KR100201008B1 - Method using glutene peptide for hyperlipemia and hypercholesterolemia - Google Patents

Abstract

The present invention relates to a method for producing gluten peptides useful for exogenous hyperlipidemia and hypercholesterolemia due to the ingestion of high fat and high cholesterol from vegetable proteins. Mature rats were fed a diet containing high fat and high cholesterol, or at the same time an experimental diet containing casein and gluten and its hydrolysates, to compare the induced levels of hyperlipidemia and hypercholesterolemia. The results showed that cluthene and protein hydrolysates were effective in reducing exogenous hyperlipidemia and hypercholesterolemia induced by high fat diet intake.

Gluten intake showed lower blood lipid levels and liver lipid content than casein intake, especially gluten hydrolyzate ingestion. Therefore, the gluten and the gluten peptide obtained by hydrolyzing the gluten are produced from corn and wheat which are inexpensive and easy to purchase. Therefore, as a functional food material useful for exogenous hyperlipidemia and hypercholesterolemia due to intake of a diet containing high fat and high cholesterol It can be widely used.

Description

Methods for using gluten peptide for the inhibition of exogenous hyperlipidemia and hypercholesterolemia

The present invention relates to a method for using a gluten peptide prepared from a vegetable protein for inhibiting hyperlipidemia and hypercholesterolemia. More particularly, the present invention relates to a method for using gluten peptide obtained by hydrolyzing vegetable protein in the suppression of exogenous hyperlipidemia and hypercholesterolemia due to ingestion of a diet containing high fat and high cholesterol.

Nutrition of food protein has been emphasized in terms of its basic functions, namely the role of nitrogen source, amino acid source, and also the number of protein metabolism functioning in the body. Therefore, in the nutritional evaluation of food proteins, quantitative evaluation of proteins and qualitative evaluation by amino acid composition have been centered.

Recently, in addition to the basic function as a source of amino acid in food protein, research results on the function of peptide derived from dietary protein have become new recognition of nutritional value of food protein. The idea of the physiological function of a peptide derived from a dietary protein is as follows. First, it is revealed that a large number of peptides having a physiological function in vivo are generated from a precursor protein. Second, as the primary structure of a protein is gradually revealed And that there is a structure similar to a physiological functional peptide in vivo in the amino acid sequence of the food protein. As an experimental approach from this point of view, it is necessary to first determine whether peptides having a physiological function in proteins can be produced by in vivo or in vitro digestive enzymes, whether the peptides produced by the enzymes and physiological functions favorable to the body Or whether peptides can be absorbed in the digestive tract.

Thus, it has been found that peptides of various molecular sizes within the gastrointestinal tract during the course of protein digestion are produced in various ways depending on the type of protein, and they perform various physiological functions. For example, it has been shown that casein phosphopeptide (CPP) is produced during digestion of casein, a milk protein, and this peptide acts as a calcium absorption promoting factor. At present, CPP is mass produced and added to food as a calcium absorption enhancer, and peptides exhibiting serum cholesterol lowering effect or peptides exhibiting an inhibitory effect of angiotensin converting enzyme (ACE) activity during soy protein ingestion are produced during digestion . In addition, peptides derived from various food proteins having physiological functions of opioids have been reported.

In general, tripeptides, which are small in molecular size, have been identified as being absorbed by the transport system separate from the amino acid transport system together with membrane digestion. As a nitrogen source of clinical nutrition (entral nutrition) The nutrition of dietary peptides has been of great interest.

