KR20130096587A - Method for manufacturing submerged-state fermented allium victorialis and composition for preventing or treating anti-diabetes or anti-diabetic complication containing fermented allium victorialis - Google Patents

Abstract

PURPOSE: A manufacturing method of submerged-state fermented allium victorialis and fermented allium victorialis are provided to increase antioxidant activity, SOD like activity, DPPH radical scavenging activity and etc and have an excellent antidiabetic effect, thereby being used for the prevention or treatment composition of diabetes or diabetic combination disease. CONSTITUTION: A manufacturing method of fermented allium victorialis comprises: a step of extracting allium victorialis; a step of filtering and concentrating the extract; a step of reacting the concentrate with enzyme by treating the concentrate with α-herbzyme; and a step which cultures the product of previous step with at least one microorganism selected from a group consisting of saccharomyces cerevisiae, aspergilus oryzea and aspergillus niger. A prevention or treatment composition of diabetes or diabetic combination disease includes fermented products which are fermented with at least one microorganism selected from a group consisting of saccharomyces cerevisiae, aspergilus oryzea and aspergillus niger.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for manufacturing fermented acidic garlic using liquid fermentation and a method for preventing or treating diabetic or diabetic complications including fermented product of acid garlic. complication containing fermented Allium victorialis}

The present invention relates to a method for producing an acidic garlic fermented product using a liquid fermentation method and a composition for preventing or treating diabetic or diabetic complication including an acidic fermented product.

Diabetes mellitus is a type of diabetes mellitus that is caused by insulin secretion due to the destruction of insulin secretion due to the destruction or function of the pancreatic β-cells that secrete in vivo insulin, The major features of the metabolic diseases are hyperglycemia and hyperglycemia. When diabetes is induced, the secretion of insulin and glucagon is disturbed, resulting in abnormal metabolism control of the body, such as protein and lipid metabolism, as well as carbohydrates, leading to various metabolic diseases. When diabetic symptoms persist for a long time, Epithelial cell membrane hypertrophy has caused many complications such as circulatory diseases.

The most important goal in the treatment of diabetes is to regulate blood sugar levels as close to normal as possible. Therapeutic methods include pharmacotherapy, diet and exercise therapy. Currently, there are α-glucosidase inhibitors, sulfonylurea preparations and biguanide preparations as oral hypoglycemic agents used in patients with type 1 and type 2 diabetes. Α-glucosidase inhibitors, Cidase inhibitors show the therapeutic effect of diabetes by reducing postprandial blood glucose and elevation of blood insulin by delaying the digestion and absorption of carbohydrates in the ingested diets. The? -glucosidase inhibitor does not induce hyperinsulinemia or hypoglycemia and promotes secretion in the small intestine of glucagon-like peptide-1, which promotes insulin secretion and inhibits glucagon secretion (Mooradian, AD, Thurman, JE, Drugs, 57, pp19-29, 1999; Baron, AD Diabetes Research and Clinical Practice, 40, ppS54-S55, 1998). Acarbose, voglibose and miglitol are currently used in clinical practice. However, long-term administration of α-glycosidase inhibitors may cause side effects such as abdominal bloating and vomiting diarrhea (Hanefeld M., Journal of Diabetes and its Complications, 12, pp228-237, 1998).

On the other hand, an acid of garlic (Allium victorialis Linne) is a lily and perennial herbaceous plant. It grows in the forests of northern region, Mt. Jiri, Mt. Seolak, Mt. Odae and Ulleungdo in Korea, and smells strong garlic in the whole plant. It has the effect of eliminating organ poisoning (臟 气 毒毒) and treating seed oil. To date, the main ingredients reported in acid garlic are sulfur compounds, methyl allyl disulfide, diallyl disulfide, methyl allyl trisulfide, which are sources of odor, 3,4-dihydro-3-vinyl-1,2-thiin, 2-vinyl-1,3- vinyl-1,3-dithiinsaponin), 1-kestose, neokestoserk, etc. (Nishimura et al., 1998; Kim et al., 1996; Lee, 2008) and astragalin astragalin, furostanol glycosides, quercetin, kaempfrol, and ferulic acid (Lee et al., 2007), and thus antioxidant activity, antimutagenicity, (Park, 1997), atherosclerosis (Kim et al., 2000), antioxidant activity, antioxidant activity Duality and cytoprotective (Ham et al., 2004), dietary gojihyeol and obesity (Choi et al., 2005) is effective and so on.

  However, the evaluation of the physiological activity of acidic garlic is still insufficient. Depending on the treatment conditions of the acidic garlic and the extraction method, the content of the active substance and the physiological activity are different (Chung, 2001).

The present invention provides a method of fermenting acidic garlic using a liquid fermentation method capable of exhibiting optimal activity for antidiabetic or antidiabetic complications.

The present invention also provides a composition for preventing or treating diabetic or diabetic complications including fermented products of acidic garlic.

The present invention relates to a method for producing a garlic sauce, Filtering and concentrating the extract; Treating the concentrate with? -Herbzyme to cause an enzyme reaction; And culturing the enzyme-reacted reactant with at least one microorganism selected from the group consisting of Saccharomyces cerevisiae, Aspergillus oryzae and Aspergillus niger, and a method for producing the fermented product of acid garlic .

The acid garlic may be obtained by powdering an acidic garlic leaf.

The concentrate may be treated with? -Herbose and distilled water may be added thereto for enzyme reaction at 40 to 60 ° C for 1 to 3 hours.

