KR20180026021A - Method of Manufacturing Kombucha with Extracts Using Green Tea and Citrus - Google Patents

Abstract

Disclosed is a method for manufacturing Kombucha using extracts of Citrus sinensis Osbeck and green tea having antioxidant ability, antimicrobial activity, anticancer activity, liver function protection and the like through inhibition of the production of active oxygen. The present invention provides the method for manufacturing kombucha using the extracts of Citrus sinensis Osbeck and green tea, which comprises the following steps: (a) mixing a Kombucha strain with black tea, firstly culturing the mixture to obtain a Kombucha culture solution and separating a pellicle from a culture medium; (b) preparing extraction solutions of the green tea and the Citrus sinensis Osbeck; and (c) mixing the Kombucha culture solution, the pellicle, and the extraction solutions of the green tea and the Citrus sinensis Osbeck and secondarily culturing the mixture to obtain the Kombucha.

Description

Technical Field [0001] The present invention relates to a method of manufacturing a green tea and citrus using citrus and green tea extracts,

More particularly, the present invention relates to a method for producing combat tea using citrus fruits and green tea extracts, and more particularly, to a method for producing combat tea using citrus fruits and green tea extracts having an antioxidant ability, antimicrobial activity, anticancer activity, The present invention relates to a method for manufacturing a comb secondary structure.

Kombucha is a fermented beverage which is fermented by the symbiotic mycelium of bacteria and yeast, known as black tea mushroom in Korea, and mainly made of black tea and sugar. Kombucha originated from ancient China and was said to have been called a "sacred tea" because of its detoxification and tonic effects. It was passed to Korea through Korea in about AD 414, and now mainly in Russia. "Tea Kvass (2), antimicrobial activity (2), antioxidant activity (2), and antioxidant activity (2). In addition, 3), anticancer effects (4), wound healing effects (5), liver function protection (6-8), etc. Jaeabalan et al. (9) measured the anticancer activity of several fractions of Combaucha extract and found strong anticancer activity in the ethyl acetate fraction. Through the purification and separation of substances, malonic acid and vitexin were found to be effective components . Srihari et al. (4) measured the anticancer activity of combat tumor using PC-3, a human prostate cancer, and found that m-RNA expression of interleukin8 and VEGF, especially of target molecules, was significantly reduced , and the activity of matrix metalloproteinases-2 and -9, which are known to be involved in the formation of neovascularization in vivo, is greatly reduced.

However, in spite of various physiological activities of kombucha, the application and utilization in industry are remarkably low. The main cause is the fermentation of kombucha through microbial symbiosis, so that the exact species and distribution of microorganisms used are established And the toxicity of the fermentation products to normal cells. In particular, the results of studies on the cytotoxicity of combusca are not consistent, and there is a lot of controversy. For example, Centers for Disease Control and Prevention (CDC) in the United States reported two clinical cases in 1995 in the state of Iowa, (10), Srinivasan et al. (11) suggested the possibility of toxicity in combatua through clinical cases of four patients who ingested combat, but in this study, Was not revealed. In another study, Bhattacharya et al. (12) reported that in a laboratory experiment, 2 ml per kg, or 1% (v / v) of distilled water, . In the present study, we found that the toxicity was not observed as a result of long-term toxicity and weight. The US FDA has also confirmed that Comvouta is produced by the Good Manufacturer's Practice (GMP) process and is suitable for consumer intake and is not toxic (13). Previously reported cytotoxic effects also include high acidity, excessive intake , And unsanitary production.

Korean Patent Laid-Open Publication No. 2015-0124258 discloses a combing tea fermentation composition. Although the present invention describes a composition having an anticancer effect by containing a green tea liquid, a culture liquid and a herbal medicine extract, it has a disadvantage in that it tends to be difficult to drink the flavor of herbal medicines even in the taste of combat tea.