Circulatory system diseases are the most prevalent among recent increasing numbers of adult diseases, and dietary treatment for their prevention and treatment is becoming popular. In particular, high levels of lipid and cholesterol in blood have been pointed out as the most important risk factors in the development and progression of circulatory diseases such as stroke, atherosclerosis and hypertension, and it is gradually becoming clear that dietary factors are involved in the regulation of the concentration. That is, serum lipid and cholesterol concentrations are mainly known to be influenced by the amount of cholesterol contained in the diet, the type and amount of fat, the type of carbohydrate, vitamins, and minerals. The relationship between the protein type and the lipid metabolism in these dietary components is not consistent according to the experimental period, experimental conditions, animal species, and animal age, but in general, serum cholesterol synergism of casein and serum of soy protein Cholesterol lowering has been reported. The mechanism of expression of the action is explained by differences in amino acid composition such as amino acid composition, lysine / arginine ratio, and sulfamic amino acid content, or lipid absorption rate depending on the protein type, and difference in lipid and cholesterol excretion amounts. In recent years, the role of the peptide on serum lipid concentration has been emphasized, in that the protein structure itself and various kinds of peptides produced during the digestion process work closely with bile, lipid, and cholesterol. In particular, it has been proposed that the effect of lowering the serum lipid and cholesterol concentration of the soy protein is the action of the polymer peptide generated during the digestion process. In other words, research results indicating the nutritional and physiological superiority of dietary peptides have been reported more than dietary protein and amino acid mixtures thereof, so research on nutrition of peptides as novel food materials or functional materials is urgently required.

In this regard, the inventors of the present invention conducted studies on peptides prepared from protein glutinase from corn gluten and wheat gluten, which are obtained as byproducts in the production of starch inexpensive and easily available corn and wheat, Has been reported to be effective as a mineral absorption promoter of calcium and iron.

The inventors of the present invention have studied the effect of ingesting gluten and gluten peptide produced by hydrolyzing vegetable protein and found that a diet containing gluten and gluten peptide is useful for exogenous hyperlipidemia and hypercholesterolemia, Thereby completing the invention.

It is an object of the present invention to provide a method of using gluten peptide prepared from vegetable protein for the inhibition of exogenous hyperlipidemia and hypercholesterolemia.

To investigate the effect of dietary gluten peptide on prevention and treatment of exogenous hyperlipidemia and hypercholesterolemia induced by dietary intake of high fat and high cholesterol, adult rats were fed a diet containing high fat and high cholesterol This study was conducted to compare the induction of hyperlipidemia and hypercholesterolemia by feeding diet containing glycerin, gluten and its hydrolyzate. In addition, mature rats were fed a hyperlipemia-inducing diet containing casein as a source of high fat, high cholesterol, and protein, and after setting up an experimental model of hyperlipidemia, they measured the effect of hyperlipidemia and hypercholesterolemia , Respectively. At this time, arterial blood, liver tissue, heart tissue and fecal matter were collected, and the total lipid and cholesterol contents of each of these samples were measured, and the following determinations were made:

1) There was no difference in dietary intake between experimental diets for 6 weeks, but weight gain was lower in gluten and hydrolyzate intake group, and there was no significant difference in weight gain during 4 weeks intake.

2) Serum total lipid concentration was highest in casein - fed group and lowest in gluten hydrolyzed group. Serum total lipid concentration was significantly influenced by nitrogen source, that is, protein type, and compared with protein, it was greatly influenced by protein hydrolyzate ingestion, and gluten and gluten hydrolyzate decreased serum lipid concentration hypolipidemic effect).

3) The effect of gluten and protein hydrolysates on serum total cholesterol concentration was almost the same as that of serum total lipid concentration, and depending on the type of protein, gluten was lower than that of protein and its hydrolyzate decreased serum cholesterol concentration (Hydrocholesterolemic effects), and the intake of protein hydrolysates showed a synergistic effect of HDL-cholesterol and an LDL-cholesterol-lowering effect.

4) The total lipid and total cholesterol contents of liver tissues were significantly decreased according to the ingestion of gluten protein or hydrolyzed protein. The total lipid and total cholesterol contents of cardiac tissues were significantly higher than those of protein type and protein hydrolyzate Were almost unaffected.

5) Total lipid and total cholesterol excretion in the diet increased significantly depending on the protein type, ie, gluten intake, but did not increase with the ingestion of protein hydrolysates.

These results indicate that gluten and protein hydrolysates are effective in reducing exogenous hyperlipidemia and hypercholesterolemia induced by high fat diet intake. Gluten intake showed lower blood lipid levels and liver lipid content than casein intake, especially gluten hydrolyzate ingestion. Therefore, the gluten peptide from the vegetable protein prepared in the present invention can be widely used as a functional food material useful for exogenous hyperlipidemia and hypercholesterolemia.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are for describing the present invention only in detail and that the scope of the present invention is not limited to these embodiments.

