TW201927326A - Method for producing fermentation product derived from green tea extract, and koji fermentation product derived from green tea extract - Google Patents

Abstract

To provide a novel product derived from green tea and a novel use thereof. Provided are: a method for producing a fermentation product derived from a green tea extract, said method being characterized by comprising fermenting the green tea extract using a koji mold; a koji fermentation product derived from a green tea extract; and a composition which comprises, as an active ingredient, the koji fermentation product derived from a green tea extract, preferably said composition being a medicinal composition or a food or beverage composition. Still preferably, the aforesaid composition is a composition for promoting adiponectin production, a composition for preventing, ameliorating or treating obesity, a skin care composition or a composition for preventing, ameliorating or treating arteriosclerosis.

Description

Method for producing fermented product derived from green tea extract, fermented fermented product derived from green tea extract, and composition containing fermented fermented product derived from green tea extract

The present invention relates to a method for producing a fermented product derived from green tea extract, a novel fermented fermented product derived from green tea extract, and an adiponectin production-promoting composition and obesity derived from the fermented fermented product as an active ingredient. Compositions for prevention, improvement or treatment.

Green tea has been used as a beverage and food since ancient times, and has high safety. Green tea contains green tea catechin, which is known to have an antibacterial effect. In order to utilize the antibacterial effect in industry, a green tea extract containing green tea catechin is contained at a high concentration, and the green tea extract is used as a raw material for oral hygiene products such as dental caries prevention or oral cleaning with improved antibacterial effect or Antibacterial masks, etc.

In addition, in recent years, various physiological functions related to green tea extract have been further explored, and research and development of new uses that utilize the physiological functions are being carried out.

For example, Patent Document 1 discloses a Helicobacter pylori remover, food, drink, or even food additive that uses tea polyphenols or tea extracts as an active ingredient, and is characterized by its interaction with a proton pump inhibitor (PPI). ).

[Prior technical literature]

[Patent Literature]

Patent Document 1: Japanese Patent Laid-Open No. 2002-068992.

Therefore, the main object of the present invention is to provide a new substance and a new use derived from green tea.

The present inventors made trial and error in order to provide new substances and new uses with green tea as a starting point. At that time, although the green tea extract had an antibacterial effect, the present inventors and others were not limited to this common sense, and they tried fermentation by adding seeds. In addition, the present inventors performed a HPLC (High Performance Liquid Chromatography) analysis on the fermented product after fermented fermented rice, and as a result, it was confirmed that the peak patterns of the two before and after the fermentation were different, indicating good results. The mash fermentation was performed. In this way, it was completely unexpected that the fermented fermented type was well performed in a green tea extract containing catechins having an antibacterial effect at a high concentration.

Furthermore, the present inventors studied the peak pattern of HPLC analysis of the obtained fermented fermented green tea extract, and as a result, the peaks of catechins known as the anti-obesity effect almost disappeared. And it is unexpected that the fermented fermented green tea extract-derived fermented adiponectin has a low concentration compared with the green tea extract before fermentation. Moreover, the present inventors performed various physiological activity tests and found that the obtained fermented fermented green tea extract can be used for the prevention, improvement, or treatment of obesity; skin beauty; the prevention, improvement, or treatment of arteriosclerosis .

The present inventors and others believe that the catechins and small molecule fermentation polyphenols (Teadenol) which are known for their anti-obesity effects have not been detected and cannot be confirmed, so they should be derived from green tea extracts. Some of the ingredients in the fermented material have their effects. From this, the present inventors have found that by using osmium, a novel green tea ferment which did not previously exist can be obtained from the green tea extract.

Based on the above circumstances, the present inventors have completed the present invention.

That is, the present invention is as described below.

[1] A method for producing a fermented product derived from green tea extract, wherein fermented green tea extract is fermented.

[2] A fermented fermented extract of green tea extract.

[3] A composition comprising a fermented fermented fermented matter derived from green tea extract as an active ingredient.

[4] The composition according to the above [3], wherein the composition is a composition for promoting adiponectin production, a composition for preventing, improving, or treating obesity, a composition for skin beauty, or A composition for preventing, improving or treating arteriosclerosis.

According to the present invention, a new substance and a new use derived from green tea can be provided.

According to the present invention, a new fermented fermented vinegar derived from green tea extract can be provided. According to the present invention, it is possible to provide a composition for promoting adiponectin production using a new fermented fermented green tea extract-derived fermented berry as an active ingredient; a composition for preventing, improving, or treating obesity; and a composition for skin beauty A composition for the prevention, improvement, or treatment of arteriosclerosis.

In addition, it is not limited to the effect described here, and it may be any effect described in this specification.

Figure 1A shows the results of HPLC analysis of standard catechins (EGC, EC, EGCG, GCG from left to right), Figure 1B shows the results of HPLC analysis of green tea extract (before fermentation), and Figure 1C shows the standard Figure 1D shows the results of HPLC analysis of small molecule fermentation polyphenol A. Figure 1D shows the results of HPLC analysis of the fermented fermented product (Production Example 3: White Bean Sprouts (Rice, Wheat Barley)), and Figure 1E shows the fermented fermented product (Production example). 5: Results of HPLC analysis of Changbai bacteria (rice gluten, wheat gluten).

Fig. 2 shows the fermented fermented product (manufacturing example 3: white bean sprouts (rice barley, wheat barley series)) obtained by subjecting green tea extract to fermented fermented green tea extract before fermentation to adiponectin of mature fat cells A graph of the impact of performance. The upper section is the result of Western blot analysis of adiponectin, and the lower section is the result of quantifying the expression of adiponectin. In addition, Cont is a control group.

Fig. 3 shows adiponectin (upper stage: Fig. 3A) and fatty acid synthase (FAS) (lower stage) in the administration group (right), non-administration group (left: Cont) of the mash fermented product administered to mice. Figure 3B). In addition, the upper section of FIGS. 3A and 3B is the result of western dot analysis of adiponectin, and the lower section is the result of quantifying the expression of adiponectin. In addition, Cont is a control group. * P <0.05 vs cont

Hereinafter, preferred embodiments of the present invention will be described. However, the present invention is not limited to the following preferred embodiments, and can be freely changed within the scope of the present invention. In addition, in this specification, unless otherwise indicated, a percentage is a mass-based expression.

1. Manufacturing method of fermented material derived from green tea extract

This embodiment is a method for producing a fermented product derived from green tea extract, which is a method for producing a fermented product using green tea extract as a starting material, and is characterized in that fermented green tea extract is fermented. Thereby, fermented fermented fermented green tea extract can be obtained.

The method for producing a fermented product derived from a green tea extract according to this embodiment includes a step of fermenting the green tea extract with mash.

In addition to the mash fermentation step, the method for producing a fermented product of the present embodiment may suitably include steps performed before and after fermentation. Examples of the pre-fermentation step include: a step of extracting green tea, a step of preparing seed mash, and a step of preparing a culture medium for mash ferment; as a step of fermentation, for example, a step of processing mash (for example, removing step),步骤 The preparation step of the fermented product (for example, the step of separating and purifying the fermented components) and the like.

In addition, the steps of the method for producing a fermented product may be incorporated into the steps of a method for producing a composition (for example, a method for producing a beverage or a food).

