KR101145181B1 - Black soybean fermentation by monascus sp. for production of body weight reducing material - Google Patents

Abstract

PURPOSE: A black soybean-fermented material using Monascaceae is provided to ensure anti-obesity activity without production of citrinin. CONSTITUTION: A method for preparing black soybean-fermented material with an anti-obesity activity comprises: a step of steaming black soybeans; a step of inoculating seed cultured liquid of M. purpureus, M. pilosus, M. ruber, or M. kaoliang into the steamed soybeans; and a step of fermenting. The black soybeans include steamed, germinated, or roasted black soybeans, or processed product thereof.

Description

Fermented black soybean using red yeast bacterium having anti-obesity activity and its preparation method {BLACK SOYBEAN FERMENTATION BY MONASCUS SP. FOR PRODUCTION OF BODY WEIGHT REDUCING MATERIAL}

The present invention relates to a black soybean fermented product using the red yeast bacterium having anti-obesity activity and a method for producing the same. More specifically, the present invention relates to a black soybean fermented product having anti-obesity activity without producing citrinin, which is a fungal toxin component obtained by inoculating and fermenting a red yeast cell culture to black soybean (black manta).

Red yeast rice (red koji) is prepared by inoculating grains of the genus Monascus, a red filamentous fungus. The bacterium has long been used as a natural food colorant and processed product in various parts of East Asia, mainly in China, as a material for promoting digestion and improving blood flow.

Recently, the second metabolite produced by Rhesus bacteria, mebinolin (or lovastatin, monacolin K, and mebacor, C ₂₄ H ₃₆ O ₅ ), is a cholesterol biosynthesis HMG-CoA ( 3-hydroxy-methyl-3-glutaryl-coenzyme) strongly inhibited reductase and reported various functionalities due to lowered lipid concentration and cholesterol synthesis (Wang IK et al., J. Agri. Food Chem. 48: 3183 -3189 2000; Endo A. et al., J. Antibiotechnol. 38: 420-422 1985; Manzoni M & Rollini M. App.Microbiol.Biotechnol. 58: 555-564 2002; Wei W. et al., J. Nutri. Biochem. 14: 314-318 2003).

However, the genus Monasker is a mycotoxin such as citrinin, which is known as a poison component of kidney and liver, along with pigment components such as monascorubramin and monascin, in addition to mevinolin as a metabolite. (Blanc PJ, Loret MO, Goma G. Biotechnol. Lett. 17: 291-294 1995; Wang YZ, Ju XL, Zhou YG. Food Microbiol. 22: 145-148 2005). This can impede the widespread use of the Monascus strain.

That is, the production of these secondary metabolites is via the same polyketide pathway initiated by acetyl-coA and malonyl-coA in cells. Biosynthesis (Hendrickson L. et al., Chem. Biol. 6: 429-439 1999; Shinizu T. et al., Appl. Environ. Microbiol. 71: 3453-3457 2005).

Recently, Wang et al. Measured the amount of citrinin in cultures of several species of Monascus. As a result, Monascus strains were detected in all samples examined at a maximum of 480 mg / L and at least 65 mg / L. The necessity of safety evaluation of all products using WP (Wang YZ, Ju XL, Zhou YG. Food Microbiol. 22: 145-148 2005) was raised.

Citrinin is a fungal metabolite that was discovered in 1931 and has been known as a fungal toxin that affects the function of the kidneys and liver. In particular, it was separated from penicillium citrinum and named as citrinin, but its presence was also present in the metabolite of Aspergillus or Monascus fungus 0.065-0.48 mg / Known as ml.

Until now, most of the culture of red yeast rice was made by submerged fermentation of liquid culture, and mainly, optimum fermentation conditions, strain detection, and production of functional products containing red yeast rice have been studied. Solid state fermentation has also been attempted as a means of obtaining some functional ingredients, including pigments, but the main grains used in the medium were mostly rice, barley and wheat were used, and soybeans were successfully fermented by S. aureus. It is known that grains that are not suitable for fermented red yeast rice are unprecedented.

