KR20030036480A - Process for preparing low molecular polymannuronate, a novel use thereof as controller of serum lipids, and functional foods and health-aid foods comprising the same - Google Patents

Abstract

PURPOSE: Provided are a method for manufacturing low molecular weight polymannuronate, novel use thereof as an improving agent of serum lipid, and a functional food and health food supplement containing the same. CONSTITUTION: Low molecular weight polymannuronate is characterized by average molecular weight of 10000-100000Da. The polymannuronate suppresses obesity, decreases the activity of GOT and GPT in serum, and reduces the content of cholesterol, phospholipid and low density lipoprotein, while increasing the content of high density lipoprotein.

Description

Process for preparing low molecular polymannuronate, a novel use describes as controller of serum lipids, and functional foods and health- aid foods comprising the same}

The present invention relates to high purity low molecular polymanuronate and its preparation, and its novel use as a serum lipid improving agent. The present invention also relates to functional foods and dietary supplements containing such low molecular polymanuronates.

Specifically, the present invention is a method for purely separating only low molecular polymanuronate by the decomposition of organic acid and solubility difference according to pH from polymer alginic acid, and low molecular polymanuronate purely separated in this way, functional food and serum containing the same It relates to a lipid-enhancing dietary supplement.

In recent years, there has been an increase in intractable cardiovascular diseases such as hypertension, arteriosclerosis, angina pectoris, myocardial infarction and thrombosis, obesity and diabetes due to excessive intake of nutrients and lack of exercise due to westernization of diet such as high fat and high protein diet. The interest in the prevention and treatment of these diseases is rapidly increasing. In particular, if the drug can be prevented and treated with foods added with materials extracted from natural substances, not chemically synthesized drugs, it will be the most desirable and safe way to reduce side effects or rejection.

Accordingly, as part of the development of bioactive food materials for a healthy diet, research and development on dietary fiber has been conducted at various angles. Dietary fiber is known to be effective against adult diseases such as constipation prevention, obesity prevention, antithrombosis, anti-arteriosclerosis, and anti-cholesterol. American Society of Clinical Nutrition, 52, 1990, 495-499; American Society of Clinical Nutrition 124, 1994, 78-83.

In particular, it is known that macromolecular alginic acid, a dietary fiber component that accounts for 20 to 30% of the cell wall-constituting polysaccharides of seaweeds (seaweed, seaweed, kelp, yam, and soybeans) in dietary fiber, has an effect on lowering cholesterol and inhibiting obesity. 26, 3, 1974, 78-83; Japanese Nutrition Journal, Vol. 33, No. 6, 1974, 273-281; 59, 5, 1993, 879-884].

Currently, many alginic acid-related products are manufactured and sold. Most of them are simply processed and extracted from alginic acid from seaweeds. Most of the alginic acid contained in these products are high molecular weight polymers (about 4 million daltons or more). to be. The polymer alginic acid is a polymer composed of only euroronate (M) and gluronate (G), and has high viscosity and low solubility in the polymer state, so that it is not easy to use it in high concentrations in foods such as beverages. Therefore, Japanese Unexamined Patent Application Publication No. Hei 6-7093 discloses the use of low molecular weight alginic acid as an additive for a functional beverage or the like. Here, the low molecular weight alginic acid means a state in which polygluronate and polymanuronate having a molecular weight of 10,000 to 900,000 Daltons are mixed. It is known that low molecular weight alginic acid has a lower viscosity and higher solubility than polymerized alginic acid, and the effect of high molecular alginic acid, such as cholesterol lowering, is increased.

