KR101759494B1 - Preparation method for sodium-reduced soy sauce with anti-hypertensive activity - Google Patents

Abstract

More particularly, the present invention relates to a method for preparing an anti-hypertensive low-salt soy sauce, wherein one or more additives selected from the group consisting of jujube extract, onion extract, pine leaf extract, mulberry leaf extract, persimmon leaf extract, To 13% by weight of an antihypertensive low salt hepatocyte having antihypertensive activity, low salt, excellent taste and storage stability.
According to the present invention, a low salt liver having excellent antihypertensive activity can be produced. The soy sauce of the present invention can reduce anxiety about vascular diseases such as hypertension as compared with the general soy sauce even if the ingestion amount is increased due to the eating habit.
In addition, it is not only excellent in taste, but also solves the problem of low salted soy sauce, that is, it can be easily decayed due to its relatively low salinity, and commercialization is very easy.

Description

Methods for the preparation of antihypertensive low-salt soy sauce (sodium-reduced soy sauce with anti-hypertensive activity)

More particularly, the present invention relates to a method for preparing an anti-hypertensive low-salt soy sauce, wherein one or more additives selected from the group consisting of jujube extract, onion extract, pine leaf extract, mulberry leaf extract, persimmon leaf extract, To 13% by weight of an antihypertensive low salt hepatocyte having antihypertensive activity, low salt, excellent taste and storage stability.

Along with the improvement of the standard of living of the people, the pattern of eating habits changed to the advanced country type, and the irregular eating habits, nutritional overgrowth, duality and deficiency state coexisted. In addition, a large number of processed foods and a lack of exercise due to a variety of adult diseases, the average life expectancy of the elderly society with an increase in the need for health promotion of the general public increased the need for special nutrition and health care is emphasized.

In contrast to the usefulness of health-functional foods, some consumers are currently using them because of the disadvantages of separately ingesting daily necessities for health supplements and the high price. Therefore, the development of high-functional convenience food for all consumers is demanded by the present society. Therefore, the present inventor has developed a convenient and highly functional premium low salt soy sauce suitable for various consumer needs as follows.

Price: By combining with the food production process, it is possible to provide low-priced food without need of separate production facilities and packaging

Convenience: Can be ingested simultaneously with food

Accessibility: Accessibility is possible by purchasing from supermarket, mart, etc.

Consumer: New concept food for all consumers targeting hypertension

On the other hand, it has been reported that aging of blood vessels induces an increase in blood pressure, and the age of onset of vascular diseases is gradually lowered due to changes in eating habits, western eating habits, and environmental factors of Koreans. Hypertension is one of the major causes of hospitalization for patients aged 60 years or older. The market for hypertension related therapies is at an enormous cost of 1.4 trillion won, which is rapidly increasing every year. Persistent exposure to hypertension means that there is an increased risk of exposure to serious and fatal diseases such as kidney failure, stroke, arteriosclerosis, peripheral vascular abnormality, and death or hospitalization due to hypertension and vascular diseases. In addition to personal medical expenses, It is becoming a problem.

According to a recent survey, the average intake of sodium in Korea is 2.4 times that of WHO's recommended maximum intake (2,000 mg), and chronic diseases such as hypertension due to excessive sodium intake are reported to increase rapidly. Therefore, the Food and Drug Administration has sought to reduce the salinity of processed foods or to develop new products with low salt. In fact, in 2011, products that reduce the salinity of noodles such as ramen have been developed and released. However, it seems that the use of products that only lowered a certain level of salinity is not likely to have a great effect because it is likely to increase the usage to meet the taste of the Korean people who eat it. Therefore, it is necessary to develop a highly functional antihypertensive low salt liver which has the effect of reducing salinity and preventing and improving hypertension.

The cause of hypertension is known as the increase of active oxygen in the living body due to excessive stress, the change of the biorhythm according to the change of the environment, and the change of the blood vessel due to the dietary change. However, what mechanism causes the cause of hypertension is clearly known It is not. In particular, the cause of essential hypertension, which accounts for more than 90% of hypertension, has not yet been clearly elucidated. However, epidemiological studies have shown that genetic factors, mental stress, and environmental factors (excessive salt intake) It is known to be a multifactorial disease caused by the action of One of the causes of essential hypertension reported so far is the abnormal activity of the renin-angiotensin system, and when angiotensin II is over-expressed in the tissues, the angiotensin II receptor that is distributed in the cell wall is stimulated and activates NADPH oxidase, It is known that reactive oxygen species (ROS) are generated and they induce abnormal proliferation and migration of vascular smooth muscle cells, thereby increasing blood pressure by affecting contraction and relaxation of blood vessels. Inhibitors of active oxygen species (skebents), which are known to be major causes of hypertension, are widely distributed in plant species. In addition, rutin and GABA (γ-AminoButyric Acid) It is known to be contained in large amounts. Accordingly, the present inventors have studied the antihypertensive function of natural materials and then developed high-functional premium foods using these useful materials.

The development of domestic low - salt soy sauce started from the influence of Japan in the mid - 1990s and has been mainly used for patients' meals, and its market is also weak. Recently, however, the use of low-salt soy sauce has been increasing since 2011 due to the progress of the reduction of intake of sodium, which is centered on the KFDA, and the improvement of people's consciousness on health. In the initial stage of the development of the low salt soy sauce, the desalination method which desalinates a certain salinity from the raw soy sauce was mainly used. However, since the total nitrogen content, which is the standard of the soy sauce, is increased by the improvement of the fermentation technique, And the taste of soy sauce is being released as a new product through the change of formulation ratio. To date, several low-salt soy sauces have been developed and launched. Among them, low-salt soy sauce emphasizes functionality through addition of minerals in order to differentiate from conventional low salt soy sauce. However, with a small amount of minerals added, There is no reality.