Korean Patent Registration No. 0482308 discloses a method for producing a combus tea drink using green tea. The present invention describes a comb-secondary fermented beverage which is prepared by mixing a green tea extract and a sugar in a black tea extract and then fermenting it, but it has a disadvantage that it is difficult to expect an efficacy higher than that of a conventional comb supplement.

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a method for manufacturing a comb secondary carcass using citrus fruits and green tea extracts, which are easy to drink, and have improved anticancer and antioxidant effects, by mixing green tea and citrus fruit extracts with existing combuecha .

In order to solve the above-mentioned problems, the present invention provides a method for culturing a microorganism, comprising: (a) mixing a microbial strain with a black tea, followed by primary culture to obtain a comb culture medium, and then separating the culture medium and a pellicle; (b) preparing green tea extract and citrus extract; And (c) a step of mixing the comb culture medium, fresh culture medium, green tea extract and citrus extract, and then culturing the mixture to obtain comb seeds, thereby producing comb seeds using citrus fruits and green tea extracts.

Also, the green tea extract may further comprise sugar.

Also, the comb secondary extract preferably further comprises acetic acid.

Also, the green tea extract is prepared by extracting 1 to 5 g of green tea with 100 to 500 ml of distilled water at a temperature of 90 to 120 ° C for 1 to 30 minutes.

Also, the citrus extract may be prepared by extracting 1 to 30 g of citrus peel from 100 to 500 mL of distilled water at a temperature of 90 to 120 ° C for 1 to 30 minutes.

(C) is performed by mixing 50 to 200 mL of a comb culture medium, 1- to 50 g of a bacterial membrane, 500 to 1200 mL of an green tea extract, and 100 to 300 mL of a citrus extract, and culturing the mixture at 10 to 40 ° C. for 5 to 20 days. To provide a secondary manufacturing method.

A comb secondary car produced by the method of any one of claims 1 to 6 is also provided.

According to the present invention, by mixing the green tea and the citrus extract to enhance the antioxidative activity of the existing comb tooth, it has a high antioxidative activity and excellent anticancer performance, and because it is easy to drink due to the mixing of the citrus extract, , Health supplements and the like.

FIG. 1 is a graph showing the results of an antioxidant activity test according to Experimental Example of the present invention,
FIG. 2 is a graph showing an experiment result of cancer cell growth inhibition according to the experimental example of the present invention,
FIG. 3 is a graph showing an anticancer activity test result for a human-derived bladder cancer cell line 5637,
FIG. 4 is a graph showing the degree of growth inhibition of bladder cancer cell line 5637 derived from human body through MTT assay,
FIG. 5 is a graph showing cytotoxicity test results using Raw 264.7 cells, a mouse macrophage cell line.

Hereinafter, preferred embodiments of the present invention will be described in detail. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. Throughout the specification, when an element is referred to as "including " an element, it means that it can include other elements, not excluding other elements, unless specifically stated otherwise.

The inventors of the present invention have noted that there is a problem that existing Kombucha has low antioxidative activity and that it is difficult to drink because it has a unique sour taste. As a result of intensive efforts to solve this problem, It has always been found that the coenzyme is increased and the consumption is also easy, leading to the present invention.

Accordingly, the present invention relates to a method for producing a microbial cell culture, which comprises (a) mixing a microbial strain with a black tea, followed by primary culture to obtain a comb secondary culture, and then separating the culture medium and a pellicle; (b) preparing green tea extract and citrus extract; And (c) culturing the comb secondary culture, fresh membrane, green tea extract, citrus extract, and secondary culturing to obtain comb secondary cells.

In the present invention, the green tea extract may further include sugar. In the case of the conventional kombucha, it has a peculiar sour taste, and it is not easy to drink because of the characteristics of the fermented tea. Therefore, it is preferable to add sugar components to suppress sour taste, and it is more preferable to add easily obtainable sugar . In this case, the amount of sugar used is preferably 50 to 150 g per 1 L of the green tea extract, more preferably 80 to 110 g per 1 L of the green tea extract.

