KR101145426B1 - Composition for improving blood flow, the preparation method thereof and health functional foods comprising thereof - Google Patents

Abstract

The present invention relates to a composition for improving blood flow, a method for preparing the same, and a health functional food comprising the same. The composition for improving blood flow of the present invention includes a fraction having a molecular weight of 100,000 dalton or less separated from a fermentation product fermented with lactic acid bacteria. .
The composition of the present invention is excellent in cholesterol synthesis inhibitory ability and bile acid binding ability to improve blood flow.

Description

Composition for improving blood flow, the preparation method thereof and health functional food comprising same

The present invention relates to a composition for improving blood flow, a method for preparing the same, and a health functional food comprising the same, and specifically, a composition for improving blood flow, which improves blood flow improving ability by inhibiting synthesis of cholesterol and having excellent bile acid binding ability, and a method for preparing the same. Health functional foods that include this.

In recent years, cardiovascular diseases caused by blood circulation disorders are the leading causes of death worldwide, including the United States, Europe and Asia. In Korea, cardiovascular disease is the leading cause of death because of the increased intake of animal foods according to westernized diets, and the increase in the level of total cholesterol and triglycerides in the blood in adults. The increase in lipid component is known to increase the viscosity of the blood or to interfere with the flow of blood flow, causing cardiovascular diseases including stroke.

In blood-related organs, plasma is composed of coagulation factors related to blood coagulation, and various interactions with blood cells such as platelets, erythrocytes, and immune cells are maintained, and each cell maintains its equilibrium. Platelets are cells that are activated when blood vessels are damaged and aggregate at the site of injury, thereby acting as a hemostatic effect. Hemostatic action is a defense mechanism to minimize the loss of blood from the damaged area and maintain the normal circulation of the blood. In normal blood vessels, homeostasis is maintained by balancing hemostatic mechanisms with inhibitory or thrombolytic reactions. However, excessive hemostatic action and formation of clots can disrupt blood flow and cause blood circulation abnormalities, leading to lesions such as thrombus. When blood clots are generated, blood circulation disorders occur in veins, resulting in swelling or inflammation, and ischemia or infarction, resulting in diseases such as myocardial infarction, stroke, and pulmonary infarction. In addition, various vascular control factors (serotonin, TXA2, PAF, etc.) are released during platelet activation, which can constrict blood vessels. When blood vessels contract, blood flow changes to prevent blood flow and platelet activation is amplified, and vascular endothelial damage progresses, thereby inhibiting normal cardiovascular function. Such hematogenous abnormalities are known to cause the development of vascular diseases such as arteriosclerosis, myocardial infarction, cardiac ischemic disease and cerebrovascular disease. (Marcus et al., 1993; Packham et al., 1994; Harker et al., 1994).

Blood flow improvement-related medicines are sold at home and abroad, but in the case of blood circulation drugs, they are simple complexes of functional materials, and some functional foods have problems due to drug addiction and side effects.

An object of the present invention to solve the above problems is to provide a composition for improving blood flow, a method for producing the same, and a health functional food comprising the same, which inhibits the synthesis of cholesterol and has excellent bile acid binding ability, thereby improving blood flow.

The present invention to achieve the above object,

It provides a blood flow improving composition comprising a fraction of less than 100,000 daltons molecular weight separated from the fermentation product fermented with lactic acid bacteria.

The composition may comprise fractions of up to 50,000 daltons in molecular weight.

The composition may comprise a fraction of molecular weight 30,000 to 50,000 dalton.

The fraction may be separated after extracting the fermentation product fermented with lactic acid bacteria with an organic solvent.

The fraction may be separated from the supernatant of the organic solvent extract of the fermentation product fermented with lactic acid bacteria.

The lactic acid bacteria is Lactobacillus acidophilus Can be.

The organic solvent may be ethanol.

The present invention to achieve another object of the present invention,

Fermentation step of inoculating lactic acid bacteria in the fermentation; And

It provides a method for producing a blood flow improving composition comprising a fractionation step of obtaining a fraction by molecular weight by ultrafiltration of the fermented product.

The method of preparing the blood flow improving composition may further include an organic solvent extraction step of extracting the fermentation product obtained by the fermentation step with an organic solvent.

