KR100858280B1 - Manufacturing method of Monascus sp., powder, gel, rice with Genistein Combined Monacolin K - Google Patents

Abstract

The present invention relates to a method for culturing red yeast bacteria having Genistein containing Monacholine K using Genistin,

It is intended to maximize the economic value by obtaining high-priced Genistin by separating high-purity Genistin from soy or bean from high-density isoflavone using solubility.

In addition, GCM (Genistein Combined Monacolin K) impregnated with glycosides genistein and monacoline K was contained in the culture of Monascus sp. It is to have an excellent effect on improvement.

Genistin, Genistein, Monacoline Cay, Red Yeast, Beans

Description

Method for culturing red yeast bacteria with genistein containing monacholine K using genistin and manufacturing method of powder, gel and rice using the same {Manufacturing method of Monascus sp., Powder, gel, rice with Genistein Combined Monacolin K}

1 is a view showing a manufacturing process of the GCM according to the present invention

Figure 2 is a graph showing the serum cholesterol content by GCM content

3 is a graph showing the LDL-cholesterol content by GCM content

Figure 4 is a graph showing the HDL-cholesterol content by GCM content

The present invention relates to a method for culturing erythrocytes using Genistin and a method for producing Genistein containing Monacoline K. More specifically, Isoflavones are prepared from soybean powder or bean curd, and the Genistein obtained by using the same is obtained in Monascus sp. The present invention relates to a method for culturing red yeast bacteria having Genistein (hereinafter referred to as GCM, Genistein Combined Monacolin K) containing Monacolin K.

The diet of modern people is caused by vascular diseases such as arteriosclerosis and angina due to excessive cholesterol production and intake, and various adult diseases.In addition, cholesterol production suppression and hyperlipidemia using safe natural materials due to side effects such as drugs through chemical synthesis Development of therapeutics is urgent.

 As a solution to this, there is a growing interest in soybean bioactive substances. Soybeans include isoflavones, protease inhibitors, folic acid, dietary fiber, saponins, vegetable sterols and phenolic substances. Estrogen-like structure has been found to have a physiological function such as phytoestrogen. Isoflavone is a compound having a molecular formula of C 15 H10 O2. Isoflavone is a glycoside that is present in a natural state of 12 kinds in total such as Genistein, Daidzein, Glycine and glucose glycosides. It is a substance which exists, and most of these isomers are Genistein and Dyzezein.

Genistin, a non-glycoside of isoflavone, has little toxic effects and has various physiological activities such as selective inhibitor of PTK (Protein Tyrosine Kinase), Topoisomerase II inhibitor, estrogen role and angiogenesis inhibitor among protein enzymes, and HMG-CoA It has been found to have a beneficial effect on hyperlipidemia by appropriately regulating genes of reductase and low density lipid protein (LDL) receptors, which has attracted considerable attention in the medical field.

By the way, since only Geniestin is present in soybeans, various methods for extracting ingredients contained in soybeans have been developed. Among them, Korean Patent Laid-Open No. 2001-0060419 discloses a mixture of soybean embryos and soybean embryos and tofu sprouts. Although the method of controlling the pH as hot water extraction by using the method and separating the separated and extracted liquid through the decanter using the adsorption process and the general process, there are still problems such as efficiency and economical efficiency.

Meanwhile, in 1979, Professor Endo Akira of Tokyo University in Japan developed Monascus SPP, a natural substance that lowers cholesterol levels. By specifically inhibiting HMG-CoA reductase, a rate-determining step, the cholesterol concentration associated with low-density lipoprotein (LDL) is lowered, lowering blood cholesterol levels and increasing high-density lipoprotein (HDL) -cholesterol levels It is known to make.

Based on the various conventional techniques described above, an object of the present invention is to separate and purify a high content of isoflavones from the sewage produced as by-products in the tofu production process, and to separate high purity zenithine using solubility, resulting in low cost. As a result, it is possible to efficiently obtain expensive Zenithin.

Another object of the present invention, by injecting the obtained Genitine into the fermentation process of Monascus sp., Genistein (hereinafter referred to as GCM, Genistein Combined Monacolin K) containing Monacolin K It is to provide a culture method to contain.

A feature of the present invention for achieving the above object is to dissolve the genus erythrocytes by dissolving genistin 0.1 to 1% by weight in 50-85% ethanol and adding 0.01-0.2% by weight to a medium adjusted to pH 5-7. One zenithine is a method for producing zenithine, in which the erythrocytes added in liquid form contain monacoline K.

