KR102274371B1 - Cudrania tricuspidata fruit-fermented vinegar with antiobesity and antihyperlipidemic effect and producing method thereof - Google Patents

Abstract

The present invention relates to a fermented cujippong fruit fermented vinegar having excellent anti-obesity and anti-hyperlipidemic effects obtained by fermenting Cuji mulberry fruit. Kujippong fruit fermented vinegar of the present invention inhibits the production of fat globules, reduces triglyceride, cholesterol and leptin, promotes the release of free glycerol, decomposes triglycerides, and reduces the expression of genes related to adipocyte differentiation, As confirmed to inhibit the activity of obesity-related enzymes, it has excellent anti-obesity and anti-hyperlipidemic effects.

Description

Cudrania tricuspidata fruit-fermented vinegar with antiobesity and antihyperlipidemic effect and producing method thereof

The present invention relates to fermented vinegar having excellent anti-obesity and anti-hyperlipidemic effects obtained by fermenting Cuji mulberry fruit and a method for preparing the same.

Cudrania tricuspidata is a deciduous broad-leaved small arboreous tree or shrub belonging to the family Moraceae, also called Gutthorn. The mulberry tree is distributed in Korea, Japan, and China, and inhabits at the 100-700 m high sunny shores, slopes, crevices, fields, and beaches, and is mainly found in mountains. There are about 10 species of Cudrania mulberry, and the leaves are mainly used as a substitute for mulberry leaves, the fruits are used to make jam or alcohol, and the bark and roots of the tree are used for medicinal purposes or as raw materials for paper.

Cujippong leaves are mainly used in oriental medicine for the treatment of eczema, mumps, tuberculosis, bruises, chronic low back pain, and acute arthritis. did. In Donguibogam, it is effective in nourishing, tonic, yin, insomnia, and fading of eyesight. The root and stem bark are recorded as being good for female diseases and have been consistently used as folk remedies. Research on these mulberry trees is also being conducted, and various efficacy studies such as lipid oxidation inhibitory action, antibacterial activity study, anticancer activity study, and antidiabetic effect have been reported.

The fruits of the mulberry tree are hard, green, and ripen in red as they become slightly soft in September to October. When the leaves are cut, a white liquid is released and the fruit is sweet.

Cuji mulberry fruit contains active ingredients such as gaba and aspartic acid, including rutin, morin, and mortine, which are flavonoids. Rutin strengthens capillaries and is known to have a good effect on diabetes and blood pressure. GABA is reported to be effective in improving anxiety, depression, insomnia, and memory as a neurotransmitter.

In spite of these effects, the fruit of Cudrania contains a large amount of moisture, so it is easily deteriorated due to high possibility of microbial contamination such as bacteria and fungi, so it must be stored frozen. If the fruit is dried to remove moisture, the original flavor disappears with discoloration. There is a problem in product development due to a decrease in product quality.

Vinegar is a fermented seasoning with the longest history. It has been developed as a seasoning that adds flavor and flavor to food, such as pickled vinegar and dressing. As the income level rises and people pursue a healthy life, the vinegar market is diversifying and growing rapidly as seasoning-oriented vinegar has been developed as a beverage. Drinking vinegar drinks are leading the vinegar market, and seasoned vinegar is also showing continuous growth. Seasoned vinegar is moving from synthetic vinegar to fermented vinegar made from fruits and grains, and increasing functionality from alcoholic fermentation to natural fermentation.

Acetic acid in fermented vinegar breaks down lactic acid to relieve fatigue, and breaks down fat to prevent obesity. Various organic acids including citric acid, vitamins, and minerals are effective in preventing adult diseases such as strengthening the body's immunity, suppressing cholesterol, and lowering blood pressure. Recently, the vinegar diet is gaining popularity not only in the United States and Japan, but also in Korea. Fermented vinegar is expanding the base of vinegar as it is used in vinegar drinks, pickled foods, vinegar dressing, and vinegar diet. Along with the growing desire for well-being, the highly functional fermented vinegar market will continue to expand, and manufacturing and sales of fermented vinegar and vinegar beverages using cultivated crops will lead to an increase in corporate sales and job creation.

Vinegar using Cuji mulberry has also been developed, and a technique for manufacturing vinegar using Cuji mulberry or its fermented product, or manufacturing vinegar using Cuji mulberry fruit extract is also known.

Korean Patent Registration No. 10-1204875 Korean Patent Publication No. 10-2015-0089129 Korean Patent Registration No. 10-1463979 Korean Patent Publication No. 10-2015-0131482 Korean Patent Registration No. 10-1693668 Korean Patent Registration No. 10-1796770 Korean Patent Publication No. 10-2018-0047239 Korean Patent Registration No. 10-1895521

An object of the present invention is to prepare Cujippong fruit fermented vinegar having excellent anti-obesity and anti-hyperlipidemic effects by fermenting Cuji-ppong fruit.

In order to achieve the above object, the present invention comprises the steps of: thawing by mixing 1.5 to 2.5 liters of hot water at 92 to 98 ° C. to 1 kg of cujippong fruit, followed by grinding to obtain cujippong juice; Mixing 0.2-0.3 kg of sugar per 1 liter of Cujippong Juice to the Cujippong Juice to increase the sugar content of Cujippong Juice; Sterilizing the kkujippong juice with increased sugar content at 85° C. for 80 to 100 minutes; Cooling the sterilized kkujippong juice to 25-30 ℃; Saccharomyces cerevisiae Fermivin (Saccharomyces cerevisiae Fermivin) main mother culture in the cooled Cujippong juice is inoculated to be 4-6% by volume of the total amount, and fermented at 23-27℃ for 9-11 days, Cujippong wine to obtain; Cooling the kkujippong wine after sterilizing for 20 to 40 minutes at 80 ~ 90 ℃; The cooled Cujippong wine and acetic acid bacteria ( Acetobacter aceti ) prepared with the isolate strain (acidity 7.0 ~ 7.4) mixed in a volume ratio of 1:0.8 ~ 1.2 and fermented at 28 ~ 32 ℃; And fermented for a total of 21-42 days while adding 230-270 ml of sterilized Cujippong wine per liter of fermented vinegar at 7-day intervals from 7 days after fermentation in a fed-batch manner to obtain Cujippong fruit fermented vinegar, anti-obesity And it provides a method for producing kkujippong fruit fermented vinegar having excellent antihyperlipidemic effect.

The method may further include inoculating Saccharomyces cerevisiae permibin and adding 250 to 350 ppm of cellulase on the second day.

In addition, the present invention provides a fermented kkujippong fruit vinegar prepared by the above method, excellent anti-obesity and anti-hyperlipidemic effect.

Kujippong fruit fermented vinegar of the present invention inhibits the production of fat globules, reduces triglyceride, cholesterol and leptin, promotes the release of free glycerol, decomposes triglycerides, and reduces the expression of genes related to adipocyte differentiation, Inhibits the activity of obesity-related enzymes. Therefore, the fermented cujippong fruit fermented vinegar of the present invention has excellent anti-obesity and anti-hyperlipidemic effects while containing useful components of the cujippong fruit.

