KR100973087B1 - Method method for manufacturing and composition for the treatment of diabetes by-products cultivation - Google Patents

Abstract

The present invention relates to a method for producing a dietary supplement for diabetic healing using agricultural by-products, and to a composition thereof, to develop and commercialize functional foods for treating hyperglycemia of diabetic patients, and to contain pagopyitol (pagopyitol) -containing situational mushrooms using agricultural by-products Products for enhancing insulin function and pazeolamin- through production / separation of active hexose correlated compound (AHCC) from mycelium, development of step-by-step purification technology of pagopyitol, and improvement of purity of D-kiro-inositol. The present invention provides a method and a composition for preparing a dietary supplement for diabetes, using agricultural by-products, which are products for delaying absorption of carbohydrates through tannin complex (PTC) processing.

By using the method for producing a dietary supplement for diabetes treatment using the agricultural a by-product as described above, and the composition thereof, it is possible to obtain an effect on the added value of farm income and agricultural products or the healing of diseases of the product.

Kidney beans, diabetes, buckwheat, mushrooms, bran

Description

METHOD METHOD FOR MANUFACTURING AND COMPOSITION FOR THE TREATMENT OF DIABETES BY-PRODUCTS CULTIVATION}

1 is a flow chart showing a method for extracting and purifying pazeolamine from kidney beans,

2 is a flowchart showing tannin component extraction in green tea,

3 is a diagram showing glucose content in water-soluble fiber eluted with four solvents in DEAE-cellulose column chromatography,

4 is a graph showing the arabinose content in the water-soluble fiber eluted from the DEAE-cellulose column,

FIG. 5 is a diagram illustrating a comparison of glucose and xylose contents in a rice bran active polysaccharide related compound (AHCC) solution fractionated with Vivaflow 200.

6 is a diagram showing changes in blood glucose in normal rats or diabetic rats,

7 is a diagram showing the concentration of serum total cholesterol and triacylglycerol in rats,

8 is a diagram showing the concentration of serum BUN and creatinine in rats,

9 shows Activities of serum GOT-GPT in rats,

10 is a diagram showing the items of liver total cholesterol and triacylglycerol in rats,

11 is a graph showing a tannin content analysis standard curve in green tea extract,

12 is a diagram showing a yield comparison of PTC precipitates according to tannin concentration;

13 is a diagram showing a comparison of PTC precipitate yield by the pH of the reaction solution,

14 is a view showing the protein content in the sample extract powder,

15 shows a comparison of the degree of inhibition of α-amylase activity of a sample protein,

FIG. 16 shows a comparison of α-amylase activity inhibition rate by concentration of PTC prototype powder; FIG.

17 is a diagram showing the hydrolysis inhibition rate of trypsin and pepsin to PTC protein,

18 is a diagram showing a particle size distribution diagram of a PTC prototype;

19 shows GOT (A) and GPT (B) activity in serum in experimental mice.

The present invention relates to a method for producing a dietary supplement for treating diabetes using agricultural by-products and a composition thereof.

Diabetes is a disease that requires prevention or control using health functional foods because it is difficult to cure with medicines and drugs. Therefore, the development of technology for manufacturing health functional foods to alleviate high blood sugar is an important task.

In recent years, the beta-glucan of the situation mushroom has been known for its special functions such as anti-cancer, diabetes control, immunity enhancement, blood pressure control and stress relief, and industrialization is rapidly progressing. As mass production became possible, resource procurement became abundant.

Moreover, it is important to develop new invention methods such as extraction, purification and hydrolysis of polymers to utilize mycelium, a by-product of situational mushrooms, which have been disposed of in the past, as a functional material for glycemic control.

In addition, pagopyritol, a special ingredient of bran, produced as a by-product during buckwheat flour milling, is a disaccharide whose main component is D-kiro-inositol, which is an important substance that acts as a powerful insulin mediator. This is an urgent situation.

In addition, grain by-products such as rice bran contain active pentozanine. Among them, arabinosilane, a water-soluble substance, is known for its cholesterol lowering, immune enhancing and anti-cancer effects. It can be called a task.

Therefore, the task of extracting β-glucan, buckwheat bran and pagopyritol from buckwheat bran, and pentozan from rice bran, and using these synergies to develop health functional foods to control blood glucose levels for diabetes It can be said to have important meanings in terms of national health.

Also, chemicals that regulate blood sugar include sulfonylureas, biguanides, and thiazolidinediones, which are difficult to manufacture and have side effects, making them unsuitable for use by patients. It is easy to develop a health functional brand because of its stability and processability such as water-soluble fiber of pentozan, a by-product of pentozan, and an inhibitor of α-glucosidase such as pazeolamine or insulin mediator such as D-kiro-inositol. can do.

In particular, the present invention has the purpose to develop a prototype by using agricultural by-products can be purchased at a low cost and can lower the production cost in the processing method can achieve an economic effect.

In the present invention, since agricultural by-products are used as an inventive material, there is an advantage of increasing the added value of agricultural products such as situation mushrooms, buckwheat and rice.

Buckwheat bran production estimated at Gangwon-do is about 1,000 to 2,000 tons per year, with the highest consumption of buckwheat in the country. However, health functional food brands using buckwheat bran are not distributed in the market.

The result of the situation mushroom cultivation in Gangwon-do was about 6,640 kg in 2001, and the production amount of Chuncheon was 4,220 kg. Therefore, if the result of the present invention is successful, the production is expected to increase rapidly.

Until now, there are many types of health foods for blood sugar control in the domestic market, but they are not effective because they are not highly functional.

Therefore, in the present invention, it is possible to increase the industrial value of the developed product by exploring the correct selection of plant components and biochemical mechanisms for controlling blood glucose levels, and further exploring the efficacy of synergism of each component.

The number of diabetic patients in Korea is about 2 million people, and because it is a disease that can increase day by day, it is urgently needed to develop a product that can be controlled as a health functional food rather than healing with drugs.

The physicochemical analysis of the structural analysis and the behavior of pagopyridol of buckwheat bran and rice bran as functional substrates in which soluble fiber contained in buckwheat bran and rice bran decomposed into low-molecular substances during the cultivation process Need to be identified.

Functional analysis by synergistic action with water-soluble cellulose in grain bran, including active polysaccharides of situational mushrooms, is required, and various functional ingredients in culture extracts must be separated and purified, and health functional foods for preventing and curing diabetes can be developed. have.

Since water soluble fiber of rice bran has been shown to have a strong blood glucose control function by delaying cholesterol lowering and absorption of carbohydrates in the diet in the intestinal tract, it is necessary to develop an airspace product with buckwheat bran.

Development of functional foods for reducing blood sugar levels of diabetes mellitus by extracting and purifying special ingredients such as situation mushrooms, buckwheat bran and rice bran listed above will be an invention project having indispensable meaning.

The invention related to buckwheat has been published by the present inventors about the physiological activity, physicochemical properties and its application value related to buckwheat type, varieties, nutritional analysis, lowering blood pressure, cholesterol lowering action.

    However, the invention and technology development related to bran, which is a by-product of buckwheat processing, have yet to be developed. Furthermore, among the bran components produced during the buckwheat flour milling and the milling process, the technology related to pagopyritol has not been invented.

In addition, in recent years, as artificial cultivation has been realized, industrial mushrooms are entering full-scale orbit, and invention and technology development are actively progressing.

However, there are no examples of functional invention using buckwheat bran as a medium in cultivation of situational mushrooms, and the invention on the development of health functional food brands using active polysaccharide-related compounds in mycelium is still insufficient in Korea.

Moreover, the invention related to the anticancer of the active polysaccharide material has been widely invented in the pharmaceutical field, but the invention of the dietary supplement related to diabetes is insignificant.

In addition, rice development is one of the important tasks as a national policy project, but it does not focus on industrial development related to rice bran, which is a by-product.

Currently, rice bran is a by-product of rice milling process and its quantity is thousands of tons nationwide, and most of it is used for livestock feed or fertilizer.

Rice bran contains active pentozan, such as arabinoxsilane, and such a substance is excellent in physiological activity such as cholesterol lowering effect as well as immune enhancement in human body, but the invention results have not been published in Korea yet.

In addition, regarding the invention of the active polysaccharide related substance, the invention relates to the extraction and use of immuno-enhanced active polysaccharide material isolated from the strain of the genus Fellinus, and analyzes the amount of carbohydrate, protein, etc. of the product purified from the situational fruiting body and analyzed the active polysaccharide. We have reported an analysis of the composition party. Extraction of active mushrooms from the situation mushroom was carried out to kill the cancer cells, and the immune-enhancing effect of the anti-cancer protein polysaccharide isolated and purified from the situation mushroom mycelium was confirmed, and it was found that the wild mushrooms had 98% anti-cancer effect. .

Fruiting hot water extract of Felinus linteus was found to have a great effect on cancer of the digestive system.Arabinose 8.2%, xylose 2.8%, glucose 61.8%, galactose 7.0%, mannose 12.4% The results of Fucose 2.9 have been published, and among the inventions related to the development of α-amylase inhibitors, there is no invention about pazeolamin in Korea.

As a state of the art related to foreign countries, the invention trend related to pagopyritol in buckwheat bran 1) The invention on the depletion of chiro-inositol and insulin resistance was realized in 1990-1993 by J. Larner et al. 2) Shinichi Kpndo, et al., Japan, discovered and announced a new agglucoside of 1D-2-O-α-D-galactosyl-chiro-inositol from zokova beans. R. Obendorf, a professor at Cornell University in the United States, suggested that in 1998, the structure of pagopyitol B1 with glycemic control in buckwheat was O-α-D-galacto-pyranosyl- (1-2) -D- The results of the invention were first published using NMR as chiro-inositol. 4) In 1998, he published the results of the invention on the effect of buckwheat bran from the milling process on bread quality. 5) In 2000, he published the results of the inventions on kiro-inositol and water-soluble carbohydrates in buckwheat seed milling and published a number of other papers on buckwheat. 6) In 1998, R. Obendorf et al. Invented that galacto-pyranosyl-chiro-inositol, related to dehydration resistance, was contained in mature buckwheat seeds. 7) In 2000, R. Obendorf et al. Announced the structure of pagopyitol A1, galactopyranosyl- (1-3) -D-chiro-inositol, by NMR. 8) In 2001, R. Obendorf et al. Determined the purification and molecular structure of two digalactosyl-kechiro-inoschols and two trigalactosyl-chiro-inositols from buckwheat seeds.

In addition, as a trend of invention related to active polysaccharides in cultured mushroom extracts, 1) In Japan, Professor Okamoto et al. In 1986 invented the Active Hexose Correlated Compound (AHCC) for the first time. 2) In 1989, Hokkaido University invented bioactive substances and functional foods using basidiomycetes. 3) In 1994, the AHCC Invention Society was established at the invention institutions and universities in Japan, mainly in Hokkaido. 4) Prof. A. Yagita presented the results of the invention on the effects of AHCC on cancer treatment at the Japan BRM Society (9th). 5) In 1996, A. Yagita et al. Invented IL-12 induction by AHCC. 6) In 1997, Professor Takai and his colleagues presented AHCC's immunotherapy for liver cancer patients. 7) Invented the effect of AHCC on cancer prevention and prescription in 1998. 8) Amino-up Chemical Co., Ltd. Nagazawha et al. Invented macrophage activation and tumor cell proliferation of A. coli culture extract AHCC. 9) Kansai University Matsuyi et al. Have published an invention on the reinhibition of AHCC administration as adjuvant therapy after hepatocellular carcinoma surgery.

In addition, as a trend of invention related to active pentozanes in grain bran, 1) Scotland A.G.J. Voragen et al. Invented arabinofuranohydrolase that degrades arabinoxsilane as a new enzyme. 2) In 1991, W.Bushuk et al. Invented pentozanes on the physical and industrial properties of bread dough. 3) In 1991, M. Gudmundsson et al. Invented the effect of water-soluble arabinoxsilane on starch aging and gelatinization. 4) In 1994, Z. Hromadkova et al. Invented the structure of arabinoxsilane using IR spectra. 5) In 1995, F. Thibault et al. Invented the extraction viscosity of arabinoxsilanes contained in wheat. 6) In 1995, W.H. Schwarz et al. Invented the side cleavage of arabinoxsilane structure. 7) In 1996, S.I. McCrae et al. Invented arabinoxsilane-degrading enzymes from the fungus family. 8) 1996, P.B. Schwarz et al. Discovered the arabinoxsilane component in barley and malt. 9) In 1997, Jan A. Delcour et al. Invented the structural properties of water-soluble arabinoxsilane from malt. 10) 1997, M.M.C. Gielkens et al. Invented arabinoxsilane decomposition by fungi. 11) 1998, J.A. Delcour et al. Invent the activity of arabinoxsilane hydrolase when grinding malt. 12) In 1998, J.A. Delcour et al. Invented the effect of low molecular weight arabinoxsilane on the physicochemical properties in baking. 13) In 1998, Maria Luz Femandez et al. Invented the effect of soluble fiber on LDL cholesterol levels in pigs. 14) In 1999, P. Aman et al. Invented the effect of protease pretreatment on arabinoxsilane fractions in DEAE-cellulose chromatography. 15) In 2000, Arja Lappalainen et al. Found Endo-xylanse isomers from the genus Trichoderma.

In addition, as the invention related to the development of the α-amylase inhibitor, 1) the invention regarding the α-amylase inhibitor is generally invented from the late 70's to early 90's, including the invention of JJMarshall et al. The present invention has been summarized based on the purification of pazeolamin and the invention of α-amylase inhibition. 2) Bowman D.E., Jaffe 'W.G. There are many reports of inventions. 3) R.H. Hopkins et al. Developed and reported the iodine staining power method in 1954 to measure α-amylase activity. Masayo KISHIDA et al. Announced in 1994 that α-amylase was inhibited by extracts from buckwheat seeds. 5) Recently, Ray Sahelian (Pharmachem llab., Inc.) has developed and distributes the brand name Pazeolamin 2250 to the international market.

An object of the present invention was made to solve the problems described above, and the final goal was to develop and commercialize functional foods for the treatment of hyperglycemia of diabetic patients. Production and separation of AHCC from pagopyritol-containing situational mushroom mycelium using agricultural by-products, development of step-by-step purification technology and improvement of purity of D-kiro-inositol, and product for enhancing insulin function and pazeolamin-tannin complex (PTC) to provide a method for producing a dietary supplement for treating diabetes using agricultural by-products, which are products for delaying absorption of carbohydrates through processing, and a composition thereof.

In order to achieve the above object, the present invention is characterized in that the method for producing a dietary supplement for treating diabetes using agricultural by-products and a composition thereof.

These and other objects and novel features of the present invention will become more apparent from the description of the present specification and the accompanying drawings.

Best Mode for Carrying Out the Invention Embodiments of the present invention will be described below with reference to the drawings.

<Example 1>

1. Development of blood sugar control material using agricultural by-products

end. Production of mycelia for development of new mushroom cultivation materials

1) The effect of buckwheat bran was added to the mushroom mushroom medium and cultured for a certain period of time.

2) Creation of optimal condition for cultivation of situation mushrooms for these mixed media and search for mycelial growth.

I. Extraction and Analysis of AHCC and Pagopyitol from Strain Mushroom Mycelium

1) Degradation of Pagopyritol during Extraction and Cultivation from Situation Mushroom Mycelium Cultured with Buckwheat and Rice Bran.

2) Purification of β-glucan, arabinoxsilane fractions from the situational mushroom culture extracts and separation of molecular weight by phenol-sulfate method to quantify glucose and xylose.

3) Analysis of constituent sugar of lyophilized powder after purification of ethanol precipitate by GC

All. Extraction, Purification and Processing of Soluble Fiber from Buckwheat Bran

1) Extract soluble cellulose from buckwheat bran by the method shown in FIG.