Wherein said saccharomyces cerevisiae comprises Saccharomyces cerevisiae KCCM 50549, said aspergillus oryzae being selected from the group consisting of Aspergillus oryzae KCCM 60241 oryzae KCCM 60241), and the Aspergillus niger is Aspergillus strain KCCM 60381 niger KCCM 60381).

The method for preparing the fermented product of the acid garlic comprises the steps of mixing the acid garlic leaf powder with a 60 to 80% ethanol aqueous solution at a ratio of 1: 7 to 15 (acid garlic leaf powder: ethanol) (W / V) Filtering and concentrating the extract; Treating the concentrate with? -Herbose and adding distilled water to the enzyme reaction at 40 to 60 ° C for 1 to 3 hours; Filtering the enzyme-reacted substance, mixing with α-herbzyme and reacting at 40 to 60 ° C. for 1 to 3 hours; And culturing the reacted substance with at least one microorganism selected from the group consisting of Saccharomyces cerevisiae KCCM50549, Aspergillus oryzae KCCM60241 and Aspergillus niger KCCM60381.

The present invention relates to a method for producing a fermentation product comprising fermentation product obtained by fermenting an acid garlic extract with? -Herbzyme and fermenting with at least one microorganism selected from the group consisting of Saccharomyces cerevisiae, Aspergillus oryzae, and Aspergillus niger, There is provided a composition for preventing or treating diabetic complications.

The production method of the fermented product of acid garlic and the fermented product of the acid garlic using the liquid fermentation method of the present invention are excellent in antioxidative activity, SOD-like activity, DPPH radical scavenging activity, nitrite scavenging ability and hydroxy radical scavenging activity, It can be used as a composition for preventing or treating diabetes or diabetic complications and can be used as a pharmaceutical composition or a health food composition.

Fig. 1 shows the measurement of the reducing sugar content of a fermented product obtained by fermenting acid garlic with liquid fermentation, and Av shows unfermented acidic garlic.
Fig. 2 is a graph showing the polyphenol content of a fermented product obtained by fermenting liquid acid fermented with acidic garlic.
FIG. 3 shows SOD-like activities of fermented products obtained by fermenting liquid acid fermented with acidic garlic.
4 is a graph showing the electron donating ability of a fermented product obtained by fermenting an acidic garlic liquid.
FIG. 5 shows the nitrite scavenging activity measured at pH 2.5 and pH 4.2 at a concentration of 1.0 mg / mL of the fermented product fermented with liquid acid fermentation.
6 is a graph showing the flavonoid content of a fermented product obtained by liquid fermentation of acidic garlic.
Fig. 7 shows the hydroxy radical scavenging activity of the fermented product obtained by fermenting acid garlic with liquid fermentation.
FIG. 8 shows the ability of the fermented product to inhibit? -Glucosidase activity according to the fermentation method and fermentation time. The control group was a 70% ethanol extract; LF-SE is a fermented product fermented by adding saccharomyces cerevisiae and alpha herbicide.
Figure 9 is a a as a measure of the blood sugar levels in diabetic SD leg (rats) induced by streptozotocin (STZ) is a mountain garlic fermented administered, roneun vehicle distilled water, is 1ml of distilled water as a control group, the control group ^* 70 % Ethanol extract (500 mg / kg) and LF-SE fermented by adding Saccharomyces cerevisiae and alpha herbicide (500 mg / kg).
10 shows the alpha hub used in the present invention.

The present invention provides a method for producing an acidic garlic fermented product using a liquid fermentation method and a composition for preventing or treating diabetic or diabetic complication including fermented product of an acidic garlic.

Hereinafter, the present invention will be described in detail.

The evaluation of the physiological activity of the acidic garlic is still in an insufficient stage. Since the content of the active substance and the degree of physiological activity are different depending on the treatment conditions and the extraction method of the acid garlic, the present invention exhibits the optimum activity as a therapeutic agent for diabetes or diabetic complications And a composition for preventing or treating diabetic or diabetic complications comprising acidic garlic produced by such a production method.

The present invention relates to a method for producing a garlic sauce, Filtering and concentrating the extract; Treating the concentrate with an? -Herbzyme to cause an enzyme reaction; And culturing the enzyme-reacted reactant with at least one microorganism selected from the group consisting of Saccharomyces cerevisiae, Aspergillus oryzae and Aspergillus niger, and a method for producing the fermented product of acid garlic .

The above-mentioned mountain garlic is distributed in Northeast Asia region, and in Korea, it grows mainly in Ulsan and northern mountains of Gangwon Province. The content of dried garlic was 7.86% in fructose, 8.89% in fructose, 8.89% in fructose and 8.89% in fructose in the stem and leaves. It contains 1.69% sucrose and high sweetness. The vitamin content of B 2 is 0.017%. Nitrogen content was 0.8% and inorganic content was the highest with 3.2% potassium. These acid garlic has been used for a long time as a herbal medicine and food, and has little toxicity and side effects, so it can be used safely for long-term use for treatment and prevention purposes.

The above-mentioned acid garlic may include all of the leaves, roots, pearls, or outposts of cultivated or naturally grown mountain garlic, and may preferably include an acid garlic leaf.

In one embodiment, the acidic garlic leaf may be cut, lyophilized, and then ground into a blender and powdered.