In order to achieve another object of the present invention, the present invention,

It provides a health functional food comprising the composition for improving blood flow.

The blood flow improving composition of the present invention inhibits cholesterol synthesis and has excellent bile acid binding ability, thereby improving blood flow improving effect.

The composition for improving blood flow of the present invention is isolated from a substance derived from nature and is harmless to the human body and has no side effects even when used for a long time.

The composition for improving blood flow of the present invention is also effective in preventing adult diseases such as atherosclerosis or stroke.

The composition for improving blood flow of the present invention can be prepared by various health functional foods and can be easily consumed.

1 is a schematic diagram illustrating a process of separating the fermented fermented product by lactic acid bacteria by molecular weight.
Figure 2 shows the standard calibration curve of bile acids.

Hereinafter, the present invention will be described in detail so that those skilled in the art can easily practice.

The present invention provides a composition for improving blood flow comprising fractions having a molecular weight of 100,000 daltons or less separated from fermented fermented medicinal products with lactic acid bacteria.

Since the composition of the present invention separates the fraction showing the best effect on improving blood flow in the fermented fermented product, it can show the blood flow improving effect even in a small amount, and it is harmless to the human body as a natural derived material and has no side effects even when used for a long time. .

The medicine is Dioscorea batatas or Dioscorea A dried herb that has been removed from the bark of japonica , dried or steamed, mainly composed of viscous polysaccharide mannan and other proteins, minerals, and steroidal saponin and phenanthrene derivatives. Antioxidant, anti-inflammatory, anti-cancer, hyperglycemic, obese, immunomodulatory It is effective on the back.

The fermented product may be obtained by fermenting the acid powder with lactic acid bacteria. Preferably, the fermented product may be obtained by inoculating lactic acid bacteria into a powdered suspension of the acid powder and water to facilitate fermentation. The powder may be a dry powder form, but is not limited thereto. The suspension may be a mixture of acid and water in a weight ratio of 1: 5 to 1:10, preferably a mixture of 1: 9 by weight. Mixing the powder and water in the above weight ratio is excellent in the suspension ability of the powder due to the addition of water and the fluidity of the suspension and high in drinking.

The lactic acid bacteria are involved in the fermentation of kimchi to give the unique flavor and carbonic acid taste of kimchi and to produce a variety of organic acids, it has the function of absorbing and decomposing cholesterol in the intestine can lower the blood cholesterol content, intestinal action, Atherosclerosis prevention, thrombolytic dissolution, etc. The lactic acid bacteria may be used in the art, but preferably Lactobacillus acidophilus , Lactobacillus amylophilus, Lactobacillus casei , Lactobacillus fermentum or Lactobacillus pentosus can be used, more preferably, Lactobacillus acidophilus can be used. The Lactobacillus acidophilus is highly resistant to bile acid (bile acid), adsorbed by intestinal epithelial cells even at low pH, and has excellent viability in the digestive tract, and has an excellent health promotion effect, and fermented fermented fermented with Lactobacillus acidophilus . Is more health-promoting than the acid itself and has superior probiotics functionality by lactic acid bacteria.

The lactic acid bacteria can be cultured to be 1.0 × 10 ⁵ to 1.0 × 10 ⁹ cfu / ㎖, and most preferably can be cultured to 1.0 × 10 ⁷ cfu / ㎖. If the number of lactic acid bacteria is less than 1.0 × 10 ⁵ cfu / ㎖ may be a slow fermentation rate, when exceeding 1.0 × 10 ⁹ cfu / ㎖ may cause the odor by the production of by-products too fast fermentation rate.

The lactic acid bacteria may be inoculated in an amount of 0.5 to 2 parts by weight based on the powder of the powder or the suspension of the powder. When inoculating less than 0.5 parts by weight, the fermentation rate may be slow, and fermentation may not proceed. When inoculating more than 2 parts by weight, excessive fermentation may occur due to excessive inoculation, resulting in excessive generation of organic acids as a by-product and off-flavor. .

