KR101097838B1 - A method for preparing fermented soymilk having improved good sensory property and Lipid lowering ability by Bacillus subtilis and Corni fructus, and fermented soymilk for prevention atherosclerosis obtained from this method - Google Patents

Abstract

The present invention relates to a method for producing fermented soy milk with improved functionality and lipid lowering ability using Bacillus subtilis and Corni fructus , and to fermented soy milk for preventing atherosclerosis obtained therefrom. Fermented soy milk may be used as a health functional food that prevents arteriosclerosis due to plasma lipid lowering, liver lipid lowering, and arterial lipid suppression effect.

Fermented soy milk, functional, lipid lowering, plasma lipid, liver lipid, lipid deposition

Description

A method for preparing fermented soymilk having improved good sensory property and Lipid lowering ability by Bacillus subtilis and Corni fructus, and fermented soymilk for prevention atherosclerosis obtained from this method}

The present invention relates to a method for producing fermented soy milk with improved functionalities and lipid lowering ability using Bacillus subtilis and cornus milk, and to a fermented soy milk for preventing atherosclerosis, specifically, to fermented soy milk treated with Bacillus subtilis and cornus milk. The present invention relates to a technology for confirming plasma lipid lowering, liver lipid lowering, and inhibiting arterial lipid deposition and providing a functional food for preventing atherosclerosis.

Cheonggukjang is a fermented food source of fermented foods in which soy protein is decomposed by the action of enzymes produced by Bacillus natto or Bacillus subtilis . Recently, it is known that Cheonggukjang has not only a protein source but also a health function, and these functions include antithrombotic activity (1), weight loss and blood pressure lowering effect (2) blood sugar lowering (3) antioxidant effect (3) and stone Mutagen suppression (4) and the like have been reported. However, Cheonggukjang is boiled and eaten, so there is a limit to always intake in everyday life. Recently, Cheonggukjang products have been developed in the form of powder or pills, and people are taking these products with water or milk. However, because the Cheonggukjang powder is not soluble in milk, we want a product that solves this problem.

On the other hand, soy milk does not contain cholesterol, lactose, gluten, etc. compared to milk has attracted the attention of people who have problems with milk intake, but because of the fishy smell of soybeans are less preferred than milk. The benefits of fermented milk have been reported to act as a probiotics in the intestine to promote health and longevity. Therefore, fermented soy milk is also thought to have a probiotic effect, and many scholars say it is Lactobacillus or Bifidobacteria alone. Or fermented soymilk with mixed fermentation (2,5), and these fermented soymilk have antioxidant effects (6), cholesterol lowering effects (7), antimutagenic and anticancer effects (8), and immunomodulatory effects (1). It is reported that there is. Therefore, in the present invention, to eliminate the hassle of ingesting the Cheonggukjang powder or Cheonggukjang pill product with milk, and to prepare a functional Cheongguk soy milk having the same efficacy.

Meanwhile, technologies related to conventional fermented soy milk include Korean Patent Application No. 2006-0052516, Korean Patent Publication No. 2006-0001656, 2005-0095770, 2005-0053555, 2003-0097264, 2001-0023143 No. 1998-032749, 1998-070641, and the like. However, all of these techniques are focused on the preparation of fermented soymilk, and none of the literature teaches or discloses the lipid lowering effects of fermented soymilk.

references

1.Heo. S., Lee. SK. and Joo. H.K. : Isolation and identification of fibrinolytic bacteria from Korean traditional cheonggukjang. Agric. Chem. Biotechnol. 41: 119-124 (1998)

2.Yang. J.L., Lee. S.H. and Song. Y.S. : Improving effect of powders of cooked soybean and cheongguljang on blood pressure and lipid metabolism in spontaneously hypertensive rats. J. Korean Soc. Food Sci Nutr. 32: 899-905 (2003)

3.Kim. M.H. and Lee. J.H. : Antioxidant materials in domestic Meju and Doenjang. J. Korea Soc. Food Nutr. 23: 604-613 (1994)

4.Park. K.Y., Moon. S.H., Cheigh. H.S. and Baik. H.S. : Antimutagenic effects of Doenjang. J. Food Sci. Nutri. 1: 151-158 (1996)

5.Ryu Sh. : Studies on antioxidative effects and antioxidative components of soybean and chingkukjang. Doctoral thesis Inje University of Korea. pp 23-122 (2002)

6.Ryu. S.H., Lee. Y.S. and Moon. G.S. : Effects of salt and soysauce condiment on lipid oxidation in broloed mackerel (Scomber japonicus). Korean J. Food Sci. Technol. 34 (6): 1030-1035 (2003)

7.Kim. K.J., Ryu. M.K. and Kim. S.S. : Chungkook-jang koji fermentation with rice straw. Korean J. Food Sci. Technol. 14 (4): 301-308 (1982)

8.Kim. S.H., Yang. J.L. and Song. Y.S. Physiological functions of Chongkukjang. Food Industry and Nutrition, 4 (2): 40-46 (1999)

Therefore, the present inventors have studied to prepare fermented soy milk with excellent functional and lipid lowering ability, and have not only succeeded in preparing fermented soy milk with Bacillus subtilis, but also produced beverages with increased functionality and lipid lowering function by adding cornus milk. In addition to confirming that it is possible to determine the effects of blood lipid lipid, hepatic lipid, intraarterial lipid deposition in atherosclerosis mice, the present invention was completed.

