KR101884298B1 - Fermentation Method for Increasing Content of Total Phenolic Compounds and ß-Glucan with Solid Fermented Doraji, Platycodon grandiflorum, using mushroom mycelia - Google Patents

Abstract

The present invention relates to a fermentation process for solidifying fermented platycodon with mushroom mycelia to increase the content of total phenolic compounds, platocidin D and betaglucan, and more particularly to a fermentation method comprising sterilizing washed platycodon, A step of inoculating a mushroom mycelium selected from Lentinula edodes KCTC 6734 and Inonotus obliquus KCTC 26147, and fermenting the mixture at a temperature of 25 ° C and a humidity of 80% by adding yeast extract to a nitrogen source, The present invention relates to a solid fermentation method of bellflower.
Platycodon fermented by mushroom mycelia of the present invention increased 1.7 to 2.5-fold and beta-glucan increased 2.5 to 32-fold, respectively, as compared to the control group in which the total phenolic compound was non-fermented. Also, the content of Platycodin D is increased in the saponin of the bellflower.

Description

FIELD OF THE INVENTION [0001] The present invention relates to fermentation methods for increasing the content of total phenolic compounds and beta-glucan by solid fermentation of bellflower with mushroom mycelium (Fermentation Method for Increasing Content of Total Phenolic Compounds and ß-Glucan with Solid Fermented Doraji, Platycodon grandiflorum, using mushroom mycelia)

In particular, the present invention relates to a method of solidifying fermented Platycodon grandiflorum with a mycelium of mushrooms, more particularly, to a method of sterilization of washed Platycodon grandiflorum, a method of sterilizing sterilized Platycodon grandiflorum, Lentinula edodes KCTC 6734 and Inonotus obliquus KCTC 26147 And a step of fermenting the yeast extract at a temperature of 25 DEG C and a humidity of 80% for 3-6 days by inoculating one mushroom mycelium with yeast extract as a nitrogen source.

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Platycodon grandiflorum is called aka gyungyeong (kikyou) and its scientific name is Platycodon grandiflorum. It is a native plant in Korea, China and Japan and has been used as a vegetable and perennial herb. The major component of Platycodin D is Platycodin D, and the bound sugar is a D-apiose, which is a pentose. Platycodin D is the major saponin in Platycodin D, The major pharmacological effects of these saponins include, but are not limited to, the following effects in animal experiments: intestinal damage, gonadal function, chewy nerve inhibitory action, acute chronic inflammation, anti-ulcer and gastric secretion inhibiting action, anticholinergic action to lower blood pressure by expanding blood vessels, Improvement action and the like were confirmed.

Recently, consumers are increasingly interested in functional health foods, and the demand for native perennial cultivars grown in Korea is increasing, and various processed products such as functional foods, soaps, cosmetics, and beverages are being developed. Bordeaux products are mainly marketed in the market as bordering segments, bamboo, juice, concentrate, concentrate, ring, juice, powder, etc. Recently, products utilizing bordering balloons have been gradually released.

Meanwhile, in the related art related to the present invention, Korean Patent Registration No. 1012907950000 discloses a method for preparing fermented Dorago cultivated with yeast or fermentation with yeast, wherein the content of platycodin D is increased, And a manufacturing method thereof. It was confirmed that the content of platycodine D was significantly increased in the case of culturing the pulverized bellflower with yeast isolated from the koji, fermenting with the koji, or fermenting with the koji after culturing with the yeast, compared with the general bellflower extract . Korean Patent Laid-Open Publication No. 10-2015-0042529 (Platycodon grandiflorum fermented composition having excellent anti-inflammatory and antioxidative activity and method for producing the same) shows that the fermented composition of mushroom mycelia of bellflower has excellent anti-inflammatory and antioxidative effects, As a food composition or a pharmaceutical composition for anti-inflammatory preventive treatment. In addition, Korean Patent Publication No. 1020140096771 discloses a pharmaceutical composition for preventing or treating contact dermatitis, which comprises platycodin D-containing platycodone extract as an active ingredient. Korean Patent Registration No. 1015160630000 Platycodin D-containing bellflower extract as an active ingredient Which is a cosmetic composition for skin moisturizing.

However, these prior arts have different technical constructions from those of the present invention.

Conventionally, in the fermentation of the bellflower using the mycelium of mushrooms, since the bellflower is frozen and then cooled, the mycelium is applied and fermented for one month or longer, so that the fermentation time is long and the process is complicated. Also, the fermentation of the fermented product was carried out using mycelium because the concentration of the saponin - based platycodin D or the antioxidant substance was increased. The present inventors intend to obtain a component having excellent immunoactivity and antioxidative activity by culturing the domestically produced native bellflower in domestic form using mushroom mycelium.