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The present invention also homogenizes the produced hongguk cell and the culture solution, and then extracted with 60-90% ethanol, and reflux-cooled and filtered with a filter to obtain genistein containing Monacholine K, and containing the Monacholine K Dissolve 1.5-3 g of lecithin in a solvent mixed with 2: 1 methanol for 1 g of genistein, remove the solvent, add 50-60 mL of water to the weight of the resulting material, and homogenize by high-speed stirring for 50-70 minutes. To prepare a suspension, 1-5 g of xanthan gum was completely dissolved in 40-60 mL of water to make a gel, and then the homogenized suspension and gelled xanthan gum were mixed with stirring to prepare a monacoline kay. It is characteristic in the manufacturing method of genistein containing.

Hereinafter, an embodiment of the present invention will be described in more detail.

First, look at the manufacturing method of the GCM according to the present invention, after extracting more than 50% isoflavones from soybeans and sesame, to obtain the genitine through the separation and purification process, and the above-mentioned zenithin cultured with the honggukyun genistein K containing Get

In addition, the above-mentioned GCM is formed in a gel form.

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1. GCM culture

(1) materials and strains

Experimental material was Genistin, which was isolated and recrystallized from isoflavones. Strain was used as Monascus pilosus (KCCM 60084), a red yeast bacterium distributed by the Korean spawn association.

(2) Red yeast  Culture medium selection

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The media most suitable for the production of erythrocytes were examined to select the most suitable media for this strain. The conditions of the medium are shown in Table 3 below, and the pHs were all adjusted to 6.0.

Kinds Composition (g / 100mL) Lean Medium (LM) Rice powder 3.0, sodium nitrate (NaNO ₃ ) 0.15, magnesium sulfate heptahydrate (MgSO ₄ · 7H ₂ O) 0.10, potassium dihydrogen phosphate (KH ₂ PO ₄ ) 0.25 YM Medium (YM) Yeast extract 0.3, malt extract 0.3, peptone 0.5, glucose 1.0 ZY Badge (GY Medium, GY) Glucose10.0, yeast extract0.2 Mizutani Medium (MM) Glucose 5.0, Peptone 2.0, Potassium Dihydrogen Phosphate (KH ₂ PO ₄ ) 0.8 Magnesium Sulphate Heptahydrate (MgSO ₄ · 7H ₂ O) 0.05, Potassium Acetate (CH ₃ COOK) 0.2 Sodium Chloride (NaCl) 0.1 Toya medium (TM) Glucose 10.0, yeast extract 3.0, malt extract 3.0, peptone 5.0

Table 3 shows the types and compositions of media.

Cultivation  And Main culture

Species culture medium was used by adding 100 mL of ultrapure water to Mizutani medium (hereinafter referred to as MM) and preserving strains for 7 days at 150 rpm in a 30 ° C incubator using the same medium. After activation, the cells were incubated for 7 days under the same conditions using a cap test tube containing 7 mL of the same medium.

In this culture, 100 mL of medium was added to a 250 mL Erlenmeyer flask and sterilized at 121 ° C. for 15 minutes, followed by a seed culture solution and incubated in an incubator at 30 ° C. for 10 days.

Growth rate and pigment content of bacteria

The growth rate of the bacteria was determined by removing the moisture from the cells grown in the medium with tweezers, and then measuring the biomass. The number of cells grown per 100 mL of the medium was expressed. The pigment content was pulverized culture medium and cells with a homogenizer for 5 minutes at 12,000rpm, 10mL was taken and 50mL of 95% ethanol was added for 15 minutes, the absorbance was measured at 500nm after filtration.

As a result of the proliferation of the hongguk strain, the weight of hongguk cultured at 30 ° C for 10 days was the highest as 26.9g / 100mL in MM, lean medium (LM), GY medium, GY) represents 15.5-17.2 g / 100 mL, respectively. In addition, YM Medium (YM) and Toya Medium (TM) showed 8.2-10.3g / 100mL, showing relatively low cell production. In this culture experiment was carried out using the MM medium.

(3) Choosing the Best Geninisine Form

To determine the morphology of Zenithine on GCM optimization, 0.5% dissolved in powder and 70% ethanol was added to the medium in the range of 0-1.0%, and 1-5 g of red yeast bacteria were added to 100 ml medium and placed in a 30 ° C incubator. Incubated for -14 days (10 days in the example of the present invention). As a result, as shown in Table 4, it was difficult to easily dissolve the powder, so that after the incubation, a certain amount of zenistine remained in the lower part, but when dissolved and added to 70% ethanol, it was dissolved in the medium and used directly in the culture of red yeast rice. It produced high pigments and aromas. As a result, the shape of the genistin was excellent in the liquid phase.

