature 26 ° C. and relative humidity of 85% to harvest the situation mushroom fruiting body at 24 months.

The cultivation method of this embodiment has already been disclosed and known by the present inventors in Korean Patent No. 179725.

Example 3 A Novel Mass Cultivation Method for Maximizing the Production of Spore Mushrooms

Seeds were cultured and fruiting bodies were grown according to Examples 1 and 2 above.

In accordance with the present invention, the environment for mass cultivation environment in a plastic house for maximizing the spore production of the situation mushroom strain was designed as follows (Table 1), and as a result, fruiting bodies were produced as follows and their characteristics were analyzed (Table 2).

Table 1.Environmental Conditions for Maximizing Spore Production

division Cultivation Temperature Relative Humidity Remarks Step 1 operation 4 ℃ ~ 6 ℃ 50-55% 6 months after spawn vaccination 2-step operation 25 ℃ ~ 27 ℃ 80-85% 12 months after spawn vaccination 3-step operation 30 ℃ ~ 35 ℃ 93-95% 24 months after spawn vaccination

Table 2. Characteristics of Green Mushroom Fruits

division Solid Medium Residue (1) Fruit body weight (2) Spore Weight (3)  β-glucan content (mg) (4) Patent Publication No. 179725 550-595 280-305 1.2 ~ 2.3 9-20 Invention 501-539 312-333 3.1 ~ 4.8 87-162

[week]. (1) to (3) are the values obtained from fruiting body cultivation for 24 months, and (4) the β-glucan content (mg) extracted from the pulverized fruiting body by pulverizing hot water, followed by filtration and drying (g). Value.

As can be seen from the experimental results of the fruiting bodies obtained according to the example of the present invention, the fruiting bodies obtained by the cultivation method already known in Example 2 are light in weight, have low spore formation, and have an average β-glucan content of 15 mg / In contrast to the g level, the fruiting bodies obtained by the novel cultivation method of the present invention not only have excellent fruiting body formation, but also have excellent sporulation which affects the β-glucan content, and thus the known cultivation method for fruiting body formation in the β-glucan content ( Compared to the inventors of the present invention (Inventive Patent No. 179725), an increase of 8 times or more can yield an average of 125 mg / g.

Comparative Example 1.

As a result of cultivation under the same environmental design conditions as in Example 3 (Table 1) using the present invention, the gold strain of mushrooms collected and stored spore weight 3.3 ~ 5.3 (g), β-glucan content 89 ~ 167 ( mg / g).

Therefore, even if the situation mushroom strains were found to be no significant difference in spore production and β-glucan content under the fruiting conditions of the present invention.

Example 4 Extraction of Anticancer Immunity

Fruiting body obtained according to the situation mushroom artificial mass cultivation method and its ethanol extract is known to be non-toxic by the Korean Patent No. 543811 already disclosed by the present inventors.

The fruiting body obtained by artificial cultivation for 2 years or more according to Example 3 was hot air dried and pulverized as shown in FIG. 2 and then filtered by hot water extraction at 110 ° C. for 96 hours, followed by hot air drying at a moisture content of 12% or less. It was pulverized again and used as a test material.

The appearance of the material was brown powder by visual inspection.

This reflects the color of the spores, the mycelium powder was found to be completely different from the gray-white appearance.

Experimental Example 1. Functional ingredient (marker ingredient) and manufacturing standard

Α-glucan was not detected in the fruiting body of the present invention, and the manufacturing standard and product requirements were as shown in Table 3 and Table 4 below.

Table 3.Functional Ingredients (Indicator Ingredients) and Manufacturing Standards

division ingredient content Remarks Indicator component β-glucan 97-162 mg / g α-glucan not detected Manufacturing Standards pb (lead)
Total arsenic
cd (cadmium)
Mercury
Escherichia coli group 1.0 (mg / kg) or less
1.0 "
0.4 "
0.3 "
negative

Korea Food & Drug Administration (2008.6.17, 2008-32, Functional Food Ingredients Certificate)

Table 4. Product Requirements

division Acknowledgment Functional content May help improve immune function (Other Functions II) Daily Intake 3.3g / day as a mushroom extract Appearance Brown powder

Co., Ltd.

Experimental Example 2 Measurement of Total Glucan and β-glucan

Total glucan was first dissolved and hydrolyzed.

The test material obtained in Example 4 was pulverized so as to pass through a 0.5 mm sieve using a mill and then contained 100 mg in a tube with a lid.

1.5 mL of concentrated hydrochloric acid was added to each tube, and a lid was added and vigorously stirred with Vortex, followed by reaction for 45 minutes in a 40 ° C. water bath.

10 mL of distilled water was added to each tube again, and the mixture was stirred with vortex again, and the lid was loosened. The tube was left for 5 minutes in a 100 ° C. water bath, the lid was closed, reacted for 2 hours, cooled to room temperature, and 10 mL of 2N KOH solution was added thereto.