That is, one object of the present invention to provide a method for producing a fermented soy milk having both functional and lipid lowering ability.

Another object of the present invention to provide a fermented soy milk as a health functional food for preventing atherosclerosis.

In a first aspect of the invention,

Provided is a method for preparing fermented soymilk with fibrin inhibitory activity using Bacillus subtilis and Corni fructus and having improved functionality and lipid lowering ability.

In the second aspect of the present invention,

Produced by the method of the first aspect, provides a fermented soy milk as a health functional food to prevent atherosclerosis by the plasma lipid lowering, liver lipid lowering, inhibiting the lipid deposition of the arteries.

Hereinafter, the present invention will be described in detail.

In the present invention, by using Bacillus subtilis and Corni fructus , fermented soybean milk with fibrin inhibitory activity and functional and lipid lowering ability can be prepared.

At this time, the subtilis bacteria and cornus milk are added so that the glucose content is 1% of the total weight, and then added to the sterilized soymilk for 15 minutes at 121-150 ° C. to be added in the order of cornus and subtilis bacteria.

That is, the concentration of the cornus oil is 10 ° Brix, the content is added to be 1% on the basis of the final concentration to improve the fibrin resolution and can also maximize the functionality.

In addition, the soymilk used may be commercially available soymilk, or selected soybeans, heat-treated, and hydrothermal grinding by adding hot water of 8 to 10 times the weight of soybean, heat-processing and extracting the soybean grinding, and then filtering it. Soymilk can also be obtained and then used.

In addition, by inoculating the Bacillus subtilis culture medium so that the Bacillus subtilis 7-8 logCFU / ml to improve the fibrin degradation and lipid lowering capacity can also be maximized. The above-described subtilis bacterium conditions are set to satisfy the conditions for fermented soy milk having a probiotic effect, in which the number of lactic acid bacteria of fermented soy milk should be at least 6 logCFL / mL and the number of these bacteria should be maintained for at least one week when refrigerated.

The Bacillus subtilis 2829PNU015 isolated from Cheonggukjang (Cheonggukjang, Sunchang, Kim Yong-soon) with excellent taste and antioxidant function was inoculated in a culture medium containing 0.8% sodium chloride to obtain shaking culture in a water bath at 37 ° C for 24 hours. desirable. Specifically, as described in the following examples, Bacillus subtilis 2829PNU015 was selected from the six products of Cheonggukjang in the Sunchang region was selected from the Cheonggukjang with high antioxidant capacity and functionalities after separating the bacteria. The process of producing fermented soymilk with Bacillus subtilis bacteria was established, and three kinds of Bacillus subtilis with high fibrin degradability were selected to prepare fermented soymilk and their antioxidant and fibrin degradability were compared. The Bacillus subtilis 2829PNU015 was deposited on March 26, 2009 (Accession No. KCCM 42923).

Then, in order to improve the functionality and functionality of Cheonggukjang soybean milk, inoculated with Bacillus subtilis soymilk added as a functional material, and lowered plasma lipid by fermenting for approximately 8 hours to reach pH 5.5 ± 0.2 at 37-40 ℃ The fermented soy milk for the prevention of atherosclerosis is improved, which lowers liver lipid and suppresses lipid deposition of arteries. In other words, apoE ^(-/-) mice were ingested with anti-arterial cured soybean soymilk for 8 weeks, and the effects of lipid lowering and atherosclerosis were compared with soymilk added with Chungkookjang powder.

As described above, the fermented soymilk prepared in the present invention may be used as a health functional food that prevents arteriosclerosis due to the effect of lowering lipid lipids, lowering liver lipids, and inhibiting lipid deposition of arteries.

Hereinafter, the present invention will be described in detail by way of examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

<Material>

-Cheonggukjang

Cheonggukjang purchased 6 finished products from Sunchang in August 2007 and used them as samples for physicochemical analysis and Bacillus isolates. As shown in Table 1, each manufacturer was S1: Moon Ok Rae, S2: Hyang Guk Woon, S3: Kim Young Sun, S4: Moon Neung Ja, S5: Oh Bok, S6: Oh Sun E.

-Soymilk

Bacillus sp. MYCO (Innobiz Myco) and KCCM (KCCM 11316), which are used as commercial strains, were purchased from Donghwa Food (Yangsan, Korea) for fermented soymilk production and used as ingredients.