<Green tea extract>

The green tea extract is obtained by extracting from tea leaves, and can be obtained by a known production method. Examples of the tea leaves include tea-made tea leaves and green tea leaves. As an example of the production method, for example, it can be obtained by self-made tea leaves and / or green tea leaves using hot water (0 ° C to 100 ° C) or a water-soluble organic solvent for extraction. Examples of the water-soluble organic solvent include lower alcohols (ethanol, 1,3-butanediol, etc.) having a carbon number of 1 to 4, and the like. One or two or more of these may be used in combination, or may be further combined with Water is mixed to make a water mixed solvent. Alternatively, a supercritical extraction method may be used. In addition, an extraction aid (for example, an organic acid such as sodium ascorbate or an organic acid salt thereof) may be added for extraction.

The aforementioned tea-making tea leaves are tea leaves obtained by making tea from Camellia genus (for example, Chinese tea (C. sinensis), Assamica (C.assamica)) or a hybrid thereof. In terms of efficiently obtaining a target fermented product, the tea leaves used in the present embodiment are preferably, for example, non-fermented green teas obtained by making green teas (e.g., sencha, Fancha, Yulu, sweet tea, roasted tea, etc.) Of tea.

Examples of green tea extracts available on the market include green tea extract BL (Accessone (Co., Ltd.)), Polyphenon (Mitsui Agriculture & Forestry (Co., Ltd.)), Teafuran (Itoen Co., Ltd.), and Sunphenon. (Taiwan Chemical Co., Ltd.), etc.

The aforementioned green tea extract preferably contains tea catechins in terms of enabling good simmering fermentation and obtaining a target fermentation product.

Regarding the main components of tea catechins, epicatechin (EC) and epigallocatechin (EGC), which is a hydroxyl group thereof, are known as gallate. Epicatechin gallate (epicatechin gallate, ECG) and epigallocatechin gallate (epigallocatechin gallate, epigallocatechin gallate) Theophylline, EGCG) four kinds, and the content is EGCG> EGC> ECG> EC in this order.

The amount of tea polyphenols in the green tea extract in this embodiment is not particularly limited. In terms of dry matter, the lower limit of the amount of tea polyphenols is preferably 30% by mass or more, more preferably 40% by mass or more. The upper limit of the amount of the tea polyphenol is preferably 60% by mass or less, and more preferably 50% by mass or less.

The amount of tea caffeine in the green tea extract in this embodiment is not particularly limited. In terms of dry matter, the upper limit of the amount of tea caffeine is preferably 10% by mass or less, more preferably 8% by mass or less. It is more preferably 6% by mass or less, and The lower limit of the amount of tea caffeine is preferably 1% by mass or more, and more preferably 3% by mass or more.

The total catechin content in the green tea extract in this embodiment is not particularly limited. In terms of dry matter, the lower limit of the total catechin content is preferably 20% by mass or more, and more preferably 30% by mass. Above, the upper limit of the total catechin amount is preferably 60% by mass or less. In addition, the amount of total catechins in the green tea extract is not particularly limited. In terms of dry matter, it is preferably 20% by mass to 60% by mass, and more preferably 30% by mass to 50% by mass. It is preferable to perform good mash fermentation and to obtain a target fermentation product efficiently.

In addition, the amount of "EGCG" in the aforementioned green tea extract is not particularly limited. The lower limit of the amount of "EGCG" in terms of dry matter is preferably 5 mass% or more, more preferably 10 mass% or more. The upper limit of the amount of "EGCG" is preferably 30% by mass or less, and more preferably 20% by mass or less. In addition, the amount of "EGCG" in the aforementioned green tea extract is preferably from 10% by mass to 30% by mass, and more preferably from 10% by mass to 20% by mass. This range can be obtained with good fermented fermentation and efficiently. In terms of target fermentation, it is better.

In addition, the ratio of EGCG to the total catechin content is preferably from 20% by mass to 60% by mass, and generally from about 30% by mass to 60% by mass.

The form of the green tea extract used in this embodiment is not particularly limited, and may be any of solid (powder or granular), semi-solid, and liquid.

<麴>

There is no particular limitation on the mash used in this embodiment, and in terms of efficiently obtaining the target fermented product, it is preferably a mash containing maggots (for example, rice gluten, tempeh, and wheat gluten).

The aforementioned rice dumplings can be used as a seed tincture when foods are fermented by using grains such as rice, soybeans, and wheat as raw materials to produce products such as Japanese sake, miso, soy sauce, and shochu. Generally speaking, the fungus used in seed mash breaks down starch or protein contained in cereals, and multiplies the produced glucose or amino acid as a nutrient source.

In view of the ease with which the target fermented substance can exert various actions and effects, it is preferable that the aforementioned tadpoles include tadpoles that can be used as rice tadpoles and / or wheat tadpoles (rice tadpoles and or wheat tadpoles).

As the aforementioned tadpole, a commercially available tadpole can be used. Examples of commercially available products include: Shirataki Komachi (8043) (Akita Imano Store), yellow small granules (8143) (Akita Imano Store), Tomomi (White Bean Sprouts) (Hagiguchi Matsunosuke Store (Shares) Co., Ltd.), 麴 species (soy bean sprouts) (Horiguchi Matsunosuke Store (Stock Co., Ltd.)) You can choose one or two or more kinds of soy sauce (Bioc (Co., Ltd.)).

The aforementioned tadpole includes a microorganism belonging to the genus Aspergillus (so-called tadpole), and examples of the tadpole include Aspergillus awamori, Aspergillus awamori ver.fumeus, Aspergillus Kawachii, and rice fungus (Aspergillus oryzae), Aspergillus oryzae ver oryzae, Aspergillus sojae, Aspergillus luchuensis, Aspergillus saitoi, and Aspergillus usami, etc., you can choose one or more of these .

Among them, in terms of efficiently obtaining the target fermented product, it is preferred that the fungus can be used as a seed fungus. Examples of the fungus used in this kind of fungus include Aspergillus kawachii and rice fungus ( Aspergillus oryzae), Aspergillus sojae, and Aspergillus awamori, etc., One or two or more kinds can be selected from these.

Rhenium is a traditional manufacturing industry that has been used in Japan since ancient times. There are liquid radon and solid radon in the radon. Generally, solid radon is used when processing grain. In this embodiment, as shown in [Examples] described below, a commercially available solid 麴 seed 麴 can be used for liquid culture, and the liquid culturing can be used to obtain the complete fermentation product of the 麴Unexpected. Thereby, the mass production of the fermented fern in this embodiment is also made easier by the liquid culture using the fermented fern.

[麴 fermentation steps]

The fermented coriander fermentation step of this embodiment is preferably carried out by culturing the coriander with a medium containing green tea extract in terms of good fermented coriander fermentation and efficiently obtaining a target fermented product.

The green tea extract-containing medium used in this embodiment can be prepared by mixing the aforementioned basic medium composition with the green tea extract.

In addition, the culture medium containing the green tea extract of the present embodiment is preferably a liquid culture medium, in terms of being able to perform good mash fermentation and in terms of efficiently producing a target fermentation product, and in addition, it is easy to recover the fermentation product. In terms of better.

The aforementioned basic medium composition is not particularly limited as long as it is a fermentable fermentable medium composition, as long as an inorganic salt, a carbon source (preferably a sugar), a nitrogen source, etc., which are generally used as a nutrient source of the nutrient, can be used.