Korean Patent Registration No. 10-0964587

The present invention has been proposed to solve the conventional problems as described above, the object of the present invention is to inoculate hongguk bacteria to black beans having excellent antioxidant activity by fermentation through a solid fermentation method, does not produce the toxin component citrinin It is to provide a method for mass production of a substance having a safe anti-obesity effect.

The present invention Pseudomonas coarse fur pure mouse (M. purpureus), Pseudomonas coarse Philo Versus (M. pilosus), Pseudomonas coarse louver (M. ruber) and Pseudomonas coarse car up hem (M. kaoliang) one or more selected from the soybean honggukgyun It provides a black bean fermented product having anti-obesity activity obtained by inoculating and fermenting the seed culture solution.

The red yeast bacillus may be M. pilosus KCCM 60084, M. pilosus IFO4480, M. ruber ATCC22080 or M. kaoliang KCCM 60154 have.

The black beans may be selected from the group consisting of black beans, crushed black beans, germinated black beans, roasted black beans, black beans mixed with whole wheat and processed products thereof.

In another aspect, the present invention,

Add black beans; And

Monascus Anka, M. purpureus , M. purlos , M. pilosus , M. ruber and Monascus kaolin M. kaoliang ) provides a method for producing a black soybean fermented product having anti-obesity activity comprising the step of inoculating and fermenting one or more hongguk species culture medium selected from.

The fermentation step may be a step of fermenting 10 to 40 days at 20 ~ 30 ℃ by adding to the black soybean seed culture medium to 1-10% by weight.

The hongguk species culture medium can be cultured by inoculating the honggukyun cultured in potato agar medium (PDA medium) in a liquid nutrient medium.

The composition of the liquid nutrient medium is a rice flour 2.0g, glucose 3.0 g, peptone _{2.0 g, KH 2 PO 4 0.8} g, MgSO 4? 7H 2 O 0.05 g, sodium nitrate, 0.2 g, NaCl 0.1 g, d- water 100 mL It may be a preparation ratio of and the medium of the initial pH 6.0.

Black bean (black tae) is a traditional soybean called Odu (한) in oriental medicine. It is known to be effective in weakening constitution, lethargy, relieving anxiety, preventing each disease, drug addiction and food poisoning. According to the agreement, black soybean seed nourish blood, communicate wind, improve vision, clear the brain and treat headache.

   In addition, black soybean is one of the food materials with high utility value, which contains a lot of antioxidant substance called anthocyanin, which contains a lot of bioactive ingredients for disease prevention and treatment functions other than nutritional function.

In the present invention, the black soybean, which has not been attempted until now, attempted the fermentation of red yeast bacteria having an anti-obesity effect without producing the fungal toxin component citrinin. First, a superior strain was selected using the strain of Monascus, and it was confirmed that citrine was not detected in the liquid nutrient medium, and the anthocyanin content instead of the rice which was previously used most as a substrate of red yeast fermentation as a natural medium. Solid fermentation was performed using high black beans. However, black soybeans may use whole wheat in addition to the use of liquid culture medium to overcome the disadvantage of very high protein content but very low starch sugar content compared to grains such as rice.

Obesity is a condition in which fat tissue is excessively accumulated in the body. Obesity is the leading cause of serious adult illness, including diabetes, arteriosclerosis, cardiovascular disease, hypertension, and hyperlipidemia. Research into the causes of obesity, treatment and prevention of obesity is being actively conducted around the world, especially research on anti-obesity functional foods.

 Black beans contain many antioxidants called anthocyanins. Anthocyanin is a pigment that is contained in black soybeans and is known to have antimicrobial, antioxidant, anticancer and anti-obesity effects.

There is also a report stating that black soybean peptides are effective in obesity (domestic patent application 10-2007-0133156).