- Bacillus bacteria

Bacillus subtilis 2805 PNU 014, Bacillus subtilis 2829 PNU 015, Bacillus subtili s 2825 PNU 016 isolated from Bacillus bacteria (see Table 1) isolated from six kinds of Cheonggukjang prepared in Sunchang area. Used in this study.

Chungkukjang
products Strains S1 Bacillus sp. 2811 PNU 010 Bacillus subtilis 2821 PNU 011 S2 Bacillus subtilis 2831 PNU 007 Bacillus licheniformis 2807 PNU 008 Bacillus sp . 2805 PNU 009 S3 Bacillus subtili s 2805 PNU 014 Bacillus subtilis 2829 PNU 015 Bacillus subtilis 2825 PNU 016 S4 Bacillus subtilis 2753 PNU 004 Bacillus subtilis 2843 PNU 005 Bacillus subtilis 2821 PNU 006 S5 Bacillus sp. 2748 PNU 001 Bacillus subtilis 2807 PNU 002 Bacillus sp. 2809 PNU 003 S6 Bacillus sp. 2815 PNU 012 Bacillus sp. 2805 PNU 013

-Functional material

It is supplied with extracts of purple sweet potato, black garlic, onion, fortified lion cucurbita, garlic, and cornus milk produced by Cheonho Foods (Busan, Korea), which is conducting joint research to produce customized fermented soy milk with enhanced function to prevent atherosclerosis. It was.

Experimental Method

Example 1 Preparation of Fermented Soymilk

Bacillus subtilis 2805 PNU 014, Bacillus subtilis 2829 PNU 015, Bacillus subtilis 2825 PNU 016 (P Univ., Busan, Korea) isolated from Cheonggukjang and MYCO (Innobiz Myco, Gyeong Ju, Korea) and KCCM (KCCM 11316) , Seoul, Korea) was inoculated in nutrient broth containing 0.8% sodium chloride, incubated in water bath at 37 ℃ for 24 hours, and then inoculated with 1% (CFU 10 ⁷ ~ 10 ⁸ ) in sterilized soymilk containing 1% glucose. Shake culture was performed at 40 ° C. for about 8 hours until pH 5.5 ± 0.2.

Functional fermented soymilk preparation is added to the sterilized soymilk so that the final concentration of 10 ^o Brix cornus milk is 1% and then inoculated with the same amount of Bacillus subtilis. The fermentation solutions are shaken at 40 ° C. until the pH reaches 5.5 ± 0.2. Normal time was about 8 hours. The resulting fermented soy milk was in a liquid phase with a higher concentration than soy milk.

Example 2. Bacillus  Fibrin Degradation Effect of Bacteria

The fibrin degradability of Bacillus strains was determined by fibrin plate method to select the best bacterium from among Bacillus subtilis 2805 PNU 014, Bacillus subtilis 2829 PNU 015 and Bacillus subtilis 2825 PNU 016 and two commercial bacteria isolated from S3 Cheonggukjang. Table 6).

Fibrin resolution of Bacillus subtilis 2829 PNU 015 was the highest among the five Bacillus strains tested with 27.2 mU plasmin / hr. The lowest thrombolytic ability was PNU 014 isolated from Cheonggukjang. Bacillus subtilis 2829 PNU 015 with high fibrin resolution was deposited on March 26, 2009 (Accession No. KCCM 42923).

Example 3 Selection of Functional Material with High Fibrin Resolution

  Fibrin degradability of the extracts of cornus, garlic, ripened garlic, purple sweet potato, and fortified wormwood, which were prepared and used as health functional foods, was observed and summarized in Table 2 below.

Specifically, cornus, garlic, aged village, purple sweet potato, and fortified mugwort concentrate were supplied from Cheonho Foods (Busan, Korea). Their concentration was adjusted to 10 ^o Brix and used for the experiment.

Fibrin degrading activity is described by Astrup et al. (Lee.SJ, Lee.KI, Rhee.SH and Park.KY: Phsiological activity in Doenjang added with various Mushrooms.Korean J. Food Cookery SCI. 20 (4): 365-370 (2004) Fibrin plate method of That is, 0.03 g of fibrinogen was added to 1 mL of 10 mM impression buffer solution, and 50 µl of 100 NIH thrombin dissolved in the same buffer was added to the solution completely dissolved at 37 ° C., and the mixture was shaken uniformly to allow the fibrinogen to gradually solidify with fibrin. The prepared plate was used for the experiment after leaving for 40 minutes at room temperature.

 Fibrin degradation activity of each sample was measured by dropping 50 μl of the sample on a previously prepared fibrin plate and incubating at 37 ° C. for 18 hours to determine the degraded area of each sample. As a control, plasmin (0.5 U / mL), a purified thrombolytic enzyme, was used. Fibrin degrading activity of each sample was represented by plasmin activity in proportion to the area of plasmin degradation.