Examples of the saccharides include sucrose, glucose, lactose, mannose, mannitol, and sorbitol. One or two or more kinds can be selected from these. As the saccharides, the content of saccharides in the culture medium of this embodiment is not particularly limited, and it is possible to perform a good fermented ferment and obtain a target fermented product. In other words, it is preferably from 0.1% by mass to 2% by mass.

Examples of the inorganic salt include sodium nitrate, sodium nitrite, potassium dihydrogen phosphate, potassium monohydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, potassium chloride, ferrous sulfate, manganese sulfate, copper sulfate, Calcium carbonate and the like may be selected from one kind or two or more kinds.

The content of the green tea extract in the medium used in this embodiment is not particularly limited, and the lower limit of the content is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, and the upper limit of the content is preferably 40% by mass or less, more preferably 30% by mass or less.

The content of the green tea extract in the culture medium of this embodiment is preferably 1% to 20% by mass, and more preferably 1% to 10% by mass. In this range, a good fermented fermented aspect and a target fermented substance can be obtained. It's better in terms of it.

The total amount of green tea catechins and the content of EGCG in the culture medium of this embodiment are not particularly limited, and can be adjusted by using the green tea extract used in the culture medium and the amount thereof.

The total amount of green tea catechins in the culture medium of this embodiment is preferably from 0.1% by mass to 5% by mass, and this range is preferable in terms of enabling good mash fermentation and obtaining a target ferment.

The content of EGCG in the culture medium of this embodiment is preferably 0.05% by mass to 2.5% by mass, and this range is preferable in terms of enabling good mash fermentation and obtaining a target ferment.

麴 is added to the aforementioned medium containing green tea extract, and the 麴 fermentation is performed. The amount of 麴 added at this time is not particularly limited. It is preferable to add it to a culture medium so that it may become 0.001 mass% or more, and the upper limit of the usage-amount may be 1 mass% or less. It is preferable to add gadolinium to the medium so as to be 0.005 mass% to 0.5 mass%, and more preferably 0.01 mass% to 0.1 mass%.

As the fermentation conditions in the present embodiment, aerobic culture conditions are preferred in terms of the ability to perform good fermentative fermentation and to obtain the target fermentation product. Preferably, methods such as shaking, aeration, and stirring are used alone or in combination for aerobic culture.

Furthermore, the culture temperature in this embodiment is not particularly limited, but is preferably 10 ° C to 40 ° C, and more preferably 20 ° C to 30 ° C.

The pH of the culture medium during the cultivation in this embodiment is preferably 4.0 to 8.0, more preferably 5.0 to 7.0, and even more preferably 5.5 to 6.5.

The culture period in this embodiment is not particularly limited, but it is preferably 5 days or more, more preferably 10 days to 30 days, and still more preferably 10 days to 20 days.

The culture form in this embodiment is not particularly limited, and may be continuous culture (perfusion culture, etc.) or batch culture. The production status of the fermented fermented fermented material during the cultivation in this embodiment can be confirmed by HPLC analysis as shown in [Example] described later, whereby the culture period, addition of culture medium components, and fermented material recovery can be set. Timing, etc.

As described above, the fermented fermented green tea extract derived from the green tea extract of this embodiment is produced in a culture medium.

In addition, the treatment method of the fermented fermented material obtained by carrying out the fermented fermenting can be performed according to a known method of treating microorganisms such as fungi. For example: Heating and / or filtering etc. sterilize or sterilize the mash. In the case of using a kind of ravioli, the kind of ravioli is highly safe, so it may contain radon directly. However, in terms of changes in flavor and the like, it is preferable to perform sterilization or sterilization. As the sterilization, it is sufficient to perform a filtration treatment generally removing fungi, and examples thereof include a treatment using a filtering aid (active clay, diatomaceous earth, etc.) or a membrane.

The method for preparing the fermented components in the obtained fermented fermented material can be performed in accordance with a known separation and purification method.

2. Fermented fermented extract from green tea extract

According to the manufacturing method of this embodiment, the fermented fermented material derived from the green tea extract of this embodiment can be obtained.

The fermented fermented fermented material derived from the green tea extract of this embodiment is as described below [Example], and no catechins and small-molecule fermented polyphenols are detected, which are novel fermented fermented ferments which did not exist in the previous green tea source. .

The components contained in the fermented fermented fermented green tea extract of this embodiment are currently under analysis. Although catechins and small-molecule fermented polyphenols have not been detected, as described later, the fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented fermented soybeans has not been detected. Therefore, it is believed that the novel ingredients are produced by mash fermentation.

The fermented fermented fermented product manufactured by the manufacturing method of this embodiment preferably has a relative retention time of 13 minutes to 14 minutes in the HPLC analysis of reverse phase column chromatography measured in the [Example] described later. The root crest is more preferably the highest crest of the crest. By using this wave peak as an index, the production state of the fermented fermented product can be judged, and the recovery efficiency of the target wave component can be improved.

Furthermore, the green tea extract-derived fermented fermented material in this embodiment The content of the catechins contained is preferably 0.1% by mass or less, and more preferably 0.05% by mass or less in terms of exhibiting performance not caused by catechins.

Furthermore, regarding the content of the small-molecule fermented polyphenols A and / or B in the fermented fermented fermented green tea extract derived from the embodiment of the present embodiment, from the standpoint of exhibiting performance not caused by small-molecule fermented polyphenols, It is preferably 0.1% by mass or less, more preferably 0.05% by mass or less, and still more preferably 0.001% by mass or less.

In addition, the fermented fermented product derived from the green tea extract of this embodiment can be expected to be used in combination with known potency components (such as catechins and / or small molecule fermentation polyphenols) or potency components that will be discovered in the future. Further improve various performance.

Furthermore, as shown in the [Example] described later, the fermented fermented product derived from the green tea extract of the present embodiment has: an effect of increasing adiponectin production; an effect of promoting adiponectin expression; an effect of promoting ATGL expression of a fat cell decomposition enzyme; Differential induction of adipocytes with less fat accumulation; transcription factor C / EBPα (CAAT / enhancer-binding proteinα; CAAT enhancer binding protein α) and PPARγ (Peroxisome proliferator-activated receptor γ); activation of peroxisome proliferators The body γ) shows a decreasing effect; an inhibitory effect of an increase in fat accumulation caused by fat cell hypertrophy; a vascular endothelial wound healing effect; a fatty acid synthesis enzyme (FAS) exhibiting an inhibitory effect.

Furthermore, it is generally known that the following effects are confirmed by increasing the production of adiponectin: (1) the effect of promoting glucose uptake without passing through the insulin receptor; (2) reducing the fatty acid in the cell to increase the sensitivity of the insulin receptor ( Sensitivity); (3) by activating liver AMP (Adenosine 5'-monophosphate; adenosine monophosphate) kinase (4) Burning effect of fatty acids; (5) Inhibition of arteriosclerosis; (6) Anti-inflammatory effect; (7) Promoting effect of hyaluronic acid synthesis in dermis.

Therefore, the fermented product derived from the green tea extract of this embodiment has an effect of increasing adiponectin production, so it can exert a blood glucose inhibitory effect, an anti-diabetic effect, a fat-reducing effect, an anti-obesity effect, a slimming effect, and an anti-hyperlipidemia Effect, anti-inflammatory effect, skin effect.