In Korea, antihypertensive inhibition studies using various natural extracts have been conducted, and blood pressure is suppressed in hypertensive rats fed directly with mushroom. However, until now, the development of products that have been applied to foods has been minimal and most of them have been developed as products of functional foods for general medicine and health supplement. As described above, these products are expensive and have an inconvenience of being taken besides meals. Therefore, from the viewpoint of steady blood pressure management, it is considered that if the blood pressure management food having safety that is harmless to the human body is made, a good effect can be obtained .

As a result, the salinity of Japanese soy sauce products was reduced from 18 ~ 20% in the 1970s to 16 ~ 18% at present. In addition, low salt and infected liver have been developed since the late 1980s and are being used for patients. In Japan, unlike in Korea, low salty liver and infected liver are classified according to salinity, and low salinity liver has 10 ~ 12% salinity and 8 ~ 10% infected liver. As the manufacturing method, there is a method of diluting compressed raw soy sauce stock solution, a method of electrodialysis of raw soy sauce with ion exchange membrane, an ion exchange resin adsorption method and a selective dialysis method. In the case of low salt soy sauce, I am using it. In Japan, sales of low-salt soy sauce and infected soy sauce are higher than those of low-salt soy sauce, and the infected soy sauce is found in the kidney sauce (Japan Soy Sauce Institute magazine, 2004) It does not seem to be a big help for your health. Therefore, it is expected that if the new concept of anti - hypertensive / anti - vascular disease premium soy sauce is developed, it will be able to obtain many people 's attention from domestic as well as foreign countries.

Thus, the present inventors have made extensive efforts to produce soy sauce having high antihypertensive activity and low flavor while having excellent flavor. As a result, it was found that jujube extract, onion extract, pine leaf extract, mulberry leaf extract, persimmon leaf extract, To 9% to 13%, it is possible to produce an antihypertensive low-salt soy sauce which exhibits excellent antihypertensive activity, is excellent in low-salt flavor, and is safe for long-term storage and distribution.

Korean Patent No. 10-0561688

Journal of the Korean Society of Food Science and Nutrition 39 (3), 331-336, (2010). Clin. Exp. Hypertens. 33 (2), 95-99, (2011).

Accordingly, it is a main object of the present invention to provide a method for producing a functional soy sauce capable of reducing vascular disease anxiety due to ingestion of soy sauce.

Another object of the present invention is to provide an anti-hypertensive low-salt liver produced by the above-described method.

According to one aspect of the present invention, the present invention provides a method for producing a plant extract, which comprises adding one or more extracts selected from the group consisting of jujube extract, onion extract, pine leaf extract, mulberry leaf extract, persimmon leaf extract, ) ≪ / RTI > salt of the present invention.

In the method of the present invention, the jujube extract is prepared by adding 5 to 15 times the weight of the dried jujube to the dried jujube and extracting the extract at 90 to 110 ° C for 30 minutes to 10 hours, The onion extract is prepared by crushing onions and extracting the juice obtained by concentrating the juice obtained so as to have a soluble solid content of 50 to 70%. The pine needle extract is prepared by pine needles To 5 to 15 times the weight of the pine leaf, and extracting the extract at 90 to 110 DEG C for 30 minutes to 10 hours. The extract is concentrated to obtain a soluble solid content of 50 to 70% And 5 to 15 times the weight of the upper leaf, and extracting the extract at 90 to 110 캜 for 30 minutes to 10 hours. The extract is then lyophilized, Water or 10 to 80% (v / v) ethanol solution of 5 to 15 times the weight of the persimmon leaf powder is added to the persimmon leaf powder obtained by pulverizing the persimmon leaves, and the extract obtained by extracting at 0 to 30 ° C for 30 minutes to 10 hours And the water content is 10 wt% or less. The extract of the Middude bark is prepared by pulverizing the Middude bark, and the water content of the Middude bark powder is 5 to 15 times the weight of the Middude bark powder or 10 to 80% (v / v ) Ethanol solution is added and the extract obtained by extracting at 0 to 30 캜 for 30 minutes to 10 hours is preferably prepared by concentrating the extract so that the water content is 10% by weight or less.

In the method for producing antihypertensive low salt soy sauce according to the present invention, 0.01 to 1.0 wt% of jujube extract, 0.05 to 5.0 wt% of onion extract, 0.01 to 1.0 wt% of pine needle extract, 0.01 to 1.0 wt. %, 0.01 to 1.0 wt% of the persimmon leaf extract, and 0.001 to 0.1 wt% of the heder leaf extract.

In the method for preparing an anti-hypertensive low-salt soy sauce according to the present invention, it is preferable to add the above-mentioned Middude bark extract, mulberry leaf extract or onion extract as an essential ingredient. It is more preferable to add all the extracts of Rhododendron bark extract, Mulberry leaf extract and onion extract.

In the method for producing antihypertensive low-salt soy sauce according to the present invention, the defatted soybean or soybean soybean is fried and then the wheat is roasted and mixed with the fried tofu in the ratio of 6: 4 to 4: 6, 20 to 25% (w / v) of saline was added in an amount of 10 to 15 times by weight to the modified meju prepared by inoculating Aspergillus oryzae and culturing at 20 to 35 ° C for 40 to 45 hours, And then fermenting the mixture at 25 to 32 ° C for 1 to 12 months, and then adding the extract to the soy sauce stock solution.

In the method for producing an anti-hypertensive low-salt liver of the present invention, it is preferable to add 4 to 6% by weight of liquid fructose and 0.4 to 0.6% by weight of a purified salt and dilute so that the saltiness of the final liver is 10 to 10.5 (wt)% Do.

In the method for producing an anti-hypertensive low-salt liver of the present invention, it is preferable to add 6 to 8% by weight of liquid fructose and 0.6 to 1% by weight of a purified salt, and dilute so that the saltiness of the final liver is 11.5 to 12%.

According to another aspect of the present invention, there is provided an anti-hypertensive low-salt liver produced by the above-described method.