The fermentation may be performed at 35 to 40 ℃ for 3 to 7 days. The lactic acid bacterium causes lactic acid fermentation, and lactic acid fermentation inhibits cholesterol synthesis and binds bile acids to produce substances that improve blood flow. When fermentation proceeds at 35 to 40 ° C. for 3 to 7 days, proper fermentation proceeds to increase blood flow improving effect. If the fermentation is out of the above range, fermentation does not proceed properly and the blood flow improving effect is insignificant or the fermentation is excessive. Can be.

As the fermented product fermented with lactic acid bacteria, the supernatant obtained by centrifuging the fermented product may be used. Centrifugation of the fermented product is separated into a supernatant and a precipitate. At this time, the active ingredient is dissolved in a large amount in the supernatant, and thus the supernatant is superior to the precipitate in cholesterol synthesis inhibitory activity and bile acid binding ability, thereby improving blood flow improving effect.

The fraction may be a fraction having a molecular weight of 100,000 daltons or less separated from the fermentation product fermented with lactic acid bacteria, preferably a fraction having a molecular weight of 50,000 daltons or less, and more preferably a fraction of a molecular weight of 30,000 to 50,000 daltons. Can be. When the fraction of a specific molecular weight range is separated from the fermentation product fermented with lactic acid bacteria, the ability to inhibit cholesterol synthesis and bile acid binding is further improved, thereby improving blood flow.

The method of fractionating the fermented product by molecular weight may be used without limitation methods known in the art, and preferably may be separated by molecular weight using ultrafiltration. Figure 1 is a schematic diagram showing the separation of the fermented fermented product by lactic acid bacteria by molecular weight. Separation by molecular weight as shown in Figure 1 can be obtained an active ingredient for improving blood flow in the fermented fermented product.

The fraction may be an organic solvent extract obtained by extracting the fermentation product fermented with lactic acid bacteria with an organic solvent according to the molecular weight. The organic solvent may be used alone or a mixture thereof selected from the group consisting of n-hexane, chloroform, ethyl acetate, n-butanol and ethanol, but is not limited thereto. Preferably, ethanol may be used. Ethanol is harmless to the human body, so it is easy to apply to food, and the ethanol extract of the fermentation improves blood flow because HMG-CoA (3-hydroxy-3-methlglutaryl-CoA) reductase inhibitory activity inhibits cholesterol synthesis. HMG-CoA reductase is one of the enzymes needed to synthesize cholesterol in the body and is responsible for the initial stage of cholesterol synthesis. Acetyl CoA is converted to HMG-CoA by HMG-CoA synthase, and the converted HMG-CoA is synthesized as final cholesterol by HMG CoA reductase. Therefore, inhibition of HMG-CoA reductase also inhibits cholesterol synthesis. In the extraction process, the organic solvent may be used in a weight ratio of the fermented product and 4: 6 to 6: 4. When the organic solvent is used in a content less than the ratio may not be extracted properly, when used in a higher ratio than the increase of the effect is not large and rather the manufacturing process may not be efficient.

The fraction may be separated from the supernatant of the organic solvent extract of the fermentation product fermented with lactic acid bacteria. When the fermented product is extracted with an organic solvent and centrifuged, the supernatant and precipitate are separated. At this time, the active ingredient is dissolved in a large amount of the supernatant, and the supernatant has better cholesterol synthesis inhibitory activity and bile acid binding ability than the precipitate. Is raised.

The present invention also provides a fermentation step; And it provides a method for producing a composition for improving blood flow comprising a fractionation step. In addition, the method may further include an organic solvent extraction step. By fermenting the powder by the above method, not only a fermentation product can be obtained that enhances the blood flow improving effect, but also a composition capable of further enhancing the blood flow improving effect by separating the main active ingredient in the powder of fermentation according to the molecular weight. .

The fermentation step is a step of fermenting by inoculating lactic acid bacteria into the acid.

Specifically, after inoculating lactic acid bacteria into the powdered suspension of the mixed powder and water, it can be stored at 35 ℃ to 40 ℃ 3 to 7 days to ferment. The lactic acid bacteria can be incubated in MRS medium at 30 to 37 ℃ to 1.0 × 10 ⁵ to 1.0 × 10 ⁹ cfu / ㎖, centrifuged broth and discarded the supernatant and diluted with sterile saline to inoculate the medicinal suspension can do. The lactic acid bacteria used at this time are as described above. The fermented product can be obtained by fermentation as described above, and the supernatant can be taken by using the fermented product itself or by centrifuging the fermented product several times for 20 to 30 minutes at 5000 to 10000 rpm.