Fungi Addition form result Solubility Cell generation Pigmentation incense Monascus pilosus, 60084 Control - + + + Monascus pilosus, 60084 powder - ++ + ++ Monascus pilosus, 60084 Liquid + ++ ++ ++

Table 4 shows the results of the culture of red yeast bacteria (Monascus Pilosus, 60084) according to the form of the addition of Genistin.

(4) Selecting Geninisine Conditions

In order to examine the conditions for adding 70% ethanol of 0.1% geninisine, 0% was added in a range of 1.0%. The higher the purity of ethanol, the easier the solubility of genistin was, but the cells and pigments were produced until dissolution with 70% ethanol, while no culture occurred in the 95% addition section. The optimum condition of the amount of ethanol dissolved therein was measured by the growth rate of the bacteria and the pigment content.

 70% ethanol dissolved genistin added amount (mL) Cell growth rate (g / 100mL) Pigment Content 0 5 + 0.05 10 + 0.1 12 ++ 0.5 20 +++ One - -

Table 5 shows proliferation and pigment content according to the amount of genistein added.

As a result, it was found that Zenithine was the best culture in the section in which 0.5 mL was dissolved in 70% ethanol.

3. GCM  Manufacturing method

After homogenizing hongguk cell and culture, extract with 60-90% ethanol, reflux and filter to obtain GCM.

4. GCM  Experiment on efficacy

(1) Effect on dietary addition

To investigate the effects of GCM on hyperlipidemia in rats, male and female rats were fed 1% cholesterol and 0.3%, 0.6% and 3% GCM with special diets. Blood was collected after free diet for about 1 month to measure serum total cholesterol, HDL, LDL and triglyceride (TG), and the effect of GCM on weight gain and food efficiency ratio (FER) was confirmed.

Laboratory Animals and Diet

 SPF rats of SD (Sprague-Dawley) strains were purchased from Orient Co., Ltd. for 7 weeks of age. Seven-week-old SD was acclimated for one week and divided into five animals in each group. The diet was AIN-76 diet. Test substance was administered by mixing 1% cholesterol 0.3%, 0.6% and 3% GCM with feed. The test group consisted of 1% cholesterol administered group (HC), 1% cholesterol (+ ester) + 0.3% GCM (0.3% GCM), 1% cholesterol (0.6% GCM) (0.6% GCM) 1% cholesterol + 3% GCM (3% GCM).

Serum Lipid Analysis

Fasting for 1 hour before blood collection, anesthesia with ether (ether) was collected in the abdominal aorta, and left for 30 minutes at room temperature, centrifuged for 20 minutes at 4 ℃, 3,000rpm. Total cholesterol, HDL-cholesterol and triglyceride (TG) of serum were measured for absorbance using an enzyme-based kit (kit, Asan Pharmaceutical, Korea) [Perkinelmer, VICTO3 family) LDL-cholesterol (LDL-cholesterol) was calculated by Friedewald formula Total cholesterol- (HDL-cholesterol + TG / 5)].

Dietary Efficiency

Dietary intake was measured twice a week and body weight was measured once per week. The food efficiency ratio (FER) was calculated by dividing the weight gain during the three week breeding period by the dietary intake during the same period.

Statistical method

After verifying the significance through Analysis of Variance (ANOVA) using SAS, Duncan's multiple range test was performed.

The experimental results are as follows.

Mortality and Clinical Observation

There were no deaths or grossly clinical signs of animals from GCM intake during the study.

Serum cholesterol

As shown in Table 6, the total cholesterol (TC) content was significantly lower in the 3% GCM group, and the content of LDL-cholesterol was also the lowest in the 3% GCM group.

The content of triglyceride / TG was the highest in the HC group and there was no significant difference between the other groups. The content of HDL-cholesterol was significantly higher as the amount of the added additive increased.

TC LDL TG HDL mean SD mean SD mean SD mean SD HC 233.76 ^{a1 ) 2)} 28.28 193.50 ^a 10.35 63.68 ^{NS3 )} 9.98 19.88 ^b 2.15 0.3% GCM 182.03 ^b 28.03 149.85 ^b 12.08 50.79 9.10 26.50 ^a 3.70 0.6% GCM 148.27 ^c 11.73 117.86 ^c 16.36 49.82 4.14 31.18 ^a 5.09 3% GCM 134.20 ^c 7.81 101.88 ^c 12.08 49.73 10.98 30.34 ^a 3.21

In Table 6, the unit is mg / dl, NS means no significance, mean means an average value, and SD represents a standard deviation.