1 is a graph showing the change in acidity of Cujippong fruit fermented vinegar according to fermentation time.
Figure 2 is the result of confirming the cytotoxicity of kkujippong fruit fermented vinegar.
3 is a result confirming the inhibitory effect on the production of adipocytes in 3T3-L1 cells of kkujippong fruit fermented vinegar.
4 is a result confirming the triglyceride reduction effect in 3T3-L1 cells of kkujippong fruit fermented vinegar.
5 is a result confirming the free glycerol release effect in 3T3-L1 cells of kkujippong fruit fermented vinegar.
6 is a result confirming the total cholesterol reduction effect in 3T3-L1 cells of kkujippong fruit fermented vinegar.
7 is a result confirming the effect of reducing leptin release in 3T3-L1 cells of kkujippong fruit fermented vinegar.
8 is a result confirming the effect of reducing the expression of fat cell differentiation-related gene expression of kkujippong fruit fermented vinegar.
Figure 9 is a graph showing the results of confirming the triglyceride decomposition ability of kkujippong fruit fermented vinegar.
10 is a result of analyzing the antihypertensive effect of cujippong fruit fermented vinegar using p-nitrophenyl butyrate as a lipolytic enzyme substrate.
Figure 11 is the result of analyzing the antihyperlipidemic effect of kkujippong fruit fermented vinegar using p-nitrophenyl palmitate as a lipolytic enzyme substrate.
12 is a result of analyzing the antihyperhemostatic effect of cujippong fruit fermented vinegar using p-nitrophenyl decanoate as a lipolytic enzyme substrate.
Figure 13 is a result of analyzing the activity of pancreatic lipase when kkujippong fruit fermented vinegar is treated.
14 is a result of confirming the residual enzyme activity of pancreatic lipase when the kkujippong fruit fermented vinegar is treated.
Figure 15 is the result of confirming the enzyme inhibitory activity of pancreatic lipase when treated with kkujippong fruit fermented vinegar.
16 is a result of analyzing the activity of α-amylase when treated with kkujippong fruit fermented vinegar.
17 is a result of confirming the residual enzyme activity of α-amylase when treated with kkujippong fruit fermented vinegar.
18 is a result confirming the enzyme inhibitory activity of α-amylase when treated with kkujippong fruit fermented vinegar.
19 is a result of analyzing the activity of β-glucosidase when treated with kkujippong fruit fermented vinegar.
20 is a result of confirming the residual enzyme activity of β-glucosidase when treated with kkujippong fruit fermented vinegar.
Figure 21 is a result confirming the enzyme inhibitory activity of β-glucosidase when treated with kkujippong fruit fermented vinegar.
22 is a kinetic analysis of the lipolytic enzyme inhibitory activity of p -hydroxybenzyl alcohol in kkujippong fruit fermented vinegar.
23 is a kinetic analysis of the glycolytic enzyme inhibitory activity of p -hydroxybenzyl alcohol in kkujippong fruit fermented vinegar.

Cuji mulberry fruit fermented vinegar with excellent anti-obesity and anti-hyperlipidemic effects of the present invention,

Obtaining Cujippong juice by mixing 1.5~2.5ℓ of hot water at 92~98℃ with 1kg of Cujippong fruit, thawing and grinding;

The step of increasing the sugar content of the kkujippong juice by mixing 0.2 to 0.3 kg of sugar per 1 liter of cujippong juice with the cujippong juice;

Sterilizing the kkujippong juice with increased sugar content at 85° C. for 80 to 100 minutes;

Cooling the sterilized kkujippong juice to 25-30 ℃;

Saccharomyces cerevisiae Fermivin (Saccharomyces cerevisiae Fermivin) main mother culture in the cooled Cujippong juice is inoculated to be 4-6% by volume of the total amount, and fermented at 23-27℃ for 9-11 days, Cujippong wine to obtain;

Cooling the kkujippong wine after sterilizing for 20 to 40 minutes at 80 ~ 90 ℃;

The cooled Cujippong wine and acetic acid bacteria ( Acetobacter aceti ) prepared with the isolate strain (acidity 7.0 ~ 7.4) mixed in a volume ratio of 1:0.8 ~ 1.2 and fermented at 28 ~ 32 ℃; and

After 7 days of fermentation, 230-270 ml of sterilized Cujippong wine per liter of fermented vinegar is added in a fed-batch manner at intervals of 7 days and fermented for a total of 21-42 days to obtain Cujippong fruit fermented vinegar.

Hereinafter, the present invention will be described in detail.

Thawed by mixing 1.5~2.5ℓ of hot water at 92~98℃ with 1kg of Cujippong fruit, and grinding the thawed Cujippong fruit to obtain Cujippong juice. At this time, it is more preferable to use hot water of about 95 ℃ hot water, and it is more preferable to mix 2 liters of hot water with 1 kg of kkujippong fruit.

The sugar content of the juice is increased by mixing 0.2 to 0.3 kg of sugar per 1 liter of kkujippong juice. It is desirable to increase the sugar content from 6°Brix to 24°Brix. It is more preferable to mix 0.22 to 0.24 kg of sugar per 1 liter of kkujippong juice.

Sterilize the kkujippong juice with the increased sugar content at 85° C. for 80 to 100 minutes. Sterilization for 90 minutes is more preferable. Sterilization using an autoclave is preferred.

Cooling the sterilized kkujippong juice to 25 ~ 30 ℃, Saccharomyces cerevisiae Fermivin (Saccharomyces cerevisiae Fermivin) is inoculated so as to be 4 ~ 6% by volume of the total amount of jumo culture. It is more preferable to inoculate so that it may become 5 volume% of the total amount. It is fermented for 9-11 days at 23-27°C after inoculation, and more preferably fermented at 25°C for 10 days. During fermentation, stir twice daily to prevent long-term exposure of the flesh to air.

Saccharomyces cerevisiae permibin may be inoculated and 250-350 ppm of cellulase may be added on the second day. As the cellulase, Aspergillus niger cellulase is more preferably used, and the cellulase is more preferably added in an amount of 300 ppm. When cellulase is added, glycoside-type useful components are eluted and the content of fragrance components increases.

Cujippong wine is obtained by the fermentation.

The kkujippong wine is sterilized at 80-90° C. for 20-40 minutes and then cooled. It is more preferable to sterilize at 85° C. for 30 minutes.

The cooled Cujippong wine and acetic acid bacteria prepared from the isolated strain ( Acetobacter aceti ) seed vinegar (acidity 7.0-7.4) is mixed in a volume ratio of 1:0.8-1.2, and acetic acid fermentation is performed at 28-32 ℃.

After 7 days of fermentation, the content of acetic acid is gradually increased while adding sterilized Cujippong wine in a fed-batch manner at a rate of 230-270㎖ per liter of fermented vinegar at 7-day intervals.

Fermented for a total of 21-42 days, Cujipon fruit fermented vinegar is obtained. Fermentation for 30 days is more preferable.

The present invention will be described in more detail with reference to the following examples. These examples illustrate the present invention, but the scope of the present invention is not limited thereto.

