KR20230009682A - A novel method for producing cordycepin using coffee and mushroom strains showing antibacterial, antifungal, immune enhancement, anticancer, and antithrombotic effects, and a composition comprising an extract containing cordycepin produced by the production method as an active ingredient - Google Patents

Abstract

In the present invention, green coffee beans with markedly enhanced cordycepin are prepared by inoculating and fermenting green coffee beans with at least one spawn selected from spawns consisting of Ganoderma lucidum, Phellinus elegans, Chaga mushroom, Zinnia mushroom, and Cordyceps sinensis, and then extracting the green coffee beans. extract containing cordycepin showing antibacterial, antifungal, immune enhancing, anticancer, and antithrombotic effects can be prepared, and since the basic base is coffee, it can be supplied in the form of a beverage containing cordycepin, which is very suitable for industrialization and commercialization. useful.

Description

A novel method for preparing cordycepin using coffee and mushroom strains showing antibacterial, antifungal, immune enhancing, anticancer, and antithrombotic effects, and a composition containing cordycepin-containing extract produced by the manufacturing method as an active ingredient {A novel method for producing cordycepin using coffee and mushroom strains showing antibacterial, antifungal, immune enhancement, anticancer, and antithrombotic effects, and a composition comprising an extract containing cordycepin produced by the production method as an active ingredient}

The present invention relates to a novel method for preparing cordycepin using coffee and mushroom strains exhibiting antibacterial, antifungal, immune enhancing, anticancer, and antithrombotic effects, and a composition containing cordycepin-containing extract produced by the manufacturing method as an active ingredient. will be.

Since cordycepin was discovered by Cunningham et al. in 1950, many studies have been conducted on its pharmacological activity. Such cordycepin inhibits m-RNA synthesis and is mainly known to have antibacterial, antifungal, immune enhancing and anticancer effects (Nature, 166, 949~954, 1950; Cancer Res., 21, 216~220, 1961 (Biochem. Biophys. Acta., 80, 640-647, 1964).

Recently, many studies on the platelet aggregation inhibitory action of cordycepin have been conducted. As an example of this, Korean Patent Registration No. 10-464876 discloses an antithrombotic composition containing cordycepin of Cordyceps sinensis.

In addition, as a method for separating and purifying cordycepin components, Korean Patent Publication No. 10-2001-0054264 discloses cordycepin for anticancer drugs separated and extracted from Cordyceps sinensis and a method for producing the same. After extracting the dried body with methanol in hot water, filtering to prepare a methanol extract, and the methanol extract was fractionated into Hexane, Chloroform, Ethyl acetate, Buthanol, and Water layers, and then assayed for each fraction to obtain an active fraction. It consists of eluting with chloroform-methanol on a silica gel column, purifying it with prep HPLC, obtaining an active peak using methanol-water (2:8), performing assay on it, and continuing to separate only this part. there is.

In addition, Korean Patent Publication No. 10-2017-0049353 discloses a method for rapidly extracting high-purity cordycepin from Cordyceps sinensis, which includes the steps of extracting Cordyceps sinensis extract from Cordyceps sinensis; Passing the cordyceps extract through porous gel column chromatography to obtain a cordycepin fraction; and purifying the cordycepin fraction through silica gel column chromatography, followed by recrystallization to obtain cordycepin.

However, the above-described method uses only Cordyceps sinensis, so it has limitations in industrialization, commercialization, and manufacturing in the form of beverages that we often drink.

1. Korean Patent Registration No. 10-464876 2. Korean Patent Publication No. 10-2001-0054264 3. Korean Patent Publication No. 10-2017-0049353

Therefore, the problem to be achieved by the present invention is a novel method for preparing cordycepin using coffee and mushroom strains showing antibacterial, antifungal, immune enhancing, anticancer, and antithrombotic effects, and a cordycepin-containing extract produced by the manufacturing method as an active ingredient It is to provide a composition that

In order to achieve the above technical problem, the present invention

Inoculating green coffee beans with at least one spawn selected from the group consisting of Ganoderma lucidum, Phellinus elegans, chaga, zinnia and cordyceps and fermenting them; and

Provided is a novel method for producing a cordycepin-containing extract using coffee and mushroom strains, comprising the step of extracting the fermented green coffee beans using water or ethanol as a solvent.

In the manufacturing method according to the present invention as described above, the green coffee beans may be germinated.

In the manufacturing method according to the present invention as described above, any one or more spawn selected from the group consisting of Ganoderma lucidum, Phellinus linteus, Chaga mushroom, Cinnamon mushroom, and Cordyceps sinensis is inoculated with yeast and then heated to 70 °C at 20 to 40 ° C. It may be cultured for 170 hours.

In the manufacturing method according to the present invention as described above, the fermentation may be performed by culturing the complex spawn inoculated into the green coffee beans at a temperature of 20 to 45 ° C. and a humidity of 35 to 70% for 12 to 120 hours. .

As described above, the manufacturing method according to the present invention may further include roasting the green coffee beans before the extraction step.