According to statistical data of the Ministry of Agriculture, Forestry and Livestock Foods in Korea, it is reported that the cultivation area of the platycodon in Korea is maintained at 600-700 ha. Domestic production of platycodon was 5,255 tons in 1991 but decreased gradually to 2,697 tons in 2002, but it started to increase again in 2006 and increased to 2.575 times in 2011 It is expected to last for several years.

Therefore, it is necessary to supply the functional ingredient of health food or medicinal ingredient using the active ingredient of the bellflower to make the material industrial.

The present invention relates to a method for sterilization of washed platycodon, a method of inoculating a mushroom mycelium selected from Lentinula edodes KCTC 6734 and Inonotus obliquus KCTC 26147, which is a mycelium of shiitake mushroom, and a yeast extract, % ≪ / RTI > at 25 DEG C for 3-6 days.

Platycodon pollen fermented by the mushroom mycelium of the present invention showed 200-250% increase in the total phenolic compounds of the antioxidant activity compared to the non-fermented control and 250-500% increase in the? -Glucan. Also, the content of Platycodin D is increased in the saponin of the bellflower.

Fig. 1 shows L. edodes and I. obliquus mycelium grown on a selective medium.
Fig. 2 shows the solid state of borage broth according to the inoculation of 2% to 4% of the mushroom mycelium with respect to the bellflower.
Figure 3 shows the content of saponin in the platycodon fermented by mushroom mycelium (control: autoclave (non-sterile broth). LFD: bellflower fermented with mycelium of shiitake mushroom IFD:
Fig. 4 shows the cell survival rate of A549 bronchial epithelial cells according to the treatment with mushroom fermented Dorago extract.
FIG. 5 shows IL-8 secretion inhibitory activity of A549 bronchial epithelial cells treated with mushroom fermented Dorago extract.

The present invention comprises a step of selecting mushroom mycelium seeds from a plurality of mushrooms for bellflower fermentation, a step of solidifying the bellflower using mushroom mycelium, and a step of extracting the active ingredient from the mushroom mycelium fermentation broth.

≪ Example 1 > Selection of mushroom mycelium for bellflower fermentation

Mycelia of the mushroom mycelia of Doraji fermented mushroom were selected from mycelium of shiitake mushroom KCTC6734, mycelium mushroom KCTC26147, mushroom mycelium KCTC6190, mushroom mycelium KCTC6283, mushroom mycelium KCTC16855 and mushroom mycelium KCTC26249. YPD, which is commonly used to grow fungi and yeasts including mushroom mycelium, was selected as a general growth medium for mushroom mycelia used for fermentation of the bellflower. The mushroom and mushroom mycelia grown on the YPD medium were selected and used for blooming. After 3 days of liquid culture at 25 ℃, mushrooms with good growth of mycelium were selected as the strains of Doraji fermentation.

When mushroom and mushroom mycelium were cultured at 25 ℃ for 3 days, it can be observed that YPD agar grows like a white mycelium extending around the inoculated stock (a piece of medium containing mushroom mycelium). When YPD broth was transferred to YPD broth and grown under the same conditions, it was found that the mycelium grows radially around the inoculated mycelial culture medium piece. Over time, the mycelium grows larger, and the new mycelium that grows out of it grows into another mass of mycelium, increasing its amount and volume.

≪ Example 2 > Strawberry solid fermentation using mycelium of mushroom and mushroom

The length of the section was cut to 2 ~ 3cm so that the surface area of the mushroom mycelium reached wide when the bellflower provided by the mountain village farming association was fermented. This was sterilized at 121 ° C for 20 minutes in a beaker. It was confirmed that the sterilized platycodon was more refined than the untreated platycodon, and the platycodon itself contained a lot of moisture, so that it easily disappeared by heating. In addition, the water from the bellflower is considered to have a bad effect on the quality of the water. Therefore, in the present invention, the bellflower removed from the bark provided by the manufacturer is used and washed twice or three times in a laboratory to be used in the experiment without cutting.

The mushroom mycelium cultured in the YPD broth was centrifuged and inoculated with 2% and 4% (v / W), as shown in Fig. The fermentation was conducted for 3 days at 80% humidity and 25 ° C set as initial fermentation conditions, and no change was observed on the third day of fermentation. In the YPD agar, when white mushroom mycelium was cultivated, it was observed that the white mycelium was grown. Therefore, if white mycelium is visible on the surface of the bellflower, fermentation can be confirmed. However, no mushroom mycelium was observed on the third day of fermentation, so the fermentation time was increased to 6 days, and a blue mold was bloomed. This shows that sterilization of bellflower was not properly performed with penicillin. In addition, the mushroom mycelium was not found in the bellflower because of insufficient nutrients required for growth of mycelium.

