KR101851195B1 - Fermented mulberry leaves, femented mulberry leaves extract and use there of - Google Patents

Abstract

The present invention provides a fermented mulberry leaf or fermented mulberry leaf extract having improved functionality by lactic acid fermentation. The fermented mulberry leaf or fermented mulberry leaf extract according to the present invention is superior in safety to synthetic chemicals since it is derived from natural crops. In addition, the fermented mulberry leaf or fermented mulberry leaf extract according to the present invention has an increased flavonol content or an improved antidiabetic activity than before fermentation. Therefore, the fermented mulberry leaf or fermented mulberry leaf extract according to the present invention can be used as a material for various dietary medicines or cosmetics, and can be particularly useful as a material for antidiabetic use.

Description

Fermented mulberry leaf, fermented mulberry leaf extract and use thereof &

The present invention relates to a fermented mulberry leaf, a fermented mulberry leaf extract, and a use thereof. More particularly, the present invention relates to a fermented mulberry leaf or fermented mulberry leaf extract having an increased content of flavonol or an improved antidiabetic activity compared to before fermentation, Lt; / RTI >

Diabetes mellitus is one of the five causes of death in Korea, and its prevalence is increasing worldwide due to the increase in obesity and lack of exercise. In Korea, the prevalence of diabetes mellitus, which was only 5.9% of the total population in 2003, is expected to increase sharply to 10.85% in 2030 (Park et al.

 Diabetes is a metabolic disorder caused by deficiency or insulin secretion of pancreatic beta cells, which modifies the metabolism of the calorie source, which causes hypertension, hyperlipidemia, obesity and arteriosclerotic vascular disorders (Lee et al. In addition to these symptoms, retinopathy, nephropathy, neuropathy, and infectious diseases can be seen as diabetic complications. (Rim. 2012), which also affects the immune system and thus has a greater effect on cell-mediated immunity. There are no treatments available to treat diabetes, and it is known as the best treatment to keep blood glucose levels at a normal level.

 In recent years, fermentation has been attracting attention as a major technology for enhancing the physiological activity of natural products. Fermentation of the active ingredients of red ginseng (Bae et al. 2003), enhancement of antioxidant activity of natural products (Kusznierewicz et al. 2008) (Han et al., 2011), and it has been proven that fermentation is an effective method for enhancing the physiological activity of natural products.

 Nuruk plays an important role as an inoculum of yeast and fermentation-related microorganisms by supplying various hydrolytic enzymes including amylase which breaks down starch raw materials such as rice in the traditional brewing of Takju and Yakju in Korea, (Jo et al. 1995). Studies on the yeast microorganisms have been carried out since the early 1900s and have been isolated and identified using various nutrient media such as bacteria, yeast and fungi. Specifically, between 1906 and 1945, various bacteria (16 genera of 4 genera), yeast (8 genera, 29 species) and filamentous fungi (12 genera, 59 species) were reported as koji microorganisms and then classified by the development of classification and identification technology (So et al., 2009; Lee et al., 2004). Isolation and identification of lactic acid bacteria from yeast plays a major role in the nutritional value of Takju as a fermented food in addition to its role in the hydrolysis of starch and alcohol fermentation in the fermentation of Takju (Jo et al. 1995)

However, studies on the increase of antidiabetic activity of mulberry leaves through fermentation using lactic acid bacteria have not been conducted until now. Regarding anti-diabetic use of mulberry leaf or mulberry leaf fermented product, Korean Registered Patent Application No. 10-1274664 discloses a beverage composition containing a fermented mulberry leaf comprising 20 to 30% by weight of mulberry leaf fermented product, 0.1 to 0.5% And 0.05 to 0.5% by weight of an apple concentrate. The fermented mulberry leaf is prepared by fermenting a mixture of mulberry leaf, brown rice extract and purified salt with aerobic lactic acid bacterium Bacillus coagulance , Or hyperglycemia in a food or beverage. Korean Patent Laid-Open Publication No. 10-2014-0066846 discloses a composition for preventing, treating or ameliorating diabetes or diabetic complications, comprising Morus alba Leaf and Cudrania tricuspidata Leaf as active ingredients. . Korean Patent Registration No. 10-0653460 discloses a mulberry leaf extract containing 0.4 wt% or more of 1-deoxynojirimycin and a fenugreek extract containing 5 wt% or more of 4-hydroxyisosuccinic acid at a weight ratio of 40:60 And a method for preventing and treating diabetes mellitus.

The present invention has been made under the background of the prior art, and an object of the present invention is to provide a fermented mulberry leaf or fermented mulberry leaf extract having an increased content of flavonol having various physiological activities or an improved antidiabetic activity.

It is also an object of the present invention to provide food-medicinal uses of fermented mulberry leaves or fermented mulberry leaf extract.

The inventors of the present invention selected fermentation strains of mulberry leaves or mulberry leaf extracts from various lactic acid bacteria isolated from yeasts and found that the specific strains significantly improved flavonol content or antidiabetic activity of mulberry leaves or mulberry leaf extract And the present invention has been completed.

In order to achieve the above object, the present invention provides a fermented mulberry leaf characterized in that the mulberry leaf is a product obtained by fermenting Pediococcus acidilactici with Pediococcus .

In order to accomplish the above object, an example of the present invention is a method for producing an extract of mulberry leaf or a mulberry leaf extract obtained by fermenting mulberry leaves with Pediococcus acidilactici to Pediococcus, acidulactici , which is a fermented mulberry leaf extract.

In order to achieve the above object, an example of the present invention provides an antidiabetic composition comprising the fermented mulberry leaf or the fermented mulberry leaf extract as an active ingredient.

The fermented mulberry leaf or fermented mulberry leaf extract according to the present invention is superior in safety to synthetic chemicals since it is derived from natural crops. In addition, the fermented mulberry leaf or fermented mulberry leaf extract according to the present invention has an increased flavonol content or an improved antidiabetic activity than before fermentation. Accordingly, the fermented mulberry leaf or the fermented mulberry leaf extract according to the present invention can be used as an effective ingredient for the promotion of hyaluronic acid production, antioxidation, prevention or treatment of malignant mesothelioma, treatment of infectious C type infection, , Stem cell function improvement, prevention or treatment of nonalcoholic fatty liver, prevention or treatment of lipid metabolic diseases, blood glucose lowering, exercise performance enhancement, pancreatic lipase inhibition, etc., It can be usefully used as a material for use.

FIG. 1 is a graph showing changes in contents of flavonol, quercetin and camempferol, when fermented with 21 kinds of lactic acid bacteria isolated from mulberry leaf extract, respectively.
Fig. 2 is a graph showing changes in contents of flavonol, quercetin and camempferol, when fermented with 21 kinds of lactic acid bacteria isolated from leaven, respectively.
FIG. 3 is an analysis of the dispersion when the factors affecting the fermentation of mulberry leaf extract were analyzed using the reaction surface method.
FIG. 4 is a graph showing the effect of the concentration of mulberry leaf extract and fermentation time on the glucose uptake inhibitory activity of the fermented mulberry leaf extract by the reaction surface method analysis.
FIG. 5 is a graph showing the effect of mulberry leaf extract concentration and fermentation temperature on the glucose uptake inhibitory activity of the fermented mulberry leaf extract by the reaction surface method analysis.
FIG. 6 is a graph showing the effect of fermentation time and fermentation temperature on the glucose uptake inhibitory activity of the fermented mulberry leaf extract by the reaction surface method analysis.
FIG. 7 is an analysis of variance when the factors affecting the fermentation of the persimmon leaf extract were analyzed using the reaction surface method.
8 is a graph showing the effect of the persimmon leaf extract concentration and the fermentation time on the lipid differentiation inhibitory activity of the fermented persimmon leaf extract by the reaction surface method analysis.
9 is a graph for predicting the effect of the persimmon leaf extract concentration and the fermentation temperature on the lipid differentiation inhibitory activity of the fermented persimmon leaf extract by the reaction surface method analysis.
10 is a graph showing the effect of the fermentation time and the fermentation temperature on the lipid differentiation inhibitory activity of the fermented persimmon leaf extract by the reaction surface method analysis.
FIG. 11 shows the results of measuring the alpha-glucosidase inhibitory activity of the fermented mulberry leaf extract prepared through mulberry leaf extract or mass fermentation.
12 shows results of measurement of glucose uptake inhibitory activity of mulberry leaf extract, fermented mulberry leaf extract prepared through mass fermentation, and fermented mulberry leaf extract prepared through mass fermentation and an equivalent amount of fermented persimmon leaf extract prepared through mass fermentation .
FIG. 13 shows measured values of lipid differentiation inhibition activity and fermented persimmon leaf extract prepared by mass fermentation, and the predicted values analyzed by the reaction surface method.