In addition, the present invention, in order to achieve another technical problem, green coffee beans with increased cordycepin content by inoculation and fermentation of one or more seed fungi selected from the group consisting of Ganoderma lucidum, Sanghwang mushroom, chaga mushroom, zinnia mushroom and cordyceps sinensis, and green coffee beans containing the same They serve roasted coffee beans.

In order to achieve another technical problem, the present invention has antibacterial, antifungal, immune enhancing, anticancer, and antithrombotic effects by using green coffee beans or an extract extracted from coffee beans containing a large amount of cordycepin prepared as described above as an active ingredient. Provides a visible composition.

The present invention inoculates green coffee beans with at least one spawn selected from spawns consisting of Ganoderma lucidum, Phellinus linteus, Chaga mushroom, Zinnia mushroom, and Cordyceps sinensis, prepares green coffee beans with markedly enhanced cordycepin, and then extracts the green coffee beans. extracts containing cordycepin showing antibacterial, antifungal, immune enhancing, anticancer, and antithrombotic effects can be prepared, and since the basic base is coffee, it can be supplied in the form of a beverage containing cordycepin, which is very suitable for industrialization and commercialization. useful.

When the composition according to the present invention is taken, the immune function of human normal cells is activated by the increased cordycepin content to inhibit the proliferation and recurrence of cancer cells, and antithrombotic, antibacterial, and antifungal effects can be obtained through inhibition of platelet aggregation, , Incidentally, it has the advantage of being able to feel the cacao scent that stands out with the soft chocolate scent, and the soft, deep and rich taste and aroma.

1 is a flowchart of a method for manufacturing green coffee beans according to the present invention.
2 to 7 are photographs of green coffee beans over time in culture after inoculation with 5 types of mushroom composite spawn according to the present invention.

Hereinafter, specific details for carrying out the present invention will be described in detail with reference to the accompanying drawings.

The present invention provides a method for producing green coffee beans with increased cordycepin content, which primarily shows antibacterial, antifungal, immune enhancing, anticancer, and antithrombotic effects, and green coffee beans prepared thereby, and an extract using the green coffee beans , Coffee beans prepared by roasting the green beans and extracts using the coffee beans are provided. Accordingly, a method for manufacturing green coffee beans having an increased cordycepin content will be first described with reference to FIG. 1 .

1 is a flowchart of a method for manufacturing green coffee beans according to the present invention in which the content of cordycepin is increased according to the present invention. First, complex mushroom spawns composed of Ganoderma lucidum, Phellinus linteus, Chaga mushroom, Zinnia mushroom, and Cordyceps sinensis will be described. .

After various studies on methods for producing green coffee beans with increased cordycepin content, one or more spawners selected from the group consisting of Ganoderma lucidum, Sanghwang mushroom, Chaga mushroom, Zinnia mushroom, and Cordyceps sinensis are selected, particularly Ganoderma lucidum, It was applied to the present invention after confirming that the use of 5 types of mushroom composite spawn fungi of Phellinus Phellinus, chaga, zinnia and cordyceps was effectively cultured and fermented in green beans to significantly increase the cordycepin content.

The five types of mushroom spawn consisting of Ganoderma lucidum, Phellinus lucidum, Chaga mushroom, Zinnia mushroom, and Cordyceps mycelium are each or two or more species simultaneously Most preferably, it is obtained by mixing and culturing, and it is preferable to mix and culture 5 species from the culture step, and when the 5 species are combined, there is no particular restriction on their ratio, but preferably based on 100 weight of ganoderma lucidum mycelium Other mycelium may be 30 to 150 weight (S100).

That is, when culturing two or more species in a complex, it is preferable to culture by mixing 30 to 150 weight of other mycelium based on 100 weight of one mycelium.

In addition, in the present invention, the mushroom spawn obtained by culturing can be directly inoculated into green coffee beans for culture and fermentation, but in order to further enhance the content of cordycepin and facilitate culture and fermentation, yeast is inoculated with mushroom spawn, and 20 to 40 It is preferable to use those cultured for 70 to 170 hours at ° C.

Any widely known technique to which the present invention belongs, such as the culture temperature, culture medium, shaking speed, and culture period of the method of culturing the mushroom spawn, can be used without particular limitation, and the culture medium, for example, in a medium rich in nitrogen and mineral sources Since it shows vigorous growth, it can be cultured in MCM medium or YMPG medium.

Next, washing and sterilization of green coffee beans to remove residual pesticides and foreign substances from green coffee beans and sterilization of green coffee beans (S200). Methods and conditions for washing and sterilizing green coffee beans may be used without particular limitation as long as they are widely known in the art to which the present invention belongs.

Next, the green coffee beans washed and sterilized in S 200 are inoculated with the composite spawn of the 5 types of mushrooms (S 300). At this time, inoculation of spawn is inoculated by shaking green coffee beans and spawn in a pot (200ml or 500ml) since the spawn of each mushroom is in liquid form. The weight ratio of green coffee beans and mushroom spawn is not particularly limited, but is preferably 1:0.05 to 0.30.