The acidic garlic extract of the present invention is extracted using a conventional extraction method such as an ultrasonic extraction method, a solvent extraction method, a filtration method and a reflux extraction method, and is not particularly limited in its kind, but water, C1-C4 lower alcohol, And may be an extract obtained by extracting with water, ethanol or a mixed solvent thereof.

When the acidic garlic is extracted using an extraction solvent, the ratio of the acidic garlic to the extraction solvent is not particularly limited, but it is preferably 1: 2 to 30 times, preferably 1: 7 to 15 times (acidic garlic extract solvent) V). ≪ / RTI >

In one embodiment, when the acid garlic leaf powder is extracted with a 60-80% ethanol aqueous solution, the ratio of the acid garlic leaf powder to the 60-80% ethanol aqueous solution is 1: 7-15 (acid / water: ethanol / The mixture can be mixed for 12 to 48 hours to obtain an extract.

After the step of filtering and concentrating the extract, the concentrated liquid is treated with? -Herbzyme to perform an enzyme reaction.

The α-herbzyme is not particularly limited in its kind, For example, a product manufactured by Korea Enzyme Co., Ltd. (Gyeongin KFI No.19) can be used (Fig. 10).

In the present invention, in order to enhance the effect of microbial fermentation, By pre-treating the? -herbzyme, the therapeutic effect of the fermentation product obtained according to the present invention on diabetes or diabetic complications can be maximized.

In the step of treating the concentrate with? -Herbzyme to effect an enzyme reaction, the mixing ratio of the concentrate to? -Herbzyme is not particularly limited, but is preferably 1? The mixture of α-herbzyme and acidic garlic extract concentrate was prepared by mixing 1: 1 to 3 (α-herbzyme: acidic garlic extract concentrate) (W / V) have.

In one embodiment of the step of reacting the enzyme, the concentrate may be subjected to an enzyme reaction at 40 to 60 ° C for 1 to 3 hours by treating the concentrate with? -Herbose and adding distilled water. Herein, the concentrate,? -Hebzyme and distilled water Is not particularly limited, but preferably a mixture of an acidic garlic extract concentrate having a volume corresponding to one to three times the weight of? -Hebbzyme and? -Herbzyme is mixed, and then 20 To 50 times the volume of distilled water may be added.

The enzyme activity may be lowered at a reaction temperature lower than the enzyme reaction temperature and the reaction may take a long time. At a temperature higher than the enzyme reaction temperature, denaturation of the enzyme may occur and the reactivity may be decreased.

The α-herbicide-treated reaction solution can be directly used for culturing microorganisms. Alternatively, α-herbzyme can be treated again to maximize the antidiabetic or anti-diabetic effect, and then used for microbial culture have.

For example, the enzyme reaction product treated first with the α-herbicide may be filtered and mixed with α-herbzyme to perform a secondary enzyme reaction. The reaction conditions of the secondary enzyme are not particularly limited, Can be reacted at 40 to 60 DEG C for 1 to 3 hours.

The reaction product obtained by the enzyme reaction can be cultured with a fermenting microorganism. The fermenting microorganism which can be used at this time is not particularly limited as it is capable of fermenting an extract of acidic garlic, and preferably produces a fermentation product having excellent antidiabetic or anti-diabetic complication activity The microorganism may include one or more microorganisms selected from the group consisting of Saccharomyces cerevisiae, Aspergillus oryzae, and Aspergillus niger, and most preferably, Saccharomyces cerevisiae.

The saccharomyces cerevisiae is not particularly limited, but preferably includes Saccharomyces cerevisiae KCCM 50549 ( S. cerevisiae KCCM 50549).

In one embodiment, the enzyme-reacted fermentation product is cultured with Saccharomyces cerevisiae. The enzyme-reacted fermentation product is sterilized and mixed with Saccharomyces cerevisiae at 25 to 40 ° C for 12 to 80 hours Lt; / RTI >

The Aspergillus duck claim may include but are not particularly limited, preferably Aspergillus duck claim KCCM60241 (A. oryzae KCCM60241).

The enzyme-reacted fermentation product is cultivated with an aspirinase agent. The enzyme-reacted fermentation product is sterilized and then mixed with Aspergillus oryzae and cultured at 20 to 35 ° C for 12 to 80 hours .

The Aspergillus niger may include, but is not limited to, Aspergillus niger KCCM 60381 ( A. niger KCCM 60381).

The enzyme-reacted fermentation product is incubated with Aspergillus niger, and the enzyme-reacted fermentation product is sterilized and then mixed with Aspergillus niger and cultured at 20 to 35 ° C for 12 to 80 hours .

The present invention relates to a method for producing an acidic garlic fermented product, comprising the steps of mixing and extracting an acidic garlic leaf powder with a 60 to 80% aqueous ethanol solution at a ratio of 1: 7 to 15 (acid garlic leaf powder: ethanol; Filtering and concentrating the extract; Treating the concentrate with? -Herbose and adding distilled water to the enzyme reaction at 40 to 60 ° C for 1 to 3 hours; Filtering the enzyme-reacted substance, mixing with α-herbzyme and reacting at 40 to 60 ° C. for 1 to 3 hours; And culturing the reacted material with at least one microorganism selected from the group consisting of Saccharomyces cerevisiae KCCM 50549, Aspergillus oryzae KCCM 60241 and Aspergillus niger KCCM 60381. .

The culture may be filtered again and then concentrated.

