KR20110130924A - Microbial-fermented green tea and a method for preparing the same - Google Patents

Abstract

PURPOSE: Microorganism fermented green tea, and a producing method thereof are provided to offer an oxygen-resistant effect, an anti-obesity effect, and an anticancer effect to users. CONSTITUTION: A producing method of microorganism fermented green tea comprises the following steps: preparing fresh green tea leaves; deactivating polyphenol oxidase through a freshness reducing process of the green tea leaves; rolling and roasting the tea leaves; secondly rolling the tea leaves before drying; injecting mixed microorganisms including aspergillus niger, saccharomyces cerevisiae, and acetobacter aceti subsp. xylinum into the dried tea leaves; packing the tea leaves injected with the mixed microorganisms before burying under sterilized sand or yellow soil, and fermenting the tea leaves at 25-35 deg C.

Description

Microbial-fermented green tea and a method for preparing the same

The present invention relates to a microbial fermented green tea, a method for producing the same and its use.

Most teas in Korea are developed mainly with green tea and black tea, and the general green tea has its disadvantages in that the harvesting time is determined, which makes it difficult to maximize labor efficiency and maximize production. On the other hand, in the case of microbial fermented tea, since the time of harvesting the tea leaves is not so important, it can be produced with a leisurely time after preparing the right tea or sulloc tea in the green tea factory, and there is an advantage that the use of labor force can be improved.

Fermented tea is classified into weakly fermented tea, semi-fermented tea (black tea, yellow tea, etc.), and post-fermented tea (boiled tea, black tea, etc.). The world's leading producers of post-fermented teas are China and Japan. The famous aerobic fermented teas in China are deliberately injured when harvesting green tea leaves, heated and roasted, and then applied with an appropriate amount of moisture to remove microorganisms in the air. The fermented tea in Boi Prefecture, which is fermented naturally, is used for fermentation tea in Japan.The fermentation monarch is made of aerobic fungi (Fungi) and black tea, which is produced mainly in Toyama, and anaerobic Lactobacillus Anaerobe, as fermentation strains. The main mountain is Awa (阿波). Among them, Boicha, which is popular at home and abroad, is inconvenient to separate leaves from Boicha circular mass, which is not easy to consume, and because it is fermented by falling bacteria in the air, it smells like a fungus or various bacteria, and contains pathogenic microorganisms. It may be suspected. In addition, the first taste has a short taste a strong taste and bitter taste is also poor.

A technique for improving these drawbacks Republic of Korea Patent Publication No. 2009-0056313 discloses a Saccharomyces My access Celebi jiae (Saccharomyces cerevisiae, ATCC9763) in green tea leaves, Casey Lactobacillus (Lactobacillus casei, KFRI000127), sub - Bacillus subtilis (Bacillus subtlis , F4041, F4163), Lactobacillus bulgarius (KFRI000344), Lactobacillus plantarum (ATCC14917) or Leuconostoc mesenteroides (KFRI 818) fermented with green tea It discloses a method for preparing fermented green tea, which comprises the step of, but only discloses a simple sensory evaluation, and there is no mention of what functionalities they have. On the other hand, Republic of Korea Patent No. 816957 is inoculated with a mixture of green tea powder, skim bran, wheat flour in a weight ratio of 3: 5: 2, respectively, inoculating Lactobacillus esidophilus, such as beneficial lactic acid bacteria, to prepare a solid fermentation product. After the inoculation, the solid fermentation product is inoculated with Saccharomyces cerevisiae, Bacillus subtilis and Bacillus coagulans, and the method of preparing a functional fermented green tea microorganism comprising the step of culturing and drying, the fermentation Green tea microorganisms are not used as functional teas, but are used as feed additives for animals and are different from the techniques of the present invention.

As described above, fermented green tea which is not only excellent sensory but also enhanced in functionality is not known yet.

The present inventors have diligently tried to develop fermented green tea with excellent organoleptic properties and improved functionality. As a result, aspergillus niger, Saccharomyces cerevisiae and acetobacter aceti zilium subspecies were added to green tea leaves. The present invention was completed by confirming that the cultured fermented tea has very high sensory and antioxidant activity and anticancer metastasis activity.

The first object of the present invention is to provide a microbial fermented green tea.

It is a second object of the present invention to provide a method for preparing the microorganism fermented green tea.

It is a third object of the present invention to provide an anti-obesity agent containing the powder or extract of the microbial fermented green tea as an active ingredient.

A fourth object of the present invention is to provide an anticancer agent containing the powder or extract of the microbial fermented green tea as an active ingredient.

A fifth object of the present invention is to provide an angiogenesis inhibitor containing the powder or extract of the microbial fermented green tea as an active ingredient.

A sixth object of the present invention is to provide a dietary supplement for preventing or inhibiting obesity containing the powder or extract of the microbial fermented green tea as an active ingredient.

A seventh object of the present invention is to provide a dietary supplement for cancer prevention and inhibition containing the powder or extract of the microbial fermented green tea as an active ingredient.

An eighth object of the present invention is to provide a method for maintaining a stable temperature and humidity by fermentation in loess or sand during fermentation of the microorganism fermented green tea.

The present invention to solve the technical problem,

Aspergillus niger, Saccharomyces cerevisiae and Acetobacter aceti subsp. After inoculating a mixed microorganism containing Xylinum, fermented green tea fermented with sucrose as a sugar source is provided.

In addition, the present invention is an embodiment,

Iii) preparing tea leaves;

Ii) inactivating the polyphenol oxidase through a desalination process;

Iii) spreading the tea mass and keeping in mind;

Iii) scavenging the cared for;

Iii) reminding;

Iii) drying the tea leaves;

Iii) Aspergillus niger, Saccharomyces cerevisiae and Acetobacter aceti subsp. Inoculating a mixed microorganism comprising Xylinum; And

Iv) packaging the tea leaves inoculated with the mixed microorganisms, and then burying them in sterilized sand or loess, and then fermenting at 25 to 35 ° C. with a humidity of 60 to 90%. .