Plant extracts ¹⁾ Fibrinolytic activity (PU / mL) Corni fructus 1.12 ± 0.20 ^a Garlic 0.28 ± 0.10 ^b Aged garlic 0.30 ± 0.05 ^b Purple sweetpotato 0.28 ± 0.05 ^b Pumpanini 0.78 ± 0.57 ^ab

As shown in Table 2, the highest fibrin degradability of cornus milk than garlic, ripened garlic, purple sweet potato, and fortified wormwood extract.

Example 4 Determination of Cornus Amount Added

In order to determine the concentration to be added during the fermentation of the selected corn cob in Example 3, corn cob at a concentration of 10 ^O Brix was added to the fermented soy milk by concentration, and the sensory test was performed on 30 people and summarized in Table 3 below.

For reference, the sensory test was conducted on 30 soybean milk, fermented soy milk, and Cheonggukjang powder for 30 graduate students in Pusan National University. Appearance, taste, aroma, texture, and preference were examined using a 9-point scale. One point is the worst case evaluation of each item and nine points is the best sensory result of the item.

Corni Fructus extracts Appearance Flavor Taste Texture Overall
acceptance One% 7.2 ± 0.9 ^a 4.3 ± 2.0 ^NS 5.6 ± 1.4 ^a 4.0 ± 1.8 ^NS 6.1 ± 1.3 ^a 2% 5.6 ± 1.3 ^b 4.3 ± 1.9 5.4 ± 2.0 ^a 4.5 ± 1.7 5.0 ± 2.1 ^ab 3% 3.9 ± 1.5 ^c 4.1 ± 1.8 4.2 ± 1.9 ^ab 4.1 ± 1.7 3.7 ± 1.9 ^bc 4% 3.5 ± 1.7 ^c 4.0 ± 2.1 4.7 ± 2.0 ^ab 5.0 ± 1.8 4.4 ± 2.0 ^bc 5% 3.2 ± 2.3 ^c 3.3 ± 1.2 3.3 ± 1.8 ^b 4.0 ± 1.5 3.1 ± 1.8 ^c

Values are mean ± SD (n = 30).

^ac Data were significantly different with one-way ANOVA followed by Duncan'smultiplerangetestatthe0.05levelofsignificance.

NS: not significant

As shown in Table 3, when the 10 ° Brix concentration of cornus oil was added at a concentration of 1% of the total weight, the best score was obtained in appearance, taste, and comprehensive evaluation.

Example 5 Cornus Milk Addition Method

Fermented soymilk containing Corni fructus (FSCC) and Corni fructus extract added fermented soymilk. ) Were compared with fermented soymilk and fermented soymilk (Cookkukjang powder (5%) added soybean milk CPAS), which is commonly used by the general public, and summarized in Table 4 below. .

Fermented
Soymilk Appearance Flavor Taste Texture Overall
Acceptance FS 7.4 ± 1.2 ^a 5.7 ± 1.3 ^ab 5.3 ± 1.5 ^a 5.1 ± 2.7 ^NS 5.5 ± 1.8 ^NS FSCC 4.2 ± 1.5 ^b 5.7 ± 1.6 ^a 5.8 ± 1.5 ^a 5.0 ± 1.6 5.8 ± 1.5 CAFS 4.1 ± 1.7 ^b 4.6 ± 1.0 ^ab 3.8 ± 1.3 ^b 5.2 ± 1.4 4.4 ± 1.2 CPAS 4.9 ± 2.7 ^b 4.1 ± 2.6 ^b 4.0 ± 2.5 ^b 5.8 ± 2.3 4.4 ± 2.2

Values were mean ± SD (n = 30).

^{a, b} Data were significantly different with one-way ANOVA followed by Duncan's multiple range test at the 0.05l evel of significance.

NS: not significant

FS: fermented soymilk with Bacillus subtilis 2829PNU015

FSCC: Fermented soymilk containing Corni fructus . The mixture solution of Corni fructus extract (1%, 10 ^o Brix) and soymilk fermented with Bacillus subtilis 2829

CAFS: Corni fructus extract added fermented soymilk. 1% Corni fructus extracts was added to the fermented soymilk with Bacillus subtilis 2829PNU015.

CPAS: Chungkukjang powder (5%) added soymilk

As shown in Table 4, the comprehensive evaluation score of the product fermented soy milk and cornus milk together was the highest. In appearance, it was darker than fermented soymilk, but the taste was improved by the addition of Cornus soybean, and the smell of Cheonggukjang was less. Therefore, the overall evaluation was the best. As a result, the method of adding cornus oil was found to be significantly better in smell and taste than in fermentation than in fermentation. In addition, it was confirmed that the odor and taste were significantly improved than the current Cheonggukjang powdered milk.