In order to lower the molecular weight of alginic acid, conventionally used methods include using acid and alkali [Haug, A., Larsen, B. and Smidsrod, O. 1966. Acta Chem. Scand., 20 (1), 183-190; Hirst, E. and Rees, D. A. 1965. J. Chem. Soc., 9, 1182-1187; Hirst, E. L., Percival, E. and Wold, J. K. 1964. J. Chem. Soc., 8, 1493-1499], a method using heat and pressure [Japanese Patent No. 6-7093, 1994; Kimura, Y., Watanabe, K. and Okuda, H. 1996. J. Ethnopharmacology. 54, 47-54, and methods using enzymes [Doubet, R. S. and Quatrano, R. S. 1984. Appl. Environ. Microbiol. 47 (4): 699-703; Dunne, W. M. and Buckmire, F. L. A. 1985. Appl. Environ. Microbiol. 50 (1): 562-567; Hansen, J. B. and Nakamura, L. K. 1985. Appl. Environ. Microbiol., 49 (4), 1019-1021; Haug, A. and Larsen, B. 1971. Carbohydr. Res., 17, 297-308; Romeo, T. and Preston, J. F. 1986. Biochemistry. 25 (26): 8385-8391; Yonemoto, Y., Murata, K., Kimura, A., Yamaguchi, H. and Okayama, K. 1991. J. of Fermen. and Bioengin. 72 (3): 152-157. However, the method of using acid and alkali is disadvantageous for industrial use because of problems of deterioration of product, corrosion of equipment, necessity of neutralizer and post-treatment. The method of producing low molecular weight alginic acid with molecular weight of 10,000 to 900,000 Daltons by heat treatment at 100 to 200 ° C. under pressure (Japanese Patent 特 開平 6-7093, 1994) has a long decomposition time and is processed at a high temperature of 100 ° C. or higher under pressure. Energy is required for high temperature and high pressure, and high cost is required. Finally, the method using the enzyme also requires a long reaction time is not suitable for industrial use.

As described above, low molecular weight alginic acid is expected to be useful in the manufacture of dietary supplements using low molecular weight alginic acid by improving the physical properties such as solubility while increasing the effect of lowering cholesterol and the like compared to the polymer alginic acid.

However, if only the active ingredient extracted directly from the low-molecular-weight alginic acid directly affects the effects such as cholesterol lowering, and can be directly used in foods, functional foods containing higher units of the active ingredient in the correct content can be prepared. The industrial expectations and effects will be enormous.

That is, previously, there was no method for separating and manufacturing only the low molecular polymanuronate, which is an active ingredient from the polymer alginic acid, with high purity, and it was not known that the low molecular polymanuronate itself is a useful substance as a serum lipid improving agent.

On the other hand, polymanuronate itself is a substance that regulates toxic substances in a method of filtering toxins from dialysis patients (U.S. Patent No. 4,689,322 to Kulbe et al.), And immune rejection in organ transplantation. In order to circumvent, only the use of US Pat. No. 5,656,468 to Dorian et al. As a coating material for cells or tissues is known.

It is a primary object of the present invention to provide a process for the preparation of high purity low molecular polymanuronates.

Another object of the present invention is to provide the use of low molecular weight polymanuronate as a serum lipid improving agent.

It is another object of the present invention to provide a functional food and health supplement containing high purity low molecular polymanuronate.

It is another object of the present invention to provide a high purity low molecular polymanuronate having the above uses.

That is, the present invention is a functional food and dietary supplement having the effect of treating and preventing cardiovascular diseases such as hypertension, arteriosclerosis, hypercholesterolemia, obesity, and diabetes by lowering fat, cholesterol, and glucose levels in blood and liver. It is intended to contribute to the promotion of human health and disease prevention by providing a useful material and a method of producing the material.

The method for producing the low molecular polymanuronate of the present invention

(1) adding an organic acid to the polymer alginic acid and heating it,

(2) adjusting the pH to 2.5 to 3.5, and

(3) recovering polymanuronate

It includes.

According to the method of the present invention, the polymer alginic acid induces partial hydrolysis into an organic acid to make a lower molecular weight alginic acid having a smaller molecular weight, and then pH control in the low molecular weight alginic acid composed of a hybrid block of polymanuronate and polygluronate. Only the polymanuronate block can be separated and manufactured using the solubility difference method.

Polymeric alginic acid as a starting material that can be used in the present invention can be obtained through appropriate extraction, neutralization and dehydration, drying under reduced pressure, and the like known in the art from brown algae samples or dry powder samples thereof present in nature.