Another embodiment of the present invention,

Iii) preparing tea leaves;

Ii) spoofing at 22-27 ° C. for 12-18 hours to activate polyphenol oxidase;

Iii) keeping in mind;

V) primary fermentation for 1 to 3 hours at a humidity of at least 95 ° C. and a temperature of 25 to 35 ° C .;

Iii) drying the tea leaves;

Iii) Aspergillus niger, Saccharomyces cerevisiae and Acetobacter aceti subsp. Inoculating a mixed microorganism comprising Xylinum; And

Iii) packaging the tea leaves inoculated with the mixed microorganisms and burying them in sterilized sand or loess, followed by secondary fermentation at a temperature of 25 to 35 ° C. and a humidity of 60 to 90%. It is about.

The present invention also provides a therapeutic agent for obesity containing the dry powder or extract of the fermented green tea as an active ingredient.

Furthermore, the present invention provides an anticancer agent containing the dry powder or extract of the fermented green tea as an active ingredient.

In addition, the present invention provides an angiogenesis inhibitor containing the dry powder or extract of the fermented green tea as an active ingredient.

In another aspect, the present invention provides a health functional food for inhibiting or improving obesity containing the dry powder or extract of the fermented green tea as an active ingredient.

In another aspect, the present invention provides a dietary supplement for cancer prevention or improvement comprising the dry powder or extract of the fermented green tea as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The first embodiment of the present invention is aspergillus niger, Saccharomyces cerevisiae and Acetobacter aceti subsp. It relates to a fermented green tea fermented with sucrose as a sugar source after inoculating a mixed microorganism containing Xylinum.

The mixed microorganism is 0.1 to 5% (w / v), preferably 0.5 to 2% (w / v), most preferably 1% (w / v) of fermentation sugar (sucrose or glucose or Honey) is inoculated with 0.5 to 10 mL, preferably 1 to 5 mL, and most preferably 2 mL of bacterial cultures having an OD ₆₀₀ of 0.01 to 1.0, preferably 0.02 to 0.1, most preferably 0.05. Although not limited, the mixed microorganism may further include other kinds of useful microorganisms, and the useful microorganism is preferably Leuconostoc mesnetroides , but is not limited thereto.

The present invention is characterized in that fermentation using sucrose as a fermentation sugar member of the mixed microorganism, glucose or honey can be used in addition to sucrose, but because it does not reach the sucrose in terms of functionality and content such as catechin and amino acid content Sucrose is the most suitable sugar member (see Tables 4 and 6). The sucrose is preferably added at 0.1 to 5% (w / v) based on the microbial culture, but is not limited thereto. If the sucrose content is too high, the culture of the microorganism itself can be suppressed, if the sucrose content is too low, there is a disadvantage that the incubation period is too long.

The inventors have searched for useful microorganisms suitable for fermentation of green tea leaves and capable of enhancing the functionality of fermented green tea. Indeed, Aspergillus niger (hereinafter abbreviated as 'A'), Saccharomyces cerevisiae (hereinafter abbreviated as 'S'), Leuconostoc mesentroides (hereinafter abbreviated as 'L'), Acetobacter aceti subsp. Five kinds of microorganisms, Xylinum (hereinafter abbreviated as 'X') and Corynebacterium glutamicum (hereinafter abbreviated as 'C'), were inoculated and fermented into green tea leaves in various combinations, resulting in functionality, catechin content and amino acids. In terms of content of mixed microorganisms containing X, in particular Aspergillus niger , Saccharomyces cerevisiae and Acetobacter aceti subsp. When using mixed microorganisms (ASX) mixed with Xylinum was excellent in terms of functionality, when using sucrose as a sugar source, it was confirmed that the amino acid content and anti-angiogenic activity is excellent (see Tables 4 and 6). Furthermore, in the case of the fermented green tea, anti-obesity activity and anticancer activity was confirmed to be remarkably superior to the jeoncha prepared in the traditional manner (see FIGS. 7 and 8).

The second embodiment of the present invention

Iii) preparing tea leaves;

Ii) inactivating the polyphenol oxidase through a desalination process;

Iii) spreading the tea mass and keeping in mind;

Iii) scavenging the cared for;

Iii) being mindful again;

Iii) drying the tea leaves;

Iii) Aspergillus niger, Saccharomyces cerevisiae and Acetobacter aceti subsp. Inoculating a mixed microorganism comprising Xylinum; And

Iii) packaging the tea leaves inoculated with the mixed microorganisms and then burying them in sterilized sand or ocher, and then fermenting them under conditions of a temperature humidity of 60 to 90% at 25 to 35 ° C.

In the above production method, the fermentation of step iii preferably lasts for 1 to 24 months, but is not limited thereto. If the fermentation period is too short, there is no difference between the normal green tea or black tea and its function, and if the effect is more than 24 months, the effect may be better, but considering the fermentation conditions, the manufacturing cost may be higher and economical may be reduced. On the other hand, in fermentation of green tea in the loess or sand is to maintain a stable temperature and humidity by remaining in the loess or sand to ferment. The mixed microorganism is 0.1 to 5% (w / v), preferably 0.5 to 2% (w / v), most preferably 1% (w / v) of fermentation sugar (sucrose or glucose or Honey) is inoculated with 0.5 to 10 mL, preferably 1 to 5 mL, and most preferably 2 mL of bacterial cultures having an OD ₆₀₀ of 0.01 to 1.0, preferably 0.02 to 0.1, most preferably 0.05. Although not limited, the mixed microorganism may further include other kinds of useful microorganisms, and the useful microorganism is preferably Leuconostoc mesnetroides , but is not limited thereto. The manufacturing method is a combination of the fermentation method of the present invention to the traditional manufacturing process of green tea, and by inactivating the polyphenol oxidase through the desalination process, while the original polyphenols possessed by the green tea leaves can be used as they are, By fermentation, the content of catechin and amino acid is much higher than that of normal green tea, which is excellent in anti-obesity, anti-cancer and anti-angiogenic activity (see Table 4, Table 6, Figures 4 to 10).