2) Purification of water-soluble cellulose by DEAE-cellulose chromatography

la. Extraction, Purification and Processing of Pagopyitol from Buckwheat Bran

1) Compare the yield of pagopyritol obtained from buckwheat bran extract with 50% ethanol and the yield of pagopyitol from culture extract.

hemp. Extraction and processing of arabinoxsilane from rice bran

1) Calculation of the yield of arabinoxsilane and β-glucan from rice bran

2) Purifying water-soluble cellulose and then comparing the yields to trace the biochemical metabolism of active polysaccharide-related substances in the culture process

bar. Development of Continuous Extraction Process of Arabinoxsilane and Pagopyitol from Buckwheat Bran

1) Develop a continuous processing system for extracting and purifying arabinoxsilane from buckwheat bran with water and then extracting the residue from pagopyitol with ethanol.

2) Exploring the possibility of industrialization by analyzing components per component by GC.

four. Specification Analysis for the Invention

1) In the process of the present invention, spectroscopic analysis of buckwheat bran soluble fiber and pagopyitol powder prototype, rice bran from water soluble fiber powder product, and situation mushroom mycelium extract powder prototype is carried out.

Ah. Component Analysis of Pagopyitol, β-Glucan, and Arabinoxsilane

1) Analysis of the content of galactose and D-chiro-inositol, components of pagopyridol obtained from buckwheat bran and culture extract

2) GC, glucose, galactose, and mannose, which are constituents of β-glucan, are determined by GC among active polysaccharide-related substances obtained from buckwheat bran, rice bran and culture extract

3) Arabinoxsilane constituents, arabinose, xylose, glucose and galactose, among the active polysaccharide-related compounds obtained by the same method, were quantified by GC.

4) Protein is analyzed by Kjeldahl method, solubility of product by general analysis method, particle size by microscopic method, and E. coli is analyzed according to Food Code.

2. Functional test of blood sugar control material

end. Sample Preparation for In vivo Functional Assay

1) Animal Feed Preparation

2) Preparation of lyophilized powder of pagopyritol purified purely from buckwheat bran

3) Preparation of Lyophilized Powder of Active Polysaccharides Isolated from Rice Bran

4) Preparation of Lyophilized Powders of Active Polysaccharide-related Compounds Fractionated from Cultivated Mushroom Culture Extracts

I. Animal Breeding and Metabolite Assays

1) Four-week-old male Wistar rats were purchased and grown for two weeks, then classified into each experimental diet group and adapted for one week in a breeding environment.

2) Induced type 1 diabetes with stereptozotocin

3) If the hyperglycemic state of the diabetic rats is maintained for 1 week, the experimental blood is administered to confirm the blood glucose change for 4 weeks.

All. Sample analysis

1) After breeding for a certain period, the rats are sacrificed to separate organs, adipose tissue and serum

2) Analysis of Biochemical Indicators in Serum

3) analysis of lipid concentration in liver

4) analysis of glucose in urine

<Example 2>

1. Development of new material for α-amylase inhibitor (AAI)

Kidney bean, a legume plant, is representative of Phaseolus vulgaris LINNE in Korea, and is widely used as food in foreign countries such as China. Recently, pazeolamin among kidney bean proteins inhibits the activity of α-amylase. Invention related to the regulation function has been made. Although there are 6 kinds of kidney beans belonging to the genus Phaseolus, in this experiment, five kinds of kidney beans known as medicinal or functional are domestic kidney beans (beans), Chinese kidney beans, Chinese white kidney beans, red beans and mung beans, and black beans, peas and buckwheat. In order to determine whether the protein and tannin complex have a glycemic control function, the following experiment was conducted.

2. Pazeolamin-tannin complex (PTC) powder processing

1) Pazeolamine Processing Method

A) Selection of new material containing pazeolamin of kidney beans

The following samples were purchased commercially in anticipation of containing pazeolamin. In other words, there are 8 kinds of domestic kidney beans, Chinese kidney beans, white kidney beans, red beans, black beans, green beans, peas, buckwheat bran.

B) Extracting Pazeolamine from Kidney Beans

Pazeolamine extraction method is shown in FIG. The powder of the kidney beans was ground to make powder, and then 1.5% of 1% saline was added to 1 kg of the raw material, followed by extraction at room temperature for 2 hours. The extract was centrifuged and tannin extract or ethanol was added to the supernatant to form a precipitate, followed by centrifugation to concentrate the supernatant, which was then lyophilized with a dialysis membrane and used as a sample.

Pazeolamine protein extract X was purified by precipitation with green tea tannin extract solution. After designing the extraction method of pazeolamine and determining the purification method, the raw materials were screened through the α-amylase inhibitory activity test.

C) Processing of tannin solution from green tea

Green tea tannin component was extracted as shown in FIG.

3. Review the yield of pazeolamin and tannin

end. Extraction of Pazeolamine

Extraction and purification process of pazeolamin from three kidney beans are shown in the following table.

Table 1. Extract Preparation of Pazeolamin from Kidney Beans

     Experiment method  Sample-1 (domestic kidney bean) Sample-2 (Chinese kidney beans)      Remarks ① Sample grinding                 ↓ 70-80mesh ② fat extraction                 ↓ Extractor / Reflux Cooling  Hexane 65-70 ℃ / 2h ③ Centrifugation                 ↓ 3,000rpm / 10min ④ Drying / Solvent Removal                 ↓ dryer / 85 ℃   Hexane removal ⑤ protein extraction                 ↓ 1% NaCl / 12hr   4 ℃ cooling room ⑥ Centrifugation                 ↓ 5,000rpm / 10min ⑦ Filter paper / filtration                 ↓ Filtration ⑧ concentration                 ↓ Brix 5-7 regulate ⑨ Centrifugation                 ↓ 5,000rpm / 10min Protein Extract               Kidney Bean Extract

 Note: Sample-1 = Chuncheon Kidney Beans, Sample-2 = Chinese White Kidney Beans

I. Extraction of Tannins by Temperature

Dried green tea leaves were purchased on the market to make powder, and then 1.5 liters of distilled water was added to 1 kg of the sample. The isolate was used as tannin extract for protein precipitation.

Table 2. Contents of Tannin Extract Preparation from Green Tea Leaves

    Experiment method Sample-1 (T-1) Sample-2 (T-2) Sample-3 (T-3) Remarks  ① Setting extraction temperature 95-100 ℃ 85-90 ℃ 80-85 ℃  ② Extraction time setting ↓ 10min  ③ cooling ↓ 4 ℃ / 60min  ④ Centrifugation ↓ 5,000rpm / 10min  ⑤ filter paper / filtration ↓ Filtration  ⑥ concentration ↓ Brix 2-3  ⑦ Centrifugation ↓ 5,000rpm / 10min  ⑧ Tannin Extract Green tea extract

All. Comparison of PTC Sediment Yields by Concentration of Tannins

The tannin extract extracted by temperature was mixed with kidney bean protein extract according to the concentration of the extract, and compared with the yield of sediment yield of pazeolamin-tannin.

la. Comparison of PTC Sediment Yield by pH of Reaction Solution

Before precipitation of the pazeolamin-tannin complex, the yield of the precipitate was compared and examined while changing the pH of the reaction solution to 2, 4, 6, 7, and 8.

4. Analysis of Protein Content in PTC Powders

Protein content measurement in kidney bean extract was quantified by Kieldahl method.

5. Avidity of α-amylase and tannin in vitro

In vitro, the protein produced when 10% α-amylase was used as the protein source and 5% tannic acid was added thereto was centrifuged and lyophilized to compare the yields of the tannic acid concentrations.

6. Carbohydrate Analysis in PTC Powders

After purifying the kidney bean extract, the total equivalent weight was measured by phenol-sulfate method to determine the sugar mixing ratio in the lyophilized powder, and the content of individual constituent sugars was analyzed by a gas chromatography.

7. Assays for Inhibiting α-amylase Activity of Pazeolamin

α-amylase breaks down α- (1,4) glucan bonds of polysaccharides of D-glucose units bound to three or more α- (1,4) forms in the digestive tract to liberate glucose for absorption. do. Therefore, the higher the blood glucose level after meals, it is necessary to reduce the amount of glucose or delay the absorption of glucose to inhibit the activity of α-amylase to control blood glucose levels.

In order to determine whether the Pazeolamin protein of kidney beans processed in the present invention inhibits α-amylase activity, a DNS method of measuring the absorbance by reducing reducing sugars with 3.5-dinitrosalicylic acid and Rochelle salt was used.

end. Reagent Preparation

The following reagents were prepared and measured.

1) Reagent A: 20mM phosphate buffer (pH 6.9) solution

2) Reagent B: 1% aqueous starch solution (substrate solution)

3) Reagent C: Preparation of Potasium Sodium Tartrate (S-2377) solution

4) Reagent D: Preparation of 96mM 3.5-dinitrosalicylic acid (DNS, FW 228.1) solution

5) E reagent: Preparation of coloring agent solution (coloring reagent)

6) Reagent F: Preparation of Maltose Standard Solution

7) G reagent: α-amylase solution preparation

I. Experiment method

Using 15 ml cap tube, 1.0 ml of B reagent (starch solution) and G reagent (α-amylase solution) were added, vortex mixed, equilibrated at 20 ° C for 30 minutes, and then E reagent (coloring agent) was added. After reacting in a boiling water bath for 15 minutes, it is immediately put in an ice cooling bath and cooled to room temperature. Distilled water was added thereto to fill the reaction solution with a total amount of 12 ml. After sufficient vortex mixing, Abs. Absorbance was measured as a control at Blank at 450 nm to calculate the value of activity.

8. Inhibition of Hydrolysis by Trypsin of PTC Powder

As mentioned above, the protein-tannin complex is formed by hydrogen bonding of the C-O group of proline constituting the protein with OH group 4 of the polyphenol B-ring. Thus, the complex molecular structure of the PT complex is generally explained by two mechanisms in which polyphenols are intercalated into protein molecules or are adopted by protein molecules. Tannin's protective role is believed to play a role. The following experiment was conducted to determine the degree of digestion of digestive enzymes.

end. Stage 1

1) Preparation of tannin sample (green tea extract) and pazeolamin sample (kidney bean extract).

2) Determination of Tannin Content in Tannin Extract and Protein Content in Pazeolamin Extract

3) The mixing ratio of the two extracts was precipitated by adding tannin extract to 1, 2, 3, 4, 5, 6 and 7 ml in 10 ml of pazeolamin extract.

4) Centrifuge the precipitate to obtain pazeolamin-tannin complex (PTC). Determination of yield of sediment and determination of tannin and protein content in sediment

5) Determination of unreacted protein content after protein-tannin complex formation

I. 2 steps

    1) Add 1-10 mg of PTC powder to a 50 ml Erlenmeyer flask and add 2 ml of distilled water.

(At this time, the content was adjusted to contain about 140 mg of protein in 1 ml. For example, if the protein is 14,4%, 1 g of powder contains 144 mg.)

2) Add 2 ml of 0.1 M Na2HPO4 / NaH2PO4 buffer pH7.6 solution

3) Dissolve 6.25 mg of trypsin in 2 ml of distilled water. Add 2 ml.

4) Hydrolyze at 26 ℃ for 3 hours in shaking incubator

5) Stop the reaction by adding 10% trichloroacetic acid to the reaction solution. At this time, shake for 1 hour and centrifuge the reaction solution.

6) The protein in the centrifuge supernatant and the precipitate was measured.

7) The rate of hydrolysis by the enzyme of pazeolamin-tannin complex (PTC) was calculated by the following formula.

Table 3. Enzymatic digestion of tannin-protein complexes

    I      Ⅱ      Ⅲ      Ⅳ      Ⅴ T-P Powder 20mg / 1ml
(10: 1) 20mg / 1ml
(10: 2) 20mg / 1ml
(10: 4) 20mg / 1ml
(10: 6) 20mg / 1ml
(10: 7) Buffer pH7.6  2 ml  2 ml 2 ml 2 ml 2 ml Trypsin 6.25mg / 2ml 6.25mg / 2ml 6.25mg / 2ml 6.25mg / 2ml 6.25mg / 2ml Hydrolysis                        26 ℃ / 3hr assay TCA addition             10% w / v TCA 5ml add → shaken for 1hr Centrifugation                       5,000 rpm / 10 min analysis                  Protein analysis in supernatant

  ※ T-P powder weight is based on 14,4% protein in pazeolamine

  ※ TCA = 10% trichloroacetic acid

9. PTC Powder Manufacturing for In vivo Bioactivity Assay

end. The powder of pazeolamin-tannin complex was added to 2 kg of kidney beans samples in a 50-liter extractor, 1% saline was added and the mixture was extracted for 3 hours with stirring, centrifuged to separate the residue, concentrated in Brix 12 range, concentrated and centrifuged again. .

Separately, the green tea leaf powder raw material was added to a 30-liter extractor, and 1.5 times of distilled water was added, followed by extraction for 10 minutes at 95-100 ° C., centrifugation, and the filtrate was concentrated and adjusted to Brix 8 to filter the membrane.

Pazeolamin extract was mixed with tannin extract at a ratio of 10: 7.5 to precipitate the pazeolamin-tannin complex, followed by centrifugation and lyophilization, which was used as an animal feed.

10. Developed Pazeolamine-tannin complex prototype

end. Emulsification of Kidney Bean Samples

42.7kg of fresh kidney beans (38% moisture) produced in Chuncheon area were purchased and milled with millstone to produce kidney bean oil.

I. Extraction and Compression Filtration

A sample (42.7 kg) oil was placed in an extractor for 1 ton, and 28% of 1% NaCl solution was added, followed by extraction while rotating at room temperature for 12 hours. 136 L of the liquid obtained by primary compression filtration of the extract was obtained.

All. High density filtration

The filtered filtrate was centrifuged with a TOMO-E centrifuge to separate solids and liquids to obtain kidney beans Ex 120 L.

la. Pazeolamine-tannin complex (PTC) processing

In addition to the kidney extract 120ℓ (Brix. 6), 107ℓ of distilled water was added to 10.7kg of green tea, and extracted at 95 ° C for 1 hour, followed by mixing 90L of green tea extract (96ℓ, Brix. 3.9) obtained by compression filtration. The mixture was allowed to settle for 48 hours at a low temperature of 4 ° C., the supernatant was removed by decantation, and the precipitate was separated by TOMO-E centrifuge again. The precipitate was lyophilized to obtain 2.914 kg of a powder product. The yield of the PTC prototype was 6.7% and the moisture content of the product was 1%.

Next, the development result according to the present invention will be described.

1. Production of mycelia for development of new materials for growing situational mushrooms

end. Situation mushroom cultivation experiment

1) Strain fungus Felinus linteus 6190 was purchased from the Korea Institute of Bioscience and Biotechnology.

2) Situation mushroom cultivation experiment was inoculated on April 4, 2004 and incubated for 45 days by adjusting the room temperature from 20 ℃ to 25 ℃.

I. Mycelial Production

1) The total weight of the medium was about 800 g per bottle, and the total dry weight of the mycelium was 140 kg for sample A and 129 kg for sample B.

2. Extraction, fractionation, purification and powder processing of AHCC from S. mushroom mycelium

end. AHCC Extraction from Mycelium

The filtrate-I and the residue of AHCC water-extracted from the mycelium in accordance with Mizuno method (Mizuno T 1999. The extraction and development of antitumor-active polysacchaarides from medical mushroom in Japan.International Journal of Medicinal Mushrooms 1. 8-29) The filtrate-II extracted with the salt and the filtrate-III extracted from the residue with 5% NaOH, respectively, were purified to compare the yields.

Table 4 shows a comparison of the yields obtained by fractionation of water-soluble fiber extracted from the rice bran-containing mycelium of Felinus linteus 6190-A and the buckwheat bran-containing mycelium of 6190-B.

As shown in Table 4, the yield of neutral AHCC eluted in water was low and the yield of acidic AHCC eluted in next alkali (NaOH) was high. The overall yield is in the range of 4-5%, so extraction with alkaline solvent can increase the yield, but the cost of the product can be increased due to technical problems such as sodium removal during purification.