<Example 1>

2 kg of frozen Cuji mulberry fruits were mixed with 4 liters of hot water at about 95° C. and thawed, and the thawed Cuji mulberry fruits were ground with a blender.

Sugar content was increased by adding 1.38 kg of sugar to 6ℓ of kkujippong juice.

Kujippong juice with increased sugar content was sterilized at 85° C. for 1 hour and 30 minutes using an autoclave.

After sterilization, after cooling to 25-30°C, the Saccharomyces cerevisiae permibin strain was inoculated to 5% by volume of the total amount and fermented at 25°C for 10 days. During the fermentation period, it was stirred twice daily to prevent long-term exposure of the flesh to air.

Saccharomyces cerevisiae permibin strain was inoculated and 300 ppm of Aspergillus niger cellulase was added on the second day.

It was fermented for 10 days to obtain Cujippong wine.

Changes in the quality of Cujippong wine during the fermentation period are shown in Table 1 below.

Saccharomyces cerevisiae
(Work) enzyme
(Work) Contents pH sugar content
(°Brix) total acidity
(%) Alcohol
(%) 0 - Saccharomyces cerevisiae pre-inoculation (curdling juice state) sampling 5.44 24.0 0.18 0.00 One 0 Sampling after enzyme addition 4.38 23.8 0.22 1.1 2 One Sampling on the 2nd day after Saccharomyces cerevisiae inoculation (rose scent) 3.94 23.0 0.66 2.2 4 3 Sampling on the 4th day after Saccharomyces cerevisiae inoculation (old watermelon flavor remains) 3.73 16.0 0.79 6.1 6 5 Sampling on the 6th day after Saccharomyces cerevisiae inoculation (increasing alcohol flavor) 3.77 12.0 0.60 11.6 8 7 Sampling on the 8th day after Saccharomyces cerevisiae inoculation 3.81 10.0 0.55 12.1 10 9 Day 10 sampling after Saccharomyces cerevisiae inoculation 3.82 9.5 0.58 14.0

As described above, the kkujippong wine (10th day) obtained by fermentation for 10 days was filtered, and the filtered kkujippong wine was sterilized at 85° C. for 30 minutes and then cooled.

Cujippong wine and acetic acid bacteria ( Acetobacter aceti ) seed vinegar (pH 7.2) prepared from the isolated strain was mixed in a volume ratio of 1:1 and acetic acid fermentation was performed in an incubator at 30°C.

After 7 days of fermentation, the acetic acid content was gradually increased by adding pasteurized Cujippong wine in a fed-batch manner at a rate of 250 ml per liter of fermented vinegar at 7-day intervals.

Fermented for 30 days, Cujippong fruit fermented vinegar was obtained.

[Experimental example]

Preparation of samples

For functional analysis of Cujippong fruit fermented vinegar, alcohol was removed from Cujippong fruit fermented vinegar fermented for 30 days with a rotary vacuum concentrator and then freeze-dried to prepare a semi-solid concentrate and provided as a sample.

<Experimental Example 1>

The change in acidity according to the fermentation time (days) of the curdling fruit fermented vinegar prepared in Example 1 was confirmed.

Total acidity was measured at 0 days, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, 56 days and 63 days of fermentation, and the results are shown in Table 2 and FIG. 1 below.

Fermentation time (days) Total acidity (%) 0 4.212 7 5.088 14 5.724 21 6.864 28 7.387 35 7.398 42 7.692 49 8.964 56 9.75 63 11.232

<Experimental Example 2>

Confirmation of cytotoxicity (measurement of cell viability)

MTT assay using 3T3-L1 cells was performed to confirm the cytotoxicity of kkujippong fruit fermented vinegar.

3T3-L1 cells were seeded in a 96-well plate at 4×10 ³ cells/well and cultured at 37° C., 5% CO _{2 in an} incubator for 24 hours. Cujippong fruit fermented vinegar as a sample was added to the cultured 3T3-L1 cells at concentrations of 0㎍/㎖, 30㎍/㎖, 50㎍/㎖, 100㎍/㎖, 300㎍/㎖, 500㎍/㎖ and 750㎍/㎖. It was diluted and incubated for 48 hours. After adding MTT solution to each well and reacting for 4 hours, the MTT solution was removed, DMSO was added, and after 15 minutes, absorbance was measured at 540 nm using an ELISA reader. The medium was treated as a control for comparison. The measured absorbance was calculated as a ratio to the control and shown in FIG. 2 .

As in the results of Figure 2, when the kkujippong fruit fermented vinegar was treated, the cell viability at each concentration was confirmed to be 100%, 102%, 103%, 103%, 100%, 95% and 87%.

<Experimental Example 3>

Confirmation of inhibitory effect on the formation of fat cells in adipocytes

In order to determine whether Cujipon fruit fermented vinegar inhibits the production of adipocytes by acting on the 3T3-L1 cell differentiation process from preadipocytes to adipocytes, after processing the Cujippong fruit fermented vinegar sample in 3T3-L1 adipocytes during differentiation. Absorbance was measured by staining adipocytes generated in 3T3-L1 cells using Oil Red O staining solution.

Insulin, DEX (dexamethasone, dexamethasone) and IBMX (3-isobutyl-1-methylxanthine, 3-isobutyl-1-methylxanthine) were used as inducers to induce intracellular fat accumulation during adipocyte differentiation. Insulin activates the expression of adipogenic genes through insulin signaling activation. DEX acts as a PPARγ and CEBPα regulator. IBMX increases the cAMP concentration in cells and activates the transcriptional activity of PPARs and CEBPs accordingly, thereby activating the expression of adipogenic genes. When an inducer such as insulin, DEX, or IBMX is added to the medium, growth stops and differentiation into adipocytes begins. The fat produced in the cells continues to form fat globules until the final stage of differentiation. As time goes on, the fat spheres combine with each other and increase in size. The triglycerides present in the fat globules are produced from 3T3-L1 pre-adipocytes It is produced during the stage of differentiation into cells.

To differentiate 3T3-L1 cells into adipocytes, 3T3-L1 cells are seeded in a 6-well plate at 1×10 ⁵ cells/well, and when 80-90% confluent, 5 μg/ml of differentiation inducer Differentiation was induced with DMEM medium containing insulin, 1 μM DEX, 0.5 mM IBMX and 10% FBS. After 2 days, it was replaced with DMEM medium containing 5 μg/ml insulin and 10% FBS, and was used for the experiment 4 days later while supplementing with DMEM medium containing 10% FBS once every 2 days.

Differentiation of 3T3-L1 cells seeded in a 6-well plate was induced for 8 days with 0㎍/㎖, 50㎍/㎖, 100㎍/㎖, and 150㎍/㎖ of kkujippong fruit fermented vinegar. After induction of differentiation, it was washed with PBS, fixed with 10% formalin solution for 30 minutes, and washed once with distilled water. After treatment with 0.3% Oil Red O solution for 1 hour, adipocytes generated in 3T3-L1 cells were stained, washed once with 60% isopropanol, and absorbance was measured at 540 nm. The medium was treated as a control for comparison.