Organic acids that may be used in the present invention include citric acid, malic acid, fish acid, lactic acid, succinic acid, tartaric acid, acetic acid and the like, but is not limited thereto. Any organic acid capable of hydrolyzing alginic acid and low molecular weight can be used in the present invention. There may be a difference in the degree of low molecular weight of alginic acid according to the type of organic acid, but the degree of low molecular weight may be controlled by adjusting the concentration or hydrolysis time of the organic acid. According to one embodiment of the present invention, the low molecular weight of acetic acid was found to be the highest at the same concentration. In one embodiment of the present invention, the concentration of the preferred organic acid is preferably in the range of 0.2 mol to 2 mol, in particular 0.2 mol to 1 mol.

The reaction of the polymer alginic acid with the organic acid is generally preferred in the temperature range of 80 to 120 ℃, in particular 95 to 105 ℃ is most suitable.

In the second step, the pH is preferably adjusted to 2.5 to 3.5. It is especially preferable to adjust to 2.8 to 3.0. If the pH is less than 2.5, the purity of the resulting polymanuronate is high, but the yield is low. If the pH is more than 3.5, the purity is low, and therefore, a range in which purity and yield are most appropriately balanced is preferable. In order to obtain more than 95% purity of polymanuronate, the pH must be lowered to 1.5 or less, but the yield is only 10-20%, which is inappropriate for practical industrial use. The purity of polymanuronate resulted in much lower purity.

The purity of the low molecular polymanuronate obtained in accordance with the invention is at least 90% high. As used herein, the low molecular weight polymanuronate means an average molecular weight of 1,000 to 100,000 Daltons. The polymanuronate of the present invention preferably has a molecular weight of 10,000 to 100,000 Daltons, more preferably 30,000 to 50,000 Daltons, and most preferably has a molecular weight of 35,000 to 45,000 Daltons.

The present inventors have found that the low molecular polymanuronate obtained as described above has better serum lipid improving function than the polymer alginic acid, low molecular weight alginic acid and low molecular polygluronate. Here, the "serum lipid improvement" function is to lower the total cholesterol level in the blood and liver, increase the level of beneficial high-density lipoprotein, lower the level of low-density lipoprotein, and control the content of phospholipid and neutral lipid to the human body. It is defined as meaning including the ability to lower GOT and GPT levels.

Geoti and zipiti are measurements of the activity of glutamic oxalotransaminases and glutamic pyruvic transaminase enzymes, respectively, and the activity of these enzymes is highly sensitive to all types of liver damage. Thus, the ability to lower geotyi and geititi levels is a measure of improved liver function.

As a result of animal experiments, it was confirmed that the serum substrate improvement effect of the low molecular polyman euronate is significantly improved compared to the low molecular weight alginic acid and the low molecular weight polygluronate, and there is no damage to the liver function even in continuous dose (the animal experiment of Example 3 below). See results). The low molecular polymanuronate of the present invention not only lowers the total cholesterol content, but also has an effect of advantageously controlling the cholesterol composition ratio, and is expected to contribute greatly to the improvement of liver function due to its excellent synergistic inhibitory effect on serum geothyroides and gephyti. do.

In addition, the present inventors have found that polymanuronate has excellent binding ability with harmful heavy metals such as cadmium, lead, and mercury, and thus has the ability to discharge these heavy metals out of the body. The binding capacity of polymanuronate with harmful heavy metal ions was found to be significantly increased compared to the polymer alginic acid.

In addition, polymanuronate is expected to have an excellent effect on constipation due to its high binding strength and water retention.

As described above, the low-molecular polymanuronate obtained in the present invention is not only high purity and excellent serum lipid improving effect, but also excellent in solubility and viscosity in water, and does not have the peculiar or odor characteristic of natural brown algae. As an additive to foods and alone, it can be usefully used for serum lipid improvement and prevention of obesity and diabetes.