In addition, in the present invention, in order to increase the efficiency of increasing the cordycepin content, mushroom spawn can be inoculated after germinating green coffee beans. The germination method is not particularly limited as long as it is widely known in the art to which the present invention belongs. An example is a method of soaking at a weight ratio of 1:1.0 to 2.5 at 36 to 68° C. for 3 to 5 hours to germinate.

Finally, when the green coffee beans inoculated with the mushroom spawn are cultured and fermented and then dried (S 400), the manufacturing method of green coffee beans with significantly increased cordycepin according to the present invention is completed.

The culture fermentation is preferably carried out for 12 to 120 hours at a temperature of 20 to 45 ° C. and a humidity of 35 to 70% by putting the inoculated green coffee beans in a fermenter, and sour fermented vinegar begins about 12 to 24 hours after the start of the culture fermentation. It is possible to visually confirm that the mushroom strain is implanted on the surface of green coffee beans with the aroma rising, and when the cultured mushroom strain blooms on the surface of green coffee beans and a strong scent of sour fermented vinegar rises, stop fermentation. It may take a wide range of time.

As an example of the culture fermentation described above, after inoculating germinated green coffee beans with 20% by weight of 5 types of composite mushroom spawn, they were placed in a fermentor and cultured and fermented at a temperature of 25 ° C and a humidity of 60%, and a photograph of the mushroom strain blooming is shown in FIG. 2 to Figure 7.

The extraction composition according to the present invention can be prepared by extracting the green coffee beans prepared as described above, or it can be prepared by extracting the coffee beans prepared by roasting the green coffee beans. There is no particular limitation as long as it is widely known in the art, and it is preferable to extract water or ethanol as a solvent.

The present invention will be described in detail through examples and experimental examples below.

< Example 1>

Ganoderma lucidum mycelium, Sanghwang mushroom mycelium, Chaga mushroom mycelium, Zinnia mushroom mycelium and Cordyceps mycelium were mixed in MCM medium at the same weight ratio, and then 5 types of mushroom composite spawn obtained by complex culture at 23 ° C for 168 hours were washed and sterilized. It was inoculated by putting it in a pot together with green coffee beans and shaking. At this time, the weight ratio of green coffee beans and 5 types of mushroom composite spawn was 1:0.2.

Subsequently, fermentation was terminated by culturing and fermenting in a fermentor at a temperature of 29 ° C to 39 ° C and a humidity of 46 to 60% to confirm the blooming of the mushroom strain, and the total fermentation time until termination was 120 hours.

< Example 2>

Same as in Example 1, except that after sterilizing green coffee beans, the weight ratio of green coffee beans and water was 1:2, and soaked at 36 ° C to 68 ° C for 3 to 5 hours to germinate. Inoculation of 5 types of mushroom composite spawn There is a difference in what

< Example 3>

It is the same as in Example 1, except that the cultured yeast was inoculated with 5 types of mushroom composite spawn, cultured at 36 ° C for 168 hours, and inoculated into green coffee beans.

< Example 4>

It is the same as in Example 1, and there is a difference in that only Ganoderma lucidum mycelium was used.

< Example 5>

It is the same as in Example 1, and there is a difference in that only Sanghwang mushroom mycelium was used.

< Example 6>

It is the same as Example 1, and there is a difference in that only the mycelium of the chaga mushroom was used.

< Example 7>

It is the same as in Example 1, and there is a difference in that only the mushroom mycelium was used.

< Example 8>

It is the same as in Example 1, and there is a difference in that only Cordyceps sinensis mycelium was used.

< Example 9>

Same as Example 1, ganoderma lucidum mycelium. There is a difference in using only three types of mycelium of Phellinus Phellinus and cordyceps mycelium.

< Example 10>

It is the same as in Example 1, and there is a difference in that only three types of mycelium of Phellinus linteus, mycelium of Chaga mushroom, and mushroom mycelium were used.

< Example 11>

It is the same as in Example 1, and there is a difference in that only three types of ganoderma lucidum mycelium, chaga mushroom mycelium, and cordyceps mycelium were used.

< Example 12>

It is the same as in Example 1, and there is a difference in that only four types of ganoderma lucidum mycelium, mycelium of Sanghwang mushroom, mycelium of chaga mushroom, and mycelium of Cordyceps sinensis were used.

< Example 13>

It is the same as in Example 1, and there is a difference in that only four types of ganoderma lucidum mycelium, mycelium of Sanghwang mushroom, mycelium of chaga mushroom, and mycelium of cauliflower mushroom were used.

< test example One: roasting Determination of Cordycepin Content in Bean Extract>

After drying the green coffee beans of the above example, the coffee beans were roasted at a low temperature of 90 ° C to 189 ° C for 21 minutes and 189 ° C to 198 ° C for 2 minutes to prepare beans, and then ground to prepare coffee bean powder. measured. The results are shown in Table 1, and the fraction obtained by passing the ethanol extract of Cordyceps sinensis through porous gel column chromatography was used as a control.

