KR20100091393A - Food for improving cholesterol and lipid metabolism - Google Patents

Abstract

PURPOSE: Food for improving cholesterol and lipid metabolism, and a producing method thereof are provided to obtain the food by solid fermenting a mixture of defatted soybean meal and mung bean powder with bacillus subtilis. CONSTITUTION: A producing method of food for improving cholesterol and lipid metabolism comprises the following steps: mixing 100 parts of defatted soybean meal by weight with 0.1~10 parts of mung bean powder and adding distilled water for sterilizing, to make sterilized mixture; forming culture fluid by cultivating bacillus subtilis for 46~48 hours at 40~42 deg C; injecting the cultivated bacillus subtilis into the mixture and fermenting for 22~24 hours; freeze-drying the fermented mixture.

Description

Food for improving cholesterol and lipid metabolism

The present invention relates to a food for improving cholesterol and lipid metabolism, and relates to a food for improving cholesterol and lipid metabolism of a mixture of skim soybean meal fermentation and green beans fermentation.

As the pattern of eating habits has been westernized due to the high industrialization and the economic level, the cause of death of Koreans is high. Cerebrovascular disease, malignant tumors, hypertension and heart disease account for a high proportion. Has been pointed out as a serious issue. In particular, increased intake of animal and purified foods is accompanied by complications such as fatty liver, arteriosclerosis, cardiovascular diseases, hyperlipidemia, hypertension. Among these, hyperlipidemia is a condition in which cholesterol or triglycerides are abnormally increased, and hypercholesterolemia is known to cause atherosclerosis and hypertriglyceridemia. According to this trend, cholesterol synthesis inhibitors to improve hyperlipidemia have been studied in various fields.

HMG-, an enzyme that converts 3-hydroxy-3-methylglutaryl coenzyme A (hereinafter referred to as HMG-CoA) into mevalonate during cholesterol biosynthesis. CoA reductase is a major regulator of cholesterol synthesis rate, and it has been reported that a decrease in HMG-CoA reductase activity in the liver decreases blood cholesterol levels by increasing the activity of LDL-receptors in the liver (Sitory, . CR Pharmacology and mechanism of action of the new HMG-CoA reductase inhibitors Pharm Res 1990, 22, 555-562;.. Qureshi, AA; Abuimeileh, N .; Din, ZZ; Elson, CE; Burger, WC Inhibition of cholesterol and fatty acid biosynthesis in liver enzymes and chicken hepatocytes by polar fractions of garlic.Lipids . 1983 , 18, 343-348; Qureshi, AA, Burger WC Peterson DM, Elson, CE: Suppression of cholesterogenesis by plant constituents: review of wisconsin contributions to NC-167. Lipids. 1985 , 20, 817-824. See description).

Most recently used as a treatment for such hyperlipidemia and hypercholesterolemia is lovastatin, simvastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and cevastatin It is a substance of statins. Statins can inhibit the synthesis of cholesterol in vivo and consequently lower the amount of cholesterol, but also involve side effects such as severe muscle pain. Therefore, studies are actively underway to find bioactive substances that inhibit cholesterol synthesis without side effects.

Meanwhile, it has been reported that proteins affect blood cholesterol levels and atherosclerosis (Iwami, K .; Sakakibara, K .; Ibuki, F. Involvement of post-digestrion hydrophobic peptides in plasma cholesterol lowering effect of dietary plant proteins. Agric. Biol. Chem . 1986 , 50, 1217; Sugano, M .; Goto, S .; Yamada, Y .; Toshida, K, M .; Hashimoto, Y .; Matsuno, T .; Kimoto, M. Cholesterol- lowering activity of various undigested fraction of soybean protein in rats.J. Nutr. 1990 , 120, 977-985; Iritani, N .; Hosomi, H .; Fukuda, H .; Tada, K .; Ikeda, H. Soybean protein suppresses hepatic lipogenic enzyme gene expression in wister fatty rats.J. Nutr. 1996 , 126, 380-388 .; Moi, MAE; Smet, RC; Terpstra, AHM; West, CE Effect of dietary protein and cholesterol on cholesterol concentration and lipoprotein pattern in the serum of chickens.J. Nut. 1982, 112, 1029-1037.), especially the concentration of blood cholesterol in soybean protein compared to casein, an animal protein. A number of studies have reported that the effect of lowering the level of blood sugar is significant (Huff, MW; Carrol, KK Effects of dietary proteins and amino acid mixtures on plasma cholesterol levels in rabbits. J. Nutr. 1980 , 110, 1676-1685 .; Park, MS; Liepa, GU Effects of dietary protein and amino acids on the metabolism of cholesterol-carring lipoproteins in rats. J. Nutr. 1982 , 112, 1892-1898 .; Sautier, C .; Doucet, C .; Flament, C .; Lemonnier, D. Effect of soy protein and saponins on serum, tissue and feces steroids in rats. Atherosclerosis. 1979 , 34, 233-241 .; Sirtori, CR; Agra, E .; Conti, F .; Gatti, E .; Mantero, O .; Tremili, E .; Sirtori, M .; Fraterrigo, L .; Tavazzi, L .; Kritxhevsky, D. Clinical experience with the soybean protein diet in the treatment of hypercholesterolemia. Am. J. Clin. Nutr. 1979 , 32, 1645-1658. Reference).

As a mechanism for lowering cholesterol in the blood, soy protein has been shown to affect the activity of HMG-CoA reductase, a cholesterol synthesis rate enzyme in the liver (Forsythe, WA Comparison of dietary casein or soy protein effects). on plasma lipids and hormone concentrations in the gerbil.J. Nutr. 1986 , 116, 1165-1171.) and reports that certain amino acid ratios reduce blood cholesterol levels (Milner, JA; Hassan, AS Species specificity of arginine). deficiency-induced hepatic steatosis.J. Nutr . 1981 , 111, 1067-1073 .; Forsythe, WA; Miller, ER; Hill, GM; Romsos, DR; Simpson, RC Effects of dietary protein and fat sources on plasma cholesterol parameters, LCAT activity and amino acid levels on tissue lipid content of growing pogs. J. Nutr. 1980 , 110, 2467-2479. Second, ingestion of soy protein increases the excretion of bile acids into the feces, and liver cholesterol is used to produce more bile acids, and cholesterol is supplied from the blood through the apolipoprotein B / E receptor to lower plasma cholesterol levels, thereby reducing the cholesterol pool in the body. (Iwami, K .; Sakakibara, K .; Ibuki, F. Involvement of post-digestrion hydrophobic peptides in plasma cholesterol lowering effect of dietary plant proteins.Agric . Biol. Chem . 1986 , 50, 1217 .; Sugano, M .; Goto, S .; Yamada, Y .; Toshida, K, M .; Hashimoto, Y .; Matsuno, T .; Kimoto, M. Cholesterol-lowering activity of various undigested fraction of soybean protein in rats. J. Nutr. 1990 , 120, 977-985.). Third, decrease in blood glucagon / insulin ratio by affecting the endocrine system (see Potter, SM Overview of proposed mechanisims for the hypocholesterolemic effect of soy. J. Nutr. 1995 , 125, 606-611) and thyroxine concentrations There are reports of increasing triglyceride and cholesterol levels in the blood (see Forsythe, WA Soy protein, thyroid regulation and cholesterol metabolism. J. Nutr. 1995 , 125, 619-623.).

