KR101072926B1 - Dietary composition for functioning low cholesterol contents in blood or liver - Google Patents

Abstract

The present invention relates to a cholesterol-lowering functional dietary composition, so that the whole soybean milk or whole soymilk hydrolyzate dietary composition exhibits a significant effect of lowering the total cholesterol content of blood and liver tissues as a result of a model animal test compared to the soymilk or soybean hydrolysate dietary composition. It is disclosed that it is excellent in regulating action.

Cholesterol, Model Animals, Soy Milk, Soy Milk Hydrolyzate, Dietary Composition

Description

Dietary composition for functioning low cholesterol content in blood or liver

The present invention relates to a cholesterol-lowering functional dietary composition of blood or liver tissue, and more particularly, to a soybean soybean called water after washing with water, which is ground soybean milk or whole soybean oil hydrolyzate obtained by hydrolysis by adding proteolytic enzyme thereto. It relates to a cholesterol-lowering functional dietary composition as an ingredient.

Soybean (Glycine max L. Merrill) is a legume, originated from Manchuria, and is known to have been cultivated in Korea since the 4th-5th century BC. Soybeans contain not only large amounts of protein (40%) and lipids (20%), but also carbohydrates and vitamin groups, and the processed products are rich in calcium, phosphorus, magnesium, and other inorganic components such as iron, zinc and copper. It is contained. Traditionally, Asian countries, including Korea, have traditionally used soy protein in the form of doenjang, soy sauce, and red pepper paste using decomposing enzymes by microbial groups to use them as food corrosion. In addition, various components contained in soybean are known to have various physiological activities such as antihypertensive effect, anti-mutation, anti-cancer, thrombolytic function.

In recent years, by ingesting a large amount of animal protein accumulates a large amount of cholesterol in the body, causing arteriosclerosis diseases, interest in the use of vegetable protein is increasing. Soybeans are receiving new attention because they are re-evaluating that soybeans can be a nutritionally superior food material as they contain high-quality proteins and high levels of essential fatty acids. Soy protein is one of the most widely used vegetable protein. It is relatively inexpensive and has a wide range of uses to enhance the functionality and nutrition of processed foods. Soy protein is not only in terms of food nutrition but also excellent in physiological activity. It plays a role in lowering the content of serum cholesterol, and it has the effects of anti-cancer effect, prevention of osteoporosis and improvement of renal dysfunction symptoms.

In general, in order to use a protein widely in foods can be modified the functional properties, the method is mainly used chemical modification and enzymatic modification. Hydrolysis of proteins with acids or alkalis increases the concentration of salts or causes harmful substances to be fished. Therefore, many modifications by enzymes have been used.Therefore, functional and nutritional value of soybeans, peanuts, cottonseeds and rapeseed proteins are increased. Enhancements have been reported, and the method used for the development of new peptide-based peptides by enzymes involves the breakdown of large proteins into small peptides and amino acids using proteolytic enzymes. Among them, a process of separating a desired peptide or hydrolysis of the peptide into amino acids is to develop a new peptide by using a reverse reaction. Proteolytic products include free amino acids, oligopeptides, and low molecular weight proteins, which have different physiological activities from the original proteins after their degradation, and thus develop new biomaterials. It has been suggested that if adjusted well, the hydrolyzate is a very effective ingredient for inhibiting aging and carcinogenesis, and the antioxidant and physiological activities are found.

The hydrolyzate of soy protein is attracting attention as a new functional food material.The function of soy protein is changed by processing conditions such as pH, salt concentration, temperature, etc. and is restricted in its use. Attempts have been made to control functionality. Thus, partial hydrolysis by enzymes has been suggested to increase the water solubility of the hydrolyzate, to change the functionality, as a new functional food. For example, it is widely used as a nutrient enhancer for soft drinks and juice drinks, and its value as a soluble protein food that can be easily digested and absorbed for patients with dysfunction of anti-nitrogen intake. Soybean protein is treated with enzymes to partially hydrolyze and new nutritional and processing functionalities are being studied. These changes in functional properties are mainly due to changes in protein structure and molecular weight due to hydrolysis by enzymes. It is known to be due to changes in protein molecules.