The extraction of the organic solvent is a step of extracting by adding an organic solvent to the fermentation product formed by the fermentation. The fermentation product or the supernatant of the fermentation product may be extracted with an organic solvent such as n-hexane, chloroform, ethyl acetate, n-butanol or ethanol, preferably ethanol to obtain an organic solvent extract, and the supernatant and precipitate of the organic solvent extract. Both improve blood flow, but the supernatant is more effective than the sediment.

The fractionation step is a step of obtaining a fraction for each molecular weight by ultrafiltration of the fermentation product or the organic solvent extract of the fermentation product. Fermentation products obtained from fermentation of fermented or organic solvent extracts of fermented products by using ultrafiltration membranes 300,000 dalton or more, 300,000 dalton or less, 100,000-300,000 dalton, 100,000 dalton or less, 50,000-100,000 dalton, 50,000 dalton or less, 30,000-50,000 dalton, The fraction according to the molecular weight can be obtained by sequentially filtering to the size of less than 30,000 daltons, 10,000 ~ 30,000 daltons, 10,000 daltons or less. In addition, the fractionation by molecular weight may be obtained by using the supernatant of the fermentation product or organic solvent extract in the fractionation step.

The present invention also provides a health functional food comprising the composition for improving blood flow. The health functional food is excellent in the ability to inhibit cholesterol synthesis and binding with bile acid, so the blood cholesterol concentration is lowered, thereby improving blood flow.

The blood flow improving composition may be added to various foods, beverages, grains, gums, teas, vitamin complexes and extracts, such as palates or health supplements, to prepare various foods according to a commonly known method. In addition, it may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like according to conventional methods. In addition, it may be formulated into a general formulation, and conventional additives used in food, such as excipients, extenders, binders, thickeners, emulsifiers, lubricants, wetting agents, suspending agents, coloring agents, flavorings, food additives, etc. Those skilled in the art can select and blend suitably.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. This is for the purpose of illustrating the invention only, and thus the scope of the invention is not limited.

< Example  1> Medicinal Fermentation  By molecular weight Fraction  purchase

Lactobacillus from the Genetic Bank of Korea Biotechnology Research Institute acidophilus KCTC3111 was incubated in 10 mL MRS broth to 1 × 10 ⁷ cfu / mL. The broth was incubated at 3000 rpm for 10 minutes to discard the supernatant, diluted in sterile saline, and then added to the powdered suspension containing powdered powder and water in a mass ratio of 1: 9, followed by fermentation at 37 ° C for 4 days. It was. All processes and preparations of fermentation remained sterile. The fermented fermented fermented 4 days was centrifuged twice at 8000 rpm for 30 minutes to separate the supernatant and precipitate. After adding supernatant and ethanol in a weight ratio of 1: 1 to a separate funnel at room temperature and mixing, the mixture was allowed to stand for 6 hours and then separated into a precipitate and a supernatant. The supernatant was collected, filtered and concentrated under reduced pressure to obtain an ethanol extract. 15 mL of the ethanol extracts were respectively dispensed into an ultrafiltration filter, and the supernatant was separated by centrifugation at 5000 rpm for 20 minutes. At this time, the ultrafiltration filter used was a centrifuge (J251, Beckman, Germany) and used as a micro centrifuge tube membrane cell (ultrafree-15, Millipore). The ultrafiltration filter had a pore size of 300,000. A total of five filters were isolated for each dalton, 100,000 dalton, 50,000 dalton, 30,000 dalton and 10,000 dalton sizes. Separation was carried out at 4 ℃ and the separated supernatant was also stored at 4 ℃ was used in the experiment.