According to the present invention, the jujube extract, the onion extract, the pine needle extract, the mulberry leaf extract, the persimmon leaf extract and the hederock bark extract have antihypertensive activity. This was confirmed by analysis of the inhibitory effect of angiotensin-converting enzyme (ACE), analysis of DPPH radical scavenging ability (antioxidant activity), analysis of total phenol content, and analysis of various factors related to angiotensin II or vascular smooth muscle cells. The above-mentioned extracts were selected on the basis of the ease of antihypertensive activity and commercialization. Among them, the extracts of Chidyuk peel, onion extract and mulberry leaf extract are more effective. According to the results of the analysis, the antihypertensive effect varies depending on the concentration of the extract. Therefore, it is preferable to prepare the liver by adding the extract to the amount added in the present invention.

The production method of the present invention can be applied to brewed soy sauce, acid-decomposed soy sauce, and mixed soy sauce. In general, brewing soy sauce is made by sterilizing brewed soy sauce liquid made by fermenting and aging Meju in salt water. Amino acid soy sauce is prepared by hydrolyzing defatted soybeans with hydrochloric acid and neutralizing and then adjusting the salinity to salt. The raw liquid and acid-decomposed soy sauce solution are mixed and sterilized. When the preparation method of the present invention is applied to brewing soy sauce, a brewing soy sauce stock solution may be prepared and the extract of the present invention may be added thereto. In this case, the salt content can be controlled by adjusting the addition amount of the extract and the addition amount . In the case of amino acid soy sauce, the method of adding the extract of the present invention and salt or the like to the defatted soybean hydrolyzate can be used. In this case, the salt content can be controlled by controlling the addition amount of the extract, salt and additives. In case of mixed soy sauce, it is possible to use the method of adding the extract of the present invention and salt, etc. after mixing the stock solution of brewed soy sauce and the raw material of acid-decomposed soy sauce at appropriate ratio. In this case, the addition amount of extract, Can be adjusted.

According to the present invention, it is possible to lower the water activity of the low-salt soy sauce by adding the liquid fructose and the refined salt separately even if the dilution drainage is slightly increased, rather than adjusting the salinity by diluting only the brewed soy sauce stock solution. safe. At this time, it is preferable to add the liquid fructose and the purified salt in the same amount according to the salinity of the final liver.

According to the present invention, a low salt liver having excellent antihypertensive activity can be produced. The soy sauce of the present invention can reduce anxiety about vascular diseases such as hypertension as compared with the general soy sauce even if the ingestion amount is increased due to the eating habit.

In addition, it is not only excellent in taste, but also solves the problem of low salted soy sauce, that is, it can be easily decayed due to its relatively low salinity, and commercialization is very easy.

FIG. 1 is a graph showing the results of ACE inhibition analysis of persimmon leaf extract, green tea extract, jujube extract, hederock bark extract, mulberry leaf extract, pine leaf extract and onion extract.
2 is a graph showing the results of ACE inhibition assay according to the combination of extracts.
FIG. 3 is a graph showing the results of analysis of the total phenol content according to the combination of the hederfly skin extract, the leaf extract, and the onion extract.
FIG. 4 is a graph showing the results of DPPH radical scavenging analysis according to a combination of a hederfly skin extract, a leaf extract, and an onion extract and combinations thereof.
5 is a photomicrograph of the morphology of smooth muscle cells of vascular smooth muscle cells used for screening natural products having antihypertensive activity.
6 is a graph showing cytotoxicity analysis results of each extract against vascular smooth muscle cells.
FIG. 7 shows the results of analysis of LRP-1 expression pattern of vascular smooth muscle cells following treatment with angiotensin II.
FIG. 8 shows the results of analysis of the effect of each extract on LRP-1 expression of vascular smooth muscle cells following treatment with angiotensin II.
FIG. 9 shows the results of analysis of ERK phosphorylation pattern of vascular smooth muscle cells following treatment with angiotensin II.
FIG. 10 shows the results of analysis of effects of each extract on ERK phosphorylation of vascular smooth muscle cells following treatment with angiotensin II.
11 is a graph showing the results of analysis of the proliferation pattern of vascular smooth muscle cells following treatment with angiotensin II.
12 is a graph showing the results of analysis of the treatment of each extract in the proliferation of vascular smooth muscle cells following treatment with angiotensin II.
FIG. 13 is a graph showing the results of re-testing of extracts showing excellent effects in the proliferation of vascular smooth muscle cells following treatment with angiotensin II.
FIG. 14 is a graph showing the results of analysis of Ca ²⁺ free phase of vascular smooth muscle cells following treatment with angiotensin II. FIG.
FIG. 15 is a graph showing the results of analysis of the Ca ^{2+ release} of vascular smooth muscle cells according to the treatment of angiotensin II.
FIG. 16 shows the results of analysis of the effect of each extract on the migration ability of vascular smooth muscle cells following treatment with angiotensin II.
FIG. 17 shows the results of analysis of HO-1 expression pattern of vascular smooth muscle cells following treatment with angiotensin II.
18 shows the results of analysis of the effect of each extract on the expression of HO-1 in vascular smooth muscle cells following treatment with angiotensin II.
FIG. 19 is a graph showing the results of ACE inhibition assay analysis of each anti-hypertensive low-salt liver of the present invention.
20 is a graph showing the results of analysis of the total phenol content of each antihypertensive low salt liver of the present invention.
21 is a graph showing the results of analysis of DPPH radical scavenging activity of each anti-hypertensive low-salt liver of the present invention.
FIG. 22 shows the results of analysis of the effect of each anti-hypertensive low salt hepatic treatment of the present invention on LRP-1 expression of vascular smooth muscle cells following treatment with angiotensin II.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

<Search for anti-hypertensive / anti-vascular disease functional natural material>

In order to develop a functional low salt hepatocyte capable of inhibiting hypertension from natural materials, the physiological activities of hypertension of various natural materials were investigated in vitro and in vivo. Therefore, natural products that are thought to have antihypertensive action were selected through the preliminary investigation. When these materials were applied to soy sauce, the type of extracts were considered to be the most appropriate, and the extracts of each material were tested for antihypertensive ability.