[Example 1]

Production of gluten hydrolyzate

To prepare the gluten hydrolyzate, 1.5 kg of corn gluten or wheat gluten was added to 1 L of water, and then 10 to 150 g of papain or bromelain was added at a temperature of 30 to 80 캜 and a pH of 4.0 to 8.0 to react. For pH control during the reaction, pH adjuster was installed in the reactor and pH 6.0 was kept constant with sodium hydroxide or hydrochloric acid. The gluten hydrolyzate prepared by reacting for 24 hours was a peptide having an acidic amino acid of 20 mol% or more and a molecular weight of about 200 to 6000 daltons based on the total amino acids. The gluten hydrolyzate was centrifuged, separated into supernatant and precipitate, and each was used as a gluten hydrolyzate supernatant and a precipitate in the following examples.

[Example 2]

Prevention of hyperlipidemia and hypercholesterolemia in gluten hydrolysates

A total of 32 male Sprague-Dawley rats weighing approximately 160 g were randomly assigned to four groups of 8 rats each. The rats were divided into four groups: high fat (18% in the diet) and high cholesterol (1% in the diet) (C), casein hydrolyzate (CH), gluten (G) and gluten hydrolyzate (GH) were fed for 6 weeks to compare the degree of induction of hyperlipidemia and hypercholesterolemia. At this time, the rats were separated and kept in a shoe-box cage. The breeding room was maintained at a temperature of 22 ± 2 ° C., a relative humidity of 65 ± 5%, and a condition of 6:00 am to 6:00 pm Respectively. In addition, the experimental diet and deionized water were fed as a complete free diet (ad libitum), and daily dietary intake and moderate body weight were recorded at regular intervals once every two days during the experiment. Also, a metabolic experiment was conducted for 4 days before the end of the experiment.

The experimental diet was a semi-purified diet, and its constituents and composition ratios are shown in Table 1 below. The composition of the experimental diet is basically following AIN-76 (American Institute of Nutrition Standards for Nutritional Studies Report, J. Nutr., 107: 1340-1348 (1977)), A high level of 18% beef tallow (manufactured by Seoul National Fisheries Co., Ltd.) Was obtained from the National Research Council (NRL) Used for purification). Nitrogen sources were 10% based on egg protein (Shinwoo Chemical, Korea) with a protein content of 82% and casein (Maeil Industry Co., Ltd., Korea) and its hydrolyzate , Korea), and gluten and its hydrolyzate were added by 10% for each type. The nitrogen content of each experimental diet was the same, and PEG (polyethylene glycol) # 4000 was included as a dietary transport marker in the digestive tract.

The animals were fasted for 12 hours before sampling and fed for 1.5 hours. One hour after the end of the dietary intake, blood was taken from the carotid artery by anesthesia with ethylether. After collecting the blood, the liver and the heart were removed, and the fat adhered to the organ was cleanly removed. The blood was removed by washing with cold saline (0.9% NaCl solution) and the water was wiped off with a filter paper to measure the weight of the living tissue , And the stools were collected for 4 days before the end of the experiment. The collected blood was stored in a refrigerator (4 ° C) for 24 hours, and then serum was obtained by centrifugation (Sorvall GLC-2B, USA) at 3000 rpm for 20 minutes. The long and minute samples were freeze-dried by freeze-dryer (Freeze-Dryer 18, Labconco, USA) and pulverized to measure dry weight. All samples were frozen at -40 ° C or below until they were used for analysis.

[Example 2-1]

Weight and Dietary Intake

The body weight change, daily weight gain, dietary intake, and feed efficiency of each experimental formula were measured and shown in Table 2 below. In Table 2, feed efficiency is the weight gain divided by the dietary intake. As shown in Table 2, there were significant differences in termination weight, body weight gain, and feed efficiency among the experimental groups, but there was no difference in the dietary intake. According to protein type, casein and casein hydrolyzate intake group showed higher body weight gain than gluten and gluten hydrolyzate intake group because of differences in amino acid composition of the two proteins.

Each analysis value by the treatment of the experimental diets was presented with mean and standard error (mean ± SE) using SAS program. (C + CH): (G + GH)] and the effect of the hydrolyzate [(C + G) :( CH + GH)] on the significance of each treatment and on the protein type (P 0.05) using a Dumcan's multiple range test.