< Example  2> Health functional drink  Produce

The functional beverage containing the fraction of molecular weight 30,000 to 50,000 dalton obtained after extracting the fermented fermented product prepared in Example 1 with ethanol was prepared in the composition shown in Table 1 below.

ingredient Content (mg) Molecular weight 30,000 to 50,000 dalton fraction 1.28 honey 522 Chioctosanamide 5 Nicotinic acid amide 10 Riboflavin Sodium Hydrochloride 3 Pyridoxine hydrochloride 2 Inositol 30 Ortsan 50 water 200

< Experimental Example  1> HMG - CoA reductase  Inhibitory activity

The HM-CoA reductase inhibitory activity was measured as follows by partially modifying the molecular weight fraction obtained in Example 1 as a sample based on Kleinsek (1981) method. Phosphoric acid buffer solution (pH 7.0) was added to 0.1 ml of the molecular weight fraction to reduce the pH change by the reagent to be added, and then 0.1 mM of 20 mM dithiotreitol (DTT, D-0632, sigma, USA), 3 mM β-NADPH ( sigma) 0.1 mL and HMG-CoA reductase 0.1 mL were added and the reaction was induced at 37 ° C. for 5 minutes. The HMG-CoA reductase was obtained by the following method. Liver from rats fed a high-fat diet for one week was extracted with ice cold buffer A (50 mM phosphate buffer pH 7.0 with 0.2 M sucrose) and 2 mM dL-dithiothreitol (DTT, D-0632, sigma aldrich chemical) per gram of liver. ) Add 2 mL, homogenize for 15 seconds with a homogenizer and centrifuge at 15000 × g for 10 minutes to obtain a supernatant, then ultracentrifuge at 100000 × g for 75 minutes, remove supernatant and white fat layer to remove microsome pellet. Recovered. Add 1 mL of buffer A (containing 50 mM EDTA) to 1 g of this pellet, wash, centrifuge at 100000 × g for 60 minutes, discard the supernatant, and buffer B (50 mM phosphate buffer pH 7.0 with 0.1 M sucrose, 2). mM DTT, 50 mM KCl, 30 mM EDTA) was added to 3 mL per 1.5 g of pellet and homogenized. Again, 7 mL of the same buffer was mixed and left at room temperature for 15-30 minutes, followed by centrifugation for 10 minutes at 100000 × g, 20 ° C. for 60 minutes, and the supernatant was used as an enzyme source and stored at -70 ° C. until use. Thus, HMG-CoA (sigma, U.S.A) was added to the sample that induced the reaction for 5 minutes, and the absorbance change of 340 nm was measured while reacting for 5 minutes, and the inhibitory activity was calculated by the following equation. The results are shown in Table 2 below. In Table 2, the control refers to the ethanol extract of the fermented fermented powder before the ultrafiltration.

HMG-CoA reductase inhibition rate (%) = ([1- (T / C)] × 100

(T: OD of sample, C: OD of blank)

Molecular weight (dalton) Inhibition activity (%) Control 63.4 More than 300,000 18.9 300,000 or less 73.0 100,000-300,000 5.7 100,000 or less 82.1 50,000-100,000 7.3 50,000 or less 88.3 30,000 ~ 50,000 95.4 30,000 or less 9.1 10,000-30,000 5.7 10,000 or less 5.3

As shown in Table 2, it can be seen that the fraction of which the molecular weight is lowered by ultrafiltration compared to the extract fermented with fermented fermentation with an organic solvent, HMG-CoA reductase inhibitory activity is excellent. That is, fractions with a molecular weight of 100,000 or less were superior to the inhibitory activity, less than 50,000 were more excellent, and showed the best inhibitory activity at 30,000 to 50,000. Therefore, it is estimated that a substance having a molecular weight of 30,000 to 50,000 has a blood flow improving effect.