Selection of candidates for natural materials

Seven materials that can be used commercially are selected among the natural materials known to have antihypertensive ability. Among these materials, the five materials that can be purchased from the market and the two extracted materials are shown in Table 1 below.

shape Candidate natural material Purchase of extract Jujube, onion, pine needles Purchase of extract powder Mulberry leaf, green tea Direct extraction Persimmon leaves, Midduk skin

At this time, the jujube extract was prepared by adding 10 times of purified water to the dried jujube weight and pressurizing and extracting the extract obtained at 100 ° C for 60 minutes by pressure filtration, concentrating the soluble solid content to 60% by using a vacuum concentrator, The extracts were prepared by crushing onion and washed onion with a grinder, filtering with a pressurized filter, filtering with a vacuum filter, and concentrating the soluble solid content to 50 to 62% after sterilization with a vacuum concentrator. 10 times of purified water was added and the extract obtained by pressure extraction at 100 ° C for 60 minutes was pressure filtered, and then the soluble solid content was concentrated to 60% by a vacuum concentrator and then sterilized. The extract of the upper leaves was 10 times Purified water was added and the extract obtained by pressure extraction at 100 ° C for 60 minutes was pressure-filtered and lyophilized. The green tea extract powder contained green tea 10 times more purified water is added to the weight, and the extract obtained by pressure extraction at 100 ° C for 60 minutes is filtered under pressure and freeze-dried.

Manufacture of persimmon leaf and midduk skin extract

In case of Persimmon leaf and Rhododendron shell, the extract was directly prepared.

The prepared persimmon leaves were dried and pulverized in a dryer at 50 ° C. for 24 hours to prepare persimmon leaf powder. The persimmon leaf powder and the 60% ethanol were mixed at a ratio of 1: 8 (w: v) Lt; / RTI &gt; The extract solution was filtered through Whatman No. 1 filter paper, and then concentrated to a moisture content of less than 10 (wt)% by a rotary vacuum concentrator at 60 ° C.

In the case of the hederhess shell, the extract was prepared by using the hederhide shell powder obtained by freeze-drying and pulverization as described above.

The yield of extract was 22.67% for persimmon leaves and 8.05% for heder - shell powder.

Analysis of anti-hypertensive ability using ACE inhibition

Method: 20 ml of 50 mM sodium borate buffer (pH 8.3) was mixed with 1 g of rabbit lung acetone powder at 4 ° C for 24 hours and then centrifuged at 4 ° C for 5 minutes to obtain an ACE enzyme solution. 50 AC of ACE enzyme solution was added to 50 희 of each sample diluted to each concentration, and preliminary reaction was carried out at 37 캜 for 10 minutes. Then, 100 25 of 25 mM HHL was added and reaction was carried out at 37 캜 for 60 minutes. The reaction was stopped by adding 250 μl of 1M HCl, and then 0.5 ml of ethyl acetate was added thereto, stirred for 30 seconds, and centrifuged (5,000 rpm, 10 min, 4 ° C) to obtain 0.2 ml of supernatant. The supernatant was completely dried at 80 ° C for 30 minutes, and 1 ml of distilled water was added thereto, and the absorbance was measured at 228 nm.

The antihypertensive ability of 7 candidate natural substances (persimmon leaves, green tea, jujube, muddoot shell, top leaf, pine leaf, onion) was analyzed by ACE inhibition. As shown in Fig. 1, the ACE inhibitory activity was measured in the order of chrysanthemum shell> top leaf> persimmon leaf> onion> jujube at an extract concentration of 10 μg / ml.

Among them, 3 kinds of materials with excellent ACE inhibition ability and easy commercialization (heder shell, onion, upper leaves) were selected, and the ACE inhibition performance according to the alone or in combination was analyzed and shown in FIG. Analysis of the extract at a concentration of 1 / / ㎖ showed no inhibitory effect on each other and no significant synergistic effect. Generally, when ACE extract was mixed with onion extracts, relatively high ACE inhibition was observed.

Total phenol content analysis

Take 1 ml of the natural product material diluted to a concentration of 1 / / ml, add 1 ml of 2% (w / v) Na ₂ CO ₃ solution, leave for 3 minutes, add 0.2 ml of 50% Folin-Ciocalteu reagent and react And allowed to stand at room temperature for 30 minutes. The mixture was centrifuged for 10 minutes at 13,400 x g, and 1 ml of the supernatant was taken and absorbance was measured at 750 nm. The total phenolic content was expressed as gallic acid equivalent (GAL) / ㎖ in comparison with the standard curve prepared using gallic acid.

Total phenolic content and antioxidative capacity of the extracts of Chrysanthemum morifolium extract, Onion extract, Mulberry leaf extract and mixtures thereof were analyzed. Total phenol contents of the sole material were 64.75 ㎍ GAE (gallic acid equilibrant) / g in the top layer, followed by onion and hederd peel (see Fig. 3). When these were mixed, the phenol content was measured as predicted according to the mixing type.

Analysis of antioxidative ability by DPPH radical scavenging ability

0.9 ml of a 0.041 mM DPPH solution was added to 0.1 ml of a natural material diluted to a concentration of 1 / / ml, followed by reaction at room temperature for 30 minutes, and the absorbance at 517 nm was measured. The DPPH radical scavenging activity of each sample was calculated by the following formula and expressed as a percentage.

×100DPPH radical scavenging ability (%) =

× 100

The DPPH radical scavenging ability of the extract was analyzed to determine antioxidant activity. As a result of analysis at a concentration of 1 / / ml, the overall tendency was found to be very similar to the total phenol content as shown in Fig. This means that the phenolic materials present in each natural product contribute greatly to the antioxidant ability.

Construction of Premature Cultured Vascular Smooth Muscle Cells

In order to screen for natural products with antihypertensive activity, a method of isolating vascular smooth muscle cells, which are closely related to hypertension, from free radicals of Rat thoracic aorta was constructed. Experiments were performed using cells between 4-8 passages while cryopreserving the early passage cells of the vascular smooth muscle cells thus isolated. No changes in morphology of primary cultured vascular smooth muscle cells following passage of culture were observed (see FIG. 5).