The acidic garlic fermented product produced by the production method of the present invention exhibits a higher total polyphenol content and total flavonoid content than the unfermented acidic garlic, and the phenolic hydroxy group of the polyphenol is a protein and other It exhibits activities such as antioxidation, anti-cancer and antimicrobial, anti-thrombotic action, hyperlipidemia and fatty liver inhibiting action through the property of binding with macromolecules, and the flavonoid plays a role of preventing lipid oxidation, active oxygen scavenging and oxidative stress, Prevention, cancer and heart disease, etc., the fermented product of the present invention can be suitable for the treatment of diabetes and diabetic complications.

In addition, the acidic garlic fermented product produced by the present invention has an increased reducing sugar content and exhibits SOD (superoxide dismutase) activity as compared with unfermented acidic garlic, thereby preventing oxidative disorders, exhibiting a high electron donating ability, inhibiting lipid oxidation , Exhibits a high elimination ability of nitrite which causes poisoning by carcinogenesis or hemoglobin oxidation in the blood, is excellent in the ability to cleave the hydroxyl radical which is an oxygen species that damages lipid oxidation and DNA, and also exhibits an inhibitory effect on? -Glycosidase, And it can be confirmed that it is excellent in the treatment of diabetes and diabetic complications.

The fact that the fermented acidic garlic of the present invention is more effective in treating diabetes or diabetic complications than the non-fermented acidic garlic as described above indicates that the selection of the strains and enzymes used in the method of producing the fermented product of acidic garlic of the present invention was excellent, Is optimized to produce acidic garlic fermentations so as to exhibit the therapeutic effect of diabetic and diabetic complications.

The present invention relates to a method for producing a fermentation product comprising fermentation product obtained by fermenting an acid garlic extract with? -Herbzyme and fermenting with at least one microorganism selected from the group consisting of Saccharomyces cerevisiae, Aspergillus oryzae, and Aspergillus niger, A composition for preventing or treating diabetic complications can be provided.

The method for producing the fermented product is not particularly limited, but may be a fermented product produced by the production method of the present invention.

The diabetic complications include, but are not limited to, diabetic retinopathy, diabetic cataract, diabetic keratopathy, diabetic neuropathy, diabetic neuropathy, diabetic peripheral neuropathy, atherosclerosis, cardiovascular disease, And diabetic vascular complications.

Diabetic complications often occur despite the normal recovery of blood sugar. The final glycation product is a protein glycosylation product accumulated in tissues in proportion to the concentration of hyperglycemia, and exhibits a specific absorbance and fluorescence. Especially, amino groups of proteins cause cross-link reaction with each other. Once the final glycation products are produced, they are not degraded even when blood glucose is normally regulated, and accumulate in the tissues, abnormally changing the structure and function of the tissue, and are known to be involved in atherosclerosis, DNA mutation, senescence, Alzheimer's disease and diabetic complications have.

The fermented product is excellent in antioxidant activity, SOD-like activity, DPPH radical scavenging activity, nitrite scavenging ability and hydroxy radical scavenging activity, as well as excellent in blood glucose control and insulin control effect, and thus is useful as a composition for preventing or treating diabetes or diabetic complications And can be used as a pharmaceutical composition or a health food composition.

The pharmaceutical compositions comprising the fermentations of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the compositions of the present invention may also be used in the form of their pharmaceutically acceptable salts and may be used alone or in combination with a compound having other pharmacological activity or in a suitable combination.

The pharmaceutical composition containing the fermented product according to the present invention may be in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, external preparation, suppository and sterilized injection solution Can be used.

Examples of carriers, excipients and diluents that can be included in the composition including the fermented product of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration may include tablets, pills, powders, granules, capsules and the like. Such solid preparations may contain one or more excipients such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the fermented product of the present invention is not particularly limited, but may be generally 1 to 500 mg / kg, preferably 100 to 300 mg / kg, but the dosage may vary depending on the condition and weight of the patient, , The type of drug, the route of administration, and the period of time, and can be appropriately increased or decreased by those skilled in the art, thereby not limiting the scope of the present invention. The above administration may be carried out once a day or divided into several times.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention provides a health functional food comprising the fermented product and the pharmaceutically acceptable food supplementary additive exhibiting the effect of preventing or ameliorating diabetes or diabetic complications. Foods to which the fermented product of acid garlic can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, and health functional foods.

It may also be added to food or beverage for the purpose of preventing or improving diabetic and diabetic complications. At this time, the amount of the fermented product in the food or drink may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml , And thus the scope of the present invention is not limited thereto.

The health functional beverage composition of the present invention is not particularly limited to other components other than those containing the fermented product as an essential ingredient at the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above . The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention, but the scope of the present invention is not limited thereto.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like.

The present invention is described in detail by the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example  >

Example  1: Preparing the mountain garlic

A. victorialis Linne used in this experiment was sampled from Ulleungdo wild and garlic grown in Gangwon-do, and was used for the experiment. After the purchase, it was washed with water to remove impurities, water was removed and then cut into a cryocooler deep freezer), lyophilized and pulverized and used as a sample for extraction.

Example  2: Optimum conditions for fermentation of acid garlic extract

Example  2-1: Collection and Separation of Fermenting Microorganisms

The spread plate method in which 1 mL of salting-acid-garlic leaf suspension is dropped on a plate medium, spreads widely on the surface of the medium using a triangular glass rod, and the suspension is treated with 1 mL in a Petri dish, The pour plate method was used to separate the fermented microorganisms from the acidic garlic.