Example 6 DPPH Scavenging Effect of Functional Fermented Soymilk

The result obtained by comparing free radical scavenging ability of functional fermented soy milk fermented with Bacillus subtilis by adding 1% of 10 ^o Brix Cornus milk to soymilk compared to fermented soymilk without adding cornus milk is summarized in FIG. 1.

Specifically, 100 μL of methanol extracts of fermented soy milk and functional fermented soy milk and 100 μL of 60 μM 1,1-diphenyl-2-picrylhydrazyl (DPPH) were added to a 96 well plate, mixed and left for 30 minutes in a cow, followed by ELISA reader. Absorbance was measured at 540 nm using (10). Methanol extract of fermented soy milk was used to extract the lyophilized sample 10 times methanol at room temperature and then completely blow off the solvent. Methanol extract obtained for the experiment was used by dissolving the concentration in ethanol. DPPH scavenging effect is expressed as a percentage (%) compared to the control without the sample.

As shown in Figure 1, the DPPH scavenging effect of the functional fermented soy milk increased concentration-dependently and significantly higher than the fermented soy milk at each experimental concentration, confirming the functionality of fermented soymilk added with cornus milk.

Example 7 Lipid Lowering Effect of Functional Fermented Soymilk in Atherosclerotic Mice

(1) experimental animals

To investigate the effects of Chungkookjang soymilk on the prevention of atherosclerosis through plasma lipid reduction, apoprotein E deficient (apo E ^(-/-) ) mouse, a transformed atherosclerosis-induced animal model, was used as a central laboratory animal (Seoul). , Korea) and used for the experiment.

The experimental animals were acclimated to the laboratory environment for 1 week, and then the weight of each group was adjusted equally so that the control group, fermented soybean milk containing Corni fructus (FSCC) group, and chungkukjang power added soybean were added. milk, CPAS) group. Cheonggukjang powder added soymilk was used as a positive control to compare the lipid-lowering and anti-arterial effects of functional fermented soymilk. There were seven animals in each experimental group.

(2) Diet and Breeding

The diet used in the experiment was an atherosclerotic diet (Atherogenic diet, AD) to induce hypercholesterolemia and was prepared according to the AIN-76 diet preparation and used as a control diet.

Functional fermented soymilk (Fermented soymilk containing Corni furctus (FSCC)) prepared for animal experiments and soymilk (Chungkukjang powder added soymilk, CPAS) containing 5% of Cheonggukjang powder were added after freeze drying. The amount of FSCC and CPAS added is 20.8 mL / 100 g body weight / day, which is 10 times the 200 mL of adult (60 Kg) milk daily. The diet was prepared at weekly intervals (100 g standard, see Table 5).

The temperature of the animal breeding room was maintained at 20 ± 1 ℃ and relative humidity at 55 ± 5%. Animals were supplied with sufficient feed and drinking water, and diets were fed at the same time each day. The animal duration was 8 weeks. After 8 weeks of breeding, mice were fasted for 24 hours before sacrifice, anesthetized with ether, and then disinfected with 70% ethanol and then opened. After the abdomen was opened, blood was taken from the inferior vena cava and heart using a sterile syringe treated with heparin. After the blood was collected, the remaining blood in the organ was removed by perfusion with PBS solution. The collected organs were washed several times with PBS, and then water was completely removed using filter paper. Each organ was weighed and stored in a -70 ° C. freezer.

Organs were homogenized with a homogenizer (Polytron, PT-MR 3100, Switzerland) and used as samples.

Ingredients Group Control ¹⁾ FSCC ²⁾ CPAS ³⁾ Casein 20 20 20 Sucrose 44 44 44 Cholesterol 1.25 1.25 1.25 Lard 10 10 10 Cellulose 5 5 5 DL-Methionine 0.3 0.3 0.3 Mineral mixture 3.5 3.5 3.5 Vitamin mixture One One One Choline bibatrate 0.2 0.2 0.2 Corn statch 15 15 15 FSCC ²⁾ 20.8 mL ⁴⁾ CPAS ³⁾ 20.8 mL ⁴⁾

¹⁾ Atherogenic diet was prepared according to the guideline of AIN-76A to which 1.25% cholesterol and 10% lard added

²⁾ FSCC: Fermented soymilk containing Corni fructus . The mixture solution of Corni fructus extract (1%, 10 ^o Brix) and soymilk fermented with Bacillus subtilis 2829

³⁾ CPAS: Chungkukjang powder was added to the soymilk to be 5%.

⁴⁾ The amount of sample added to the diet was 10 times of the one serving of soymilk (200 mL) consumed by adults (60kg)

(3) plasma lipid lowering effect

Functional in vitro soybean milk, which was found to have a significantly high DPPH scavenging effect in vitro , was prepared and fed to genetically modified Apo E knockout mice for 8 weeks, and compared with the weight and weight of major organs. It summarized in 6.