Hereinafter, terms used in the present invention will be described.

As used herein, the terms "pharmaceutically acceptable" and "pharmaceutically acceptable" are intended to mean not significantly irritating the organism and not interfering with the biological activity and properties of the administered active substance.

As used herein, the term "prophylactic " means any action that inhibits the symptoms of, or delays the progression of, a particular disease (e.g., obesity or diabetes) upon administration of the composition of the present invention.

The term "treatment" as used herein refers to any action that improves or alleviates the symptoms of a particular disease (e. G., Obesity or diabetes) upon administration of the composition of the present invention.

The term "improvement" as used in the present invention means all actions that at least reduce the degree of symptom associated with the condition being treated.

The term "administering" as used herein is meant to provide any desired composition of the invention to an individual by any suitable method. The term " individual " means any animal such as a human, a monkey, a dog, a goat, a pig, or a mouse having a disease in which symptoms of a specific disease can be improved by administering the composition of the present invention.

The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit or risk rate applicable to medical treatment, including the type of disease, severity, activity of the drug, The time of administration, the route and rate of excretion of the drug, the duration of the treatment, factors including drugs used simultaneously and other factors well known in the medical arts.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a fermented mulberry leaf having an increased content of flavonol or an improved antidiabetic activity. The fermented mulberry leaf according to an example of the present invention is characterized in that mulberry leaves are added to Pediococcus with Pediococcus acidilactici . Pediococcus acidilactici , which is a lactic acid bacterium used for fermenting the mulberry leaves, is isolated from the yeast, and when considering an increase in the flavonol content of the fermented mulberry leaves or an improvement in the anti-diabetic activity, Pediococcus in the oocyte acidilactici ) C2-17 (Accession No .: KFCC 11662P). When the fermented mulberry leaf is prepared, the fermentation temperature is preferably 33 to 39 ° C, more preferably 35 to 38 ° C, in view of an increase in the flavonol content of the fermented mulberry leaves or an improvement in the anti-diabetic activity. The fermentation time of the fermented mulberry leaf is preferably 37 to 57 hours, more preferably 38 to 56 hours, in view of an increase in the flavonol content of the fermented mulberry leaves or an improvement in the anti-diabetic activity.

One aspect of the present invention provides a fermented mulberry leaf extract having an increased content of flavonol or an improved antidiabetic activity. The fermented mulberry leaf extract according to an example of the present invention is characterized in that mulberry leaves are added to Pediococcus with Pediococcus acidilactici or mulberry leaf extract is fermented with Pediococcus acidilactici in Pediococcus . In lactic acid bacteria is Lactococcus Phedi O that is used to ferment the mulberry leaf extract, mulberry leaf or CD rakti when (Pediococcus acidilactici is isolated from the yeast. When considering the increase of the flavonol content of the fermented mulberry leaf extract or the improvement of the anti-diabetic activity, Pediococcus acidilactici C2-17 (accession number: KCCM KFCC 11662P). When the fermented mulberry leaf extract is prepared, the fermentation mulberry leaf extract may have an increased flavonol content or an improved antidiabetic activity. The fermentation mulberry leaf extract preferably has a temperature of 33 to 39 ° C, more preferably 35 to 38 ° C. The fermentation time in the preparation of the fermented mulberry leaf extract is preferably 37 to 57 hrs, more preferably 38 to 56 hrs, in view of an increase in the flavonol content of the fermented mulberry leaf extract or an improvement in antidiabetic activity. The mulberry leaf extract concentration during fermentation of the mulberry leaf extract is preferably 1.2 to 1.9 wt% based on the total weight of water in consideration of an increase in flavonol content or an improvement in antidiabetic activity of the fermented mulberry leaf extract, And more preferably 1.4 to 1.8% by weight.

In order to obtain the fermented mulberry leaf extract from mulberry leaves in the present invention, an extraction process is required. As the extraction method, a conventional extraction method known in the art can be used, for example, a solvent extraction method. The extraction solvent that can be used when preparing the fermented mulberry leaf extract using the solvent extraction method includes water, a lower alcohol having 1 to 4 carbon atoms (for example, methanol, ethanol, propanol and butanol), or a mixture thereof, Propylene glycol, 1,3-butylene glycol, glycerin, acetone, diethyl ether, ethyl acetate, butyl acetate, dichloromethane, chloroform, hexane and mixtures thereof, Is preferred. When water is used as the extraction solvent, the water is preferably hot water. When an alcohol is used as an extraction solvent, the alcohol is preferably a lower alcohol having 1 to 4 carbon atoms, and the lower alcohol is more preferably selected from methanol or ethanol. In addition, when the functional alcohol is used as the extraction solvent, the alcohol content is preferably 50 to 90%. It is obvious to those skilled in the art that the fermented mulberry leaf extract can be obtained not only by using the above-mentioned extraction solvent but also by using other extraction solvent, which exhibits substantially the same effect. For example, decompression by carbon dioxide, extraction by supercritical extraction with high temperature, extraction by extraction using ultrasound, separation by ultrafiltration membrane with constant molecular weight cutoff value, various chromatography (size, charge, hydrophobic or affinity The active fraction obtained through various purification and extraction methods, such as separation using the above-described method for separation according to sex, is also included in the extract of the present invention. The supercritical fluid extraction refers to supercritical fluid extraction. Generally, the supercritical fluid is extracted from the liquid and the liquid when the gas reaches the critical point at high temperature and high pressure. (J. Chromatogr. A. 1998; 479: 200-205). In addition, it has been reported that the supercritical fluid has a polarity similar to that of a nonpolar solvent. Carbon dioxide is a supercritical fluid with both liquid and gaseous nature, with the operation of supercritical fluid equipment reaching its critical pressure and temperature, resulting in increased solubility in fat-soluble solutes. When supercritical carbon dioxide passes through an extraction vessel containing a certain amount of sample, the lipophilic substance contained in the sample is extracted into supercritical carbon dioxide. When the supernatant carbon dioxide containing a small amount of cosolvent is passed through the sample remaining in the extraction vessel after extracting the lipid-soluble substance, components that were not extracted by pure supercritical carbon dioxide can be extracted. The supercritical fluid used in the supercritical extraction method of the present invention can effectively extract an active ingredient by using a mixed fluid in which a cosolvent is further mixed with supercritical carbon dioxide or carbon dioxide. These co-solvents may be used alone or in admixture of two or more selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane and diethyl ether. Most of the extracted samples contain carbon dioxide. Since the carbon dioxide is volatilized into air at room temperature, the extract obtained by the above method can be used as a cosmetic composition, and the cosolvent can be removed by a reduced pressure evaporator. In addition, the ultrasonic extraction method is an extraction method using energy generated by ultrasonic vibration. Ultrasonic waves can destroy an insoluble solvent contained in a sample in a water-soluble solvent. Due to the high local temperature, Since the kinetic energy of the reactant particles is increased, sufficient energy required for the reaction is obtained. By inducing the high pressure by the shock effect of the ultrasonic energy, the mixing effect of the substance contained in the sample and the solvent is enhanced, thereby increasing the extraction efficiency. As the extraction solvent which can be used for the ultrasonic extraction method, one or a mixture of two or more selected from the group consisting of chloroform, ethanol, methanol, water, ethyl acetate, hexane and diethyl ether can be used. The extracted sample is recovered by vacuum filtration, and the filtrate is recovered, and the extract is removed by a vacuum evaporator and freeze-dried to obtain an extract.