In addition, the polymanuronate obtained in the present invention can also be used as a substance for coating on cells or tissues in order to modulate toxic substances and to avoid immunorejection in organ transplantation.

The low molecular polymanuronate of the present invention can be used as a main ingredient or additives and auxiliaries of foods in the manufacture of various functional foods and dietary supplements.

As used herein, the term "functional food" means a food product having improved functionality of a general food product by adding polymanuronate to the general food product. Functionality can be roughly divided into physical properties and physiological functions. The polymanuronate of the present invention has inherent viscosity and binding properties with heavy metal ions as physical properties, and hyperlipidemia prevention functions such as cholesterol lowering ability and liver as physiological functions. It has been shown to have a lowering ability of geotyi and gepiti activity in relation to function improvement. Therefore, when the polymanuronate of the present invention is added to a general food, the physical properties and physiological function of the general food will be improved, and the present invention generally defines a food with such an improved function as a "functional food". For example, when the polymanuronate of the present invention is added to secondary processed foods such as ham to increase the viscosity of the food and to improve functions such as hyperlipidemia prevention and obesity prevention, such secondary processed foods are functional foods. It is called.

Distinguishing from functional foods, "health supplement" or "special dietary supplement" in the present specification is a health food manufactured by adding polymanuronate to a general food or encapsulating only polymanuronate. It is interpreted to mean that it has a certain health effect. That is, the health supplement means that it has a specific pharmacological function, but unlike general medicine, it has the advantage that there are no side effects that may occur when the long-term use of the drug as a food raw material.

For example, dietary functional foods may be manufactured using the function of the dietary efficiency of polymanuronate (that is, weight gain inhibiting effect). In addition, functional enhancement foods using liver function enhancement function, it may be possible to manufacture a liver function protective drink to remove hangovers.

In addition, the polymanuronate of the present invention is applicable to the manufacture of dietary supplements for hyperlipidemia patients or dietary supplements having a cholesterol lowering and control effect for preventing hyperlipidemia.

It is applicable to various fields such as dietary fiber drink for preventing constipation, cholesterol lowering functional bread, ramen, and margarine production.

Functional foods and dietary supplements containing low molecular polymanuronate according to the present invention preferably contain from 0.01 to 100% by weight of low molecular polymanuronate. Particularly preferred contents are different depending on the food group to be added, but in general, it is about 0.01 to 5% when prepared as a drink, about 10 to 50% when added to noodles, and 40 to 100% when prepared as a dietary supplement. Depending on the food group to be applied in and out can be used in various ways.

The low molecular polymanuronate dry powder according to the present invention is mixed so as to include 1 to 50% by weight in general wheat flour to form a polymanuronate mixed wheat flour, which is functional as a method for preparing a conventional ramen and bread using the wheat flour. Low molecular polymanuronate-containing ramen and bread can be prepared.

Dietary foods in capsule and tablet form containing the low molecular polymanuronate of the present invention as a main component and optionally containing conventional additives can be produced by a conventional manufacturing process.

In addition, the use of low-molecular polymanuronate can be used as a functional drink by adding it to beverages in the food industry, and it can be added to foods high in fat or cholesterol, such as hams, and processed and added to noodles. It can be applied to, and can be used as a seasoning mixed with other seasonings and salt of ribs, etc. It can be used in various powder forms or dissolved in water depending on the needs.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to examples of the present invention, but it is obvious that the present invention is not limited by the following examples.

Example 1

1. Preparation of low molecular polymanuronate

About 60 grams of alginic acid (molecular weight: about 1.3 million daltons) was mixed with 600 ml of an organic acid solution having the concentrations shown in Tables 1 to 6, followed by stirring, and the time shown in the table below at about 100 ° C. Is a functional relationship, so the higher the concentration of organic acid, the shorter the time required). The low molecular weight alginate polymanuronate and polygluronate mixing blocks were adjusted to pH 2.8 to 3.0 with each organic acid and centrifuged to separate the supernatant and the precipitate. At this time, since the supernatant is the polymanuronate fraction and the precipitate is the polygluronate fraction, the supernatant is taken and neutralized by addition of 1 molar sodium carbonate to the supernatant, and ethanol is added to 50% to form a precipitate. Centrifugation gave a precipitate.