< Experimental Example  2> Bile acid  Measurement of binding capacity

Bile acid is formed from cholesterol as a precursor and is known to be involved in the digestion and excretion of lipids. Bile acids or bile salts include cholic acid, deoxycholic acid, taurocholic acid and glycocholic acid. The bile acid is a precursor of cholesterol, and when the acid-enzyme product is combined with bile acid and finally discharged, the concentration of cholesterol in the blood is lowered. Thus, the method according to Camire's method (1993) targeting bile acid binding capacity according to the molecular weight prepared in the above example is as follows. Was measured. 2 mL of 0.1N HCl (Dae-jung, Korea) solution was added to 1 mL of the sample, followed by shaking for 1 hour in a constant temperature water bath. The pH was adjusted to 7.0 with 1N NaOH (Dae-jung-Korea) solution. In addition, 4 mL of 0.1 M phosphate buffer prepared with cholic acid (Sigma, USA), deoxycholic acid (Sigma, USA), taurocholic acid (Sigma, USA) and glycocholic acid (Sigma, USA) to 31.25 μmol / mL, respectively. 5 mL of a solution of 10 mg / mL of porcine pancreation (Sigma, USA) was added to 0.01 M phosphate buffer, and the mixture was stirred for 1 hour in a constant temperature bath. 2 mL of 1.33 M phosphoric acid (Sigma, USA) was added to the stirred solution, followed by centrifugation at 26,890 × g for 10 minutes, and the supernatant was taken. The remaining residue was added with 0.01 M phosphate buffer, followed by mixing and centrifugation. All collected. After adjusting the pH of the supernatant to 7.0, add 0.28 mL of the above solution and 3 mL of distilled water to the test tube, and add the test reagent (nitroamide dinucleotide (NAD), nitro blue tetrazolium salt (NBT), diaphorase, 3a-hydroxysteroid dehydrogenase) 0.5 mL was added and reacted for 5 minutes. Then, the reaction was stopped by adding 1.33M phosphoric acid, and the absorbance was measured at 530 nm. The bile acid binding force was calculated through the bile acid standard calibration curve, and the results are shown in Table 3 below. At this time, the standard calibration curve of the bile acid is shown in FIG. 2.

Molecular size Binding capacity ( μ M) Cholic acid Deoxycholic acid Taurocholic acid Glycocholic acid Control 7.6 17.2 7.8 8.4 More than 300,000 3.0 2.8 1.1 0.9 300,000 or less 10.6 18.0 9.2 10.1 100,000-300,000 1.7 0.3 1.1 1.3 100,000 or less 10.9 21.2 11.2 10.0 50,000-100,000 1.2 0.1 1.3 1.8 50,000 or less 10.8 17.7 16.2 15.3 30,000 ~ 50,000 11.5 21.4 16.8 15.6 30,000 or less 1.0 1.9 0.7 0.2 10,000-30,000 0.3 0.7 0.3 0.2 10,000 or less 0.0 0.1 0.3 0.1

As shown in Table 3, the bile acid binding capacity was also superior to the fraction of molecular weight 100,000 or less than the extract of the fermented product extracted with ethanol, the fraction of molecular weight 30,000 to 50,000 was the best. However, fractions less than 30,000 had little adsorption capacity for all bile acids.

As described above, the fractions obtained by fermentation of the fermented fermented with lactic acid bacteria by molecular weight have a molecular weight of 100,000 or less, preferably 50,000 or less, and more preferably 30,000 to 50,000. It is expected to be an active ingredient that indicates the activity of improving blood flow.

Claims (10)

A blood composition for improving blood flow, comprising a fraction having a molecular weight of 100,000 daltons or less separated from a fermentation product fermented with lactic acid bacteria. The method of claim 1,
Food composition for improving blood flow, characterized in that the composition comprises a fraction having a molecular weight of 50,000 dalton or less. The method of claim 1,
Food composition for improving blood flow, characterized in that the composition comprises a fraction of molecular weight 30,000 to 50,000 dalton. The method of claim 1,
The fraction is a blood flow improvement food composition, characterized in that the fermented product fermented with lactic acid bacteria separated from the organic solvent extract extracted with an organic solvent. The method of claim 1,
The fraction is separated from the supernatant of the organic solvent extract of the fermentation product fermented with lactic acid bacteria, blood flow improving food composition. The method of claim 4, wherein
The organic solvent is a food composition for improving blood flow, characterized in that the ethanol. The method of claim 1,
The lactic acid bacteria is Lactobacillus acidophilus food composition for improving blood flow, characterized in that. Fermentation step of inoculating lactic acid bacteria in the fermentation; And
Ultrafiltration of the fermentation product obtained in the fermentation step of producing a fraction for obtaining a fraction for each molecular weight, characterized in that the method for producing a composition for improving blood flow. The method of claim 8,
After the fermentation step, the organic solvent extraction step of extracting the fermentation product obtained by the fermentation step with an organic solvent further comprising the step of producing a composition for improving blood flow. A health functional food comprising a composition for improving blood flow of any one of claims 1 to 7.

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