The third embodiment of the present invention

Iii) preparing tea leaves;

Ii) spoofing at 22-27 ° C. for 12-18 hours to activate polyphenol oxidase;

Iii) keeping in mind;

V) primary fermentation for 1 to 3 hours at a humidity of at least 95 ° C. and a temperature of 25 to 35 ° C .;

Iii) drying the tea leaves;

Iii) Aspergillus niger, Saccharomyces cerevisiae and Acetobacter aceti subsp. Inoculating a mixed microorganism comprising Xylinum; And

Iii) packaging the tea leaves inoculated with the mixed microorganisms and burying them in sterilized sand or loess, followed by secondary fermentation at a temperature of 25 to 35 ° C. and a humidity of 60 to 90%. It is about. In the above production method, the second fermentation of step ⅶ preferably lasts for 1 to 24 months, but is not limited thereto. If the fermentation period is too short, there is no difference in function between the conventional green tea or black tea, If it is more than 24 months, the effect may be better, but considering the fermentation conditions, the manufacturing cost may be higher and the economics may be reduced. On the other hand, in the secondary fermentation of the green tea in the ocher or sand is to maintain a stable temperature and humidity by remaining in the ocher or sand in the fermentation. The mixed microorganism is 0.1 to 5% (w / v), preferably 0.5 to 2% (w / v), most preferably 1% (w / v) of fermentation sugar (sucrose or glucose or Honey) is inoculated with 0.5 to 10 mL, preferably 1 to 5 mL, and most preferably 2 mL of bacterial cultures having an OD ₆₀₀ of 0.01 to 1.0, preferably 0.02 to 0.1, most preferably 0.05. Without limitation, the mixed microorganism may further include other kinds of useful microorganisms, and the useful microorganism may include Leuconostoc. mesnetroides is preferred but not limited thereto. The method of preparing the fermented green tea combines the fermentation method of the present invention with the conventional method for producing black tea, and in addition to the step of activating the polyphenol oxidase in place of the agar working, the polyphenol is oxidized to produce an oxidized polyphenol. This gives a different flavor than green tea. After the first fermentation, the secondary fermentation using the mixed microorganism used in the present invention, it can be seen that it shows an excellent anti-angiogenic action compared to the conventional right tank (see Fig. 4).

The fourth embodiment of the present invention relates to an anti-obesity agent containing the dry powder or extract of the fermented green tea as an active ingredient.

As a result of administering the fermented green tea extract of the present invention to a rat fed a high fat diet, the tail vein administration resulted in a weight loss effect of 14.8% for females and 27.5% for males, compared to the control group (group of jeoncar extract administration), respectively. It is better than the effect of XENAL ™ (component name: Ollystat), which is 9.2% and 18.8% (see FIG. 8). In addition, as a result of examining the hemostatic components of these rats, the total cholesterol content was not significantly different in the experimental group and the control group, but HDL cholesterol was higher in the experimental group (see FIG. 10), which was higher than the control group. It is superior to the lower positive control group (Olistat administration group). Triglyceride was confirmed to be lowered to the positive control level in the experimental group (see Fig. 10). The present inventors treated the fermented green tea extract of the present invention to 3T3-L1 adipocytes, which are adipocytes, to determine what mechanism the anti-obesity effect of the fermented green tea extract of the present invention is caused, the expression level of PPARγ As a result, it was confirmed that the expression level of PPARγ was suppressed rapidly in the case of the right tank, whereas the expression level of the fermented green tea of the present invention was sharply reduced (see FIG. 9). Therefore, the fermented green tea of the present invention seems to exhibit anti-obesity effect through the mechanism of inhibiting the differentiation into adipocytes by inhibiting the activity of PPARγ.

The fifth embodiment of the present invention relates to an anticancer agent containing the dry powder or extract of the fermented green tea as an active ingredient. Gastric cancer cells (SNU-719) and cervical cancer cells (SNU-1005) were fed into nude mice to confirm the anticancer activity of the present invention, and the fermented green tea extracts of the present invention and tailed tea extracts as control groups were 300 respectively through the tail vein. As a result of daily administration for 8 weeks by μl, the extract of right jeoncha did not significantly inhibit the proliferation of cancer cells, while the fermented green tea of the present invention was confirmed to cause necrosis of gastric cancer cells and uterine cancer cells (see FIG. 7).

A sixth embodiment of the present invention relates to an angiogenesis inhibitor containing the dry powder or extract of the fermented green tea as an active ingredient.

The inventors performed cell adhesion experiments and angiogenesis assays that are related to the metastasis of cancer cells. Angiogenesis inhibition experiments were performed via Matrigel analysis. As a result, compared with the conventional fermented tea and right tea, the microbial fermented green tea of the present invention was very high in the neovascularization inhibitory activity, especially in the case of fermented green tea fermented ordinary green tea 4 It was confirmed that it reached about twice (see FIG. 4). Furthermore, the cell adhesion inhibitory ability associated with angiogenesis was confirmed. As a result of comparing the inhibitory effect of the U937 cells treated with the concentration of the Uchacha extract (control) and the microorganism fermented green tea extract of the present invention to HUVEC cells, While the cell adhesion inhibitory effect of the right tank was insignificant, it was confirmed that the cell adhesion inhibitory effect of the microbial fermented green tea extract of the present invention was significantly high (see FIG. 6). In order to confirm the mechanism through which the anti-angiogenic action of the fermented green tea is made, the right tea extract and the fermented green tea extract of the present invention were treated with HUVEC cells, respectively, and then Akt expression levels were analyzed by Western blot. The extract slightly inhibited Akt expression, while the fermented green tea extract of the present invention was found to inhibit Akt expression in a concentration-dependent manner (see FIG. 5). Therefore, the fermented green tea extract of the present invention controls Akt, which is present under VEGFR2, VE-cadherin, β-catenin, and PI3 kinase, thereby preventing NF-κB from activating by transmitting signals to NF-κB (neovascular vessels). It is judged that the formation of is suppressed.