Cytotoxicity test of natural hyperhidrosis candidate

As a result of treating natural products with vascular smooth muscle cells at different concentrations, cytotoxicity of the natural product itself was confirmed by MTT assay. As a result, it was confirmed that cytotoxicity was not observed in most of the natural products as shown in FIG. 6, , And cytotoxicity was observed at a concentration of 100 μg / ml.

Effect of Natural Products on Increased Expression of LRP-1 Protein by Angiotensin II

Angiotensin II, a major hypertensive factor in vivo, has been implicated in the expression of low density lipoprotein receptor-related protein (LRP) -1, which is known to be hypertensive or hypercholesterolemic, . The expression of LRP-1 was determined by angiotensin II in a time- and concentration-dependent manner. The expression of LRP-1 was maximal in the group treated with 1 μM angiotensin II, and the angiotensin II- (Fig. 7). The change in expression was observed after 1 hour of treatment, and after 24 hours of treatment, the expression increased to a maximum, and then continued until 48 hours.

Therefore, in order to examine the effects of antihypertensive agents against the expression of LRP-1 by angiotensin II, each natural product was treated with vascular smooth muscle cells at different concentrations for 24 hours and then treated with 500 nM angiotensin II for 24 hours LRP-1 on the expression of LRP-1. As shown in FIG. 8, the expression of LRP-1 increased by angiotensin II was observed in 100 쨉 g / ml onion treatment group, 0.1 쨉 g / ml Midock treatment group, 10 쨉 g / ml second leaf treatment group, And it was confirmed that it was inhibited in 0.1 占 퐂 / ml persimmon treated group. However, green tea and jujube extract did not affect the expression of LRP-1 by angiotensin II.

Effect of Natural Products on ERK Activation by Angiotensin II

Activation (phosphorylation) of extracellular signal-regulated kinase (ERK) is an important signaling protein in the mechanism of angiotensin II-induced hypertension. Therefore, in order to investigate the effect of natural products on the activation of ERK, which is an important hypertension-related signal transduction system, an increase in phosphorylation of ERK was observed temporarily after 5 minutes of treatment with 500 nM of angiotensin II to vascular smooth muscle cells. (See Fig. 9).

Therefore, 100 μg / ml onion, 0.1 μg / ml midduk, 10 μg / ml upper leaf, 100 μg / ml pine leaf, and 0.1 μg / ml persimmon leaf extract, which are natural products showing the inhibitory effect of angiotensin II on the expression of LRP- After treatment with vascular smooth muscle cells for 24 hours, angiotensin II was treated for 5 minutes to examine the effect of natural products on phosphorylation of ERK. In contrast to previous studies on the expression of LRP-1, the results showed that phosphorylation of ERK increased by angiotensin II was suppressed only slightly in the lobed group, but other candidate natural substances inhibited the phosphorylation of ERK No effect was observed (see Fig. 10). These results suggest that natural products may exhibit antihypertensive action by suppressing other signal transduction pathways not through ERK.

Effect of Natural Products on Cell Proliferation of Vascular Smooth Muscle Cells

Hypertension and vascular smooth muscle cell proliferation are known to be closely related to each other. Therefore, we examined the effect of angiotensin II, which is an important hypertensive agent of blood vessels, on the proliferation of vascular smooth muscle cells. As a result, the proliferative capacity was increased from 24 hours after 500 nM of angiotensin II treatment, It was confirmed that the proliferation was increased (see Fig. 11).

As a result of examining the effect of natural products on the proliferation of angiotensin II-induced vascular smooth muscle cells, it was confirmed that the effect of inhibiting cell proliferation was significantly inhibited in the group treated with onion, middock, upper leaf, pine leaf and persimmon leaf extract 12). Onion extract and pine needle extract inhibited the proliferation of vascular smooth muscle cells in a concentration-dependent manner. However, in the upper leaves, persimmon leaves, and mithodok extract, cell proliferation was further promoted at a high concentration (see FIG. 12).

As a result, the effect of onion, middark, and upper leaves, which are the most effective inhibitors of cell proliferation, was further confirmed. As a result, angiotensin II-induced cell proliferation was observed in 100 μg / ml onion, 0.1 μg / (See Fig. 13).

Angiotensin II process Vascular smooth muscle cells Ca ₂ ⁺  Inhibitory Effect of Natural Products on Glass

It is known that when calcium is liberated from blood vessels due to various causes and deposited on the wall of blood vessels, it accelerates the calcification of blood vessels, thereby reducing the contraction and relaxation function of blood vessels, and is associated with the onset of hypertension. Therefore, in order to discover natural products that can inhibit hypertension, we examined natural products that can inhibit angiotensin II - induced calcium release in vascular cells. When using fura-2 that the binding affinity of the Ca ₂ ⁺ very high, measurement of very fine Ca ₂ ⁺ concentration change is not combined with Ca ₂ ⁺ in the fluorescent spectrophotometer 380nM, When a combination of two wavelength at 340nM as a result of measuring the Ca ₂ ⁺ concentration in accordance with the change ratio, angiotensin by II treatment it was confirmed that after 200 seconds occurs is Ca ₂ ⁺ free, non-ionic detergent let the addition of the digitonin fura-2 is Ca ₂ ⁺ and complete After 400 seconds. However, the treatment with EGTA, a chelator, revealed that fura-2 completely disappeared with Ca ₂ ⁺ and the signal disappeared (see FIG. 14).

On the other hand, after using fura-2 by pre-treatment of each natural materials with a 100 second, it processes the angiotensin II at 200 seconds results confirm the free Ca ₂ ^+, the group treated only with angiotensin II at 200 cho glass Ca ₂ ⁺ . However, it was confirmed that Ca ₂ ^{+ release} was inhibited in the group treated with angiotensin II after pretreatment with onion extract and leaf extract (see FIG. 15). In the group pretreated with the thyroid, persimmon leaf and pine leaf extract, results were obtained that did not affect Ca ₂ ^{+ release} by angiotensin II (see FIG. 15).