In the present invention, the comb supplement extract may further include acetic acid. Although the sourness of the Sukbuka extract is strong, the sour taste is reduced by mixing the green tea and the citrus extract, and it can be altered accordingly. Therefore, it is more preferable to add an acetic acid component to keep the taste constant and increase the storage period. At this time, the amount of acetic acid to be added is preferably in the range of 100 to 1500 쨉 l per 1 L of the supernatant extract, more preferably 400 to 600 쨉 l per 1 L of the supernatant extract.

In the present invention, the green tea extract may be prepared by extracting 1 to 5 g of green tea with 100 to 500 ml of distilled water at a temperature of 90 to 120 ° C for 1 to 30 minutes. The citrus extract may contain 1 to 30 g of citrus peel as 100 To 500 ml of distilled water at a temperature of 90 to 120 ° C for 1 to 30 minutes. The green tea and citrus extract can be prepared by a desired method depending on the purpose of use of the sour taste of kombucha, but it is preferable to carry out the extraction by the above method.

In the present invention, the step (c) may be carried out by mixing 50-200 mL of the comb culture medium, 1-50 g of the bacterial membrane, 500-100 mL of the green tea extract, and 100-300 mL of the citrus extract and incubating the mixture at 10 to 40 ° C for 5 to 20 days .

The present invention also relates to a comb secondary manufactured by any one of the methods of claims 1 to 6.

Hereinafter, a specific embodiment of the present invention will be described.

Sample Processing

The supernatant was used as a supernatant after centrifugation. The supernatant was filtered through a syringe filter unit (pore size 0.45 μm, EMD Millipore Inc. Billerica, MA, USA) And then diluted with RPMI-1640 (Sigma-Aldrich Co., St. Louis, Mo., USA). Citrus bloom extract was extracted with 10 g of hot water for 10 minutes at a rate of adding 200 mL of distilled water to 10 g of citrus pear. The green tea extract was extracted with hot water for 10 minutes at a rate of 300 mL of distilled water added to one tea tea bag (1.5 g, Jeju, Korea). Each sample was filtered by filter unit (0.45 μm, EMD Millipore Inc.) as necessary to inhibit cell growth.

In this experiment, Kombucha is a local domestic Kombucha, which is sold in the private sector and sold through the Internet (www.auction, com).

Statistical processing

This experiment was repeated three times or more independently. The results were analyzed by t-test and ANOVA analysis using the SPSS Version 18.0 Package Program (IBM, New York, NY, USA) for statistical analysis. Duncan's multiple range test to verify the significance between the treatment groups.

Example  One

To 100 mL of the supernatant obtained from the pre-culture, 500 μL of acetic acid was added to 700 mL of green tea extract, 20 g of pellicle, 200 mL of citrus pine (Jeju, Korea) and incubated at 30 ° C for 10 days.

Comparative Example  One

100 g of the culture obtained from the pre-cultivation and 20 g of the pellicle were added to 900 mL of the extract (900 mL) of the green tea (East Green tea tea, Seoul, Korea) and incubated at 30 ° C for 10 days (Comparative Example 1) Respectively.

Experimental Example 1  : Cell culture

The B16F10 mouse skin malignant tumor cell line, 5637 human bladder cancer cell line and mouse macrophage Raw 264.7 cell line used in the experiment were purchased from Korean Cell Line Bank (KCLB, Seoul, Korea) and cultured in RPMI-1640 (Sigma-Aldrich Co.) FBS (Gibco Inc. San Francisco, CA, USA) and 1% Penicillin (Gibco Inc.) were added and cultured at 37 ° C and 5% CO 2.

