KR20020048778A - Alismatis Rhizoma fermented wine and process for prepatration therof - Google Patents

Abstract

PURPOSE: Provided is Alismatis Rhizoma fermented wine which has excellent aroma and color and its preparation process. Alismatis Rhizoma has excellent antibacterial activity, antioxidation effect and immuno activity. CONSTITUTION: The manufacturing method comprises the steps of; soaking rice in water then steaming it; inoculating Aspergillus awamori thereinto then culturing them at 30-55 deg.C to manufacture koji; supplying water to the koji then followed by inoculating yeast into and fermenting it to prepare raw spirit; soaking Alismatis Rhizoma then steaming it: mixing the steamed Alismatis Rhizoma with the first-fermentate and water; fermenting the mixture at 20-40 deg.C then followed by filtering and sterilizing it; and adding sugar thereto at 2-7%.

Description

Taxus fermented wine and its manufacturing method {Alismatis Rhizoma fermented wine and process for prepatration therof}

The present invention relates to a taxi fermentation wine and a method for producing the same. More specifically, the present invention incubated with black bacteria, and then prepared a jumogi inoculated in the taxis having antimicrobial, antioxidant and immuno-activating effect, and the fermentation of liquor fermented with excellent color, flavor and taste produced by alcohol fermentation and its preparation It is about a method.

Alismatis Rhizoma , an Alismataceae plant, is a remnant group differentiated during the evolution of monocotyledonous plants, with about 13 genera and 90 species distributed worldwide. Fujian, Jiangxi, Sichuan, Guitian and It is also produced in Yunnan province, and in Korea, it is produced in the Jeonnam Suncheon region, which accounts for more than 50% of its production, in Muan, Gurye, and Sangju, Gyeongsangbuk-do, and the taxa used as a herbal medicine is Alisma plantago- aquatica L. var. orientale Samuels) is mainly grown and produced. The constituents of taxa are known as 25% starch, 7% protein, furfural, resin, essential salts, alisol A, B, C, and acetates and acetates of triterpenoids. Such taxa is known as a medicine that removes moisture, helps the intestine, and strengthens energy. It is contained a lot of potassium (K), and when the taxi is taken, potassium (K) in the urine is significantly increased. Taxa has also been reported to reduce blood cholesterol, lower blood pressure and blood sugar, and have an effective interpolation effect on hepatotoxicity induced by carbon tetrachloride. In addition, recently, the tack powder is used as a coating agent for athlete's foot athletes, it is thought that a variety of physiologically active substances present in the tack. However, the actual research results on physiological activity of taxis are very insufficient and the development of health foods using them is insufficient.

The present inventors have completed the present invention by identifying the physiological activity such as the effect on the antibacterial, antioxidant activity and immune response control action of the taxi cab produced in Suncheon area by focusing on the above points.

Accordingly, it is an object of the present invention to provide a fermented liquor. Another object of the present invention is to provide a method for preparing a taxin fermented wine.

1 is a schematic diagram showing an embodiment of a preferred manufacturing method of the present invention for each process.

Figure 2 is a photograph of a plate medium showing the growth inhibition ring produced after the addition of the tax water extract to the test strain to determine the antimicrobial activity of the tax extract of the present invention.

Figure 3a is a graph showing the growth inhibitory effect of the Bacillus subtilis strain of the present invention tack extract.

Figure 3b is a graph showing the growth inhibitory effect of Pseudomonas aerignosa strain of the present taxa extract.

Figure 3c is a graph showing the growth inhibitory effect of E. coli strains of the present invention tack extract.

Figure 3d is Salmonella typhymuriu of the present invention taxa extract ( Salmonella typhymuriu-

m ) It is a graph showing the growth inhibitory effect of the strain.

Figure 3e is a graph showing the growth inhibitory effect of the Vibrio parahaemo liticus strain of the present taxa extract.

Figure 4 is Salmonella typhymuriu according to the process of extracting the taxa ( Salmonella typhymuriu

m ) electron micrograph.

Figure 5 is a graph showing the change in peroxide value according to the addition of the tack extract (100 μl) during linoleic acid automatic oxidation for 7 days at 50 ℃.

Figure 6 is a graph showing the change in peroxide value according to the concentration of the tax extract during the automatic oxidation of linoleic acid for 7 days at 50 ℃.

Figure 7 is a graph showing the change in TBA value when the present invention is treated with a rat extract.

Figure 8a is a graph showing the effect on the proliferation of splenocytes when the present invention is treated to splenocytes.

Figure 8b is a graph showing the effect on the proliferation of B cells when the present invention is treated to B cell tack extract.

Figure 9a is a graph showing the effect of the present invention tack extract on T cell proliferation in response to homologous antigen.

