KR101083356B1 - Microorganisms for Improving Blood Stream, and functional food composition including the same - Google Patents

Abstract

The present invention relates to a strain that is effective in improving blood flow isolated from kimchi, and more particularly, to a strain that is isolated from kimchi and has a thrombus lytic function, chitin degrading function, and cholesterol inhibiting function.

According to the present invention, strains having thrombolytic ability, chitin degradation ability and cholesterol lowering ability are respectively selected from the lactic acid bacteria and yeast strains isolated from kimchi, and functional strains can be prepared according to the functionalities of the strains. Therefore, the use of the strain according to the present invention can effectively produce a functional food having a blood flow improving function.

Kimchi for blood flow improvement, thrombolysis, chitin decomposition, cholesterol inhibition, lactic acid bacteria, yeast

Description

Microorganisms for Improving Blood Stream, and functional food composition including the same}

The present invention relates to a strain for improving blood flow, and more specifically, to isolate from kimchi, to prepare a functional food having a blood flow improving function, including kimchi as a strain having a thrombolytic function, chitin decomposition function and cholesterol lowering effect. The present invention relates to a strain for improving blood flow.

As a biological component, blood has a variety of important functions such as transporting and buffering oxygen, nutrients, and wastes, maintaining body temperature, controlling osmotic pressure and ion balance, maintaining constant moisture, controlling liquids, maintaining and controlling blood pressure, and protecting the body. have. Therefore, the functional abnormality of the blood causes a variety of diseases, in particular, cerebrovascular disease or heart disease resulting from abnormal hemostatic function to minimize blood loss in the damaged blood vessels are fatal to life and serious sequelae. At present, cardiovascular diseases are rapidly increasing with the development of society and the aging of the population, and the sum of the mortality rates is higher than that of malignant tumors.

In general, blood circulation, i.e., blood flow, may be caused by (1) high lipid and hyperglycemia, resulting in poor blood flow, (2) increased fluidity of blood cells, i.e., decreased flexibility or adhesion of red blood cells and white blood cells, or (3) ) It is affected by the action of increasing the aggregation ability of platelets and lowers.

Blood must remain fluid in normal blood vessels and solidify quickly in nonecdothelial veins, stopping blood loss from damaged blood vessels. In addition, intravascular thrombus (thrombus), that is, blood clots formed by clotting of blood in an active cardiovascular system, the mechanism of thrombolytic action is activated to prevent the formation of thrombus and restore fluidity. However, due to various factors, such a mechanism causes abnormalities and appears as a pathological phenomenon of the normal hemostasis mechanism is called thrombosis. Blood clots cause ischemic damage to tissues or organs by reducing or blocking the blood flow, leaving the blood vessels to become embolus, which is carried away in the bloodstream. Embolism is a generic term for clumps (including solids, liquids, and gases) that block blood vessels, but most of them come from blood clots. Infarcts usually result from thromboembolic closure in the blood vessels. Mortality from infarction of the heart, lungs and brain is higher than that of various cancers and infectious diseases.

Many studies have been conducted for the removal of these blood clots. In particular, thrombolytic activity by plasminogen activator such as streptokinase, urokinase, and tPA (tissue-type plasminogen activator) is injected intravenously. Therapies to activate the fibrinolytic system have been used universally for over 30 years. However, these drugs are not specific for blood clots and have side effects such as systemic haemorrhage during treatment, as well as oral administration and are very expensive. To this end, various studies on economic and oral administration of thrombosis treatments have been attempted, and a preparation for increasing thrombolytic ability in blood by direct oral administration or in combination with vascular injection has been developed. Currently, as a thrombotic agent that can be administered orally, six thrombolytic enzymes isolated from earthworms are known, which are also restricted in Korea.

To date, a number of foods and food ingredients that have the potential to improve blood flow have been reported. For example, black vinegar, pickled plum pickles, etc. are mentioned as a close foodstuff. In addition, Japanese Patent Laid-Open No. 7-138168 reports that polar solvent extract of fish bile improves blood flow. In Japanese Unexamined Patent Publication No. 2002-97143, it has been reported that glucosamine salt or glucosamine derivative improves blood clot prevention or blood flow. However, the improvement of blood flow is mainly due to the action related to the fluidity of blood, and there are no foods or pharmaceutical compositions incorporating components having the effect of improving blood flow and improving the strength and elasticity of blood vessels, that is, protecting the blood vessels. not. On the other hand, Japanese Laid-Open Patent Publication No. 2000-135071 discloses that the wine lee extract has a superior effect to the polyphenol-containing extract with respect to the action of strengthening blood vessels.

In addition, the study of thrombolytic enzymes in fermented foods, especially in Korea and Japan, is noteworthy in that it targets foods that can be consumed directly. Natto (natto), a traditional Japanese soybean fermented food, and shiokara (pickled food) are known to have thrombolytic enzymes, and production strains were isolated and enzymes were purified. Among them, nattokinase isolated from natto. It is reported that the enzyme can increase the thrombolytic ability in vivo even after oral administration.Therefore, in Korea and Japan, isolates of strains that secrete thrombolytic enzymes from Doenjang and Cheonggukjang, which are traditional fermented foods based on soybean, are used for thrombolysis. Sales of healthy food products are increasing rapidly (Sumi, H., et al., Experimentia, 43, 1110, 1987; Bioindustry, 7, 725, 1990; Korean Patent Application No. 1996-75523).