In addition, soy peptides, glubulins, isoflavones and saponins are produced during fermentation of soybeans (Choi, YB; Sohn, HS Isoflavone content in Korean fermented and unfermented). .... soybean foods Korean J. Food Sci Technol 1998, 30, 745-750 see) fibrinolytic function (Hso, S .; Lee, SK ;. Joo, HK Isolation and identification of fibrinolytic bacteria from Korean traditional chugkookjang J. Korean Soc.Agric.Chem.Biotechnol . 1998 , 41, 119-124.), Anti-cancer (see Kwon, EY Standardization of Chungkookjang preparation and its cancer preventive effect.MS Thesis.Pusan National University, Pusan. 2000. ), antioxidant And anti-arterial effect (Yang, JL Antiatherogenic effect of chonkukjang. Dissertation of Ph. D., Pusan National University, Pusan. 2000. ), and the ability to improve lipid metabolism due to glycemic control and decrease of blood triglycerides and cholesterol ( Kang, MJ Antibiabetic effect of soy pinito and C hongkukjang: Animal studies and clinical trials.Dissertation of Ph. D., Inje University, Gimhae. 2004 . In particular, the viscous material produced during the fermentation process of soybean bacterium is composed of gamma-poly-glutamic acid (γ-PGA) and levan, a fructose polymer, and γ-PGA combines calcium with phosphorus to form insoluble salts. It prevents the formation and increases calcium solubility, and Levan showed blood lipid reduction effect.

Soybean is the most commonly consumed soybean in Korea, and it is widely used as an alternative source of nutrition for meat. It is also widely used as food such as fermented products, germinated products, and processed foods. It is also widely used as a raw material such as soap. Increased availability of commercially produced defatted soybean grit, soybean protein concentrate, soybean protein isolate and textured soybean protein as new protein foods Doing.

In particular, defatted soybean meal is a by-product produced by soybean oil production, which is made from soybean with the skin removed, has relatively low fiber, has a high protein content, and has a lipid content of 1% or less.

In one embodiment of the present invention, cholesterol and lipid metabolism in vitro and in vivo for a mixture of fermented skim soybean meal fermented with B. subtilis strains and natural fermentation products having excellent HMG-CoA reductase inhibitory activity. The comparative search for improvement ability is intended to provide a use as a new food material having the ability to improve cholesterol and lipid metabolism of these fermentations.

In one embodiment of the present invention Bacillus subtilis ( Bacillus) subtilis) fermented soybean meal, and the cleaning of Bacillus subtilis by subtilis (Bacillus Subtilis ) provides a food for improving cholesterol and lipid metabolism, including a mixture of fermentation products.

In a preferred food according to one embodiment of the present invention, the mixture is Bacillus subtilis of mung beans ( Bacillus subtilis) of defatted soybean meal fermented by the Bacillus subtilis (Bacillus subtilis ) may be included in an amount of 0.1 to 10 parts by weight based on 100 parts by weight of the fermented product.

In the foodstuff according to one embodiment of the present invention, the mixture is a mixture of steamed skim soybean meal and mung bean powder, Bacillus subtilis ( Bacillus subtilis ), which may be obtained by solid fermentation, specifically, 0.1 to 10 parts by weight of mung bean powder to 100 parts by weight of defatted soybean meal, and sterilization and steaming by adding distilled water to prepare a sterilized mung bean-added defatted soybean meal solution; In a sterile soy flour solution Bacillus subtilis (Bacillus subtilis ) to incubate for 46 to 48 hours at 40 ~ 42 ℃ to prepare a culture solution; And after inoculating the culture solution in sterilized mung bean added skim soybean meal solution may be obtained by performing a step of fermentation for 24 to 26 hours at 40 ~ 42 ℃.

In the foodstuff according to one embodiment of the present invention, the mixture may be a lyophilisate.

In the foodstuff according to one embodiment of the present invention, the mixture may be obtained by further performing a lyophilization process after the fermentation process.

According to an embodiment of the present invention by adding mung bean to degreasing soybean (DSG) Bacillus subtilis bacteria ( Bacillus fermented product obtained subjected to a solid-state fermentation using sbutlis) was confirmed excellent cholesterol control in the resulting cholesterol adsorbability searching cholesterol improved performance and HMG-CoA reductase inhibitory activity and HepG2 cells ability on the in vitro, also in the HepG2 cells It was confirmed that apolipoprotein AI, AII activity increase and apolipoprotein CIII activity inhibitory effect is excellent.

In addition, the content of tyrosine and slime is more than 20 times higher and the protease activity and the α-amylase activity are high.

In addition, the changes in cholesterol content in the body were measured to improve cholesterol and lipids, and in particular, plasma and liver cholesterol and TG contents were decreased, and fecal cholesterol and TG contents were increased. In addition, cholesterol and lipid metabolism were regulated by inhibiting HMG-CoA reductase activity in the liver.

Therefore, the mixture of skim soybean meal fermentation and mung bean fermentation according to an embodiment of the present invention has an effect of improving cholesterol and lipid metabolism, thereby can be expected to be useful as a functional food natural material related to cholesterol and lipid metabolism improving ability.

Hereinafter, the present invention will be described in detail.

In one embodiment of the present invention Bacillus subtilis ( Bacillus) subtilis) fermented soybean meal, and the cleaning of Bacillus subtilis by subtilis (Bacillus Subtilis ) provides a food for improving cholesterol and lipid metabolism, including a mixture of fermentation products. In other words, the mung bean of Bacillus subtilis (Bacillus subtilis) fermented soybean meal, and the cleaning of Bacillus subtilis by subtilis (Bacillus subtilis ) and the use as a food to improve cholesterol and lipid metabolism using a mixture of fermented products.

Best decided in cholesterol and synergistic effect of improving lipid metabolism resulting from the mixture of the two fermented product is the mixture of green beans Bacillus subtilis (Bacillus subtilis) of defatted soybean meal fermented by the Bacillus subtilis (Bacillus subtilis ) 0.1 to 10 parts by weight based on 100 parts by weight of the fermentation.

Defatted soybean meal of Bacillus subtilis (Bacillus of Bacillus with respect to the green beans fermented by subtilis) subtilis (Bacillus subtilis) fermentation of skimmed soybean meal and the water content reaches its end of Bacillus subtilis by subtilis (Bacillus subtilis ) can be difficult to expect the effect of addition to reduce the effect of improving cholesterol and lipid metabolism expressed by the fermentation alone. Preferably the mixture of mung Bacillus subtilis (Bacillus subtilis) of defatted soybean meal fermented by the Bacillus subtilis (Bacillus subtilis ) may be included in an amount of 0.1 to 5 parts by weight based on 100 parts by weight of the fermented product.

Mixtures of these two fermentations can be obtained in a variety of ways, but rather than mixing and fermenting skim soybean meal and green beans, respectively. The mixture of the defatted soybean and mung bean powder increase, Bacillus subtilis (Bacillus solid fermentation with subtilis ) affects the production of enzymes and the increase in the content of viscous substances during fermentation. Tillis ( Bacillus) subtilis ) and a method of solid fermentation.

More specifically, first, 0.1 to 10 parts by weight of mung bean powder is mixed with 100 parts by weight of skim soybean meal, and sterilized and cooked by adding distilled water to prepare a sterilized mung bean-added skim soybean meal solution.

In this case, the amount of distilled water may be 0.1 to 10 parts by weight based on 100 parts by weight of degreased soybean meal.

Sterilization and cooking can be carried out using autoclave, At 120 ~ 121 ℃ After 15-16 minutes of cooking, a mixed solution of sterile defatted soybean meal and mung bean powder (sterile mung bean-added defatted soybean meal solution) can be obtained.

The sterilized mung bean-added defatted soybean meal solution is left to cool to 50-60 ° C. for transition to the fermentation process.

Apart from this, a process of strain culture for carrying out fermentation is carried out, specifically, Bacillus subtilis ( Bacillus) in a sterile soybean powder solution. subtilis ) may be cultured at 40 to 42 ° C. for 46 to 48 hours to prepare a culture solution.

In this case, in addition to the sterilized soybean powder solution, black soy crushed powder, MRS medium, and the like may be used, and Bacillus subtilis ( Bacillus subtilis) may be used. subtilis ) If the starter bacteria of the strain can be cultured is not particularly limited thereto.

Then, after inoculating the culture solution in sterilized mung bean added skim soybean meal solution and fermentation at 40 ~ 42 ℃ for 24 to 26 hours. The inoculation concentration of the culture solution may be 1 to 2%.