Table 4. Yield Comparison of AHCC Filtrate Extracted and Fractions from Strain Mushroom Mycelium

Felinus Linteus 6190-A (320 g) Felinus Linteus 6190-B (320 g)    Fractionation Yield (g) yield(%)    Fractionation Yield (g) yield(%)    Filtrate-Ⅰ     2.33    0.73    Filtrate-Ⅰ     1.20    0.40    Filtrate-Ⅱ     4.80    1.50    Filtrate-Ⅱ     2.60    0.81    Filtrate-Ⅲ     8.65    2.70    Filtrate-Ⅲ     7.60    2.38      Total    15.78    4.93      Total    11.20    3.50

I. Glucose Content in AHCC Powders Extracted from Buckwheat Bran Mycelium

The glucose content in the powder processed by extracting AHCC from the Felinus linteus 6190 mycelium is measured by the phenol sulfate method, as shown in Table 5 below.

Table 5. Glucose content in AHCC extracted from buckwheat bran-containing mycelia

   Mycelium AHCC Powder Glucose Content (%)   Buckwheat bran mycelium extract powder               45.5   Filtrate-Ⅰ Extract Powder               16.7   Filtrate-II Extract Powder                7.8   Filtrate-III Extract Powder               20.9

As shown in Table 5, the glucose content of the buckwheat bran-containing mycelium AHCC powder was slightly higher than that of the rice bran AHCC powder, according to the glucose standard curve.

The glucose content in the AHCC extract powders for the filtrates I, II, and III was the highest as the glucose content was 20.9% in the powder fractionated with NaOH solution, and β-glucan was well eluted in alkali. Next, the fraction eluted in water was relatively high (16.7%) and the lowest in sodium oxalate was 7.8%.

At the industrial level, it is considered economical and hygienic to use water as an extraction solvent, and it is necessary to create a large amount of extraction process to increase the yield.

All. Xylose Content in Water-soluble Fibers Extracted from Buckwheat Bran

The xylose content of water-soluble fiber powder and buckwheat bran-containing mycelium extract AHCC powder extracted by the method of Nap and Ng as a raw material of buckwheat bran is indicated by the Orcinol method. Is the same as

Table 6. Xylose Content in Mycelial Extracts and Fractions Containing Buckwheat Bran

  Buckwheat bran mycelium water soluble fiber          Xylose Content (%)   Buckwheat bran mycelium extract powder             2.49   Buckwheat bran mycelium extract             5.37   Buckwheat bran filtrate-Ⅰ             1.42   Buckwheat bran filtrate-Ⅱ             0.75   Buckwheat bran filtrate-Ⅲ             3.09

As shown in Table 6, the xylose content in the powder processed by extracting water-soluble fiber from buckwheat bran was 2.49%, and the content of arabinoxsilane was determined to be low, and the whole water-soluble fiber was extracted from mycelium for processing animal feed. Xylose content in the side was 5.37%, which was high. When the powder was dissolved in water and fractionated with three kinds of solvents, the xylose content in the filtrate was 1.42% in the water extract, and 0.75% in the filtrate extracted with sodium oxalate, which was measured at a slightly lower amount, and extracted with 5% sodium hydroxide. The most of the filtrate was 3.09%.

As for the content of xylose in buckwheat bran AHCC, it can be seen that the water-soluble fiber of grains is eluted in alkali as in the glucose content pattern in the mycelium extract.

3. Extraction and Purification of Soluble Fiber from Buckwheat Bran

end. Glucose Content in Water-soluble Fibers Extracted from Buckwheat Bran

The invention of water-soluble fiber in grain bran has been found by Dr. M. Gohneum of the Department of Medicine, Drew University, USA to extract and purify arabinoxsilane from rice bran, which is a highly immune substance. In the present invention, the yield and column purification of water-soluble cellulose extracted with water as a solvent, and the glucose content in the powder extracted from buckwheat bran were measured by the phenol-sulfate method. Is the same as

 Table 7. Free sugar content in buckwheat bran soluble fiber powder

      Glass sugar       Sugar content (%)           Remarks      Glucose         35.80      Arabinos          1.60 Arabinos: xylose ratio
1: 1.89      Xylose          2.49

As shown in Table 7, the glucose content in the water-soluble fiber powder extracted and purified from buckwheat bran and lyophilized was 35.8%, which was measured in a relatively large amount. Whether the non-starch polysaccharide consisting of glucose is β-glucan is to be confirmed later with an analysis kit.

As a pento residue component in water-soluble fibrin, arabinose was 1.6% and xylose was 2.49%. Is determined. The overall content was in the 4% range.

I. Purification of Buckwheat Soluble Fiber

Water-soluble fiber was extracted from buckwheat bran, and enzyme-treated with α-amylase, amyloglucosidase, and the like. Then, the powder processed by the general method was dissolved in distilled water and purified by DEAE-cellulose column chromatography.

The sample solution was adsorbed onto the column resin and then eluted with distilled water first, eluted with 0.025M NaBO2.4H2O in the second, eluted with 0.4M NaOH in the third, and finally with saturated NaBO2.4H2O. The elution amount is 2ml for 1 minute in 50-ml 14-24 tubes, and the glucose content is measured by phenol-sulfate method, arabinose and xylose are reacted by Orcinol method, and glucose is measured at 490 nm by spectrophotometer. Arabinos and xylose were measured at 670 nm to determine the content of water soluble fibrin.

4. Purification and powder processing of pagopyridol from buckwheat bran

end. Pretreatment for Sucrose Purification in Pagopyritol Extract

When extracting buckwheat bran with 50% ethanol, the extraction extract powder of 6.4g is obtained in 100g of total carbohydrates, including 55% of sucrose, 40% of pagopyitol, and other ingredients. It is reported to contain 5% (Obendorf, Cornell University, USA). As such, more than half of the sucrose in the extract X is a problem in the process of processing diabetes control health food. Therefore, an experiment was conducted on the pretreatment process to remove sucrose.

In order to elute the sucrose in buckwheat bran, a method of washing with heat at 50 ° C., immersing in water at room temperature for 24 hours, and extracting with waring blander were performed. Table 8 shows the results of analyzing the free sugar in the sample obtained by the pretreatment by GC.

Table 8. Free Sugar Content in Pagopyritol Extract by Extraction Pretreatment Conditions

 Unit: concentration (g / 100g)

sample Processing conditions Ara Xyl Rham Man DCI Gal Glu SUM  S1 Buckwheat bran-Washed at 50 ℃ for 45 minutes
Extracted with 50% EtOH   1.56   0.76   2.09   2.39   4.31   5.44   8.25  24.80  S2 Buckwheat bran-Washed at 50 ℃ for 45 minutes
Filtrate-Powder   1.08   0.55   1.01   0.97   5.66   5.69  10.70  25.66  S3 Buckwheat bran-24 hours at room temperature
Extracted with 50% EtOH-Powder   1.00   0.52   1.46   1.56   4.67   4.21   4.84  18.26  S4 Buckwheat bran-24 hours at room temperature
Filtrate-Powder   0.64   0.54   0.79   1.00   5.83   5.80   5.63  20.23  S5 Buckwheat bran-Waring blander extraction-
Centrifuge- Powder   1.05   0.00   1.02   0.97   4.72   4.32  10.42  22.50  S6 Buckwheat bran-Waring blander 50%
EtOH Extraction-Treatment   0.59   0.00   0.73   0.00   5.22   5.37   6.67  18.58

Ara: arabinose; Xyl: xylose; Rham: Rhamnose; Man: mannose; DCI: D-kilo-inositol; Gal: galactose; Glu: Glucose

As shown in Table 8, when the pretreatment was performed in six ways, the glucose content in S1 X was 8.25 g, which was measured at a relatively high value. This suggests that glucose is contained in the extract because sucrose is a disaccharide combined with glucose and fructose.

In particular, when the buckwheat bran of S1 was extracted with 50% ethanol from the waring blander, 4.31% of D-kiro-inositol and 5.44% of galactose showed that the sources of these two free sugars were derived from pagopyitol in the extraction solution. Can be. That support supports the evidence that pagopyitol is a combination of D-chiro-inositol and galactose, and sometimes isomers such as digalactosyl-D-chiro-inositol with two galactoses. Since it is not preferable that sucrose is contained in the material obtained by extracting from the above results, in this invention, the decomposition operation of a sucrose and the pretreatment process of removing are needed. Table 8 shows the measured values of free sugars in the pagopyitol extract with different pretreatment conditions. As shown in Table 9, the pretreatment experiment to remove free sugar through another pretreatment process showed somewhat higher values than the measurements described in Table 8.

Table 9. Free Sugar Content in Pagopyitol Extract Extracts by Pretreatment Conditions

Unit: concentration (g / 100g)

sample          Pretreatment conditions Ara Xyl Rham Man DCI Gal Glu  SUM  S1 Supernatant after 40% alcohol treatment (water 8.01%) 0.72 0.29 1.21 0.00 8.55 8.02 14.89 33.68  S2 40% sediment after alcohol treatment (moisture 17.6%) 0.24 0.00 0.24 0.00 1.66 1.90  3.49  7.53  S3 Supernatant after low temperature precipitation (12h) (water 4.13%) 0.62 0.16 1.11 0.00 8.05 8.05 15.12 33.11  S4 Precipitation after Low Temperature Precipitation (12h) (Moisture 0.96%) 0.16 0.00 0.00 0.00 2.08 1.65  3.37  7.26  S5 Supernatant after heating at 80 ° C (moisture 3.66%) 0.84 0.17 1.49 0.00 9.57 9.03 16.26 37.36  S6 Precipitation after heating at 80 ℃ (0.99% of water) 0.14 0.00 0.00 0.00 1.49 1.10  2.62  5.35

Ara: arabinose; Xyl: xylose; Rham: Rhamnose; Man: mannose; DCI: D-kilo-inositol; Gal: galactose; Glu: Glucose

Pretreatment S1 to S6 were measured in relatively high amounts of free sugar in the supernatant samples under extraction conditions. From these results, the glucose is water-soluble, it can be determined by the gel filtration method. However, D-kiro-inositol and galactose are well soluble in 50% ethanol, but suggest that they are likely to be lost because they dissolve in small amounts in hot water.

I. Purification of Pagopyridol by Column Chromatography

After extracting the pagopyritol, the lyophilized powder was dissolved in distilled water, and the celite was sufficiently mixed. Then, the supernatant was centrifuged with amberlite XAD 400, a strong alkali resin, and amberlite XG-7, a strong base resin. / W) to fill the glass column, adsorb the sample and elute with distilled water to remove the free sugar. The pagopyritol was eluted with 50% ethanol as a solvent, and then concentrated under reduced pressure and lyophilized.

5. Extraction and Processing of Water-soluble Fiber from Rice Bran

end. Glucose Content in Water-soluble Fibers

Table 10 shows the values of glucose content measured by phenol-sulfate method after preparing water-soluble fiber powder through an enzyme treatment and centrifugation in the process of extracting water-soluble fiber from rice bran.

Table 10. Glucose Content in Water-soluble Fiber of Rice Bran

   Glass sugar kind       content(%)     Glucose         22.40     Arabinos          1.83     Xylose          1.76

As shown in Table 10, the glucose content was 22.4%, which was lower than the buckwheat bran soluble fiber powder, and the arabinos content was 1.83%. The content of xylose was 2.76%, which was comparable to that of the buckwheat soluble fiber.

I. Glucose Content in Soluble Fibers Fractionated by Solvent in DEAE-Cellulose Columns

The glucose content of the 50 ml collected solution eluted from the column with distilled water, 0.025M NaBO2, 0.4M NaOH solution and saturated NaBO2 · 4H2O solution, respectively, was determined by phenol-sulphate method. One content is shown in FIG. Solvent-1: water in FIG. 3; Solvent-2: 0.025 M NaBO ₂ .4H ₂ O; Solvent-3: 0.4M NaOH; Solvent-4: saturated NaBO ₂ .4H ₂ O. Arabinose measured by the Orcinol method is shown in Figure 4 a comprehensive pattern for the value compared with each solvent recovery. 4 solvent-1: distilled water; Solvent-2: 0.025 M NaBO ₂ .4H ₂ O; Solvent-3: 0.4M NaOH; Solvent: saturated NaBO ₂ .4H ₂ O.

The migration of water soluble fiber in the DEAE-cellulose column was the most eluted at 300 ml and the elution tendency began to gradually decrease at 400 ml as it increased at 200 ml.

The behavior of water soluble fiber eluted with 0.025 M sodium borate was highest in the initial 200 mL and gradually decreased from 300 mL.

The behavior of water-soluble cellulose eluted with 0.4 M sodium hydroxide was the highest at the initial 100 ml, gradually decreased, and the overall content was low.

The glucose content eluted with saturated sodium borate solution was highest and gradually decreased at the initial stage of 100 ml.

As described above, the elution tendency of the soluble fibers by solvent in the column is not constant according to the type of solvent, and among the four solvents, the most soluble fibers are eluted in water, and the next is eluted in saturated sodium borate solvent. The amount was relatively high in sodium hydroxide solvent.

As shown in FIG. 3, the highest glucose content was found in the experimental sphere eluted with 150 ml of distilled water, followed by saturated sodium borate solvent, and the other two solvents showed comparable values. It is determined that the water-soluble fiber extracted from rice bran contains a large amount of non-starch polysaccharide comprising glucose as a structural unit, which is estimated to be β-glucan.

All. Trace content of arabinose by solvent in DEAE-cellulose column

As described in the preceding paragraph, the contents of comparing the arabinos measured in the liquids eluted with the four solvents are as shown in FIG. 4. The arabinose content in the solution of the soluble fiber from the DEAE-cellulose column with distilled water was analyzed to be much lower than that of glucose, and the amount of the collected solution was around 400 ml, which is slower than the flow rate of the soluble fiber. It is estimated to be low arabinoxsilane. The arabinose content in water-soluble fiber eluted with 0.025 M sodium borate was analyzed to be extremely low, ranging from 0.11% to 0.47%. The overall pattern of arabinose content in water-soluble fiber eluted with 0.4 M sodium hydroxide was analyzed to be lower than at 0.025 M sodium borate. The arabinose content in the solution eluted with saturated sodium borate solution was the highest in the initial 100 ml and gradually decreased.

As described above, distilled water was the best among the four solvents in the process of purifying the water-soluble fiber extracted from the rice bran in the DEAE-cellulose column, and the next eluted in saturated sodium borate solution. Since the glucose content of soluble cellulose is higher than that of arabinose, hexane content of rice bran is estimated to be higher than that of pentozanine. As shown in FIG. 4, the sugars in the solutions eluted with the four solvents are analyzed and the patterns of the values are collectively shown.

la. Contents of Glucose and Arabinoxsilane in Fractions by Gel Filtration Solvent

Table 11 shows the content of glucose and arabinose fractionated by solvent in the DEAE-cellulose column.

Table 11. Glycosyl Compound Content in Rice Bran Soluble Fibers Fractionated from DEAE-Cellulose Columns

unit : (%)

Glycosyl residues Distillation fraction 0.25M NaBO2 0.4M NaOH Saturated NaBO2   Glucose     31.10      24.10     7.60     22.50   Arabinos      3.82       1.52     0.71      2.06

As shown in Table 11, 6 g of AHCC powder prepared from rice bran was dissolved in water, adsorbed onto a DEAE-cellulose column, and the total amount of glucose in the solution fractionated with different solvents was the highest as 31.1% in the solution fractionated with distilled water. 0.025M NaBO2.4H2O 24.1% and saturated NaBO2.4H2O were 22.5%, but 0.4M NaOH solution was the lowest at 7.6%. Arabinos was as low as one-tenth of glucose, most of which was 3.82% in distilled water and 0.71% in 0.4M NaOH solution.

hemp. Fraction test by molecular weight of water-soluble cellulose

The glucose and xylose contents of the solution collected by distillation shoe from DEAE-Celuulose column were collected by Vivaflow 200 modules by molecular weight (100kD or more, 50kD ~ 100kD, 10kD ~ 50kD, 5kD ~ 10kD). Measured.