Black soybean fermentation product using red yeast bacteria according to the present invention has the effect of providing a fermentation product having anti-obesity activity without producing the toxin component citrinin.

Figure 1 shows the total polyphenol content of black soybean fermentation with different fermentation methods and conditions, the data is the average value and standard deviation of three measurements.
Figure 2 shows the electron donating activity (DPPH scavenging ability) of the black soybean fermentation with different fermentation methods and conditions, the data is the average value and the standard deviation of three measurements.
Figure 3 shows the SOD-like activity of the black soybean fermentation with different fermentation methods and conditions, the data are three times the average value and the standard deviation.
Figure 4 shows the difference in the monacholine content of black soybean fermentation with different fermentation methods and conditions, the data is the average value and standard deviation of the measurement results of three replicates.
Figure 5 shows the effect of obesity in the diet of the high-fat diet animal model, the data is the mean value and standard deviation of the measurement results of three replicates.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, the following examples are merely to illustrate the present invention is not limited to the following examples.

Example 1 Materials and Methods

Monascus pilosus KCCM 60084, Monascus pilosus IFO4480, Monascus ruber ATCC22080, and Monascus kaoliang KCCM 60154 were used. Black beans and whole wheat were harvested and used in the silver harvest in 2010.

Example 2 Spawn Culture

First, all the strains were incubated at 28 ° C. for 4 days using PDA medium (potato dextrose agar, Difco, MI, USA), and then S-INAE medium (rice flour 2.0 g, glucose 3.0 g, peptone 2.0 g, KH ₂ PO _4). In a cap test tube containing 0.8 g, MgSO ₄ ˜7H ₂ O 0.05 g, sodium nitrate 0.2 g, NaCl 0.1 g, d-water 100 mL, pH 6.0), the above-mentioned strains were implanted into a shaking incubator at 30 ° C. 150 rpm) was incubated for 10 days, and then inoculated in 1 L of the same medium and maintained at 30 ° C. in a Jar Fermenter (Value kio-Reactor, kiotron, Korea) for 10 days and used as a starter.

Example 3 Preparation of Hongguk Black Soybean Fermented Product

After washing black beans, immerse them in 1 ~ 3 times of water for 10 ~ 16 hours, then remove excess water and then increase or increase the soaked beans by crushing them. Black beans were prepared using a method of increasing the amount of 60% of steaming or bean soaking, sprouting, pulverizing, and cooking.

5 to 10% of red yeast seed cultured in S-INAE medium was added to the sample prepared as described above, fermented at 24 to 30 ° C. for 10 to 50 days, and then hot-air dried at 40 to 60 ° C. to grind red fermented soybean black fermented product. Got it.

As a control, the black bean was cooked for 90 minutes without adding water to the soy flour pulverized into a 100 mesh size.

Fermented products obtained according to various fermentation methods and conditions are shown in Table 1 below.

[Table 1]

Example 4 Hot Water Extraction

The hot water extract was prepared with the black soybean fermented product obtained in Table 1, and was used as a total polyphenol and flavonoid content and an assay for antioxidant activity. Hot water extraction used a method known in the art.

Example 5 Total Polyphenol Content

Polyphenols have a strong antioxidant power, which prevents or cures various diseases caused by oxidation of lipid components in human tissues. Therefore, the present invention was prepared by fermenting erythrocytes using black soybeans containing a lot of anthocyanins known to have strong antioxidant effects, anti-cancer and anti-obesity effects, and investigated the total polyphenol content.

According to the method of Dewanto et al. (Dewanto V, Xianzhong W, Liu RH. 2002. Processed sweet corn has higher antioxidant activity.J Agric Food Chem 50: 4959-4964.) 2% in 100 μL of the solution, 2 mL of sodium carbonate and 50 After adding 100 μL of% Folin-Ciacalteu reagent, the absorbance was measured at 720 nm, and the content was calculated by the calibration curve of gallic acid (Sigma-Aldrich, St. Louis, MO, USA).