According to the above method, a mixture solution of mung bean fermentation product and skim soybean meal fermentation product may be prepared, and the mixture solution may be used as a food as it is, or may be used as food in the form of a lyophilized product with a drying process such as lyophilization. have.

Hereinafter, the experiment to support the use as a food for promoting cholesterol and lipid metabolism of the mixture of mung bean fermentation and skim soybean meal fermentation according to one embodiment of the present invention to confirm the effect.

However, in the following examples, in order to easily prepare for the characteristics and effects of the mixture of mung bean fermented product and skim soybean meal fermentation, a comparison with the case of skim soybean meal fermentation alone is carried out, but this is only for defatted soybean meal fermentation and mung bean fermentation. It is only intended to estimate the change in the effect of the mixing with and the use of the skim soybean meal fermentation as a food to promote the improvement of cholesterol and lipid metabolism should not be understood.

<Examples>

1. Reagents and Instruments

1) Reagent

Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), antibiotics, and trypsin-EDTA used for cell culture were purchased from Gibco BRL (Gibco BRL, Grand island, N.Y., USA).

Total cholesterol measurement kit was purchased for measurement of cholesterol control ability by Asan Pharmaceutical (Seoul, Korea), and apolipoprotein-AⅠ, apolipoprotein-AⅡ, and apolipoprotein-CIII were used for Academy Bio-Medical ) And anti-goat IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, USA).

Folin-phenol reagent used in measuring the tyrosine content is Junsei Chemical Co. (Tokyo, Japan) was used. NADPH, HMG-CoA, mevalonlactone were purchased from Sigma (ST. Louis. MO., USA) for the measurement of HMG-CoA reductase activity of the liver. Other reagents and organic solvents were also tested using first-class reagents.

2) appliance

Instruments used in this experiment were UV / visible spectrophotometer (Unikon 922, Kontron, Italy), incubator (Model 3154, Forma Scientific, USA), ultracentrifuge (Model 695-7, Hitachi, Japan), microplate spectrophotometer (Molecular Devices, Spectra max 340PC, USA), microplate shaker (Finemixer SH2000, FINEPCR, Korea), deep freezer (CLN-50VW, Nihon freezer, Japan), glass teflon homogenizer (Glascol, 099C K44, USA), pH meter (model 691, Metrohm, Swiss).

2. Sample Preparation

1) Preparation of natural material extract

Nine natural products including edible mushrooms, spices, herbal ingredients and mung beans used in this experiment are shown in Table 1, and curry flowers and mung beans were purchased at the hypermarket, and the remaining ingredients except curry flowers and mung beans were Daegu Yangnyeong Market. The dried thing was purchased and used. In order to remove impurities, the sample was washed with water and dried, and 10 times (w / v) of 80% methanol was added thereto, and the mixture was left for 24 hours and extracted three times. The extract was filtered using a filter paper (Whatman No. 3, England) and concentrated using a rotary vacuum evaporator (R-3000, Buchi Switzerland) at 55 ° C., followed by freeze drying.

2) Raw material processing and fermented product manufacturing

Defatted soybean meal (abbreviated LA Defatted Soybean Grit, hereinafter DSG) is ADM Company (Decatur, IL, USA) by was used, with strains purchased from the (main) NUC holds strain Bacillus sub-blocks scan of the electron Bio Research BN - Experiment using NUC1 (KCCM10839P).

Referring to the manufacturing process of the fermentation with reference to Figure 1, first, the process of producing a single DSG fermentation, the distilled water was added to the DSG raw material 2.5 times the weight ratio of the raw material and then a high-pressure sterilizer for 15 minutes at a steam. The cooked DSG was cooled to 50-60 ° C. at room temperature. On the other hand in a sterile soy flour solution B. subtilis BN - NUC1 gain (KCCM10839P) for 46 ~ 48 hours to the culture medium at 40 ~ 42 ℃, B. subtilis BN at a DSG solution the increase it - NUC1 (KCCM10839P) 2 After inoculating to%, it was fermented at 40-42 ° C. for 24 to 26 hours to obtain a DSG fermented product (hereinafter referred to as FD).

Meanwhile, in the preparation of the mixed fermentation product of skim soybean meal and mung beans, first, distilled water is added to the DSG raw material at 2.5 times the weight ratio, and then the powdered green soybean is added by varying the amount of 2.5 to 10 parts by weight based on the DSG raw material weight. After dissolving, the mixture was cooked in a high pressure sterilizer at 120-121 ° C. for 15-16 minutes and then cooled.

Meanwhile, B. subtilis BN - NUC1 (KCCM10839P) was incubated at 40-42 ° C. for 46-48 hours in a sterilized soybean powder solution to obtain a culture medium, which was then added to B. subtilis BN - NUC1 (KCCM10839P). After inoculation to 2%, fermentation was carried out at 40 to 42 ° C. for 22 to 24 hours to obtain a mung bean-added DSG fermentation product (hereinafter referred to as FDM).

Each fermentation was lyophilized and used for the following experiment.

No. Korean name Scientific name One. Gold Scutellaria baicalensis Georgi 2. Oyster Mushroom Sarcodon aspratus 3. Curry flower Brassica oleracea L. var. 4. Donkey Angelicae Gigantis Radix 5. Situation Mushroom Pellinus Linteus 6. licorice Glycyrrhiza uralensis Fisch 7. Chicory Cichorium intybus L. 8. Molokia Corchorus olitorius L. 9. green gram Phaseolus radiatus

3.  In vitro  Cholesterol Regulatory Capability by Using System

1) Total cholesterol adsorption capacity

Total cholesterol adsorption capacity was measured using Asan Pharmaceutical Total Cholesterol Kit (AM 202-K) by enzyme method. The lyophilized fermented product was dissolved in 10-fold distilled water, and 30 μg of cholesterol was added to 1 mL of the fermented product, and the mixture was mixed well at 25 ° C. for 20 minutes. After centrifugation for 10 minutes at rpm, the supernatant was taken, reacted with enzyme solution for 5 minutes at 37 ° C, and absorbance was measured at 500 nm (Soh, HS; Kim, CS; Lee, SP A new in vitro assay of cholesterol adsorption by food). and microbial polysaccharides J. Med. Food. 2003, 6, 225-230.

2) HMG-CoA reductase inhibitory activity

Enzyme sources of liver tissue are described by Hulcher et al. (Hulcher, F .; Oleson, WH Simplified spectrophotometric assay for microsomal HMG-CoA reductase by measurement of coenzyme A. J. Lipid Res. 1973, 14, 625-631. And some modifications). 4 g of 0.1 M potasium phosphate (pH 7.4) was added per g of isolated liver tissue, ground and centrifuged at 600 × g for 10 min at 4 ° C or below, and then the supernatant was collected. Centrifuged for 20 minutes. The supernatant was again used for ultrasomal separation at 105,000 × g, 4 ° C. for 1 hour to obtain a microsomal fraction as a precipitate. Fractions were stored at 80 ° C. until used in experiments in portions.

In the measurement method, 20 μL of a sample, 0.5 mM phosphate buffer (pH 7.0), 20 μm DTT 100 μL, 3 mM NADPH 100 μL, and 100 μL enzyme source were added to a 1 mL cuvette. The temperature of the reaction solution was kept constant at 37 ° C., preincubated for about 10 minutes, and then 100 μL of 3 mM HMG-CoA was added to start the enzyme reaction. At the same time as the reaction started, the absorbance change was recorded for 5 minutes at 340 nm. Inhibitory activity of HMG-CoA reductase was calculated by the following equation.

HMG-CoA reductase inhibition activity (%) =

 T: ΔO.D. of sample C: ΔO.D. of blank

3) cell culture

HepG2, a human-derived liver cancer cell line used in this experiment, was distributed from Korea Cell Line Bank, and 37 ° C incubator containing 5% CO ₂ using DMEM (Dulbecco's modified eagle medium) medium containing 10% FBS and 1% antibiotics. Passage was incubated once every two days.