Figure 9b is a graph showing the degree of inhibition of T cells in response to the tax extract of the present invention homologous antigen.

Figure 10 is a graph showing the effect of the present invention taxa extract on the cytotoxicity of T cells in response to homologous antigen.

Figure 11 is a photographic representation of the fermentation liquor product of the present invention.

12 is a graph showing the alcohol content change during the second fermentation period of the present invention fermentation taxa.

Figure 13 is a graph showing the change in the amount of carbon dioxide (CO ₂ ) generated during the secondary fermentation period of the present invention fermentation taxa.

Figure 14 is a graph showing the change in total acid content during the secondary fermentation period of the present invention fermentation taxa.

15 is a graph showing a change in the reducing sugar content during the second fermentation period of the present invention fermentation taxa.

Figure 16 is a graph showing the change in free sugar content during the secondary fermentation period of the present invention fermentation taxa.

17 is a graph showing the sensory evaluation results of the present invention fermentation strain.

The object of the present invention is to prepare a taekta extract to check the antimicrobial activity against food poisoning and pathogenic microorganisms, the antioxidant effect and immune activity against linoleic acid and liver lipids, and then inoculated and cultured in rice cooked with Aspergillus awamori koji koji Koji was prepared, watered to the koji and inoculated with yeast ( Saccharomyces cereviceae ) and fermented for 48 hours to prepare a hair seedling . Prepared and added sugar in various concentrations to achieve the palatability of 20 sensory evaluation personnel by comparing and respectively.

Hereinafter, the configuration of the present invention will be described.

The present invention comprises the steps of preparing a taxi extract using methanol, ethanol and water to investigate the antibacterial, antioxidant and immune activity of the taxi; Measuring antimicrobial activity against food poisoning and pathogenic microorganisms to investigate the antimicrobial activity of the taxa extract, and examining the morphological changes of the microorganisms according to the treatment of the taxa extract; Irradiating with respect to the in vitro antioxidant activity and lipid peroxidation inhibiting liver functions for the linoleic acid in order to examine the antioxidant activity of the extract Alismataceae; Effects of Taxa Extract on Immune Cell Proliferation, Effects of Macrophage Cells on Nitric Oxide Production, Proliferative Response to Homologous Antigens, Inhibition of T Cell Cytokine Secretion by Homologous Antigens, and T Cell Killer Action on Homologous Antigens Investigating; After soaking rice for about 2 hours and then watering and increasing, inoculate with black rice (Aspergillus awamori) to incubate for 48 hours at 45 ℃ to prepare a koji (water) and yeast (Saccharomyces cereviceae) Inoculating the primary fermentation for 48 hours to prepare a hair seed; 2 hours after the taxi was called, water saving and steaming were added, and the first fermented product and water were added thereto, followed by fermentation at 30 ° C. for 6 days, followed by filtration and sterilization to produce products; Investigating the change of alcohol content during the second fermentation period of the present invention fermentation taxa; Investigating the change in the amount of carbon dioxide generated during the second fermentation period of the taxi fermentation strain of the present invention; Investigating the change of the total acid content during the second fermentation period of the taxi fermentation strain of the present invention; Investigating the change of the reducing sugar and free sugar content during the second fermentation period of the taxi fermentation strain of the present invention; Investigating the organic acid content of the present invention fermentation taxa; Investigating the inorganic component content of the present invention fermentation taxa; Investigating the free amino acid content of the present invention fermentation taxa; It consists of a step of comparing the preference degree by sensory evaluation by the five-point method of the present invention fermented liquor with 0, 2, 5% of sugar.

Alismatis Rhizoma of the present invention purchased and used harvested in Suncheon, Jeonnam in 1998. Alismatis Rhizoma extract used to measure antimicrobial, antioxidant and immune activity was added at a rate of 1500 ml per 100 g of taxa using solvents such as methanol, ethanol and water, and methanol and ethanol using a reflux cooler. Silver 80 ℃, water was prepared by extracting at 100 ℃ 2 hours and concentrated to 0.5ml per g of taxi.

The strains Bacillus subtilis , Pseudomonas aeruginosa , Escherichia coli , Salmonella typhimurium , Escherichia coli O-157 and Vibrio parahaemolyticus were used for antimicrobial testing. Nutrient broth (agar) was used for the culture of various microorganisms, and 3% NaCl was added for Vibrio parahaemoliticus .