In addition, the fermented product derived from the green tea extract of this embodiment has the function of promoting the fat cell decomposition enzyme ATGL and inhibiting the expression of fatty acid synthesis enzyme (FAS), so it can exert the function of promoting lipolysis, reducing fat, and anti-obesity ( Especially the decomposition of body fat).

The fermented product derived from the green tea extract of this embodiment has the following effects: induction of differentiation into adipose cells with little fat accumulation; reduction of the expression of transcription factors C / EBPα and PPARγ; increased fat accumulation caused by hypertrophy of adipocytes Inhibition effect, so it can exert fat accumulation reduction effect, fat reduction effect, anti-obesity effect (especially the body fat accumulation inhibition effect).

In addition, the fermented product derived from the green tea extract of the present embodiment has a healing effect on vascular endothelial trauma, so it can exert an anti-atherosclerotic effect.

In addition, the fermented product derived from the green tea extract of this embodiment has catalase as an antioxidant enzyme and HO-1 (Hemeoxygenase-1; heme oxygenase-1) as an antioxidant enzyme. Reduction of expression and inhibition of NOX4 (nicotinamide adenine dinucleotide phosphate oxidase 4) and Tumor Necrosis Factor α (tumor necrosis factor α) Sub-alpha)). This can play a role in reducing the adverse oxidative stress in the body.

In this way, the fermented fermented fermented product derived from green tea extract of the present embodiment has various effects as described above (hereinafter also referred to as "adiponectin production increasing effect" or "effect of fermented fermented product"), so it can be used for obesity, It can be used for the prevention, improvement or treatment of diabetes, arteriosclerosis, hyperlipidemia (lipid abnormality), high blood pressure. In addition, it can be used to improve or maintain the skin quality and skin oxidative stress for the purpose of skin beauty. Furthermore, for patients with these diseases or symptoms or their potential patients, the fermented fermented green tea extract derived from this embodiment is effective.

In addition, as shown in Examples to be described later, the fermented fermented product of green tea extract derived from the present embodiment has an advantage that excellent effects can be obtained even in a simple administration form such as oral ingestion or oral administration. Furthermore, the fermented fermented product derived from the green tea extract of this embodiment can be appropriately selected to be powdery or liquid, and therefore, it can be used for ingestion or oral administration for beverages, foods, pharmaceuticals, and feeds. The form is easy to use, and it is also easy to provide beverages, foods, and the like containing the fermented fermented green tea extract derived from this embodiment in a liquid, solid, tablet, or jelly form. The fermented fermented fermented green tea extract derived from the green tea extract of this embodiment has an advantage that it can easily exert effects on adult or adult animals.

In addition, this embodiment can be used for both therapeutic purposes and non-therapeutic purposes.

The so-called "non-therapeutic purpose" does not include the concept of medical behavior (that is, treatment of the human body by treatment). Examples include health promotion and beauty behavior.

"Improvement" means: an improvement in a disease, symptom, or condition; Prevention or delay of deterioration of condition or condition; reversal, prevention or delay of progress of disease or symptom.

The "prevention" refers to the prevention or delay of the onset of a disease or symptom of a subject, or a reduction in the risk of a disease or symptom of a subject.

Therefore, the fermented fermented green tea extract derived from the green tea extract of this embodiment can be used as an agent or composition for increasing adiponectin production, a composition for increasing adiponectin production, etc. (hereinafter also referred to as "The preparation or composition of this embodiment"), and can also be used to manufacture such preparations or compositions.

The preparation or composition of the present embodiment described above is in a case where an animal including a human (such as a pet) ingests the preparation or the composition itself, or an animal including a human (such as a pet) is administered the preparation or the composition itself In this case, various effects such as the above-mentioned adiponectin production increasing effect are exerted. In addition, the aforementioned preparations or compositions may be used for pharmaceuticals, quasi-drugs, beverages, foods, or feeds for the purpose of animals or animals other than humans, or may be formulated for use in such pharmaceuticals, quasi-drugs, or beverages. , Food or feed materials or preparations.

In addition, the beverages and foods include beverages, foods, functional foods, foods for patients, and foods for specific health care, which are labelled as necessary with the above-mentioned anti-obesity as the concept. These beverages and foods can be distinguished from ordinary beverages and foods by their functional labels.

Furthermore, by blending the fermented fermented green tea extract derived from the green tea extract of this embodiment into the previous drink or food, it can also be distinguished from the previous drink or food. Extract-based fermented tea products (such as tea leaves, tea bags, instant tea, tea drinks, products with tea extract (pastry, noodles, etc.), etc.).

In addition, the dosage form of the above-mentioned medicinal product (including quasi-drug) containing the fermented fermented product of green tea extract according to this embodiment may be a tablet, capsule, granule, powder, syrup, intravenous injection, or intramuscular injection. , Suppositories, inhalants, subcutaneous absorbents, eye drops, nasal drops, wet dressings, patches, ointments, lotions, creams, oral preparations (brushing agent, liquid toothbrush, mouthwash, gum massage Cream, oral ointment, lozenge for mouthwash, lozenge for the mouth, lozenge, etc.). The administration form may be any of oral administration (internal use) and parenteral administration (external use, injection).

In addition, when preparing the various pharmaceutical preparations, the fermented fermented green tea extract derived from the green tea extract of this embodiment may be separately prepared, or other pharmaceutically acceptable excipients, binding agents, and extenders may be appropriately combined. Agents, disintegrating agents, surfactants, lubricants, dispersants, buffering agents, preservatives, flavoring agents, perfumes, coating agents, carriers, diluents, medicinal ingredients other than the present invention, and the like. Among these administration forms, a preferred form is oral administration, and a liquid preparation for oral administration can be prepared by a conventional method by adding a flavoring agent, a buffer, a stabilizer, and the like.

Regarding the content of the fermented fermented green tea extract-derived fermented matter of the present embodiment in the preparation for oral administration, in general, the solid content concentration is preferably 0.01% by mass or more, and more preferably 0.1% by mass or more. It is preferably 20% by mass or less, and more preferably 10% by mass or less. In addition, it is preferably 0.1% by mass to 10% by mass, and more preferably 0.5% by mass to 5% by mass.

As the form of the aforementioned food containing a fermented product derived from green tea extract of this embodiment, in addition to refreshing water, tea-based drinks, tea products, instant tea, Coffee drinks, fruit juice drinks, soft drinks, fruit juices, jellies, wafers, biscuits, breads, noodles, sausages, and other foods, as well as various foods such as nutritious foods, can also be listed in the same form as the above-mentioned oral preparations (tablets) , Capsules, syrups, etc.).

The above plants or their extracts can be prepared separately for various forms of food, or other food materials or solvents, softeners, oils, emulsifiers, preservatives, incense, stabilizers, colorants, antioxidants, humectants can be appropriately combined. , Tackifier, and other active ingredients other than the present invention.

Regarding the content of the fermented material derived from the green tea extract of this embodiment in the beverage, food, or feed, in general, the solid content concentration is preferably 0.01% by mass or more, and more preferably 0.1% by mass or more. It is preferably 10% by mass or less, and more preferably 5% by mass or less. In addition, it is preferably 0.01% by mass to 10% by mass, and more preferably 0.5% by mass to 5% by mass.