Weight change

As shown in Table 7, there was no significant difference between the groups.

In Table 7, the unit is g.

Food efficiency ratio: FER )

As shown in Table 8, there was no significant difference between the HC group and the 3% GCM-administered group, but the 0.3% GCM group and 0.6% GCM group were significantly reduced than the HC group.

Group FER ^{1 )} HC 0.247 ± 0.016 0.3% GCM 0.197 ± 0.019 0.6% GCM 0.225 ± 0.015 3% GCM 0.247 ± 0.022

In Table 8, FER is weight gain / diet intake.

(2) effect on oral administration

Laboratory Animals and Diet

SPF rats of SD (Sprague-Dawley) strains were purchased from Orient Co., Ltd. for 7 weeks of age. Seven-week-old SD was acclimated for one week and divided into five animals in each group. The diet was AIN-76 diet. Test substance was administered by mixing 1% cholesterol with feed. The test group consisted of the negative control group (NC), 1% cholesterol-treated group (HC), 1% cholesterol + 0.165 g GCM / kg (HC1 × GCM), 1% cholesterol + 0.330 g GCM / kg (HC2 × GCM), 1 % Cholesterol + 0.825 GCM / kg (HC5 × GCM) administration group.

Serum Lipid Analysis

Fasting for 1 hour before blood collection, anesthesia with ether (ether) was collected in the abdominal aorta, and left for 30 minutes at room temperature, centrifuged for 20 minutes at 4 ℃, 3,000rpm. Serum total cholesterol, HDL-cholesterol and triglyceride were measured by absorbance (Perkinelmer, VICTO3 family) using an enzyme-based kit (kit, Asan Pharmaceutical, Korea), and LDL-cholesterol was Friedewald type (HDL-cholesterol + TG / 5)].

Dietary Efficiency

Dietary intake and body weight were measured daily during the test period. The food efficiency ratio (FER) was calculated by dividing the weight gain during the test period by the dietary intake during the same period.

Statistical method

SPSS 12.0K was used to analyze all the data in the study by one-way batch ANOVA test.

The experimental results will be described below.

Mortality and Clinical Observation

There were no deaths or grossly clinical signs of animals from GCM intake during the study.

Serum cholesterol: TC )

As shown in FIG. 1, the total cholesterol content of the negative control group (NC) and the HC group treated with 1% cholesterol was 72.83 ± 7.59mg / dl and 231.33 ± 53.17mg / dl, respectively, about 3 times higher by the cholesterol diet. In the test groups of HC1 × GCM, HC2 × GCM, and HC5 × GCM, 181.41 ± 14.68mg / dl, 135.33 ± 11.07mg / dl and 114.40 ± 6.51mg / dl, respectively. There is a significant difference. In particular, the higher the concentration of the test substance tends to significantly decrease the serum cholesterol content.

Triglyceride ( Triglyceride  : TG)

The triglyceride content of the negative control group (NC) and HC group treated with 1% cholesterol was 72.43 ± 24.67 mg / dl and 45.95 ± 9.65mg / dl, respectively, which tended to be slightly decreased by cholesterol diet, but there was a significant difference between the two groups. none. In the test groups of HC1 × GCM, HC2 × GCM, and HC5 × GCM, 54.59 ± 22.01mg / dl, 28.41 ± 9.70mg / dl, and 26.20 ± 5.45mg / dl, respectively. There was a significant decrease in triglyceride content.

LDL-cholesterol content

As shown in FIG. 2, the LDL-cholesterol content of the negative control group (NC) and the HC group treated with 1% cholesterol was 20.59 ± 6.25 mg / dl, respectively, about 5 times increased by the cholesterol diet. In the test group of HC1 × GCM, HC2 × GCM, and HC5 × GCM, 144.82 ± 14.03mg / dl, 99.76 ± 8.46mg / dl, 77.49 ± 13.64mg / dl, respectively. There is a significant difference.