[Example 2-2]

Serum total lipid and triglyceride concentration

The total serum lipid and triglyceride concentrations were measured and are shown in Table 3 below. At this time, the total lipid level of the blood was measured by the method of Fringe and Dunn (Fringe, CS and Dunn, RM, Am. J. Clin. Patho., 53: 89-92 (1980) (Colorimetric method based on sulfo-phospho vanillin reaction) and serum triglyceride levels were assayed by the method of Biggs et al. (See Biggs, HG et al., Clin. Chem., 21: 437-441 (1975)).

As shown in Table 3, serum total lipid concentration was significantly different among the experimental groups, but there was no significant difference in triglyceride. Serum total lipid concentrations were significantly higher in the caseine group than in the other groups and there was no statistical significance in the other three groups. In particular, gluten hydrolyzate ingestion group showed the lowest value, more than 40% reduction compared to casein intake group, and 22% reduction compared to gluten protein intake group. On the other hand, as a result of examining the ingestion effect of the experimental group according to the type of protein, it was significantly lower than that of casein and hydrolyzate thereof by about 74% in gluten and its hydrolyzate ingestion group, The serum lipid levels were significantly lower in the protein intake group. In addition, the effect of hydrolysates on the protein was examined, and the hydrolyzate group showed a significant decrease of about 25% as compared with the protein group. Therefore, it was judged that there is a preventive effect of gluten hydrolyzate on hyperlipidemia induced by high cholesterol - containing high fat diet due to the effect of reducing the concentration of gluten protein and protein hydrolyzate to total serum lipid concentration.

[Example 2-3]

Serum total cholesterol and cholesterol concentration

(LDL) concentration and athero-index (AI) were calculated according to Friedwald's formula, and the results are shown in Table 4 below. . Serum total cholesterol levels were measured by the method of Zlatkis and Zak (Zlatkis, A. and Zak B., Anal. Biochem., 29: 143-146 (1969) (Manufactured by Yeongdong Pharmaceutical Co., Ltd., Korea) using an enzyme method.

As shown in Table 4, the serum total cholesterol concentration was significantly different between the protein and its hydrolyzate intake group, but it was not different depending on the protein type. In particular, the casein hydrolyzate intake group showed a significant decrease by about 15%, compared with casein, but the gluten hydrolyzate intake group also showed a tendency to decrease, but the statistical significance was not recognized. There was no significant difference in the intake of casein gluten.

The effect of diet on serum HDL cholesterol concentration was significantly higher (P 0.05) in the hydrolyzate intake group than in the protein intake group. In particular, when the gluten hydrolyzate ingested, casein or gluten intake group Compared to the control group. Given the fact that HDL cholesterol is known as a lipoprotein that has an inhibitory effect on the risk factors of circulatory system diseases, it is expected to reduce the risk factors of cardiovascular diseases such as protein hydrolysates, especially gluten hydrolysates ingestion, arteriosclerosis and hypertension I could.

LDL - cholesterol concentrations were significantly lower in the hydrolyzate - fed group than in the protein - fed group (p0.05), as measured by total cholesterol, HDL - cholesterol, and triglyceride. However, there was no statistically significant difference in the gluten consumption group among the protein types compared to the casein intake group.

Taken together, these results suggest that the intake of protein hydrolyzate decreases the serum total cholesterol level and increases the HDL cholesterol level, which may lower the atherosclerotic index significantly.

[Example 2-4]

Lipid content in liver tissue

Total lipid, total cholesterol, and triglyceride content per liver tissue weight, dry weight, and dry weight were measured and are shown in Table 5 below. The cholesterol and triglyceride contents of tissues were determined by extracting total fat from tissues and minus using the method of Folch et al. (Folch et al., J. Biol. Chem., 226: 497-502 (1957) Were analyzed in the same manner as post-serum.

As shown in Table 5, the weight of raw tissue, dry weight, total lipid content, total cholesterol content, and triglyceride content were significantly different among the experimental groups. The weight of liver was significantly different according to the protein type. There was no difference between casein - fed group and protein - hydrolyzate group. Total lipid, total cholesterol and triglyceride contents of gluten group were significantly lower than those of casein group. The total lipid and triglyceride contents of the hydrolysates were not significantly different in the hydrolyzate group compared to the protein group, but the hydrolyzate group was lower than the protein group. Thus, protein type and hydrolyzate intake were found to have an overall effect on lipid metabolism in liver tissue.