The obtained precipitate was dissolved in a minimum amount (about 200 ml) of distilled water, and again adjusted to pH 2.8 to 3.0 with each organic acid and centrifuged. The lower layer was neutralized with 1 molar sodium carbonate and then reprecipitated with the same amount of ethanol and centrifuged to separate low molecular polymanuronate.

2. Determination of Molecular Weight of Low Molecular Polymanuronate

The molecular weight of the low molecular polymanuronate of the present invention uses Sepharose Ciel-4 ratio (Sepharose CL-4B) and Sepharose Ciel-6 ratio (Sepharose CL-6B), column chromatography (Ø12 mm × 97.6 cm) , Pullulan (Pullulan, Shodex standard P-82) was measured as the molecular weight standard. The molecular weight of the polymanuronate prepared in the present invention was 46.1 kilodaltons.

3. Purity test of low molecular polymanuronate

Purity and composition analysis of the separated low molecular polymanuronate was performed by dissolving the low molecular polymanuronate in 1% triethylamine solution and then Whatman patil with 0.02 molar potassium phosphate buffer (pH 4.6) containing 5% methanol. It was analyzed as high performance liquid chromatography using Whatman Partisil 10-SAX anion exchange column (250 × 4.6 mm id). Purity test was performed by comparing the elution pattern of each of the elution pattern on the basis of the chromatogram analyzed by high-performance liquid chromatography under the same conditions as the sample analysis, using the gluuronic acid lactone and manuronic acid lactone (manufactured by Sigma) as a standard product. . The purity of the polymanuronate prepared in the present invention was 1 hour-91%, 3 hours-93% and 5 hours-96% for each hydrolysis time.

Example 2 Results of Decomposition of Polymer Alginic Acid Under Various Decomposition Conditions by Various Organic Acids

Table 1 shows the results of hydrolysis for the same time using various kinds of organic acids. The degree of low molecular weight was different according to the type of organic acid, and hydrolysis with acetic acid at the same concentration was the lowest. Each yield tended to be nearly similar around 80%.

Correlation between Organic Acids Used and Molecular Weights of Polymanuronates Produced Organic acid (0.4 mol) Response time (hours) Molecular weight (kilodaltons) Citric Acid Apple Acid Lactic Acid 3333333 24.053.237.633.835.433.17.5

* The reaction is carried out at a constant temperature of 100 ℃.

Table 2 shows the results of hydrolysis during the same time using various concentrations (0.2 to 1.0 mole) of acetic acid. The degree of low molecular weight of alginic acid was higher with higher concentration of organic acid. However, even when a low concentration of acetic acid was used, the molecular weight of the produced polymanuronate was about 40,000 Daltons, which was about 1/30 of the original alginic acid molecular weight.

Correlation between acetic acid concentration and molecular weight of polymanuronate produced Acetic Acid Concentration (Mall) Response time (hours) Molecular weight (kilodaltons) 00.20.40.60.81.0 033333 1,283.040.07.53.81.90.6

* The reaction is carried out at a constant temperature of 100 ℃.

When the same concentration of organic acid was used but only hydrolysis time was different, the degree of low molecular weight was measured for acetic acid, malic acid, fish acid and citric acid, respectively, and the results are shown in Tables 3 to 6. Hydrolysis showed high molecular weight as the reaction time increased from 10 to 240 minutes. In particular, it was found that low molecular weight occurred rapidly during the initial reaction time (10 to 60 minutes).

Correlation between Reaction Time and Acetyl Polymanuronate Molecular Weight in Acetic Acid Acetic Acid Concentration (Mall) Response time (minutes) Molecular weight (kilodaltons) 0.4 01020405560120180240 1,283.0462.1185.6109.043.232.823.77.54.4

* The reaction is carried out at a constant temperature of 100 ℃.