On the other hand, high cholesterol causes the arteriosclerosis disease to be deposited on the walls of blood vessels, causing loss of elasticity of blood vessels, narrowing the inner diameter, and blocking the flow of blood. Lactobacillus acidophilus bacteria were used for the cholesterol lowering ability of the lactic acid bacterium, and many experiments were performed. In addition, chitosan, a degradation product of chitin, has the effect of blocking the human circulation ring of cholesterol by combining with bile acids in the intestine and lowering the density of low-density lipoprotein (LDL) when absorbed into the bloodstream.

Kimchi fermentation, a traditional fermented food in Korea, has been reported to be involved in various strains, and lactobacillus bacteria include Leuconostoc mesenteroides , Lactobacillus plantarum , and Lactobacillus ( Lactobacillus plantarum ). Lb has been reported as Lactobacillus abbreviation) ( Lb.brevis ), Streptococcus faecalis , Pediococcus cerevisiae , and the like. Leu. Mesenteroides is the main fermenting bacterium of kimchi and Lb. plantarum is the main cause of rancidity. If the functions of these bacteria can be used to check the functionalities of these bacteria, not only can they produce healthier functional foods, but they can also provide excellent disease prevention and treatment effects by continuously eating delicious foods. will be.

Under such technical background, the present inventors have isolated and identified lactic acid bacteria and yeast isolated from kimchi to find a strain among them, in particular, to improve blood flow, thus leading to the present invention.

After all, it is an object of the present invention to provide a strain having a large number of colonies among the strains isolated from kimchi with good blood flow improvement (thrombolytic, chitin degradation and cholesterol lowering) effect.

Another object of the present invention is to provide a functional food composition having a thrombolytic ability, chitin degradability and cholesterol lowering ability.

Another object of the present invention is to provide a functional kimchi having thrombolytic ability, chitin degradation ability and cholesterol lowering ability.

In order to achieve the object of the present invention, the present invention provides Enterococcus faecalis SKD1019: KCTC18144P to produce a fibrin degradation ring.

In another aspect, the present invention provides Lactobacillus paraplantarum ( Lactobacillus paraplantarum : WL 3; accession number KCTC18142P).

The present invention also provides Weissella cibaria (WP 3; accession number KCTC18141P).

In another aspect, the present invention provides Leuconostoc kimchi (KR 8; accession number KCTC18143P).

In another aspect, the present invention provides Leuconostoc mesenteroides (YRWM11; accession number KCTC18145P).

In another aspect, the present invention Lactobacillus brevis:; provide (Lactobacillus brevis YRWP4 accession No. KCTC18147P).

The present invention also provides Lactococcus lactis (YRWM1; accession number KCTC18146P).

The present invention also provides Saccharomyces cerevisiae (OBP4).

According to the present invention, strains having thrombolytic, chitin and cholesterol inhibitory properties were isolated and identified from lactic acid bacteria and yeast strains isolated from kimchi, respectively, to select strains effective for improving blood flow, thereby completing the present invention. Therefore, by using the blood flow improving strain according to the present invention it is easy to manufacture a functional food having a blood flow improving effect, in particular can be easily and efficiently produced functional kimchi with blood flow improving effect.

As the strain of the present invention is effective in improving blood flow, it is isolated from kimchi as follows.

That is, the present invention provides Enterococcus faecalis SKD1019, which is effective for thrombolysis to generate fibrin degradation rings.

In addition, the present invention is Lactobacillus paraplantarum ( Lactobacillus paraplantarum : WL 3; Accession No. KCTC18142P), Weissella cibaria (WP 3; Accession No. KCTC18141P), Leuconostoc kimchi (KR 8; Accession No. KCTC18143P), Leuconostoc mesenteroides (YRWM11; Accession No. KCTC18145P), Leuconostoc mesenteroides (YRWSP4), Lactobacillus sakei (Yactsococcus lactis ), Lactococcus lactis YRWSM4), Enterococcus faecalis SKD1019, at least one strain selected from the group consisting of Rhodotorula mucilaginosa (YRWP1) and Saccharomyces cerevisiae (JBD4). Chitin-degrading functional strains are provided.