The glucose content of the fraction obtained by passing the purified solution by the membrane size of Vivaflow 200 was the highest at 33.1% and below 5,000 Dalton, and the highest was 22.6% and above 100,000 Dalton.

The content of xylose, a constituent of arabinoxsilane, was analyzed to be generally lower than the amount of glucose. Among them, the content of xylose was the highest in the range of 2% at the molecular weight of 5,000 Dalton or less and the equivalent value at the rest of the molecular weight.

The AHCC solution fractionated by molecular weight in the preceding paragraph was put in a dialysis membrane and dialyzed with distilled water for 3 days. Distilled water was exchanged every 10 hours and the dialyzed sample was centrifuged again and the contents of glucose and xylose were determined for the clear solution.

Table 12. Glucose content in rice bran AHCC fractionated by molecular weight

Buckwheat bran (arabinoksilane) content(%) 100KD or more 14.2 50KD-100KD  6.4 10KD-50KD  7.1 5KD ~ 10KD  6.8 5KD or less  5.5

Table 13. Xylose Content in Rice Hull AHCC Fractionated by Molecular Weight

Buckwheat bran (arabinoksilane) content(%) 100KD or more 0.48 50KD-100KD 0.29 10KD-50KD 0.24  5KD ~ 19KD 0.46 5KD or less 0.18

Tables 12 and 13 show the results of measuring glucose in AHCC solution extracted from rice bran and fractionated by molecular weight. In general, the molecular weight content was low in the crude sample, which is presumed to be lost in the centrifugation of ammonium sulphate which precipitated at low temperature during purification.

As shown in FIG. 5, the glucose content in the powder collected at a molecular weight of 100,000 or more was the highest as 14.2% and generally the equivalent below. The content of xylose was determined to be extremely low compared to the glucose content, which is considered to be lost in the purification process.

6. Development of Continuous Extraction System of Arabinoxsilane and Pagopyitol from Buckwheat Bran

In the process of extracting arabinoxsilane from buckwheat bran by a new method, it is heated for 90 minutes at 40 ° C. without heating with hot water and homogenized with water to obtain arabinoxsilane. Here, a continuous extraction method was developed to obtain pagopyitol by extracting the remaining residue by centrifugation with 50% ethanol.

In order to explore the industrialization possibility of such a method, the processing yield of arabinoxsilane and the yield of pagopyridol and the constituent sugar in these powders will be analyzed.

7. Lab of Example 2. Spec analysis of intermediate products obtained from the system

end. Processing yield of arabinoxsilane

Yields were obtained for the AHCC extract powder and the situation mushroom mycelium AHCC extract powder from arabinoxsilane extract powder and rice bran from buckwheat bran.

Arabinoxsilane is a representative material of non-starch water-soluble cellulose, which is generally extracted from grains, and the powder yield is different for each site. R.J. Henry et al. Averaged 6.90% of pentozanes from wheat prostates and 2.62% from endosperm. However, from Barley prostate, it was reported that 10.4% of pentozan and 11.1% of β-glucan were obtained.

In the present invention, it was found that the water-soluble fiber from buckwheat bran obtained a high yield of 11.0%, which is generally in agreement with the literature value. However, the yield of AHCC obtained from pilot plant process was 3.07% and 4,70%.

I. Processing yield of β-glucan

After harvesting from mycelia grown for 45 days in medium containing 30% of buckwheat and rice bran, the amount of the processed powder was 3.07% for buckwheat bran mycelium and 4.70% for rice bran. Low values were obtained with 2 content. The reason for this is considered to be that the mycelium raw material contains a large amount of lignin eluted from the shrub or any debris in the extract which is not treated with enzyme.

All. Content of D-Kiro-Inositol in Pagopyitol Powder

Since pagopyritol is a combination of galactose and D-chiro-inositol, no standard sample has yet been submitted. Therefore, it is reasonable to use gasochromatography based on the analysis of chiro-inositol. Extracting the pagopyritol from buckwheat bran in various ways to measure the content of D-chiro-inositol in its powder is shown in Table 14 below.

Table 14. Free Sugar Content in Pagopyitol Extract Extraction by Extraction Conditions

Unit: concentration (g / 100g)

sample         Extraction conditions Ara Xyl Rham Man DCI Gal Glu SUM  S1 Room temperature, 40% ethanol supernatant 0.72 0.29 1.21 0.00 8.55 8.02 14.89 33.68  S2 Room temperature, 50% ethanol supernatant 0.62 0.16 1.11 0.00 8.77 8.05 15.12 33.83  S3 80 ° C, 50% ethanol supernatant 0.84 0.17 1.49 0.00 9.57 9.03 16.26 37.36

Ara: arabinose; Xyl: xylose; Rham: Rhamnose; Man: mannose; DCI: D-kilo-inositol; Gal: galactose; Glu: Glucose

Although there are many methods for extracting pagopyitol, the ratios of D-chiro-inositol and galactose in each of the four types extracted in the present invention were approximated to 1: 1. The highest content here was 9.55% as a supernatant after heating to 80 ° C. The method of extracting 50% ethanol for 3 minutes with a waring blender was similar to that obtained by Obendorf et al in the US, which obtained about 4.5% of pagopyitol and 5.22% in this experiment. Extracting with 40% ethanol yielded 8.55%, yielding higher yield than 50% ethanol.

8. In vivo experiments for functional assays

end. Breeding of Animals

Four-week-old male wistar rats were purchased from Harlan Sprague Dawley (Indianapolis, USA) through Polars International, and controlled in temperature (20-24oC) and dark time (20: 00-08: 00). After three weeks of adaptation, the animals were divided into six groups for each diet by the ingot method. The experimental group was divided into 1 normal group and 5 diabetic groups. Mycelial extract (MPB) was classified into a diet group.

Diabetes induction of experimental animals is as follows. The animals were fasted for 20 hours and then injected intraperitoneally with a certain amount (50 mg / kg body weight) of streptozotocin (Sigma, USA). Diabetes induction was confirmed by using a hand-held blood glucose meter (Lifescan, USA) by collecting whole blood from the tail vein. One week after the induction of diabetes, Harlan Teklad, USA was fed to determine whether hyperglycemia was maintained. The diet was changed to an experimental diet for five weeks. Each diet was fed for 35 days, fasted for 3 hours, and sacrificed with scaffold. Experimental diet was prepared according to AIN-93G diet composition, four experimental samples were added to the basic diet at 5% level in place of corn starch. Body weight change, dietary intake and water intake during the experiment were recorded at regular times every day.

I. Blood glucose change measurement

The blood glucose change for 6 weeks was measured by fasting the animal for 3 hours twice a week for 3 hours, collecting whole blood from the tail vein, and using a portable blood glucose meter (Lifescan, USA). It is expressed as the weekly blood sugar value.

All. Experimental Analysis

1) Organ and Serum Separation

The animals were fasted for 3 hours, sacrificed with the head, and blood was collected. After collection, liver, kidney, pancreas and spleen were separated and washed with 0.9% cold saline to remove blood and the respective weight was recorded. Serum was obtained by collecting whole blood for 1 hour at room temperature after centrifugation and centrifuging at 3,000 rpm for 20 minutes and stored at -30 ° C until analysis.

2) Biochemical Analysis of Serum

Total cholesterol (Total Cholesterol), Triglyceride, Blood Urea Nitrogen (BUN), Creatinine and GOT / GPT concentrations in serum were measured using the respective diagnostic kits (Asan Pharmaceutical, Seoul).

3) Measurement of lipid concentration in soy sauce

Total cholesterol and triglyceride concentrations in the liver were extracted by Folch et al., And then solubilized with 1% triton X-100 according to Carr et al. Soluble soy fat was measured by enzyme method respectively.

4) urine analysis

For 24 hours, the urine of the animals was carefully collected to avoid mixing with feces, and then the volume was measured and pH was checked using a pH meter. In addition, in order to check the amount of glucose discharged into the urinary tract for 1 day, the collected urine was filtered and diluted to an appropriate concentration, and then measured using a glucose diagnostic kit (Asan Pharmaceutical, Seoul).

5) Experimental result analysis

Statistical analysis was performed by one-way analysis to verify the difference in dietary effects according to the amount of each sample added, and significant differences were tested at the p <0.05 level for the mean values according to the LSD test.

la. result

1) weight change

Table 15 shows the weight change and dietary intake of experimental animals during the experiment. There was no difference in dietary weight between the two groups, but after streptozotocin (STZ) treatment, the weight loss of diabetic group was faster than that of normal group. In the diabetic group, the weight gain tendency was increased in the experimental diet group compared to the control group, especially in the buckwheat bran extract administration group (DBBE) and buckwheat bran-containing medium mushroom mycelium extract administration group (DMPB). However, there was no statistically significant difference. Dietary intake increased significantly in the diabetic group compared to the normal control group, but decreased significantly in the rice bran extract group (DRBE).

Table 15. Dietary Intake and Body Weight of Normal and Diabetic Rats for 6 Weeks

Early middle final Dietary intake
(g / day) g NC 252 ± 9.1 272 ± 8.3 468 ± 17.8 27.8 ± 1.14 DC 246 ± 8.3 237 ± 13.6  212 ± 22.5 **   38.7 ± 1.15 b ** DBBE 243 ± 8.0 248 ± 14.0 258 ± 32.2   38.3 ± 0.80 b DRBE 254 ± 8.5 243 ± 8.0 232 ± 20.3   33.4 ± 1.79 a DMPR 248 ± 12.0 233 ± 10.4 233 ± 15.0    36.2 ± 1.25 ab DMPB 247 ± 7.1 242 ± 8.1 255 ± 28.6    35.5 ± 1.06 ab

NC: Normal AIN 93 Dietary Supply
DC: Diabetic AIN 93 Dietary Supply
DBBE: AIN 93 dietary supplement containing 5% BBE (buckwheat bran extract) in diabetes
DRBE: Supply AIN diet containing 5% RBE (rice bran extract) of diabetic group
DMPR: AIN dietary supplement containing 5% MPR (Felinus linteus mycelium extract cultured in rice bran containing medium)
DMPB: AIN dietary supplement containing 5% MPB (Felinus linteus mycelium extract cultured in buckwheat bran-containing medium)
Each value represents 6 mean ± SE (n = 6).
a, b Values with different superscript letters are significantly different within diabetic group (p <0.05)

** P <0.01 vs. normal control group.

2) changes in blood sugar

Figure 6 shows the weekly blood sugar changes according to the experimental diet. For one week after the induction of diabetes, rodent forages were fed and the blood glucose level was significantly increased in the diabetic group compared to the normal group. After starting feeding each experimental diet based on AIN-93G, there was a difference in blood glucose between the three groups and the difference was more pronounced at the fourth week. The glycemic effect of diet was the highest in DRBE group and similar in DBBE, DMPR and DMPB group.

3) Serum fat concentration and biochemical indicators

Figure 7 shows the concentration of total cholesterol and triglycerides in the serum of experimental animals. Serum total cholesterol concentration was significantly higher in diabetic group than in normal group, but it was confirmed that dietary formula decreased due to feeding. In particular, the effect of dietary diet on serum cholesterol reduction was significant in DBBE, DMPR and DMPB groups. Serum triglyceride levels were significantly decreased in the experimental diet group except the DRBE group compared to the diabetic control group. Neutral lipid reduction effect of DBBE, DMPR and DMPB was stronger than that of total cholesterol.

Figure 8 shows the concentration of blood urea nitrogen (BUN) and creatinine in the serum of the experimental animals. The serum urea nitrogen (BUN) was found to be increased significantly in the diabetic group compared to the normal group. However, the 5% DBBE, DRBE, DMPR and DMPB diets effectively reduced the concentration of BUN in elevated serum due to diabetes induction. Serum creatinine levels were significantly higher in the diabetic group than in the normal group. Increasing creatinine levels in the diabetic group tended to be decreased by the administration of the experimental diet. In particular, dietary supplementation of DMPR reduced creatinine levels in serum to significant levels compared to controls.

The activity of GOT and GPT in serum was significantly increased in the diabetic group compared to the normal group (FIG. 9). This increase in GOT and GPT levels in the diabetic group was significantly recovered due to the administration of RBE, MPR and MPB. However, dietary addition of BBE had no significant effect on the activity of GOT and GPT.

4) fat content in soy sauce

Figure 10 shows the total cholesterol and triglyceride content in the liver of the experimental animals. The diabetic control group showed higher hepatic cholesterol content than the normal control group. The accumulation of hepatic cholesterol due to diabetes was reduced to 5%, similar to that of the normal control group by the administration of BBE, RBE, MPR and MPB. The content of triglyceride in liver was higher in the diabetic control group than in the normal control group. Interestingly in the diabetic group, the experimental diet group showed lower triglyceride content than the normal control group.

5) Weight comparison of organs in the body

Table 16 shows the weights of organs in the body of the experimental animals. The liver and kidney weights were increased by 72.3% (P <0.05) and 32.9% (P <0.01) in the diabetic control group, respectively. The increase in hepatic weight due to diabetes was recovered by the diet in the DBBE group (32.3%), DRBE group (20.7%), DMPB (39.8%) and DMPR group (37.9%). No dietary effect of test samples on weight of kidney was observed.

Pancreatic and spleen weights were decreased by 25.9% (P <0.05) and 48.9% (P <0.01), respectively. The decrease in pancreatic weight due to diabetes was recovered by the respective diets in the DBBE group (85.7%), DRBE group (25.1%), DMPB (49.8%) and DMPR group (57.1%). Spleen atrophy was recovered in the DBBE group (26.0%), DRBE group (15.3%), DMPB (17.4%) and DMPR group (32.5%).

6) Urinary Analysis

Table 17 shows the water intake, daily urine volume and glucose output of experimental animals. It was confirmed that the amount of water ingested and the volume of urine recovered during the 24 hours increased dramatically in the diabetic control group compared to the normal control group. Increased daily water intake and urine volume due to diabetes were reduced by dietary administration of RBE, MPB and MPR.

The concentration of free glucose per unit volume of urine was significantly higher in the diabetic control group than in the normal control group. However, considering the total urine output, the total amount of glucose released through the urine for 24 hours was significantly reduced in the RBE and MPR groups.

The urine pH was neutral in the normal control group and DMPR group, and the pH was slightly increased in the diabetic control group, DBBE group and DRBE group. In contrast, the pH of urine in the DMPB group was found to be neutral. However, urine pH was within normal range in all experimental animals.

Table 16. Organ Weights in Normal and Diabetic Rats

liver kidney Pancreas spleen g NC 3.54 ± 0.040 2.86 ± 0.098 1.08 ± 0.096 0.94 ± 0.099 DC 6.10 ± 0.485 * 3.80 ± 0.197 ** 0.80 ± 0.043 * 0.48 ± 0.054 ** DBBE 5.27 ± 0.317 4.09 ± 0.255 1.04 ± 0.150 0.60 ± 0.051 DRBE 5.57 ± 0.363 3.61 ± 0.129 0.87 ± 0.064 0.55 ± 0.057 DMPR 5.08 ± 0.297 3.78 ± 0.149 0.94 ± 0.081 0.56 ± 0.027 DMPB 5.13 ± 0.297 3.85 ± 0.151 0.96 ± 0.163 0.63 ± 0.082

 Each value represents the mean ± S.E. (n = 6) of 6 animals.

* P <0.05 vs. normal control group. ** P <0.01 vs. normal control group.