Examples of the feed include feeds for small animals such as rabbits, rats, and mice, feeds for pet foods such as dogs, cats, and the like, which can be adjusted to the same form as the above-mentioned foods.

The intake amount of the fermented material derived from the green tea extract of this embodiment can be changed according to the type, weight, sex, age, state, or other factors of the subject. The dosage, route, interval, and amount or interval of administration can be appropriately determined by the practitioner. For adults, it is converted into dry matter every day. Generally speaking, it is preferably 1 mg / 60 kg body weight or more, and more preferably 2 mg / 60 kg body weight. Above, and more preferably 1000 mg / 60 kg or less, more preferably 500 mg / 60 kg or less, and even more preferably Below 200mg / 60kg body weight. In addition, it is preferably 1 mg / 60 kg body weight to 2000 mg / 60 kg body weight, more preferably 2 mg / 60 kg body weight to 1000 mg / 60 kg body weight, and still more preferably 2 mg / 60 kg body weight to 5000 mg / 60 kg body weight.

In this embodiment, the subject of administration or ingestion is not particularly limited as long as there is a person who needs or desires administration or ingestion, whether it is a patient or a healthy person. For example, an insufficient exercise, fear of obesity, or diabetes is preferred. Middle-aged and elderly, men and women who value skin, etc.

In addition, this embodiment is applicable not only to obese patients, but also to those who are prone to gain weight, or those who desire to maintain an appropriate weight.

In addition, this embodiment can be applied not only to patients with diabetes or insulin resistance, but also to those who do not suffer from diabetes or insulin resistance, such as those who have high postprandial hyperglycemia but no abnormal blood glucose on fasting. Fasting blood glucose is not a problem, but those who want to reduce postprandial hyperglycemia.

In addition, this embodiment can be applied to a patient with hypertension or a person who desires to prevent hypertension or reduce the risk of the disease.

Furthermore, it can be applied to a subject who needs enhanced vascular protection, or a subject who desires enhanced vascular protection.

This embodiment can also take the following forms.

[1] A method for producing a fermented product derived from green tea extract, wherein fermented green tea extract is fermented.

It is preferred that the aforementioned tadpole includes a microorganism belonging to the genus Aspergillus, and further preferably that the rice tadpole-type tadpole and / or wheat tadpole-type tadpoles.

Preferably, the aforementioned green tea extract is 1 to 30% by mass in a liquid culture medium. More preferably, the total amount of green tea catechins in the liquid culture medium is 0.1 mass The content of 5% to 5% by mass and / or the content of EGCG is 0.05% to 2.5% by mass.

[2] A fermented fermented extract of green tea extract. Preferably, this fermented fermented material is a fermented material derived from the green tea extract obtained by the manufacturing method as described in said [1]. The target of this fermented fermented material is preferably a healthy person or a healthy animal, an adult, or an adult animal.

[3] A composition or a preparation containing the fermented fermented fermented meat according to the above [2] as an active ingredient. It is preferably a pharmaceutical composition, a beverage, a food composition, a feed composition or a preparation.

[4] The composition or preparation according to the above [3], wherein the composition is the following composition: a composition for promoting adiponectin production; or a composition for preventing, improving, or treating obesity; or for skin beauty A composition; or a composition for the prevention, improvement, or treatment of arteriosclerosis; or a composition exhibiting an inhibitory effect on fatty acid synthase (FAS).

[5] The composition or preparation according to the aforementioned [4], wherein the aforementioned composition is a composition having the following effects: for increasing adiponectin production; for promoting adiponectin expression; for promoting ATGL expression of a fat cell decomposition enzyme; For inducing differentiation into fat cells with little fat accumulation; for reducing the expression of transcription factors C / EBPα and PPARγ; for inhibiting the increase of fat accumulation caused by hypertrophy of adipocytes; for healing of vascular endothelial wounds; or for fatty acid synthesis enzymes ( FAS) showed an inhibitory effect.

[6] A fermented fermented product derived from green tea extract, used for promoting adiponectin production; for preventing, improving, or treating obesity; for skin beauty; or for preventing, improving, or treating arteriosclerosis; or a fatty acid synthesis enzyme (FAS) shows an inhibitory effect. The fermented fermented material derived from the green tea extract obtained by the manufacturing method of said [1] is preferable.

[7] A fermented fermented extract derived from green tea extract, which increases adiponectin production For promoting the expression of adiponectin; for promoting the expression of ATGL for fat cell decomposition enzymes; for inducing differentiation into fat cells with little fat accumulation; for reducing the expression of transcription factors C / EBPα and PPARγ; caused by hypertrophy of adipocytes Inhibition of increased fat accumulation; or for healing of vascular endothelial wounds; or inhibition of fatty acid synthase (FAS).

[8] A fermented fermented green tea extract derived from the green tea extract according to [2], which is used for medical purposes or non-medical purposes for any of the following purposes.

Promotion of adiponectin production; prevention, improvement or treatment of obesity; skin beauty; or prevention, improvement or treatment of arteriosclerosis; promotion of adiponectin performance; promotion of ATGL performance of adipose cell decomposition enzyme; differentiation to fat cells with less fat accumulation Induction; decreased expression of transcription factors C / EBPα and PPARγ; inhibition of increased fat accumulation caused by hypertrophy of adipocytes; healing of vascular endothelial trauma; or inhibition of expression of fatty acid synthase (FAS).

The fermented fermented material derived from the green tea extract obtained by the manufacturing method of said [1] is preferable.

[9] Use of a fermented fermented green tea extract derived from the green tea extract for producing the composition or preparation of the above-mentioned [3] or [4].

[Example]

Hereinafter, the present invention will be described in more detail based on examples. The embodiment described below represents an example of a representative embodiment of the present invention, and the present invention is not limited to the following embodiments.

<Manufacturing method of fermented fermented material derived from green tea extract>

Green tea extract

As the green tea extract, green tea extract BL (trade name: Accessone Co., Ltd.) was used (green tea polyphenols were 46.5 mass%, green tea catechins were 40.11 mass%, EGCG content was 13.6 mass%, and green tea coffee was 5.2 quality%).

In addition, in the specifications of the green tea extract BL, the moisture content is 5% or less according to the Karl Fischer method. In addition, tea polyphenols can be measured by the iron tartrate colorimetric method, and the total amount of green tea catechins, EGCG content, and green tea caffeine can be measured by HPLC.

2. Cultivation method

100 mL of a liquid culture medium composed of the culture medium shown in Table 1 was dispensed into a 500 mL capacity Sakaguchi flask preliminarily added with a silicone stopper and subjected to dry heat sterilization, and then subjected to autoclave sterilization according to a conventional method. After leaving to cool to room temperature, 1 cup of various kinds of crickets (Table 2, 20 mg to 60 mg) were directly planted with a spatula. After planting the bacteria, the cells were cultured at 25 ° C. for 17 days with shaking at 120 oscillations / min.

After incubation, the filter cells (Advantec No. 2) were used to remove the bacteria by suction filtration, and the culture supernatant was used for subsequent experiments of physiological activity tests.