Soy milk is a representative soybean processed product with high digestibility and protein utilization of soybean. It is well absorbed, rich in essential amino acids, rich in essential fatty acids, rich in minerals such as iron, phosphorus, potassium, arteriosclerosis and lipid metabolism. Improvement, nerve function, anti-aging and isoflavones are effective in preventing breast cancer, and demand is increasing day by day due to nutritional excellence and low cholesterol content. On the other hand, when calcium is essential for growing children and the elderly, the soy protein of soy milk and calcium bind and precipitate, making it unsuitable for the nature of beverages. Due to precipitation of calcium salts, practicality is poor. However, the treatment of protease, which was used to improve the functional properties of proteins, may increase calcium intolerence due to a decrease in protein molecular weight and a change in protein structure, which may solve the calcium deficiency problem of soy milk. There is a report.

Therefore, the development of biological materials through functional control suitable for processing purposes and conditions using soy protein is required.

In the present invention, the soybean milk is crushed by adding 5 to 9 times of water to the soybean soaked after washing, adding the hydrolyzate of soybean milk to protein soybean milk and shaking for 30 to 90 minutes at 30 to 60 ℃. After hydrolysis, heat treatment was performed at 100 ~ 145 ℃ for 15 seconds ~ 10 minutes.

The soybean milk and the soybean oil hydrolyzate as the test material of the present invention are disclosed in detail in Korean Patent Application No. 10-0673433. However, the various physiological activities and novel uses of these soybean and soybean hydrolysates have never been disclosed.

An object of the present invention is to determine the cholesterol lowering function of blood or liver tissue of soybean milk and soybean milk hydrolyzate using a model animal.

Another object of the present invention is to provide a cholesterol lowering functional dietary composition.

The object of the present invention comprises the steps of animal design and experimental diet composition;

Analyzing various biomarkers related to lipid metabolism, such as fasting blood glucose and plasma lipids of experimental animals;

The biomarker analyzed was used to evaluate cholesterol lowering function of blood or liver tissue.

The soybean milk and whole soybean hydrolyzate of the present invention not only greatly reduce the plasma total cholesterol concentration, but also greatly reduce the total cholesterol content of liver tissue. Plasma and liver tissue have an excellent effect on lipid metabolism.

Animal Experiment Design and Experimental Diet

(1) experimental animals

The experimental animals were purchased from 50 Orient C57BL / 6N mice from Korea Orient Bio Co., Ltd. They were acclimated to the AIN-76 diet for 1 week (Table 1) and then assigned to group 5 by a randomized method (FIG. 1).

(2) experimental diet group design

The dietary group was assigned to a total of five dietary groups (FIG. 1) with a control group and a dietary experimental group having the compositions shown in Table 2, and the dietary composition of each dietary group is shown in Table 3. The diet group consists of a negative control group and a soybean milk, a hydrolyzed soybean milk and a whole-soybean milk. ), And experimental groups supplemented with hydrolyzed whole-soybean milk, were given to these animals for six weeks.

(3) disclosure materials and experimental methods

Soy milk and whole soymilk samples as test materials were provided in powder form from Keimyung Fudex Co., Ltd.

After animal breeding was completed for 12 hours fasting, the ether was aspirated by primary anesthesia, ketamin-HCl was intramuscularly injected second anesthesia, and fasting blood was collected from the inferior vena cava. Collected blood was collected directly in a test tube treated with heparin, and centrifuged at 1000 × g, 4 ° C. for 15 minutes to separate plasma and stored at −70 ° C. until sample analysis. Organ tissues of each experimental animal were rinsed several times in PBS (phosphate buffered saline) solution, and then weighed by removing surface moisture. Organ tissues were collected separately for enzyme activity measurement and lipid quantification, quenched in liquid nitrogen and stored at -70 ° C until sample analysis, and biomarkers were analyzed from biological samples.

Sample analysis method

(1) fasting blood sugar and weight measurement

After 12 hours of fasting every week, blood glucose was collected using a glucose analyzer.

(2) Measurement of glycated hemoglobin (HbA1c) concentration

A portion of whole blood was used to measure glycated hemoglobin (HbA1c) concentration using a glycated hemoglobin autoanalyzer (Roche, Switzerland).