The fermented mulberry leaf or fermented mulberry leaf extract according to the present invention has a remarkable increase in flavonol content such as quercetin, kaempferol, myricetin and the like compared to the mulberry leaf or mulberry leaf extract before fermentation, (Korean Patent Laid-Open Publication No. 10-2006-0078292), antioxidant (Korean Patent Publication No. 10-2015-0051624), prevention or treatment of malignant mesothelioma 10-2013-0124640), C type infectious viral infection treatment (Korean Patent Laid-open Publication No. 10-2014-0102227), antiviral improvement of fish (Korean Patent Publication No. 10-2015-0015694) , Improvement of stem cell function (Korean Patent Publication No. 10-2013-0136158), prevention or treatment of nonalcoholic fatty liver (Korean Patent Publication No. 10-2012-0096328), prevention of lipid metabolism diseases (Korean Patent Laid-Open Publication No. 10-2011-0100882), blood glucose lowering (Korean Patent Publication No. 10-2011-0100880), exercise performance enhancement (Korean Patent Laid-open Publication No. 10-2014-0048402) 2003, Abdelmoaty et al. 2010; Zang et al. 2011; Hana et al., 2015), which are useful for the treatment of pancreatic lipase inhibition (Korean Patent Publication No. 10-2015-0101788), antidiabetic or anti-obesity (Vessal et al. It can be used as a medicine or a cosmetic material, and in particular, can be used as a material for antidiabetic use.

One aspect of the invention provides an anti-diabetic composition. The antidiabetic composition according to an embodiment of the present invention includes the fermented mulberry leaf or the fermented mulberry leaf extract as an active ingredient. In addition, the antidiabetic composition according to one embodiment of the present invention may further comprise fermented persimmon leaves or fermented persimmon leaves extract in addition to fermented mulberry leaf or fermented mulberry leaf extract as an effective ingredient. The persimmon leaf fermentation is when the CD rakti Phedi O Lactococcus a persimmon leaf (Pediococcus acidilactici) or Phedi O Lactococcus pen soil three mouse (Pediococcus pentosaceus ), and the fermented persimmon leaf extract was prepared by adding Persimmon leaf to Pediococcus to Pediococcus acidilactici) or Pedy five years Tosa Caucus pen mouse (Pediococcus pentosaceus) CD to extract or persimmon leaf extract of the fermented product to Phedi O caucus to rakti when (Pediococcus acidilactici) or Phedi O Lactococcus pen soil three mouse (Pediococcus pentosaceus ). In addition, Pediococcus acidilactici in Pediococcus used for fermenting the persimmon leaves or persimmon leaves extract is preferably used in an amount sufficient to increase the flavonol content of the fermented persimmon leaf or fermented persimmon leaf extract or to improve the antidiabetic activity, In Pediococcus, Pediococcus acidilactici ) C2-17 (Accession No .: KFCC 11662P). In addition, Pediococcus pentosaceus used for fermenting the persimmon leaves or persimmon leaves extract is preferably used as a fermented persimmon leaf or fermented persimmon leaf extract in consideration of an increase in flavonol content or an improvement in antidiabetic activity, Pediococcus ( Pediococcus) pentosaceus ) C2-3 (KFCC 11661P). When Pediococcus acidilactici is used for fermentation of the persimmon leaves or persimmon leaf extract, the fermentation temperature may be an increase in the flavonol content of the fermented persimmon leaves or fermented persimmon leaf extract or an improvement in antidiabetic activity The temperature is preferably 35 to 39 DEG C, more preferably 35 to 38 DEG C. In addition, when Pediococcus acidilactici is used in the Pediococcus to ferment the persimmon leaves or persimmon leaf extract, the fermentation time may be increased by increasing the flavonol content of the fermented persimmon leaves or fermented persimmon leaf extract or improving the antidiabetic activity Preferably 37 to 57 hours, and more preferably 38 to 56 hours. In order to ferment the persimmon leaf extract, Pediococcus was added to Pediococcus acidilactici is used, the concentration of the persimmon leaf extract in fermentation is preferably 1.2 to 1.9% by weight based on the total weight of water in consideration of an increase in the flavonol content of the fermented persimmon leaf extract or an improvement in anti-diabetic activity, And more preferably 1.4 to 1.8% by weight. In order to ferment the persimmon leaves or persimmon leaves, Pediococcus pentosaceus is used, the fermentation temperature is preferably from 32 to 39 ° C, more preferably from 33 to 38 ° C, in view of an increase in the flavonol content of the fermented persimmon leaf or the fermented persimmon leaf extract or an improvement in the anti-diabetic activity. In addition, when Pediococcus pentosaceus is used to ferment the persimmon leaves or persimmon leaf extract, the fermentation time may be increased by increasing the flavonol content of the fermented persimmon leaves or fermented persimmon leaf extract or improving the antidiabetic activity Is preferably 25 to 60 hr, more preferably 30 to 55 hr. In order to ferment the persimmon leaf extract, Pediococcus When pentosaceus is used, the concentration of the persimmon leaf extract in fermentation is preferably 1.0 to 2.0% by weight based on the total weight of water in consideration of an increase in the flavonol content of the fermented persimmon leaf extract or an improvement in anti-diabetic activity, And more preferably 1.4 to 1.8% by weight.

The antidiabetic composition of the present invention may be formulated into a pharmaceutical composition, a food additive, a food composition (in particular, a functional food composition), a feed additive or the like depending on the intended use or aspect and may be a fermented mulberry leaf or fermented mulberry leaf extract And the like can be adjusted in various ranges depending on the specific form of the composition, the purpose of use, and the manner of use.

When the antidiabetic composition of the present invention is a pharmaceutical composition, the content of the active ingredient such as fermented mulberry leaf or fermented mulberry leaf extract is not particularly limited and may be 0.01 to 99% by weight, preferably 0.5 to 50% by weight, By weight, more preferably 1 to 30% by weight. In addition, the pharmaceutical composition according to the present invention may further include additives such as a pharmaceutically acceptable carrier, excipient or diluent in addition to the fermented mulberry leaf or the fermented mulberry leaf extract. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition of the present invention may further contain one or more known active ingredients having antidiabetic effect such as blood glucose lowering, blood glucose control, prevention or treatment of diabetes, as well as fermented mulberry leaf or fermented mulberry leaf extract. The pharmaceutical composition of the present invention can be formulated into a formulation for oral administration or parenteral administration by a conventional method, and can be formulated into a pharmaceutical composition such as a filler, an extender, a binder, a wetting agent, a disintegrant, Diluents or excipients. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention may be administered orally or parenterally to a mammal including a human according to a desired method. Examples of the parenteral administration method include external dermal application, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, Intravenous injection or intra-thoracic injection. The dosage of the pharmaceutical composition of the present invention is not limited as long as it is a pharmacologically effective amount and is not limited as long as it depends on the body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, Varies. The usual daily dose of the pharmaceutical composition of the present invention is not particularly limited, but is preferably 0.1 to 2000 mg / kg, more preferably 1 to 1000 mg / kg, based on the active ingredient, fermented mulberry leaf or fermented mulberry leaf extract Mg / kg, and may be administered once a day or divided into several times.

In addition, when the antidiabetic composition of the present invention is a food composition, the form includes a pill, powder, granule, infusion, tablet, capsule, or liquid. Examples of the food include meat, sausage, bread, chocolate, candy, There are various kinds of soups, confectioneries, pizza, ramen noodles, other noodles, gums, dairy products including ice cream, soups, drinks, tea, functional water, drinks, alcoholic beverages and vitamin complexes. . In the food composition of the present invention, the content of the active ingredient such as fermented mulberry leaf or fermented mulberry leaf extract is 0.01 to 50% by weight, preferably 0.1 to 25% by weight, more preferably 0.5 to 10% by weight based on the total weight of the composition , But is not limited thereto. The food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient in addition to fermented mulberry leaves or fermented mulberry leaf extract. In addition, the food composition of the present invention can be used as a food composition containing various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, , A carbonating agent used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The above-mentioned natural carbohydrates are sugar alcohols such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Natural flavors such as tau Martin and stevia extract, and synthetic flavors such as saccharin and aspartame may be used as the flavor.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.

1. Isolation of lactic acid bacteria from koji and preparation of seed culture

Nuruk prepared in the conventional manner was diluted with sterilized water, and the diluted solution was plated on an MRS medium (Difco, USA) containing 1.5% agar and then cultured at 37 ° C for about 24 hours. After isolating the colonies, the strains were cultured on the medium, and 21 kinds of lactic acid bacteria were isolated. Table 1 below shows a total of 21 kinds of lactic acid bacteria isolated from yeast.