3.HPLC analysis conditions

The culture supernatant was filtered by a 0.22 μm disc filter, and then subjected to component analysis by HPLC analysis of reverse phase column chromatography. The analysis conditions are as follows.

<Analysis conditions>

Column: reverse phase column: TSK gel ODS-80Ts (5μm.4.6 × 250mm)

Mobile phase: (A) 1.0% acetic acid

(B) Acetonitrile

Gradient: A: B = 90: 10 (0min) → 20: 80 (45min)

Flow rate: 0.6ml / min

Detection: UV (Ultraviolet; Ultraviolet) 280nm

Injection volume: 20 μL

Standard 1: Mixture of catechin standard products epigallocatechin (EGC), epicatechin (EC), epigallocatechin gallate (EGCG), epicatechin Gallate (ECG) 0.025mg / mL each

Standard 2: Small molecule fermentation polyphenol A 0.025mg / mL

Regardless of the kind of cricket used, catechins or other green tea components that are visible in the culture medium at the beginning of the culture are almost disappeared due to the culture, and only compounds with visible peaks from the relative retention time of 13 minutes to 14 minutes are detected. It is unclear whether there is a single compound or a plurality of compound systems with respect to this peak, and analysis is currently underway. No matter what kind of pupa is used, the peaks of catechins and small-molecule fermented polyphenols in the culture solution after pupa fermentation are all 0.01 mg / mL or less, but the peaks of these 13 minutes to 14 minutes are not detected. When the height is set to 1.00, the catechins and small-molecule fermented polyphenols are 0.005 or less.

In addition, regarding the amount of polyphenols in the non-phytobacterial culture medium and each supernatant after the culture, gallic acid was used as a standard product, and the amount obtained by the Folin-Ciocalteu method was large. The amount of phenol was measured. In each of the supernatants after the culture, as in the result of HPLC analysis, the amount of polyphenols was significantly reduced by assimilation. When the amount of polyphenols in the non-plant culture medium was set to 100%, it was at least 13%.

In addition, FIG. 1 shows the analysis results of Production Example 3 (White Bean Sprouts) (FIG. 1D) and Production Example 5 (Luteobacterium) (FIG. 1E), which are representative culture supernatants (the vertical axis of the chromatogram is Same scale).

Production Example 3 (White Bean Sprouts) and Production Example 5 (Langbulls) were used as rice cockles and wheat cockles.

In addition, FIG. 1A shows the analysis results of catechins of Standard 1; FIG. 1B shows the analysis results of green tea extract (before fermentation); and FIG. 1C shows the analysis results of small molecule fermentation polyphenol A of Standard 2. .

<Experimental method of physiological activity>

Wound healing experiment

Pig-derived vascular endothelial cells were seeded at 3.5 × 10 ⁵ cells / dish in a 3.5 cm dish and allowed to attach for 7 hours. After simultaneous culture in a medium containing 1% FBS (Fetal Bovine Serum; fetal bovine serum) for 17 hours, a final concentration of 50 μM of Hydroxytyrosol (Hydroxytyrosol) and a fermentation product with a final concentration of 1/1000 dilution were added. After adding the compound, a simulated damage was made using a 200 μL wafer front end on the bottom of the dish, and observed and photographed under a microscope. After 24 hours of shooting again, the number of cells existing within the range of the original damage was counted to evaluate the degree of repair of the damage. The shooting system is a three-field shooting for each dish.

2.3T3-L1 cells (mouse embryo fibroblasts) culture and compound addition

High glucose DMEM (Dulbecco's Modified Eagle's medium; Dulbecco's modified Eagle's medium) was used to culture 3T3-L1 cells and cultured at 37 ° C and 5% CO ₂ . Culture with DMEM containing 10% fetal bovine serum until confluent was reached. After 2 days, it was replaced with a differentiation induction medium (including insulin (Insulin), DEX (dextran; dextran), IBMX (3-Isobutyl-1- Methylxanthine; 3-isobutyl-1-methylxanthine), 10% FBS in DMEM) and cultured for 2 days. Then, in the presence of insulin and 10% FBS, culture was repeated while changing the medium repeatedly every 2 days. Cells around day 12 from the start of differentiation induction were used as models for mature adipocytes, and cells around day 18 were used as models for hypertrophic adipocytes. In an experiment to evaluate the effect of the compound on the differentiation from 3T3-L1 cells to mature adipocytes, the compound was added together with insulin after 2 days from the change to the differentiation induction medium. In experiments to observe the effect of compounds on mature adipocytes, compounds were added to mature adipocytes after 12 days and cultured for 24 hours. Furthermore, in an experiment for reviewing the effect of changing to mastified adipocytes, a compound was added to the cells after 12 days, and the medium was cultured for 6 days while repeatedly changing the medium in the presence of the compound. After the above treatment, oil red O staining and western ink dot analysis were performed.

3. Oil red O staining

Oil Red O is dissolved at a concentration of 1 g / 200 mL (99% isopropanol) to prepare a preservation solution, and is diluted to 60% with water when in use. The cells after each stimulation were washed 3 times with PBS (Phosphate Buffered Saline; phosphate buffer solution) (-), 10% formalin solution was added and fixed for 30 minutes. After washing with water 3 times, 60% isopropyl alcohol was added and left for 5 minutes, and then oil red O solution was added to stain the lipid. After 20 minutes, it was washed three times with water, and oil red O pigment was extracted by adding DMSO (Dimethyl sulfoxide; dimethyl sulfoxide), and recovered in a 1.5 mL tube. The supernatant was collected after centrifugation at 10,000 rpm for 10 minutes, and the absorbance at 540 nm was measured and quantified.

4. Modulation of total lysate

After each cell was cultured, it was washed twice with PBS (-) and immediately stored at -80 ° C. The cells were then lysed on ice, and Lysis buffer (50 mM HEPES (hydroxyethyl piperazineethane sulfonic acid; hydroxyethyl piperazine ethane sulfonic acid) pH 7.5, 50 mM NaCl, 1 mM EDTA (Ethylenediaminetetraacetic acid) was added. ), 50% glycerol (Glycerol), 100mM NaF, 10mM Sodium pyrophosphate, 1% Triton X-100, 1mM NaVO4, 1mM PMSF (Phenylmethanesulfonyl fluoride; benzyl) Sulfasulfonium fluoride), 10 μg / mL Antipain, 10 μg / mL Leupeptin, 10 μg / mL Aprotinin) 150 μL and let stand for 5 minutes, strip with a spatula Cells were recovered into 1.5 mL tubes. The cells were allowed to stand on ice for 15 minutes, and centrifuged at 14,000 rpm for 15 minutes, and then the supernatant was recovered.

5. Electrophoresis and antigen-antibody reaction

The BCA (Bicinchoninic Acid) protein assay kit (Pierce) was used to determine the protein concentration of the recovered samples, and 20 μg of the protein was supplied to 7.5%, 10%, 12.5%, 15% acrylamide gel SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis; Sodium dodecyl sulfate polyacrylamide gel electrophoresis). After electrophoresis, transfer to PVDF (Polyvinylidene fluoride) film (Millipore) with 5% BSA (Bovine Serum Albumin; Bovine Serum Albumin) / TBS (Tris-buffered saline; Tris buffered saline)- T (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween20) was used to block various primary antibodies. After the membrane was washed with TBS-T for 15 minutes, the secondary antibody that recognized each primary antibody was used for antigen-antibody reaction. The film was cleaned again with TBS-T for 15 minutes. In the detection of the band, Chemi-lumi one (Nacalai Tesque) or Clarity Max Western ECL Substrate (BIO-RAD) was used, and C-DiGit Blot Scanner (LI-COR) ) And detect chemiluminescence caused by HRP (Horse radish peroxidase; horseradish peroxidase). For antibody dilution, 5% BSA / TBS-T was used. The dilution ratio, reaction temperature, and time are based on the protocol attached to each antibody.