Mice (BALB / c, C57BL / 6) used to investigate the immune activity of taxa were purchased from the Korea Experimental Animal Center (negative group Chungbuk), and 8-12 weeks old females were used. It was bred while supplying tea distilled water. RPMI 1640 medium, antibiotic antimicrobial was used Gibco BRL (Grand Island, NY, USA) products, FCS (fertal calf serum) was used PAA products. LPS and 2-ME (2-mercaptoehtan

ol), NaHCO 3, N-1-naphthyl-ethylen-diamine and sulfanamide were used by Sigma. In addition, the reagents used in the cytotoxicity assay (Cyto Tox 96). Non-radioactive Cytotoxicity Assay and Reagents Used to Measure Cell Proliferation (Cell titer96) Aqueous One Solution Cell Proliferation Assay was used for Promega, and the antibodies required for cytokine measurement were used for Pharmingen. Sephadex G-10 used for B cell separation was also used by Pharmingen. As a cell line for cell culture, RAW264.7, a mouse mononuclear / macrophage cell line, was purchased from Korea Cell Line Bank (Seoul National University College of Medicine). Medium for the cell culture was used by adding NaHCO ₃ 2g / L, penicillin 100unit / ml, streptomycin 100mg / ml to RPMI 1640 medium.

The alcohol content of the present invention fermented liquor is 8 to 10%, preferably 9%. In addition, the concentration of sugar added to reduce the bitter taste of the taxi fermentation liquor is 2 to 7%, preferably 5%.

The HPLC used for the measurement of reducing sugars and free sugars of the present invention fermentation liquor was from 00 company (please fill in the manufacturer's name and model name), and the ICP used for inorganic component analysis was from 00 company (please fill in the manufacturer's name and model name). Used. The amino acid automatic analyzer used for free amino acid measurement of taxa fermentation strain of the present invention was used by 00 company (please fill in manufacturer name and model name).

Preferred manufacturing method of the present invention fermented liquor is shown in FIG.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Experimental Example 1: Investigation of antimicrobial effect of taxa extract

In order to investigate the antimicrobial effect of taxa extracts, the effects of antimicrobial activity, microbial growth, and microbial morphology were investigated.

The measurement of antimicrobial activity modified Farag's method. That is, the growth medium containing 1.5% of agar is thinly spread on the bottom of a petri dish, and the growth medium containing 0.6% of agar is poured on it to make a double flat medium, and then each strain is placed on the flat medium. Smeared. The strain was placed on a plate medium coated with a certain amount of the tackifier extract 0.8cm in diameter filter disk (disc) and incubated for 24 hours at 36 ℃, the resulting growth inhibition was measured to investigate the antibacterial activity.

Microbial growth was measured using the liquid medium dilution method. Inoculate 5 ml of growth medium into the test tube and inoculate 1% of the cell culture cultured up to the middle of the logarithmic season, and then add tac extract extract to 30 to 200 µl per 5 ml of medium and incubate at optimum temperature of each strain. The extent of growth was measured by absorbance at 660nm using a spectrophotometer.

The morphological changes of microorganisms by Alixmatis Rhizoma extracts were treated with food extract microorganisms and their morphological changes were observed by electron microscopy. Incubate Salmonella typhimurium as a decay microorganism in a culture medium for 48 hours, add 100 microliters of taxa extract per ml of culture strain to the culture strain, leave for 3 hours, and centrifuge to isolate the cells. 0.05M phosphate buffer The cells were diluted with and fixed to 0.45 μl membrane filter. Soak overnight in 5% glutaaldehyde (glutaaldehyde) solution, wash with sterile water, and then immerse in 30 ~ 100% ethanol one by one in order to dehydrate, immerse in isoamylacetate for about 30 minutes to dry and electron microscope The photographing sample was prepared and observed.

Experimental results, as shown in Figure 2 and Table 1, the antimicrobial activity of the taxa extract, water extract showed stronger antimicrobial activity than methanol and ethanol extract. Also had antimicrobial activity appeared strongly to all tested strains except for Escherichia coli O-157 strain clear zone (clear zone) of Alismataceae extract of each test strain in Vibrio parahaemolyticus, Bacillus subtilis, Salmonella typhimurium, Pseudomonasaeruginosa and Escherichia coli 18.0, 16.0, 16.0, 13.0, and 11.5mm, respectively, and the antimicrobial activity of Vibrio parahaemolyticus was the strongest.

Antimicrobial Activity of Taxa Extracts Strain Methanol extract Ethanol extract Water extract Bacillus subtilis 14.0 13.5 16.0 Pseudomonas aerignosa 14.0 14.0 13.0 E.coli 11.0 11.0 11.5 Salmonella typhymurium 14.0 13.5 16.0 E.coli O-157 8.0 8.0 8.0 Vibrio parahaemolyticua 16.0 16.5 18.0 (Note) Unit: mm

As a result of investigating the effect of Alismatis Rhizoma extract on the growth of microorganisms, as shown in Figs. 3a to 3e, the growth of the bacteria was inhibited even by adding 30 μl of the Taxa extract per 5 ml of the medium. The growth of the bacteria was inhibited by more than 50% compared to the control, and the growth of the bacteria was almost suppressed when 200μl was added.