Transfer the reaction solution to a 100mL flask, wash the residue of the tube with 200mM acetic acid supernatant (PH5.0), allow to mix, shake to mix thoroughly, and filter the reaction solution using Whatman GF / A glass fiber filter. The supernatant was used as a test solution by centrifugation for a minute.

[Total Glucan Content Measurement]

0.1 mL of the experimental solution was transferred to a glass test tube, 1000u / mL of exo-1,3-β-glucanase and 20u / mL of β-glucosidase mixed solution were added thereto, and the mixture was allowed to react for 60 minutes at 40 ° C. after stirring the vortes. After adding mL to the test tube and reacting at 40 ° C. for 20 minutes, the reaction solution was measured for absorbance at 510 nm.

[α-glucan measurement]

100 mg of the ground sample was immersed in a tube with a lid, and 2 mL 2M KOH was added to each tube and stirred in a cold / water bath for 20 minutes using a magnetic stirrer bar.

8 mL of 1.2 M acetic acid supernatant (PH 3.8) was added during stirring, followed by mixing 0.2 mL of Amyloglucosidase (1630 u / mL) and Invertase (500 u / mL) 50% glycerin mixture, followed by 40 ° C water bath. The vortex was stirred in a 40 ° C. water bath to react for 30 minutes, and the reaction solution was centrifuged at 1,500 G for 10 minutes to use the supernatant as a test solution.

0.1 mL of the test solution was taken and transferred to a glass test tube. 3.0 mL of a GOPOD solution was added to each test tube and reacted at 40 ° C. for 20 minutes.

[Calculation of β-glucan Content (mg / g)]

The total glucan (%)-α-glucan (%) × 10 = β-glucan content.

Total Glucan (5) = ΔE × F × (100 / 0.1) × (1 / 1,000) × (100 / W) × 162/180

= ΔE × (F / W) × 90

                   DELTA E: Absorbance difference (reaction liquid absorbance-blank test absorbance)

                   F: Glucose Standard Solution (1.00 ~ mg / mL) Conversion Factor

                   100 / 0.1: Volume of final application of the test solution taken (0.1mL)

                             Dilution factor for 100 mL

                   1 / 1,000: Unit conversion factor in ug mg

                   100 / W: Sample amount (mg) and% conversion factor

                   162/180: β-glucan conversion factor

α-glucan (%) = ΔE × F × (10.3 / 0.1) × (1 / 1,000) × (100 / W) × 162/180

= ΔE × (F / W) × 9.27

ΔE: absorbance difference

                   F: Glucose Standard Solution Conversion Factor

                   103: final volume of the test solution taken (0.1mL)

                             Dilution factor for 10.3 mL

                   1 / 1,000: Unit conversion factor in ug mg

                   100 / W: Sample amount (mg) and% conversion factor

                   162/180: β-glucan conversion factor

Experimental Example 3. Measurement of Immune Activity of Hot Water Extract of Situation Mushroom

When immune cells are activated, such as NK cell activation or lymphocyte counting, the cell's DNA synthesis is first increased and the cell volume is increased, and secondly, the proliferation of division is accelerated as effector cells. Function.

The primary increased cells are called lymphoblastoma, and the schizophrenic effect is called the lymphoblastogenic effect.

In this experiment, mouse splenocytes were isolated and cultured in a medium to which the test material obtained in Example 4 was added, and the volume increase of immune cells was measured using a flow cytometer. It is shown as a proliferative effect of leukocyte cells.

Place a 100-stainless steel mesh on a 45 mm diameter petri dish, add 5 mL of RPMI 1640 medium, transfer the BALB / C mouse spleen, lethal to the cervical distal bone, into a mesh, finely chopped with surgical scissors, and bottom of a 15 mm diameter glass container. Lightly rub with a crushed tissue. Subsequently, the cells passed through the net were piped up and down several times, washed with RPMI 1640 medium, cooled for 5 minutes in an ice bath, and only the upper cell suspension was transferred to a new container.

After centrifugation, 1 mL of 0.83% ammonium chloride was added to the splenocytes, suspended, and left for 3 minutes in a 37 ° C. water bath to dissolve erythrocytes. Spleen leukocyte suspensions were prepared by counting with a hemocytometer and diluting to 4 × 10 6 cells / mL.

The spleen leukocyte suspension obtained above was washed once with RPMI 1640 medium without Phenol Red, and then, 50 mL was added to a 96-well culture plate, and 5 mg / mL each was separated from the test material obtained in Comparative Example 1 for each well. The mixture was added to the sphere and incubated for 48 hours in a 37 ° C., 5% CO ₂ incubator.