Each value was calculated as a ratio (%) to the absorbance of the control, and the results are shown in FIG. 3 . Each value in FIG. 3 represents the mean±SD for three measurements.

As shown in the results of Figure 3, in the cell group that was not treated with the Cujippong fruit fermented vinegar sample, the accumulated fat cells in the adipocyte differentiation-inducing cells increased to 265% compared to the total adipocytes, and in the group treated with the Cujipon fruit fermented vinegar, 240 %, 195% and 142%.

<Experimental Example 4>

Confirmation of triglyceride reduction effect

Most fat globules are composed of proteins such as triglyceride and perilipin A. The effect of reducing triglycerides constituting fat globules was confirmed as follows when Cujippong fruit fermented vinegar was treated.

4 is a result confirming the inhibitory effect of kkujippong fruit fermented vinegar on triglycerides in 3T3-L1 cells.

Triglyceride content was performed according to the quantification method indicated in the assay kit. 3T3-L1 cells seeded in a 6-well plate were induced to differentiate for 8 days with 0㎍ / ㎖, 50㎍ / ㎖, 100㎍ / ㎖ and 150㎍ / ㎖ kkujippong fruit fermented vinegar samples. After induction of differentiation, wash twice with PBS, add reagents according to the method indicated in the assay kit, and incubate at room temperature, and then measure the absorbance at 550 nm using an ELISA reader to check the triglyceride level with the assay kit did. The medium was treated as a control for comparison. Each value was calculated as a ratio (%) to the control and shown in FIG. 4 . Each value in FIG. 4 represents the mean±SD for three measurements.

As can be seen in FIG. 4 , the triglyceride content increased to 210% in the adipocyte group that was not treated with kkujippong fruit fermented vinegar. On the other hand, it decreased to 185%, 163%, and 133% in the group treated with Cujipon fruit fermented vinegar.

<Experimental Example 5>

Confirmation of free glycerol release effect

In order to confirm the degree of decomposition of triglycerides, the free glycerol content was measured.

Triglycerides, which are triglycerides accumulated in adipocytes, are decomposed into free fatty acids and free glycerol, secreted into blood vessels, and moved to the liver for accumulation. It is known that the degradation of triglycerides in adipocytes is mainly mediated by hormone-sensitive lipase, which is considered an important step in regulating the intracellular fat content. The content of free glycerol produced by the decomposition of triglycerides in 3T3-L1 adipocytes is one of the factors that can indirectly confirm the degree of decomposition of triglycerides in adipocytes.

Differentiated 3T3-L1 cells were treated with 0㎍/㎖, 50㎍/㎖, 100㎍/㎖, and 150㎍/㎖ concentration of Kujippong fruit fermented vinegar samples and cultured for 48 hours. The cultured medium was placed in a tube and heated at 70° C. for 10 minutes to inactivate the enzymes released from the cells. 50 μl of medium was added to the glycerol reagent, reacted for 1 minute, and then absorbance was measured at 540 nm. At this time, the amount of protein was quantified with a BCA kit to correct the number of cells. The medium was treated as a control for comparison. Each value was calculated as a ratio (%) to the control and shown in FIG. 5 . Each value in FIG. 5 represents the mean±SD for three measurements.

As can be seen in Figure 5, the release of free glycerol was increased to 117%, 137%, 159% when the kkujippong fruit fermented vinegar sample was treated.

<Experimental Example 6>

Confirmation of Total Cholesterol Reduction Effect

Total cholesterol (total cholesterol), such as triglyceride (triglyceride), phospholipid (phospholipid), such as an abnormally increased concentration and maintained at a high concentration causes the occurrence of diseases such as hyperlipidemia. After induction of differentiation in pre-adipocytes, the content of total cholesterol produced in adipocytes treated with the sample was measured.

Total cholesterol content was performed according to the quantification method indicated in the assay kit.

After the differentiated 3T3-L1 cells were treated with 0㎍/㎖, 50㎍/㎖, 100㎍/㎖, and 150㎍/㎖ concentration of kkujippong fruit fermented vinegar samples and washed with PBS to make a cell lysate. After treatment according to the quantification method indicated in the assay kit, the cholesterol content was measured using an ELISA reader. The medium was treated as a control for comparison. Each value was calculated as a ratio (%) to the control and shown in FIG. 6 . Each value in FIG. 6 represents the mean±SD for three measurements.

As can be seen in FIG. 6 , the cholesterol content was increased to 165% in the adipocyte group that was not treated with the Cujippong fruit fermented vinegar sample. Cholesterol content was reduced to 150%, 139%, and 118% in the group treated with Cujipon fruit fermented vinegar sample.

<Experimental Example 7>

Confirmation of the effect of reducing leptin release

Leptin is a protein mainly produced and secreted by adipocytes and controls obesity by increasing energy consumption through various metabolic activities. It is known that as the amount of fat accumulation in adipocytes increases, the secretion of leptin increases, and it is also reported that the secretion of leptin increases in proportion to fat accumulation in adipocytes in vitro.

Leptin content was performed according to the quantification method indicated in the assay kit.

Differentiated 3T3-L1 cells were treated with 0㎍/㎖, 50㎍/㎖, 100㎍/㎖, and 150㎍/㎖ concentration of Kujippong fruit fermented vinegar and treated according to the quantification method indicated in the assay kit, followed by an ELISA reader was used to measure the leptin content. The medium was treated as a control for comparison. Each value was calculated as a ratio (%) to the control and shown in FIG. 7 . Each value in FIG. 7 represents the mean±SD for three measurements.

As can be seen in FIG. 7 , the leptin content increased to 205% in the adipocyte group that was not treated with the Cujippong fruit fermented vinegar sample. The leptin content was reduced to 168%, 147%, and 123% in the group treated with the Cujipon fruit fermented vinegar sample.

<Experimental Example 8>

Confirmation of the effect of reducing the expression of genes related to adipocyte differentiation

In order to determine the mechanism by which the reduction in fat accumulation by the fermented cujippong fruit vinegar was induced, the cujippong fruit fermented vinegar was treated in the process of inducing the differentiation of pre-adipocytes and the expression of related proteins was confirmed.

The process of differentiation from adipocytes into morphologically and biochemically mature adipocytes is achieved through mutual regulation of various factors such as hormones, cytokines, and transcription factors. When receiving, expression of transcription factors CCAAT/enhancer-binding protein (C/EBP)β and C/EBPδ are induced, and these induce the expression of peroxisome proliferator-activated receptor (PPAR), a major regulator of adipogenesis. It is known to advance differentiation. Among the transcription factors involved in the differentiation process, the most well-known C/EBP family and PPAR family promote adipocyte differentiation by interacting with adipocyte gene regulatory regions. C/EBPa regulates energy homeostasis by increasing before expression of various adipose tissue-specific genes during differentiation, and PPAR is a major regulator of adipogenesis, including leptin, FAS (fatty acid synthase), and aP2 (fatty acid binding protein). Regulates the expression of adipogenic genes.