Example  2-2: Collection and Separation of Fermentation Microorganisms

Saccharomyces cerevisiae KCCM50549 ( Saccharomyces cerevisiae KCCM50549), Aspergillus age KCCM60381 (A. niger KCCM60381) and Aspergillus duck the KCCM60241 (A. oryzae KCCM60241) were collected and separated fermentation strains, the medium used to culture the attention of each zymogen Malt Extract Agar Respectively.

Example  2-3: Growth of fermenting bacteria according to fermentation condition

Example  2-3-1: Growth of fermenting bacteria depending on the concentration of extract

The saccharomyces cerevisiae KCCM50549 previously activated in a plate medium adjusted to have an acidic garlic extract concentration of 5, 10, 20, 30 and 40% in the medium was adjusted to a concentration of 10 ⁹ to 10 ¹¹ according to the strain Next, 0.1 mL of each culture dish was treated and plated. The culture was put into an incubator adjusted to 25 ± 1 ° C, and after 3 days, the number of colonies in the culture dish was counted per concentration to calculate the degree of bacterial growth by the number of colonies per mL. mL was calculated by multiplying the dilution factor by the number of colonies in the culture dish.

N = a x u

a: the dilution factor of the treated strain, u: the number of colonies present in the culture dish

On the other hand, the fermentation fungus Aspergillus oryzae KCCM60241 and Aspergillus niger KCCM60381 were inoculated by punching each strain into a plate medium treated with 5, 10, 20, 30 and 40% of the acid garlic extract in the medium After incubation for 3 days in an incubator adjusted to 25 ± 1 ° C, the size of the microflora was measured to determine the degree of bacterial growth.

In the case of Saccharomyces cerevisiae KCCM50549, the growth of the strain was decreased with increasing the concentration of the extract in the medium, but not at the concentration of 40%. On the other hand, in the case of Aspergillus oryzae KCCM60241, the concentration of the extract in the medium was good up to 10%, but the growth was slowed gradually at the above concentration and did not grow at the concentration of 40%. Aspergillus niger strain KCCM60381 also tended to be similar to that of Aspergillus oryzae strain KCCM60241, whereas aspergillin strain strain KCCM60381 did not grow at all at the 30% concentration.

Therefore, when the above-mentioned strains were used for fermentation, the strains were found to have the best growth at a concentration of 5 to 10% of the extract although there was a slight difference depending on the strains. Therefore, the concentration of the extract suitable for fermentation was determined to be 5 to 10% .

Example  2-3-2: Before and after fermentation  Reducing sugar ( Reducing sugar ) content

Reducing sugar content of fermented garlic was measured by DNS method (Kim et al., 2007). 1 g of fermented garlic powder was crushed with 100 mL of ultrapure water, filtered and diluted to 100 mL with ultrapure water and diluted 100-fold to prepare a sample solution. To 1 mL of the sample solution was added a DNS reagent (7.5 g DNS, 14.0 g NaOH , 126.1 g Rochelle salt, 5.4 mL of phenol and _{5.9 g Na 2 S 2 O 5} / distilled water 1 L) a 1 mL mix for 5 minutes cooling in cold water flowing after 15 minutes of reaction in the boiling water, the distilled water in the following reaction solution 3 mL And the absorbance was measured at 546 nm using a UV-Vis spectrophotometer. The reducing sugar content was calculated from the calibration curve of glucose.

Fig. 1 shows the results of measurement of reducing sugar in liquid fermented acidic garlic. The acid garlic fermented with Aspergillus oryzae and Aspergillus niger did not show a significant difference according to fermentation time from 59.0 to 65.0 mg% of reducing sugar, The fermented cesserabicia showed a tendency to decrease reducing sugar with increasing fermentation time. The content of reducing sugar of fermented garlic was higher than that of reducing sugar of 36.30 mg% of non - fermented acidified garlic, indicating that the reducing sugar content was increased by fermentation. On the other hand, depending on the concentration of fermented product, the reducing sugar content was low at the concentration of 5% rather than 30%.

Example  3: liquid fermentation method

To 150 g of lyophilized acidic garlic leaf powder was added 1,500 mL of 70% EtOH, and the mixture was extracted at room temperature for 24 hours at a dilution ratio of 1:10 (W / V) of acid garlic leaf powder and solvent. After the first filtration with gauze, the filtrate was subjected to secondary filtration using a filter paper (Whatman No. 2.), concentrated using a rotary vacuum evaporator on a 45 ° C water bath, and 5 mL of the concentrate and α-herbzyme Three grams of 100 mL of distilled water was added, and the mixture was leached at 50 ° C for 2 hours. Then, the mixture was firstly filtered with gauze, and then mixed with an α-Hub enzyme, followed by enzymatic reaction at 50 ° C. for 2 hours. After the enzymatic reaction, the cells were sterilized at 95 DEG C for 10 minutes and then the fermentation strain was cultured. The culture was filtered with gauze and filter paper, concentrated using a reflux evaporator on a 45 ° C water bath, and then placed in a -45 ° C deep freezer for 12 hours, and then lyophilized in a freeze dryer. .

Example  4: Fermented  Physiological Activity Test

The antioxidant activity of the fermented product of acid garlic was determined by the total polyphenol content, superoxide dismutase (SOD) activity, electron donating activity (EDA), nitrite scavenging ability, total flavonoid content, hydroxy radical scavenging Activity was measured.