The content of cordycepin was analyzed using HPLC as follows.

Device: Waters (Waters e2695 module, Waters 2998 PDA)

Column: Waters Spherisorb?? 10㎛ ODS2 column (4.6 × 250㎜ Analytical)

Flow rate: 1 ml/min

Wavelength: 254nm

Mobile phase: 0.01M KH2PO4 in 15% methanol

Cordycepin content (mg/g) control group 0.0012 Example 1 12.3 Example 2 14.7 Example 3 16.2 Example 4 5.8 Example 5 5.9 Example 6 5.6 Example 7 5.8 Example 8 5.9 Example 9 7.5 Example 10 7.8 Example 11 8.1 Example 12 10.7 Example 13 10.9

Looking at the results in Table 1 above, it was confirmed that the content of cordycepin significantly increased compared to the control group. In particular, it was found that the difference in the content of cordycepin was very large in the example using 5 types of complex spawn.

< test example 2: Flavor and taste evaluation of bean extract>

After preparing the extract using the same powder as the coffee bean powder used in Test Example 1, 10 experienced people (5 males and 5 females in their 20s and 30s) with a lot of experience in the field were recruited as a sensory test panel. According to the point scale method, a comprehensive evaluation of burnt taste, bitter taste, sour taste, aftertaste, and body was investigated, and the results are shown in Table 2. As a control group, green coffee beans without any treatment were roasted and used in the same manner as in Test Example 1.

Sensory Comprehensive Assessment control group 2.4 Example 1 4.1 Example 2 4.4 Example 3 4.9 Example 4 3.2 Example 5 3.2 Example 6 3.1 Example 7 3.2 Example 8 3.5 Example 9 3.3 Example 10 3.4 Example 11 3.4 Example 12 3.8 Example 13 3.8

Even in the results of Table 2, it was confirmed that the extract according to the present invention was evaluated excellently, and in particular, for the extract according to the present invention, a comprehensive evaluation was given to feel the cacao flavor, soft, deep and rich taste and aroma that stands out in the soft chocolate flavor.

<Test Example 3: Evaluation of anticancer effect using HepG2 and HeLa cell lines>

After drying the green coffee beans of the above example, the hot water extracted extract and the green coffee beans of the above example were dried, and then roasted at a low temperature of 90 ° C to 189 ° C for 21 minutes and 189 ° C to 198 ° C for 2 minutes to examine the anticancer effect of the hot water extract. Tumor cells at the cellular level using HeLa cell line, a human cervical carnoma cell line, and HepG2 cell line, a human hepatocellular carcinoma cell line, obtained from ATCC (American Type Culture Collection) A proliferation inhibitory effect was observed.

In order to observe the tumor cell proliferation inhibitory effect of the extract, a widely used MTT assay was used.

10% FBS, 1% L-glutamine, 1% antibiotics/antimycotics (penicillin at 10,000 U/ml, amphothecin D at 25 μg/ml or streptomycin at 10 mg/ml) were added to HeLa cell line inactivated at 56°C. It was cultured for 24 hours while maintaining a humidified 5% CO2 condition at a temperature of 37°C in DMEM medium. Next, 1ⅹ10 ⁵ HeLa cells per 1 ml were inoculated into a 24-well culture plate, cultured for 12 hours, and unattached cells were washed three times with sterile phosphate buffer, and the extract was treated at a concentration of 500 μg/ml. After 48 hours of culture in a humidified 5% CO ₂ constant temperature reactor at a temperature of 37 ℃.

4 hours before the end of the culture, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (hereinafter referred to as MTT) was added to each well in which cancer cells were cultured (final concentration 0.5 μg/ml) was added. Next, 100 ml of a solution in which hydrochloric acid was added to a final concentration of 0.04N in isopropanol was added and mixed at room temperature for about 20 minutes, and then absorbance was measured at a wavelength of 550 nm using an ELISA reader.

HepG2 cells were also carried out in the same manner as the above MTT method, and the tumor cell proliferation inhibitory effect was evaluated from the measured cancer cell viability. The results are shown in Table 3, and the control group was a coffee bean extract roasted at a low temperature under the same conditions as the dried green bean extract without any treatment.