Table 17. Water Absorption and Urine Analysis in Rats

Water intake
(Ml / 24 hr) Urine volume
(Ml / 24 hr) Urine Glucose
(g / ㎗) Glucose excretion
(g / 24 hr) pH NC  33 ± 1.7  11 ± 1.1 0.13 ± 0.021 0.013 ± 0.0011 7.01 ± 0.15 DC 173 ± 23.3 b ** 160 ± 23.2 b ** 6.23 ± 0.307 ab ** 9.671 ± 0.9820 c ** 7.59 ± 0.17 b * DBBE 165 ± 10.3 b 136 ± 3.3 ab 6.69 ± 0.010 b 9.075 ± 0.1835 bc 7.59 ± 0.23 b DRBE 131 ± 6.1 ab 114 ± 9.6 a 6.52 ± 0.122 ab 7.445 ± 0.6786 ab 7.24 ± 0.26 b DMPR 137 ± 6.0 ab 127 ± 6.0 ab 6.57 ± 0.149 ab 8.293 ± 0.3380 abc 7.01 ± 0.36 ab DMPB 123 ± 8.3 a 113 ± 8.3 a 6.07 ± 0.227 a 6.793 ± 0.3891 a 6.39 ± 0.39 a

Each value represents 6 mean ± SE (n = 6).
a, b, c Values with different superscript letters are significantly different within diabetic group (p <0.05)

delete

** P <0.01 vs. normal control group.

hemp. Result Analysis

Streptozotocin (STZ) is known to cause diabetes by selectively destroying β-cells in the pancreas, thereby lowering insulin secretion. When the STZ was administered to the experimental animals, symptoms such as weight loss, increased drink intake, and increased urine volume appeared. In addition, a significant decrease in insulin secretion causes a disturbance in metabolism, which is involved in the storage of energy, leading to a rapid rise in fasting glucose levels and the release of large amounts of glucose through the urine.

In the present invention, Wistar rats were administered STZ for 6 weeks after inducing diabetes, and compared with the normal group, the weight loss, dietary intake and beverage intake were increased in the diabetic group. In addition, blood glucose levels in dietary and fasting conditions increased rapidly, and daily urination and glucose emissions increased significantly. However, the symptoms of diabetes mellitus 5% level of buckwheat bran extract (BBE), rice bran extract (RBE), rice bran-containing culture medium mycelial extract (MPR) and buckwheat bran-containing culture medium extract (MPB) for 5 weeks It was alleviated by supplementary supplementation.

Diabetes is known not only to cause carbohydrate metabolism but also to raise triglycerides, VLDL-cholesterol and LDL-cholesterol levels higher than normal when not controlled. The present invention also confirmed an increase in serum lipid concentration by diabetes-induced diabetic buckwheat bran extract and situation by reducing total cholesterol and triglyceride concentration and serum triglyceride reduction effect in serum by BBE, MPB and MRP diet. Lipid improvement effect was confirmed by the components of the mushroom mycelium extract. However, these lipid-improving effects were not observed by RBE administration.

BUN (bood urea nitrogen) is metabolized by the protein ingested in the diet, most of which is excreted by the kidneys as urea. However, if the abnormal function of the kidney is not well excreted and remains in the blood and the concentration of urea nitrogen (BUN) is increased. In the present invention, the BUN concentration was significantly increased in the diabetic group compared to the normal group, and the BUN level was significantly decreased by the administration of BBE, RBE, MPR and MPB. Creatinine is also a by-product of protein used as energy in the body, such as BUN, and is released into the blood and excreted from the kidneys into the urinary tract, which is an indirect biochemical indicator of the functional status of the kidneys. In the diabetic group, the creatinine concentration tended to decrease in the extract group, especially in the MPB group. Serum GOT and GPT levels were significantly increased in the diabetic group compared to the normal group, but were significantly reduced by the administration of RBE, MPR and MPB.

Sochor et al. Reported that hepatic damage and enlarged kidneys were observed in STZ-induced diabetic rats. In the present invention, the weight of liver and kidney was significantly higher in the diabetic group than in the normal group, and the dietary effect of reducing the weight of the liver by administration of BBE, RBE, MPR and MPB was observed.

In general, the induction of diabetes is known to not only destroy the islet cells of the pancreas but also weaken immunity. In the present invention, it was confirmed that the weight of the pancreas and spleen of the experimental animals due to diabetes-causing was reduced to a remarkable level. However, the administration of BBE, RBE, MPR, and MPB effectively alleviated the atrophy of the pancreas and spleen compared to the diabetic control group, especially the BBE and MPB administration group containing buckwheat bran.

In conclusion, dietary supplementation of 5% buckwheat bran extract (BBE), rice bran extract (RBE), rice bran-containing medium status mycelia extract (MPR) and buckwheat bran-containing medium status mycelia extract (MPB) It effectively alleviated the severe abnormalities of carbohydrate metabolism in induced rats and showed protective effect on liver, pancreas and spleen. In particular, the administration of BBE and MPB containing buckwheat components decreased the lipid content in serum and liver, which prevented the increase of lipid concentration in the body due to diabetes and increased the utilization of glucose. Samples used in the invention of Example 1 of the present invention is not in the state of purification has been completed and the research on the best purification method that can increase the purity of the indicator material is currently in progress. Nevertheless, the samples used in the present study lowered the blood sugar and lipid content of the diabetic rats to a significant level and showed a protective effect on organs such as the pancreas and spleen.

In Example 2, protein extraction, green tea extraction, pazeolamin-tannin complex (PTC) prototyping, blood glucose control in vivo experiments on PTC complex, and eight samples including five kidney beans, black beans, peas, and buckwheat In vitro experiments on α-amylase activity inhibition on the prototype, α-amylase-tannin complex experiments, and hydrolysis of digestive enzymes on PTC prototypes were carried out.

1. Development of new α-amylase inhibitors from agricultural samples

end. Comparison of Extraction Yields of Sample Protein Precipitated with Ethanol

In order to select the raw material of kidney bean, pazeolamine, which is a protein, strongly inhibited the enzyme activity against α-amylase, and compared the extraction yield by purchasing 8 kinds in the market as shown in Table 18 in order to select a material having good protein yield.

Table 18. Pazeolamine Powder Yield from Raw Material Extraction

                                                            (unit : %)

      Raw material name             Yield of Pazeolamine by Sedimentation Sample (%)   65% ethanol   90% ethanol   (Total yield)   Remarks ① Domestic purple bean beans      5,72      2,00      7,72 ② Chinese wild bean beans      5,75      1,15      6,90 ③ White Kidney Beans      6,20      1,62      7,82 ④ Red beans      3,40      2,39      5,79 ⑤ Black Bean (Sur Tai)      4,20      1,77      5,97 ⑥ Mung bean      3,70      3,10      6,80 ⑦ Peas      4,30      6,05     10,35 ⑧ Buckwheat bran      3,81      1,75      5,56 Average      4.64      2.48      7.11

As shown in Table 18, the protein extraction yield was 65% ethanol precipitate was higher than 90% ethanol. Among them, peas were the highest with a total yield of 10.35% and yielded 6.05% in 90% ethanol compared to the yield obtained in 65% ethanol (4.30%).

Next, white kidney beans were 6.20% in 65% ethanol, 1.62% in 90% ethanol and 7.82% in total, which showed higher yields than other raw materials.

The difference in protein yield according to the ethanol concentration is believed to be due to the protein molecular weight. In general, when the ethanol concentration is low, the protein having a high molecular weight is estimated to precipitate. Therefore, pea extract protein seems to contain many small molecular weight.

The yield averages of the eight samples obtained by the ethanol precipitation method were 65% ethanol precipitate yield 4.64%, which was about 2 times higher than the 90% ethanol precipitate yield 2.48%. The total yield of ethanol precipitates was 7% on average.

The purification method for the protein was determined to have a significant effect on the inhibition of enzyme activity, so the yield of pazeolamin protein was measured for the fractionation and purification of these proteins at pH 6.3 and pH 4.5.

I. Yield Comparison of Sample Proteins Precipitated at pH 4.3 and pH 6.4

After extracting the protein from the eight samples centrifuged and the precipitate produced when adjusted to pH 6.4 and pH 4.3 by centrifugation again the yield of the pazeolamine powder is shown in Table 19.

As shown in Table 19, the yield when the sample extract was precipitated to pH 4,3 was 1.08% on average, which was lower than the average yield of protein precipitated at pH 6.4. In the precipitate obtained at pH 6.4, red beans were the highest (4.54%), followed by 4% yield in pea and white kidney beans.

The yield average value is estimated to be about 6 times higher than that obtained at pH 4.3, 1.08%, and it is estimated that there are many globulins with a molecular weight of 60,000. The yield was 4.31% on average, about 3% less than the ethanol precipitation method.

Table 19. Precipitation protein yield by pH

                                                               (unit %)

      Sample Name           Protein yield by pH Remarks   pH 4.3 / yield  pH 6.4 / yield Yield total   Domestic purple beans        1.28 3.12 4.40   Chinese Communal Kidney Beans 1.58 2.80 4.38   Chinese White Bean 0.61 4.01 4.62         red bean 0.26 4.54 4.80        green gram 0.45 1.85 2.30       Black Beans 1.49 2.57 4.06       pea 0.35 4.20 4.55        buckwheat 2.65 2.69 5.34       Average 1.08 3.22 4.31

All. Pazeolamine-tannin complex (PTC) prototype processing

1) Green Tea Extract Preparation and Tannin Content Analysis

10 times of water was added to the dried green tea leaf powder, and the extraction temperature was extracted / separated by 80-90 ° C, 90-95 ° C and 95-100 ° C, and the total polyphenols in the extract prepared in the Brix 4 range were concentrated. Quantified values are shown in Table 20. At this time, the standard curve created for analyzing the tannin content is shown in FIG.

2) Extracts from green tea samples were measured for 10 minutes at 80-90 ℃, 90-95 ℃ and 95-100 ℃ temperature ranges.

As shown in Table 20, when the green tea was extracted by temperature, the tannin content was 13.16% at 95-100 ° C, 12.69% at 90-95 ° C, and 11.72% at 80-85 ° C. It can be seen that.

Table 20. Tannin Content in Green Tea Extracts Extracted by Temperature

    Extraction temperature (℃)                     Tannin yield (%)        Mg / ml        g / 100 ml       % / 100g       95-100        0.658        0.0658        13.16       90-95        0.635        0.0635        12.69       80-90        0.586        0.0586        11.72

la. Processing yield of pazeolamin-tannin complex (PTC) powder

1) Comparison of Yield of Pazeolamin-Tanin Complex Powder by Tannin Concentration

Sediment yield of pazeolamin-tannin complex (PTC) produced when the concentration of tannin extract was added differently to 10 ml of pazeolamin extract is shown in FIG. 12.

As shown in FIG. 12, the yield of precipitate of the pazeolamin-tannin complex was found to increase with the concentration of tannin. In other words, when the protein content of pazeolamin extract was 68.5%, the formation rate of PTC precipitate gradually increased from 10: 1 to 10: 1 with the polyphenol content of 13.2% of tannin extract. In general, the same trend was observed. Therefore, it can be seen that the binding ratio of pazeolamin-tannin is suitably in the range of 60-70% of the concentration of tannin in the protein solution. However, if the protein concentration is high, the tannin concentration is expected to increase.

For comparative experiments, the PTC precipitate produced when calf serum albumin (BSA) 10% solution and tannic acid 13% solution were mixed in the same ratio, the yield tended to increase according to the amount of tannic acid addition, but the yield of sediment extract The yields were lower than those obtained from the green tea extract.

2) Processing yield of pazeolamine-tannin powder precipitated by pH range

When the pH of the tannin extract is added to the pazeolamine extract, the yield of the PTC precipitate is as follows.

As shown in FIG. 13, the yield of PTC precipitate by pH was 8.3% at pH 7.0, and was 7.4% at pH 6 and 7.1% at pH 8, so that complex formation by interaction between pazeolamine and tannin was generally neutral. It is estimated to work well in the vicinity of the solution, which is 1.3% at pH 2.0.

I. In vitro α-amylase and tannin binding capacity assay

In Example 1, the binding product of BSA and tannic acid was mentioned in the production of pazeolamin-tannin complex. As one of the glycemic control methods, to investigate whether polyphenol component is defective with enzyme protein in vivo, the experiment of α-amylase and amyloglucosidase, green tea extract, and tannic acid was performed in vitro. Ann E. Hagerman et al. Reported that β-1,2,3,4,5, -pentagalloyl-D-glucose, in which five molecules of gallic acid bind to one molecule of glucose, binds to proteins such as BSA in vitro. Based on the experimental results of forming complex precipitates, it is assumed that there is another pathway for reducing glucose absorption by inhibiting starch digestion by generating digestive enzymes and tannins in vivo. Therefore, the pazeolamin-tannin complex may have a possibility of breaking hydrogen bonds in the small intestine, and then the free polyphenol component combines with the carbohydrate digestive enzyme to form a new tannin-enzyme complex. It can be assumed that the α-amylase-tannin complex regulates blood glucose indirectly by interrupting the digestive action of starch.

Therefore, the present invention was tested whether the interaction between the α-amylase and tannic acid was obtained as follows. When a 10% tannic acid solution and 5% α-amylase solution were prepared and reacted at the ratio shown in Table 21, the yield of precipitates of 223 mg (89.2%) at A: T = 10: 5 was easily obtained. It can be seen that they combine.

At a ratio of 10: 3, a yield of 179 mg of sediment was obtained, resulting in 119.3%, which is believed to form a complex of 100% BSA and tannic acid. When 5% tannic acid was reacted, the yield of the complex was lower as the concentration of tannin decreased from 152 mg at 10: 3 to 168 mg at 10: 5. By combining the amylo-1,6-glucosidase 5% solution with the 5% tannic acid solution, 252 mg at 10: 1 ratio, 380 mg at 10: 3 ratio, and 477 mg at 10: 5 were obtained. -About 2 times higher results than amylase complex. This result is probably due to the appropriate structural conditions or high molecular weight of the protein structure of amyloglucosidase to bind to tannin.

Table 21. Sediment yield by interaction of carbohydrate digestive enzymes with tannins

Enzyme precipitants    Alpha-amylase (5%) Amylo-1,6-glucosidase (5%)  E-T complex ratio 10: 1 10: 3 10: 5 10: 1 10: 3 10: 5  10% tannic acid, PTC (mg)     3    179   223     -     -     -  5% tannic acid, PTC (mg)     3    152   168    252   380    477

Based on the above results, it is estimated that tannins in the body can be easily combined with digestive enzymes such as α-amylase.

All. Carbohydrate Assay in Pazeolamin-Tanin Complex

1) Analysis of Total Sugar Content in PTC Powder by Phenol-Sulfate Method

To determine how much sugar is contained in the PTC protein powder, the results of analysis by the phenol sulfate method are shown in Table 22 below. In the development of this prototype, it is not preferable to contain a lot of carbohydrates because it is intended for blood sugar control.

As shown in Table 22, the sugar content of the protein precipitate obtained by the addition of 65% ethanol and 90% ethanol from the sample extract was 1.59% and 3.15% on average, containing a very small amount, and in general, 65% in the 90% ethanol extract powder. It was analyzed to contain about three times more than the ethanol extract powder. The sugar content in the extract powder is an important data because it has an absolute effect on the measurement of α-amylase activity. Fortunately, the sugar content in all the samples extracted is less than 5%, and the sugars measured here are considered to be soluble fibers precipitated simultaneously with the protein, so it is unlikely to affect the glycemic control function when the product is developed. .

Table 22. Comparison of Sugar Content Analysis in Ethanol Precipitate Powders to Sample Extracts

     Raw material                  Sugar content (%) Simple extract
yield(%) 65% ethanol
precipitate 90% ethanol
precipitate  ① Domestic kidney beans         2.76         4.10         30.5  ② Chinese kidney beans         2.69         3.78         28.5  ③ Chinese White Beans         3.58         3.23         30.0  ④ Red beans         0.80         3.24         22.5  ⑤ Black beans         1.36         3.20         26.0  ⑥ Mung bean         0.50         2.85         14.0  ⑦ Peas         0.69         4.31         26.7  ⑧ Buckwheat bran         0.31         0.52         10.5        Average         1.59         3.15         24.2

2) Assay of monosaccharide content mixed in pazeolamin-tannin powder

Table 23 shows the results of analyzing the sugar content in the Pazeolamin powder extracted from eight samples by gas chromatography.

As shown in Table 23, the constituent sugars in the pazeolamin powder extracted from the kidney bean sample were analyzed to contain a trace amount even if they do not or generally contain monosaccharides. Therefore, the sugar content in the powder analyzed by the phenol-sulfate method is considered to contain a small amount of water-soluble fiber, such as non-starch polysaccharides.