In addition, the present invention is Lactobacillus paraplantarum ( Lactobacillus paraplantarum : WL 3), Weissella cibaria (WP 3), Leuconostoc kimchi ( KR 8), Lactobacillus brevis ( Lactobacillus brevis : YRWP4; accession number KCTC18147P), Lactobacillus brevis (Lactobacillus brevis: YRWP6), Lactobacillus Cuba Charters (Lactobacillus curvatus: YRWP7), Lactobacillus Cocos lactis (Lactococcus lactis: YRWM1; accession number KCTC18146P), Lactobacillus four K (Lactobacillus sakei: YRWM3 ), Lactobacillus del breather key (Lactobacillus delbreiki: YRWM4), flow Pocono stock mesen steroid (Leuconostoc mesenteroides: YRWM7), Lactobacillus brevis (Lactobacillus brevis: YRWM8), Lactobacillus del breather key (Lactobacillus delbreiki: YRWM10), flow Pocono Leuconostoc mesenteroides (YRWM11), Leuconostoc citreum (YRWE1), Lactobacillus brevis ( Lactobacillus) brevis: YRWE4), current Kono Stock Sheet reum (Leuconostoc citreum: YRWSP1), Lactobacillus nose profile Russ (Lactobacillus coprophilus: YRWSP2), Lactobacillus brevis (Lactobacillus brevis: YRWSP6), Lactobacillus Cocos lactis (Lactococcus lactis: YRWSM6), Lactobacillus sakei (YRWSM10), Leuconostoc citreum (YRWSM12), Leuconostoc citreum (YRWSE4), Enterococcus faecalis ( Enterococcus faecalis SKD1019), Saccharoma a bicyclic (Saccharomyces cerevisiae: OBP1), Saccharomyces My process in serenity bicyclic (Saccharomyces cerevisiae by: OBP4; Accession No. KCTC18148P), Rhodotorula mucilaginosa (OBE1), Rhodotorula mucilaginosa (OBD1), Rhodotorula mucilaginosa (OBD4) strains consisting of one group selected from the group consisting of: Provided is a cholesterol inhibiting functional strain containing more.

The strains were deposited on October 21 to the Korea Center for Biological Resources (KCTC), an international organization under the Budapest Convention, and the accession numbers are listed in parentheses.

Method for isolating and identifying the functional strain according to an embodiment of the present invention is as follows.

Separation of thrombolytic strains is performed by mixing 0.4 ~ 0.6% in 30 ~ 60mM buffer solution, preferably 50mM sodium phosphate buffer solution (pH 7.4) with respect to thrombolytic proteins such as fibrinogen, etc. in a thermostat at 35 ~ 40 ℃. After complete dissolution for about an hour, mix into a suitable medium (e.g. 1.5% MRS agar, pH 6.0-7.4) at a ratio of 6-8: 2-4 (v / v) (e.g. 7: 3 (v / v) )) And 10 units of thrombolytic protease (e.g. thrombin) per 10 ml of the thrombolytic protein solution to form fibrin clot of uniform thickness, and then allowed to stand at room temperature for 1 to 2 hours and then separated from kimchi. After inoculating a lactic acid bacterium and culturing at 30-40 ° C. for 12-30 hours, it was observed whether or not the decomposition ring was formed.

According to one embodiment of the present invention, chitin-degrading strain separation used colloidal chitin for chitin resolution.

Chitin digestion strain separation method by adding a 60 ~ 90% weak acid solution to the crude chitin at a rate of 5 ~ 15g / 100mL and stirring at 0 ~ 10 ℃ for 20 ~ 30 hours,

Suspending the colloidal chitin precipitate obtained by adding distilled water to the solution in distilled water,

Neutralizing by adding a basic solution to the suspension and washing with distilled water 3-4 times,

Preparing a plate by adding the colloidal chitin to 0.3-0.8% of the agar medium, and

The plate was inoculated with the strain isolated from kimchi, specifically, 10 ml of crude chitin (practical grade from Crab shells, Wako) added 100mL of 85% phosphoric acid (phosphoric acid) and stirred for 24 hours at 4 ℃ Then, the white colloidal chitin precipitate obtained by adding distilled water was suspended in distilled water, neutralized by adding a small amount of 5N NaOH, and then washed with distilled water for 34 times.

To measure the chitin-degrading activity of lactic acid bacteria, a colloidal chitin was added to MRS agar to 0.6% to prepare a plate.

Plates were prepared by adding colloidal chitin to 0.4% of PDA agar for measuring the chitin degradation activity of yeast.

Separating strains cultured in plate medium on the prepared plate was inoculated with a wooden rod and incubated at 30 ° C. for 3 days. 0.1% Congo red (Fisher) solution was added to the plate, left at room temperature for 30 minutes, washed with 1M NaCl for 15 minutes, and the chitin resolution was measured with or without degradation rings.

According to one embodiment of the present invention, the cholesterol-lowering strain is isolated by inoculating a lactic acid bacterium cultured in agar medium in a liquid medium and incubating for 12-30 hours at 25-35 ° C.,

30 to 70 μl of the culture solution was added to 50 mL of the liquid medium, followed by incubation for 20 to 30 hours at 25 to 35 ° C.,

Centrifuging the cultured strain at 3500-6500 rpm at 2-6 ° C. for 5-15 minutes to separate the supernatant and