As a result, as shown in Figure 1, the total polyphenol content of the black bean fermentation according to the present invention was higher than the control group B3. Specifically, the total polyphenol content was 30 mg or more per g of fermentation in B2M3, B2M4, B3ME, and B3MG, respectively, and 20 mg or more in B1M2, B1M3, B1M4, B2M1, and B3MH, respectively, and B1M1, B3, B3MD, B3MF, and MPM. In case of more than 10 mg appeared.

Example 6 electron donating ability

Electron donating ability is used as an indicator to prevent oxidation by the binding of hydrogen ions (H +) to active oxygen radicals in vivo, the higher the value shows higher antioxidant activity.

0.4 mM DPPH (1,1diphenyl-2-picryl-hydrazyl) solution in 0.2 mL of the sample extract according to Blois (Blois MS. 1958. Antioxidant determination by the use of a stable free radical.Nature 181: 1198-1200.) 0.8 mL was added, mixed with vortex, left for 10 minutes, and the absorbance was measured at 520 nm. The activity was calculated by the formula, electron donating ability (%) = 100-[(OD of sample / OD of control) x 100].

Figure 2 shows the change in the electron donating ability of the black soybean fermentation according to the present invention, the electron donating ability of the hot water extract of black soybean fermentation per mL showed the lowest tendency in B3 used as a control. This suggests that strong electron donating ability in fermented black soybean extract can be a good material for anti-obesity and liver function improvement effect.

Example 7 Superoxide Dismutasee (SOD) -like Activity

SOD is an enzyme that is involved in the conversion of free radicals into hydrogen peroxide, produced by XO or in drug abuse, ultraviolet light, smoking, fatigue, and various diseases. The activity exhibited by a substance that is not an enzyme but has the same activity as SOD in vivo is called SOD-like activity. Hydrogen peroxide produced by this activity also undergoes strong acid oxidation, but becomes toxic by conversion to water by catalase present in the body. Therefore, SOD-like activity is also used as an indicator against oxidative disorders.

According to the method of Martin et al. (1987), a predetermined amount of a sample was added to 2.94 mL of 0.1 mM EDTA-containing 50 mM phosphate buffer (pH 7.4), warmed at 25 ° C. for 5 minutes, and then reacted for 5 minutes with 5 mM hematoxylin solution to measure absorbance at 560 nm. . Activity was calculated as 1 unit to inhibit the automatic oxidation of hematoxylin 50%.

In Figure 3 was investigated the SOD-like activity of black soybean fermentation extract using Monascus fungus. As a result, there was no SOD-like activity in the control group, and showed the highest activity in B2M2 and B3MG. This phenomenon indicates that a new SOD-like activity is produced during black bean fermentation.

In B2M2, the total polyphenol content was slightly decreased compared to B2M3, B2M4, and B3MG, but electron donating ability and SOD-like activity were almost equal or higher.

The SOD (superoxide dismutase) -like activity per mg / mL of black soybean fermentation was slightly lower than that of B2M2, B3ME, B3MH, or MPM, but the activity per mg of polyferol was significantly higher than that of MPM. .

Example 8 Extraction and Quantitation of Monacholine-K from Fermented Black Soybean

Monacholine-K was extracted from the sample of Table 1 and its content was measured and compared. Since monacholine-K is a substance that inhibits cholesterol synthesis, a high content of this substance is considered to be high in obesity inhibitory activity.

8-1: Test Method and Analyzer Conditions for Extracting Monacholine-K

Sample pretreatment conditions and analyzer conditions for the content test were tested under the conditions described in the criteria and test methods set forth in Table 2 below.