After incubation, 0.1 mL of N-methyl dibenzopyran methyl sulfate solution dissolved in 1 mg / mL of XTT in 1640 / mL RPMI 1640 medium without phenol red and 1.25 mM in PBS was mixed immediately before use. After the addition, the cells were incubated for 8 hours in a 5% CO ₂ incubator.

After the incubation, the absorbance was measured at 490 nm using an ELISA reader.

Table 5. First Experimental Results of XTT Proliferation

                                                                    (unit: %)

division OD ₄₉₀ Lymphoblastic lymphoma effect (pharmaceutical OD ₄₉₀ / control OD ₄₉₀ ) x100 One Control group 0.172 100 2 The present invention 0.483 281 3 Comparative Example 1 0.408 237

Table 6. Second Experiment Result of XTT Growth

                                                                    (unit: %)

division OD ₄₉₀ Lymphoblastic lymphoma effect (pharmaceutical OD ₄₉₀ / control OD ₄₉₀ ) x100 One Control group 0.187 100 2 The present invention 0.521 279 3 Comparative Example 1 0.492 263

Table 7. Third Experiment Result of XTT Growth

                                                                   (unit: %)

division OD ₄₉₀ Lymphoblastic lymphoma effect (pharmaceutical OD ₄₉₀ / control OD ₄₉₀ ) x100 One Control group 0.824 100 2 The present invention 1.592 193 3 Comparative Example 1 1.489 181

Example 5 Confectionery

95 weight% of biscuit composition was added to the kneader with water and kneaded at 100 rpm, and 5 weight% of the brown powder in which the present invention β-glucan was added, and kneaded again to cut into circular shapes. 90 at 340 ° C. in a conventional bakery oven. Baked sugar-free biscuits were prepared for a second time (the present invention 1).

Example 6 Biscuit Preparation

Add 95% by weight of biscuit composition to the kneader with water and knead it at 80 rpm, add 5% by weight of brown vulcanized with β-glucan of the present invention, and knead it again. Biscuits were prepared (invention 2).

Example 7 Dumpling Preparation

After adding 330 g of water to 1 kg of semi-strong powder and kneading at 75 rpm, 20 g of salt and 33 g of the brown powder of the present invention are added and blended, and then kneaded for 12 minutes with a noodle mixer to prepare dumpling skin and aged at 25 ° C. for 20 minutes. Baked dumplings were prepared with a conventional dumpling stuffing (product of the present invention 3).

Experimental Example 4. Sensory Evaluation Results

Ten panel samples randomly compared with the ready-made products (H confectionery, K biscuits, P dumplings) having biscuits, biscuits, and dumplings prepared according to Examples 5 to 6 were each evaluated by a five-point evaluation method. The panel test was performed.

Sensory test results are shown in Table 8.

Table 8. Sensory Test Results

division Color (appearance) Texture Flavor bitter Texture (chewy) General Symbol H company product 3.2 3.3 3.1 2.8 3.5 3.2 This invention (1) 4.3 4.5 4.5 4.5 4.3 4.4 K company 3.3 3.1 3.2 3.4 3.3 3.4 This invention (2) 4.5 4.4 4.7 4.4 4.4 4.7 P company product 3.5 3.3 3.2 2.1 3.5 3.1 This invention (3) 4.8 4.9 4.8 4.3 4.5 4.5

Experimental results show that the biscuits, biscuits, and dumplings of the present invention have a good brown reaction and excellent flavor, which promotes texture and appetite with better color, flavor, and bitter taste. In terms of the degree of preference, it was found to be superior to the existing products, thereby improving immunity enhancement effect and increasing palatability.

The present invention is a very useful invention in the health food industry by providing an immuno-functional food containing a large amount of β-glucan spores formed spores with a large amount of spores formed as an active ingredient and a hydrothermal extract excellent in immune function.

1 is a photomicrograph of germ cell spores formed on the situation mushroom fruiting body produced according to a preferred embodiment of the present invention.

Figure 2 is a schematic diagram of a process from obtaining a large amount of β-glucan brown powder directly from the situation mushroom fruiting body produced according to a preferred embodiment of the present invention.

Claims (2)

Inoculating the liquid culture of the ephedra mushroom spawn in a solid medium in a plastic pot and then artificially cultivating it in a plastic house. 55%, 80 ~ 85% at 25 ℃ ~ 27 up to 6 months thereafter, 93 ~ 95% at 30 ℃ ~ 35 ℃ up to 12 months afterwards, maximizing sporulation The immunity is characterized by the accumulation of 87-162 mg of β-glucan per unit g, which can be obtained by drying, coarse crushing together the spores formed with mushrooms and then hot water extraction at 110 ° C for at least 96 hours. Functional brown powder. Immune function improvement health supplement foods, characterized in that containing the immune functional brown powder of claim 1.

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