Table 23. Constituent Monosaccharide Content Analyzed by Gas Chromatography

 sample Arabinos Xylose Rhamnose Mannose Galactose Glucose  SUM  Domestic kidney beans    1.77    0.00     1.14    0.00     0.00    0.00  2.91  Chinese Kidney Beans    0.88    0.00     0.85    0.00     0.00    0.00  1.73  White Kidney Beans    1.29    0.00     0.64    0.00     1.52    0.00  3.45  red bean    0.00    0.00     0.00    0.00     0.00    0.00  0.00  Black Beans    0.00    0.00     0.00    0.00     0.00    0.00  0.00  green gram    0.21    0.00     0.00    0.00     0.00    0.00  0.21  pea    0.24    0.00     0.00    0.00     1.42    0.92  2.58  Buckwheat bran    0.87    0.40     0.00    1.55     1.83    0.00  4.65  Average    0.65    0.05     0.33    0.19     0.59    0.11  1.94

la. Assays for Inhibiting α-amylase Activity of Sample Proteins

1) Comparison of protein content in sample extract powder

In Figure 14, the protein content in the eight samples (literature value; Korea Food Analysis Table, Ministry of Health and Welfare Food and Drug Safety Division. Food 80 Ingredients Table, Japan Women's Nutrition and Multicultural Publishing Department) and the protein in the powder obtained by precipitation with ethanol in the extract extracted from the sample The content was compared and displayed. Buckwheat contained 13.8% of the raw materials. The protein in the powder samples obtained from the sample was 81.33% of buckwheat and 52.47% of peas. Domestic purple kidney bean contains 10% of protein in the original sample, but 63.3% of the protein from the extracted sample contains 20% protein and 60.92% of the protein from the extracted sample is concentrated. It can be seen.

Although the values in the literature differ slightly depending on the water content, the protein content analysis in the prototype in this experiment was carried out to investigate the effectiveness of pazeolamin on the inhibition of α-amylase activity.

2) Comparison of α-amylase Activity Inhibition of Pazeolamin

Pajeolamine inhibited the enzyme activity when 5 mg of each of the prototypes of the protein powder prepared by precipitating 65% ethanol in each sample extract was added to the α-amylase reaction solution based on soluble starch and reacted at 37 ° C. for 10 minutes. The value of comparative analysis of the ability to do is shown in FIG. 15.

As shown in FIG. 15, the highest inhibitory activity of α-amylase among the eight samples was measured by 15.7% of domestic purple kidney beans and 15.3% of black soybean extract, followed by 13.9% of Chinese purple kidney beans and 13.3% of mung bean extract. Was measured. The other four sample extracts had an activity inhibition rate of less than 10%. From these results, four samples, including domestic kidney beans, black beans, Chinese purple beans, and mung beans, are considered to be of value in developing enzyme inhibitory samples. These data will be the basic data to investigate the enzyme activity inhibition rate of sediment powder extracted from domestic kidney beans for domestic agricultural resource utilization. It is believed that it may be useful to determine whether to indirectly inhibit the activity of α-amylase in the small intestine by forming.

3) Inhibition of enzyme activity by purification process of PTC powder

As shown in Fig. 15, the supernatant obtained by dissolving the processed protozoa was selected as the best sample in the enzyme activity search for 8 samples, dissolved in a buffer, reprecipitated, and re-precipitated again in the buffer. Purification process of the dialysis powder and the like to increase the specific activity and search for industrial values are shown in Table 24 below.

As shown in Table 24, the protein content of the PTC prototype processed from domestic purple kidney bean extract was 68.63%, and the initial activity inhibition rate for α-amylase was 6.1%, whereas the precipitate was dissolved in the buffer again and centrifuged after 65%. The protein in the powder precipitated with ethanol was 66.2%, and the enzyme inhibition rate was 12.9%, which was about doubled. The supernatant dissolved in the buffer was dialyzed for 48 hours, and the protein content of the lyophilized powder was 51.01%, and the α-amylase inhibition rate was 34.9%, which was about three times higher than the previous step. It was found to increase.

Table 24. Enzyme activity search by purification process of PTC powder

    Refining step by step   Protein content (%)   α-amylase enzyme activity inhibition rate (%)   PTC prototype        68.63               6.1   Primary purification of the solution        66.17              12.9   Dialysis of solution        51.01              34.9

However, from the industrial point of view, it is economical to treat a large amount of dialysis, so it would be better to develop an inexpensive refining process such as reprecipitation of sediment or ion exchange column.

4) Assay of activity inhibition rate of α-amylase by concentration of PTC powder

10 g of each of the 8 enzymes, the highest in the enzyme inhibition rate of domestic and Chinese purple beans, was treated with hexane to remove fat, followed by hydrolysis of polysaccharides during extraction with 1 liter of phosphate buffer and α-amylase. After the precipitation of pazeolamine with tannin, the enzyme activity inhibition rate was measured for each concentration of the PTC prototype obtained by lyophilizing the precipitate as shown in FIG. 16.

As shown in FIG. 16, the powders of PTC powders prepared from two kidney bean samples were dissolved in buffer so as to be a 1% protein solution, and treated at concentrations of 0.5 mg to 50 mg for α-amylase activity inhibition rate. Plot to compare trends. The enzyme activity inhibition rate according to the concentration of domestic purple kidney bean PTC powder and Chinese kidney bean PTC powder showed the same tendency. At 10 mg, which was doubled at the initial concentration of 0.5 mg, there was a sharp increase, indicating an inhibition rate of about 65%.

hemp. Inhibitory Effects of Tannins on Trypsin and Pepsin Activity

In vitro experiments were conducted to determine how tannin in the complex inhibits enzyme activity when the pazeolamin-tannin complex (PTC) prototype is hydrolyzed by trypsin and pepsin in the body. Same as FIG. 17.

Figure 17 is a three-hour assay by adding trypsin and pepsin to the PTC powder obtained by precipitating pazeolamin and tannin by a certain ratio, and centrifuged to quantify the protein content remaining in the precipitate to remain hydrolyzed protein and This is a plot of difference in quantity.

Inhibition of protein hydrolysis in the PTC powder by trypsin was 51% in 10: 3 and 67% in 10: 7.5 compared to 67% in 10: 1, respectively. The degree of hydrolysis is in the range of 50%, and the hydrolysis of the enzyme to the protein of the PTC powder is estimated to be about half protected by tannin. Such a problem could be a good basis to refute what a medical practitioner claims to be without glycemic control by hydrolysis of stomach acid or enzymes.

Therefore, the PTC hydrolysis tendency of trypsin is generally better to set the degree of protein degradation in the 50% range in PTC powder mixed with more than 50% tannin. The tendency of pepsin to hydrolyze PTC was slightly lower than that of trypsin, and it was found to be hydrolyzed in the range of 48% regardless of the proportion of tannin, especially 50% even at low concentration of 10: 1. It is interpreted that the structural position of the protein in the PTC powder is confined to the three-dimensional binding position with pepsin due to its disadvantage compared to trypsin.

bar. Binding Properties and In vivo Biological Activities of PTC Powders

1) Characteristics of Pazeolamin-Tannin Interaction

Edwin Haslam et al. Have an amino acid proline containing a hydroxyl group bound to B-rings such as epicatechin-3-O-gallate and epigallocatechin-3-O-gallate. It is explained by inducing precipitation by hydrogen bonding with the C═O group of the residue. Therefore, tannin has been studied as a very important task in the food industry because of its ability to bind any protein.

Edwin Haslam et al. Have a pore of 10-15 nm in diameter in spherical micellars with α and β-substructures of protein-tannin complexes, which are soluble in protein, and intercalation of polyphenol molecules within these pores. To induce precipitation.

On the other hand, E. Haslam et al. Proposed that polyphenols hydrogen bond to polar groups or ketoimide groups exposed to the surface of proteins to induce precipitation and other hydrophobic interactions between protein molecules and polyphenol molecules. It has been announced that it generates. Based on the mechanism for the PTC sediment as described above, the beer industry, the beverage industry, and other food industries are used for the purpose of processing the product in a clean state by removing the sediment turbidity in the product.

Starting with the invention that tannins bind to proteins in saliva to form a key component of the astringent taste, there have been many inventions related to tannin-protein interaction for the purpose of protein removal in the beer industry. In addition, it inhibits the digestion of proteins and analyzes the content of tannins and binding strength with proteins, and is used as a factor to find out the differences between plant species.

In this experiment, contrary to this application method, PTC is processed to process functional foods with high physiological activity in vivo. Therefore, the production conditions of PTC products are established using the above-mentioned protein-tannin complex formation theory. In this case, the safety of digestive enzymes in the body and the hydrogen bonds of PTC products are broken down by body fluids, which indirectly inhibits starch hydrolysis by forming new enzyme-tannin complexes with digestive enzyme proteins such as α-amylase in the intestine. A mechanism can be assumed.

2) Biological Activity of Pazeolamin-Tanin Complex (PTC) in Vivo

Pazeolamine in kidney beans is one of β-globulin and has a molecular weight of about 60,000. It has been shown to inhibit rapid starch hydrolysis of α-amylase in animals and to control the rapid increase in blood glucose levels. It has been found to be effective in blood purification and treatment of diabetes mellitus due to the absorption of cholesterol.

In this experiment, the tannin component was extracted from green tea, and the complex with pazeolamin, the main protein in kidney bean extract, was precipitated. As a result, fasting blood glucose was lowered to 85 mg / dL compared to the control value of 100 mg / dL after 6 weeks of diet, and the blood glucose level was lowered to a level comparable to that of the control. In addition, the effects on other fatty livers were reduced, and it showed that the development value as a functional food for glycemic control of diabetic disease was shown by showing good results close to the normal level in the control of fat tissue and serum cholesterol level. For detailed experimental results, refer to the contents of the animal test results.

four. Assay of pazeolamin-tannin complex prototype

As described above, the kidney bean extract and the green tea extract were reacted with 4: 3 to form a precipitate, and the spectroscopic analysis values for the 2.9 kg PTC powder obtained therefrom are shown in Table 25 below.

1) In the recovery rate, when the target value was set to 10, the yield of pajeolamine-tannin complex (PTC) prototype was 7.7%, which was 77%.

Table 25. Spec Analysis Table for PTC Prototypes

   Evaluation item    unit In all items
Share Development target
(%) Found
(%) Target value / actual value
ratio(%) Assessment Methods  1. Recovery rate    %        15      10     7.7       77 Gravimetric method  2. Protein    %        30      50    68.6      137 AOAC  3. Tannin    %        15      10    13.0      130 AOAC  4. Solubility    %        10      40    30.0       75  Food Code  5. Particle size   Μm         5     100   125.0      125 Standard  6. Coliform Count   individual        10       0     0.0      100  Food Code  7. Ash    %         7       7     6.1       87 AOAC  8. Chromaticity   HV / C         8    2.0 / 3.0   2.4 / 4.8       50 Colorimeter  9. Total       100

    2) In the protein content, the protein content in the PTC prototype was 68.6% compared to the target value of 50%, which was able to exceed the target value by obtaining 137% of the protein.

     3) The tannin content of the green tea raw material was 10% of distilled water and extracted for 10 minutes at 95-100 ° C.

     4) Solubility Solubility was only 30% when PTC prototype was dissolved in buffer or water, but this is due to the structural aspect of PTC powder. It is interpreted that it is because it is combined. It was difficult to dissolve, but the degree of dispersion in water was large.

5) Particle size was classified in eight stages from 600 µm to 75 µm in terms of the weight of the powder, and the result was obtained as shown in FIG.

As shown in FIG. 18, the most common was 600% mesh, 0%, 0.5% at 355㎛, 19.6% at 212㎛, and 41.0% at 125㎛.

      6) The number of E. coli is indicated as 0 individuals in the Korean Food Code. The results of this prototype sterilization by UV light are 0 and E. coli is not checked. 7) The ash content in the PTC powder was set at 7.0%, but it was 6.1% in the prototype, which contained about 87% in the target value.

delete

       8) When the chromaticity is expressed as the ratio of the color hue, the contrast (value) and the chroma (chroma), the higher the chroma, the smaller the contrast. This PTC prototype is dark brown and has a high color, which is dark. This cause is interpreted to be due to the mixture of tannins such as green tea pigments by extracting the green tea at 95-100 ℃ for 10 minutes.

2. In vivo Pagopyritol and PTC Functional Assay

end. Breeding of experimental animals and preparation of experimental diet

Four-week-old male Zucker lean rats and fatty rats were purchased from Harlan Sprague Dawley (Indianapolis, USA) through Polars International, with temperature (20-24oC) and photoperiod (dark time, 20:00 -08: 00). After adjusting for 3 weeks in the control room was controlled by the weight of the blood sugar and control group was classified into 8 control groups and 7 dietary groups, respectively. The experimental group was divided into 1 normal weight control group, 1 obese weight control group, and 4 obese body weight group. Obese body weight group was divided into Fa-0.2 powder administration group, Fa-1.0 powder administration group, Fa-0.5 drink administration group and Fa-0.5 GP drink administration group.

Experimental animals were fed dietary supplement (Harlan Teklad, USA) for 3 weeks after acquisition to check whether obesity and blood sugar increase were induced and changed to experimental diet for 7 weeks after free feeding of water or drink and diet.

In the Examples 1 and 2, the D-chiro-inositol fraction of the pagopyritol fraction used in the animal diet was about 2.07 times higher in D-chiro-inositol content compared to the sample of Example 1, and the ratio with glucose Samples were used, which were reduced to 1/5 to significantly improve the purity of the indicator.

Table 26. D-Kiro-inositol content in pagopyitol fraction

                                                                (g / 100g)

DCI Galactose Glucose DCI / Gal DCI / Glu First year  7.37  8.18 15.93 0.90 0.46 Second year 15.24 14.52  6.63 1.05 2.30

The experimental diet was prepared according to the AIN-93G diet composition and the feed composition is shown in Table 27. Fa-0.2 and Fa1.0 groups were added to the diet diet pagopyritol, Fa-0.5D and Fa-0.5PD group was fed by dissolving pagopyritol in drinking water to the level of 0.5%. Body weight change, dietary intake and water intake during the experimental period were recorded at regular times every day.

Table 27. Composition of Experimental Diet

ingredient (%) LC / FC Fa-0.2 Fa-1.0 Fa-0.5D Fa-0.5PD Casein   20.0   20.0   20.0   20.0   17.0 Corn starch   39.7486   39.5486   38.7486   39.7486   39.7486 Dexified Corn Starch   13.2   13.2   13.2   13.2   13.2 Sucrose   10.0   10.0   10.0   10.0   10.0 Soybean oil    7.0    7.0    7.0    7.0    7.0 Alphacell, rest of non-nutrients    5.0    5.0    5.0    5.0    5.0 Mineral Mixture (AIN-93G-MX)    3.5    3.5    3.5    3.5    3.5 Vitamin Mixture (AIN-93-VX)    1.0    1.0    1.0    1.0    1.0 L-cystine    0.3    0.3    0.3    0.3    0.3 Colin Bittarate    0.25    0.25    0.25    0.25    0.25 t-butylhydroquinone    0.0014    0.0014    0.0014    0.0014    0.0014 Pagopyitol      -    0.2    1.0      -      - * PTC      -      -      -      -    3.0 Total (%)  100 100  100 100 100

* PTC: Pazeolamin-Tanin Complex

LC: slim control AIN 93 dietary feed;
FC: obese control AIN 93 dietary benefit;

F-Fa0.2: AIN 93 dietary supplement with 0.2% pagopyitol in obese group
F-Fa1.0: AIN 93 dietary supplement with 1% pagopyitol in obese group
F-Fa0.5D: AIN 93 Dietary Benefit with Obesity Drinks (0.5% Pagopyitol)
F-Fa0.5PD: AIN 93 Dietary Benefit with Obese Group 3% PTC and Drink (0.5% Pagopyitol)
A value represents the mean ± SE (n = 8 or 7).