Example  4-1: Total polyphenol content measurement

Total polyphenol content was measured by Folin-Denis method (1915). After making the fermented product of acid garlic into mg / g, 0.2 mL of Folin-ciocalteau reagent was added to 0.4 mL of the sample, mixed thoroughly, and reacted at room temperature for 3 minutes. After reacting for exactly 3 minutes, 0.4 mL of a saturated solution of Na ₂ CO ₃ was added and allowed to stand at room temperature for 1 hour, and the absorbance was measured at 625 nm using a UV / VIS spectrophotometer (UV-1250, Shimadzu, Japan). The polyphenol content was obtained by preparing a standard calibration curve using tannic acid as a standard material.

Fig. 2 shows the contents of phenolic compounds of acidic garlic fermented at 30% and 5% concentrations, wherein the contents of the treated strains at 30% concentration were 36.70 to 58.93 mg / g Aspergillus, 38.19 ~ 69.67 mg / g and saccharomyces cerevisiae 42.26 ~ 84.48 mg / g, respectively. The content of phenolic compounds in liquid fermented acid garlic was 84.48 mg / g, which was the highest in the samples fermented with Saccharomyces cerevisiae for 72 hours. At the concentration of 5%, the contents of Aspergillus strain 28.56 ~ 33.37 mg / g, Aspergillus oryzae 39.66 ~ 41.88 mg / g and the concentration of Saccharomyces cerevisiae 36.70 ~ 27.45 mg / g The concentration of phenolic compounds was higher than that of 5% concentration at 30% concentration.

Example  4-2: peroxidation enzyme ( Superoxide dismutase , SOD Activity measurement

SOD-like activity was measured by measuring the amount of pyrogallol produced by the method of Marklund and Marklund (1974), which catalyzes the conversion of active oxygen species to hydrogen peroxide (H ₂ O ₂ ). After adding 0.2 mL of the sample at a constant concentration, add 3 mL of Tris-HCl buffer (50 mM Tris aminom-mthane + 10 mM EDTA, pH 8.5) and 0.2 mL of 7.2 mM pyrogallol and incubate at room temperature for 10 minutes The reaction was quenched by the addition of 1 mL of 1N HCl and the absorbance at 420 nm was measured. The SOD-like activity was expressed as a percentage (%) of absorbance difference between the addition of the extract and the addition of no extract

Fig. 3 shows the SOD-like activity of liquid fermented acidic garlic. The SOD-like activity of the strains was 35.31 ~ 58.34% of Aspergillus niger, 23.72 ~ 52.74% of Aspergillus oryzae, 30.65% of Saccharomyces cerevisiae To 67.66%. The fermentation time decreased with 48 hours fermentation, but increased with increasing fermentation time. The highest activity was found in 67.66% of the fermented product for 72 hours of Saccharomyces cerevisiae.

SOD - like activity of fermented garlic was higher than that of 26.64% in non - fermented garlic cultivated in Gangwon province.

SOD - like activity was higher at 30% concentration than at 5% concentration, and increased with increasing treatment concentration.

Example  4-3: Electronic Donor ( Electron donating activity , EDA ) Measure

The electron donating ability was measured according to the method of Choi et al. (2006). That is, 900 μl of a 100 μM DPPH solution dissolved in ethanol and 100 μl of a sample solution were mixed and stirred well. After the reaction mixture was reacted in a dark place for 30 minutes, the absorbance was measured at 517 nm. The hydrogen electron donating ability was evaluated by repeating each experiment three times and then calculating the degree of decrease in absorbance of the control by the following equation.

4 shows the electron donating ability of liquid fermented acidic garlic by DPPH scavenging ability. DPPH radical scavenging activity of each strain was found to be as follows: Aspergillus agar strain 48.69 ~ 52.17% at 30% concentration, Aspergillus oryzae 45.09 ~ 53.38% As the fermentation time increased, the radical scavenging ability tended to increase and the highest scavenging ability was obtained at 72 hours fermentation. Especially, 72 hours fermented product using Saccharomyces cerevisiae strain was 55.93% Which is the highest level. The radical scavenging ability of non - fermented acid garlic was slightly increased as the fermentation time was longer than that of 46.22%. At the concentration of 5%, Aspergillus strain 42.07 ~ 66.81%, Aspergillus strain 55.86 ~ 59.53%, Saccharomyces cerevisia 54.37 ~ 60.34%, indicating that the concentration of 5% was slightly higher than that of 30% appear.

Example  4-4: Nitrite Scatters

Nitrite scavenging activity was measured by the method of Kato et al. (1987). That is, 1 mL of 1 mM NaNO 3 was added to 2 mL of the diluted sample solution, and the pH was adjusted to 2.5 and 4.2 using 0.1 N HCl, and the volume of the reaction solution was adjusted to 10 mL. This solution was reacted at 37 ° C for 1 hour. Take 1 mL of the reaction mixture, add 5 mL of 2% acetic acid and 0.5 mL of Griess's reagent, vortex and incubate at room temperature for 15 minutes. Were measured. As a control, 0.1 mL of distilled water was added instead of Griess reagent, and the nitrite scavenging ratio was calculated by the same method as above, and the nitrite scavenging ratio when the extract was added or not was compared with the BHT treatment.