In conclusion, the lipid metabolism in liver was affected by protein type and nitrogen type, especially the hydrolysates ingestion, and it was judged that there was a decrease effect on the liver lipid content according to the ingestion of gluten.

[Example 2-5]

Lipid content in heart tissue

The total lipid, total cholesterol, and triglyceride content per weight of the tissue, dry weight, dry weight of the heart tissue are shown in Table 6 below. At this time, the cholesterol and triglyceride contents of tissues were analyzed in the same manner as in Example 2-4.

As shown in Table 6, there was no difference in heart weight between the experimental groups, and total lipid and triglyceride contents were significantly different among the experimental groups. That is, there was no significant difference depending on the protein type, but the hydrolyzate supplementation group was significantly lower than the protein supplementation group (p0.05). In particular, the total lipid content of cardiac tissue was significantly lower during ingestion of gluten hydrolyzate. On the other hand, triglyceride was significantly lower in the hydrolyzate group than in the protein group.

[Example 2-6]

Fractional lipid excretion

Total daily lipid, total cholesterol, and triglyceride contents were measured according to the experimental diets, and are shown in Table 7 below. The cholesterol and triglyceride contents of the fats were analyzed in the same manner as in Example 2-4.

As shown in Table 7, there was no significant difference between the experimental diet groups. Total lipid was significantly different among the experimental groups, and total lipid excretion was significantly lower in casein group than in the other groups. Total cholesterol excretion was significantly higher and total lipid excretion was also increased by gluten intake. Total lipid, total cholesterol and triglyceride excretion tended to increase slightly in the hydrolyzate supplementation group compared to the protein supplement group, but did not show any significant difference. Therefore, it was found that total lipid, total cholesterol and triglyceride excretion showed an increasing effect according to ingestion of gluten protein.

[Example 3]

Effect of Gluten Hydrolyzate on Hyperlipidemia and Hypercholesterolemia in High Fat Diet

32 rats (male Sprague-Dawley rats) having an average body weight of about 160 g were fed a hyperlipemia-inducing diet containing casein in a high fat, high cholesterol and protein source for 4 weeks to establish an experimental model rat for hyperlipidemia. And 4 experimental groups were divided into 4 groups as follows: 1) For 4 weeks, the treatment effect of hyperlipidemia was measured and examined. The constituents and composition ratio of the experimental diet, the breeding conditions, the sampling, the sample analysis and the statistical analysis were carried out in the same manner as in Example 2.

[Example 3-1]

Weight and Dietary Intake

The weight change, daily weight gain, dietary intake and feed efficiency of each experimental formula are shown in Table 8 below. As shown in Table 8, there was no difference in body weight and dietary intake between the experimental groups, and there was no difference in the effect of protein type and hydrolyzate consumption.

[Example 3-2]

Serum total lipid and triglyceride concentration

Serum total lipid and triglyceride concentrations are shown in Table 9 below.

As shown in Table 9, serum triglyceride concentrations were not significantly different among the experimental groups. That is, serum triglyceride concentration was not influenced by protein type and nitrogen type. However, the casein intake group showed the highest lipid concentration, and the lowest lipid concentration in the gluten hydrolyzate intake group showed the overall trend to be the same as that of Example 2-2. However, in the present Example, four kinds of experimental diets were fed for 4 weeks after the high fat casein diet was fed for 4 weeks to induce hyperlipidemia. Therefore, in Example 2-2 in which the experimental diet was fed for 6 weeks from the beginning The results and their effects should be evaluated differently. Therefore, it was judged that the gluten hydrolyzate for hyperlipidemia induced by high cholesterol - containing high fat diet had a therapeutic effect because of the effect of reducing the concentration of gluten protein and protein hydrolyzate to total serum lipid concentration.

[Example 3-3]

Serum total cholesterol and lipoprotein cholesterol concentration

Table 10 below shows serum total cholesterol, high density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol (LDL) concentration and atherosclerotic index.