4) Determination of Total Cholesterol Content Using Cell Lines

The total cholesterol content using cell lines was measured by Park et al. (Park, SC; Noh, YH; Koo, JH Effect of ginseng componets on content of cholesterol and activity of Acyl CoA: Cholesterol Acyltransferase in Hep G2 cells cultured in cholesterol rich medium. Korean J. Ginseng Sci. 1995 , 19, 212-218. HepG2 cells were aliquoted into 5 × 10 ⁵ cells / mL in DMEM (FBS 2%) medium in a 6-well plate (corning, USA), incubated for 24 hours, washed with PBS, and then 10 μg / Extracts were added to DMEM (FBS 0%) medium containing mL of cholesterol and incubated for 8 hours. After taking the supernatant, the cells were washed once with PBS containing 1% ethanol, washed three times with PBS without ethanol, and then lysed. The total cholesterol content was determined by enzymatic method. The absorbance was measured using a kit (AM 202-K), and the protein was measured by Lowry method (Lowry, OH; Rosebrough, NH; Farr, AL; Randall, RJ Protein measurement with folin phenol reagent.J. Biol. Chem . 1951 , 193, 265-275.) To calculate the amount of cholesterol.

5) Apolipoprotein secretion search

Apolipoprotein secretion was detected by enzyme-linked immunosorbent method (Sambrook, J .; Russell, DW Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press. 2001. ). First, HepG2 cells were dispensed at 5 × 10 ⁵ cells / mL in high glucose DMEM medium, and after 24 hours of treatment, the cultures were dispensed at 50 μl / well in a 96-well plate (Corning, USA) at 4 ° C. It was left to stand. After 12 hours, incubated for 1 hour at 37 ° C. in 5% skim milk, and the first and second antibodies were reacted. Finally, after reacting with the coloring reagent 1,2-pheylenediamine (100 μl / well) for 30 minutes, the absorbance was measured at 492 nm.

4. Physicochemical Characterization of Fermented Products

1) pH measurement

  pH was measured using a pH meter after taking 20 g of the sample and dissolved in 80 mL of distilled water.

2) Tyrosine content measurement

In order to measure the degree of peptide production of fermented products, the content of tyrosine in fermented products was measured using Folin phenol reagent (Matsushita, S .; Iwami, N .; Nitta, Y. Colorimetric estimation of amino acids and peptides with the Folin phenol reagent.Anal. Biochem . 1966, 16, 365-371. 0.7 mL of 0.44 M TCA (trichloroacetic acid) was added to 0.7 mL of the sample solution diluted five-fold with distilled water and reacted at 37 ° C. for 30 minutes, followed by centrifugation at 15,000 rpm for 10 minutes to remove the precipitate. . 2.5 mL of 0.55 M Na ₂ CO ₃ and 0.5 mL of phenol reagent were sequentially added to 1 mL of the recovered supernatant, followed by reaction for 30 minutes in a 37 ° C. constant temperature water bath. After cooling at room temperature, the absorbance of the reaction solution was measured at 660 nm with a spectrophotometer.

3) Determination of hydrolase activity

Enzyme activity was divided into α-amylase and protease to measure the titer. After adding 95 mL of 0.02 M phosphate buffer (pH 7.0) to 5 g of fermentation, the mixture was shaken at room temperature and centrifuged (15,000 rpm, 15 min). The enzyme solution was prepared from and the enzyme activity was measured. 1 mL of 1% soluble starch (0.02 M phosphate buffer, pH 7.0) was used as a substrate of α-amylase. After adding 1 mL of the prepared enzyme solution and reacting at 37 ° C. for 30 minutes, the reaction was stopped with 10 mL of 1 M acetic acid, added with 2 mL of iodide solution (0.005% I ₂ + 0.05% KI), and then absorbed at 660 nm. Was measured and the blank OD value was reduced by 10% to 1 unit and converted into 1 g of sample. In the blank test as a control, a sample solution prepared by inactivating a previously prepared enzyme solution by boiling it at 100 ° C. for 30 minutes was used (Park, JM; Oh, HI Changes in microflora and enzyme activities of traditional Kochujang meju during fermentation.Korean J. Food Sci.Technol 1995, 27, see 56-62).

The activity of protease was determined by Anson's method (Kim, HJ; Lee, JJ; Cheigh, MJ; Choi, SY Amylase, protease, peroxidase and ascorbic acid oxidase activity of Kimchi ingredients.J. Food Sci. Technol. 1998, 30, 1333- 1338.). Add 0.35 mL of 0.6% casein solution and 0.35 mL of enzyme solution to the e-tube as a substrate, and react in a constant temperature water bath (37 ℃, 10 min), stop the reaction by adding 0.7mL of 0.44 M TCA solution, and stop at 37 ℃ for 30 minutes. Let it stand. Centrifuge the reaction solution (15,000 rpm, 15 min), add 1 mL of filtrate, 2.5 mL of 0.55 M Na ₂ CO ₃ , and 0.5 mL of 3-fold diluted Folin reagent, react for 30 minutes at 37 ℃, and absorbance at 660 nm. Was measured. Under this reaction condition, the amount of enzyme that liberates 1 ug of tyrosine in 1 minute was 1 unit.

4) consistency measurement

45 mL of distilled water was added to 5 g of the sample fermentation product, and homogenized to prepare a sample filtered using a sieve (sieve, 0.99 mm). 13 mL of the filtrate was taken and measured using a measuring cup DG43 by mounting a spindle (Rotor DG43 DIN 53544 Titan, Germany) on a cylindrical viscometer (HAAKE RheoStress 1, Germany). Shear velocity (γ) was measured at 20 ℃ by measuring the viscosity in the range of 1-100 s ^-1 and the viscosity index was measured by the power law model (46).

Power law model: σ = K · γ n

σ is the shear stress (Pa), K is the consistency index (Pa · s ⁿ ), γ is the shear rate (s ^-1 ), and n is the flow behavior index.

5) Determination of Viscous Content

Distilled water was added to 5 g of the sample, homogenized, filtered using a filter medium, and centrifuged at 10,000 rpm for 20 minutes to obtain a supernatant. Solids were precipitated by adding 2 times the volume of iso-propanol to the recovered supernatant, and then the solids were recovered, washed with 80% ethanol and dried in a vacuum hot air dryer at 60-70 ° C. for one day. ).

5. In vivo  Screening of Lipid Metabolism and Cholesterol Improvement Using System

1) Animal test plan and experimental diet

The experimental animals were purchased from Orient Co., Ltd. (Pusan, Korea) 24 rats of Sprague-Dawley species 5 weeks old (100-150 g). The breeding room temperature was maintained at 23 ± 2 ℃, relative humidity 60 ± 5%, and was kept in a breeding room with a contrast of 12 hours. Adapted to provide pellet lab chow diet for the first week, then divided into four groups by randomized block design.

As shown in Table 2, the experimental diet was divided into the normal diet group (CON group), the high fat diet group (HF group), the high fat diet group with 20% FD (HF-FD group), and the high fat diet with 20% FDM. It was divided into the added group (HF-FDM group) and reared for 4 weeks. Six rats of each group were used, and a high fat diet was supplied by adding 10% lard, 1% cholesterol and 0.25% sodium cholate to the basic diet. The composition of the experimental diet is shown in Table 3, where drinking water and feed were supplied in sufficient amounts.

group N Dietary form CON 6 Normal diet HF 6 High fat diet ¹⁾ HF-FD 6 High Fat Diet + 20% FD HF-FDM 6 High fat diet + 20% FDM ²⁾

^{1) High} fat diets include 1% cholesterol, 10% lard, 0.25% sodium cholate in the normal diet.