Strain
Control Number Strain name Strain
Control Number Strain name A-3-72 Lactobacillus curvatus B-3-10 Klebsiella pneumoniae B-0-24 Lactococcus lactis B-3-21 Enterococcus hirae B-0-9 Lactobacillus curvatus B-3-22 Lactobacillus curvatus A-30-10 Pediococcus acidilactici A-0-18 Pediococcus acidilactici B-0-14 Weissella paramesenteroide C-2-17 Pediococcus acidilactici B-0-23 Enterococcus avium A-3-2 Enterococcus lactis B-3-11 Klebsiella pneumoniae B-3-1 Enterococcus hirae A-0-1 Pediococcus acidilactici B-0-13 Leuconostoc citreum A-3-71 Pediococcus pentosaceus C-1-3 Pediococcus pentosaceus A-3-1 Pediococcus pentosaceus C-2-3 Pediococcus pentosaceus B-3-19 Klebsiella pneumoniae

Twenty-one kinds of lactic acid bacteria isolated from the koji were inoculated into 100 ml of MRS broth (Difco, USA) in an amount of 1% (w / v), and incubated at 37 ° C and 100 rpm in a shaking incubator SI- , UNIVERSAL SCIENTIFIC CO., LTD) for 24 hours to prepare a seed culture.

2. Selection of lactic acid bacteria suitable for fermentation of mulberry leaf or mulberry leaf extract

(1) Preparation of mulberry leaf extract and fermented mulberry leaf extract

The dried mulberry leaf powder was added to water corresponding to about 30 times the weight of the mulberry leaves and subjected to hot water extraction at 100 ° C for 9 hours. Thereafter, the extract was passed through a filter net to obtain a filtrate, which was then concentrated in a vacuum concentrator. Thereafter, the concentrated liquid was lyophilized to obtain a mulberry leaf extract, which was used as an analytical sample while being stored at -70 ° C.

The culture broth of the lactic acid bacteria was centrifuged to recover the cell pellet, and the recovered cell pellet was suspended in 10 ml of distilled water to prepare a cell suspension.

1 g of mulberry leaf extract (corresponding to 1% by weight) was dissolved in 100 ml of distilled water, and 1 ml of the cell suspension was inoculated. The mulberry leaf extract was fermented by incubating at 37 ° C for 72 hours. The fermentation broth of mulberry leaf extract was centrifuged at 12,000 rpm and 4 ° C for 20 minutes to separate the supernatant, and the supernatant was lyophilized to obtain a fermented mulberry leaf extract.

(2) Analysis of flavonol content of fermented mulberry leaf extract

200 mg of the fermented mulberry leaf extract obtained above was dissolved in 1 ml of distilled water and filtered through a 0.22 μm polyvinylidene difluoride (PVDF) syringe filter and used as an analytical sample. Flavonol contents of quercetin and kaempferol were analyzed by HPLC of a photo diode array (PDA) system equipped with an ODS-column (XBridge, 250 mm × 4.6 mm, 5 μm, Waters Corp.). As mobile phase A, 45% acetonitrile containing 2% acetic acid was used as mobile phase B, and the concentration of B solvent was increased from 100% for 25 minutes in the initial 50% The concentration gradient method was used. The flow rate of the solvent was 1 ml / min, the wavelength of the detector was 355 ㎚, the sample injection amount was 10 쨉 l, and the column oven was maintained at 30 캜 during the analysis.

FIG. 1 is a graph showing changes in contents of flavonol, quercetin and camempferol, when fermented with 21 kinds of lactic acid bacteria isolated from mulberry leaf extract, respectively. In FIG. 1, the X-axis represents the control number of the lactic acid bacteria used for the fermentation of the mulberry leaf extract, and the Y-axis represents the amount of flavonol contained in 100 g of the fermented mulberry leaf extract in mg. In Fig. 1, "Control" represents a mulberry leaf extract which has not undergone fermentation treatment. As shown in FIG. 1, flavonol such as quercetin and kaempferol was present in very small amounts in the mulberry leaf extract itself, and the value was expressed as zero. As shown in FIG. 1, when the mulberry leaf extract was fermented with 21 kinds of lactic acid bacteria isolated from the koji, the content of quercetin and kaempferol was increased in all the fermented mulberry leaf extract, -17 Pediococcus to Pediococcus The significantly higher acidilactici) as fermented to increase the content of gwereu paroxetine (quercetin), and campaign-roll (kaempferol) mulberry leaf extract obtained from the fermentation.

(3) Selection and deposit of lactic acid bacteria suitable for fermentation of mulberry leaf extract

Based on the results of Flavonol content analysis of the fermented mulberry leaf extract, the lactic acid bacteria suitable for the fermentation of the mulberry leaf extract were Pediococcus acidylactici ) C-2-17 was deposited on April 28, 2016 and deposited with KCCM 80111 at the Korea Microorganism Conservation Center (3F, 45 Junglim 2- ga ga , Hongje - mun , Seodaemun-gu, Seoul, Korea). In addition, the Pediococcus acidilactici ) C-2-17 was changed to a patent deposit on May 18 and received deposit number KFCC 11662P.

3. Selection of lactic acid bacteria suitable for fermentation of persimmon leaves or persimmon leaves

(1) Preparation of Persimmon Leaf Extract and Fermented Persimmon Leaf Extract

The dried persimmon leaf powder was added to water corresponding to about 30 times the weight of the persimmon leaves and subjected to hot water extraction at 100 ° C for 9 hours. Thereafter, the extract was passed through a filter net to obtain a filtrate, which was then concentrated in a vacuum concentrator. Then, the concentrated liquid was lyophilized to obtain a persimmon leaf extract, which was used as an analytical sample while being stored at -70 ° C.

The culture broth of the lactic acid bacteria was centrifuged to recover the cell pellet, and the recovered cell pellet was suspended in 10 ml of distilled water to prepare a cell suspension.

1 g of lyophilized persimmon leaf extract (corresponding to 1% by weight) was added to 100 ml of distilled water and dissolved. 1 ml of the cell suspension was inoculated and cultured at 37 ° C for 72 hours to ferment the persimmon leaf extract. The persimmon leaf extract was centrifuged at 12,000 rpm and 4 ° C for 20 minutes to separate the supernatant, and the supernatant was lyophilized to obtain a fermented persimmon leaf extract.

(2) Analysis of flavonol content of fermented persimmon leaf extract

200 mg of the fermented persimmon leaf extract obtained above was dissolved in 1 ml of distilled water and filtered through a 0.22 μm polyvinylidene difluoride (PVDF) syringe filter and used as an analytical sample. Flavonol contents of quercetin and kaempferol were analyzed by HPLC of a photo diode array (PDA) system equipped with an ODS-column (XBridge, 250 mm × 4.6 mm, 5 μm, Waters Corp.). As mobile phase A, 45% acetonitrile containing 2% acetic acid was used as mobile phase B, and the concentration of B solvent was increased from 100% for 25 minutes in the initial 50% The concentration gradient method was used. The flow rate of the solvent was 1 ml / min, the wavelength of the detector was 355 ㎚, the sample injection amount was 10 쨉 l, and the column oven was maintained at 30 캜 during the analysis.

Fig. 2 is a graph showing changes in contents of flavonol, quercetin and camempferol, when fermented with 21 kinds of lactic acid bacteria isolated from leaven, respectively. In FIG. 2, the X axis represents the control number of the lactic acid bacteria used for fermentation of the persimmon leaf extract, and the Y axis represents the amount of flavonol contained in 100 g of the fermented persimmon leaf extract in mg. In Fig. 2, "Control" represents a persimmon leaf extract which has not undergone fermentation treatment. As shown in FIG. 2, when the persimmon leaf extract was fermented with 21 kinds of lactic acid bacteria isolated from the koji, the contents of quercetin and kaempferol were increased in most fermented persimmon leaves extracts. In particular, Pediococcus ( Pediococcus sp. The content of quercetin and kaempferol in the fermented persimmon leaves obtained by fermentation with pentosaceus was remarkably high. The persimmon leaf extract was treated with Pediococcus ( Pediococcus) pentosaceus) was compared to the persimmon extract is not a fermentation process in a fermentation persimmon leaf extract content of the obtained fermented gwereu paroxetine (quercetin) and campaign roll (kaempferol) were each increased 1.82-fold and 1.67-fold with. On the other hand, analysis of the lipolysis inhibitory activity by the method described below showed that 24.22% of the persimmon leaves without fermentation treatment had Pediococcus penicosus pentosaceus ) showed 29.05% of the fermented persimmon leaves extract.