HepG2 cell line viability (%) HeLa cell line viability (%) control green beans 91.2±1.2 89.4±1.1 Control roasted beans 91.5±1.3 90.5±0.9 Example 1 green beans 24.1±1.3 23.4±1.3 Example 1 Roasting 24.5±1.2 24.9±1.3 Example 2 green beans 20.1±1.3 20.5±1.2 Example 2 Roasting 20.8±1.3 21.1±1.3 Example 3 Green Beans 17.2±1.1 17.1±1.3 Example 3 Roasting 17.9±1.3 17.3±1.2 Example 4 Green Beans 44.1±1.1 44.3±1.3 Example 4 Roasting 44.5±1.3 44.8±1.3 Example 5 Green Beans 43.9±1.2 43.1±1.1 Example 5 Roasting 43.9±1.4 43.8±1.3 Example 6 Green Beans 44.2±1.3 44.9±1.2 Example 6 Roasting 44.3±1.2 45.1±1.3 Example 7 Green Beans 44.1±1.2 44.9±1.2 Example 7 Roasting 43.9±1.3 17.9±1.2 Example 8 Green Beans 44.4±1.3 17.9±1.2 Example 8 Roasting 44.9±1.2 17.9±1.2 Example 9 Green Beans 35.1±1.2 35.4±1.3 Example 9 Roasting 36.7±1.3 36.2±1.1 Example 10 Green Beans 36.4±1.2 36.7±1.3 Example 10 Roasting 36.9±1.2 37.5±1.2 Example 11 Green Beans 34.1±1.3 35.1±1.2 Example 11 Roasting 34.9±1.1 36.5±1.1 Example 12 green beans 31.4±1.3 31.8±1.3 Example 12 Roasting 32.1±1.1 32.7±1.2 Example 13 Green Beans 30.9±1.1 31.4±1.1 Example 13 Roasting 30.9±1.2 31.6±1.3

Looking at the results of Table 3, it was confirmed that the survival rate of cancer cell lines of HepG2 and HeLa cells was significantly reduced in the extract of the present invention compared to the control group, and the survival rate of cancer cells reached 17 to 45%, and the effect increased as the complex spawn was used. could know that From the above test results, it can be seen that the tumor cell proliferation inhibitory effect of the example extracts according to the present invention is excellent.

<Test Example 4: Evaluation of anticancer effect using test animals>

The anti-cancer effect of the extract used in Test Example 3 was examined in the test animal stage.

As test animals, male Balb/c mice, 5 weeks old and weighing 25 to 30 g, were purchased from Daehan Bio Link and used. Feed and water were freely consumed during the test period, and the temperature of the animal laboratory was maintained at 22±1° C. and humidity 55±5%. In addition, the light-dark cycle in the test room was adjusted so that day and night were repeated every 12 hours.

Sarcoma cells (sarcoma-180) preserved by subculture for 1 week in the peritoneal cavity of Balb/c mice were used as test tumor cells. That is, sarcoma-180 cells cultured for 1 week in the abdominal cavity of the test animal were taken together with ascites, and tumor cells were separated by centrifugation (1,200 rpm, 10 min) together with phosphate buffered saline (PBS).

The separated cells were resuspended in PBS, centrifuged again to remove the supernatant, and then a tumor cell suspension was made so that the concentration was 1.0 × 10 6 cells/ml, and 1 ml each was intraperitoneally injected and used for the test while transplant preservation.

The sample was prepared using sterilized PBS, and the dose was 40 mg per kg of mouse. When not administered, it was used while being stored in a freezer.

10 test animals per group, subcutaneously transplanted 0.2㎖ (5×10 ⁶ cells/mouse) of the tumor cell suspension subcutaneously stored in the test room at intervals of 1 week into the left groin of the test animals, and one week later The sample solution was administered intraperitoneally once daily for 4 weeks.

On the 35th day after transplantation of tumor cells, solid tumors produced by killing were removed, their weight was measured, and the tumor growth inhibition ratio (IR: %) was calculated according to Equation 1 below.

<Equation 1>

(In Equation 1, Cw is the average tumor weight of the control group, and Tw is the average tumor weight of the treatment group)

In addition, the measurement results are shown in Table 4 below using the group administered with only sterile PBS instead of the extract of Test Example 3 as a control group.

Tumor growth inhibition rate (%) control group 0.8±1.2 Example 1 green beans 22.1±1.3 Example 1 Roasting 22.5±1.2 Example 2 green beans 23.2±1.3 Example 2 Roasting 23.5±1.3 Example 3 Green Beans 25.1±1.1 Example 3 Roasting 25.2±1.3 Example 4 Green Beans 15.1±1.1 Example 4 Roasting 15.5±1.3 Example 5 Green Beans 15.9±1.2 Example 5 Roasting 15.9±1.4 Example 6 Green Beans 15.2±1.3 Example 6 Roasting 15.3±1.2 Example 7 Green Beans 15.1±1.2 Example 7 Roasting 15.9±1.3 Example 8 Green Beans 15.4±1.3 Example 8 Roasting 15.9±1.2 Example 9 Green Beans 17.1±1.2 Example 9 Roasting 17.7±1.3 Example 10 Green Beans 17.4±1.2 Example 10 Roasting 17.9±1.2 Example 11 Green Beans 17.4±1.3 Example 11 Roasting 17.8±1.1 Example 12 green beans 19.5±1.3 Example 12 Roasting 20.2±1.1 Example 13 Green Beans 20.1±1.1 Example 13 Roasting 19.5±1.2

From the results in Table 4, it was confirmed that the tumor growth inhibition rate in the extract of the present invention was 15% to 25% compared to the control group, and the effect increased as the complex spawn was used. From the above test results, it can be seen that the tumor growth inhibitory effect of the example extracts according to the present invention is excellent.