Lysis buffer was added to the treated 3T3-L1 cells to separate intracellular proteins, and then the proteins were quantified with a BCA kit. After loading on SDS-PAGE and electrophoresis, the protein was transferred to a PVDF membrane using a transfer buffer. After blocking with 5% non-fat skim milk solution, primary and secondary antibodies were attached. After the antibody reaction was completed, the membrane was treated with an ECL detection kit, exposed to an X-ray film and developed, and the expression level was confirmed using the generated band. The results are shown in FIG. 8 .

As shown in the results of Figure 8, PPARγ was increased 6.2 times compared to pre-adipocytes, and 4.8 times and 4.8 times when the Cujipon fruit fermented vinegar samples were treated with concentrations of 50 μg/ml, 100 μg/ml, and 150 μg/ml. , the expression level decreased by 2.9 times. In the case of CEBPα, it was increased by 4.1 times compared to whole adipocytes, and expression levels decreased by 2.9 times, 2.0 times, and 1.2 times when the fermented vinegar samples of Cujipon fruit were treated with concentrations of 50 μg/ml, 100 μg/ml, and 150 μg/ml. . In the case of LPL, it was increased by 6.0 times compared to whole adipocytes, and expression levels were decreased by 3.1 times, 3.3 times, and 1.8 times when the Cujipon fruit fermented vinegar samples were treated with concentrations of 50 μg/ml, 100 μg/ml, and 150 μg/ml. . In the case of FAS, it was increased by 6.7 times compared to whole adipocytes, and when Cujipon fruit fermented vinegar was treated at concentrations of 50 μg/ml, 100 μg/ml, and 150 μg/ml, the expression level decreased by 6.0 times, 5.1 times, and 2.4 times.

<Experimental Example 9>

Check triglyceride resolution

Tributyrin plate assay was used for the tributyrin plate assay for triglyceride suppression and decomposition ability of the fermented vinegar of Cujippong fruit of the present invention.

In the tributyrin plate assay, a tributyrin plate is prepared by diluting a 1% tributyrin solution to an appropriate amount to a final volume of 5 ml with a 2% agar solution, and a 3 mm diameter well is placed on the tributyrin plate. This is a method to analyze the lipolytic activity of the sample by adding samples of various concentrations and reacting them at 37°C, then measuring the clear zone.

As the experimental sample, fermented Kujippong fruit fermented vinegar at concentrations of 5 μg/ml, 10 μg/ml, 20 μg/ml, 50 μg/ml and 100 μg/ml was used. Distilled water (DW) was treated as a control for comparison.

A tributyrin plate was prepared by mixing a 1% tributyrin solution and a 2% agar solution to a final volume of 5 ml. After making wells with a diameter of 3 mm on a tributyrin plate and immersing the sample in the wells, incubated at 37° C. for 30 minutes. After incubation, the clear zone was measured (mm) to analyze the lipolytic activity of the sample. The results are shown in FIG. 9 . Each value in FIG. 9 represents the mean±SD for three measurements.

As confirmed in FIG. 9, Cujipon fruit fermented vinegar contains triglycerides 1.7±0.1 mm at 5 μg/ml, 1.9±0.2 mm at 10 μg/ml, 2.0±0.2 mm at 20 μg/ml, 50 μg/ml at 2.4±0.3 mm and 2.6±0.1 mm at 100 μg/ml were resolved. The triglyceride decomposition ability of Cujipon fruit fermented vinegar increased in proportion to the concentration, and the resolution was the highest at 2.6mm at the highest concentration.

<Experimental Example 10>

Confirmation of antihyperhemostatic effect using lipolytic enzyme substrate

In order to analyze the antihyperlipidemic effect of the fermented kkujippong fruit vinegar of the present invention, the lipolytic and inhibitory effect was confirmed by a lipolytic activity assay using a lipolytic enzyme substrate.

The lipolytic activity assay is a lipolytic enzyme substrate with a concentration of 10 mM p-nitrophenyl butyrate, p-nitrophenyl palmitate, and p-nitrophenyl decanoate (p-nitrophenyl). decanoate), put samples of various concentrations in the substrate solution, react at 60°C for 15 minutes, and measure p-nitrophenol (PNP) at 405 nm for 10 minutes to measure V _max value or OD It is a spectrophotometric assay of lipolytic activity analyzed with a value of 405.

As the experimental sample, fermented Kujippong fruit fermented vinegar at concentrations of 5 μg/ml, 10 μg/ml, 20 μg/ml, 50 μg/ml and 100 μg/ml was used. For comparison, the negative control was treated with distilled water (DW), and the positive control was treated with lipase.

The sample was put into a 10 mM solution of p-nitrophenyl butyrate, p-nitrophenyl palmitate and p-nitrophenyl decanoate at a concentration of 10 mM, which is a substrate of lipolytic enzyme, and reacted at 60° C. for 15 minutes. Then, the sample was reacted at 405 nm for 10 minutes. The released p-nitrophenol (PNP) was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA) and analyzed as a V _max (mU/min) value.

The result when p-nitrophenyl butyrate was used as a substrate is shown in FIG. 10, the result when p-nitrophenyl palmitate is used is shown in FIG. 11, and the result when p-nitrophenyl decanoate is used is shown in FIG. 12 . Each value in FIGS. 10 to 12 represents the mean±SD for three measurements.

The efficacy of the negative control treated with distilled water only was 0 Vmax (mU/min), the efficacy of the positive control treated with lipase was 0.22±0.01 mU/min in p-nitrophenyl butyrate, and 0.37±0.05 mU in p-nitrophenyl palmitate. /min, measured as 0.33±0.03 mU/min in p-nitrophenyl decanoate.

When p-nitrophenyl butyrate was used as a substrate, Cuji mulberry fermented vinegar was 0.0134±0.0014 mU/min at a concentration of 5 μg/ml, 0.0299±0.0016 mU/min at a concentration of 10 μg/ml, and 0.1447 at a concentration of 20 μg/ml. The activity was 0.2335±0.0354 mU/min at the concentration of ±0.0428 mU/min and 50 μg/ml, and 0.2912±0.0458 mU/min at the concentration of 100 μg/ml.

When p-nitrophenyl palmitate was used as a substrate, the fermented cucurbita fruit vinegar was 0 ± 0 mU/min at a concentration of 5 μg/ml, 0.0037 ± 0.0009 mU/min at a concentration of 10 μg/ml, and 0.0089 at a concentration of 20 μg/ml. At a concentration of ±0.0012 mU/min and 50 μg/ml, the activity was 0.0104±0.0014 mU/min, and at a concentration of 100 μg/ml, 0.0191±0.0016 mU/min.

When p-nitrophenyl decanoate was used as a substrate, Cujipon fruit fermented vinegar was 0±0mU/min at a concentration of 5㎍/ml, 0±0mU/min at a concentration of 10㎍/ml, and 0 at a concentration of 20㎍/ml. The activity was 0.0059±0.0013 mU/min at the concentration of ±0 mU/min and 50 μg/ml, and 0.0071±0.0010 mU/min at the concentration of 100 μg/ml.