²⁾ Fermented product obtained by adding 2.5 parts by weight of powder mung beans to 100 parts by weight of DSG.

ingredient Dietary Composition (%) CON HF HF-FD HF-FDM Corn starch ^a 56.95 46.95 41.15 40.45 Casein ^b 20 20 7.8 8.1 Corn oil 7.0 7.0 7.0 7.0 Sucrose 10 10 10 10 DL-methionine 0.3 0.3 0.3 0.3 Cellulose 5.0 5.0 5.0 5.0 Mineral mixturel 3.0 3.0 1.7 1.7 Vitamin mixture 1.0 1.0 1.0 1.0 Lard - 10 10 10 Cholesterol - One One One Sodium cholate - 0.25 0.25 0.25 FD powder - - 20 - FDM powder - - - 20 Total (%) 103.25 104.5 105.2 104.8

^a Starch and ^b casein were adjusted by the protein and carbohydrate content in the FD and FDM powders.

2) Experiment animal treatment and sample collection

The dietary intake was calculated by subtracting the dietary amount from the dietary amount once a week, and the weight was measured at a certain time every week. In the last three days of the breeding period, the feces were collected, dried, and stored frozen.

In order to observe the lipid and cholesterol changes in the blood during the breeding period, blood was collected by the clavicle venous blood collection method before the breeding, and the plasma was separated by centrifugation. The separated plasma was measured for lipid and cholesterol concentrations using an automatic blood analyzer.

Rats fasted 12 hours after 4 weeks of breeding were anaesthetized with ether. Blood was collected from the abdominal aorta with a heparinized syringe, left for 30 minutes, and centrifuged at 3,000 rpm for 15 minutes to separate plasma. In addition, liver, kidney, spleen, epididymal fat, and abdominal fat were also extracted, rinsed with saline, and then drained and weighed. The separated plasma and tissues were quenched with liquid nitrogen and stored at -70 ° C until analysis. The separated plasma was used to measure neutral lipid and cholesterol content, and liver tissue was used to measure lipid content and enzyme activity in liver.

3) Analysis of triglyceride and cholesterol content in plasma

Total cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol content of plasma separated by parking lot were measured by automatic blood analyzer. At this time, the triglyceride in serum was measured using a kit for measuring triglyceride, total cholesterol content kit for total cholesterol content, and HDL-cholesterol content kit for HDL-cholesterol content.

LDL-cholesterol was calculated by the following formula using the values of total cholesterol, HDL-cholesterol and triglyceride obtained from the analysis (Friedewald, WT; Levy, RI; Fredrickson, DS Estimation of the concentration of low-density lipoprotein). cholesterol in plasma, with out use of the preparative ultracentrifuge. Clin. Chem. 1972 , 18: 499-502).

LDL-cholesterol = T-cholesterol-(HDL-cholesterol + TG / 5)

4) Intrahepatic HMG-CoA reductase Activity Assay

4 g of 0.1 M potassium phosphate (pH 7.4) is added per 1 g of extracted liver tissue, ground and centrifuged at 600 × g for 10 min at 4 ° C or below, and the supernatant is taken. Centrifuged for a minute. The supernatant was ultracentrifuged again at 105,000 × g for 1 hour at 4 ° C., and the precipitate microsomal fraction was used for the measurement. HMG-CoA reductase activity was measured by Hulcher's method (Hulcher, F .; Oleson, WH Simplified spectrophotometric assay for microsomal HMG-CoA reductase by measurement of coenzyme A. J. Lipid Res. 1973, 14, 625-631. ) Was used. After mixing 150 μM HMG-CoA and 2 mM NADPH with 500 μg microsome fraction, 1 mL was charged with 0.1 M triethanolamine, 0.02 M EDTA (pH 7.4) buffer and reacted at 37 ° C. for 30 minutes. Then, 20 μL of 0.01 M sodium arsenite was added and reacted at room temperature for 1 minute or longer. Then, 100 μL of 2 M citrate (pH 3.5) and 3% Na-tungstate buffer solution were added and reacted at 37 ° C. for 10 minutes. The reaction was centrifuged at 13,000 rpm for 10 minutes at 4 ° C, and 200 μL of 2 M Tris (pH 10.6) and 100 μL 2 M Tris (pH 8.0) were mixed in 1 mL of the supernatant and reacted for 3 minutes at room temperature. 3 mM 5,5-dithiobis 2-nitrobenzoic acid (DTNB), 0.1 M triethanolamine, and 0.2 M EDTA (pH 7.4) buffer were added and mixed well, and then the absorbance change was measured at 412 nm for 4 minutes. At this time, the enzyme activity was expressed as nmole the amount of CoA-SH produced by 1 mg of microsome protein for 1 minute. And protein quantification was performed by Lowry et al. (Lowry, OH; Rosebrough, NH; Farr, AL; Randall, RJ Protein measurement with folin phenol reagent. J. Biol. Chem . 1951 , 193, 265-275.) The standard calibration curve using albumin as standard protein solution was calculated and its amount was calculated.

5) Lipid analysis of liver tissue

Extraction of liver tissue lipids and determination of neutral lipids and cholesterol concentrations were performed by Folch et al. (49) and Sale et al. (Sale, FO; Marchesine, SP; Fishman, H .; Berra, BA Sensitive enzymatic assay for determination of cholesterol in lipid extracts). Anal. Biochem. 1984 , 347-350.).

0.5 g of liver tissue was chopped and 10 mL of Folch solution (chloroform: methanol, 2: 1, v / v) was added to extract lipids from the tissue. The extract was mixed with liver extract filtered with Whatman filter paper (No. 2) and 0.5 mL H ₂ SO ₄ 3 mL in a separatory funnel. After leaving for a certain time, the separated lower layer was placed in a new tube, and Folch solution was added so that the total amount was 10 mL. 10 μL of this extract was used for triglyceride analysis. 500 μL of 10 mL extract was added to a new tube and dried at room temperature overnight. Then, 50 μL of Triton X-100: chloroform solution (1: 1, v / v) was added to vortex and 450 μL of chloroform was added. Diluted. Triton X-100 was used to remove turbidity caused by the development of tissue lipid dissolved in chloroform. 10 μL of this solution was used to measure liver cholesterol. For liver cholesterol and triglyceride analysis, 10 μL of gastric extract was dried at room temperature, 1.5 mL of enzyme solution was added, and the resultant was reacted for 10 minutes in a water bath at 37 ° C., and cholesterol was measured at 500 nm and neutral lipid at 550 nm. .

6) Determination of lipid content in feces

Triglycerides and cholesterol in feces were determined by the Folch method (Folch, J .; Lees, M .; Sloane-Stanley, GH A simple method for isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957 , 226, 497 Plasma triglycerides were extracted by McGowan et al. (McGowan, MW; Artiss, JD; Strandbergh, DR; Zak, B. A peroxidase-coupled method for the colorimetric determination of serum triglycerides. Clin. Chem). 1983, 29: 538-542.) And the plasma total cholesterol was measured according to the enzyme method (Allain, CC; Poon, LS; Chan, CSG Enzymatic determination of total serum cholesterol.Clin. Chem). 1974, 20: 470-475, see) was quantitated using the application in a Asan pharmaceutical total cholesterol measurement kit (AM 202-K).

7) Observation of pathological changes in liver, abdominal fat and epididymal fat

To observe the pathological changes in the tissues of the experimental animals, a portion of the liver, abdominal and epididymal fats of each group were removed, fixed in 10% neutral buffered formalin for 24 hours, and then dehydrated, clear and infiltrated using an automatic paraffin infiltrator. After proceeding to format the paraffin, using a rotary cutting machine to prepare a paraffin section of 4 μm thickness. Paraffin sections were deparaffinized to xylene and functioned by descending alcohol, followed by hematoxylin-eosin (H & E) staining. After staining, the sections were dehydrated and cleared and encapsulated with Canadian balsam and observed under an optical microscope.

8) Statistical Analysis

The experimental results were analyzed by variance analysis using SAS program (53) and then tested for significant differences between samples at p <0.05 level by Duncan's multiple range test.

Looking at the results of the various search and measurement performed by the above method as follows.