N (%) = [1 - (A -C) / B] 100

N: nitrite scavenging ability, A: absorbance of 1 mM NaNO ₂ added sample after stand for 1 hour, B: absorbance of 1 mM NaNO ₂

Fig. 5 shows the nitrite scavenging activity at pH 2.5 and pH 4.2 at a concentration of 1.0 mg / mL of the fermented product fermented with acidic garlic.

At a concentration of 30% and a pH of 4.2, the scavenging activity was found to be 2.89 to 5.33% in Aspergillus strain, 3.07 to 10.15% in Aspergillus oryzae and 1.72 to 7.04% in Saccharomyces cerevisiae, The highest value was obtained at 48 hours of fermentation time, and decreased at higher fermentation time than that of Aspergillus oryzae at 7.10 ~ 9.50%, Aspergillus oryzae at 5.57 ~ 22.93% and Saccharomyces cerevisiae at 10.84 ~ 31.80%.

At 5% concentration, the Aspergillus nigrum at pH 2.5 was 10.59% of the fermented product for 48 hours at a pH of 2.5, and the fermented product was abruptly reduced from the fermentation product for 72 hours at the maximum. The aspergillus oryzae was 9.49 to 2.58% Serabicia was 11.56 ~ 2.97%, and the fermentation time was prolonged. The higher the concentration of fermented product, the higher the nitrite scavenging ability.

pH 2.5 was higher than pH 4.2 depending on pH change and decreased with increasing pH. The strains showed the highest abatement ability by 31.80% in the fermentation condition of Saccharomyces cerevisiae for 48 hours. The nitrite scavenging ability of non - fermented acid garlic showed higher nitrite scavenging ability of Aspergillus oryzae and Saccharomyces cerevisiae compared to the maximum of 19.18% in leaf water extract at pH 2.5, It can be seen that BHT has relatively high scavenging ability compared with 45.18% of scavenging ability.

Example  4-5: Determination of total flavonoid content

Total flavonoid content in the samples was measured by spectrophotometric method developed by Zhishen et al. (1999). That is, 1 mL of sample was added to a test tube containing 4 mL of distilled water, and after 5 minutes, 0.3 mL of 5% NaNO _{2 and} 0.3 mL of 10% AlCl ₃ were added in order. In the control group, 1 mL of distilled water was added instead of the sample. Six minutes after the start time, 2 mL of 1 M NaOH was added, and 10 mL of distilled water was added to make 10 mL. The amount of flavonoid was expressed in terms of catechin equivalent (CE) mg per L by measuring absorbance at a wavelength of 510 nm.

FIG. 6 shows the content of flavonoids in liquid fermented acidic garlic at a concentration of 1 mg / mL. The flavonoid content of fermented acidic garlic fermented product was 30.3% at 10.38 to 13.49 mg / mL of Aspergillus niger at 30% The flavonoid content of the fermented product fermented at 5% concentration was 52.07 ~ 66.81 mg / mL for Aspergillus niger, The content of flavonoids was 55.86 ~ 59.53 mg / mL for Saccharomyces cerevisiae and 49.05 ~ 60.34 mg / mL for Saccharomyces cerevisiae, indicating a very high content of flavonoids in the fermentation at a concentration of 5% than 30%. In liquid fermentation, fermented product showed higher content of flavonoids than acid fermented unfermented acid 4.43 mg / mL.

Example  4-6: Hydroxy Radical  ( Hydroxy radical ) Measurement of scavenging activity

Hydroxy radical scavenging activity was measured by modifying the method of Halliwell et al. (1997). That is, 345 μl of a 10 mM PBS solution containing 2.5 mM 2-deoxy-D-ribose was mixed with 50 μl of the diluted sample, and then 1 mM FeCl ₃ and 1.04 mM EDTA solution 50 Mu] l, 1 mM ascorbate 50 [mu] l and 0.1 MH ₂ O ₂ 5 &lt; / RTI &gt; After culturing at 37 ° C for 10 minutes, 500 μl of 2.8% trichloroacetic acid and 250 μl of 1% 2-thiobarbituric acid were added and the mixture was heated at 95 ° C for 8 minutes. After the reaction was cooled, the absorbance was measured at 532 nm.

Figure 7 shows the hydroxy radical scavenging activity at a concentration of 1 mg / mL of liquid fermented acidic garlic.

In the 30% concentration fermentation product, the activity of each strain was as follows: Aspergillus niger 41.98 ~ 53.83%, Aspergillus oryzae 55.00 ~ 60.38%, Saccharomyces cerevisiae 51.17 ~ 59.02% Activity showed a tendency to decrease and showed high scavenging activity in the fermented product for 24 hours. 5%, the aspergillus niger strain 71.16 ~ 79.59%, the aspergillus oryzae 73.41 ~ 77.90%, the saccharomyces cerevisiae 72.66 ~ 77.53% And higher in fermented water.

Compared with unfermented acidic garlic, the scavenging activity of liquid fermented product showed higher scavenging activity than non - fermented garlic under most conditions.

Example  5: Antidiabetic  Effect verification

Example  5-1: Measurement of α- Glucosidase  Activity inhibition experiment

The α-glycosidase activity of the sample is assayed by a method of Watanabe et al. (1997) using a 96-well plate. That is, a solution of α-glycosidase (50 μl, 0.7 U) was added to a control solution containing 5 mg / 50 μl of distilled water or 5 mg / 50 μl distilled water in a control solution containing Acarbose , And the absorbance at 405 nm is measured with an Anthos 2020 microplate reader (Cambridge, England) (zero time absorbance). Next, 50 μl of a substrate solution (5 mM p-nitrophenyl-α-D-glucopyranoside in 0.1 M phosphate buffer, pH 7.0) was added to each well containing α-glycosidase and a sample or α-glycosidase and acabos And carefully mixed well. After 5 minutes of addition of the substrate, the absorbance at 405 nm was measured to determine the? -Glucosidase activity.