Experimental Example  2 : ORAC assay

The ORAC value of the common comb secondary of Comparative Example 1 and the citrus / green tea comb secondary of Example 1 were compared by the method of Ou et al. (14). The samples, reference materials and reagents used in this experiment were diluted in 75 mM phosphate buffer (pH 7.4). AAPH (150 mM) was incubated at 37 ° C for 15 minutes immediately before the experiment. Then, 25 μL of the diluted sample and 120 μL of fluorescein (40 nM) were mixed in a 96-well plate and AAPH (150 mM) Was added. The excitation was measured at 485 nm and the emission was measured at 520 nm for 2 minutes at 37 ° C for 1 hour using a fluorescence microplate reader (Spectra Max i3, Molecular Devices, Sunnyvale, CA, USA). The measured values were obtained by substituting the area under curve (AUC) of each sample into the standard curve of trolox.

The antioxidative capacity of comb (carboxy) (K) of Comparative Example 1 and Comb (carboxy) supplemented with the extract of citrus and green tea of Example 1 were compared. The antioxidative capacity of each sample was compared by measuring the oxygen radical absorbance capacity (ORAC), and the results are shown in FIG. Previous studies have shown that combobacillus is superior in protecting cells against free radicals due to its high antioxidant capacity. Bhattacharya et al. (12) measured the antioxidant capacity after combing the mice in a diabetic model mouse. The results showed that the inhibition of glutathione in hepatocytes was significantly inhibited in the mice fed the unfermented tea There is one. The antioxidative capacity of the common fumaric acid was about 3 times higher than that of the common fumaric acid. The increase of the antioxidant activity of the fumaric acid contained in the citrus / Is present. In fact, the antioxidative effects of citrus peel and green tea have been reported in many previous studies (15-17).

Experimental Example  3: Cytotoxicity experiment ( MTT assay )

In order to measure the degree of inhibition of proliferation of B16F10 skin cancer cell line, 5637 human bladder cancer cell line and Raw 264.7 mouse macrophage cell according to the citrus / green tea comb (CK) treatment of Example 1, each cell line was divided into 3x103 cells / well in a 96- And cultured for 24 hours at different concentrations of CK. Then, 10 μL of 5 μg / mL MTT (Thiazolyl blue tetrazolium bromide) (Sigma Aldrich Co.) reagent was added to each well, and the mixture was reacted for 1 hour. After removing the medium, 100 μL of DMSO was added to each well. The absorbance was measured. The proliferation rate of the cells was expressed as a percentage of the absorbance of the control (untreated group).

First, the anticancer activity of each component was measured on the basis of the result that the antioxidative power of the kombucha containing the citrus peel and the green tea extract of Example 1 was much higher than that of the common comb negative of Comparative Example 1. Green tea extract, citrus bloom extract, and mixture thereof were measured by MTT assay. Each sample was treated for 24 hours at a concentration of 0 to 20 μL / mL and expressed as a percentage of the no-added group as shown in FIG. MTT assay showed that the anticancer effect of green tea extract was higher than that of Tangerine bark extract and showed much higher anticancer activity than the single treatment group of each component. In particular, the citrus / green tea mixture inhibited the growth of more than 80% of skin malignant tumor cell lines at a concentration of 20 uL / ml. This shows a remarkable synergistic effect when compared with the single effect of each extract. It can be seen that each component inhibits the growth of cancer cells by different mechanisms

The anticancer activity against the human bladder cancer cell line 5637 was also measured, and the results are shown in FIG. The anticancer activity of the mixture of green tea extract was similar to that of green tea extract. Based on these results, anticancer activity against bladder cancer is mainly attributed to green tea components. As for the bladder cancer cell line, more than 60% of the cells were killed at a concentration of about 20 μL / mL, and the IC50 indicating 50% cell death was present between 10 and 20 μL / mL. Compared to the results for advanced skin cancer cells, it can be deduced that the action mechanism of the extract is different between 5637 bladder cancer cells and B16F10 skin cancer cells, considering that no synergistic effect was observed in 5637 human bladder cancer cell lines.