As shown in the above results, the antihyperlipidemic effect of fermented cucurbita fruit vinegar increased in proportion to the concentration only in the p-nitrophenyl butyrate substrate, and showed an activity of 0.29 mU/min at the highest concentration.

<Experimental Example 11>

Confirmation of inhibitory effect on obesity-related enzyme activity

The effect of inhibiting obesity-related enzyme activity of kkujippong fruit fermented vinegar of the present invention was confirmed as follows.

As obesity-related enzymes, pancreatic lipase, which is an indicator of digestion and absorption of fat, and amylase and glucosidase, which are indicators of digestion and absorption of sugar, were used.

The ability to inhibit enzyme activity was calculated by the following formula:

Inhibitory activity (%) = [(absorbance of control without sample - absorbance of control with addition of sample) / absorbance of control without sample] × 100

1. Pancreatic lipase (PL) enzyme inhibitory ability analysis

The enzyme activity inhibition ability was analyzed by treating the sample with pancreatic lipase, which is an index for digestion and absorption of fat.

As the experimental samples, fermented vinegar samples from Kujippong fruit at concentrations of 5 μg/ml, 10 μg/ml, 20 μg/ml, 50 μg/ml and 100 μg/ml were used. For comparison, the negative control was treated with distilled water (DW), and the positive control was treated with human-derived pancreatic lipase.

(1) Enzyme activity

Enzyme by adding 10mU of human-derived pancreatic lipase to 10mM Tris-HCl (pH 6.8) containing 10mM 3-(N-morpholino)propanesulfonic acid [3-(N-morpholino)propanesulfonic acid, MOPS) and 5mM CaCl ₂ A solution was prepared. Human-derived pancreatic lipase enzyme (10 mU) was pretreated with various samples at room temperature for 10 minutes. After pretreatment for 10 minutes, the mixture was added to 3 mM p-nitrophenyl butyrate to initiate a reaction and reacted at 37° C. for 30 minutes. After incubation, the activity of the reaction mixture was confirmed by reading the absorbance value at 405 nm with a microplate reader. The enzyme activity was expressed as 1 mU of the activity capable of decomposing 1 nM of p-nitrophenyl butyrate per minute. The results are shown in FIG. 13, and each value in FIG. 13 represents the mean±SD for three measurements.

As in the results of Figure 13, as a result of treatment with kkujippong fruit fermented vinegar and measuring the absorbance at 405 nm, 2.42 ± 0.03 at 5 μg/ml, 2.33 ± 0.06 at 10 μg/ml, 2.21 ± 0.03 at 20 μg/ml, Absorbances of 2.11±0.04 at 50 μg/ml and 1.24±0.03 at 100 μg/ml were confirmed.

(2) Residual enzyme activity

Based on the absorbance value, when the enzyme activity of 10 mU of pancreatic lipase was 100%, the remaining enzyme activity was confirmed by comparing the enzyme activity when each sample was added (%). The results are shown in FIG. 14, and each value in FIG. 14 represents the mean±SD for three measurements.

As shown in the results of Figure 14, when 10mU of pancreatic lipase enzyme activity was 100%, when Cujipon fruit fermented vinegar was treated at a concentration of 5㎍ / ㎖, the residual activity was 98.55±5.05%, 10㎍ / ㎖ concentration If the residual activity is 94.89±3.57%, when treated at a concentration of 20 μg/ml, the residual activity is 90.18±4.78%, when treated at a concentration of 50 μg/ml, the residual activity is 85.97±4.11%, when treated at a concentration of 100 μg/ml The residual activity was 49.18±2.15%.

(3) enzyme inhibitory effect

By analyzing the residual enzyme activity, the lipolytic enzyme inhibitory effect of Kujippong fruit fermented vinegar was analyzed. The results are shown in FIG. 15, and each value in FIG. 15 represents the mean±SD for three measurements.

15, the lipolytic enzyme inhibitory effect of Cujipon fruit fermented vinegar was 1.45±5.05% at a concentration of 5 μg/ml, 5.11±3.57% at a concentration of 10 μg/ml, and 9.82±4.78 at a concentration of 20 μg/ml. %, 14.03±4.11% at a concentration of 50 μg/ml, and 50.82±2.15% at a concentration of 100 μg/ml. As such, the lipolytic enzyme inhibitory effect of Cujipon fruit fermented vinegar was 1-51%, which was lower than the inhibition rate of 90% of the lipolytic enzyme inhibitor Lipstatin.

2. α-Amylase (α-Amylase) enzyme inhibitory ability analysis

The enzyme activity inhibition ability was analyzed by treating the sample with α-amylase, which is an index for digestion and absorption of sugar.

As the experimental samples, fermented vinegar samples from Kujippong fruit at concentrations of 5 μg/ml, 10 μg/ml, 20 μg/ml, 50 μg/ml and 100 μg/ml were used. For comparison, the negative control was treated with distilled water (DW), and the positive control was treated with 10 mU human α-amylase.

(1) Enzyme activity

α-amylase was treated with samples of various concentrations at room temperature for 10 minutes. After reacting by adding 1% starch to the treated mixture, 0.1% DNS was added and reacted at 100° C. for 5 minutes. After the reaction was cooled at -20°C for 1 minute, the glycolytic enzyme activity of the reaction solution was checked by reading the absorbance value at 540 nm with a microplate reader and comparing it with a standard product. The results are shown in FIG. 16, and each value in FIG. 16 represents the mean±SD for three measurements.

As in the result of FIG. 16, as a result of treatment with Cujipon fruit fermented vinegar and measuring absorbance at 540 nm, 0.2665 ± 0.0092 at 5 μg/ml concentration, 0.2613 ± 0.0052 at 10 μg/ml concentration, 0.2523 ± at 20 μg/ml concentration Absorbances of 0.2255±0.0056 at 0.0062 and 50 μg/ml concentrations and 0.1933±0.0068 at 100 μg/ml concentrations were confirmed. The absorbance of the negative control treated with distilled water (DW) instead of the sample was 0.1330±0.0122, and the absorbance of human α-amylase as a positive control was 0.2959±0.0086 at 10mU concentration.

(2) Residual enzyme activity

Based on the absorbance value, when the enzyme activity of 10 mU of α-amylase was 100%, the remaining enzyme activity was confirmed by comparing the enzyme activity when each sample was added (%). The results are shown in FIG. 17, and each value in FIG. 17 represents the mean±SD for three measurements.

As shown in the results of Figure 17, when 10mU of human-derived α-amylase enzyme activity was 100%, when Cujipon fruit fermented vinegar was treated at a concentration of 5㎍/㎖, the residual activity was 81.96±0.02%, 10㎍/㎖ concentration Residual activity was 78.73±2.55% when treated with 20㎍/㎖ concentration, residual activity was 73.21±2.05% when treated with 50㎍/㎖ concentration, and residual activity was 56.75±2.76% when treated with 50㎍/㎖ concentration, 100㎍/㎖ concentration When treated with , the residual activity was 36.99±2.51%.

(3) enzyme inhibitory effect

The residual enzyme activity was analyzed to analyze the α-amylase inhibitory effect of Kujippong fruit fermented vinegar. The results are shown in FIG. 18, and each value in FIG. 18 represents the mean±SD for three measurements.