Anti-angiogenic inhibitors of the present invention are anti-cancer, anti-metastatic, as well as all diseases associated with angiogenesis, such as rheumatoid arthritis, psoriasis, diabetic retinopathy, atherosclerosis, exudative macular degeneration, prematurity retinopathy, neovascularization It can be used for the treatment and prevention of glaucoma, iris angiogenesis, corneal angiogenesis, hepatitis, sickle cell retinopathy and pterygium.

A seventh embodiment of the present invention relates to a dietary supplement for inhibiting or improving obesity containing a dry powder or extract of the fermented green tea as an active ingredient. The anti-obesity activity of the fermented green tea of the present invention has been described above. Health functional foods for inhibiting or improving obesity of the present invention can be prepared in all kinds of formulations used in health functional foods such as tablets, pills, powders, granules, drinks, gels, suspensions.

Eighth embodiment of the present invention relates to a health functional food for cancer prevention or improvement containing the dry powder or extract of the fermented green tea as an active ingredient. Anti-cancer and anti-angiogenesis inhibitory effect of the fermented green tea of the present invention is as described above.

Formulation:

The anticancer agent, anti-obesity agent or angiogenesis inhibitor of the present invention may be administered by selecting a suitable formulation among the following formulations according to the condition of the patient and the degree of disease:

Injections; Pill; Capsules including hard capsules, soft capsules, sustained-release capsules and enteric capsules; Elixirs; Foamed tablets, uncoated tablets, film coated tablets, dragees, multilayered tablets, sustained-release tablets, sustained-release film coated tablets, sustained-release dragees, sustained-release multilayered tablets, enteric tablets, enteric film-coated tablets, enteric sugar-coated tablets, sublingual tablets, enteric film-coated tablets Tablets; Granules, suspensions; Liquid formulations; Cream; Syrups; Ophthalmic solvents; Ointments; Ophthalmic ointments; Gels; Patches; Cataplasmase; Suppositories; Trocheses; Granules, sustained-release granules, enteric granules; Liquid formulations; Eye drops; emulsion; Suspensions; Spirits; Fragrances; Limonadease; Extractive agent, including tincture, soft extract agent, dry extract agent and liquid extract agent; Injections; Perfusate; Warning agent; Rooshonse; Lining agent; Aerosol agents; Cream; Pasta preparations; Implants and internal devices; Sponges and dressings; Sprays and inhalants; Patching agents; Oral medication.

Route of administration:

The anticancer agent, anti-obesity agent or angiogenesis inhibitor of the present invention may be administered by any of the following routes of administration depending on the condition of the patient and the extent of the disease:

Oral administration, intramuscular administration, intravenous administration, subcutaneous administration, intraarterial administration, intramedullary administration, intraperitoneal administration, inhalation, topical surface application.

Effective dose:

The anticancer agent, anti-obesity agent or angiogenesis inhibitor of the present invention is preferably administered at a dosage of 0.01 to 500 mg / kg / day, depending on the condition of the patient and the type and extent of the disease, and 0.5 to 100 mg / kg / day. More preferably, the dosage is not limited thereto.

Fermented green tea of the present invention can be produced irrespective of the harvesting time of the green tea leaves, and the functionality is not inferior to traditional green tea and black tea, and because it has excellent antioxidant, anti-obesity, anti-cancer and anti-angiogenic effects, Not only can be used as a tea substitute, but also can be used as an anti-obesity agent and an anticancer agent, which can bring great economic benefits to tea farmers.

1 is a flow chart of a conventional green tea manufacturing process.
Figure 2 is a flow chart of a conventional black tea manufacturing process.
3 is a graph showing the catechin content of the microbial fermented tea of the present invention.
Figure 4 is a graph showing the anti-angiogenic effect of the microbial fermented tea of the present invention.
Figure 5 is a photograph showing the effect on Akt expression of the right tank (A) and microbial fermentation tea (B) of the present invention.
Figure 6 is a graph showing the cancer cell adhesion inhibitory activity of the right tank (A) and microbial fermentation tea (B) of the present invention.
Figure 7 is an animal experiment showing the anticancer activity of the right tank (A) and the microbial fermentation tea (B: gastric cancer cells, C: uterine cancer cells) of the present invention.
8 is a graph showing the anti-obesity effect of the control, olistat and microbial fermentation tea of the present invention:
A: Female,
B: Male,
Control group (F): control female,
X-FM: Orliste Female,
X-MM: Orliste Male,
ASXS: Microbial fermented tea of the present invention.
Figure 9 is a photograph showing the expression inhibitory activity of PPAR-γ of the right tank (A) and microbial fermentation tea (B) of the present invention.
10 is a graph showing changes in animal blood components (A: total cholesterol, B: HDL cholesterol and C: triglycerides) according to administration of the control group of the present invention, the microorganism fermentation tea of the present invention and Olistat.

Hereinafter, the present invention will be described through specific examples. This embodiment is intended to illustrate the present invention, but the scope of the present invention is not limited to this embodiment.