As a result of investigating the morphological changes of the microorganisms according to the extract of the taxa extract, as shown in FIG. 4, after treating the taxa water extract with Salmonerlla typhimurium , the surface structure of the cells was severely compared with the control group. Seo et al. (Seo Kwon Il, Sang Won Lee, Ki Ho Ho: Korean Agricultural Products Storage and Distribution Association (1999), 6 (1)), treated with E. coli O-157 extracts of cornus water, were observed by electron microscopy. It was reported that the cell membrane function was disrupted in the cells treated with the extract and the contents of the cells were leaked out of the cells, thereby inhibiting the growth of the cells. It was reported that the structure was severely collapsed.These phenomena are also the result of the exposure of lysates or cell components due to the destruction of the cell wall and cell membrane of microorganisms by taxi water extract. I think.

Experimental Example 2: Investigation of Antioxidant Effect of Taxa Extract

The in vitro antioxidant and hepatic lipid peroxidation inhibitory effects on linoleic acid of Alismatis Rhizoma were investigated. In order to measure the antioxidant effect of taxa extract, 1 g of linoleic acid, 10 ml of ethanol, and a predetermined taxa extract were added to a Erlenmeyer flask, and 25 ml of 0.2 M phosphate buffer was added and stored at 37 ° C. for a period of time. Then, the reaction solution was transferred to a separatory funnel, and 25 ml of chloroform was added thereto, followed by extraction two to three times. 25 ml of acetic acid and 1 ml of saturated KI solution were added to the chloroform extract, which was allowed to stand in the dark for 5 minutes, and then 50 ml of distilled water was added and titrated with a 1/100 N Na ₂ S ₂ O ₃ solution. Taxi extract was added methanol, ethanol, and water extract, respectively, and BHT 0.1% was added and stored at 50 ° C. for 7 days to measure peroxide value.

Lipid peroxidation inhibitory effect of Taxa extract was investigated in vitro using liver homogenate in rats. Phosphate buffer solution (phospha-

After homogenization with te buffer, pH 7.4), 1M of hydrogen peroxide (H ₂ O ₂ ), 50 mM iron sulfate (FeSO ₄ ), and 0.05 ml of taxa extract were added to the homogenate, and stored at 42 ° C. for 72 hours. thiobarbituric acid reactive substances) was measured.

As a result, as shown in FIG. 5, when the methanol extract, ethanol extract, and water extract 100 μl were added, the peroxide value was lower than that of the control, and the peroxide value of the water extract was smaller than that of the methanol and ethanol extracts, but was 0.1% BHT. A little higher than when added. In addition, as a result of measuring the peroxide value after adding the water extracts of the taxi water at the concentrations of 50, 100, 150 and 200 μl, as shown in FIG. 6, all of them appeared to be very low compared to the control, and when 150 μl was added, it was stored until 5 days. Almost no peroxide was produced, which was about the same as adding 100 μl of 0.1% BHT. Kwon et al. (Kwon Oh-Geun, Son Jin-Chang, Sang-Chul Kim, Sang-Cheol Kim, Pyo-Sang, Park, Seung-Woo: Korean Society of Agricultural Products and Storage (1998), 5 (281)) showed that the antioxidant activity of linoleic acid in the ethyl acetate fraction of methanol extract was The comparison group reported that BHT was 96.2%, and Seo et al. (Seo, Kwon-Il, Sang-Won Lee, and Ki-Ho Yang: Korean Agricultural Products Storage and Distribution Association (1999), 6 (1)) showed that the extracts of linusic acid were 10, 30 and After adding 50 μl and storing at 50 ° C. for 7 days, the peroxide value was measured to be 42, 30 and 25 meq / kg compared to 187 meq / kg of the control, which showed a significant antioxidant effect. Addition of only μl showed significant antioxidant effect.

The TBA value of liver lipids in rats was 3597 μmol malondialdehyde (MDA) / g liver in the control group without the addition of tax water extract as shown in FIG. 7, and the TBA value of the test groups to which the water extract was added was 1443 μmol MDA / g liver. All showed lower anti-oxidant effect than the control.