Effect of Natural Products on Cell Migration Ability of Angiotensin II-induced Vascular smooth muscle Cells

A wound healing assay was performed to investigate the effect of natural extracts on the migration ability of vascular smooth muscle cells, which are implicated in hypertension. When angiotensin II was treated with vascular smooth muscle cells, it was confirmed that the cell migration ability was increased compared with the control group. However, the cells were pretreated for 24 hours with 100 μg / ml of onion or 10 μg / (See Fig. 16). However, in the group treated with 0.1 μg / ml of healdocyte, cell migration ability by angiotensin II was not affected (see FIG. 16).

Effect of Natural Products on Heme Oxygenase (HO) -1 Expression

To examine the effect of natural products on the expression of HO-1, a powerful antioxidant protein in blood vessels, we examined the expression of HO-1 over time by treating 500 nM of angiotensin II with vascular smooth muscle cells. As a result, And the expression of HO-1 was strongly inhibited by angiotensin II after 24 hours of treatment, but recovery was obtained after 48 hours (see FIG. 17).

On the basis of these results, the expression of HO-1 was examined by Western blotting after treatment of onion, middark, and leaf extracts at various concentrations and then treated with angiotensin II for 24 hours. As a result, in the group treated with 10 μg / II, the inhibition of the expression of HO-1 was restored. However, the decrease in expression of HO-1 by angiotensin II was not recovered in the group treated with 100 μg / ml of onion or 0.1 μg / ml of midodocus 18).

Establishment of manufacturing method of natural material extract

Finally, we tried to establish a method for the production of the extract from the selected natural materials, which is not commercialized. The recovered powder was freeze - dried and the yield of 14.10% was obtained. The growth of the midduch is from March to April, and the composition of the shell varies depending on the growing season and conditions. The midduck used in this experiment is the midduck harvested from March to May. Extracts were prepared by using water and 70% ethanol in the milled products of Midduk shell powder. As shown in Table 9, when water was extracted with water, the yield was higher than when using 70% ethanol. It is considered that this is a positive result in terms of environmental impact and cost. In the future, it is recommended to directly add water without drying process and extract it within 24 hours at room temperature or 50 ℃ .

Extraction solvent yield water 13.77 70% ethanol 11.13

<Development of low salt soy sauce>

Manufacture of raw soy sauce brewing

Brewing soy sauce stock solution was prepared by the following process to obtain brewing soy sauce stock which is the main ingredient of low salt soy sauce formulation. Increase the defatted soybean and wheat chain to stir 5: 5 (wt) ratio after Aspergillus is Aspergillus duck material (Aspergillus mixed in the oryzae (2.0 x 10 &lt; ⁹ &gt; / g of porphyrin) was added to the total amount of 0.2 (wt)% and then mixed well and cultured at 20 to 35 DEG C for 40 to 45 hours. To minimize the killing of the germs caused by heat generated during the culture, the culture was treated at 18 hours and 30 hours after the culture. The prepared meju was prepared by adding 12.5 times (saline) of 23% saline to the fermentation tank and fermenting the fermentation at 25 to 32 ℃ for 4 months.

Development of low salt soy sauce formulation ratio

The salinity of low salted soy sauce is 12 ~ 12.5%, which is currently being launched in Korea, and aims to develop low salinity soy sauce with lower salinity or similar salinity than these products. A sample of 8 low - salt soy sauce samples was prepared by controlling the blending ratio of brewing soy sauce and liquid fructose ratio based on 10% salinity. In case of too low salinity, consumers may have problems such as microbial contamination during long - term use. Therefore, 8 kinds of low salt soy sauce samples were prepared based on 12% salinity in consideration of product safety. In addition to high salt intake, hyperglycaemic foods may cause serious health problems and thus reduce sugar content. After the preference test for these products, the blending ratio of 10% salted low salted liver and 12% salted low salted liver were finally determined.

Mixing ratio of 10% salted and low salt soy sauce (based on 1.60% brewed soy sauce solution, unit: wt%) Ingredients One 2 3 4 5 6 7 8 Brewed Soy Sauce 60 60 60 60 60 60 56 58 Liquid fructose 0 One 2 3 4 5 5 5 Enzyme treatment Stevia 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Weekly 5 5 5 5 5 5 5 5 Purified salt 0 0 0 0 0 0 0.48 0.14 essence 34.95 33.95 32.95 31.95 30.95 29.95 33.47 31.81

Salinity 12% Mixed ratio of low salt soy sauce (based on brewed soy sauce concentrate TN 1.60%, unit:% by weight) Ingredients One 2 3 4 5 6 7 8 Brewed Soy Sauce 65 65 65 65 67 67 70 70 Liquid fructose 8 8 8 7 8 7 8 7 Enzyme treatment Stevia 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Weekly 3 3.5 4 3 3 3 3 3 Purified salt 0.95 0.95 0.95 0.95 0.61 0.61 0.10 0.10 essence 23.00 22.50 22.00 24.00 21.34 22.34 18.85 19.85

Analysis of physicochemical characteristics of low salinity 10% low salt soy sauce

The total nitrogen, Brix, net extract, specific gravity, pH, salinity, and chromaticity of the product were analyzed for the commercialization of low salted soy sauce with 10% salinity. In addition, water activity which can be a criterion for the occurrence of microbial contamination during distribution and use of soybeans was analyzed and used for selecting the blending ratio considering palatability and safety.

The sensory evaluation of these prototypes was carried out by Sacred Heart Master Food and Mongolian Foods Lab. The results showed that when the sugar content (liquid fructose) was below 3%, the sensation of salty taste was too strong and the taste was poor. The sample showed good salinity similar to that of the existing 15.5% salinity, while the sample of 6 (60% of brewed soy sauce concentrate 56%), 7 (58% of brewed soy sauce 58%) and 8 . However, these samples are low salt and low-sugar products compared to conventional soy sauce, and they may be a problem in long-term use or product manufacturing. Therefore, water activity, which is a measure of contamination possibility, was measured in advance of the possibility of microbial contamination (Table 3).