HDL-cholesterol content

HDL-cholesterol content is shown in Figure 3 negative control group (NC) and 1% cholesterol treated HC group 37.75 ± 4.32 mg /, respectively, 27.06 ± 8.61 mg / dl is slightly reduced by cholesterol diet There is a trend but no significant difference between the two groups. In the test group of HC1 × GCM, HC2 × GCM and HC5 × GCM, there was no significant difference from HC group as 25.67 ± 7.34mg / dl and 31.68 ± 8.24mg / dl, respectively.

Weight change

Body weight gain was not significantly different during the experimental period as shown in Table 9 (P> 0.05).

In Table 9 the unit is g.

Food efficiency ratio (FER)

Dietary efficiency was also not significantly different as shown in Table 10 (P> 0.05).

5. Preparation of Liposomal GCM

1 g of GCM and 2 g of lecithin were dissolved in a 250 mL round flask, and chloroform: methanol was dissolved in a 2: 1 (v / v) solvent, and then the solvent was removed by an evaporator. 47 mL of water was added thereto, followed by stirring at high speed for 60 minutes with a homogenizer to make a homogenized suspension. In another 250 mL beaker, 2 g of xanthane gum is completely dissolved in 48 mL of water to make a gel. The above materials are mixed with stirring to complete the 1% gel GCM.

Gel GCM thus produced can be used in a variety of functional cosmetics, osteoporosis therapeutics and the like.

In addition, the cultured red yeast bacteria and the medium may be dried to 110% or less moisture content in the oven to prepare a powder.

In addition, the rice is processed using the hongguk bacteria and culture medium having the GCM prepared as described above, look at the process, soaking the white rice soaked in water enough to immerse 8-12 hours to be more than 45% moisture content Then, after 10-20 minutes increase at 100-140 ℃, 5-10% by weight of the rice and the culture medium with GCM and GCM added thereto, incubated for 10-14 days in a 20-40 ℃ incubator to rice It is to contain the GCM component.

According to the present invention configured as described above, it is possible to maximize the economic benefits by establishing a separation technology of low-cost glycoside genistein by separation and solubility, and simultaneously having a non-glycoside isoflavone and monacoline K using Monukolin K. Acquisition of GCM will ensure the production of more efficient hyperlipidemic functional food production technology.

In addition to soybeans, it is possible to separate isoflavones, which are the main functional components of soybeans, from bean curd, which is a by-product of the tofu production process, which is important in terms of cost savings of raw materials and utilization of waste resources.

In addition, the generated GCM can be used as an agent for improving hyperlipidemia and heart disease through cholesterol inhibition, and is effective in treating osteoporosis through osteoclast promotion of monacoline K and osteoclast inhibition of genistein.

In addition, the genistin extraction process of the present invention is to separate the glycoside isoflavones by column chromatography, which is mainly used as a method for separating isoflavones from conventional legumes, and then Compared to the method of obtaining an aglucone isoflavone using an enzyme, a high concentration of glycoside isoflavone, zenistine, can be separated more effectively by using a difference in solubility of ethanol aqueous solution.

Claims (8)

delete delete delete Dissolve 0.1-1% by weight of Genistin in 50-85% ethanol and add 0.01-0.2% by weight to the medium adjusted to pH 5-7 to cultivate the red mussel bacterium so that the red mucus has genistein containing Monacholine K. A method for culturing red yeast bacteria having Genistein containing Monacholine K. The method according to claim 4, wherein the genistin is added in a liquid phase. Genistein with Monacholine K, characterized in that the honggyun bacteria having Genistein containing Monacholine K obtained in any one of claims 4 or 5 and the medium is dried in an oven to 110% or less moisture content. Powder manufacturing method having a. 1.5 g of lecithin was dissolved in a solvent in which chloroform: methanol was mixed in a volume ratio of 2: 1 with respect to 1 g of red yeast powder having Genistein containing Monacholine K, and the solvent was removed using an evaporator. 50-60 mL of water is added to the weight of the resulting material, and then stirred at high speed for 50-70 minutes to prepare a homogenized suspension. After dissolving 1-5 g of xanthan gum in 40-60 mL of water to make a gel, A method for producing a gel having genistein containing Monacholine K, characterized in that the homogenized suspension and gelled xanthan gum are mixed with stirring to prepare a gel. The white rice was immersed in water to immerse it in water for 8-12 hours to make the moisture content 45% or more, and then the mixture was cooked at 100-140 ° C. for 10-20 minutes, and then obtained in any one of claims 4 or 5. 5-10% of hongguk bacteria with genistein containing monacoline K and medium were added to the weight of rice, and cultured in an incubator at 20-40 ° C. for 10-14 days to have genistein. Rice manufacturing method.

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