One. In vitro  Cholesterol Regulatory Capability by Using System

1) HMG-CoA reductase inhibitory activity of natural products

In order to improve cholesterol and lipid metabolism, we investigated the inhibitory ability of HMG-CoA reductase, which acts as an important regulatory enzyme in cholesterol synthesis system, for nine natural products known to lower cholesterol for selection of natural products to be added during DSG fermentation.

As a result of detecting the inhibitory ability of HMG-CoA reductase by extracting nine selected natural products with 80% methanol and treating them at a concentration of 100 mg / mL, all nine selected natural products showed excellent inhibition over 70% as shown in FIG. In particular, the final sample was edible and phaseolus radiatus , which has the highest inhibitory activity of more than 90%.

In Fig. 2, I denotes golden, II denotes a mushroom, III denotes a curry flower, IV denotes a donkey, V denotes a situation mushroom, VI denotes a licorice, a thin chicory, a thin Molochia, and a thin green bean.

3) HMG-CoA reductase inhibitory activity

HMG-CoA reductase is a regulatory enzyme that acts in the cholesterol biosynthesis step and mediates the synthesis of mevalonic acid, an intermediate step in the biosynthetic pathway of sterols and isoprenoid compounds.

HMG-CoA reductase inhibitory activity by the sample is shown in FIG. Referring to this, the FD showed inhibitory activity of about 25% and the highest inhibitory activity of about 42% in the FD-Mung bean group added with 2.5% green beans. Specifically, the FD-Mung bean group containing 5 and 10% of mung beans showed that the inhibitory activity decreased as the amount of mung beans added increased. These results indicate that the content of mung bean fermentation in the mixture of fermentation is within 2.5 parts by weight based on 100 parts by weight of skim soybean meal fermentation in terms of HMG-CoA reductase activity. In this way, it is known that inhibition of HMG-CoA reductase activity inhibits cholesterol biosynthesis, so FD and FDM are expected to be excellent materials for inhibiting cholesterol synthesis.

4) Determination of total cholesterol content using HepG2 cell line

As a result of measuring the condition that cholesterol is best introduced into HepG2 cells, when the concentration of cholesterol was incubated at 10 μg / mL for 8 hours, the intracellular cholesterol inflow rate was the highest.

Intracellular cholesterol content by the sample is as shown in FIG.

Referring to this, first, the control group and the experimental group administered only cholesterol (10 μg / mL) added FD-Mung bean containing 2.5, 5, 10% of FD and mung bean to cholesterol-adding medium (10 μg / mL). Cholesterol content of HepG2 cells was examined under high cholesterol conditions by treating each of them to a concentration of mg / mL for 8 hours. As a result of quantifying cholesterol in HepG2 cells cultured in each medium condition and calculating the average value of protein, FD-Mung bean group containing 2.5 and 5% of mung beans significantly lowered the intracellular cholesterol level compared to the control group. It was. In particular, the lowest cholesterol content was found in the FD-Mung bean group containing 2.5% mung beans. That is, when fermentation was performed by adding 2.5% of green beans, cholesterol control ability was increased than when it was not added.

5) Secretion of Apolipoproteins A, II and CIII

In terms of improving lipid status in the blood, total cholesterol, especially LDL cholesterol and triglyceride levels are lowered, but the increase in HDL cholesterol is also very important.

The function of apolipoprotein is in the transport of fat and maintenance of lipoprotein structure, and its role is an activator of enzymes involved in lipoprotein metabolism, and it is necessary for lipids in lipoproteins to be metabolized to liver, blood vessel wall and adipose tissue. Department of Biochemistry, College of Medicine, Seoul National University, 1987 , 116.).

Among them, apolipoprotein AⅠ is not only an important lipoprotein constituting chylomicron and HDL cholesterol, but also activates LCAT, and apolipoprotein AII is an important component protein of HDL cholesterol (58). In particular, the increase in apolipoprotein AI and apolipoprotein AII is known to be proportional to the increase of HDL2, which plays a role in anti-arteriosclerosis (Taskinen, MR; Kahri, J .; Koivisto, V .; Shepherd, J .; Dackard, CJ Metabolism of HDL apolipoprotein AI and AII in type I (insulindependent) diabetes mellitus.Diabetologia . 1992 , 35: 347-356. The apolipoprotein CⅢ is VLDL, IDL, is to suppress LPL as a constituent protein of HDL known to have the ability to increase blood cholesterol (Gotto, AM;. Pownall, HJ;. Havel, RA Introduction to the plasma lipoproteins Methods Enzymol 1986, 128, 3-40.

Searching for apolipoprotein secretion of the FD-Mung bean group to which FD and mung beans were added at 2.5, 5, and 10%, the secretion of apolipoproteinA I, apolipoprotein AII, and apolipoprotein CIII was 100, respectively, as shown in FIGS.

First, in the case of apolipoprotein AⅠ, the amount of secretion was increased when Mung was added than FD. Especially, FD-Mung bean group with 2.5% showed the highest secretion. Apolipoprotein AⅡ also showed the highest secretion rate in the FD-Mung bean group containing 2.5% mung bean. On the other hand, the secretion of apolipoprotein CIII was the lowest in the FD-Mung bean group containing 2.5% of green beans. Therefore, the FD-Mung bean group containing 2.5% of mung beans increased the apolipoproteins A and A II, which constitute HDL, which acts as an anti-arteriosclerosis factor, and secreted apolipoprotein CIII, which constitutes VLDL, IDL, and HDL. It was confirmed that inhibition.

As a result of searching for cholesterol control ability in vitro , FD itself had cholesterol control ability, and when fermented with 2.5% of green beans, it was confirmed that cholesterol control ability was better. However, when the amount of mung beans added to 5, 10% more cholesterol control ability was found to decrease. Therefore, the physicochemical characteristics of FD-Mung bean group added 2.5% of green beans, which had the best effect with FD, were analyzed for lipid metabolism and cholesterol improvement in vivo .

2. Physicochemical Characterization of Fermented Products

1) Search for Physicochemical Properties of FD and FDM

The results of measuring the pH, tyrosine content and hydrolase activity of DSG fermentation (FDM) added 2.5% mung bean, which had the highest cholesterol inhibitory effect on FD and in virto fermented DSG, are shown in Table 4 below.

The pH of the DSG was 6.48 and the fermented FD and FDM rose to 6.91 and 6.96, respectively.

In order to measure the degree of peptide production of DSG fermentation, the results of measuring tyrosine showed that DSG was about 95.6 mg%, and the fermented FD and FDM were increased by about 20 times to 1893.42 and 1973.96 mg%, respectively. It was found that the content increased more than this FD.

Protease is known to play a most important role in forming taste fermented soybean water by digesting the protein of soybean (Lee, KH; Lee, HJ ;.. Chung, MK Studies on Chungkookjang on the changes of soybean protein in manufacturing Chungkookjang J Korean Agric.Chem. 1971, 14, 191-200. The protease activity was higher in FDM and 482.46 and 507.83 unit / g in FD and FDM, respectively, and the α-amylase activity in both FD and FDM was 24.38 and 38.29 unit / g, respectively. High. These results suggest that the addition of an appropriate amount of mung beans promoted the growth of the strain and increased the activity of the hydrolase.

2) Searching consistency and viscosity content of FD and FDM

Viscous , a fermentative metabolite of Bacillus strains, consists of γ-polyglutamic acid (PGA) polymerized with approximately 5,000 glutamic acids and a fructan-type levan, a polymer of fructose (Lee, YL, Kim, SH; Choung, NH). Tim, MH A study on the production of viscous substance during the Chungkookjang fermentation. J. Korean Agirc. Chem. Soc . 1992, 5, 202-209. These viscous substances have an important influence on the quality characteristics of products, and in particular, they have various physiologically active functions and are functional materials that are attracting attention as foods and medicines as well as cosmetics (Choi, HJ; Kunioka, M. Preparation conditions and swelling equilibria of hydrogel prepared by (γ-irradiation from microbiol poly (γ-glutamic acid)). radiat.Phys.Chem. 1995 , 46, 175-179.