Water activity and BRIX of low salinity 10% low salt liver (Judgment: 0.85 or less ○, 0.85 or more ×) Sample Water activity Brix Judgment Remarks One 0.859 26.02 × Brewed soy sauce 60%, liquid fructose 0% 2 0.857 27.09 × Brewed soy sauce 60%, liquid fructose 1% 3 0.855 27.48 × Brewed soy sauce 60%, liquid fructose 2% 4 0.853 27.62 × Brewed soy sauce 60%, liquid fructose 3% 5 0.852 28.51 × Brewed soy sauce 60%, liquid fructose 4% 6 0.849 28.97 ○ Brewed soy sauce 60%, liquid fructose 5% 7 0.861 27.83 × Brewed soy sauce 56%, liquid fructose 5% 8 0.850 28.59 ○ Brewed soy sauce 58%, liquid fructose 5%

As a result, samples 7 and 8 (brewing soy sauce juice 58%) and 8 (brewing soy sauce juice 60%), which are the standard measure of inhibition of microbial contamination in soy sauce, were found to have a water activity of 0.85% Was the best. However, even if the water activity is less than 0.85%, it can be used as a domestic product for long-term use by purchasing soy sauce, because there is a risk of microorganisms in use. Therefore, it is sold as small-sized disposable small-capacity product or soy sauce- It was judged to be good.

The physicochemical properties of Sample 7 are shown in Table 6 below.

product name Total nitrogen Brix Net extract importance pH Salinity Chromaticity Water activity Low salt soy sauce
(Sample 7) 0.93 28.59 18.56 1.113 4.79 10.03 11 0.850

Analysis of physicochemical characteristics of low salinity 12% low salt soy sauce

The total nitrogen, Brix, net extract, specific gravity, pH, salinity and chromaticity of low salted soy sauce adjusted to 12% salinity were analyzed. Water activity was analyzed and used for selecting the mixture ratio considering palatability and safety.

Physicochemical characteristics and water activity of low salt salted 12% salted soy sauce Sample Total nitrogen Brix Net extract importance pH Salinity Chromaticity Water activity One 1.12 32.85 20.95 1.145 4.80 11.90 11 0.829 2 1.11 32.85 20.93 1.142 4.80 11.89 11 0.823 3 1.11 33.03 21.14 1.142 4.80 11.89 11 0.813 4 1.11 32.22 20.33 1.142 4.80 11.89 11 0.833 5 1.13 33.12 21.26 1.144 4.79 11.86 11 0.810 6 1.14 32.40 20.54 1.143 4.79 11.86 11 0.834 7 1.18 33.74 21.85 1.147 4.79 11.89 11 0.805 8 1.19 33.19 21.30 1.146 4.80 11.89 11 0.807

As a result, all eight samples were found to be more than 0.850% safe from microbial contamination in terms of water activity. As a result of sensory evaluation for these 8 samples, 1, 4, and 6 showed good acceptance. Among them, 4 samples were the best. In view of the differences in production batches of these samples, samples (see Table 8) were prepared at the blending ratios of Samples 1, 4 and 6 using the stock solution of brewed soy sauce manufactured by Mongolian Food Co., (See Table 9). The results of sensory evaluation (Sacred Heart Master and Mongolian Food Co., Ltd.) Sample 4 showed the highest degree of preference and no problem in water activity. Therefore, it was concluded that the preparation rate of the antihypertensive / anti - vascular disease premium low salt hepatocyte should be adjusted to the mixing ratio of Sample 4.

Mixing ratio of samples 1, 4 and 7 (TN 1.58%, using brewing soy stock solution of Mongolian Food Co., unit: wt%) Ingredients One 4 6 Brewed Soy Sauce 65 65 67 Liquid fructose 8 7 7 Enzyme treatment Stevia 0.05 0.05 0.05 Weekly 3.0 3.0 3.0 Purified salt 0.95 0.95 0.95 essence 23.00 24.00 22.34

Chemical Product Characteristics and Water Activity of Secondary Samples (Using Brewed Soy Sauce Liquid from Mongolian Food Co., Ltd.) Prototype Total nitrogen Brix Net extract importance pH Salinity Chromaticity Water activity One 1.03 32.86 20.98 1.144 4.85 11.88 11 0.820 4 1.03 32.18 20.39 1.142 4.85 11.79 11 0.830 6 1.05 32.49 20.79 1.140 4.86 11.70 11 0.824

Establishment of low salt soy sauce manufacturing process

After sterilization for 30 minutes at 93 ℃ and natural cooling to 30 ℃, the precipitate was precipitated for 3 days by addition of precipitating agent, Cobolox, Lt; / RTI &gt; In the subsequent mass production, the low-salt soy sauce mixture was sterilized with a heat exchanger sterilizer at 93 ° C, cooled to 30 ° C, added with 0.1% of cobolock, precipitated for 3 days, filtered with paracellum, precipitated for 2 days, The manufacturing process of packing method was established.

<Development of anti-hypertensive low-salt soy sauce>

Seven samples were prepared by adding or extracting the extracts of Chrysanthemum morifolium extract, Lycopersicon esculentum L. and onion extracts, which were found to have antihypertensive effect, to 12% low salting liver. Samples for 15.5% and 12% salinity 15% and 12% low salt liver control were also prepared for functional testing in vitro and in vivo. The physicochemical properties of total nitrogen, Brix, net extract, specific gravity, pH, salinity, and chromaticity of these products were analyzed.

Production of antihypertensive low salt soy sauce

A sample was prepared at the blending ratio shown in Table 10 below. Samples 1 and 2 are control samples for the assay of antihypertensive bioactivity.