As shown in the results of FIG. 18, the α-amylase inhibitory effect of Cujipon fruit fermented vinegar was 18.04±0.02% at a concentration of 5 μg/ml, 21.27±2.55% at a concentration of 10 μg/ml, and 26.79±2.05 at a concentration of 20 μg/ml. %, 43.25±2.76% at a concentration of 50 μg/ml, and 63.01±2.51% at a concentration of 100 μg/ml. As such, the α-amylase inhibitory effect of the fermented vinegar of Cujipon fruit increased in proportion to the concentration, and the highest concentration showed an inhibitory effect of 63%. This showed a somewhat higher inhibitory effect than the α-amylase inhibitory rate of about 60% of the α-amylase inhibitor, acarbose, at a concentration of 10 mg/ml.

3. β-glucosidase enzyme inhibitory ability analysis

The enzyme activity inhibition ability was analyzed by treating the sample with β-glucosidase, which is an indicator of sugar digestion and absorption.

As the experimental samples, fermented vinegar samples from Kujippong fruit at concentrations of 5 μg/ml, 10 μg/ml, 20 μg/ml, 50 μg/ml and 100 μg/ml were used. For comparison, the negative control was treated with distilled water (DW), and the positive control was treated with 0.1U β-glucosidase.

(1) Enzyme activity

An enzyme solution was prepared by adding β-glucosidase to 10 mM PBS (phosphate-buffered saline, pH 6.8). 0.1U β-glucosidase was pretreated with samples of various concentrations at room temperature for 10 minutes. 3 mM p-nitrophenyl-β-D-glucopyranoside was added to the treated mixture and reacted at 37° C. for 10 minutes. After the reaction, the glycolytic enzyme activity of the reaction solution was checked by reading the absorbance value at 405 nm with a microplate reader. The enzyme activity was expressed as 1 mU of the activity capable of decomposing 1 nM of p-nitrophenyl-β-D-glucopyranoside per minute. The results are shown in FIG. 19, and each value in FIG. 19 represents the mean±SD for three measurements.

As in the result of FIG. 19, as a result of treatment with Cujipon fruit fermented vinegar and measuring the absorbance at 405 nm, 0.315 ± 0.006 at 5 μg/ml concentration, 0.285 ± 0.016 at 10 μg/ml concentration, and 0.273 ± at 20 μg/ml concentration Absorbances of 0.191±0.008 at concentrations of 0.008 and 50 μg/ml and 0.174±0.010 at concentrations of 100 μg/ml were confirmed.

(2) Residual enzyme activity

Based on the absorbance value, when the enzyme activity of 0.1 U of β-glucosidase was 100%, the remaining enzyme activity was confirmed by comparing the enzyme activity when each sample was added (%). The results are shown in FIG. 20, and each value in FIG. 20 represents the mean±SD for three measurements.

As shown in the result of Figure 20, when 10mU of human-derived α-amylase enzyme activity was 100%, the residual activity was 99.19±0.25%, 10㎍/㎖ concentration when treated with fermented kkujippong fruit vinegar at a concentration of 5㎍/㎖ Residual activity was 89.72±3.37% when treated with 20㎍/㎖ concentration, residual activity was 86.01±1.08% when treated with 50㎍/㎖ concentration, and residual activity was 59.95±1.36% when treated with 50㎍/㎖ concentration, 100㎍/㎖ concentration When treated with , the residual activity was 54.66±2.06%.

(3) enzyme inhibitory effect

By analyzing the residual enzyme activity, the β-glucosidase inhibitory effect of Kujippong fruit fermented vinegar was analyzed. The results are shown in FIG. 21, and each value in FIG. 21 represents the mean±SD for three measurements.

As shown in the result of FIG. 21, the β-glucosidase inhibitory effect of the fermented vinegar of Cujipon fruit was 0.81% at a concentration of 5 μg/ml, 10.28% at a concentration of 10 μg/ml, 13.99% at a concentration of 20 μg/ml, and 50 μg 40.05% at the /ml concentration and 45.34% at the 100㎍/ml concentration. As such, the β-glucosidase inhibitory effect of kkujippong fruit fermented vinegar was 1-45%, which was lower than that of the β-glucosidase inhibitor, cyclophellitol, about 90%.

<Experimental Example 12>

Analysis of inhibition of obesity-related enzyme activity using kinetics

It analyzed the dynamics (kinetics) of the lipase and glycolytic enzyme inhibitory activity of the hydroxy benzyl alcohol (p -Hydroxybenzyl alcohol) - kkujippong fruit vinegar fermentation within the active ingredients, p.

As targets for body fat reduction-related anti-obesity activity, a mechanism for inhibiting fat digestion and absorption and a mechanism for inhibiting sugar digestion and absorption were selected. The kinetics of glucosidase, which is an index of digestion and absorption of sugar, and pancreatic lipase, which is an index of digestion and absorption of fat, were selected. By analyzing the activity, an inhibition pattern for the target was derived.

Kinetics analysis uses the Lineweaver-Burk plot to obtain the Michaelis-Menten constant (Km), the maximum velocity (Vmax), and the catalytic rate constant constants) (Kcat), Kik, and Kiv inhibition constants are derived, and based on these values, inhibition characteristics (competitive, noncompetitive, uncompetitive, or mixed inhibition type) ] was determined.

1. Cujippong Fruit Fermented Vinegar p -Kinetic analysis of lipolytic enzyme inhibitory activity of hydroxybenzyl alcohol

p - hydroxybenzyl alcohol is the concentration that inhibits the activity of a lipolytic enzyme derived from animals _50% 2.34μM (IC 50), and the concentration to inhibit the activity of human-derived lipase _50% 3.70μM (IC 50) of was analyzed as

Km (substrate concentration), Vmax (maximum reaction rate), Kcat (conversion substrate concentration per hour), Kcat/Km were analyzed through kinetic analysis of the activity of p-hydroxybenzyl alcohol to inhibit lipolytic enzyme, and inhibition pattern analysis was performed. For this, Kik (low constant of substrate), Kiv (low constant of reaction rate), and Kik/Kiv values were derived using this, and the results are shown in FIG. 22 and Table 3.

The Reinweaver-Burk plot was used for the inhibition of the target enzyme, and the plot was 10 μg (0.8 μM) or 20 μg (1.6 μM) of HBA when HBA, an inhibitor for human pancreatic lipase, was not added (control). It was expressed as 1/velocity (1/V) versus 1/substrate (1/S) for the case of adding . In Table 3, V _max and K _m values were calculated according to the Lineweaver-Burk from the data shown in FIG. 22 , and each value represents the mean±SD for three measurements.

enzyme HBA
(μM) K _m
(mM) V _max
(mU/min) K _cat
(s-1) K _cat / K _m
(mM/s) K _ik K _iv K _ik / K _iv human origin
pancreas
lipase 0 0.620
±
0.020 0.120
±
0.004 1.15
±
0.04 1.839
±
0.013 0.578
±
0.352
mM 0.052
±
0.004
mM 11.14 0.8 0.547
±
0.026 0.070
±
0.003 ^** 0.67
±
0.03 ^** 1.223
±
0.005 ^** 1.6 0.509
±
0.063 ^* 0.040
±
0.002 ^** 0.38
±
0.02 ^** 0.753
±
0.070 ^**

(Compared to the HBA-free group (0 μM), *P < 0.05, **P < 0.01.)