<Results of physiological activity test>

<Adiponectin expression increase confirmation test>

The results obtained using the fermented fermented green tea extract-derived fermented fermented green fermented fermented fermented fermented with various kinds of fermented as shown in Table 2 as described above are shown below.

In mature fat cells, the fermented fermented product of Production Example 1 to Production Example 6 was added so that the final concentration was diluted to 1/1000, and cultured for 24 hours, and then prepared Cell lysates were quantified by Western blot method for adiponectin protein expression. Cont represents the control group without added compounds. As the internal standard for western blot analysis, α-Tubulin is used.

The fermented fermented green tea extract-derived fermented fermented product (Production Example 1 to Production Example 6) obtained by subjecting a green tea extract to fermentation by using a variety of species all increased the expression level of adiponectin in mature adipocytes.

<Confirmation test of vascular endothelial cell wound healing promotion effect>

To the vascular endothelial cells derived from pigs, the fermented product of Manufacturing Example 5: Changbai bacteria (rice barley, barley barley) was added so that the final concentration was diluted to 1/1000, and the wound healing effect was evaluated 24 hours later. Cont represents the control group without added compounds.

The fermented fermented product (manufacturing example 5: Rhizoctonia glutinosa (rice barley, wheat barley series)) shows the wound healing promoting effect of vascular endothelial cells.

<Adiponectin expression increase confirmation test: before and after fermentation>

In mature adipocytes, diluted to a final concentration of 1/1000 Fermented product (manufacturing example 3: white bean sprouts (rice noodles, wheat barley)) and its pre-fermentation material were added, and after 24 hours of culture, cell lysates were prepared, and the adiponectin protein was expressed by western blotting method The amount is quantified. Cont represents the control group without added compounds. As an internal standard for western blot analysis, alpha-tubulin is used. The analysis results are shown in FIG. 2.

麴 Fermented products (Production Example 3: White Bean Sprouts (rice gluten, wheat gluten)) increase the expression of adiponectin. In contrast, the tea extract before fermentation under the same conditions (1/1000 dilution) is not It shows the effect of increasing the expression of adiponectin. This means that the newly generated compounds from catechins by controlling fermentation by microorganisms have stronger adiponectin production than catechins.

<Enzyme ATGL expression increase confirmation test>

Add fermented fermented product to mature adipocytes so that the final concentration is diluted to 1/1000 (Production Example 3: White Bean Sprouts (rice noodles, wheat barley series), Production Example 5: Changbai bacteria (rice barley, wheat barley series )). After culturing for 24 hours, the cell lysate was prepared, and the protein expression of the enzyme ATGL related to lipolysis was quantified by Western blotting method. Cont represents the control group without added compounds. As an internal standard for western blot analysis, alpha-tubulin is used.

Fermented fermented product (Production Example 3: White Bean Sprouts (rice noodles, barley vinegar), Production Example 5: Rhizoctonia oleracea (rice noodles, barley vinegar)) shows the enzymes that cause fat cells to undergo lipolysis In contrast, the ATGL showed a tendency to increase, whereas small-molecule fermentation polyphenol A (0.3 μM) did not show this effect.

<Confirmation test for inducing differentiation into fat cells with little fat accumulation>

In adipose precursor cells, the fermented fermented product is added so that the final concentration is diluted to 1/1000 within 8 days from the beginning of the induction of differentiation (Production Example 3: White Bean Sprouts (Rice and Wheat Barley), Production Example 5: Changbai Bacteria (rice barley, wheat barley series)), adding small molecule fermentation polyphenol A at a final concentration of 0.3 μM, and using oil red O to stain and quantify fat accumulated in cells. Cont represents the control group without added compounds.

Fermented fermented product (Production Example 3: White Bean Sprouts (rice noodles, barnyard grasses), Production Example 5: Rhizobium (rice noodles, barnyard grasses)) inhibits fat accumulation during differentiation from 3T3-L1 cells to mature adipocytes . This result means that when the fat precursor cells differentiate into adipocytes in vivo, the fermentation product may produce adipocytes with less fat accumulation.

<Confirmation test for increasing expression of transcription factors C / EBPα and PPARγ>

In fat precursor cells, 2 days after the induction of differentiation, at the final concentration The fermented fermented product was added so that it might be diluted to 1/1000 (Production Example 3: White bean sprouts (rice noodles, wheat barley series), Production Example 5: Changbai bacteria (rice barley, wheat barley series)), and after 8 days of cultivation, the preparation Cell lysates were quantified by Western blotting method for protein expression of transcription factors C / EBPα and PPARγ related to adipogenesis. Cont represents the control group without added compounds. As an internal standard for western blot analysis, alpha-tubulin is used.

The fermented fermented product (manufacturing example 3: white bean sprouts (rice barley, barnyard barley series), manufacturing example 5: rhubarb (rice barley, barley barley series)) is related to the differentiation from 3T3-L1 cells to mature adipocytes The expression of the transcription factors C / EBPα and PPARγ decreased. This result confirms that the fermentation product locally inhibits fat accumulation during differentiation from 3T3-L1 cells to mature adipocytes.

<Confirmation test of inhibition of increase in fat accumulation due to adipocyte hypertrophy>

The fermented fermented product was added to mature adipocytes 12 days after the start of differentiation so as to be diluted to a final concentration of 1/1000 (Production Example 3: White Bean Sprouts (Rice and Wheat Barley), Production Example 5: Changbai Fungus ( Miyu, Maiji))), after 6 days of culture, make Oil red O was used to stain and quantify the accumulated fat in the cells. Each Cont at 12 days and 18 days represents a control group to which no compound was added.

The amount of intracellular fat is greatly increased by continuous culture of mature adipocytes. This increase was locally suppressed by the fermented fermented product (Production Example 3: White Bean Sprouts (Rice Roe, Barley Root), Manufacturing Example 5: Rhodobacteria (Rice Roe, Barley Root)). This result shows the possibility that the fermentation product inhibits further increase in fat accumulation caused by adipocyte hypertrophy in vivo.

<Adiponectin and lipolytic enzyme ATGL expression confirmation test>

The fermented fermented product was added to mature adipocytes 12 days after the start of differentiation so as to be diluted to a final concentration of 1/1000 (Production Example 3: White Bean Sprouts (Rice and Wheat Barley), Production Example 5: Changbai Fungus ( Rice noodles, wheat noodles))), and after 6 days of culture, oil red O was used to stain and quantify the fat accumulated in the cells. Each Cont at 12 days and 18 days represents a control group without compound addition.