TABLE 2

8-2: MK and MKA Content Test in Fermented Black Soybean Extract

Preparation of Linearity Test Solution (MK):

The test solution was prepared by accurately taking 5 mg of Monacolin K lactone form (ChromaDex Inc. USA) and using 50 mg of MeOH as the standard solution. A standard solution (100 μg / ml each) was diluted with MeOH to prepare 6 concentrations of test solution. The concentrations in the test solution were 24.54, 9.82, 4.91, 2.45, 1.23, 0.49 μg / ml. The test solutions were each filtered through a 0.45 μm membrane filter and 20 μl were taken and analyzed by injection into HPLC.

 Preparation of MKA by akaline hydrolysis of MK Standards:

Preparation of Linearity Test Solution (MKA):

The test solution preparation was made into 100 g / ml of the solution (MK) obtained by reaction, and it was set as the test solution. A test solution (100 μg / ml each) was diluted with 50% EtOH to prepare a test solution at 7 concentrations. The concentration in the test solution was 24.27, 9.71, 4.85, 2.43, 1.21, 0.49 μg / ml. Each test solution was filtered through a 0.45 μm membrane filter and 20 μl was taken and analyzed by injection into HPLC.

Figure 4 shows the result of measuring the content by extracting Monacholine-K. As shown in Figure 4, Monacholine-K was not detected in the control group B3. In FIG. 4, indicated by M-2 is inoculated with 10% by weight of 10% by weight of seeded Monascus pilosus KCCM 60084 incubated at 28 ° C. for 30 days. Rather than using this method, it can be seen that monacoline-K is more detected in fermented black soybeans using the present invention.

Example 9 Obesity Effect in Experimental Animals

9-1: Experimental Animals and the Basic Diet

   In order to investigate the effect on the anti-obesity effect of black soybean red yeast ferment prepared in Example 3, the experimental animals were born on May 9-10, 2011, the average weight of 21-24 g, CrljOrli; CD1 (ICR ) SPF / VAF outbred mice were purchased from Orient Bio Co., Ltd. (Seongnam-si, Gyeonggi-do, Korea). The basic diet is 5L79 Rat / Mouse Formula 18% (PMI Nutrition LLC, Po Box 19798 Brentwood Mo63144, USA), crude porotein (minimum) 18%, crude fat (minimum 5%, fiber (maximum) 5%, ash (maximum) 8% and carbohydrate 64% were used.

9-2: Experiment

Experimental animals adapted to the environment for 7 days were randomly divided into 6 groups, which were normal group (basic feed group: NC), and high-fat diet control group (37.54% of Raad in the basic diet, and fat contained in the basic feed: HC ), HC + Biji feeding group (37.54% Ride and 2% biji: Biji), HC + MA feeding group (37.54% Ride and B. inoculated with 10% by weight of Monascus pilosus KCCM 60084 spawn incubated at 28 ° C for 30 days It was divided into five groups: 2% fermented products: MA), HC + B3MG-treated group (37.54% of the Ride and 2% B3MG fermented products: B3MG). Table 3 shows the components of Biji, MA, and B3MG in terms of fiber.

  [Table 3]

9-3: Breeding Method

Experimental diet was 50g at 10 am every day except for the remaining amount was the dietary amount, water was supplied daily tap water. The diet of the experimental group was prepared for the amount to be supplied for 10 days, and kept fresh at 4 ° C. while refrigerated. The kennel was made of stainless steel, and the temperature and humidity were adjusted to 22 ± 2 ° C and 60 ± 5%, and the contrast was maintained from 6:00 Am to 6:00 Pm.

9-4: Weight gain, dietary intake, drinking water intake and dietary efficiency

   Body weight, diet and drinking water intake were measured at regular times every day throughout the entire experimental period. The food efficiency ratio (FER) was calculated by dividing the weight gain during the same period by the food intake during the same period.

9-5: Statistics processing

All experimental results were expressed as mean and standard deviation of 6 experimental animals, and significance test was performed using Duncan's multiple range using the Statistical Package for Social Sciences, Version 12, SPSS Inc., Chicago, IL, USA test was performed.