(3) plasma lipid analysis

Plasma total cholesterol and triglyceride concentrations were determined by measuring the absorbance at 500 nm and 550 nm, respectively, which were reacted with enzyme reagents of total cholesterol and triglyceride solution (Asan Pharmaceutical Kit) using the enzyme method.

Plasma free fatty acid concentrations were measured with an Enzymatic non-esterified fatty acid (NEFA) kit.

Plasma HDL-cholesterol was isolated by dextransulfate-MgCl2 precipitation and quantified by the same method as the measurement of total plasma cholesterol.

(4) Measurement of liver tissue triglyceride and total cholesterol concentration

Hepatic lipids were extracted by Folch method, and the turbidity was removed by mixing 0.5% Triton X-100 3mM sodium cholate, an emulsifier, in the enzyme solution and measured by the same method as plasma cholesterol and triglyceride.

(5) Measurement of enzyme activity related to lipid metabolism

① Measurement of 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase activity

Microsome isolation, an enzyme source, was carried out with a slight modification of Hulcher et al. (1986), and the protein was quantified by Bradford method. Enzyme activity was measured by Shapiro et al. (1974) using [14C] HMG-CoA. Activity was expressed in pmol, the amount of mevalonate produced by 1mg of microsome protein per minute reaction.

② Acyl-coenzyme A: cholesterol acyltransferase (ACAT) activity measurement

ACAT activity was measured by a modified and supplemented method of Erickson et al. (1986). The amount of cholesteryl oleoate produced by 1 mg of microsome protein for 1 minute by treating [14C] acetyl-CoA in liver tissue microsome as an enzyme source was expressed as pmol.

(6) Measurement of antioxidant enzyme activity of red blood cells and liver tissue

① Determination of Superoxide Dismutase (SOD) activity: Marklund's method (1974) was used to develop pyrogallol color by alkaline oxidation. The enzyme activity unit was determined as the amount of protein that inhibits the automatic oxidation of pyrogallol solution by 50% without adding the enzyme solution.

② Catalase activity measurement: Using the method of Abei (1974), the concentration of H2O2 was determined by the change in absorbance of H2O2 and the molar absorption coefficient of H2O2, and then the enzyme activity was calculated using decreased H2O2 nmol / min / mg protein.

③ Determination of Glutathione Peroxidase (GSH-Px) Activity: Measure the absorbance of NADPH at 340 nm when oxidized glutathione is reduced by glutathione reductase and NADPH by Paglia and Valentine's method (1967). Enzyme units were expressed as the amount of enzyme that produced 1 nmol of oxidized NADPH for 1 minute.

(7) Determination of paraoxonase (PON) activity in plasma

Modified and applied methods such as Mackness. Plasma and microsome fractions, enzymes, were used to determine the amount of para-on decomposition product ρ-nitrophenol for 90 seconds at 25 ° C and 405 nm.

Statistical processing

The experimental results were analyzed using the SPSS package program, and ANOVA was performed to test the significance of the mean difference between the groups. Multigroup differences were tested at the level of P <0.05 or higher by Duncan's multiple range test, and the results were expressed with standard error (SE).

Effect of Dietary Supplementation on Body Weight, Dietary Intake, and Organ Weight (Table 4)

(1) The average daily dietary intake was higher in the soymilk supplement group than in the control group, the soybean milk group, and the soybean hydrolyzate group. On the other hand, the weight gain was significantly lower in the soymilk hydrolyzate group, whole soymilk group, and soybean hydrolyzate group than the control group and soymilk group. In addition, the dietary efficiency was significantly lower in the soy milk group, soy milk hydrolyzate group, whole soybean milk group, whole soybean hydrolyzate group than the control group (Fig. 2).

The weight of kidney and muscle was significantly higher in soymilk hydrolysis, soybean milk and soybean hydrolysis supplement group than the control group. In contrast, the adipose tissue had significantly lower soymilk hydrolysis, soybean milk and soybean hydrolysis supplementation groups than the control group, regardless of the site.

The changes in body weight gain during the 6-week experimental diet supplemented with C57BL / 6N mice are shown in FIG. 3. Weekly weight changes were similar in all groups at the beginning of the experiment, but over time, the soymilk hydrolyzate group, whole soymilk group, and soybean hydrolyzate group remained lower than the control group and soymilk group. It was lower than the control and soymilk groups.