I. Blood glucose change measurement

Fasting blood sugar for 7 weeks was fasted for 10 hours at base line, 3 weeks and 6 weeks, and then centrifuged by collecting whole blood from tail vein. It measured using. Feeding blood glucose of experimental animals was measured 1 hour after meal completion at 1, 3, and 6 weeks of dietary supplementation. Oral Glucose Tolerance Test (OGTT) was performed by fasting the test animals for 12 hours and administering glucose at a constant concentration through oral and measuring blood glucose at 0, 30, 60, and 120 minutes, respectively.

All. Experimental Analysis

1) Organ and Serum Separation

The animals were fasted for 5 hours, sacrificed with the head, and blood was collected. After collection, the liver, kidney, pancreas, spleen, abdominal fat tissues and epididymal fat tissues are rapidly isolated and 0.9% cold saline. Washed to remove blood and recorded each weight. Serum was obtained by collecting whole blood for 1 hour at room temperature after centrifugation and centrifuging at 3,000 rpm for 20 minutes and stored at -30 ° C until analysis.

2) Biochemical Analysis of Serum

Total cholesterol (Total Cholesterol), Triglyceride, Blood Urea Nitrogen (BUN) and GOT, GPT concentrations in serum were measured using the respective diagnostic kits (Asan Pharmaceutical, Seoul).

3) Analysis of Experiment Results

Statistical analysis was performed by one-way analysis to verify the difference in dietary effects according to the amount of each sample added and tested for significant differences at the p <0.05 level for the mean values according to the LSD test.

la. Experiment result

1) weight change, dietary intake and beverage intake

Table 28 shows the body weight change, dietary intake and beverage intake of experimental animals during the experiment. There was a significant difference in the initial weights of the normal weight group and the obese group, and there was no difference between the diet groups in the obese group. The weight gain was significantly higher in the obese group than in the normal weight group during the 7-week experimental period. In the obese group, the pagopyitol-administered group was higher than the control group, and the pagopytolitol and PTC combination group were the lowest. The weight gain of the pagopyitol drink and PTC combination group was almost the same as the normal weight group. The dietary intake was significantly higher in the obese group than in the normal weight group, but the F-Fa0.5PD group was similar to the normal weight group. The intake of beverage was the highest in the obese group and the lower in the F-Fa1.0 and F-Fa0.5PD groups.

Table 28. Dietary Intake and Body Weight of Normal and Diabetic Rats for 7 Weeks.

Group Initial weight weight (g) Weight gain
(g) Dietary intake
(g / day) Drink intake
(Ml / day) LC (n = 8) 282 ± 3.1 122 ± 9.3 22.5 ± 2.06 26.0 ± 1.45 FC (n = 8) 374 ± 11.3 *** 202 ± 13.8 *** b 31.6 ± 0.41 *** b 39.1 ± 4.78 * b F-Fa0.2 (n = 7) 371 ± 9.8 239 ± 16.1bc 32.1 ± 0.91b 31.2 ± 1.46ab F-Fa1.0 (n = 7) 370 ± 16.4 250 ± 10.1c 31.0 ± 0.74 b 28.3 ± 1.29a F-Fa0.5D (n = 7) 372 ± 16.9 235 ± 22.8bc 32.5 ± 0.84b 35.0 ± 2.00ab F-Fa0.5PD (n = 7) 373 ± 11.3 130 ± 13.1a 25.9 ± 0.77a 27.0 ± 0.89a

a, b Values with different superscript letters are significantly different within diabetic group (p <0.05)

* P <0.05, ** P <0.01. *** P <0.001 vs. lean control group.

2) Fasting and Feeding Glucose

Table 29 shows the serum glucose concentrations in the fasted state of the test animals. Serum fasting glucose levels were significantly higher in the obese group than in the normal weight group, and there was no difference among the obese groups. Dietary supplementation 3-week fasting glucose concentration was decreased by pagopyritol administration, especially in the 1.0% group and the PTC group. After 6 weeks of experiment, fasting glucose concentration was significantly decreased by pagopyritol and PTC administration, and the 1.0% group and the PTC group were lower than the normal group.

Table 29. Fasting Glucose Levels

group Base line 3 weeks later 6 weeks later LC (n = 8) 100 ± 4.2 110 ± 2.2 100 ± 2.3 FC (n = 8) 121 ± 6.7 * 136 ± 14.0b 144 ± 17.8 * b F-Fa0.2 (n = 7) 116 ± 3.2 119 ± 8.6ab  99 ± 7.1a F-Fa1.0 (n = 7) 113 ± 4.6 108 ± 7.2a  87 ± 1.9a F-Fa0.5D (n = 7) 116 ± 4.3 117 ± 8.6ab 102 ± 9.0a F-Fa0.5PD (n = 7) 121 ± 5.4  97 ± 3.5a  83 ± 3.5a

 a, b Values with different superscript letters are significantly different within diabetic group (p <0.05)

 * P <0.05, vs. lean control group.

After one week of feeding, the fed blood glucose level was significantly higher in the obese group than in the normal weight group. In particular, pagopyritol and PTC coadministration group showed lower feeding blood glucose than normal weight group, so pagopyritol and PTC were effective in synergistic function. After 3 weeks of feeding, the obesity control group's feeding blood sugar level was about 3.5 times that of the normal weight group, which was significantly higher than the feeding blood sugar level (about 1.4 times) after 1 week. Significant increases in dietary blood glucose with dietary periods may be due to, for example, increased obesity and feeding glucose disorders in Zucker fatty rats. The trend of feeding glucose in the obese control group was maintained after 6 weeks of diet. Feeding blood sugar elevation in the obese group was significantly reduced by dietary supplementation of pagopyritol. Feeding hypoglycemic effect of pagopyritol was effectively exhibited even at 0.2% level, and when combined with PTC, hyperglycemia was improved to the level of normal weight group.

Table 30. Feed Glucose Levels

group 1 week later 3 weeks later 6 weeks later LC (n = 8)  79 ± 1.6  85 ± 3.7  84 ± 2.3 FC (n = 8) 109 ± 3.7 *** d 294 ± 26.9 *** c 265 ± 21.9 *** c F-Fa0.2 (n = 7)  90 ± 5.6b 248 ± 22.7bc 172 ± 17.2b F-Fa1.0 (n = 7)  93 ± 3.2bc 210 ± 16.1b 155 ± 16.1b F-Fa0.5D (n = 7) 102 ± 3.4bc 231 ± 16.7b 188 ± 20.8b F-Fa0.5PD (n = 7)  65 ± 2.8a 131 ± 13.7a  87 ± 8.7a

a, b, c, d Values with different superscript letters are significantly different within diabetic group (p <0.05)

 *** P <0.001, vs. lean control group.

3) Oral glucose tolerance test (OGTT)

Oral glucose tolerance test is a test to check the degree of glucose clearance in the blood at 30, 60 and 120 minutes by oral glucose loading after 12 hours of fasting, and confirmed the difference between the diet group in the normal weight group, the obese group and the obese group. In order to carry out. At the beginning of the diet, the normal group showed the highest blood glucose level at 30 minutes of glucose load and almost normal blood glucose level after 60 minutes, whereas the obese control group did not recover to the basal state after 60 minutes, thus increasing glucose intolerance. The increase in glucose intolerance in the obese group was more severe in OGTT during the 3rd week of dietary experiments and showed hyperglycemia even after 60 minutes of glucose loading. Increasing glucose intolerance in the obese group was significantly improved by dietary supplementation of pagopyitol. The effect of pagopyritol on improving glucose intolerance was highest in the 1.0% group and the PTC group, and also in the 0.2% powder group and the 0.5% group. In the OGTT performed at 5 weeks, the glucose intolerance of the obese group was higher than the 3rd week, and it was confirmed that the effect of improving the glucose intolerance of pagopyitol was further enhanced (Table 31, Table 32, and Table 33).

Table 31. Oral glucose tolerance test results at baseline

Group 0 min. 30 min. 60 min. 120 min. LC (n = 8) 56 ± 3.2  95 ± 5.2  69 ± 3.1 53 ± 4.4 FC (n = 8) 88 ± 2.8 *** 158 ± 11.8 *** 124 ± 4.8 *** 93 ± 3.7 *** F-Fa0.2 (n = 7) 78 ± 3.3 155 ± 13.9 110 ± 7.5 80 ± 4.3 F-Fa1.0 (n = 7) 76 ± 5.2 155 ± 12.5 122 ± 19.9 82 ± 4.5 F-Fa0.5D (n = 7) 87 ± 4.4 163 ± 9.4 123 ± 15.1 87 ± 10.5 F-Fa0.5PD (n = 7) 82 ± 3.2 159 ± 18.2 118 ± 11.8 79 ± 6.4

 *** P <0.001, vs. lean control group.

Table 32. Results of oral glucose tolerance test at 3 weeks

Group 0 min. 30 min. 60 min. 120 min. LC (n = 8) 67 ± 1.9  96 ± 8.2  83 ± 3.3 63 ± 3.9 FC (n = 8) 65 ± 5.2 146 ± 14.4 ** 149 ± 19.6 ** b 78 ± 5.7 * F-Fa0.2 (n = 7) 57 ± 8.1 126 ± 19.7 112 ± 11.5a 70 ± 3.5 F-Fa1.0 (n = 7) 55 ± 1.3 138 ± 10.0  97 ± 6.2a 75 ± 7.8 F-Fa0.5D (n = 7) 57 ± 2.6 129 ± 18.5 104 ± 10.5a 74 ± 2.7 F-Fa0.5PD (n = 7) 63 ± 2.4 123 ± 13.3  97 ± 6.0a 80 ± 3.6

 a, b Values with different superscript letters are significantly different within diabetic group (p <0.05)

 ** P <0.01, vs. lean control group.

Table 33. Results of oral glucose tolerance test at 5 weeks

Group 0 min. 30 min. 60 min. 120 min. LC (n = 8) 61 ± 2.7 112 ± 7.3  95 ± 8.6  71 ± 4.6 FC (n = 8) 68 ± 4.6ab 176 ± 12.3 *** c 158 ± 9.6 *** c 108 ± 11.4 ** b F-Fa0.2 (n = 7) 66 ± 6.5ab 139 ± 5.3ab 109 ± 4.5ab  85 ± 5.4a F-Fa1.0 (n = 7) 60 ± 4.0a 146 ± 9.7abc 117 ± 5.4ab  81 ± 6.4a F-Fa0.5D (n = 7) 74 ± 4.3b 158 ± 11.0bc 131 ± 7.1b  97 ± 4.9ab F-Fa0.5PD (n = 7) 64 ± 1.4ab 122 ± 13.4a 105 ± 7.2a  82 ± 3.7a

a, b, c Values with different superscript letters are significantly different within diabetic group (p <0.05)

 ** P <0.01, *** P <0.001, vs. lean control group.

4) Fat and BUN Concentrations in Serum

The concentrations of total cholesterol (TC), HDL-cholesterol (HDL-C), triacylglycerol (TG) and HDL-C / TC ratio in serum are shown in Table 34.

The serum TC concentration was about 4 times higher in the obese control group than in the normal weight group, indicating that Zucker fatty rats exhibited severe hypercholesterolemia with impaired glycemic control. The concentration of serum HDL-C was proportional to the amount of TC and the obese control group was the highest, indicating that the ratio of HDL-C to TC was similar among the experimental groups. As described above, hypercholesterolemia in the obese group was markedly improved by dietary supplementation of pagopyitol, especially in the 0.5% beverage group. In addition, the effect of pagopyridol lowering blood cholesterol concentration was stronger when combined with PTC.

Serum TG levels tended to be high in the obese group, but no change was observed by pagopyitol administration. However, the combination of pagopyritol and PTC significantly increased neutral lipid concentrations, as opposed to significantly lowering the concentration of TC in serum.

The serum urea nitrogen (BUN) was significantly higher in the obese group than in the normal group, but was significantly improved by the feeding of pagopyitol.

Table 34. Serum Lipids and BUN Experiments

Group TC HDL-C HDL-C / TC TG BUN mg / ㎗ mg / ㎗ % mg / ㎗ mg / ㎗ LC (n = 8) 106 ± 5.2  46 ± 3.03 44 ± 1.7 208 ± 29.2 13.8 ± 0.32 FC (n = 8) 423 ± 27.8 *** c 189 ± 12.2 *** c 45 ± 3.1 251 ± 12.5a 19.0 ± 0.52 *** c F-Fa0.2 (n = 7) 366 ± 13.1b 168 ± 14.4bc 46 ± 2.5 252 ± 19.8a 17.5 ± 0.82bc F-Fa1.0 (n = 7) 324 ± 11.7b 143 ± 3.5b 44 ± 2.6 242 ± 16.3a 17.1 ± 0.73b F-Fa0.5D (n = 7) 318 ± 14.4b 150 ± 10.8b 47 ± 3.0 227 ± 33.5a 13.9 ± 0.68a F-Fa0.5PD (n = 7) 227 ± 11.3a 110 ± 7.1a 48 ± 1.7 378 ± 47.1b 18.6 ± 0.92bc

a, b, c Values with different superscript letters are significantly different within diabetic group (p <0.05)

 ** P <0.01, *** P <0.001, vs. lean control group.

5) GOT and GPT activity in serum

Serum GOT and GPT activity was higher in the obese group than in the normal group and increased more significantly as the experimental period progressed (FIG. 19). The increase in serum GOT and GPT activity in obese group was markedly reduced by pagopyritol administration and the effect of PTC combination was enhanced.

6) Weight comparison of organs in the body

Table 35 shows the weights and relative weights of the organs in the body of the experimental animals. The liver weight was significantly increased in the obese group compared to the normal weight group, and the liver tissue was found to be severe fatty liver condition. Hypertrophy with fatty liver in the obese group was improved by administration of pagopyritol, and more significant effects were obtained with the combination of PTC. Kidney weight of experimental animals showed a similar pattern to that of liver, and the dietary effect of pagopyitol was dependent on the dose.

The pancreas weight was markedly small in the obese group but increased in proportion to the concentration of pagopyritol. On the other hand, the weight of the spleen was not significantly different between the normal weight group and the obese group. In the combination of pagopyritol and PTC, the weights of the pancreas and spleen were similar to those of normal weight.

Table 35. Organ mass of slender Zucker rats and obese Zucker rats

group liver theocracy Pancreas spleen Mass g (g / 100g weight, relative mass) LC
(n = 8) 12.2 ± 0.40
(3.02 ± 0.054) 2.52 ± 0.06
(0.63 ± 0.015) 0.94 ± 0.07
(0.23 ± 0.015) 0.56 ± 0.02
(0.14 ± 0.008) FC
(n = 8) 34.0 ± 1.89 *** c
(5.94 ± 0.399 *** c) 3.35 ± 0.11 *** b
(0.58 ± 0.016c) 0.76 ± 0.04 * a
(0.13 ± 0.005 *** a) 0.62 ± 0.02
(0.11 ± 0.005 ** ab) F-Fa0.2
(n = 7) 30.5 ± 0.88bc
(5.03 ± 0.232b) 3.22 ± 0.13b
(0.53 ± 0.022abc) 0.79 ± 0.03ab
(0.13 ± 0.008a) 0.64 ± 0.02
(0.11 ± 0.003ab) F-Fa1.0
(n = 7) 28.0 ± 1.63b
(4.51 ± 0.214ab) 3.04 ± 0.14b
(0.49 ± 0.022ab) 0.86 ± 0.04ab
(0.14 ± 0.006a) 0.62 ± 0.02
(0.10 ± 0.002a) F-Fa0.5D
(n = 7) 29.0 ± 0.77b
(4.81 ± 0.226b) 3.31 ± 0.18b
(0.55 ± 0.034bc) 0.83 ± 0.03ab
(0.14 ± 0.004a) 0.57 ± 0.04
(0.09 ± 0.005b) F-Fa0.5PD
(n = 7) 19.0 ± 0.97a
(3.78 ± 0.193b) 2.40 ± 0.05a
(0.48 ± 0.007a) 0.90 ± 0.07b
(0.18 ± 0.011b) 0.59 ± 0.04
(0.12 ± 0.009b)

a, b, c Values with different superscript letters are significantly different within diabetic group (p <0.05)

* P <0.05, ** P <0.01, *** P <0.001, vs. normal control group.