Instead of the beaker, the test material (test sieve, diameter: 7 cm, sieve size: 300 μm) was sterilized with the bellflower and used in the experiment. Sterilized at 121 DEG C for 20 minutes, cooled and sterilized again under the same conditions. As a result, unlike the previous experiment, the growth of other microorganisms such as blue mildew was not observed in addition to the mushroom mycelium, indicating that the sterilization was completely completed.

In the previous experiment, 0.002% (w / w) yeast extract was added to the mycelium at the time of inoculation, because the mushroom mycelium was not found in the fermented bellflower using the mushroom mycelium. 2% (v / W) of shiitake mushroom and mushroom mycelia were inoculated into the sterilized bellflower broth, and mushroom mycelia grew on the bellflower surface.

≪ Test Example 1 > Antioxidant content analysis of fermented Doradas

Antioxidant contents of fermented bellflower were analyzed by total phenol and total flavonoids. The pretreatment of the sample was performed by freezing and drying the frozen Doradji using the shiitake mushroom and the mushroom mycelium, and microwave and hot water extraction were performed using methanol as a solvent. This was used as a sample. Using the mycelium of shiitake mushroom, solidified fermented bellflower was heated at 50 ℃ for 24 ± α hours, dried and powdered, and extracted 3 times with distilled water for 1 hour. This was concentrated and lyophilized to prepare a sample.

≪ Analysis of total phenolic compounds >

The content of polyphenol compounds was measured by modifying the Folin-Denis method (Foloin and Denis, 1912). That is, 6 mL of distilled water was added to 0.1 mL of the extract, and 0.5 mL of folin-ciocalteu's phenol reagent was added thereto in order. After stirring, 1.5 mL of a saturated solution of Na ₂ CO _{3 was} added, and the mixture was sufficiently stirred. After allowing to stand at room temperature for 1 hour, absorbance was measured at 765 nm (UV / VIS spectrophotometer, UV-1650PC, Shimadzu Co., Kyoto, Japan). The total phenolic compound content of the sample extract was calculated from the standard calibration curves obtained by preparing gallic acid as a standard. The fermented bellflower was frozen and dried, and methanol was extracted by microwave and heat - extraction with an extraction solvent, respectively. The fermented bellflower was used to analyze the antioxidant content of the fermented bellflower.

It can be seen that the content of total phenolic compounds is much higher than that extracted by microwave heating. The amount of the phenolic compound in the bellflower is increased by 170% to 250% by fermentation although there is a difference in the content depending on the extraction method. As the bellflower is fermented by mycelium of mushrooms, the amount of the phenolic compound, which was present as a polymer in the bellflower, is decomposed and increased.

<Total flavonoid analysis>

The analysis of flavonoid compounds was performed according to the total flavonoid test described in the Health Functional Food Cycle. That is, 1.5 mL of ethanol, 0.1 mL of 10% aluminum nitrate solution, 0.1 mL of 1 M potassium acetate solution and 2.8 mL of distilled water are added to 0.5 mL of the extract, and the mixture is sufficiently stirred. Blank tests of standard substances and samples for each concentration were made by adding 0.1 mL of distilled water instead of 10% aluminum nitrate solution. After standing at room temperature for 40 minutes, the absorbance of the liquid layer is measured at 415 nm (UV / VIS spectrophotometer, UV-1650PC, Shimadzu Co., Kyoto, Japan).

This also shows a higher total flavonoid content than the heat extracted from the microwave. The total flavonoid content of fermented bellflower showed a tendency to decrease compared to the control group, and it was found that the amount of fermented bellflower was remarkably decreased especially by using the mycelium of mushroom. It appears that fermentation completely decomposes the flavonoid material or converts it to another substance such as a phenolic compound.

<Analysis of content of saponin of fermented Doragoji>

The content of saponins in the fermented bellflower of mushroom mycelium was analyzed by Platycodin D, Platycodin D2 and Deapio-paltycodin D among the bellflower saponins. The results are shown in FIG. Platycodin D, which is the main saponin which is the pharmacological action of the bellflower, increased in both the mushroom and the mushroom mycelial fermentation. Platycodin D content was 1.2 mg / g and 1.3 mg / g, respectively, And it was confirmed that it was increased more in the mycelial fermentation broth. In addition to Platycodin D, Platycodin D2 and Deapio-paltycodin D, which are related to Platycodin D, were found to be slightly altered but not greatly differentiated. It is considered to be a strain that can produce more than bellflower saponins.

(1-3) (1-4) β-glucan and (1-3) (1-6) β-glucan analysis

The fermented bellflower was freeze-dried and pulverized into a sample. Staphylococcus aureus was sterilized twice at 121 ° C for 20 minutes as a control for fermented bellflower. (1-3) (1-4) β-glucan and (1-3) (1-6) β-glucan were analyzed using the Megazyme kit.