Experimentalal
group ¹⁾ Body weight
gain (%) ²⁾ Relative organ weight (mg / body weight) Liver Heart Lung Kidney Spleen Testis Control 15.71 ± 3.11 ^NS 1.45 ± 0.24 ^NS 0.14 ± 0.02 ^NS 0.20 ± 0.03 ^NS 0.37 ± 0.07 ^NS 0.15 ± 0.07 ^NS 0.17 ± 0.01 ^NS FSCC 18.07 ± 6.99 1.61 ± 0.37 0.15 ± 0.04 0.18 ± 0.03 0.38 ± 0.06 0.13 ± 0.05 0.18 ± 0.02 CPAS 20.50 ± 5.88 1.47 ± 0.23 0.17 ± 0.03 0.20 ± 0.02 0.43 ± 0.07 0.15 ± 0.03 0.16 ± 0.02

Values are mean ± SD

NS: not significant

FSCC: Fermented soymilk containing Corni fructus . The mixture solution of Corni fructus extract (1%, 10 ^o Brix) and soymilk fermented with Bacillus subtilis 2829

CPAS: Chungkukjang powder (5%) added soymilk

¹⁾ Control group: mouse fed atherogenic diet (AD, 1.25% cholesterol diet),

FSCC group: mouse fed AD containing FSCC (33.3 mL / kg BW),

CPAS group: mouse fed AD containing CPAS (33.3 mL / kg BW)

²⁾ (final weight-initial weight) / final weight × 100

As shown in Table 6, the weight of the mice and the weight of the organs were not significantly different from those of the control group fed the atherosclerosis diet and the positive control group fed the soymilk containing 5% of the Cheonggukjang powder. Functional fermented soymilk was confirmed to have no problems with intake.

Furthermore, the results of comparing the lipid lipid concentrations of the mice ingesting functional fermented soymilk with the mice ingesting soymilk added with Cheonggukjang powder are summarized in Table 7 below.

Specifically, the collected blood was centrifuged at 3,000 rpm for 15 minutes at 4 ° C to separate plasma.

① Total cholesterol concentration measurement

Plasma total cholesterol concentration was measured by a kit reagent (Total cholesterol AM 202-K, Asan Pharmaceutical, Korea) for quantification by enzyme method (12). 3 mL each of the reaction reagents prepared (0.01 mL of enzyme reagent, ie AM 157S-2 dissolved in 1 bottle of enzyme reagent solution) were added to 0.02 mL of the sample, mixed with vortex, and reacted for 5 minutes in a water bath at 37 ° C. Was measured. Using the standard cholesterol solution, the absorbance was measured by the same method as described above, and then the absorbance value of the sample was substituted into the following equation to determine the cholesterol concentration of plasma.

Cholesterol(㎎/㎖100) = x 300

Cholesterol (mg / ml 100) = x 300

② Triglyceride concentration measurement

Triglyceride concentration was measured with a kit reagent for triglyceride measurement (Triglyceride AM 157S-K, Asan Pharmaceutical) (13). Experimental method was prepared in the same manner as the sample preparation for measuring total cholesterol. Using a triglyceride standard solution, the color was measured for 10 minutes in the same manner as above, and the absorbance at 540 nm was measured.

Triglyceride(㎎/㎖100) = x 300

Triglyceride (mg / ml 100) = x 300

③ Measurement of cholesterol concentration of high density lipoprotein

Cholesterol concentration in HDL cholesterol was measured using a kit reagent (HDL Cholesterol AM 203-K, Asan Pharmaceutical) by enzyme method. In the experimental method, 0.2 mL of the separated solution was added to 0.2 mL of the separated plasma, mixed well, and allowed to stand at room temperature for 10 minutes. After centrifugation at 3,000 rpm for 10 minutes at 4 ° C, 3 mL of the reaction reagents prepared by taking 0.1 mL of the supernatant were added. The standard solution was prepared in the same way as cholesterol, mixed with a vortex mixter, reacted at 37 ° C for 5 minutes, and absorbance was measured at wavelength 490 nm within 60 minutes. .

HDL-choesterol(㎎/㎖100)= x 50 x 2

HDL-choesterol (mg / ml 100) = x 50 x 2

④ Measurement of cholesterol concentration of low density lipoprotein

Cholesterol concentration of low density lipoprotein was calculated by Friedewald based on the following equation.

LDL-Cholesterol = Total Cholesterol-(HDL-Cholesterol + Triglyceride / 5)

Experimentalal
group ³⁾ Lipids (mg / dL) Total-c HDL-C LDL-C Triglyceride Control 1013.7 ± 228.6 ^a 43.6 ± 9.0 ^NS 930.3 ± 225.6 ^a 228.1 ± 92.8 ^NS FSCC 748.3 ± 107.3 ^b 61.0 ± 24.4 648.5 ± 124.3 ^b 193.8 ± 37.2 CPAS 861.3 ± 105.7 ^ab 44.3 ± 10.0 778.1 ± 102.9 ^ab 194.4 ± 72.0

Values were mean ± SD

NS: not significant

¹⁾ See the legend of Table 6

^{a, b} Data were significantly different with one-way ANOVA followed by Duncan's multiple range test at the 0.05 level of significance.