Experimental Example 3: Determination of Immune Activity of Taxa Extract

Effects of Alismatis Rhizoma Extract on the Proliferation of Immune Cells, Effects of Macrophage Cells on Nitric Oxide Production, Proliferative Response to Homologous Antigens, Inhibition of T Cell Cytokine Secretion by Homologous Antigens, and T Cells Against Homogenous Antigens The inhibitory effect of killer cells was investigated. To separate the splenocytes of the mice, the mice (BALB / c) were sacrificed with cervical distal bone, sterilized with alcohol, placed on the dissection, and the spleen was removed by dissecting the right flank. The spleen is made into single cells using tweezers, washed three times with washing medium, and diluted in an appropriate amount of 10% FCS-RPMI 1640 medium. The isolated splenocytes were diluted in 10 ml of 10% FCS-RPMI 1640, and 40 µl anti-Thy1.2 ascites were added thereto and incubated at 4 ° C. for 60 minutes. Thereafter, the mixture was centrifuged at 4 ° C and 1200rpm for 6 minutes, diluted in 5ml of FCS-RPMI 1640, and incubated for 45 minutes in a 37 ° C water bath by adding 250µl rabbit complement. Then, centrifugation was repeated twice for 6 minutes at 4 ° C. and 1200 rpm with 10 ml washing medium, diluted in 500 μl of 10% FCS-RPMI 1640 medium, and passed through a Sephadox G-10 column. Only cells were purified. Separation of T cells was diluted in 1 ml of 10% FCS-RPMI 1640 and the splenocytes were separated into nylon wool column and incubated for 60 minutes in 37 ℃, 5% CO2 incubator. After incubation, the nylon wool column was washed with media preheated to 37 ° C., and only non-adherent T cells were purified and used as alloreative T cells.

Splenocytes, purely isolated B cells, or purely isolated T cells were cultured in 96 well plates with allogenic splenocytes (C57BL / 6) in 1, 10, 100, 1000 ㎍ / ml concentrations. Next, the proliferation of each cell was measured on day 2 and day 3 and day 4. Measure the cell titer96 15 μl of cell titer was added to 100 μl of the culture solution on day 2, 3, and 4 using Aqueous One Solution Cell proliferation Assay (promega), followed by incubation for 4 to 8 hours, followed by 490 nm. OD value was measured at.

To investigate the effects of taxa extract on nitric oxide production in macrophages, macrophages were treated with 1, 10, 100, and 1000 µg / ml concentrations, or when LPS and IFN-γ were added. The concentration of nitric oxide produced was measured. Stable nitrogen monoxide (NO) oxide, NO ₂ ^- (nitrite) was measured using the Griess reaction. Incubate the macrophage line (RAW264.7) under appropriate conditions for 48 hours, add 100 µl of the culture supernatant to a 96well plate, and add Greiss reagent (0.1% N -naphthyl-ethylendiamine in H ₂ O + 1% sulfanilamide in 5% H ₃ PO ₄ ) was added in the same amount and reacted for 10 minutes, and then the absorbance was measured at 570 nm with a microplate reader (Titertek Multiscan Plus, Finland). The concentration of nitrate was calculated by comparison with a standard curve obtained by diluting sodium nitrite from 32 μM to 0.25 μM in 2-fold.

In order to investigate the effects of taxa extract on the proliferative response by homologous antigens, cell number determination experiments were performed to induce homologous antigens. Splenocytes of mouse C57BL / 6 treated with MMC, which are target cells, and T cells (T cell of mouse BABL / c), which were isolated from splenocytes with effector cells, were cultured together for 3 days. The conditions showing the proliferative response were confirmed.

In order to determine the cause of the proliferation of T cells caused by allogeneic antigens significantly inhibited by the taxa extract, the amount of cytokines produced by T cells in response to allogeneic antigens was measured. The amount of cytokines secreted when T cells reacted was recovered after 24 hours of supernatant cultured with splenocytes and taxa, and IL-2, IL-4, IFN-γ, IL- contained in the supernatant. The amount of 10 ELISA (enzyme linked immounso-

It was measured by an rbent assay kit. At this time, the limit of measurement of cytokine concentration was 10 pg / ml.

Splenocytes were isolated from C57BL / c mice using the splenocyte separation method, diluted in 2ml of 10% FCS-RPMI medium, and 200mg of mytomycin C was added to 2mg / ml. (water bath) was treated for 25 minutes. Then, wash repeatedly with centrifuged sedimentation for 5 minutes at 4 ℃ and 1200rpm for 5 minutes with washing medium.

Used as ic splenocytes or target cells.