(3) Selection and deposit of lactic acid bacteria suitable for fermentation of persimmon leaf extract

Based on the results of Flavonol content analysis of the fermented persimmon leaf extract, it was confirmed that Pseudococcus pentosaceusPediococcus pentosaceus) C-2-3 was selected and deposited with KCC Microbiological Conservation Center (3F, 45 Junglim 2-ga Road, Seodaemun-gu, Seodaemun-gu, Seoul, Korea) on Apr. 28, 2016 and deposited with the deposit number KCCM 80110. In addition, Pediococcus pentosaceus (Pediococcus pentosaceus) The safety deposit of C-2-3 was changed to a patent deposit on May 18 and was granted deposit number KFCC 11661P.

Pediococcus acidilactici C-2-17 was selected for Pediococcus as a lactic acid bacterium suitable for fermentation of persimmon leaf extract. It was transferred to the Korea Microorganism Conservation Center (3F, 45 Junglim 2- ga road, Seodaemun- On Apr. 28, it was deposited with safety and granted the deposit number KCCM 80111. In addition, a safe deposit of Pediococcus acidilactici C-2-17 in Pediococcus was changed to a patent deposit on May 18 and was granted accession number KFCC 11662P.

4. Center Synthesis Planning Experiment and Reaction surface method  Analysis of Optimum Fermentation Conditions of Mulberry Leaf Extracts

(1) Establishment of fermentation experiment conditions by central synthetic design method

In order to investigate the optimal fermentation conditions to maximize the glucose uptake inhibitory activity of the fermented mulberry leaf extract, the fermentation experiment conditions were set using a central composite design (CCD), and Design Expert S / W ver 7.1.6 And the results were analyzed. Specifically, mulberry leaf extract concentration, fermentation time, and fermentation temperature were used as independent variables and coded in five steps of -α, -1, 0, 1, α according to the central synthetic design method. Respectively. Dependent variables affected by independent variables were glucose uptake inhibitory activity, and were used for regression analysis using the mean value after 3 repeated measurements. Table 2 shows the setting conditions for independent variables for applying the central synthetic design to the fermentation experiment of mulberry leaf extract, and Table 3 below shows the fermentation conditions of the mulberry leaf extract actually set using the central synthetic design method.

Five-step code -α -One 0 One alpha Concentration of mulberry leaf extract (% by weight) 0.66 One 1.5 2 2.34 Fermentation time (hour) 11.73 24 42 60 72.27 Fermentation temperature (℃) 29.95 32 35 38 40.05

* Mulberry leaf extract concentration: Based on the weight of distilled water in which the mulberry leaf extract is dissolved in the fermentation test of the mulberry leaf extract

* [alpha]: 1.682

Experiment No. Fermentation condition Concentration of mulberry leaf extract (% by weight) Fermentation time (hour) Fermentation temperature (℃) One One 24 32 2 2 24 32 3 One 60 32 4 2 60 32 5 One 24 38 6 2 24 38 7 One 60 38 8 2 60 38 9 0.66 42 35 10 2.34 42 35 11 1.5 11.73 35 12 1.5 72.27 35 13 1.5 42 29.95 14 1.5 42 40.05 15 1.5 42 35 16 1.5 42 35 17 1.5 42 35

(2) Method for measuring glucose uptake inhibiting activity

Glucose uptake into the blood from small intestinal cells using CaCo-2 cells and 2-NBDG [2- (N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino-2-deoxygluocose] Caco-2 cells were plated in 96-well plates and placed in a CO ₂ incubator at 37 ° C for 13 days at 2.5 × 10 4 cells / 100 μl of a sample solution (solvent: distilled water) at a concentration of 1 mg / ml, 2-NBDG solution of 100 μM in concentration (solvent: : Dimethyl sulfoxide, DMSO) and 400 μl of PBS solution were added and reacted for 2 hours at 37 ° C. The reaction solution was removed and the cells were washed 3 times with cold PBS (phosphate buffered saline) solution, and then 2-NBDG Was measured with a spectrophotofluorometer (excitation: 485 nm, emission: 535 nm). The fluorescence intensity of glucose The Bradford protein assay was performed as follows to compensate for the effect of cell concentration on the measurement of water inhibition activity: Cells were collected from wells of the fluorescence intensity-measuring plate and lysed at -20 ° C for 1 hour or more BSA (bovine serum albumin) was performed using supernatant obtained by centrifugation at 12,000 rpm for 20 minutes at 4 ° C. The absorbance of the reaction solution was measured at 595 nm. And converted according to the following formula.

Glucose uptake (%) = [Fs / Fc] x 100

Glucose absorption inhibitory activity (%) = 100-glucose absorption ability

Fs: Fluorescence intensity of the specimen treated

Fc: Fluorescence intensity of the sample without treatment

* Quercetin was used as a positive standard.

(3) Analysis of optimum condition of fermentation of mulberry leaf extract using response surface method (RSM) analysis

Table 4 shows glucose absorption inhibition activity measured from the fermented mulberry leaf extract after the experiment according to the fermentation experiment conditions of the mulberry leaf extract set using the central synthetic design method.

Experiment No. Fermentation condition Glucose absorption inhibitory activity
(%, Experimental value) Mulberry leaf extract concentration
(weight%) Fermentation time
(hour) Fermentation temperature
(° C) One One 24 32 6.27 2 2 24 32 0 3 One 60 32 2.29 4 2 60 32 4.37 5 One 24 38 13.15 6 2 24 38 22.21 7 One 60 38 27.04 8 2 60 38 19.06 9 0.66 42 35 5.21 10 2.34 42 35 13.72 11 1.5 11.73 35 19.67 12 1.5 72.27 35 28.81 13 1.5 42 29.95 22.64 14 1.5 42 40.05 28.61 15 1.5 42 35 35.52 16 1.5 42 35 34.66 17 1.5 42 35 36.44

As a result of the analysis of the glucose uptake inhibitory activity value of the fermented mulberry leaf extract obtained from various fermentation conditions, the following two-regression relationship was established between the glucose uptake inhibitory activity and three important fermentation process independent variables .

Glucose absorption inhibitory activity (%) = 35.99 + 0.82 x A + 1.94 x B + 5.75 x C - 1.09 x A x B + 0.66 x A x C + 1.29 x B x C - 10.78 x A ² - 5.55 x B ² - 5.06 x C ²

[A: Mulberry leaf extract concentration (% by weight), B: Fermentation time (hr), C: Fermentation temperature (占 폚)

FIG. 3 is an analysis of the dispersion when the factors affecting the fermentation of mulberry leaf extract were analyzed using the reaction surface method. As shown in FIG. 3, the constants of C, A ² and B ² are statistically significant factors in the second-order regression equation. In addition, since R-square (coefficient of determination) is 0.8200 in FIG. 3, it can be seen that the experimental value for the glucose uptake inhibitory activity of the fermented mulberry leaf extract is almost the same as the predicted value from the second-order regression model equation.

Tables 5 and 6 below show the reaction inhibition activity of the fermented mulberry leaf extract according to various fermentation conditions of mulberry leaf extract by the reaction surface method. FIG. 4 is a graph showing the effect of the concentration of mulberry leaf extract and the fermentation time on the glucose uptake inhibitory activity of the fermented mulberry leaf extract by the reaction surface method analysis. In FIG. 4, "A" is a contour plot between mulberry leaf extract concentration and fermentation time, and "B" is a three-dimensional reaction surface curve between mulberry leaf extract concentration and fermentation time. FIG. 5 is a graph showing the effect of the concentration of mulberry leaf extract and the fermentation temperature on the glucose uptake inhibitory activity of the fermented mulberry leaf extract by the reaction surface method analysis. 4, "A" is a contour plot between mulberry leaf extract concentration and fermentation temperature, and "B " is a three-dimensional reaction surface curve between mulberry leaf extract concentration and fermentation temperature. 6 is a graph showing the effect of the fermentation time and the fermentation temperature on the glucose uptake inhibitory activity of the fermented mulberry leaf extract by the reaction surface method analysis. In FIG. 4, "A" is a contour plot between fermentation time and fermentation temperature, and "B" is a three-dimensional reaction surface curve between fermentation time and fermentation temperature.