FIG. 8 shows the fermentation method and the ability of the fermented product to inhibit? -Glycosidase activity according to fermentation time. The inhibitory activity of α-glycosidase activity of each sample was 26.7%, 21.6%, 11.6%, and 10.6% in LF-SE 5 days, LF-SE 7 days, The LF-SE 5 day treatment showed the same activity as the α-glycosidase inhibitor acarbose.

Example  5-2: SD rat Of the sample Antidiabetic experiment  (Type 1 diabetes)

Male SD rats (6-7 weeks, 300-350 g) were purchased from Samtako (Osan) and the standard feed citrate buffer was prepared as follows. In a 12 hr light-and-dark cycle control system, the temperature (23 ± 2 ° C) and humidity (50 ± 10%) were controlled while feeding 25.5 ml of 0.01 M citric acid and (Samtako) We adapted to the environment for one week and assigned group by average weight. After 7 days of breeding, 1 mL of STZ (Streptozotocin) [65 mg / kg body weight; STZ solution was prepared with 0.01 M citrate buffer (pH 4h) before use and stored on ice]. The pH of the solution was adjusted to 100 mL. The solution was filtered through a syringe filter (0.22 ㎛), adjusted to pH 4.6, and stored frozen.

Animals were fed with water and feed for 4 hours after STZ administration, and then fed again after 4 hours. After 72 hours, blood glucose was measured (fasting blood glucose for 10 hours) and animals with a blood glucose level of 250 mg / dL or more were diabetic rat ).

After STZ administration, free diet was administered until the start of the experiment for one week. Oral administration of the sample after fasting (10 hours) and oral administration of glucose (3 g / kg) in distilled water (1 mL) Glucose was dissolved in a constant temperature water bath at 40 ° C. The concentrations of each sample were measured using a control (1 mL PBS), saccharomyces cerevisiae Fermented extracts (200 mg / kg) (6 per each treatment) were orally administered. After 30 minutes of sample treatment, glucose (3 g / kg) dissolved in 1 mL of distilled water was orally administered. After 0, 30, 60, 120 and 180 minutes of glucose administration, blood was drawn from the tail vein and stored in heparin-treated culture tubes to measure blood glucose.

As shown in Figure 9, streptozotocin-induced diabetic rats were treated with control (1 mL of PBS), control (70% ethanol extractives), LF-SE for 5 days (200 mg / kg BW, (0 g / kg) was treated 30 minutes after the treatment with the sample (500 mg / kg) after 10 hours of fasting, The values obtained by subtracting the blood glucose levels after 60, 120, and 180 minutes were numerically expressed, and the result that the acidic garlic treatment inhibited the uptake of the initial glucose as compared with the control group was shown in FIG. The fermented product of acid garlic inhibited glucose uptake over time, and the LF-SE 5-day fermented extract, which showed -15.7 as compared with the control group of -15.3, is considered to be suitable as a substance inhibiting glucose uptake.

Claims (6)

Extracting acid garlic;
Filtering and concentrating the extract;
Treating the concentrate with α-herbzyme to perform enzymatic reaction; And
Culturing the enzyme-reacted reactant with one or more microorganisms selected from the group consisting of Saccharomyces cerevisiae, Aspergillus aurise and Aspergillus niger;
Mountain garlic fermentation method comprising a. The method of claim 1, wherein the acid garlic is a powder of the acid garlic leaves, acid garlic fermentation method. The method of claim 1, wherein the concentrate is treated with α-hubzyme and distilled water is added to carry out enzymatic reaction at 40 to 60 ° C. for 1 to 3 hours. The method of claim 1, wherein the Saccharomyces cerevisiae is Saccharomyces cerevisiae KCCM50549 ( Saccharomyces cerevisiae KCCM50549), and the Aspergillus duckling agent Aspergillus duckling KCCM60241 ( Aspegillus oryzae KCCM60241), wherein the Aspergillus niger comprises Aspergillus niger KCCM60381 ( Aspergillus niger KCCM60381). The method of claim 1,
Extracting the mixed acid garlic leaf powder with an aqueous 60 to 80% ethanol solution in a ratio of 1: 7 to 15 (acid garlic leaf powder: ethanol) (W / V);
Filtering and concentrating the extract;
Treating the concentrate with? -Herbose and adding distilled water to the enzyme reaction at 40 to 60 ° C for 1 to 3 hours;
Filtering the enzyme-reacted substance, mixing with α-herbzyme and reacting at 40 to 60 ° C. for 1 to 3 hours; And
Incubating the reacted material with one or more microorganisms selected from the group consisting of Saccharomyces cerevisiae KCCM50549, Aspergillus aurise KCCM60241 and Aspergillus niger KCCM60381;
Mountain garlic fermentation method comprising a. Prevention of diabetes or diabetic complications comprising fermentation of wild garlic extract with α-Herbzyme and fermented with one or more microorganisms selected from the group consisting of Saccharomyces cerevisiae, Aspergillus aurise and Aspergillus niger Or a therapeutic composition.

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