<Test Example 5: Immune Function Enhancement Effect Using Cell Lines>

In order to examine the immune function enhancing effect of Test Example 1, the activation of macrophages, which are immune cells involved in both reactions of innate immunity and acquired immunity, and particularly play a pivotal role in non-specific immunity, was measured.

In this test, the increase in the activity of lysosomal enzymes was measured by acid phosphatase assay (APL) using U937 cells (obtained from ATCC, Inc., American Growers Association).

When the activity of acid phosphatase when the extract of Test Example 3 was not treated was set to 1 and the treatment concentration was 200 μg / ml, the relative ratio of the activity of acid phosphatase was measured, and the results are shown in Table 5. The control group was a coffee bean extract roasted at a low temperature under the same conditions as the dried green bean extract without any treatment.

Acid phosphatase activity relative ratio control green beans 0.3 Control roasted beans 0.3 Example 1 green beans 1.7 Example 1 Roasting 1.7 Example 2 green beans 1.6 Example 2 Roasting 1.8 Example 3 Green Beans 2.1 Example 3 Roasting 2.0 Example 4 Green Beans 1.1 Example 4 Roasting 1.3 Example 5 Green Beans 1.2 Example 5 Roasting 1.2 Example 6 Green Beans 1.3 Example 6 Roasting 1.2 Example 7 Green Beans 1.2 Example 7 Roasting 1.3 Example 8 Green Beans 1.3 Example 8 Roasting 1.2 Example 9 Green Beans 1.4 Example 9 Roasting 1.5 Example 10 Green Beans 1.5 Example 10 Roasting 1.5 Example 11 Green Beans 1.4 Example 11 Roasting 1.5 Example 12 green beans 1.5 Example 12 Roasting 1.4 Example 13 Green Beans 1.4 Example 13 Roasting 1.3

Looking at the results of Table 5, it was confirmed that the immune function enhancing effect was significantly increased in the extract of the present invention compared to the control group. It can be seen that the immune function enhancing effect of the extracts of the examples according to the invention is excellent.

<Test Example 6: Immune function enhancement effect using test animals>

The immune function enhancing effect of the extract of Test Example 3 was examined in the test animal stage.

As test animals, male Balb/c mice, 5 weeks old and weighing 25 to 30 g, were purchased from Daehan Bio Link and used. Feed and water were freely consumed during the test period, and the temperature of the animal laboratory was maintained at 22±1° C. and humidity 55±5%.

In addition, the light-dark cycle in the test room was adjusted so that day and night were repeated every 12 hours.

The sample was prepared using sterilized physiological saline, and the dose was 20 mg per kg mouse, and was used while being stored in a refrigerator when not administered.

600 μl (1 mg/ml) of the sample pretreated in the test laboratory was intraperitoneally injected every day with 5 animals per group. After 30 hours, the mouse was decapitated to obtain blood, and macrophages were obtained by peritoneal washing and applied to the test.

Macrophages recovered from the abdominal cavity of mice were re-dispersed in RPMI-1640 medium to reach 1×10 ⁶ cells/ml, and 200 μl of this dispersion was dispensed into each well of a 96-well plate, followed by CO2 incubation at 37°C for 2 hours. After fixing the macrophages to the wells, RPMI1640 complete medium (with 10% FBS) was added to each well and cultured for 24 hours. was dissolved and the amount of lysosomal phosphatase secreted at this time was measured by the absorbance at 405 nm with an ELISA reader to evaluate the degree of activity of macrophages, and the results are shown in Table 6. The administered group was used as a control group.

Activation of macrophages control group 100±1.2 Example 1 green beans 122.2±1.3 Example 1 Roasting 121.5±1.2 Example 2 green beans 123.5±1.3 Example 2 Roasting 123.1±1.3 Example 3 Green Beans 125.1±1.1 Example 3 Roasting 125.2±1.3 Example 4 Green Beans 115.4±1.1 Example 4 Roasting 115.1±1.3 Example 5 Green Beans 115.6±1.2 Example 5 Roasting 115.6±1.4 Example 6 Green Beans 115.2±1.3 Example 6 Roasting 115.3±1.2 Example 7 Green Beans 115.1±1.2 Example 7 Roasting 115.9±1.3 Example 8 Green Beans 115.4±1.3 Example 8 Roasting 115.9±1.2 Example 9 Green Beans 117.1±1.2 Example 9 Roasting 117.7±1.3 Example 10 Green Beans 117.4±1.2 Example 10 Roasting 117.9±1.2 Example 11 Green Beans 117.4±1.3 Example 11 Roasting 117.8±1.1 Example 12 green beans 119.5±1.3 Example 12 Roasting 120.2±1.1 Example 13 Green Beans 120.1±1.1 Example 13 Roasting 119.5±1.2

Looking at the results of Table 6, it was confirmed that the effect of enhancing the activity of macrophages in the extract of the present invention was remarkably increased compared to the control group. It can be seen that the immune function enhancing effect of the extracts of the examples according to the invention is excellent.