The consistency and viscosity of the extracts filtered by diluting FD and FDM with 10-fold water are shown in Table 4 below.

In DSG, the consistency or viscosity content was very low or not measured. In FD, the consistency value was less than 0.966 Pa · s ⁿ and the viscosity content was 20 times higher than that of DSG. In particular, FDM fermented with mung beans had a consistency value of 1.679 Pa · s ^{n, which} was higher than that of FD, and the viscous content was about 3% higher than that of FD. Therefore, when fermented by adding mung beans to DSG, it was confirmed that the production of viscous substances is increased.

Sample ¹⁾ Consistency index
(Pas ⁿ ) Viscous substance
content (%) D 0.003 ND FD 0.966 23.67 FDM 1.679 26.67

¹⁾ D: Defatted soybean meal FD: Defatted soybean meal FDM: Defatted soybean meal fermented with green beans

ND: Not Detected

3. Effects of FD and FDM on Cholesterol and Lipid Metabolism

1) Effect on weight gain and dietary intake

To confirm the lipid metabolism and cholesterol-improving effects of FD and FDM, the weight change of animals reared for 4 weeks with diets containing 1% cholesterol, lard 10% and sodium cholate 0.25% in SD rats is shown in Table 5 below. .

After 4 weeks, weight gain in each group was significantly higher in HF group than in control group. In the high fat diet, FD and FDM added body weights were lower than the HF group, but there was no significant decrease. Feed efficiency was also not significantly different between groups.

County ¹⁾ Weight loss
(g / 4 weeks) FER ²⁾ CON 155.90 ± 15.0 ^b 22.31 ± 2.08 ^ns HF 198.48 ± 23.65 ^a 28.02 ± 4.01 HF-FD 189.24 ± 18.42 ^ab 30.42 ± 4.34 HF-FDM 193.80 ± 20.08 ^a 27.30 ± 1.06

The values are mean ± S.D. (n = 6).

Values with a common superscript letter within the same column are not significantly different (ρ <0.05).

¹⁾ Groups are as shown in Table 2 above.

²⁾ FER: food efficiency ratio = body weight gain / food intake

 ns: not significant

2) Effect on long-term weight

The organ weight per unit weight is shown in Table 6 below.

In the experiment comparing the weight of organs in terms of weight per 100 g body weight, the liver weight was significantly increased in the HF group compared to the control group. This may be due to the cholesterol diet, and liver weight also appears to be increased due to the concentration of triglycerides and total cholesterol in the liver. In contrast, the HF-FD and HF-FDM groups showed reduced weight compared to the HF group, and in particular, the HF-FDM group showed significantly lower liver weight than the HF group. This reduces the fat deposition of soy by treating FD and FDM, resulting in reduced weight, and it appears that FDM, a fermented product with green beans, further reduces fat deposition in soy.

Lee et al. (Rhee, SJ; Park, HK Changes of lipid concentration and histochemical observation in liver of rats fed high fat diet.See J. Nutr . 1984, 17, 113-125.) If it is not normally discharged, it accumulates in the liver, and it is said that the liver is enlarged. Thrley et al. (Thrley, E .; Armstrong, NC; Wallace, JM; Gilore, WS; Mckelvey-matin, JV; Allen, TM; Strain, JJ Effect) of cholesterol feeding on DNA damage in male and female syrian hamsters.Ann. Nutr.Merab. 1990 , 43, 48-51.) also cause hepatomegaly in long-term feeding of cholesterol, resulting in excess fat intake and lipid metabolism It has been reported that fat deposits in hepatic tissue. This study also confirmed that fat was deposited in the liver tissues in the HF group compared to the control group.

However, the final weights of the spleen, kidney, epididymal fat and abdominal fat, except for liver, were not significantly different in all groups, but were highest in the HF group and the percentage of epididymal fat, which is considered as a measure of abdominal fat and abdominal obesity. Was not significant, but the weight was decreased in the FD and FDM groups.

(unit:%) CON HF HF-FD HF-FDM Soy sauce 3.25 ± 0.17 ^c 6.21 ± 0.60 ^a 5.56 ± 0.69 ^ab 5.60 ± 0.41 ^b spleen 0.20 ± 0.01 ^ns 0.28 ± 0.08 0.27 ± 0.06 0.27 ± 0.09 kidney 0.20 ± 0.01 ^ns 0.26 ± 0.08 0.27 ± 0.06 0.27 ± 0.09 Epididymal fat 0.81 ± 0.25 ^ns 1.15 ± 0.43 1.04 ± 0.20 1.04 ± 0.19 Abdominal fat 1.29 ± 0.63 ^ns 1.42 ± 0.43 1.28 ± 0.40 1.16 ± 0.26

The values are mean ± S.D. (n = 6).

Values with a common superscript letter within the same column are not significantly different (ρ <0.05).

ns: not significant

3) plasma triglyceride and cholesterol concentration

Changes in plasma triglyceride and cholesterol contents in plasma at weeks 2 and 4 during high-fat diet and FD- and FDM-ingested rats were measured after blood collection from the clavicle vein. .

(Lee, HJ Effects of dietary casein. Soy protein and mixed protein on body lipid components of rats.Master thesis, Sung-shin women's University, Seoul, 1986 Is known as. The plasma triglyceride content was significantly increased in the HF group compared to the control group, but the neutral lipid content was significantly decreased in the FD and FDM groups, especially in the FDM group fermented with green beans. It showed a low triglyceride content (FIG. 9A).

Plasma total cholesterol concentration was significantly increased in the HW group as compared to the control group in the 2nd week as shown in B) of FIG. 9, but in the 4th week FD and FDM added cholesterol content was significantly higher than the HF group Reduced.

Increasing LDL cholesterol increases oxLDL content, a very important risk factor for cardiovascular disease (Parthasarathyl, S .; Santanam, N .; Auge, N. Oxidized low-density lipoprotein, a two-faced janus). in coronary artery disease. Biochem. Pharmacol . 1998, page 56, 279-284.). Plasma LDL cholesterol content was significantly increased in all HF groups compared to the control group, but the FD and FDM groups in the high-fat diet significantly reduced the LDL cholesterol content in comparison with the HF group. Lowest LDL-cholesterol content in the diet group (Fig. 10 A)).

In contrast, the HDL cholesterol content in plasma was significantly increased in the HF group compared to the control group, HDL cholesterol was increased in the high fat diet and the FD group, and HDL cholesterol was significantly increased in the FDM group (Fig. 10B). )). HDL cholesterol is known to be effective in improving and preventing arteriosclerosis by lowering cholesterol in blood by transporting cholesterol accumulated in peripheral tissues or blood to cholesterol esters, and maintaining a negative correlation with LDL cholesterol content. (Lee, HJ Effects of dietary casein.soy protein and mixed protein on body lipid components of rats.Master thesis, Sung-shin women's University, Seoul, 1986 .; Fernandez, ML; Mcnamara, DJ Regulation of cholesterol and lipoprotein metabolism in guinea pogs mediated by dietary fat quality and quantity.J. Nutr. 1991, 121: 934-943.). Therefore, FD and FDM are expected to be involved in the prevention of hypercholesterolemia by decreasing the LDL cholesterol content increased by the high fat diet and increasing the HDL cholesterol content.