The content of β-glucan in the mushroom mycelia was higher than that of (1-3) (1-4) β-glucan in (1-3) (1-6) β-glucan in all the broodstock fermented with mushroom mycelia The contents of (1-3) (1-4) β-glucan and (1-3) (1-6) β-glucan in the mushroom mycelial fermentation broth were 1.7 ± 0.016% (w / (1-3) (1-4) β-glucan and (1-3) (1-6) β-glucan in the mushroom mycelial fermentation broth were 0.92 ± 0.041% (w / Were 3.5 ± 0.003% (w / w) and 1.1 ± 0.036% (w / w), respectively. (1-3) (1-4) β-glucan fermented with mushroom mycelium rather than non-fermented bellflower is about 250-500%, (1-3) (1-6) β-glucan is 270-320% And it is considered to be an increase by mycelium of mushrooms.

<IL-8 inhibitory activity of bronchial epithelial cells of fermented bell pepper>

The human bronchial epithelial cell line, type II human alveolar epithelial cell line (A549), was purchased from the Korean Cell Line Bank (Seoul, South Korea) and cultured in Dulbecco's modified Eagle's medium (DMEM) medium FBS, penicillin (10,000 U / (P / S) manufactured by Life Technologies Co. (gibco ^® , Grand Island, NY, USA) was used. IL-1b, TNF-α, and IFN-γ were used to express inflammatory genes (IL-8) in A549 cell lines (BioLegend, San Diego, CA, USA). IL-8 was measured by enzyme-linked immunosorbent assay (ELISA) kit (BioLegend, USA).

Cell Culture: 80-90% under antibiotic-free DMEM medium (100 units / ㎖ penicillin, 100 ㎎ / ㎖ streptomycin) and 37 ℃ by the addition of 10% fetal bovine serum (FBS) , 5.0% CO 2 conditions confluent (3 ~ 4 days). The cells were subcultured and used for the experiment.

Measurement of IL-8 concentration by ELISA To determine the amount of IL-8 secreted in the supernatant by ELISA kit (BioLegend, USA), A549 cells (100 mm culture dish 1 x 10 ⁵ cells / mL) After incubation for 24 hours at a concentration of 100 ng / ml each containing 100 μg / ml of TNF-α and IFN-γ and containing all samples with a cell viability exceeding 90% by CCK-8 assay, the supernatant was collected The inhibitory effect of fermented Dorado was calculated by measuring the amount of protein secretion at 450 nm or 570 nm by the IL-8 ELISA kit provided.

<IL-8 inhibitory activity of bronchial epithelial cells of fermented bell pepper>

To elucidate the anti-inflammatory effects of the mushroom fermented Doradji extract by using the type II human alveolar epithelial cell line (A549), which is a human bronchial epithelial cell line, A549 bronchial epithelial cells were isolated from the extracts by CCK-8 assay The sample throughput was determined to be 0.5 mg / mL, the highest concentration showing survival rate (FIG. 4).

The protein secretion of IL-8 in A549 bronchial epithelial cells at a concentration of 0.5 mg / mL of mushroom fermented bell pepper extract was about 9.8 ug / mL (9% reduction), slightly less than 10.1 ug / mL (7% reduction) (Fig. 5-2). As a result of the CCK-8 assay, the mushroom fermentation broth was less toxic to the cells than the raw platelets, The inflammation-inhibiting effect is considered to be large (Fig. 5).

After extracting the total phenolic compound and beta-glucan from the above-mentioned solid fermented dorado with hot water or a lower alcohol, it can be made into a food such as a beverage, a ring, a powder, a capsule, or a health functional food.

Platycodon fermented by the mushroom mycelium of the present invention showed an increase of 200-250% in total phenolic compounds and 250-500% in? -Glucan over non-fermented control. In addition, the content of Platycodin D is found to increase in the saponin of the bellflower, which is industrially applicable.

Claims (7)

(One). Sterilizing the washed raw broccoli;
(2). Culturing mycelium of Lentinula edodes KCTC 6734 and mycorrhizal mycelium Inonotus obliquus KCTC 26147 in YPD medium;
(3). 2% (v / w) of mycelium of Lentinula edodes KCTC 6734 and 2% (v / w) of Inonotus obliquus KCTC 26147, mycelia of mushroom mycelia, cultured in step (2) Adding 0.002% (w / w) of yeast extract to solid fermentation for 3-6 days at a temperature of 25 DEG C and a humidity of 80%
Wherein the solid fermentation is carried out by cultivating the sterilized platycodon in a water-repellent sieve. delete delete delete delete delete delete

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