Effect of Dietary Supplementation on Blood Glucose

The initial fasting blood glucose levels of each diet group began at similar levels. The concentration change measured every two weeks during the six-week experimental period was as shown in FIG. 4. At 2 weeks, the control group showed higher fasting glucose levels than all the soymilk-related test groups, and at 6 weeks, the fasting blood glucose levels of the soymilk hydrolyzate, pre- soymilk, and soybean milk hydrolysates were significantly lower than those of the soymilk group. In particular, the soymilk hydrolyzate group and the whole soymilk group were maintained at lower blood sugar level than the other groups during the entire experimental period, and were significantly lower than the control group and the soymilk group at the final 6 weeks. The blood glucose levels of all diet groups at 6 weeks were in the normal range, but the soymilk and soymilk hydrolyzate supplemented groups were significantly lower than those of the control or soymilk group, indicating that their hypoglycemic effect was excellent even in the normal state. This suggests that even in healthy and semi-healthy people, soymilk hydrolyzate and soybean milk can suppress blood sugar rise.

Effect of Dietary Supplementation on Glycated Hemoglobin (HbA1c)

After providing the experimental diet for 6 weeks, the glycated hemoglobin (HbA1c) concentration was measured, and there was no significant difference in all groups. Therefore, it was found that the test diet provided for 6 weeks in the normal state but not the diabetic level did not affect the glycated hemoglobin change (Table 5).

Effect of Dietary Supplementation on Plasma Lipid Concentration

Plasma total cholesterol concentrations were significantly lower in all test supplementation groups than in the control group, and especially in the soymilk group than the soymilk and soymilk hydrolysates. That is, the soybean milk substance was found to have an excellent blood cholesterol lowering effect. Neutral lipid concentrations were also significantly lower in all test groups than in the control group. In other words, soy milk, soy milk hydrolyzate, whole soybean milk, and soybean milk hydrolyzate have been shown to have lowering cholesterol and neutral lipids in blood. Especially, the total cholesterol lowering effect of soy milk and soy milk hydrolyzate is higher than soy milk and soy milk hydrolyzate Evaluated as excellent.

In general, plasma free fatty acid concentrations, which are known to increase in diabetic conditions, were significantly lower in all test groups than in controls (Table 6).

The plasma neutral lipid concentration change for 6 weeks is shown in FIG. 5. Soymilk group, soymilk hydrolyzate group, and soybean milk group showed lower plasma triglyceride concentrations than the control group. Especially, the soymilk hydrolyzate group was significantly lower than other groups.

Plasma total cholesterol levels were increased in all groups at 2 weeks and significantly decreased in the test supplement group except at 4 weeks. At 6 weeks, plasma total cholesterol concentration was significantly decreased in the test group compared to the control group. In particular, it was noted that the soymilk and soymilk hydrolyzate groups were significantly reduced compared to the soymilk and soymilk hydrolyzate groups, and the plasma cholesterol concentrations of these two diet groups were lower than those at week 0.

Effect of Dietary Supplementation on Hepatic Lipid Concentration

Hepatic lipid concentrations were similar to those of plasma total cholesterol. The total cholesterol content of liver was significantly decreased in the whole soybean milk and soybean hydrolyzate groups compared to the control group. Neutral lipid content was also significantly decreased in the whole soybean milk and whole soymilk hydrolysis group.

Therefore, supplementation of whole soymilk and soybean hydrolyzate was evaluated as the most effective in reducing plasma lipids and hepatic lipid concentrations . Since the amount of dietary fiber in whole soybean milk has already been corrected in the course of diet, the lipid lowering effect of whole soybean milk is believed to be due to other functional ingredients rather than its own dietary fiber.

Effect of Dietary Supplementation on Enzyme Activity Related to Lipid Metabolism

HMG-CoA reductase and ACAT activity, the enzymes related to cholesterol metabolism of liver tissue, were measured as shown in FIG. 6. HMG-CoA reductase activity, a cholesterol biosynthetic rate enzyme, was significantly increased in all test groups except for the soybean hydrolyzate group. ACAT is an enzyme that plays an important role in maintaining cholesterol homeostasis by catalyzing cholesterol esterification. The soymilk hydrolyzate group increased significantly compared to other groups.