As shown in Table 10, serum total cholesterol was significantly higher in the caseine-fed group and lowest in the gluten hydrolyzate-fed group. When considering the effects of protein type and hydrolyzate, the ingestion effect of gluten or protein hydrolyzate was drastically reduced by 55% to 69% as compared with casein intake group. This effect indicates that the gluten hydrolyzate can be used for the treatment of hypercholesterolemia induced by high fat diet.

The effect of empirical formula on the serum HDL cholesterol concentration was found to be increased by 40% compared to casein or gluten supplementation in the ingestion of gluten hydrolyzate. Since HDL cholesterol is known as a lipoprotein that has an inhibitory effect on the risk factors of circulatory system diseases, it could be expected that the effect of protein hydrolysates, especially gluten hydrolysates, on the risk factors of cardiovascular diseases such as arteriosclerosis and hypertension could be expected.

On the other hand, the LDL - cholesterol concentration was not significantly different between the groups, but the LDL - cholesterol concentration was lower in the hydrolyzate group than in the protein - intake group. In addition, according to the type of protein, gluten intake group showed a tendency to be lower than that of casein intake group, but there was no statistically significant difference.

Taken together, these results suggest that the intake of protein hydrolyzate decreases the serum total cholesterol level and increases the HDL cholesterol level, which may lower the atherosclerotic index significantly.

[Example 3-4]

Lipid content in liver tissue

The total lipid, total cholesterol, and triglyceride content per gram of tissue weight, dry weight, dry weight of liver tissue are shown in Table 11 below. There was no difference in liver weight among the experimental groups, and total lipid, total cholesterol, and triglyceride were significantly different among the experimental groups. Protein type did not show any difference, but hydrolyzate intake group was significantly lower than protein intake group. Therefore, it was concluded that the effect of hydrolyzate supplementation was reduced by ingesting more than 4 weeks.

[Example 3-5]

Lipid content in heart tissue

Total lipid, total cholesterol, and triglyceride content per unit weight, dry weight, dry weight of heart tissue are shown in Table 12 below.

As shown in Table 12, only the triglyceride groups showed significant differences among the experimental groups, and the triglyceride levels were lower in the gluten group than in the caseine group. There was no significant difference in the lipid content of cardiac tissue by protein source and nitrogen type among experimental groups. Therefore, the protein type does not affect the lipid content of the heart generally, and the hydrolyzate effect is not expected to be expected in the 4 week feeding period.

[Example 3-6]

Fractional lipid excretion

The daily excretion, total lipid, total cholesterol, and triglyceride content according to the experimental diets are shown in Table 13 below.

As shown in Table 12, there was a significant difference in total lipid and triglyceride excretion among the experimental groups, with each excretion being lowest in the casein intake group and highest in the gluten hydrolyzate intake group. In addition, total lipid and triglyceride showed significant differences according to protein type, and when they ingested gluten and its hydrolyzate, each excretion amount was higher than casein and its hydrolyzate. Total cholesterol did not show any significant difference, but gluten - fed group showed higher excretion rate than casein - fed group. Therefore, it was concluded that total lipid, total cholesterol and triglyceride excretion were increased by the ingestion of gluten protein.

As described and demonstrated in detail above, the present invention provides a method of using gluten peptide prepared from vegetable protein for inhibiting exogenous hyperlipidemia and hypercholesterolaemia. Gluten and its hydrolyzed gluten peptides are produced from corn and wheat which are inexpensive and easy to purchase. Therefore, it is widely used as a functional food material useful for exogenous hyperlipidemia and hypercholesterolemia due to intake of a diet containing high fat and high cholesterol .

Claims (4)

A method of using a gluten peptide obtained by hydrolyzing a vegetable protein with an enzyme, wherein the acidic amino acid is at least 20 mol% based on the total amino acid and the molecular weight reaches about 200 to 6000 daltons for the inhibition of exogenous hyperlipidemia and hypercholesterolemia. The method according to claim 1, wherein the vegetable protein is at least one substance selected from the group consisting of corn, wheat gluten and corn zein. The method according to claim 1, wherein the enzyme is papain or bromelain. The method according to claim 1, wherein the hydrolysis is carried out at a temperature of 30 to 80 DEG C and a pH of 4.0 to 8.0 for 20 to 30 hours.