Example

The strains used were as follows:

Aspergillus niger (hereinafter abbreviated as 'A'), Saccharomyces cerevisiae (hereinafter abbreviated as 'S'), Leuconostoc mesentroides (hereinafter abbreviated as 'L'), Acetobacter aceti subsp. Xylinum (hereinafter abbreviated as 'X') and Corynebacterium glutamicum (hereinafter abbreviated as 'C'). All of the above strains were distributed from KCTC in the Korea Research Institute of Bioscience and Biotechnology, located at 111, Gwahak-ro, Yuseong-gu, Daejeon.

The strain was used alone or mixed, glucose (G), sucrose (S), honey (H) was used as the sugar source. On the other hand, the application of the fermentation method of the present invention to the manufacturing process of the general green tea (woojeon tea) is denoted by W in front, and the application of the fermentation method of the present invention to the manufacturing process of black tea, which is a general fermented tea is indicated by the F in front.

In combination with microorganisms, for example, Aspergillus niger alone is expressed as W-AG when glucose is applied to green tea as a sugar source , and it is expressed as W-AG, and mixed microorganism and sucrose of Aspergillus niger and Saccharomyces cerevisiae are applied to black tea as a sugar source. In case of fermentation, it is indicated as F-ASG. On the other hand, as a control, no microorganisms and sugars were added (Control), glucose was added as a sugar source without adding microorganisms (Con-G), and sucrose was added as a sugar source without adding microorganisms (Con-S). And only honey was added as a sugar source without adding microorganisms (Con-H).

Examples of the combination of microorganisms are shown in Table 1 below.

Manufacture of Green Tea:

(1) manufacture of ordinary green tea

Normal green tea is prepared through the following process (FIG. 1):

Extracting tea leaves → polyphenol oxydase ( inactivation of enzymes) → unfolding tea masses → beware → beating → beware → dividing the grade → drying → storing

(2) manufacturing of black tea

Normal black tea is prepared through the following process (Fig. 2):

Extracting tea leaves → Natural forgery (22-27, 12-18 hours, weight loss 20-30%: ( polyphenol oxydase: activation of enzymes) → Keep in mind fermentation chamber (humidity 95% or more, 30, 1-3 hours) → Drying → Save

2 g of each dried green tea or black tea leaf prepared by the above process was inoculated with 4 mL of a bacterial culture solution of OD ₆₀₀ 0.05 containing 1% (w / v) of fermentation sugars (sucrose, glucose, honey) on a medium basis. After that, it was put into a cylindrical mold and molded into a cylindrical shape, then packaged in Hanji, buried in sterilized sand or ocher soil in a styrofoam box, and then placed in a fermentation chamber at 30 ° C. and 80% humidity for 6 months. Fermented. After fermentation was completed, each tea was dried, and then poured into 100 mL of boiling water per 2 g of dried tea leaves, and the solids were removed to prepare an extract for subsequent experiments.

Experimental Example:

Fermented green tea prepared as described above was subjected to the sensory test and analysis of various biological activities as follows.

General way:

Culture of Cell Lines:

① Culture of HUVECs and U937 cells

Human umbilical vein endothelial cells (HUVECs) were purchased from Young Science (Korea) and used 3-5 passages. U937 human monocyte cell line (American Type Culture Collection, Rockville, USA) was incubated at 37 ° C. with 5% CO ₂ feed with 10% FBS added to RPMI-1640 medium (Life Technologies, USA) for cell adhesion testing. .

② Culture of cancer cell line

Cancer cells such as gastric cancer (SNU-719) and uterine cancer (SNU-1005) were distributed from the Korea Cell Line Bank (KCLB), located in Seoul National University College of Medicine, 28 Yeongun-dong, Jongno-gu, Seoul, Korea. 10% FBS) was incubated at 37 ° C. under 5% CO ₂ feed conditions.

Experimental Example  1: sensory evaluation

For the fermented green tea prepared according to the above example, a sensory evaluation was performed on seven specialized panels to assign four points of color, aroma, astringent taste, and a total rating on a scale of 1 to 10 points on a minimum of 0 to 10 points. . The results are shown in Table 2 below.

Experimental Example  2: component analysis

Analysis of the content of various catechins to the control and fermented green tea of Examples 1 to 45 was performed. Specifically, acetonitrile was used as a solvent. Specifically, 2 g of the fermented tea leaves were leached into 100 mL for 20 to 40 minutes to mix well the fertilized extract with the same volume of acetonitrile, followed by the analysis conditions shown in Table 3 below. The analysis results are shown in Table 4 below.

Analysis condition HPLC Model  LC-10Avp, Shimadzu Co., JAPAN column  Phenomenex Luna 5μC18 Temperature  30 ℃ Detector  UV-VIS (270 nm) Analysis interval  45 min / sample Mobile phase A: acetonitrile containing 0.1% (v / v) formic acid
B: 0.1% (v / v) formic acid containing water Flow rate  1 ml / min