4) HMG-CoA reductase activity in liver tissue

Hepatic HMG-CoA reductase is an important regulator of cholesterol biosynthesis, and its inhibition is known to inhibit cholesterol synthesis in cells. HMG-CoA reductase inhibitors are widely used as hypocholesterolemic agents that can most effectively lower blood cholesterol, especially LDL cholesterol (Fujioka, T .; Nara, F .; Tsujita, Y .; Fukushige, J .; Fukami, M .; Kuroda , M. The mechanism of lack of hypocholesterolemic effects of pravastatin sodium, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, in rats.BBA . 1995 , 1254, 7-12.). In general, the mechanism by which satin-based drugs lower plasma total cholesterol and LDL cholesterol is known to inhibit the activity of HMG-CoA reductase in the liver, thereby inhibiting cholesterol biosynthesis, thereby promoting the expression of LDL receptor in the liver, thereby lowering blood lipid concentration. (Igel, M .; Sudhop, T .; vonBergmann, K. Metabolism and drug interactions of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins). Eur. J. Clin. Pharmacol. 2001, 57, 357-364 ; Paoletti, R .; Corsini, A .; Bellosta, S. Pharmacologicl interactions of statins.Atherosclerosis Supplements. 2002, 3, 35-40.).

The activity variation of HMG-CoA reductase is shown in FIG. 11. HMG-CoA reductase activity was significantly increased in the HF group compared to the control group, but significantly decreased in the HF-FD and HF-FDM groups. Increasing HMG-CoA reductase activity in high fat diet group is thought to be a result of high fat and high cholesterol diet, and the decrease of blood cholesterol content by FD and FDM addition is important for HMG, which is an important enzyme for endogenous cholesterol synthesis. This may be due to the inhibition of CoA reductase activity.

5) Effect on the lipid content of liver tissue

It is known that the intake of high fat or cholesterol causes abnormal lipid metabolism in the liver tissue, which increases the weight of the liver and increases the lipid and cholesterol content in the liver by the deposition of lipids.

Lipid content variation of the liver is as shown in FIG. Cholesterol content was significantly increased in the HF group compared to the control group, while HF-FD group showed a lower cholesterol level than the HF group, but the HF-FDM group significantly decreased the cholesterol content. In the case of triglyceride content, the content was increased in HF group and similarly decreased in HF-FDM group.

6) Effect on Fecal Lipid Concentration

Excretion of total cholesterol and triglycerides into feces is shown in Fig. It is shown in 13. The fecal cholesterol content was not significantly different in the HF group compared to the control group, but cholesterol excretion was significantly increased in the HF-FD and HF-FDM groups, and cholesterol concentration was the highest in the HF-FDM group. Neutral lipid concentration was increased in HF group compared with control group, but neutral lipid excretion was significantly increased in HF-FD group and HF-FDM group. Neutral lipid excretion was significantly higher in HF-FDM group. High. In other words, the addition of FD and FDM to the high fat diet increased the amount of total cholesterol and neutral lipid excretion in feces compared to the HF group.

Therefore, FD and FDM are thought to be a material that can be involved in improving lipid metabolism in the body by regulating the content of cholesterol and neutral lipid in the body.

7) Pathological changes of liver, abdominal fat and epididymal fat

Morphological analysis of liver tissues of each group is shown in FIG. 14. Compared with the control group, the HF group, which had high fat intake, had the most accumulation of fat globules and the largest size of fat globules. In contrast, the HF-FD group and the HF-FDM group were found to reduce the size of fat globules than the HF group. In particular, the HF-FDM group was observed to be more effective than the HF-FD group.

Morphological analysis of abdominal fat cells and epididymal fat cells of each group is shown in FIGS. 15 and 16, respectively. First, the size of abdominal adipocytes was larger in HF group than in control group, but in the HF-FD group and HF-FDM group, the size of adipocytes was reduced again. And HF-FDM groups were reduced than adipocyte size of HF group.

As a result, the fact that the deposition of lipids in hepatocytes or adipocytes due to the intake of high-fat diets is reduced by the addition of FD and FDM is thought to be due to the components of viscous substances and peptides produced during fermentation.

Figure 1 schematically shows the manufacturing process of the skim soybean meal fermentation and the manufacturing process of the skim soybean meal fermented with green beans.

Figure 2 is a bar graph showing the results of searching for HMG-CoA reductase inhibitory activity of 80% methanol extract of edible plants,

I is golden, II is Twill mushroom, III is curry flower, IV is Angelica, V is situational mushroom, VI is licorice, young chicory, young Molokia, young green bean.

FIG. 3 is a bar graph showing the results of evaluation of cholesterol adsorption degree for the group (FD-Mung bean.) In which green beans were added at 2.5%, 5% and 10% for FD and DSG, respectively.

Figure 4 is a bar graph showing the results of evaluating the HMG-CoA reductase inhibitory activity against the group (FD-Mung bean.) Added Mung beans 2.5%, 5% and 10% for FD and DSG, respectively.

5 is a bar graph showing the results of measuring the intracellular cholesterol content by each sample.

6 to 8 shows the results of apolipoprotein A I secretion from HepG2 cells by the group (FD-Mung bean.) In which Mung beans were added 2.5%, 5% and 10% for FD and DSG, respectively (FIG. 6), apolipoprotein A II secretion search results (Fig. 7) and apolipoprotein CIII secretion search results (Fig. 8).

9 and 10 show the high-fat diet and week 2 and 4 during the rearing of rats fed the group (FD-Mung bean.) Supplemented with 2.5%, 5% and 10% green beans for FD and DSG, respectively. Neutral lipid and cholesterol content changes in the field after the plasma from the clavicle vein is measured and shows the results of the separation, Figure 9 is for the triglyceride (A) and total cholesterol (B) content, Figure 10 Shows the HDL cholesterol (A) and LDL cholesterol (B) content.

Figure 11 shows the changes in HMG-CoA reductase activity in the liver after four weeks of dietary method according to each diet.

Figure 12 shows the lipid content of the liver after diet for four weeks according to each diet method.

Figure 13 shows the amount of excretion of total cholesterol, triglycerides by fractionation after dieting for 4 weeks according to each diet method.

Figure 14 shows the results of morphological analysis of liver tissues after dieting for 4 weeks according to each diet (H & E staining. X 400).

Figure 15 shows the results of morphological analysis of abdominal fat cells after 4 weeks of diet according to each diet (H & E staining. X 400).

Figure 16 shows the results of morphological analysis of epididymal adipocytes after 4 weeks of dietary treatment (H & E staining. X 400).

Claims (7)

Of mung Bacillus subtilis (Bacillus subtilis) fermented soybean meal, and the cleaning of Bacillus subtilis by subtilis (Bacillus food for improving cholesterol and lipid metabolism, comprising a mixture of fermentations by subtilis ). The method of claim 1 wherein the mixture of mung Bacillus subtilis (Bacillus subtilis) of defatted soybean meal fermented by the Bacillus subtilis (Bacillus subtilis ) foods for improving cholesterol and lipid metabolism, characterized in that it comprises 0.1 to 10 parts by weight based on 100 parts by weight of the fermented product. The method of claim 1 or 2, wherein the mixture of mung Bacillus subtilis (Bacillus subtilis) of defatted soybean meal fermented by the Bacillus subtilis (Bacillus Subtilis ) foods for improving cholesterol and lipid metabolism, characterized in that it comprises 0.1 to 5 parts by weight based on 100 parts by weight of the fermented product. The method of claim 1, The mixture is The mixture of the defatted soybean and mung bean powder increase, Bacillus subtilis (Bacillus subtilis ) foods for improving cholesterol and lipid metabolism, which are obtained by solid fermentation. The method according to claim 1 or 3, The mixture is Mixing 0.1 to 10 parts by weight of mung bean powder to 100 parts by weight of degreased soybean meal and sterilizing and cooking by adding distilled water to prepare a sterilized mung bean-added defatted soybean meal solution; In a sterile soy flour solution Bacillus subtilis (Bacillus subtilis ) at 40-42 ° C. for 46-48 hours Culturing to prepare a culture solution;  Foodstuff for improving cholesterol and lipid metabolism, characterized in that obtained by inoculating the culture solution in sterilized mung bean added skim soybean meal solution is fermented at 40 ~ 42 ℃ 22-24 hours. The food for improving cholesterol and lipid metabolism according to claim 1, wherein the mixture is a lyophilized product. The food for improving cholesterol and lipid metabolism according to claim 4, wherein the mixture is obtained by further performing a lyophilization process after the fermentation process.

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