KOH and ethanol (2: 3 (v / v)) to the supernatant is added to 5 times the volume of the supernatant is a separation method of cholesterol inhibition strain comprising the step of maintaining for 5 to 15 minutes at 50 ~ 70 ℃, specifically As an example, lactic acid bacteria cultured in MRS agar medium were inoculated with a wooden rod in 5 mL of MRS broth, and then cultured for 24 hours at 30 ° C., followed by soluble cholesterol (polyoxyethanyl-cholesterol sebacate, sigma) [cholesterol (polyoxyethanyl- cholesterol sebacate, Sigma)] 50 μl of MRS broth containing 500 μg was incubated for 24 hours at 30 ° C., and the cultured strain was centrifuged (4 ° C., 4000 rpm, 10 min) in 0.5 mL of supernatant. After separation, 2 mL of 5% KOH and 3 mL of 95% ethanol were added and maintained at 60 ° C. for 10 minutes. After cooling, 5 mL of hexane was added and mixed, followed by addition of 3 mL of distilled water, followed by well mixing. The mixture was left at room temperature for 15 minutes to separate 2.5 mL of the obtained hexane layer. The separated hexane layer was separated with nitrogen gas (N2). After evaporation at room temperature using gas), add 4 mL of O-phthalaldehyde solution (0.5mg O-phthalaldehyde / glacial acetic acid 1mL) and leave for 10 minutes, then slowly add 2 mL of concentrated sulfuric acid and mix well. After 10 minutes, the absorbance was measured at 550 nm, and the treatment group in which the cholesterol content in the medium was reduced to 50% or less was selected.

In the present invention, in order to confirm the function of improving blood flow, thrombolytic ability, chitin resolution (cholesterol lowering chitosan production) and cholesterol lowering ability were confirmed.

In order to induce fermentation as a dominant bacterium in the kimchi flora having a natural flora in the present invention, the strains were selected as a strain for improving blood flow by selecting a strain with a high population number of 106 to 8 cfu / mL from the original Kimchi isolate.

In addition, the lactic acid bacteria and yeast strains for improving blood flow according to the present invention can be frozen and used as a powdered product with a lyophilizer.

In the present invention, by mixing the lactic acid bacteria and yeast can be used in food to produce a functional food having a function of improving blood flow, in particular by using it in the production of kimchi can improve the blood flow improvement function of kimchi.

When kimchi is described as an example, the kimchi is prepared according to a general kimchi production method with reference to FIG. 6. That is, according to one embodiment of the present invention, the step of mixing the blood flow improving seed powder according to the present invention with kimchi seasoning, mixing the kimchi seasoning with pickled cabbage, and the cabbage mixed with the seasoning 5 ~ 10 ℃ Provided is a method for producing blood flow improving functional kimchi, comprising the step of aging at low temperature.

The present invention also provides a strain set for producing general kimchi containing only functional lactic acid bacteria and a strain set for manufacturing old paper containing a mixture of functional lactic acid bacteria and yeast.

The set of strains of general kimchi uses a mixed strain that can antagonize with kimchi strains from nature by selecting from each representative lactic acid bacteria found in kimchi. The antagonistic conditions are intended to inoculate strains of various genus so that the remaining strains can prevail even if a strain is inoculated with a condition that makes a predominant strain inoculate a large amount of the initial inoculation amount to 106 / g or more. The strain set of the old paper is the same as the normal kimchi strain set, but by adding yeast to reduce the organic acid produced by lactic acid bacteria gave a less sour old paper effect.

The present invention also comprises the steps of identifying a functional strain from kimchi and

It provides a method for separating functional seed from kimchi comprising the step of obtaining a purified product of a specific functional strain from the functional strain group.

In addition, another object of the present invention is the lactic acid bacteria in the lactic acid bacteria strains and the yeast strains of the genus 5 lactic acid strains and yeast strains isolated from the kimchi secondary passage in the YRS liquid medium, respectively, and MRS (lactic acid bacteria) medium and YM ( Inoculating 1% (for example, 0.5 ml in 500 ml) liquid medium to the yeast and yeast malt extraction medium for culture of filamentous fungi,

Incubating at 28-32 ° C. for 12-16 hours with 1-3 × 10 9 cfu / mL of yeast (1-6 × 10 8 cfu / mL),

Centrifuging the culture to make precipitate pellets, and

The pellet-like cells were suspended in 10% skim-reducing oil in a 10% (50 mL) dose of the stock solution (lactic acid bacteria count; 1-10 × 1010 cfu / mL, yeast bacteria count; 1-10 × 109 cfu / mL) and frozen. It provides a method for producing a powder spawn comprising the step of drying (lactic acid bacteria number; 1 ~ 10 × 1011 ~ 12 cfu / g, yeast bacteria; 1 ~ 10 × 1010 ~ 11 cfu / g).

Hereinafter, the contents of the present invention will be described in more detail with reference to Examples. However, these examples are described by way of example for understanding the contents of the present invention and should not be construed that the scope of the present invention is limited to these examples.

Selection of Kimchi

The types of kimchi used in the experiment for the isolation of strains were two kinds of young radish kimchi (Yulmumul Kimchi 1; Kimchi refrigerator 1 month ripening water kimchi, homemade at home, Yeolmu Kimchi 2; Kimchi refrigerator 1 month ripening kimchi, home made), Chinese cabbage Kimchi 3 kinds (Chinese cabbage kimchi 1; Kimchi refrigerator 6 months of mature organic kimchi, Jeonbuk organic acid, Chinese cabbage kimchi 2; Kimchi refrigerator 6 months of aged green jade kimchi, Chinese cabbage kimchi 3; 1 month ripened at 5 ℃ Kimchi). This is described by the method of one embodiment, but is not limited thereto.