As shown in the results of FIG. 22 and Table 3, when p -hydroxybenzyl alcohol was not treated, the Km, Vmax, Kcat, and Kcat/Km values were sequentially 0.620±0.020mM, 0.120±0.004mU/min, 1.15±0.04 /s, 1.839±0.013 mM/s. When 0.8 μg of p -hydroxybenzyl alcohol was treated under the same conditions, the Km, Vmax, Kcat, and Kcat/Km values were 0.620±0.020mM, 0.120±0.004mU/min, 1.15±0.04/s, 1.839±0.013mM in order. /s, when 1.6 μg of p -hydroxybenzyl alcohol was treated under the same conditions, Km, Vmax, Kcat, and Kcat/Km values were sequentially 0.620±0.020mM, 0.120±0.004 mU/min, 1.15±0.04/s, 1.839 ±0.013 mM/s.

When the Kik, Kiv, and Kik/Kiv values are derived based on the Km, Vmax, Kcat, and Kcat/Km values, when p -hydroxybenzyl alcohol is present at a high concentration, the Kik, Kiv, and Kik/Kiv values are 0.578±0.352mM in that order. , 0.052±0.004 mM, 11.14.

When the Kik and Kiv values are determined, the inhibitor properties can be determined by deriving the Kik/Kiv values. If the Kik/Kiv value is 1~2, it is determined as uncompetitive type, if it is 2~5, it is determined as mixed competitive type, and if it is 5 or more, it is determined as competitive or noncompetitive type. ) to be determined.

Considering that the Kik/Kiv value is 11.14, it can be seen that p -hydroxybenzyl alcohol inhibited human-derived lipolytic enzyme with competitive or noncompetitive type or uncompetitive type. have.

2. Cujippong fruit fermented vinegar p -Kinetic analysis of glycolytic enzyme inhibitory activity of hydroxybenzyl alcohol

The concentration at which p-hydroxybenzyl alcohol inhibits the activity of glycolytic enzyme by 50% was analyzed to be _{9.08 μM (IC 50 ).}

Km (substrate concentration), Vmax (maximum reaction rate), Kcat (conversion substrate concentration per hour), Kcat/Km were analyzed through kinetic analysis of the activity of p-hydroxybenzyl alcohol to inhibit glycolytic enzymes, and inhibition pattern analysis was performed. For this purpose, Kik (low constant of substrate), Kiv (low constant of reaction rate), and Kik/Kiv values were derived using this, and the results are shown in FIG. 23 and Table 4.

The Reinweaver-Burk plot was used for the inhibition of the target enzyme, and the plot was 10 μg (0.8 μM) or 20 μg (1.6 μM) of HBA when HBA, an inhibitor for human pancreatic lipase, was not added (control). It was expressed as 1/velocity (1/V) versus 1/substrate (1/S) for the case of adding . In Table 4, V _max and K _m values were calculated according to the Lineweaver-Burk from the data shown in FIG. 23 , and each value represents the mean±SD for three measurements.

enzyme HBA
(μM) K _m
(mM) V _max
(mU/min) K _cat
(s-1) K _cat / K _m
(mM/s) K _ik K _iv K _ik / K _iv α-glucosidase 0 1.18
±
0.03 0.079
±
0.001 2658.9
±
60.5 2252.3
±
3.8 0.112
±
0.008
μM 0.052
±
0.002
μM 2.16 0.8 1.09
±
0.02 ^** 0.064
±
0.001 ^** 2162.5
±
29.7 ^** 1975.5
±
2.5 ^** 1.6 0.93
±
0.02 ^** 0.050
±
0.001 ^** 1692.0
±
36.9 ^** 1813.5
±
3.4 ^**

(Compared to the HBA-free group (0 μM), *P < 0.05, **P < 0.01.)

As shown in the results of FIG. 23 and Table 4, when p -hydroxybenzyl alcohol was not treated, the Km, Vmax, Kcat, and Kcat/Km values were sequentially 0.18±0.03mM, 0.079±0.001mU/min, 2658.9±60.5 /s, 2252.3±3.8 mM/s. When 0.8 μg of p -hydroxybenzyl alcohol was treated under the same conditions, Km, Vmax, Kcat, and Kcat/Km values were sequentially 1.09±0.02mM, 0.064±0.001mU/min, 2162.5±29.7/s, 19755.5±2.5 mM /s, when p -hydroxybenzyl alcohol 1.6㎍ was treated under the same conditions, Km, Vmax, Kcat, Kcat/Km values were 0.93±0.02mM, 0.050±0.001mU/min, 1692.0±36.9/s, 1813.5 in that order. ±3.4 mM/s.

Considering that the Kik/Kiv value was 2.16 and 1/Km and 1/Vmax were increased, p -hydroxybenzyl alcohol inhibited glycolytic enzymes with mixed competitive type or non-competitive inhibitory characteristics. can be known

Claims (3)

a) Obtaining Cujippong juice by mixing 1.5~2.5ℓ of hot water at 92~98℃ with 1kg of Cujippong fruit, thawing, and grinding;
b) mixing 0.2 to 0.3 kg of sugar per 1 liter of kkujippong juice to the kkujippong juice to increase the sugar content of kkujippong juice;
c) sterilizing the kkujippong juice with increased sugar content at 85° C. for 80 to 100 minutes;
d) cooling the sterilized kkujippong juice to 25-30 ℃;
e) Saccharomyces cerevisiae Fermivin (Saccharomyces cerevisiae Fermivin) inoculated so that 4-6 volume % of the total amount of Saccharomyces cerevisiae Fermivin in the cooled Cujippong juice, and fermented for 9-11 days at 23-27 ℃ obtaining Cujippong wine;
f) cooling after sterilizing the kkujippong wine at 80-90° C. for 20-40 minutes;
g) mixing the cooled kkujippong wine and acetobacter aceti seed seed of acidity 7.0-7.4 in a volume ratio of 1:0.8-1.2 and fermenting at 28-32°C; and
h) fermenting for a total of 21 to 42 days while adding 230-270 ml of sterilized Cujippong wine per liter of fermented vinegar at 7-day intervals from 7 days after fermentation in a fed-batch manner to obtain Cujippong fruit fermented vinegar;
The step e) is inoculated with Saccharomyces cerevisiae permibin and, on the second day, aspergillus niger cellulase (Aspergillus niger cellulase) further comprising the step of adding 250 to 350 ppm, anti-obesity and anti-hyperlipidemic effect A method of producing fermented vinegar from Cujippong fruit. delete A fermented cucurbiton fruit fermented vinegar with excellent anti-obesity and anti-hyperlipidemic effects, prepared by the method of claim 1.

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