Combination ratio of anti-hypertensive low-salt liver (unit:%) Ingredients 1 ^* 2 ^** 3 ^** 4 ^** 5 ^** 6 ^** 7 ^** 8 ^** 9 ^** Brewed Soy Sauce 65 65 65 65 65 65 65 65 65 Liquid fructose 7 7 7 7 7 7 7 7 7 Enzyme treatment Stevia 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Weekly 3 3 3 3 3 3 3 3 3 Thyme shell extract - - 0.01 - - 0.01 0.01 - 0.01 Leaf extract - - - 0.1 - 0.1 - 0.1 0.1 Onion extract - - - - 0.5 - 0.5 0.5 0.5 Purified salt 4.45 0.95 0.95 0.95 0.95 0.95 0.95 0.95 0.95 essence 20.50 24.00 23.99 23.90 23.50 23.89 23.49 23.40 23.39

*: Salinity 15.5%, **: Salinity 12.0%

Analysis of physicochemical properties of antihypertensive low salt liver

The physicochemical properties of the prepared samples are shown in Table 11 below.

Physicochemical properties of anti-hypertensive low-salt liver Prototype Total nitrogen Brix Net extract importance pH Salinity Chromaticity One 1.04 34.66 19.09 1.164 4.56 15.57 11 2 1.05 32.33 20.03 1.144 4.56 12.03 11 3 1.06 32.45 20.42 1.145 4.53 12.03 11 4 1.06 32.45 20.40 1.145 4.53 12.05 11 5 1.05 32.51 20.48 1.146 4.53 12.03 11 6 1.05 32.45 20.41 1.146 4.51 12.04 11 7 1.05 32.56 20.52 1.147 4.51 12.04 11 8 1.05 32.62 20.58 1.147 4.52 12.04 11 9 1.06 32.62 20.59 1.146 4.52 12.03 11

Antihypertensive effect, total phenol content, antioxidant ability analysis of antihypertensive low salt liver

Functionality was analyzed for nine samples. Each sample was diluted 100-fold and each analysis was performed with a filtrate through a 0.2-μm filter paper. As shown in FIG. 19, anti-hypertensive activity measured by ACE inhibition was slightly increased in 12% low salted soy sauce compared with 15% general soy sauce, and further increased when natural material was added. Especially, the highest ACE inhibitory effect was observed in the case of the mixture of the hederock shell and the onion extract, which was increased by more than 10% compared to 15% of normal soy sauce by absolute value and about 30% by ACE inhibition by relative value.

The results of analysis of total phenol content and DPPH radical scavenging ability are shown in FIGS. 20 and 21, respectively. Also, 12% low salted soy sauce was slightly increased compared with 15% general soy sauce, and it was further increased when natural material was added. In particular, the highest phenol content was obtained in the case of mixed healdocarp and onion extract, and the DPPH radical scavenging ability was similar in all natural material-containing soy sauce.

Anti-hypertensive effect of low salt liver containing antihypertensive agent

The effect of angiotensin II on the expression of LRP-1, an important antihypertensive marker, was examined by using low-salt liver samples containing antihypertensive agents. As a result, the expression of LRP-1 by angiotensin II was inhibited by a low salt liver containing onion, Showed a tendency to decrease in the added vascular smooth muscle cells (see Fig. 22).

Establishment of manufacturing process of anti-hypertensive premium low salt liver

After mixing, the mixture was sterilized at 93 ° C and spontaneously cooled to 30 ° C. Then, the spirits were added to each mixture at a ratio of 0.1%, and the mixture was allowed to stand for 3 days to precipitate the precipitate . After filtration by paracell filtration, only the supernatant was recovered. Cobolox was added to adsorb and remove macromolecular substances such as proteins produced during soy sauce production.

Claims (9)

(1) to (6), wherein the weight ratio of the defatted soybean and roasted wheat flour is from 6: 4 to 4: 6. w / v) saline, fermented and aged at 25 to 32 ° C for 1 to 12 months to prepare a soy sauce stock solution,
In the brewing soy sauce stock solution
An onion extract obtained by crushing onions and juicing and concentrating the juice obtained so that the content of soluble solids is 50 to 70%; Extracting the extract obtained by lyophilizing the extract obtained by adding water 5 to 15 times the weight of the upper leaves to the upper leaves and extracting the extract at 90 to 110 캜 for 30 minutes to 10 hours; And water or 10 to 80% (v / v) ethanol solution of 5 to 15 times the weight of the heald crust powder is added to the heald crust powder obtained by crushing the heald crust, followed by extraction at 0 to 30 ° C for 30 minutes to 10 hours The extract obtained is concentrated to a water content of 10% by weight or less; And one or more extracts selected from the group consisting of salicylic acid and salicylic acid are prepared and prepared with a salinity of 10 to 12% (w / v)
A method for preparing a low salt soy sauce having the effect of preventing and / or improving hypertension, which comprises adding the extract of Chrysanthemum morifolium as an essential ingredient. delete The method according to claim 1,
The onion extract may contain 0.05 to 5.0% by weight,
The topical extract comprises 0.01 to 1.0% by weight,
Wherein the extract is added in an amount of 0.001 to 0.1% by weight based on the total weight of the extract. delete The method according to claim 1,
A method for preparing a low salt soy sauce having the effect of preventing and / or improving hypertension, which comprises essentially adding the extract of Chrysanthemum morifolium peel, Lobstaff extract and onion extract. delete The method according to claim 1,
4 to 6% by weight of liquid fructose and 0.4 to 0.6% by weight of purified salt are added,
And diluting the resulting soy sauce so that the salinity of the final soy sauce is 10 to 10.5%. The method according to claim 1,
6 to 8% by weight of liquid fructose and 0.6 to 1% by weight of purified salt are added,
And diluting the resulting soy sauce so that the salinity of the final soy sauce is 11.5 to 12%. A low salt soy sauce having the effect of preventing and improving hypertension produced by the method of any one of claims 1, 3, 5, 7, and 8.

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