Example 1: Preparation of Lactic Acid Bacteria Sample

Grind ripe kimchi with a sterile blender, take 1 g, dilute it continuously with physiological saline solution, and then use Lgosa modified medium BSBS (mLBS plate), P. leucine stock separation medium Inoculate on PES plate, KF-Streptococcus plate (Pedococcus and Enterococcus) and YM plate (YM plate) for 1 ~ 2 days at 30 ~ 35 ℃ Incubated. Five strains of different colonies were selected from well-formed plates. Among them, Lactobacillus paraplantarum WL3, Lactococcus lactis YRWM1, Weissella cibaria WP3, Leuconostoc mesenteroides YRWM11, Enterococcus faecalis SKD1019 were finally selected. Identified as an API Plus (bioMerieux, France) program.

Example 2: Preparation of Yeast Sample

A yeast sample was prepared by the method corresponding to Example 1.

Example 3 Measurement of Thrombus Solubility of Lactic Acid Bacteria and Yeast Isolated from Kimchi

Thrombolytic activity was measured by the fibrin plate method. 10 g of fibrinogen (Sigma) 0.6 g of 50 mM sodium phosphate buffer (pH 7.4) was completely dissolved in a thermostat at 37 ° C. for about 2 hours, and the fibrinogen solution was dissolved in 1.5% MRS agar (pH 7.4) and 7: 3 (pH 7.4). v / v) and added 10 units of thrombin (Sigma) per 10 mL of the fibrinogen solution to form a fibrin clot of uniform thickness, and then allowed to stand at room temperature for about 1 hour. Inoculated with lactic acid bacteria isolated from kimchi through 1 and incubated at 37 ℃ for 24 hours to observe whether the clear zone (clear zone) generation is shown in Table 1 the results. According to Table 1, it was confirmed that Enterococcus faecalis SKD1019 was a strain capable of dissolving blood clots because the clear ring was formed by melting fibrin (Table 1).

In yeast, strains with thrombolytic ability could not be isolated (Table 2).

 Fibrin Resolution of Yeast Isolated from Kimchi Strain name Scientific name Cluster amount (CFU / mL) Fibrin resolution YRWP1 Rhodotorula mucilaginosa 2.0 × 10 ⁶ - YRWD1 Candida ciferrii 9.0 × 10 ⁵ - YRWD4 Candida ciferrii 2.5 × 10 ⁵ - YRWD7 Saccharomyces cerevisiae 2.0 × 10 ⁵ - OBP1 Saccharomyces cerevisiae 5.0 × 10 ⁶ - OBP4 Saccharomyces cerevisiae 3.0 × 10 ⁶ - OBM1 Saccharomyces cerevisiae 1.1 × 10 ⁵ - OBM4 Saccharomyces cerevisiae 1.8 × 10 ⁵ - OBM9 Saccharomyces cerevisiae 1.3 × 10 ⁴ - OBE1 Rhodotorula mucilaginosa 2.4 × 10 ⁶ - OBD1 Rhodotorula mucilaginosa 1.5 × 10 ⁷ - OBD4 Rhodotorula mucilaginosa 1.3 × 10 ⁶ - JBP1 Rhodotorula mucilaginosa 8.5 × 10 ⁵ - JBP4 Candida ciferrii 5.4 × 10 ⁶ - JBD1 Saccharomyces cerevisiae 3.0 × 10 ⁵ - JBD4 Saccharomyces cerevisiae 1.3 × 10 ⁶ - JBD7 Saccharomyces cerevisiae 4.0 × 10 ⁵ -

-; Can not generate crack

Example 4 Measurement of Chitin Resolution of Lactic Acid Bacteria and Yeast Isolated from Kimchi

Colloidal chitin was used to measure chitin resolution. 100 mL of 85% phosphoric acid was added to 10 g of crude chitin (Crab shells, Wako) and stirred at 4 ° C. for 24 hours. Then, a white colloidal chitin precipitate obtained by adding distilled water was suspended in distilled water and a small amount of 5N NaOH was added thereto. After neutralization it was used to wash 3-4 times with distilled water.

Plates were prepared by adding colloidal chitin to 0.6% MRS agar (Merk) to measure chitin degradation activity of lactic acid bacteria, and 0.4% to PDA agar (Difco) to measure chitin degradation activity of yeast. The plates were prepared by addition. Kimchi-separated lactic acid bacteria samples and yeast samples isolated in Example 1 and Example 2 cultured in the plate was inoculated with a wooden rod and incubated for 3 days at 30 ℃. 0.1% Congo red: Fisher solution was added to the plate, left at room temperature for 30 minutes, washed with 1M NaCl for 15 minutes, and the chitin resolution was measured with or without a clear ring. The results are shown in the lactic acid bacteria samples and yeast samples in Table 3 and Table 4, respectively.