<Test Example 7: Weight loss effect using test animals>

As test animals, 3-week-old male Sprague-Dawley rats were purchased and divided into two groups: a normal diet group and a high-fat diet group.

The normal diet group supplied 11.7% of the total calories as fat with AIN-76A diet #100000 (Dyets Inc., Bethlehem, PA, USA), and the high-fat diet group used beef tallow as a fat source to provide AIN -76 High-fat diet #100496 (Dyets Inc., Bethlehem, PA, USA) was fed with 40% of total calories as fat.

The test animals were fed a normal diet and a high-fat diet for 6 weeks from 4 weeks of age to 10 weeks of age, and a diet containing 3% of the weight of each test sample extract was supplied to the high-fat diet for 4 weeks from 10 weeks of age to observe the effect. .

The test animals were bred separately one by one, and water and food were supplied without restriction. During the test period, food intake and body weight were measured twice a week. Food efficiency ratio (FER) was calculated by dividing the weight gain during the test period by the food intake during the test period, with the total test period from the test meal supply date to the sacrifice date, and the results are shown in Table 7.

dietary efficiency normal diet 8.14 high fat diet 12.10 Example 1 green beans 8.04 Example 1 Roasting 8.04 Example 2 green beans 7.98 Example 2 Roasting 8.01 Example 3 Green Beans 7.52 Example 3 Roasting 7.68 Example 4 Green Beans 8.42 Example 4 Roasting 8.41 Example 5 Green Beans 8.43 Example 5 Roasting 8.41 Example 6 Green Beans 8.43 Example 6 Roasting 8.43 Example 7 Green Beans 8.43 Example 7 Roasting 8.45 Example 8 Green Beans 8.43 Example 8 Roasting 8.46 Example 9 Green Beans 8.17 Example 9 Roasting 8.17 Example 10 Green Beans 8.18 Example 10 Roasting 8.20 Example 11 Green Beans 8.19 Example 11 Roasting 8.21 Example 12 green beans 8.15 Example 12 Roasting 8.16 Example 13 Green Beans 8.15 Example 13 Roasting 8.15

Looking at the results of Table 7, the extract of the present invention did not significantly increase in weight compared to the high-fat diet of the control group and was almost similar to the normal diet of the control group. The result of further weight loss was confirmed. From the above results, the significant weight loss effect of the extract of the present invention was confirmed.

<Test Example 8: Adipocyte size reduction effect using test animals>

In each test group of Test Example 7, when they were 16 weeks old, 4 rats were randomly selected and sacrificed, and blood and organs were collected.

The average size of adipocytes was measured by cutting the excised epididymal adipose tissue about 30 times with scissors, then using a medium containing 4% (wt/vol) albumin and 1.5 mg/ml collagenase (Sigma, USA) 199 (Gibco BRL) after homogenization at 37 ° C. for 60 minutes, the diameter of about 30 cells was measured using a microscope to obtain the resulting value, and the results are shown in Table 8 below.

Adipocyte size (㎛) normal diet 10.73±3.03 high fat diet 18.72±4.11 Example 1 green beans 8.46±1.15 Example 1 Roasting 8.44±1.14 Example 2 green beans 8.27±1.13 Example 2 Roasting 8.34±1.11 Example 3 Green Beans 8.07±1.15 Example 3 Roasting 8.11±1.13 Example 4 Green Beans 10.17±1.15 Example 4 Roasting 10.13±1.14 Example 5 Green Beans 10.21±1.15 Example 5 Roasting 10.17±1.17 Example 6 Green Beans 10.20±1.15 Example 6 Roasting 10.19±1.14 Example 7 Green Beans 10.19±1.14 Example 7 Roasting 10.22±1.15 Example 8 Green Beans 10.22±1.15 Example 8 Roasting 10.22±1.16 Example 9 Green Beans 9.75±1.12 Example 9 Roasting 9.74±1.11 Example 10 Green Beans 9.81±1.13 Example 10 Roasting 9.90±1.11 Example 11 Green Beans 9.89±1.11 Example 11 Roasting 9.84±1.12 Example 12 green beans 9.32±1.11 Example 12 Roasting 9.34±1.12 Example 13 Green Beans 9.30±1.11 Example 13 Roasting 9.32±1.11

Looking at the results of Table 8, the extract of the present invention significantly reduced the size of fat cells compared to the high-fat diet of the control group and was almost similar to the normal diet of the control group. The reduction result was confirmed. From the above results, it was confirmed that the extract of the present invention had a significant effect of reducing the size of adipocytes.

<Test Example 9: Change in blood cholesterol content>

Using the blood collected in Test Example 8, the content of cholesterol was measured by an enzyme colorimetric method using a kit (Sigma Chemical Co. (St. Louis, MO)), and the results are shown in Table 9.