Experiment No. Fermentation condition Glucose absorption inhibitory activity
(%, Predicted value) Mulberry leaf extract concentration
(weight%) Fermentation time
(hour) Fermentation temperature
(° C) One 2.00 60.00 38.00 23.98 2 1.03 26.80 32.47 12.36 3 1.50 42.00 35.00 35.99 4 1.00 60.00 38.00 23.19 5 1.97 26.80 32.47 14.56 6 1.03 26.80 37.53 19.19 7 1.00 60.00 32.00 10.41 8 1.97 26.80 37.53 23.47 9 2.00 60.00 32.00 8.57 10 1.50 55.20 37.55 63.46 11 1.17 56.63 35.39 30.03 12 1.62 24.48 36.69 29.71 13 1.29 51.87 35.14 33.59 14 1.48 58.35 32.59 24.34 15 1.66 40.39 36.44 36.60 16 1.04 49.17 34.98 26.31 17 1.91 36.87 33.08 22.88 18 1.02 46.51 37.05 26.90 19 1.38 50.95 34.93 34.80 20 1.84 37.03 36.59 32.59

Experiment No. Fermentation condition Glucose absorption inhibitory activity
(%, Predicted value) Mulberry leaf extract concentration
(weight%) Fermentation time
(hour) Fermentation temperature
(° C) 21 1.07 50.83 32.37 18.38 22 1.25 32.66 32.96 24.58 23 1.56 56.74 35.52 34.72 24 1.77 58.79 34.46 28.18 25 1.66 50.06 37.80 36.48 26 1.81 37.10 35.31 32.15 27 1.64 35.47 33.71 30.78 28 1.39 40.58 34.05 32.86 29 1.17 45.08 32.74 24.11 30 1.19 39.92 34.05 28.83 31 1.14 37.69 37.44 29.48 32 1.64 52.28 37.44 36.55 33 1.57 24.20 34.59 27.98 34 1.42 50.27 33.71 31.74 35 1.75 48.88 33.18 27.60 36 1.16 55.03 33.81 26.32 37 1.88 42.00 37.78 31.88 38 1.91 43.79 32.91 22.39 39 1.42 27.25 32.36 22.14

As shown in Tables 5 and 6, the optimum fermentation conditions for maximizing the glucose uptake inhibitory activity of the fermented mulberry leaf extract are as follows.

* Mulberry leaf extract concentration: 1.66 wt%

* Fermentation time: 40.39 hr

* Fermentation temperature: 36.44 ℃

5. Center Synthesis Planning Experiment and Reaction surface method  Analysis of Optimum Fermentation Conditions of Persimmon Leaf Extracts

(1) Establishment of fermentation experiment conditions by central synthetic design method

In order to investigate the optimum fermentation conditions to maximize the lipid differentiation inhibitory activity of the fermented persimmon leaf extract, the fermentation experiment conditions were set using the central composite design (CCD) and the Design Expert S / W ver 7.1.6 And the results were analyzed. Specifically, the concentration of persimmon leaf extract, fermentation time, and fermentation temperature as independent variables were coded in five steps of -α, -1, 0, 1, α according to the central synthetic design method. Respectively. Dependent variables affected by independent variables were lipid differentiation inhibitory activity, and were used for regression analysis using the mean value after 3 repeated measurements. Table 7 shows the setting conditions for the independent synthetic parameters for applying the central synthetic design method to the fermentation experiment of the persimmon leaf extract. Table 8 below shows the fermentation conditions of the persimmon leaf extract actually set using the central synthetic design method.

Five-step code -α -One 0 One alpha Persimmon leaf extract concentration (% by weight) 0.66 One 1.5 2 2.34 Fermentation time (hour) 11.73 24 42 60 72.27 Fermentation temperature (℃) 29.95 32 35 38 40.05

* Persimmon leaf extract concentration: Based on the weight of distilled water in which the mulberry leaf extract is dissolved in the fermentation test of the mulberry leaf extract

* [alpha]: 1.682

Experiment No. Fermentation condition Persimmon leaf extract concentration (% by weight) Fermentation time (hour) Fermentation temperature (℃) One One 24 32 2 2 24 32 3 One 60 32 4 2 60 32 5 One 24 38 6 2 24 38 7 One 60 38 8 2 60 38 9 0.66 42 35 10 2.34 42 35 11 1.5 11.73 35 12 1.5 72.27 35 13 1.5 42 29.95 14 1.5 42 40.05 15 1.5 42 35 16 1.5 42 35 17 1.5 42 35

(2) Method for measuring lipid differentiation inhibition activity

 1) Culture of 3T3-L1 adipose precursor cells

The 3T3-L1 cell line was purchased from Korean Cell Line Bank (KCLRF, Korean Cell Line Research Foundation). Cells is 10% bovine calf serum (BCS, Gibco) , 1% penicillin streptomycin (Gibco) a Dulbecco's modified Eagle's medium was added (DMEM, Gibco) into a 3T3-L1 cell line to the culture medium temperature, and 5% CO for 37 ℃ ₂ Lt; / RTI >

2) Measurement of survival rate of 3T3-L1 adipose precursor cells

To exclude toxic effects of fermented persimmon leaves extract on 3T3-L1 adipose precursor cells, only viable cells were identified during cell proliferation using Cell Counting Kit-8 (CCK-8). Cells were seeded at a density of 5 × 10 ⁴ cells / ㎖ in a 96-well plate and incubated at 37 ° C and 5% CO ₂ for 48 hours. Sample solutions (0.5, 1, 2, 4 mg / Ml; prepared by dissolving the fermented persimmon leaf extract in distilled water) was further added and cultured for 24 hours. After incubation, 10 μl of CCK-8 solution was added. After 2 hours, 10 μl of 1N HCl solution was added to stop the reaction, and the degree of color development was measured by absorbance at 450 nm.

3) Differentiation and drug treatment of 3T3-L1 adipose precursor cells

The 3T3-L1 cell line was cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco) at a concentration of 2 × 10 ⁵ cells / ml using a 24-well plate, The solution was prepared by dissolving the fermented persimmon leaf extract in distilled water at a concentration of 0.5 mg / ml with a cell viability of about 80% or more. At the start of differentiation (day 0), the cells were changed to DMEM medium containing 10% FBS, 1% penicillin / streptomycin, 1 μg / ml insulin, 1 μM dexamethasone, 115 μg / ml isobutyl methylxanthine and 2 μM rosiglitazone, Lt; / RTI > After 4 days, the medium was changed to DMEM medium containing 10% FBS, 1% penicillin / streptomycin, and 1 μg / ml insulin. After 6 days, the medium was replaced with medium lacking insulin and cultured until the 8th day. In addition, the fermented persimmon leaf extract was treated at the same concentration each time when the differentiation medium was changed. Epigallocatechin gallate (EGCG, 0.1 mM) was used as a positive control.

4) Oil Red O dyeing

  To observe the effect of 3T3-L1 adipocyte differentiation on adipocyte differentiation and fat accumulation, each well was washed twice with DPBS (Dulbecco's Phosphate buffered saline) solution and 0.5 ml of 60% isopropanol solution And then allowed to stand for 5 minutes. After removing 60% isopropanol aqueous solution, it was fixed with 10% formalin solution, 0.5 ml of Oil Red O solution was added, and stained for 20 minutes. After staining, the staining solution was removed, washed four times with distilled water, and then observed under a microscope. For quantitative analysis, fat was eluted in 100% isopropanol and absorbance was measured at 500 nm using a spectrophotometer. The lipid differentiation inhibitory activity of the samples was calculated according to the following formula.