The ratio of target cells (allogeneic spleen cells, 1 × 10 ⁷ , 5 × 10 ⁶ / well) and effector cells (Effetor cell, T cells, 1 × 10 ⁶ / well) is 10: 1 or 5 : into a final volume of 96well round bottom plate 1 so as to 100㎕ were incubated 8 hours for 4 ℃, then centrifuged 5 minutes in the precipitation 250g 37 ℃, 5% CO ₂ incubator. However, 45 minutes before the incubation time was 8 hours, the target maximum and volume correcting control (Lysis Buffer) was diluted 10-fold and added to the cell solubility to the maximum. After incubation, centrifuged for 5 minutes at 250 g at 4 ° C. Then, 50 µl of the supernatant was transferred to a 96-well plate on a flat bottom, and 50 µl substrate mix buffer was added thereto, and the aluminum foil was covered at room temperature for 30 minutes. After the reaction, 50 μl stop soultion was added and the OD was measured at 490 nm using a microplate reader. The amount of lactate dehydrogenase (LDH) in the supernatant was measured. The degree of killer cell activity was shown. The degree of killer cell activity was calculated using the following equation.

% CYTOTOXICITY = Expermental-Effector Spontaneous-Target Spontaneous × 100 Target Maximum-Target Spontaneous

As a result of measuring the proliferation of cells by the concentration of taxa extract in the spleen cells of the mouse by concentration, as shown in FIG. 8a, the concentration of the splenocytes in the experimental group treated with the extract was increased in a concentration-dependent manner as shown in FIG. Could. As a result of measuring the induction of B cell proliferation of taxa by separating only B cells contained in splenocytes, as shown in FIG. 8B, proliferation was increased in a concentration-dependent manner as in splenocytes, especially at a concentration of 1000 ㎍ / μl. It was found that there was an excellent synergistic effect.

As a result of investigating the effect of taxa extract on nitric oxide (NO) production of macrophage, as compared to the untreated control group, as shown in Table 2, the experimental group treated with taxa extract alone by concentration and LPS were treated together with LPS. None of the experimental groups induced carbon monoxide (NO) production in macrophages. However, the experimental group treated with 1ng / ml of IFN-γ showed a tendency to decrease slightly at low concentrations of the taxa extract, but it was found that the production of nitric oxide (NO) was significantly increased at high concentrations. In other words, it can be seen that the taxa extract has a synergistic effect on the production of nitrogen monoxide (NO) induced by IFN-γ at high concentrations such as 1000 µg / µl.

Effects of Taxa Extracts on Nitric Oxide Production in Macrophage Cell RAW 264.7 Taxa Extract Stimulant 0 LPS (1 μg / ml) IFN-γ (1 ng / ml) 0 4.17 ± 1.57 3.81 ± 1.25 5.47 ± 1.74 One 2.90 ± 0.13 3.12 ± 0.08 2.86 ± 0.23 10 2.87 ± 0.24 2.66 ± 0.19 3.50 ± 0.20 100 3.02 ± 0.10 3.02 ± 0.71 6.43 ± 0.01 1000 4.49 ± 1.47 5.87 ± 2.62 21.44 ± 3.04 Taxa extract unit: µg / ml

As a result of investigating the effect of taxa extract on the proliferative response by homologous antigen, as shown in FIG. 9A, when the same number of target cells (5 × 10 ⁵ cells / well), the number of effect cells was 1 × 10 ⁶ cells per well. Maximum proliferative response was seen at 3 days after culture. Therefore, as a result of measuring the proliferative response on day 3 after cultivation by treating 0.01, 0.1, 1, 10 ㎍ / ml taxa extract under the same conditions, as shown in FIG. It was found that the reaction was suppressed. That is, it showed a growth inhibitory effect of about 50% at 1 μg / ml.

As a result of measuring the amount of cytokines produced by T cells, T cells responding to homologous antigens produced IL-2, IL-10 and IFN-γ, as shown in Table 3. It was found that the production of IL-2, IFN-γ and IL-10 was suppressed in the experimental group added. Therefore, it is thought that the taxa extract inhibits cleavage proliferation by inhibiting cytokine production of T cells in response to homologous antigens.

Effects of Taxa Extracts on Cytokine Secretion of T Cells Responding to Homologous Antigens Experimental conditions Cytokines IL-2 IFN-γ IL-4 IL-10 T cell <10 94.3 ± 17.2 <10 200.0 ± 21.2 T cell + spleen 372.4 ± 4.2 644.3 ± 12.1 104 ± 13.3 430.0 ± 148.5 T Cell + Spleen + Taxa Extract 316.4 ± 3.7 422.9 ± 23.2 <10 205.0 ± 70.7 (Note) Cytokine Unit: pg / ml

As a result of investigating the effect of taxa extract on T cell apoptosis on allogeneic antigen, as shown in FIG. 10, T. agar extract completely inhibited T cell apoptosis on homologous antigen at low concentration of 1㎍ / ml And it was found. Therefore, the extract of Taeksa inhibited the proliferation by inhibiting the cytokine production of T cells against homologous antigens, and also inhibited the killer cell activity against homologous antigens of T cells.