Total Cholesterol (mg/dl) normal diet 60.71±1.21 high fat diet 73.10±1.11 Example 1 green beans 61.24±1.15 Example 1 Roasting 61.18±1.14 Example 2 green beans 60.29±1.13 Example 2 Roasting 60.17±1.11 Example 3 Green Beans 58.14±1.15 Example 3 Roasting 58.29±1.13 Example 4 Green Beans 67.17±1.15 Example 4 Roasting 67.13±1.14 Example 5 Green Beans 67.21±1.15 Example 5 Roasting 67.17±1.17 Example 6 Green Beans 67.20±1.15 Example 6 Roasting 67.31±1.14 Example 7 Green Beans 66.19±1.14 Example 7 Roasting 66.58±1.15 Example 8 Green Beans 66.25±1.15 Example 8 Roasting 66.22±1.16 Example 9 Green Beans 65.75±1.12 Example 9 Roasting 65.74±1.11 Example 10 Green Beans 64.81±1.13 Example 10 Roasting 64.90±1.11 Example 11 Green Beans 64.89±1.11 Example 11 Roasting 64.84±1.12 Example 12 green beans 63.12±1.11 Example 12 Roasting 63.19±1.12 Example 13 Green Beans 63.30±1.11 Example 13 Roasting 63.32±1.11

Looking at the results of Table 9, the total cholesterol content of the extract of the present invention was significantly reduced compared to the high-fat diet of the control group and was almost similar to the normal diet of the control group. reduced results were observed.

In addition, the precipitation reagent dextran sulfate (dextran sulfate) MgCl ₂ After precipitating LDL and VLDL using a kit (Sigma, USA), the cholesterol content of the supernatant was colorimetrically quantified to measure HDL cholesterol. The decrease in cholesterol content in was accompanied by a decrease in LDL cholesterol.

As described above, the present invention prepares green coffee beans with markedly enhanced cordycepin by inoculating and fermenting green coffee beans with one or more spawners selected from spawners consisting of Ganoderma lucidum, Phellinus linteus, Chaga mushroom, Zinnia mushroom, and Cordyceps sinensis. Then, by extracting the green coffee beans, an extract containing cordycepin showing antibacterial, antifungal, immune enhancing, anticancer, and antithrombotic effects can be prepared. It is very useful for industrialization and commercialization.

When the composition according to the present invention is taken, the immune function of human normal cells is activated by the increased cordycepin content to inhibit the proliferation and recurrence of cancer cells, and antithrombotic, antibacterial, and antifungal effects can be obtained through inhibition of platelet aggregation, , Incidentally, it has the advantage of being able to feel the cacao scent that stands out with the soft chocolate scent, and the soft, deep and rich taste and aroma.

Claims (10)

Inoculating green coffee beans with at least one spawn selected from the group consisting of Ganoderma lucidum, Phellinus elegans, chaga, zinnia and cordyceps and fermenting them; and
A novel method for producing a cordycepin-containing extract using coffee and mushroom strains, comprising the step of extracting the fermented green coffee beans using water or ethanol as a solvent.
The method according to claim 1, wherein the green coffee beans are germinated.
The method of claim 1 or claim 2, wherein at least one spawn selected from the group consisting of Ganoderma lucidum, Sanghwang mushroom, Chaga mushroom, Zinnia mushroom, and Cordyceps sinensis is inoculated with yeast and then maintained at 20 to 40 ° C for 70 to 170 hours. Method for producing a cordycepin-containing extract, characterized in that cultured.
The cordycepin according to claim 1 or 2, wherein the fermentation is performed by culturing the complex spawn inoculated into the green coffee beans at a temperature of 20 to 45 ° C and a humidity of 35 to 70% for 12 to 120 hours. Method for preparing the containing extract.
The method for preparing an extract containing cordycepin according to claim 1 or 2, further comprising roasting the green coffee beans before the extraction step.
Green coffee beans in which cordycepin content is increased by inoculation and fermentation of one or more seed fungi selected from the group consisting of Ganoderma lucidum, Sanghwang mushroom, Chaga mushroom, Zinnia mushroom, and Cordyceps sinensis.
The green coffee beans with increased cordycepin content according to claim 6, wherein the green coffee beans are germinated.
Coffee beans with increased cordycepin content prepared by roasting green coffee beans by inoculating and fermenting at least one spawn selected from the group consisting of Ganoderma lucidum, Sanghwang mushroom, Chaga mushroom, Zinnia mushroom, and Cordyceps sinensis.
Inoculating green coffee beans with at least one spawn selected from the group consisting of Ganoderma lucidum, Phellinus elegans, chaga, zinnia and cordyceps and fermenting them; and
A composition containing cofisepin exhibiting antibacterial, antifungal, immune enhancing, anticancer, and antithrombotic effects by including the extract prepared through the step of extracting the fermented green coffee beans using water as an active ingredient.
Inoculating green coffee beans with at least one spawn selected from the group consisting of Ganoderma lucidum, Phellinus elegans, chaga, zinnia and cordyceps and fermenting them;
Roasting the fermented green coffee beans to prepare coffee beans: and
A composition containing cofisepin showing antibacterial, antifungal, immune enhancing, anticancer, and antithrombotic effects by including an extract prepared through the step of extracting coffee beans using water or ethanol as an active ingredient.

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