      As shown in Table 7, the plasma total cholesterol concentration of the functional fermented soymilk group was reduced by 26.2% compared to the control group who consumed the atherosclerosis diet (p <0.05), and about 15.0% significantly compared to the positive control group added to the Chungkookjang powder. It decreased to the enemy (p <0.05). Therefore, the concentration of low density lipoprotein also decreased functional fermented soy milk by 30.0% compared with the control group and 16.7% compared with the addition of Cheonggukjang powder as a positive control. The LDL-C lowering effect of the functional fermented soy milk group was significantly (P <0.05) compared with the control group, but there was no significant difference with the soymilk added with the Cheonggukjang powder. Thus, fermented soybean milk lowered plasma cholesterol and significantly lowered the concentration of low-density lipoprotein, a major cause of atherosclerosis.

(4) hypolipidemic effect

   The hepatic lipid lowering effect of functional Chungkook soybean milk was observed after 8 weeks ingestion in genetically modified Apo E knockout mice and summarized in Table 8 below.

Specifically, liver was added to a mixture of chloroform and methanol 2: 1, homogenized with a homogenizer, left for 2 hours, and centrifuged at 3,000 rpm for 30 minutes to use the supernatant as a lipid extract. The total cholesterol and triglyceride concentrations of the liver were measured by the same method as that used for the measurement of plasma lipids.

Lipids (mg / g tissue) Group ¹⁾ Total cholesterol Triglyceride Control 6.1 ± 1.0 ^a 47.5 ± 12.5 ^NS FSCC 4.9 ± 0.6 ^b 38.7 ± 12.6 CPAS 4.9 ± 0.8 ^b 36.7 ± 6.3

¹⁾ See the legend of Table 6

^{a, b} Data were significantly different with one-way ANOVA followed by Duncan's multiple range test at the 0.05 level of significance.

As shown in Table 8, the cholesterol lowering effect was observed as in the plasma, but the neutral lipid lowering effect was not observed. This effect showed a similar effect to the intake of the Chungkukjang powder added to soy milk.

(5) inhibiting lipid deposition in arteries

    The lipid deposition of arterial soybean soybean milk was ingested into genetically modified Apo E knockout mice for 8 weeks and the results are summarized in Table 9 below.

Specifically, the heart obtained from apoE-/-mice was placed in PBS for 10 minutes, then in 4% paraformaldehyde for 5 minutes, and immediately removed. And the heart was stored at -70 ℃ using OCT compound fixed in 10% neutral formalin for one day. The heart was sectioned in 3 trillion 8 μm series from the aorta with a cryostat maintained at -20 ° C. The resulting fat lesions were stained with Oil red O and hematoxylin was used as a counterstain. Stained lip lesions were measured using a computer-assisted morphometry system (Axio Vision, Carl Zeiss, Canada) and the area was calculated using an image analysis program.

Group ³⁾ Lesion area (mm ² ) Control 0.56 ± 0.08 FSCC 0.27 ± 0.23 ^*** CPAS 0.46 ± 0.25 ^#

¹⁾ The area stained with Oil red O staining is confirmed by the area analyzer equipped with a computer.

^2,3) See the legend of Table 6

*** p <0.001 by t-test between control and FSCC

# p <0.05 by t-test between FSCC and CPAS

As shown in Table 9, the lipid deposition area was significantly suppressed compared to the control, the effect was higher than the soymilk added with Chungkookjang powder, but there was no significant difference.

In summary, the present invention as described above is as follows:

1. Physicochemical Characteristics and Antioxidant Activity of 6 Products of Cheonggukjang, Sunchang Area

The pH, acidity, total bacterial count, amino nitrogen (NH ₃ -N) concentration and functionalities of 6 products of Cheonggukjang in Sunchang were high in S1, S2, and S3, and the free radical scavenging ability of S3 was significantly higher than that of other products. .

2. Establishment of conditions for producing anti-arterial functional fermented soymilk

1) Three types of Bacillus isolates isolated from S3 Cheonggukjang, Bacillus subtili s 2805 PNU 014, Bacillus subtilis 2829 PNU 015, Bacillus subtilis 2825 PNU 016 and two commercially available bacteria, MYCO (Innobiz Myco, Gyeong Ju, The highest fibrin resolution of Bacillus subtilis 2829 PNU 015 was found to be 27.2 mU plasmin / hr among the five Bacillus strains (KCCM 11316, Seoul, Korea).

2) Optimum conditions for the production of fermented soymilk were inoculated with 1% (CFU 10 ⁷ ~ 10 ⁸ ) concentration of Bacillus subtilis in soy milk and incubated for about 8 hours until pH 5.5 ± 0.2 at 40 ° C.