Chitin Resolution of Yeast Isolated from Kimchi Strain name Scientific name Cluster amount (CFU / mL) Chitin resolution YRWP1 Rhodotorula mucilaginosa 2.0 × 10 ⁶ + YRWD1 Candida ciferrii 9.0 × 10 ⁵ - YRWD4 Candida ciferrii 2.5 × 10 ⁵ - YRWD7 Saccharomyces cerevisiae 2.0 × 10 ⁵ - OBP1 Saccharomyces cerevisiae 5.0 × 10 ⁶ - OBP4 Saccharomyces cerevisiae 3.0 × 10 ⁶ - OBM1 Saccharomyces cerevisiae 1.1 × 10 ⁵ - OBM4 Saccharomyces cerevisiae 1.8 × 10 ⁵ - OBM9 Saccharomyces cerevisiae 1.3 × 10 ⁴ - OBE1 Rhodotorula mucilaginosa 2.4 × 10 ⁶ - OBD1 Rhodotorula mucilaginosa 1.5 × 10 ⁷ - OBD4 Rhodotorula mucilaginosa 1.3 × 10 ⁶ - JBP1 Rhodotorula mucilaginosa 8.5 × 10 ⁵ - JBP4 Candida ciferrii 5.4 × 10 ⁶ - JBD1 Saccharomyces cerevisiae 3.0 × 10 ⁵ - JBD4 Saccharomyces cerevisiae 1.3 × 10 ⁶ + JBD7 Saccharomyces cerevisiae 4.0 × 10 ⁵ +

Example 5 Measurement of Cholesterol Inhibitory Activity of Kimchi Isolated Lactic Acid Bacteria and Yeast

In order to measure cholesterol inhibitory activity of lactic acid bacteria and yeast, the amount of cholesterol remaining in the medium was measured after incubating the lactic acid bacteria and yeast in MRS medium to which cholesterol was added. Lactobacillus cultured in MRS agar medium was inoculated with 5 mL of MRS broth with wooden rods, and then cultured for 24hr at 30 ° C. Put each other and incubated for 24 hours at 30 ℃ again. The strain cultured in the medium was centrifuged (4 ° C., 4000 rpm, 10 min) to separate 0.5 mL of the supernatant, and then, 2 mL of 5% KOH and 3 mL of 95% ethanol were added and maintained at 60 ° C. for 10 minutes. After cooling, 5 mL of hexane (hexane) was added and mixed, 3 mL of distilled water was added again, mixed well, and the obtained hexane layer was left at room temperature for 15 minutes to separate 2.5 mL of the obtained hexane layer. The separated hexane layer was evaporated at room temperature using nitrogen gas (N2 gas), 4 mL of OPA (O-phthalaldehyde) solution (0.5 mg OPA / 1 mL glacial acetic acid) was added and left for 10 minutes. 2 mL of concentrated sulfuric acid was slowly added thereto, mixed well, and after 10 minutes, absorbance was measured at 550 nm. The results are shown in Tables 5 and 6 for lactic acid bacteria and yeast, respectively.

Cholesterol Inhibitory Activity of Lactic Acid Bacteria Isolated from Kimchi Strain name Scientific name Cluster amount (CFU / mL) Cholesterol inhibition WL3 Lactobacillus paraplantarum 4.5 × 10 ⁸ + WP3 Weissella cibaria 2.8 × 10 ⁷ + KR8 Leuconostoc kimchii 5.4 × 10 ⁸ + YRWP4 Lactobacillus brevis 6.6 × 10 ⁷ + YRWP6 Lactobacillus brevis 3.3 × 10 ⁷ + YRWP7 Lactobacillus curvatus 3.0 × 10 ⁶ + YRWM1 Lactococcus lactis 4.0 × 10 ⁶ + YRWM3 Lactobacillus sakei 2.0 × 10 ⁶ + YRWM4 Lactobacillus delbreiki 2.3 × 10 ⁶ + YRWM7 Leuconostoc mesenteroides 2.3 × 10 ⁵ + YRWM8 Lactobacillus brevis 4.7 × 10 ⁵ + YRWM10 Lactobacillus delbreiki 3.2 × 10 ⁶ + YRWM11 Leuconostoc mesenteroides 6.5 × 10 ⁶ + YRWE1 Leuconostoc citreum 1.8 × 10 ⁷ + YRWE4 Lactobacillus brevis 4.4 × 10 ⁷ + YRWSP1 Leuconostoc citreum 1.9 × 10 ⁷ + YRWSP2 Lactobacillus coprophilus 3.9 × 10 ⁷ + YRWSP4 Leuconostoc mesenteroides 1.6 × 10 ⁷ - YRWSP6 Lactobacillus brevis 8.0 × 10 ⁶ + YRWSP7 Lactobacillus sakei 4.3 × 10 ⁷ - YRWSM1 Lactococcus lactis 1.2 × 10 ⁷ - YRWSM4 Lactococcus lactis 1.1 × 10 ⁷ - YRWSM6 Lactococcus lactis 5.0 × 10 ⁶ + YRWSM7 Lactococcus lactis 2.4 × 10 ⁷ - YRWSM10 Lactobacillus sakei 2.2 × 10 ⁷ + YRWSM12 Leuconostoc citreum 1.1 × 10 ⁷ + YRWSE4 Leuconostoc citreum 5.0 × 10 ⁶ + YRWSE7 Lactobacillus brevis 4.3 × 10 ⁶ - YRWSE8 Lactobacillus brevis 2.2 × 10 ⁶ - YRWSE10 Leuconostoc mesenteroides 5.5 × 10 ⁶ -