(%) = 100 - [Fs / Fc] x 100

Fs: Absorbance of the experimental group treated with the sample

Fc: Absorbance of the sample not treated

(3) Analysis of optimum condition of fermentation of persimmon leaf extract using response surface method (RSM) analysis

Table 9 below shows the lipid differentiation inhibition activity values measured from the fermented persimmon leaves extract obtained after the experiment was conducted according to the fermentation experiment conditions of the persimmon leaf extract set using the central synthetic design method.

Experiment No. Fermentation condition Lipid differentiation inhibitory activity
(%, Experimental value) Persimmon leaf extract concentration
(weight%) Fermentation time
(hour) Fermentation temperature
(° C) One One 24 32 30.94 2 2 24 32 33.53 3 One 60 32 32.70 4 2 60 32 35.30 5 One 24 38 35.89 6 2 24 38 34.00 7 One 60 38 31.29 8 2 60 38 33.18 9 0.66 42 35 27.17 10 2.34 42 35 32.82 11 1.5 11.73 35 32.70 12 1.5 72.27 35 33.18 13 1.5 42 29.95 33.53 14 1.5 42 40.05 36.71 15 1.5 42 35 37.07 16 1.5 42 35 38.18 17 1.5 42 35 37.18

As a result of the analysis of the lipid differentiation inhibition activity value of the fermented persimmon leaf extract obtained from the various fermentation conditions, the following secondary regression relationship was established between the inhibitory activity of lipid differentiation and the independent variables of three important fermentation processes .

(%) = 37.41 + 1.08 x A - 0.079 x B + 0.53 x C + 0.47 x A x B - 0.65 x A x C + 1.12 x B x C - 2.43 x A ² - 1.39 x B ² - 0.62 x C ²

[A: Persimmon leaf extract concentration (% by weight), B: Fermentation time (hr), C: Fermentation temperature (占 폚)

FIG. 7 is an analysis of variance when the factors affecting the fermentation of the persimmon leaf extract were analyzed using the reaction surface method. As shown in FIG. 7, the constants of A, A ² , B × C and B ² are statistically significant factors in the second-order regression equation. Also, since the R-square (coefficient of determination) is 0.9066 in FIG. 7, it was found that the experimental value for the lipid differentiation inhibitory activity of the fermented persimmon leaf extract was almost identical to the predicted value from the second-order regression model equation.

The following Tables 10 and 11 are values predicted by the reaction surface method for the lipid differentiation inhibitory activity of the fermented persimmon leaf extract according to fermentation conditions of various persimmon leaves extracts. FIG. 8 is a graph showing the effect of the persimmon leaf extract concentration and fermentation time on the lipid differentiation inhibitory activity of the fermented persimmon leaf extract by the reaction surface method analysis. In FIG. 8, "A" is a contour plot between the concentration of persimmon leaf extract and fermentation time, and "B" is a three-dimensional reaction surface curve between the concentration of persimmon leaf extract and fermentation time. 9 is a graph showing the effect of the persimmon leaf extract concentration and the fermentation temperature on the lipid differentiation inhibitory activity of the fermented persimmon leaf extract by the reaction surface method analysis. 9, "A" is a contour plot between the persimmon leaf extract concentration and the fermentation temperature, and "B " is a three-dimensional reaction surface curve between the persimmon leaf extract concentration and the fermentation temperature. 10 is a graph showing the effect of the fermentation time and the fermentation temperature on the lipid differentiation inhibitory activity of the fermented persimmon leaf extract by the reaction surface method analysis. In FIG. 10, "A" is a contour plot between fermentation time and fermentation temperature, and "B" is a three-dimensional reaction surface curve between fermentation time and fermentation temperature.

Experiment No. Fermentation condition Lipid differentiation inhibitory activity
(%, Predicted value) Persimmon leaf extract concentration
(weight%) Fermentation time
(hour) Fermentation temperature
(° C) One 1.50 42.00 35.00 37.41 2 1.97 26.80 37.53 35.29 3 2.00 60.00 38.00 33.20 4 2.00 60.00 32.00 35.68 5 1.97 26.80 32.47 33.83 6 1.00 60.00 38.00 31.40 7 1.03 26.80 37.53 35.05 8 1.00 60.00 32.00 31.28 9 1.03 26.80 32.47 31.54 10 1.97 57.40 37.68 34.16 11 1.58 59.02 33.92 36.43 12 1.99 42.24 37.57 35.60 13 1.60 48.83 37.45 36.91 14 1.87 36.65 36.26 36.74 15 1.02 58.23 37.91 32.11 16 1.68 56.96 35.44 36.49 17 1.02 39.38 34.83 34.09 18 1.54 31.16 36.51 37.43 19 1.63 39.94 37.70 37.44 20 1.22 48.67 37.73 35.67

Experiment No. Fermentation condition Lipid differentiation inhibitory activity
(%, Predicted value) Persimmon leaf extract concentration
(weight%) Fermentation time
(hour) Fermentation temperature
(° C) 21 1.19 54.41 37.22 34.66 22 1.11 25.35 32.15 31.80 23 1.09 35.08 34.92 34.80 24 1.69 52.80 33.27 37.05 25 1.50 38.69 33.74 36.96 26 1.76 45.78 32.07 36.74 27 1.37 53.57 35.38 36.27 28 1.51 35.70 37.12 37.62 29 1.02 53.54 34.06 33.03 30 1.01 25.98 32.62 32.35 31 1.48 59.08 36.89 35.45 32 1.23 57.93 33.11 34.53 33 1.07 50.70 36.66 34.23 34 1.93 57.42 33.95 36.06 35 1.15 45.74 32.25 34.11 36 1.09 26.60 35.99 34.85 37 1.03 46.81 36.87 34.32 38 1.54 40.11 33.93 37.18 39 1.78 26.56 32.23 34.51

As shown in Table 10 and Table 11, optimum fermentation conditions for maximizing the lipid differentiation inhibitory activity of the fermented persimmon leaf extract are as follows.

* Persimmon leaf extract concentration: 1.51 wt%

* Fermentation time: 35.70 hr

* Fermentation temperature: 37.12 ℃

6. Preparation and Characterization of Fermented Extracts by Mass Fermentation

(1) Mass-Fermentation of Mulberry Leaf Extract Using Bioreactor

The fermented mulberry leaf extract was prepared by fermenting the mulberry leaf extract in lyophilized form in a 3.5 L capacity bioreactor and centrifuging and lyophilizing. The process conditions for fermentation of mulberry leaf extract are as follows.

* Fermentation Strain: Pediococcus with Pediococcus acidilactici ) C2-17 (Accession No .: KCCM 80111)

* Inoculum amount: 1% (v / v)

* Mulberry leaf extract concentration: 1.66 wt%

* Fermentation time: 40.39 hr

* Fermentation temperature: 36.44 ℃

(2) Mass-Fermentation of Persimmon Leaf Extract Using Bioreactor

The persimmon leaf extract was lyophilized in a 3.5 L capacity bioreactor and centrifuged and lyophilized to produce a fermented persimmon leaf extract. The process conditions for fermentation of Persimmon leaf extract are as follows.

* Fermentation Strain: Pediococcus pentosaceus C2-3 (KCCM 80110)

* Inoculum amount: 1% (v / v)

* Persimmon leaf extract concentration: 1.51 wt%

* Fermentation time: 35.70 hr

* Fermentation temperature: 37.12 ℃

(3) Measurement of alpha-glucosidase inhibitory activity of fermented mulberry leaf extract prepared by the fermentation

Glucosidase of fermented mulberry leaf extract prepared by mass-fermentation of mulberry leaf extract using 3 mM p-nitrophenyl-α-D-glucopyranoside and 0.25 unit α-glucosidase Inhibitory activity was measured. Specifically, 50 μl of α-glucosidase and 50 μl of a sample solution (prepared by dissolving fermented mulberry leaf extract in distilled water, and a concentration of fermented mulberry leaf extract of 5 mg / ml and 10 mg / ml) And reacted for 15 minutes. As a positive control, acabose was used instead of fermented mulberry leaf extract. 100 μl of 3 mM p-NPG was added to the reaction solution, followed by further reaction at 37 ° C for 10 minutes. Then, 750 μl of 0.1 M sodium carbonate solution was added to terminate the reaction. Then, the absorbance was measured at 405 nm using a spectrophotometer and alpha-glucosidase inhibitory activity (or alpha-glucosidase inhibitory activity) was calculated using the following equation.