Correlation between Reaction Time and Molecular Weight of Polymanuronate in Malic Acid Malic Acid Concentration (Mall) Response time (minutes) Molecular weight (kilodaltons) 0.4 0204060120180240 1,283.0569.0446.1234.2123.953.224.0

* The reaction is carried out at a constant temperature of 100 ℃.

Correlation between Reaction Time and Molecular Weight of Polymanuronate Produced in Fisheries Fisheries concentration (mall) Response time (minutes) Molecular weight (kilodaltons) 0.4 0204060120180240 1,283.0465.4354.8162.882.537.615.4

* The reaction is carried out at a constant temperature of 100 ℃.

Correlation between the Reaction Time and the Polymanuronate Molecular Weight Produced in Citric Acid Citric Acid Concentration (Mall) Response time (minutes) Molecular weight (kilodaltons) 0.4 0204060120180240 1,283.0452.4332.8154.078.524.013.2

* The reaction is carried out at a constant temperature of 100 ℃.

Example 3 Effect Experiment of Low Molecular Polymanuronate [Animal Experiment]

1. Experimental Materials and Methods

(1) Preparation of experimental diet: The composition of the basic diet, cholesterol diet and experimental diet is shown in Table 1. The cholesterol diet is prepared by reducing the amount of sucrose by adding 1% cholesterol to the basic diet, and the experimental diet is 1% cholesterol, 5% low molecular polymanuronate, and polygluronate from the amount of sucrose of the basic diet. 5% and polymanuronate and polygluronate were prepared by reducing the amount corresponding to 2.5% each, respectively.

Composition of Experimental Feed (g / kg) Dietary Ingredients Laboratory animals Basic diet Control Polymanuronate group Polyman euronate + polygluronate group Polygluronate Group Caseinradyukonoilmineralvitaminchlorinecholinecholesterolcholate sodium choline polymanuronatepolygluronatesugar 1808020408.520000669.5 1808020408.52102.500657 1808020408.52102.5500607 1808020408.52102.52525607 1808020408.52102.5050607

(2) experimental animals

The experimental animals used in this experiment were Sprague Dawley (SD) 4 week old male rats (purchased from the Korea Institute of Laboratory Animals). This was made for each of the experimental animal group of Table 7 (total 5 groups) 10 animals for 5 weeks as the experimental feed of the composition.

At this time, the breeding conditions of experimental animals were raised for 5 weeks in an animal control room where the temperature was 22 ± 2 ℃ and the humidity was 65 ± 3% RH, and then blood was collected to separate serum, cholesterol, neutral lipid and phospholipids of liver and liver. And the inhibitory effect of low density lipoprotein was analyzed and examined.

The content of cholesterol, triglyceride, phospholipid and low density lipoprotein was kit reagent [manufactured by Shinyang Chemical Co., Ltd.], and the food grade of experimental animals was used.

* (3) Total Cholesterol and Free Cholesterol: Total Cholesterol and Free Cholesterol in serum and soy sauce extract samples take 100 μl of serum and sample extracts and measure Cholesterol CII-Test Kit (Shinyang Chemical Co., Ltd.) It measured using the free cholesterol C-test kit reagent (made by Shinyang Chemical Co., Ltd.) for free cholesterol measurement.

(4) Neutral Lipids and Phospholipids: Neutral lipid concentrations were taken from 100 μl of serum and soy extract samples, and neutral lipid G-test kit reagent (Shinyang Chemical Co., Ltd.) was used. 100 μl was taken and measured using a phospholipid C-test kit reagent (manufactured by Shinyang Chemical Co., Ltd.).

(5) High density lipoprotein- and low density lipoprotein-cholesterol: The concentration of high density lipoprotein-cholesterol in serum and soy sauce is obtained by taking 100 μl of serum and soy sauce extract sample, respectively, and the high density lipoprotein-cholesterol C-test kit reagent (manufactured by Shinyang Chemical Co., Ltd.). The low density lipoprotein-cholesterol was expressed by subtracting the concentration of high density lipoprotein-cholesterol from the total cholesterol concentration.