Component analysis result (unit: ppm) Example Catechin Types Sum GC ^* EGC C EC  EGCG  CGC ECG CG Control group 1 56.74 1052.21 29594.5 533.61 6189.05 12.24 958.09 28.66 38,425.10 Control 2 427.94 342.84 30231 385.8 2771.42 3.84 349.23 9.94 34,522.01 Control group 3 261.89 1630.44 26469 694 8983.08 18.5 1284.2 33.94 39,375.05 Control 4 470.31 526.32 29352.5 390.29 3344.01 6.68 522.84 16.7 34,629.65 One 57.91 664.9 20776 371.48 4371.22 7.57 673.78 13.3 26,936.16 2 20.72 372.07 22216 401.8 2828.75 3.49 368.94 5.79 26,217.56 3 228.6 1092.04 24809 467.82 6225.31 10.08 928.86 22.82 33,784.53 4 190.79 1151.21 22961 479.87 6606.3 9.5 944.01 19.86 32,362.54 5 270.48 1645.93 23436 722.09 3536.38 4.34 1282.15 11.2 30,908.57 6 36.39 778.58 26996 410.29 9001.78 14.02 475.93 25.51 37,738.50 7 260.14 1467.06 25972 598.36 7607.51 12.24 1165.35 27.02 37,109.68 8 75.65 1439.43 28044 623.8 3432.56 5.09 473.32 12.43 34,106.28 9 335.34 1782.45 29087.5 699.76 9068.57 16.92 1300.25 27.99 42,318.78 10 50.56 899.45 26677 478.17 5705.79 10.26 827.78 24.93 34,673.94 11 24.46 648.89 21308 413.74 3317.29 4.84 457.78 15.63 26,190.63 12 386.49 1692.75 34677.5 673.7 9414.55 20.57 1379.37 39.71 48,284.64 13 93.91 521.49 25748 389.24 4041.8 7.12 549.9 15.96 31,367.42 14 321.1 164.31 29476 319.73 1209.18 1.76 165.56 7.25 31,664.89 15 249.28 1291.05 24310 541.53 7456.17 15.22 1085.12 27.53 34,975.90 16 311.1 1563.24 27905.5 555.27 773.3 13.54 1139.49 29.06 32,290.50 17 353.85 244.03 26483 325.31 2051.81 2.94 294.81 9.57 29,765.32 18 294.49 1461.31 29131 628.36 8485.77 17.48 1241.84 30.53 41,290.78 19 272.47 1190.44 24941.5 464.42 6021.91 12.27 925.37 24.57 33,852.95 20 330.27 270.55 22785.5 267.56 2209.15 3.14 315.01 9.21 26,190.39 21 284.44 1497.37 28839.5 668.39 7743.76 16.96 1163.46 29.06 40,242.94 22 73.28 1074.15 26940 578.61 7221.18 12.05 1070.6 28.31 36,998.18 23 342.91 551.16 28449.5 347 4609.96 6.92 621.02 17.57 34,946.04 24 335.19 1644.25 25480.5 630.77 8709.63 18.05 1294.79 32.72 38,145.90 25 56.46 835.82 19275 464.24 5703.4 11.25 850 21.91 27,218.08 26 408.1 276.42 28699 275.38 1768.4 2.59 234.1 7.83 31,671.82 27 331.75 1444.76 29641.5 601.68 8645.44 17.84 1340.93 38.11 42,062.01 28 302.72 1078.9 24125.5 481.57 7675.1 17.04 1083.94 37.08 34,801.85 29 386.28 1290.53 45232 581.68 7441.73 17.44 1213.2 38.91 56,201.77 30 367.21 1253.08 49591.5 534.73 7724.12 18.32 1180.92 37.43 60,707.31 31 312.91 1016.21 57750.5 463.67 6577.6 16.68 1012.07 36.52 67,186.16 32 354.43 1028.91 59709.5 412.61 6285.96 18.83 1019.1 38.63 68,867.97 33 282.67 1057.5 51796.5 457.03 6444.98 15.17 956.05 31.49 61,041.39 34 344.57 1131.41 55390.5 488.07 6156.64 21.38 1008.8 42.54 64,583.91 35 322.08 1126.28 56228.5 459.63 6603.39 18.43 1050.91 38.48 65,847.70 36 374.14 1120.23 52430.5 475.99 6786.92 20.85 1176.83 45.92 62,431.38 37 319.35 988.66 53354 427.28 6184.78 16.26 943.07 34.95 62,268.35 38 357.46 1024.31 58658.5 370.27 6846.65 17.88 1063.29 40.28 68,378.64 39 331.92 976.24 54180.5 409.34 6014.25 17.95 940.31 37.06 62,907.57 40 355.15 1120.97 57910 474.09 6848.75 20.46 1121.12 42.91 67,893.45 41 358.04 1215.8 54203.5 511.02 7339.17 19.1 1226.84 42.28 64,915.75 42 333.61 1044.72 58605 453.05 6544.63 19.49 1095.04 40.55 68,136.09 43 387.58 948.75 52419 389.2 5874.86 37.81 999.29 59.8 61,116.29 44 361.65 1068.44 54022.5 485.79 7040.57 18.62 1089 39.12 64,125.69 45 359.96 1044.7 58506 470.57 6664.21 19.28 1147.5 39.94 68,252.16

* Abbreviation

GC: Gallocatechin; EGC: Epigallocatechin; C: Catechin hydrate; EC: Epicatechin; EGCG: Epigallocatechin gallate; CGC: Gallocatechin gallate; ECG: Epicatechin gallate; CG: Catechin gallate

Experimental Example 3: Amino Acid Component Analysis

In the sensory evaluation, an analysis of amino acid content was performed on samples with high scores of 35 or more. The specific method is as follows:

First, 20 mL of ethanol was added to 2 g of the sample, homogenized with a homogenizer for 10 minutes, centrifuged at 3000 rpm for 20 minutes, and 10 mL of 75% ethanol aqueous solution was added to the residue, followed by homogenization for 10 minutes with a homogenizer, followed by 3000 Centrifugation for 20 minutes at rpm. The combined supernatant was concentrated under reduced pressure, dissolved in distilled water, and 20 mg of sulfosalicylic acid was added thereto, left at 4 ° C. for 1 hour, followed by centrifugation at 3000 rpm for 20 minutes, followed by filtration through a membrane filter (0.2 μm). Quantitative analysis was performed using an amino acid autoanalyzer (S433-H) under the same conditions as 5. The analysis results are shown in Table 6.