Inhibition ratio (%) = {(Xb-Xa) / Xb} x 100

Xb: Absorbance of the reaction solution subjected to the sample treatment

Xa: Absorbance of reaction solution without sample treatment

FIG. 11 shows the results of measuring the alpha-glucosidase inhibitory activity of the fermented mulberry leaf extract prepared through mulberry leaf extract or mass fermentation. In Fig. 11, "mulberry leaf (extract)" represents an experimental group treated with hot water extract of mulberry leaf, and "mulberry leaf (fermentation) " represents an experimental group treated with fermented mulberry leaf extract prepared by fermenting a large amount of hot water extract of mulberry leaf. As shown in FIG. 11, the fermented mulberry leaf extract showed higher alpha-glucosidase inhibitory activity than the mulberry leaf extract and acabose, which is a positive control substance.

(4) Measuring glucose uptake inhibitory activity of fermented mulberry leaf extract prepared by mass fermentation

The glucose uptake inhibitory activity of the mulberry leaf extract, the fermented mulberry leaf extract prepared through mass fermentation, the fermented mulberry leaf extract prepared through mass fermentation and the fermented persimmon leaf extract prepared by mass fermentation in the same amount, Method (see Fermentation Experiment by Central Composite Design). 12 shows results of measurement of glucose uptake inhibitory activity of mulberry leaf extract, fermented mulberry leaf extract prepared through mass fermentation, and fermented mulberry leaf extract prepared through mass fermentation and an equivalent amount of fermented persimmon leaf extract prepared through mass fermentation . In Fig. 12, "Control" represents a control group without sample treatment, "Quercetin" represents an experimental group treated with a positive standard substance Quercetin, "mulberry leaf extract" And "Mulberry leaf-fermented mixture (5: 5)" represents the experimental group treated with the fermented mulberry leaf extract prepared by mass-fermenting the hot water extract of mulberry leaf. The experimental group treated with the same amount of mulberry leaf extract and fermented persimmon leaf extract was shown. In Fig. 12, the Y axis represents the relative glucose uptake ability of the other experimental groups, with the control group not treated with the sample being 100, and it is interpreted that the glucose uptake inhibiting activity is higher when the grafting ability is lower. As shown in FIG. 12, the mixture of the fermented mulberry leaf extract prepared by mass-fermentation or the fermented mulberry leaf extract prepared by fermentation in a large amount and the fermented persimmon leaf extract showed higher glucose absorption inhibitory activity than the mulberry leaf hot water extract, (Quercetin).

(5) Verification of predicted value of fermented persimmon leaf extract prepared by mass fermentation

The lipid differentiation inhibitory activity of the fermented persimmon leaf extract prepared through the mass fermentation was measured by the same method as that described above (see the fermentation experiment by the central synthetic method) and compared with the predicted value analyzed by the reaction surface method. FIG. 13 shows measured values of lipid differentiation inhibition activity and fermented persimmon leaf extract prepared by mass fermentation, and the predicted values analyzed by the reaction surface method. In Fig. 13, "persimmon leaf fermented product" represents fermented persimmon leaf extract prepared through mass fermentation. As shown in FIG. 13, there is no significant difference between the actual measured value and the predicted value.

7. Production of pharmaceutical composition containing fermented mulberry leaf extract and the like

In the preparation of the following pharmaceutical composition, the fermented mulberry leaf extract can be replaced with a fermented mulberry leaf, fermented persimmon leaf or fermented persimmon leaf extract.

<7-1> Manufacture of powder

20 mg of fermented mulberry leaf extract

Lactose 100 mg

10 mg of talc

The above components were mixed and packed in airtight bags to prepare powders.

<7-2> Preparation of tablets

10 mg of fermented mulberry leaf extract

Corn starch 100 mg

100 mg of milk

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

<7-3> Preparation of capsules

10 mg of fermented mulberry leaf extract

3 mg of crystalline cellulose

15 mg of milk

Magnesium stearate 0.2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; 7-4 >

10 mg of fermented mulberry leaf extract

Lactose 150 mg

100 mg of glycerin

Xylitol 50 mg

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<7-5> Preparation of granules

15 mg of fermented mulberry leaf extract

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

<7-6> Preparation of injection

10 mg of fermented mulberry leaf extract

Sodium metabisulphite 3.0 mg

Methyl paraben 0.8 mg

0.1 mg of propylparaben

Sterile sterilized water for injection

After mixing the above ingredients, 2 ml of the mixture was filled in an ampoule and sterilized to prepare an injection.

8. Preparation of food composition containing fermented mulberry leaf extract and the like

In the preparation of the following food composition, the fermented mulberry leaf extract can be replaced with a fermented mulberry leaf, fermented persimmon leaf or fermented persimmon leaf extract.

<8-1> Manufacture of Flour Food

To 100 parts by weight of wheat flour, 0.5 part by weight of fermented mulberry leaf extract was added to wheat flour, and bread, cake, cookies, crackers and noodles were prepared using this mixture.

<8-2> Manufacture of dairy products

To 100 parts by weight of milk were added 0.5 part by weight of fermented mulberry leaf extract to milk, and various dairy products such as butter and ice cream were prepared using the milk.

<8-3> Manufacturing of Sunshine

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The grains, seeds and fermented mulberry leaf extracts prepared above were blended in the following proportions.

(30 parts by weight of brown rice, 17 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

Fermented mulberry leaf extract (1 part by weight),

(0.5 part by weight),

(0.5 parts by weight)

<8-4> Manufacture of health drinks

The ingredients such as liquid fructose (0.5 g), oligosaccharides (4 g), sugar (2 g), salt (0.5 g) and water (77 g) and 1 g of fermented mulberry leaf extract were mixed homogeneously, Glass bottles, plastic bottles, and so on.

<8-5> Manufacture of vegetable juice

2 g of fermented mulberry leaf extract was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice.

<8-6> Manufacture of fruit juice

1 g of fermented mulberry leaf extract was added to 1,000 ml of apple or grape juice to prepare fruit juice.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Therefore, the scope of the present invention should be construed as including all embodiments falling within the scope of the appended claims.

Claims (13)

delete delete delete delete A composition comprising an extract of fermented mulberry leaves as an active ingredient,
The fermented mulberry leaf extract can be obtained by extracting a mulberry leaf or a mulberry leaf extract obtained by fermenting Pediococcus acidilactici C2-17 strain (Accession No: KFCC 11662P) with Pediococcus acidilactici in Pediococcus acidulactici , C2-17 strain (Accession No .: KFCC 11662P)
Wherein the fermentation temperature is 33 to 39 DEG C and the fermentation time is 37 to 57 hours.
delete delete 6. The antidiabetic composition according to claim 5, wherein the fermentation temperature is 35 to 38 DEG C and the fermentation time is 38 to 56 hours.
[6] The antidiabetic composition according to claim 5, wherein the concentration of the mulberry leaf extract in the fermentation of the mulberry leaf extract is 1.2-1.9 wt% based on the total weight of water.
[10] The antidiabetic composition according to claim 9, wherein the mulberry leaf extract has a concentration of 1.4 to 1.8% by weight based on the total weight of water when the mulberry leaf extract is fermented.
11. The composition according to claim 5 or 8 to 10, wherein the composition further comprises a fermented persimmon leaf extract,
The fermented persimmon leaf extract is an extract of fermented persimmon leaf with Pediococcus acidilactici or Pediococcus pentosaceus in Pediococcus or a persimmon leaf extract with Pediococcus acidilactici Or fermented with Pediococcus pentosaceus . &Lt; Desc / Clms Page number 15 &gt;
12. The method according to claim 11, wherein the Pediococcus acidilactici used in fermenting the persimmon leaves or the persimmon leaves extract is Pediococcus acidilactici C2-17 (accession number: KFCC 11662P) &Lt; / RTI &gt;
12. The method according to claim 11, wherein the Pediococcus pentosaceus used for fermenting the persimmon leaves or persimmon leaf extract is Pediococcus pentosaceus C2-3 (KFCC 11661P) &Lt; / RTI &gt;

Images