7) Comparison of abdominal and epididymal fat mass

The weight and relative weight of the abdominal fat and epididymal fat of the test animals are shown in Table 36. The epididymal fat mass increased approximately 2.63 times in the obese group compared to the normal weight group. The epididymal fat mass was not decreased by pagopyritol alone but decreased significantly when co-administered with PTC. The abdominal fat mass of experimental animals was found to be similar to the pattern of epididymal fat mass. The administration of pagopyitol did not affect the total amount of abdominal and epididymal fats, which are the major storage tissues of body fat, but the relative weight to body weight was significantly decreased. The administration of pagopyitol increased weight in the obese group and did not increase fat mass, resulting in a decrease in body fat percentage.

Table 36. Fat mass in slender Zucker rats and obese Zucker rats

Group EFT AFT SUM (EFT + AFT) g (g / 100g BW) g (SUM / BW,%) LC (n = 8) 7.5 ± 0.48
(1.85 ± 0.098) 13.1 ± 1.21
(3.24 ± 0.291) 20.6 ± 1.48
(5.1 ± 0.34) FC (n = 8) 19.7 ± 1.11 *** ab
(3.41 ± 0.135 *** a) 50.5 ± 1.70 *** b
(8.76 ± 0.210 *** b) 70.2 ± 2.67 *** b
(12.2 ± 0.30 *** c) F-Fa0.2 (n = 7) 20.4 ± 1.28b
(3.33 ± 0.166a) 48.2 ± 1.75b
(7.91 ± 0.214a) 68.6 ± 2.52b
(11.2 ± 0.25ab) F-Fa1.0 (n = 7) 19.0 ± 1.08ab
(3.06 ± 0.105a) 47.7 ± 1.69ab
(7.71 ± 0.162a) 66.7 ± 2.56ab
(10.8 ± 0.19a) F-Fa0.5D (n = 7) 18.3 ± 1.35ab
(3.02 ± 0.179a) 47.8 ± 1.31b
(7.88 ± 0.105a) 66.1 ± 2.58ab
(10.9 ± 0.27a) F-Fa0.5PD (n = 7) 16.7 ± 0.76a
(3.31 ± 0.129a) 43.0 ± 1.24a
(8.55 ± 0.122b) 59.7 ± 1.85a
(11.9 ± 0.22bc)

EFT: epididymal adipose tissue, AFT: abdominal adipose tissue, BW: body weight

a, b, c Values with different superscript letters are significantly different within diabetic group (p <0.05)

*** P <0.001, vs. normal control group.

Dietary supplementation of pagopyritol in Zucker obese mice gained weight but decreased adipose tissue fraction. The correlation between the weekly feeding blood glucose and the normal weight group and the obese group showed a positive correlation with r = 0.3365 (n = 44, P = 0.0294). However, the correlation analysis of the obese group except the PTC combination group showed a negative correlation with r = -0.5982 (n = 28, P = 0.00098), indicating that weight gain due to the administration of pagopyridol was a result of postprandial blood sugar control. It is considered to be. On the other hand, the pagopyritol drink and PTC combination group showed a significantly different pattern of dietary intake, weight gain and feeding blood glucose, showing a different pattern from the dietary effect of pagopyritol alone administration. These results are believed to be due to the dietary effects of PTC, such as dietary restrictions and delay in carbohydrate absorption in addition to the effect of pagopyritol.

Serum glucose concentrations in fasted and fed obese rats were significantly lowered by administration of pagopyitol alone and in combination with pagopyritol + PTC. D-chiro-inositol, a major functional ingredient of pagopyridol, is a component of inositol phosphoglycan (IPG), a mammalian cell membrane component, and glucose-related enzymes such as pyruvate dehydrogenase phosphatase and glycogen synthase. It has been reported through human and animal experiments that functions as an insulin mediator that activates them to promote oxidative energyification and tissue storage metabolism of glucose. In addition, there was a significant decrease in D-chiro-inositol content in body tissues compared to normal group in patients with insulin-independent diabetes (NIDDM), insulin resistance and each model animal. It has been reported to have a close correlation with. Therefore, hypoglycaemic effect of pagopyritol may be exerted by constituent D-Kiro-inositol. The hypoglycaemic effect of pagopyritol was the strongest in the 1% group, but the 0.2% group also showed similar efficacy as the 1% group over the diet. These results show that the glycemic control effect by pagopyritol can be sufficiently exerted when consumed for one month or more at a small dose. Obese rats significantly increased glucose intolerance with elevated fasting and feeding blood glucose. Increasing glucose intolerance in the obese group was indicative of increased insulin resistance, and this symptom was markedly improved by the combination of pagopyritol and PTC. The co-administration of pagopyritol and PTC is effective in improving glucose in the blood due to the increase in obesity and insulin resistance by synergistic effect of pagopyritol's insulin sensitivity, PTC's delay in carbohydrate absorption and dietary restriction. It is feed.

While dietary supplementation of pagopyritol and PTC significantly reduced blood cholesterol levels, PTC administration elevated levels of triglycerides. Increasing the concentration of neutral lipid by PTC administration in male obese rats is thought to be due to the incorporation of isoflavones in the pazeolamin fraction isolated from the legume. Isoflavones, which are classified as phytoestrogens, are known to have a beneficial effect on women, while increasing the amount of fat in large amounts or ingested by men. Therefore, effective dose should be considered in order to take advantage of synergistic effects by using PTC in combination with pagopyritol.

The liver of obese rats was significantly increased in weight and severe fatty liver compared to the normal weight group. In addition, the activity of GOT and GPT in serum increased rapidly in proportion to the hepatic weight. As a result of correlation analysis between the relative weight to weight of liver and the activity of GOT and GPT in the normal weight group and the obese group, GOT was r = 0.7385 (n = 44, P = 5.8 × 10-10) and GPT was r = 0.7248 A close positive correlation was confirmed with (n = 44, P = 1.3 × 10-8). In the correlation analysis of serum GOT and GPT activity with feeding blood glucose at week 6, GOT was r = 0.7850 (n = 44, P = 8.9 × 10-10) and GPT was r = 0.7286 (n = 44, P = 4.5 x 10 &lt; -8 &gt; Therefore, the improvement effect of pagopyitol on fatty liver formation and hepatic weight increase due to obesity is thought to be a result of the glycemic control effect by increasing insulin sensitivity of pagopyritol.

Dietary supplementation of pagopyitol reduced the increased body fat percentage in obese mice. In the correlation analysis of body fat ratio and feeding blood glucose, r = 0.4776 (n = 44, P = 0.0014) was applied when all the subjects were applied. High correlation was shown by r = 0.6927 (P = 4.0 × 10-10). Therefore, the decrease of the body fat ratio by the administration of pagopyritol may act as a factor to further enhance its blood glucose control function.

In conclusion, 0.2-1.0% pagopyritol intake significantly reduced blood glucose levels in fasting and feeding conditions and significantly improved glucose intolerance in insulin-resistant obese model rats. Pagopyritol also enhanced blood glucose control by improving fatty liver and reducing body fat percentage. Therefore, pagopyitol isolated / purified from agricultural by-products could be usefully used as a dietary supplement for diabetes treatment.

A comprehensive review of the above is as follows.

1. Theoretical Approach to Glycemic Control Function of Pazeolamin-Tanin Complex (PTC)

end. When natural protein is mixed with tannins contained in the plant, it results in the formation of complexes and turbidity of beer or beverage products.In addition, protein-tannin complexes in which tannins in foods combine with amylase in saliva are often expressed. It is due to the invention has been much progress.

I. Edwin Haslam et al. Have published a theory that polyphenol molecules intercalate into the pores in sub-micellar particles of globular proteins to form complexes. Karl J. Siebert et al. Explained that when the protein molecular weight is small, polyphenol molecules are surrounded on the main surface of the protein to form a complex, and the binding pattern is different due to the weak correlation between the protein and the polyphenol.

All. Ann E. Hagerman et al., When reacting BSA with pentagalloyl glucose (PGG) and epicatechin, respectively, epicatechin complexes more sensitively than PGG at nmol levels, and this protein-tannin (TP) interaction is poly It is suggested that the molecular structure of phenol is greatly affected.

la. Haruo Kawamoto et al. Found that TP complex formation produced complexes differently depending on the type of protein at pH conditions. For example, at pH 4.0, protein precipitates are rapidly produced at a low initial TP ratio of about 1: 5 and at pH 7.0. It has been reported that precipitates are formed in the high 1:40 range.

hemp. In this experiment, eight samples (such as kidney beans) and green tea leaf extract Ex were processed at a ratio of 10: 7.5 to process pazeolamin-tannin complex (PTC) to determine whether it is worth developing as a blood glucose control functional food in vivo. In vivo and in vitro experiments were performed for several assays.

2. Blood Glucose Control in Experimental Animals Raised with In vivo PTC Prototype

end. Zucker rat 8-week-old male oxus was purchased in vivo for prototyping and induced fasting and feeding blood glucose elevation with spontaneous and insulin-resistant rats, followed by normal weight control group, obesity control group and Zucker obesity (Buchitol drink + pazeola). Min- tannin complex (PTC)] was fed in the diet for 6 weeks, fasting blood glucose was lowered to 80 mg / 에서는 in the prototype group compared to 100 ㎎ / 의 of the normal weight control group, and also in the blood glucose level of the control group It fell to the value equivalent to 80 mg / dl. Serum cholesterol was about 200 mg / dL compared to normal body weight 100 mg / dL but lowered by half compared to obese control 400 mg / dL and serum GOT and GPT activities were significantly lower than those of other groups.

I. These results showed good results in the function of lowering blood sugar level to the level expected in the in vivo experiment on the above-mentioned pazeolamin-tannin complex (PTC) prototype, and the growth condition of the animal and visceral hypertrophy were not found. It is believed that it is worth developing as a glycemic control food. The results showed that the neutral lipid in serum was slightly elevated, but this phenomenon may be due to the flavonoids contained in the green tea extract. The improvement of the cause is preliminary if the temperature is controlled to 85-90 ℃ when the green tea is extracted. Neutral lipid metabolism seems to be possible.

3. Inhibition of α-amylase Activity of In vitro Pazeolamin-Tanin Complex

end. To date, kidney bean protein is called pazeolamin (extracted from Phaseolus vulgaris L.), which is a water-soluble protein with a molecular weight of about 60 kDa, known as β-globurin. In this experiment, an average yield of 4.5% protein was obtained from 65% ethanol precipitate of the extract extracted at low temperature with water solvent and 2.5% yield from 90% ethanol, yielding more than 7.0% protein yield from two concentrations of ethanol precipitate. It was possible to obtain a protein yield of more than 7.0% from two concentrations of ethanol precipitates. In general, it is assumed that 65% ethanol precipitate contains a lot of protein of 60 kDa or more.

I. The protein content of the PTC prototype powder is 63% on average, and is estimated to contain about 37% of other tannins, water soluble fiber, and ash. Inhibitory activity of α-amylase activity of ethanol-extracted protein was highest in Korean purple bean extract (15.7%), 15.3% in black bean extract, and 13% in Chinese purple bean and mung bean extract, respectively. However, among the eight samples, white kidney beans, peas, red beans, and buckwheat extracts showed low α-amylase activity inhibition of less than 10% on average.

All. In order to develop a glycemic control prototype aimed at the processing of α-amylase inhibitors in this experiment, Pazeolamin-tannin complex (PTC) powder was processed from kidney bean protein extract and green tea tannin extract, which were used for several functional in vitro. Was assayed. Purification of the powder produced from the powdered precipitate obtained by mixing green tea extract Ex at 10: 7.5 ratio with purified protein Ex extracted from domestic purple kidney beans in two stages increased α-amylase activity inhibition by 34%. It was found that the enzymatic hydrolysis of the PTC powder by trypsin and pepsin was suppressed to about 50% due to inhibition of the enzymatic activity of tannin contained in the prototype.

4. Value of PTC's Development of Functional Food Brand for Glucose Control

end. The new findings found in this experiment were that the tannin component of PTC powder interacted with proteins of digestive enzymes such as α-amylase and amyloglucosidase, which are digestive enzymes in vitro, to form new Enzyme-tannin complexes (ETCs). It is conceivable that enzymes that hydrolyze may be able to indirectly inhibit activity in vivo.

I. If the α-amylase inhibitor of PTC products is used to control blood glucose levels normally, and the synergistic effect of pagopyridol, D-kiro-inositol, mediates insulin metabolism, these mixed products will be effective in controlling blood glucose. Could be.

In conclusion, the PTC powder, which is a complex of pazeolamine and tannin, inhibited α-amylase activity in terms of its function and delayed the production and absorption of glucose, resulting in blood glucose levels. Evidence for the control of blood glucose levels was found, especially when pagopyridol beverages were ingested in combination with PTC prototypes in vivo. Therefore, the PTC prototype developed in the present invention has a brand development value because it is judged to be suitable as a functional food for glycemic control for diabetics.

As mentioned above, although the invention made by this inventor was demonstrated concretely according to the said Example, this invention is not limited to the said Example and can be variously changed in the range which does not deviate from the summary.

As described above, according to the manufacturing method and composition of the diabetic cure health functional food using the agricultural by-product according to the present invention, the following effects are obtained.

1) The number of diabetic patients in Korea is close to 2 million, and the number is expected to increase in the future, which is a good image for consumers. It is used to control diabetes processed from mushroom mycelium and grain bran soluble fiber and buckwheat oligosaccharide. Health functional food has a large ripple effect.

    2) Since blood glucose control products contain active polysaccharides, oligosaccharides, and proteins as main ingredients, it is possible to improve reliability by developing capsules or tablets that can prevent hydrolysis by hydrochloric acid in the stomach.

3) Pagopyridol contained in buckwheat bran is extracted and purified as oligosaccharide and can be used as a beverage as one of daily foods.

4) From the technical point of view, the economic and industrial ripple effects are significant, such as the direct ripple effect of developed products, as well as the improvement of national health by the added value of farm income and agricultural products in charge of raw material production, or the effect on the healing of diseases of products.

5) Buckwheat bran is sold in the range of 15,000 won per 15kg as a buckwheat flour extender because there is no special consumption.

6) Situation mushroom mycelium has been discarded for many years, but since the invention was used in Japan as a resource for β-glucan, it is sold in won per kilogram, so the price of buckwheat and rice bran may increase. This can increase economics by increasing farm income.

7) As one of the ways to increase the added value of rice, the development of products that make the best use of the functionality of rice bran is the priority. By developing a technology that can utilize rice bran, which has been used as a feed, for the treatment of diabetes Increasing the added value of rice contributes to vitalizing rural economy.

8) Provides products that can support synergistic effects such as immune enhancement, diet effect, cholesterol lowering effect, arabinoxane with blood sugar control function.

9) Developing health functional foods for the treatment and prevention of diabetes by using bran, which is a by-product produced during the buckwheat milling process, can create an economic utilization plan to increase the added value of buckwheat.

10) By using rice bran as a raw material for the treatment and prevention of diabetes, it is possible to increase the economic added value of rice and increase farm income.

11) By using buckwheat bran and rice bran as raw materials for cultivating situation mushrooms, it is possible to grow mycelia with high functional activity and use them as processed raw materials for the treatment and prevention of diabetes.

Claims (11)

A method for producing a dietary supplement for diabetes treatment using agricultural by-products, comprising extracting, fractionating, purifying and powdering an active polysaccharide-related compound (AHCC) from a buckwheat bran-containing situation mushroom mycelium. The method of claim 1, wherein the extraction is performed with water, ammonium oxalate or 5% aqueous sodium hydroxide solution. delete delete delete delete delete delete delete delete delete

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