Amino Acid Autoanalyzer Operating Conditions Device model  S433-H (SYKAM) column  Cation Separation Column (LCA K07 / Li) Column size  4.6 × 150 mm Column temperature  37 ~ 74 ℃ Flow rate  Buffer 0.45 / min, reagent 0.25 / min Buffer pH range  2.90-7.95 Absorption wavelength  440 nm and 570 nm

Amino acid content Example Fermented tea Sensory evaluation score Amino Acid Content (mg / 100g) ^* Comparative Example 1 Con-g 39 389.3 Comparative Example 2 Con-s 36 382.5 Comparative Example 3 Con-h 37 519.0 Comparative Example 4 W-control 31 59.0 16 ASLCG 30 15.8 17 ASLCS 30 88.2 18 ASLCH 34 206.1 28 XG 30 553.5 29 XS 33 629.2 30 XH 40 862.2 33 AXH 37 115.2 34 SXG 36 184.8 40 ASXG 32 255.2 41 ASXS 39 554.6 42 ASXH 39 271.3 43 ASLXG 41 369.7 44 ASLXS 41 511.1 45 ASLXH 38 283.0

^* : 100g is the dry leaf weight of fermented tea

Experimental Example 4: Investigation of Antioxidant Capacity

Antioxidant activity of the fermented tea of the present invention was determined indirectly based on the catechin content. The result is Acetobacter aceti subsp. Xylinum strains (X) were fermented with three substrates and were found to have high antioxidant power in XS and XH fermented teas. Acetobacter aceti subsp. The fermentation difference of the strain containing the Xylinum strain (X), that is, AX, SX, LX series and ASX, ASLX series was found to have a high content of catechin (FIG. 3). In conclusion, Acetobacter aceti subsp. Xylinum Strain It is believed to enhance the taste and antioxidant power of fermented tea.

Experimental Example 5: Angiogenesis Analysis

Inhibition of neovascularization of fermented tea prepared by applying the fermentation method of the present invention after the process of manufacturing green tea and black tea for five kinds of fermented teas with high antioxidant power centered on the tasty fermented teas mentioned above The experiment was performed. Specifically, 150 μl of matrigel was coated on a 24 well culture plate and incubated for 37 to 1 hour for solidification. HUVECs were separated from the wells using trypsin-EDTA, and then 25,000 cells / well per coated matrigel plate. Resuspend. After the administration of the extract of the development tea to the cells by dose and incubated for 4 hours at 37 ℃ 5% CO ₂ conditions. The length of the formed tube network was randomly divided into five sectors of the plate under a microscope and photographed with a digital camera (Nikon, Coolpix), the image was converted into pixels, and analyzed by NIH image program (FIG. 4). As a result, when microorganism fermented black tea and right tea, the microbial fermentation of teat tea rather than black tea showed a strong anti-angiogenic effect that can have anti-cancer and anti-obesity effects. Among ASXS, Angiogenesis inhibitory ability was most excellent, ASLXS was also less than ASXS, it was found to be superior to the conventional jeoncha or black tea. In the case of XH tea, the highest score was recorded in the sensory evaluation, antioxidant evaluation (catechin content) and amino acid content. However, the angiogenesis inhibitory activity was less than that of the control group, either the right tea or black tea, and was excluded from the subsequent functional analysis. In the case of XS, it was superior to conventional tea or black tea in terms of antioxidant evaluation, amino acid content and angiogenesis inhibitory activity, but it was not good in terms of sensory evaluation and was excluded from subsequent functional analysis (Tables 2, 4 and 6 and FIG. 4).

Experimental Example 6: Analysis of angiogenesis mechanism

As shown in Example 4, the microbial fermentation tea of the present invention was shown to have angiogenesis inhibitory activity. Thus, in order to determine what mechanism the anti-angiogenic activity is expressed through, the expression level of Akt in cell signaling mechanisms related to angiogenesis was investigated. Specifically, it was found that the expression level of Akt was suppressed in a concentration-dependent manner in the cells to which the microorganism fermentation tea (ASXS) of the present invention was administered (FIG. 5). It controls Nkt downstream of vascular endothelial growth factor receptor 2 (VEG2), VE-cadherin, β-catenin, and PI3 kinase, which is an intracellular signaling material, thereby preventing NF-κB from being transmitted to NF-κB. Is not activated and formation of neovascularization is inhibited. EGCG, a component of green tea, has been shown to inhibit the signal transduction of VEGFR2, VE-cadherin, β-catenin, and PI3 kinase. It has been shown to inhibit Akt signaling.

Experimental Example 7: Cell adhesion assay

U937 human monocyte cell line (American Type Culture Collection, Rockville, MD) was prepared using RPMI-1640 medium containing 2 mM L-glutamine (Life Technologies), 10 mM HEPES, 100 units / mL of penicillin and 100 μg / mL of streptomycin. Technologies, Grand Island, NY) was added to 10% FBS and incubated at 37 ℃, 5% CO ₂ conditions. The U937 cells were centrifuged at 100 rpm for 5 minutes, concentrated to 10 ⁶ cells / mL, and then redistributed into 24 well plates, and then cultured for 20 hours with or without the extract of the development car as needed. . Human umbilical vein endothelial cells (HUVECs) and U937 cells were cultured in 24-well plates. Subsequently, U937 cells treated with the developmental tea extract were added to IL-1β and HUVECs treated with the extract, then incubated at 37 for 30 minutes, washed three times with 1% FBS-PBS, and then divided into five fields per well. After dividing the image with a digital camera, the actual cells were counted by using an image analysis software (Scion Image program), and then the average value of the number of cells in five regions was calculated as the number of attached cells. As a result, the cell adhesion inhibitory effect of the fermented green tea (ASXS) of the present invention was significantly higher than the cell adhesion inhibitory effect of the right tank (Fig. 6).