Experimental Example  4: Anticancer effect of CK on human bladder cancer cell line 5637

Based on the results of the anticancer activity measurement of each of the citrus extract and green tea extract of the components used for the fermentation of CK, Combaux containing two components (Example 1) was cultured for 10 days. After this, the culture solution was sterilized with a 45 μm filter, and the degree of growth inhibition of the bladder cancer cell line was measured by MTT assay. The result was as shown in Fig. CK showed a higher anticancer activity against 5637 bladder cancer cell lines than K, and showed a dose - dependent anticancer activity. Interestingly, however, the IC50 value was increased when compared to anticancer measurements of single components. This may be due to the fact that the bioavailability of EGCG, which is known to exhibit strong anticancer activity among green tea components, decreases with time (18). It is believed that the functional ingredient of green tea and citrus can be developed into a generic chewing gum, which can promote the development of physiologically active beverages, and further studies should be conducted to ensure the chemical stability of each ingredient.

Experimental Example  5: Human Kidney origin  Toxicity to pseudo-normal cells

Based on the inhibition of growth of cancer cells, cytotoxicity was confirmed using Raw 264.7 cells, an immortalized mouse macrophage cell line, to examine whether the growth inhibition of cancer cells was caused by general cytotoxicity. The results were as shown in Fig. K or CK showed low overall cytotoxicity against Raw 264.7 cells and CK had lower cytotoxicity than K. Unlike bladder cancer and skin cancer cell lines, Raw 264.7 cells were significantly less cytotoxic at 100 μL / ml. From the above results, it was confirmed that the anticancer activity of the kombucha fermented with the citrus and green tea extracts was higher than that of the common combus subspecies, and the synergistic effect of the green tea extract and the citrus pus extract was confirmed , Toxicity test using mouse macrophages showed that toxicity to normal cells would be low. Based on the above results, one of the important points to be secured for the industrialization of microbial fermentation functional beverage, including the antioxidant effect of green tea and citrus extract, is toxicity to general cells, and Raw 264.7 macrophages In this study, the degree of toxicity to normal cells was also examined using Raw 264.7 cells, a mouse macrophage, on the basis of a general cytotoxicity assay (19). As a result, CK was not cytotoxic even at about twice the concentration applied to cancer cells. Although more experimental data should be obtained, the results of this study suggest that CK could be developed as an anti-cancer functional beverage.

The preferred embodiments of the present invention have been described in detail above. It will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Accordingly, the scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning, range, and equivalence of the claims are included in the scope of the present invention Should be interpreted.

Claims (7)

A method for manufacturing a comb secondary carcass using citrus fruits and green tea extracts comprising the steps of:
(a) a step of mixing a subspecies of Pseudomonas aeruginosa with a black tea, followed by primary culturing to obtain a comb secondary culture, and then separating the culture medium and a pellicle;
(b) preparing green tea extract and citrus extract; And
(c) mixing the comb culture supernatant, a fresh membrane, a green tea extract, and a citrus extract, and then secondary culturing to obtain comb secondary cells.
The method according to claim 1,
Wherein the green tea extract further comprises sugar.
The method according to claim 1,
Characterized in that the comb secondary extract further comprises acetic acid.
The method according to claim 1,
Wherein the green tea extract is prepared by extracting 1 to 5 g of green tea with 100 to 500 mL of distilled water at a temperature of 90 to 120 ° C for 1 to 30 minutes.
The method according to claim 1,
Wherein the citrus extract is prepared by extracting 1 to 30 g of citrus peel into 100 to 500 mL of distilled water at a temperature of 90 to 120 ° C for 1 to 30 minutes.
The method according to claim 1,
Wherein the step (c) comprises culturing the cells at 10 to 40 ° C for 5 to 20 days by mixing 50 to 200 ml of a comb culture medium, 1- to 50 g of a bacterial membrane, 500 to 1200 ml of a green tea extract, and 100 to 300 ml of a citrus extract, Gt;
6. A comb-bicycle produced by any one of claims 1-6.

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