+; 50% reduction in cholesterol content in the medium,

-; Less than 50% of cholesterol content in the medium

Cholesterol Inhibitory Activity of Yeast Isolated from Kimchi Strain name Scientific name Cluster amount (CFU / mL) Cholesterol inhibition YRWP1 Rhodotorula mucilaginosa 2.0 × 10 ⁶ - YRWD1 Candida ciferrii 9.0 × 10 ⁵ + YRWD4 Candida ciferrii 2.5 × 10 ⁵ - YRWD7 Saccharomyces cerevisiae 2.0 × 10 ⁵ + OBP1 Saccharomyces cerevisiae 5.0 × 10 ⁶ + OBP4 Saccharomyces cerevisiae 3.0 × 10 ⁶ + OBM1 Saccharomyces cerevisiae 1.1 × 10 ⁵ - OBM4 Saccharomyces cerevisiae 1.8 × 10 ⁵ - OBM9 Saccharomyces cerevisiae 1.3 × 10 ⁴ + OBE1 Rhodotorula mucilaginosa 2.4 × 10 ⁶ + OBD1 Rhodotorula mucilaginosa 1.5 × 10 ⁷ + OBD4 Rhodotorula mucilaginosa 1.3 × 10 ⁶ + JBP1 Rhodotorula mucilaginosa 8.5 × 10 ⁵ - JBP4 Candida ciferrii 5.4 × 10 ⁶ - JBD1 Saccharomyces cerevisiae 3.0 × 10 ⁵ - JBD4 Saccharomyces cerevisiae 1.3 × 10 ⁶ - JBD7 Saccharomyces cerevisiae 4.0 × 10 ⁵ +

+; 50% reduction in cholesterol content in the medium,

-; Less than 50% of cholesterol content in the medium

Example 6 Preparation of Strain Powder Having Blood Flow Improvement Function

Based on the results measured in Examples 3, 4 and 5, the blood flow improving functional seed was selected, and the seed powder was prepared through the manufacturing process of FIG. 1. That is, the lactic acid bacteria cultured in secondary passages were inoculated in 500 mL of MRS medium, and the bacterial counts at 30 ° C. for 16 hours; The cells were incubated to 109 cfu / mL. The culture was centrifuged to make precipitate pellets, and the cells on the pellets were suspended in 50 mL of 10% skimmed reduced oil (bacterial; x1010 cfu / mL) and lyophilized (microbial number; x1011 cfu / g). Lyophilization was allowed to freeze by leaving the cells suspended in skimmed reduced oil in a -70 ° C freezer (Deep freezer, DF9017, Ilshin) for at least 12 hours and then dried in a freeze dryer (Freeze dryer, DF5512, Ilshin) for 48 hours.

Using the powdered strains prepared as above, the mixed seed set was divided into general kimchi and dried paper according to the functionalities as shown in Table 7. The characteristics of the final selected strains are shown in Table 8, and the yeast was added to the seed for preparing dried paper. This is to give it a less sour effect like yeast with increased yeast.

The spawn of cholesterol lowering and chitin-degrading function is set of 4 strains; The spawn of cholesterol lowering, chitin-degrading, and thrombolytic-function is set of 5 strains; Cholesterol-lowering and chitin-degrading functions for the old paper were 5 strains; For old paper, the spawn of cholesterol lowering and chitin-degrading function consisted of 6 strain sets (Table 7).

Mixed spawn set selected for manufacturing functional foods (kimchi) for improving blood flow Kimchi Functional Spawn set General Kimchi Lower cholesterol
Chitin decomposition Lactobacillus WL3 , Lactococcus YRWM1 , Weissella WP3 , Leuconostoc YRWM11 Lower cholesterol
Chitin decomposition
Thrombus resolution Lactobacillus WL3 , Lactococcus YRWM1 , Weissella WP3 , Leuconostoc YRWM11 , Enterococcus SKD1019 Old paper Lower cholesterol
Chitin decomposition Lactobacillus WL3 , Lactococcus YRWM1 , Weissella WP3 , Leuconostoc YRWM11, Saccharomyces cerevisiae OBP 4 Lower cholesterol
Chitin decomposition
Thrombus resolution Lactobacillus WL3 , Lactococcus YRWM1 , Weissella WP3 , Leuconostoc YRWM11 , Enterococcus SKD1019), Saccharomyces cerevisiae OBP4

Final Selection Strain Characteristics Strain name Thrombus resolution Chitin resolution Cholesterol lowering ability Weissella cibaria WP3 - + + Lactobacillus paraplantarum WL3 - + + Leuconostoc kimchi KR8 - + + Enterococcus faecalis SKD1019 + + - Leuconostoc mesenteroides YRWM11 - + + Lactoccus lactis YRWM1 - - + Lactobacillus brevis YRWP4 - +/- + S.cerevisiae OBP4 - - +

The mixed seed set for general kimchi prepared according to Tables 7, 8 is shown in the photo in Figures 3 and 4. Figure 3 is a photograph of a seedling set for producing general kimchi consisting of Lactobacillus, lactococos, wasella and leuconosstock, Figure 4 is a seedling for producing general kimchi consisting of Lactobacillus, Lactococcus, Wyella, leuconosstock and Enterococcus. It is a picture of the set.