Example 1: Preparation of the present invention fermented liquor

1st step: cultivation

The raw rice was washed, soaked 80g in water for 2 hours and then water-saved for 1 hour. The soaked rice was put in a see-through lamp and the mixture was increased for about 1 hour. The steamed rice was removed by lumps and cooled to about 35 ° C., inoculated with black pepper ( Aspergillus awamori ), and incubated at 45 ° C. for 48 hours to prepare koji. Cozy production of this process aims to produce yeast by breeding yeast in rice.

Second Process: Primary Fermentation

In order to actively proceed the secondary fermentation for the production of fermented liquor was cultured and yeast was prepared juju. That is, 104 g of water was added to 80 g of koji prepared in the first step, and 18 ml of yeast ( Saccharomyces cereviceae ) was inoculated to ferment for 48 hours to prepare a main hair .

Third Process: Secondary Fermentation

80 g of the taxi was soaked in water for 2 hours and then water-saved for 1 hour and then increased. 200 g of water was added to 80 g of the increased taxa, and the primary fermentation product prepared in the second step was added together and fermented at 30 ° C. for 6 days. After the fermentation was terminated, the filtrate was obtained by filtration and sterilized using 0.45 membrane filter and then commercialized as shown in FIG. 11.

Experimental Example 4 Change of Alcohol Content during Fermentation

The changes of alcohol content during the second fermentation were investigated by mixing the primary and fermented talc with the fermented black soup. Alcohol quantification was performed by accurately taking 100 ml of the sample and placing it in a distillation flask when the distillate received about 70 ml (about 20 minutes) into the volumetric flask. After distilled water was added to distilled water to make 100ml, shake well and mix well, and after about 15 ℃, alcohol content (volume%) was measured by alcohol. When it could not be found at 15 ℃, it was calibrated using the temperature calibration table. As a result, as shown in FIG. 12, the alcohol content of the first 3% was increased as the alcohol content increased over the fermentation period, reaching 9.1% on the 6th day of fermentation, and thereafter, there was no change.

Experimental Example 5 Carbon Dioxide (CO) during Fermentation ₂ Change in generation

Changes in carbon dioxide emissions during the second fermentation of liquefied liquor were investigated. Carbon dioxide generation (please specify the carbon dioxide measurement method) was measured using.

As a result, as shown in FIG. 13, the amount of carbon dioxide (CO ₂ ) generated during the second fermentation period of the ethanol of the taxi was 3.2g / 80g, and gradually increased as the fermentation period elapsed, and became 14.98g / 80g on the 6th day of the fermentation. There has been little change since then.

Experimental Example 6: Change of Total Acid Content during Fermentation

The total acidity was quantified during the second fermentation period of the taxi fermentation strains. Total acid quantification was converted to succinic acid by titrating 10 ml of the sample with 0.1N-NaOH. As a result, as shown in FIG. 14, the total acid content, which was 0.07% for the first time, gradually increased as the fermentation period elapsed, and became 0.19% on the 6th day of fermentation, and there was little change thereafter.

Experimental Example 7: Changes of Reducing Sugars and Free Sugars in Fermentation

The changes of reducing sugar and free sugar contents were investigated during the second fermentation of liquefied liquor. Reducing sugar content was quantified by the DNS method and expressed in terms of glucose. In addition, the content of free sugar was centrifuged at 3000 rpm for 20 minutes for a certain amount of the sample, and the supernatant was membrane filtered with a 0.25 μm filter. The filtrate was passed through a Sep-Pak C ₁₈ cartridge (cartidge) to remove the pigment and polymer material was analyzed by HPLC. For HPLC analysis, 0.5% of glucose, fructose and sucrose were used as a special solution, and the sample peak area and the standard sugar peak area were compared and the sugar content in the sample was calculated. As a result, as shown in Figure 15, the reducing sugar content was 7.1% at the beginning of the second fermentation of the present invention fermentation liquor of the present invention gradually decreased with the passage of the fermentation period and became 1.9% on the 6th day of fermentation, after which almost no change was observed. There was no. As a result of free sugar analysis of the present invention fermentation liquor, as shown in FIG. 16, most of the glucose was, and other than that contained a small amount of maltose, fructose and sugar, the content of the second fermentation period was lower than the initial fermentation, but in the case of glucose Was decreased from 0.53% to 0.3% at 6 days of fermentation. The rest of the free sugar was very small.