Experimental Example 8: Anticancer Activity Analysis

In order to confirm that the microbial fermented tea of the present invention exhibits anticancer activity in animal experiments, the tail vein injection was performed in the nude mouse transplanted with the right-handed hot water extract of the microorganism fermented tea and the control of the present invention, and then the growth of cancer cells was observed. It was. Specifically, gastric cancer cells (SNU-719) and cervical cancer cells (SNU-1005) were respectively implanted subcutaneously in nude mice, and 300 μl of the lactose extract of the microbial fermentation tea (ASXS) of the present invention was injected into the tail vein for 8 weeks. Injections were made daily (FIG. 7). As a result, the fermented tea extract of the present invention was shown to cause necrosis of cancer cells, it was found that the anticancer activity is excellent. On the other hand, in the case of the right tank, the growth of the tumor cells was partially inhibited, but the necrosis of the tumor cells was not induced.

Experimental Example 9: Investigation of anti-obesity activity

In order to confirm that the fermented green tea of the present invention has anti-obesity activity, a high-fat diet was applied to the SD rats, and 500 μl of the extract of the selected microbial fermented tea was injected into the tail vein to measure weight loss and dissection. The fat accumulated in the liver and intestine was examined. At this time, as a positive control group, Zenical ™ (ingredient name: orlistat) was used. As a result, females showed a decrease of 14.8% compared to the control group (physiological saline administration group) in 22 days of breeding, Olistat showed a 9.2% effect, and males showed a decrease of 27.5% compared to the control group. Olistat showed an effect of 18.8% (FIG. 8). Both ASXS and the positive control group showed better effects on males, which is believed to be due to the males eating more food than the females.

Experimental Example 10 Identification of Mechanism Indicating Antiobesity of Microbial Fermented Tea

In order to elucidate the anti-obesity mechanism of the microbial fermented tea of the present invention, the expression level of the signaling protein in 3T3-L1 adipocytes was investigated. Specifically, as a result of investigating the expression level of PPARγ in 3T3-L1 cells, the expression level of PPARγ was lowered in the concentration-dependent manner in the hot water extract of the right-hand car, whereas the expression was rapidly increased when the microorganism fermented tea (ASXS) of the present invention was treated. It was confirmed that the level dropped (Fig. 9).

Experimental Example 11: Blood Component Analysis

Glucose, LDL, HDL, cholesterol, triglycerides and the like were measured from blood collected from the heart of rats to which the microbial fermented tea extract of the present invention was administered, and compared with the control group to which the same amount of saline was administered (FIG. 10). As a result, the total cholesterol content did not change significantly, but HDL was significantly higher in rats to which the microbial fermented tea extract of the present invention was administered, which was a positive control group (olistat) with little change in HDL content. Contrasts sharply with Triglycerides, on the other hand, showed a similar degree of degradation as Olistat. The results show that the microbial fermented tea of the present invention is effective in improving obesity and preventing cardiovascular diseases by increasing the content of healthy HDL and lowering the content of unhealthy LDL and triglycerides.

Claims (14)

Aspergillus on Green Tea Leaf niger , Saccharomyces cerevisiae and Acetobacter aceti subsp. Fermented green tea fermented with sucrose as a sugar source after inoculating a mixed microorganism containing Xylinum. The fermented green tea according to claim 1, wherein the mixed microorganism is inoculated at a concentration of OD ₆₀₀ 0.01 to 1.0. The fermented green tea according to claim 1, wherein the sucrose is added at 0.01 to 5% (w / v) per microbial culture. The fermented green tea of claim 1, wherein the mixed microorganism additionally comprises Leuconostoc mesnetroides . Iii) preparing tea leaves;
Ii) inactivating the polyphenol oxidase through a desalination process;
Iii) spreading the tea mass and keeping in mind;
Iii) scavenging the cared for;
Iii) reminding;
Iii) drying the tea leaves;
Iii) Aspergillus niger, Saccharomyces cerevisiae and Acetobacter aceti subsp. Inoculating a mixed microorganism comprising Xylinum; And
Iii) a method of manufacturing the fermented green tea according to claim 1, comprising the step of packaging the tea leaves inoculated with the mixed microorganisms and sterilizing them in sterilized sand or loess, and then fermenting the same at a temperature of 25 to 35 ° C and a humidity of 60 to 90%. . Iii) preparing tea leaves;
Ii) spoofing at 22-27 ° C. for 12-18 hours to activate polyphenol oxidase;
Iii) keeping in mind;
V) primary fermentation for 1 to 3 hours at a humidity of at least 95 ° C. and a temperature of 25 to 35 ° C .;
Iii) drying the tea leaves;
Iii) Aspergillus niger, Saccharomyces cerevisiae and Acetobacter aceti subsp. Inoculating a mixed microorganism comprising Xylinum; And
Iii) packaging the tea leaves inoculated with the mixed microorganisms and burying them in sterilized sand or loess, followed by secondary fermentation at a temperature of 25 to 35 ° C. and a humidity of 60 to 90%. Manufacturing method. A therapeutic agent for obesity, comprising the dry powder or extract of fermented green tea of claim 1 as an active ingredient. 8. The therapeutic agent for obesity according to claim 7, wherein the extract is extracted with fermented green tea using water, a lower alcohol of C ₁ to C ₄ or an aqueous solution of the alcohol. An anticancer agent comprising the dry powder or extract of fermented green tea of claim 1 as an active ingredient. 10. The therapeutic agent for obesity according to claim 9, wherein the extract is extracted using fermented green tea as water, a lower alcohol of C ₁ to C ₄ or an aqueous solution of the alcohol. Angiogenesis inhibitor comprising the dry powder or extract of fermented green tea of claim 1 as an active ingredient. 12. The angiogenesis inhibitor according to claim 11, wherein the extract is extracted using fermented green tea as water, a lower alcohol of C ₁ to C ₄ or an aqueous solution of the alcohol. Fermented green tea powder or extract of claim 1 as an active ingredient, obesity prevention or improvement health functional food. The health functional food for cancer prevention or improvement comprising the fermented green tea powder or extract of claim 1 as an active ingredient.

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