The expression levels of adiponectin and lipolytic enzyme ATGL in the cells are greatly reduced by continuous culture of mature adipocytes. This reduction was locally suppressed by the fermented fermented product (Production Example 3: White Bean Sprouts (Rice Roe, Barley Root), Production Example 5: Rhodobacteria (Rice Roe, Barley Root)). The results show that the fermented fermented product can reduce the production of adiponectin, which is a superior hormone caused by hypertrophy of adipocytes associated with obesity, and the reduction of lipolysis ability caused by the reduction of ATGL production.

<Confirmation test for reduction in expression of antioxidant enzyme catalase and heme oxygenase-1 (HO-1)>

The fermented fermented product was added to mature adipocytes 12 days after the start of differentiation so as to be diluted to a final concentration of 1/1000 (Production Example 3: White Bean Sprouts (Rice and Wheat Barley), Production Example 5: Changbai Fungus ( Rice glutinous rice and wheat glutinous rice)), after 6 days of culture, the cell lysate was prepared, and the protein expression of oxidase catalase and heme oxygenase-1 (HO-1) was performed by Western blot method. Quantitative. Each Cont at 12 days and 18 days represents a control group without compound addition. As an internal standard for western blot analysis, alpha-tubulin is used.

The expression of the antioxidant enzymes catalase and heme oxygenase-1 (HO-1) in the cells was greatly reduced due to the continuous culture of mature adipocytes. This reduction was significantly improved by the fermented fermented product (Production Example 3: White Bean Sprouts (Rice Root, Barley Root), Manufacturing Example 5: Changbai Fungus (Rice Root, Barley Root)). Hypertrophy of adipocytes produces reactive oxygen species as an inflammatory response. Reduced performance of antioxidant enzymes due to hypertrophy means that The number of raw active oxygen species is further increased.降低 Fermentation products can improve the performance of antioxidant enzymes. The reduction is related to the body's ability to reduce this undesirable oxidative stress cycle.

<Confirmation test for inhibition of increase in expression of enzyme NOX4 and inflammatory factor TNFα related to active oxygen production>

The fermented fermented product was added to mature adipocytes 12 days after the start of differentiation so as to be diluted to a final concentration of 1/1000 (Production Example 3: White Bean Sprouts (Rice and Wheat Barley), Production Example 5: Changbai Fungus ( Rice glutinous rice and wheat glutinous rice)), after 6 days of culture, the cell lysate was prepared, and the Western blot method was used to quantify the protein expression of the active oxygen generating enzyme NOX4 and the inflammatory factor TNFα. Each Cont at 12 days and 18 days represents a control group without compound addition. As an internal standard for western blot analysis, alpha-tubulin is used.

The expression of the enzyme NOX4 and the inflammatory factor TNFα, which are related to the production of reactive oxygen species in the cell, has increased significantly due to the continuous culture of mature adipocytes. The increase in TNFα is derived from the fermented fermented product (manufacturing example 3: white bean sprouts (rice noodles, barley noodles), Preparation Example 5: Inhibition of Changbai bacteria (rice barley, wheat barley). In addition, the increase in NOX4 was suppressed by the fermentation product of Production Example 2: White Bean Sprouts (rice glutinous rice series), but this effect was not seen in the fermentation product of cultivating cultivar (rice gluten, rice gluten rice). The increased expression of TNFα and NOX4 caused by hypertrophy directly leads to an increase in reactive oxygen species. Inhibiting the performance of the reactive oxygen species means reducing the oxidative stress in the body caused by the hypertrophy of fat cells associated with obesity.

<Administration of Fermented Products in Mice: Adiponectin Performance Promotion and Fatty Acid Synthesis Enzyme (FAS) Performance Inhibition of Adipose Tissue>

Animal experiment:

Mature female ICR (Institute of Cancer Research) mice (SLG Japan) were divided into fermented product administration groups (6 animals) and non-administration groups (6 animals). A freeze-dried product of a fermented culture supernatant prepared using Changbai bacteria (Liuliu) was administered to the administration group at 10 mg / kg body weight / day for 10 days (Production Example 5). Dosing is based on the edible powder feed MF powder (Japan SLG Co., Ltd. The company) mixes the powder of the dried product with free ingestion. After 10 days, adipose tissue was removed and stored at -80 ° C.

． Tissue-modulated proteins:

After dissolving the adipose tissue stored at -80 ° C on ice, a portion was cut out, and Lysis buffer (50mM HEPES pH7.5, 50mM NaCl, 1mM EDTA, 50mM NaF, 1mM NaVO4, 25 mM β-glycerophosphate, 200 μM dithiothreitol, 0.02% Triton X-100) were homogenized, centrifuged at 14,000 rpm for 30 minutes, and the supernatant was recovered. After centrifugation at 14,000 rpm for 15 minutes, the recovered supernatant was used as a final sample.

． Electrophoresis and antigen-antibody reaction:

The BCA Protein assay kit (Pierce) was used to determine the protein concentration of the recovered samples, and 20 μg of the protein was supplied to a 10% acrylamide gel SDS-PAGE. After electrophoresis, it was transferred to a PVDF film (Millipore) and blocked with 5% BSA / TBS-T (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween20) to react various primary antibodies. After the membrane was washed with TBS-T for 15 minutes, the secondary antibody that recognized each primary antibody was used for antigen-antibody reaction. The film was cleaned again with TBS-T for 15 minutes. In the detection of the band, Chemi-lumi one (Nacalai Tesque) or Clarity Max Western ECL Substrate (BIO-RAD) was used, and C-DiGit Blot Scanner (LI-COR) ) To detect chemiluminescence caused by horseradish peroxidase (HRP). For antibody dilution, 5% BSA / TBS-T was used. The dilution ratio, reaction temperature, and time are based on the instruction manual attached to each antibody.

． result:

The bands of adiponectin and fatty acid synthetase (FAS) from Western blots are quantified, and those quantified are shown in FIG. 3. For quantification, β-actin was used as an internal standard. As indicated by the intensity of the band, the performance of the adiponectin of the fermented product-administered group compared with the non-administered group showed a significantly higher value. In contrast, the performance of FAS is that the fermentation product administration group shows a low value. This result indicates that the fermentation product not only has the effect of increasing the expression of adiponectin in cultured adipocytes, but also has the effect at the individual level. Furthermore, it is considered that the fermentation product has the ability to inhibit lipid synthesis by reducing the performance of FAS.

This is a green tea extract-derived fermented fermented product that has the effect of promoting the expression of adiponectin in adipose tissue of mice and the effect of inhibiting the expression of fatty acid synthase (FAS). In addition, the fermented fermented ferment derived from green tea extract is obtained by oral administration to promote adiponectin performance and fatty acid synthetase performance, and is used for ingestion of beverages, drinks, foods, etc., orally for medicines, etc. During administration, various symptoms, diseases, or states such as obesity, diabetes, hyperlipidemia, and hypertension can also be prevented, improved, or treated. In addition, the fermented fermented fermented green tea extract is very effective for adults with so-called geriatric diseases or many potential patients.

Claims (4)

A method for producing a fermented product derived from green tea extract, wherein fermented green tea extract is fermented. A fermented fermented extract of green tea extract. A composition containing the fermented fermented fermented green tea extract as an active ingredient. The composition according to claim 3, wherein the composition is a composition for promoting adiponectin production, a composition for preventing, improving, or treating obesity, a composition for skin beauty, or preventing or improving arteriosclerosis Or therapeutic composition.