Experimental Example 8: Organic Acid Content of the Talc Fermented Wine of the Invention

In the present invention, the organic acid content of the fermented liquor was analyzed by HPLC by removing the oil and fat components with hexane, membrane filtration with a 0.45 μm filter, and removing the pigment and protein components with Sep-pak C ₁₈ . It was. As a result, the organic acid of the present invention fermented liquor was present in a large amount of succinic acid and lactic acid as 54.8 and 15.7 mg%, respectively, as shown in Table 4, citric acid and malic acid. (Malic acid) was 13.2 and 15.7 mg%, respectively, which was relatively small compared to succinic acid and lactic acid.

Organic Acid Content of the Talc Fermented Wine of the Invention Organic acid Citric acid Malic acid Succinic acid Lactic acid content 13.2 15.7 54.8 56.7 [Example] unit mg%

Experimental Example 10: Inorganic Component Content of Talc Fermented Wine of the Present Invention

The analysis of the content of the inorganic component in the second fermentation of the taxi fermentation strain of the present invention was carried out in 100 ml of the sample solution, 25 ml of a decomposition agent (HClO ₄ : H ₂ SO ₄ : H ₂ O ₂ = 9: 2: 5, v / v). The solution was slowly heated at low temperature, decomposed in a hot plate until completely colorless, and filtered using Whatman No. 2 filter paper. The filtrate was normalized to 100 ml and analyzed using ICP. As a result, as shown in Table 5, the total inorganic component content of the present invention fermented liquor was 41.15 ppm, and the content of potassium (K) was the most as 22.30 ppm. Next, the content of calcium (Ca) was 18.51 ppm, and the rest was trace amount.

Inorganic Component Content of Taxa Fermented Wine of the Present Invention Inorganic ingredients Calcium (Ca) Potassium (K) Magnesium (Mg) Iron (Fe) Copper (Cu) Total amount content 18.51 22.30 3.30 0.97 0.07 45.15 (Note) Unit: ppm

Experimental Example 11: Free amino acid content of the present invention

The change in the free amino acid content of the present invention fermented liquor was investigated. In the analysis of free amino acid, 30 ml of ethanol was added to 10 ml of the sample, and the protein was allowed to settle overnight at room temperature. The dried supernatant was diluted by adding 10 ml of citric acid buffer (citrate buffer) at pH 2.2, and the filtrate was filtered with a 0.45 μm filter. As a result, as shown in Table 6, the total free amino acid content of the present invention fermented liquor was 385.33 mg%. valine) and alanine contents were 43.52, 38.43, 36.98, 36.57, 35.20 and 34.23 mg%, respectively. Histidine was the lowest detected as 9.32 mg%.

Free amino acid content of the present invention fermented liquor Peak number Amino acid RT content One Threonine 28.050 14.06 2 Serine 29.725 20.06 3 Aspartic acid 32.983 24.50 4 Glutamic acid 34.625 43.52 5 Proline 47.967 36.98 6 Glycine 49.467 27.34 7 Alanine 50.975 34.23 8 Valine 56.625 35.20 9 Isoleucine 70.950 21.69 10 Leucine 73.525 36.57 11 Tyrosine 80.733 24.74 12 Lysine 111.750 18.69 13 Histidine 115.042 9.32 14 Arginine 129.800 38.43 Total volume 385.33 [Note] unit mg%

Example 2: sensory evaluation of the present invention fermentation strain

The sensory evaluation of the present invention fermented liquor was performed. In the sensory evaluation, sensory personnel, composed of 20 college students and graduate students of the School of Food and Nutrition, Sunchon National University, were scored by the five-point method, which scored the best, five points, the lowest, and one point for color, aroma, bitterness, and overall taste. . As a result of the experiment, as shown in FIG. 17, both the color and the fragrance of the present invention fermentation liquor showed good results. However, bitter taste was strong and 5% of sugar was added. As a result, bitterness was decreased and sweetness was increased.

As described above, the present invention has an effect of providing a tax fermentation liquor having excellent color and taste by inoculating fermented with sackcloth after increasing the antimicrobial activity, antioxidant effect, and immune activity of the present invention. The present invention is a very useful invention in the brewing industry.

Claims (2)

(a) after the rice is soaked, water-saving and increasing the inoculation of inoculated Asukgill bacteria ( Aspergillus awamori ) to incubate at 30-55 ℃ to prepare a koji (b) watering the koji of step (a) Then inoculating yeast and subjecting it to primary fermentation to produce a hair seed; (c) After calling the texas, water is saved, and then the mixture is mixed with the primary fermented product and water of step (b), and then fermented at 20 to 40 ℃ for secondary fermentation, followed by filtration and sterilization. Taxa fermentation liquor manufacturing method characterized in that consisting of the step of forming a product. A fermented liquor according to claim 1, characterized in that the alcohol content is 8 to 10%.