3) DPPH scavenging activity, total antioxidant activity (TEAC), and LDL oxidation inhibition activity of fermented soybean milk were significantly higher than that of unfermented soymilk (p <0.05). Among them, Bacillus subtilis 2829 PNU 015 showed the best antioxidant effect (p <0.05).

4) The fibrin resolution of cornus milk was 33.4 mU plasmin / hr when the six functional materials (garlic, cornus, purple sweet potato, black garlic, onion, fortified lion cucurbita) were compared to enhance the functionality and functionality of fermented soymilk. It was high and the highest in the 1% addition concentration.

5) The fermented soybean milk fermented with 1% cornus milk increased DPPH scavenging activity 28.0%, O ₂ ^- scavenging activity 22.8% and nitrite scavenging activity at a concentration of 1,000 ㎍ / mL compared to the uncooked sorghum fermented soymilk (p). <0.001) Functionality and functionality were enhanced.

3. ApoE ^(-/-)  Lipid-lowering Effect of Fermented Soymilk with Anti-arteriosclerosis in Mice

Soymilk fermented with Bacillus subtilis 2829 PNU 015 (Accession No. KCCM 42923) by adding 1% cornus oil was lyophilized and added to atherosclerosis feed for 8 weeks to examine the lipid lowering effect. The experimental group consisted of the control group fed the atherosclerosis diet, the anti-arteriosclerosis functional fermented milk group, and the soymilk group 3 added with the Cheonggukjang powder used as a positive control group, and there were 7 animals per group.

1) There were no significant differences in weight gain and liver, kidney, lung, testis and spleen weights between the control group and the functional fermented soy milk group and Cheonggukjang powdered soy milk group.

2) Plasma lipid concentrations were increased by more than 1013.7 mg / dL and 930.3 mg / dl of total cholesterol and LDL-C by arteriosclerosis diet with 1.25% cholesterol, but hypercholesterolemia was induced by addition of functional fermented soymilk, respectively. , 30.3% and Chungkookjang powder decreased by 15.4% and 16.4%, respectively (p <0.05). However, no effect of improving plasma triglyceride and HDL-C levels was observed.

3) The lipid concentrations of liver were 19.7% and 19.4% lower than those of the control fermented soymilk group and Chunggukjang powder added group, respectively (p <0.05). However, triglyceride levels did not differ significantly between groups.

4) The lipid oxidation degree (TBARS) of liver was significantly lower in functional fermented soymilk group and cheongukjang powder added group, 23.6% and 15.4%, respectively, than the control group ( p <0.05).

5) Fecal fat concentrations in the functional fermented soymilk group were 7.2% and 9.5% lower than those of the control group, but there was no significant difference.

In the above experiments, the fermented soybean milk was found to have higher free radical scavenging ability and fibrin resolution than soymilk. The effect of the agglomerated milk added fermented soybean milk is superior to that of the soybean milk added to the soybean paste powder, which is thought to have an anti-arteriosclerosis effect by lowering the plasma cholesterol concentration during long-term intake.

The present invention is the result of research conducted by the Ministry of Education, Science and Technology and the Korea Industrial Technology Foundation (M-020-20070131134352).

1 is a graph measuring the DPPH free radical activity of soy milk added to Cornus milk and inoculated and fermented with Bacillus subtilis according to an embodiment of the present invention. In the drawings, the abbreviation FS refers to soymilk fermented with Bacillus subtilis 2829PNU015, and FSCC refers to soymilk inoculated and fermented by adding Corni fructus extracts (1%, 10 ^o Brix) to FS.

Claims (7)

Adding Bacillus subtilis and Corni fructus so that the glucose content is 1% of the total weight; After the addition, the step of increasing the steam for 15 minutes at 121 ~ 150 ℃ sterilized soy milk in the order of the addition of cornus milk and Bacillus subtilis, characterized in that it comprises a ; Way. delete The method of claim 1 , wherein the concentration of the cornus milk is 10 ° Brix, the content is a fermented soymilk production method characterized in that the addition of 1% based on the final concentration to improve the fibrin resolution. According to claim 1 , Fermented soybean milk production method characterized in that the inoculation by adding the subtilis culture medium so that the concentration of the Bacillus subtilis 7-8 logCFU / ml. The method of claim 4, wherein the Bacillus subtilis culture is obtained by inoculating Bacillus subtilis of Accession No. KCCM 42923 isolated in Cheonggukjang in a culture medium containing 0.8% sodium chloride, shaking culture for 24 hours at 37 ℃ in a water bath. Fermented soy milk production method. The method of claim 4, wherein the soymilk inoculated with Bacillus subtilis is fermented to reach pH 5.5 ± 0.2 at 37-40 ° C. A fermented soy milk for preventing atherosclerosis, which is prepared by the method of claim 1 and has an effect of lowering lipid lipids, reducing liver lipids, or inhibiting arterial lipid deposition.

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