The mixed seed set for kimchi tied according to Table 7, 8 is shown in Figure 5 and Figure 6. Figure 5 is a photograph of a seedling set for the production of old paper consisting of Lactobacillus, Lactococcus, Wyella, Leukonostock and Saccharomyces cerevisiae, Fig. 6 is Lactobacillus, Lactococcus, Wyella, Leukonostok, Enterero It is a photograph of the seed paper set for manufacturing the old paper which consists of cocos and Saccharomyces cerevisiae.

Example 7 Preparation of Kimchi with Blood Flow Improvement Function

Blood flow improving functional kimchi was prepared using the mixed seed set prepared in Example 6. 5 g (1011 cfu / g) of the mixed spawn set powder to prepare a kimchi for improving blood flow of 100 kg. When Kimchi is prepared, 5g of spawn powder is added to 100kg of Kimchi so that the initial bacterial count is 106cfu / g or more.The spawn powder is added to seasonings and used to add kimchi. Maintaining often results in culling of naturally occurring strains (FIG. 6).

The acidity of kimchi fermented with the selected seed was shown in Table 9, and the pH of the used kimchi was high.

PH of Kimchi Prepared with Cholesterol Lowering, Chitin, and Thrombolytic Spawn Storage temperature, ℃ Effective date 0 10 20 30 General Kimchi Old paper General Kimchi Old paper General Kimchi Old paper General Kimchi Old paper 5 5.7 5.7 4.7 5.0 4.2 4.3 4.0 4.2 10 5.7 5.7 4.3 4.4 3.9 4.1 3.7 3.9

Figure 1 shows a process for preparing a blood flow improving strain according to the invention into a powder.

Figure 2 is Lactobacillus paraplantarum ( Lactobacillus paraplantarum: WL 3: KCTC18142P), Lactococcus YRWM1: KCTC18146P, Weissella ( Weissella WP3: KCTC18141P) and Leukonostok ( Leuconostoc YRWM11P) manufactured by KC1818 made of KTC18 A picture of the spawn set.

Figure 3 is a Lactobacillus para Plan tarum (Lactobacillus paraplantarum: WL 3: KCTC18142P ), Lactococcus (Lactococcus YRWM1: KCTC18146P), Wi Cellar (Weissella WP3: KCTC18141P), current Kono Stock (Leuconostoc YRWM11: KCTC18145P) and Enterococcus Cocos (Enterococcus faecalis SKD1019: KCTC18144P) is a photograph of a set of spawns for general kimchi production.

Figure 4 shows Lactobacillus paraplantarum (WL 3: KCTC18142P), Lactococcus YRWM1: KCTC18146P, Weissella WP3: KCTC18141P, Leuconostoc YRWM11: KCTC18145P and FIG. ( Saccharomyces cerevisiae : OBP4; accession number KCTC18148P) It is a photograph of the seedling set for manufacture of the old paper.

Figure 5 shows Lactobacillus paraplantarum (WL 3: KCTC18142P), Lactococcus YRWM1: KCTC18146P, Weissella WP3: KCTC18141P, Leuconostoc YRWM11: KCTC 18145P, It is an old photographic species of a set consisting of Saccharomyces cerevisiae (OBP4; Accession No. KCTC18148P), Enterococcus faecalis SKD1019: KCTC18144P and Saccharomyces cerevisiae : OBP4; Accession No. KCTC18148P.

6 is a kimchi production process.

Claims (27)

delete delete delete delete delete delete delete delete delete delete delete delete delete Lactobacillus paraplantarum ( Lactobacillus paraplantarum : WL 3; KCTC18142P), Weissella cibaria (WP 3; KCTC18141P), Leuconostoc mesenteroides (YRWM11; KCTC18145P), and Lactobacillus Lactococcus lactis : YRWM1; KCTC18146P) A set of spawn for producing general kimchi for improving blood flow. The spawn set for producing general kimchi for improving blood flow according to claim 14, further comprising Enterococcus faecalis SKD1019: KCTC18144P. The spawn set for producing general kimchi for improving blood flow, according to claim 14 or 15, which has a cholesterol lowering effect. The spawn set for producing general kimchi for improving blood flow, according to claim 14 or 15, which has a chitin decomposition effect. 16. The seed set for producing kimchi for improving blood flow, according to claim 15, which has a thrombolytic effect. 16. The seed set according to claim 14 or 15, wherein the strain is isolated from kimchi for production of general kimchi for improving blood flow. delete delete delete delete delete delete delete delete

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