9-6: weight gain, dietary intake, dietary efficiency and defecation

   Obesity-induced body weight change during the seven weeks of breeding the experimental animals according to the dietary composition and experimental classification of Table 2 is shown in FIG. The results of the weight gain, dietary intake, and dietary efficiency are shown in Table 4 below. During the seven-week breeding period, the normal control group (NC) continued to gain weight, while the obese control group (HC) fed a high-fat and high-cholesterol diet after obesity induction showed a rapid increase until four weeks, but since then, weight gain has been slow. Biji group gained weight from the beginning of salary and showed significant weight gain at about 4 weeks, but since then, the difference with HC group began to narrow and showed similar weight to HF group at 7 weeks. It was. However, the red yeast fermented black soybean group (B3MG) showed similar weight gains to the HF group until 2 weeks, but began to show a difference afterwards, and showed similar weight to the NC group at 7 weeks. The average weight gain per week calculated from the difference between the seventh week weight and the initial weight was 4.66 g in HC group, and 3.77 g and 3.75 g in fermented BJG and BJG, respectively, compared to HC group. Reductions of 19.1% and 19.5% were observed, and for B3MG, 3.25 g showed a 30.3% reduction. In the case of B3MG, the average daily dietary intake was 31.63 g in the NC group, but there was no significant difference in the HC, Biji and MA groups from 27.38 to 34.98 g.

In the present invention, the B3MG group showed a higher weight loss rate than the B3MG group, although the HC3 and Biji and MA groups did not show any significant difference in dietary intake or defecation.

TABLE 4

9-7: Liver weight and abdominal fat content

Table 5 shows the results of measuring the liver weight (%) and the abdominal fat content of the rats fed the experimental diet for 7 weeks after inducing obesity with a cholesterol-containing high fat diet. Liver weight (%) per body weight was significantly increased in all experimental groups compared to NC group, and B3MG was significantly decreased in comparison with HC group.

TABLE 5

In the above experimental results, especially in the B3MG group, compared to the HC group, the significant reduction in weight gain and dietary efficiency as well as weight per liver weight (%) without lowering the dietary intake, suggesting that the extract of Hong Kong bacteria can reduce and prevent obesity. Able to know. In other words, fermented black soybean fermented with excellent anti-obesity effect by fermenting black soybeans with red yeast.

Claims (8)

delete delete delete Add black beans; And
The increase in the soybean Pseudomonas coarse fur pure mouse (M. purpureus), Pseudomonas coarse Philo Versus (M. pilosus), Pseudomonas coarse louver (M. ruber) and Pseudomonas coarse car up hem (M. kaoliang) one or more selected from honggukgyun Inoculating and fermenting the seed culture solution;
Including steps
The hongguk species culture medium is a method for producing a black soybean fermentation product having an anti-obesity activity, characterized in that the culture was inoculated in a liquid nutrient medium (S-INAE) cultured in potato agar medium (PDA medium).
The method of claim 4, wherein the black bean is selected from the group consisting of black beans, crushed black beans, germinated black beans, roasted black beans, and processed products thereof.
The fermentation step of the black bean fermentation having anti-obesity activity, characterized in that the fermentation of the hongkuk seed culture medium in a weight ratio of 1-10% by weight to the black soybean for 10 to 40 days at 20 ~ 30 ℃ Method of producing water.
delete The method of claim 4 wherein the composition of the liquid nutrient medium (S-INAE) is rice flour 2.0 g, glucose 3.0 g, peptone _{2.0 g, KH 2 PO 4 0.8} g, MgSO 4? 7H 2 O 0.05 g, sodium nitrate 0.2 g, NaCl 0.1 g, d-water 100 mL preparation ratio and a method for producing a black bean fermented product having anti-obesity activity, characterized in that the medium of the initial pH 6.0.

Images