In general, the activity of these enzymes that regulate cholesterol homeostasis has been changed sensitively to blood cholesterol concentrations. Therefore, hepatic cholesterol synthesis and esterase activity were increased in positive soymilk, soymilk hydrolyzate, whole soymilk and soymilk hydrolysates supplemented by positive feedback action according to homeostatic control results.

Effect of Dietary Supplementation on Antioxidant Enzyme Activity (Table 7)

Among the antioxidant antioxidant activities, SOD activity was the lowest in the control group, and the soymilk group, soymilk hydrolyzate group, soymilk hydrolyzate group, and soymilk hydrolyzate group supplemented with test substance showed a significant increase compared to the control group.

Hepatic catalase activity was significantly lower in the test group than in the control group. The soybean hydrolyzate group in the test group had significantly higher catalase activity than the rest.

Glutathione peroxidase (GPx) activity in liver was lower in all test groups than in control.

Superoxide dismutase (SOD) activity in erythrocytes was in the order of soy milk group, control group, whole soybean hydrolyzate group, whole soymilk group, and soymilk hydrolyzate group.

Erythrocyte Catalase activity was significantly lower in the whole soymilk group and the highest in the soymilk hydrolyzate group.

Soymilk group showed significantly lower GPx (glutathione peroxidase) activity of erythrocytes, and the soymilk hydrolyzate and soymilk group were higher than the control group.

In the case of erythrocytes, SOD activity was highest in the soymilk hydrolyzate group, and catalase and GPx (glutathione peroxidase) activities were higher in the soymilk hydrolyzate group and the whole soymilk group than the control group, respectively.

Effect of Dietary Supplementation on Paraoxonase Activity in Plasma

Paraoxonase activity, which prevents the oxidation of low-density lipoprotein (LDL), was significantly higher in the LBS group than in the control group.

Soy milk, soy milk hydrolyzate, whole soy milk, whole soy milk hydrolyzate diet supplement of the present invention showed a significant lipid lowering effect compared to the control group, especially soybean milk, soy milk hydrolyzate, soybean milk, soy milk hydrolyzate, blood sugar In addition to the excellent control effect, soymilk hydrolyzate, soybean milk, soybean milk, soybean milk hydrolyzate supplement is excellent because it is a very useful invention in the functional health food industry.

1 is an experimental design of the efficacy of soy milk, soy milk hydrolyzate, whole soybean milk, whole soybean hydrolyzate.

Figure 2 is a graph showing the effect of adipose tissue as a result of experiments by adding soymilk, soymilk hydrolyzate, whole soymilk, and soymilk hydrolyzate to the diet in mouse C57BL / 6N.

Figure 3 is a graph showing the effects on the body weight over time as a result of experiments by adding soymilk, soymilk hydrolyzate, whole soymilk, and soymilk hydrolyzate to the diet to the mouse C57BL / 6N.

4 is a picture showing the effect on the plasma glucose concentration of the mouse over time as a result of experiments with the dietary group to which the test substance is added to the mouse C57BL / 6N.

Figures 5a and 5b is a picture showing the changes over time in the triglyceride (Fig. 5a) and total cholesterol (Fig. 5b) content of the mouse plasma as a result of experiments in the diet group to which the test material is added to the mouse C57BL / 6N.

6 is a diagram showing the mouse liver lipid concentrations as a result of experiments with the diet group to which the test substance was added to the mouse C57BL / 6N.

Figures 7a and 7b is a graph showing the effect of HMG-COA reductase (7a) and ACAT activity (7b) of the liver as a result of experiments in the diet group to which the test substance is added to C57BL / 6N.

8 is a graph showing the effect of plasma paraoxonase enzyme activity as a result of experiments with the dietary group to which the test substance was added to mouse C57BL / 6N.

Claims (2)

5 ~ 9 times of water is added to soybeans soaked with water, and protein hydrolase is added to the ground soybean milk. After hydrolysis by shaking for 30 ~ 90 minutes at 30 ~ 60 ℃, it is hydrolyzed at 100 ~ 145 ℃ for 15 seconds ~ 10 minutes. Cholesterol lowering functional dietary composition comprising the hydrolyzate of the heated soybean oil as an active ingredient. delete

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