(6) Glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activity: Kit reagent for measuring GOT and GPT activity by taking 100 μl of serum (Shinyang Chemical Co., Ltd.) ) Was measured.

(7) Statistical treatment: The experimental results were calculated for each experimental group by statistical processing, and the significance between each experimental group was tested by Duncan's multiple test at p <0.01.

2. Anti-obesity Effect of Low Molecular Polymanuronate

5% of low molecular weight polymanuronate was mixed with the animal's diet for 5 weeks to see the weight loss effect. As shown in Table 8, the low molecular weight polymanuronate addition group of the present invention effectively increased the weight gain compared to the control group. It was found that suppression.

Dietary Efficiency During the Breeding Experiment Experimental group Weight gain Feed intake Dietary efficiency Basic diet 197.2 429.1 0.46 Control group ^{* 1} 212.6 433.8 0.49 Polymanuronate group ^{* 2} 199.0 446.8 0.44 Polyman euronate + polygluronate group ^{* 3} 201.6 453.4 0.44 Polygluronate Group ^{* 4} 200.1 442.1 0.45

* 1 Basic diet + 1% cholesterol

* 2 Basic diet + Cholesterol 1% + Polymanuronate 5% diet group

* 3 Basic diet + Cholesterol 1% + Polymanuronate 2.5% + Polygluronate 2.5%

* 4 Basic diet + polygluronate 5% diet group

3. Cholesterol-lowering effect of low molecular polymanuronate

The 5% low molecular weight polymanuronate of the present invention was administered to experimental animals for 5 weeks, and then serum and liver were separated to compare cholesterol, triglyceride, and low density lipoprotein contents, which are known as the causative agents of adult disease. The cholesterol content of serum and hepatic lipids of all experimental diet groups was significantly lower than that of the cholesterol diet group, and the lowering effect of the low molecular polymanuronate diet group was remarkable, and the polymanuronate and polygluronate mixtures The effect of the diet group was superior to the polygluronate diet group. From this result, it was confirmed that the diet of polyman euronate has a greater effect of reducing cholesterol in blood and liver than the diet of polygluronate. Serum cholesterol content was reduced by 46% and hepatic cholesterol content was reduced by 59% by the low molecular polymanuronate diet of the present invention.

Serum and liver cholesterol lowering effect Experimental group Serum Cholesterol (mg / ㎗) Soy Cholesterol (mg / g) Basic diet 35.1 ± 1.3 7.4 ± 0.2 Control group ^{* 1} 284.2 ± 3.6 35.6 ± 0.3 Polymanuronate group ^{* 2} 153.3 ± 2.7 14.7 ± 0.2 Polyman euronate + polygluronate group ^{* 3} 207.5 ± 3.3 19.6 ± 0.2 Polygluronate Group ^{* 4} 218.8 ± 3.4 22.1 ± 0.3

* 1, * 2, * 3, * 4; See [Table 8]

4. Effect of Low Molecular Polymanuronate on Neutral and Phospholipids

As shown in Table 10 and Table 11, the amount of triglyceride in serum was the highest in the cholesterol diet group, the lowest in the low molecular polymanuronate diet group, and the other experimental diet diet group. The content was similar to that of the basic diet group. Hepatic lipids, like serum, had the highest cholesterol and low molecular weight polymanuronate diets. The amount of phospholipid was highest in the cholesterol diet group and lowest in the basic diet group in both serum and liver. In the experimental diet diet group, the phospholipid content was lower than the cholesterol diet group, and the effect was the most significant in the low molecular polymanuronate diet group. The low molecular weight polymanuronate, the product of the present invention, reduced the content of neutral lipids and phospholipids in the serum by 42% and 48%, respectively. Soy sauce decreased 35% and 40%, respectively.

Serum and hepatic triglyceride lowering effect Experimental group Serum triglyceride (mg / dl) Soy triglyceride (mg / g) Basic diet 62.5 ± 3.4 42.3 ± 1.3 Control group ^{* 1} 93.3 ± 4.2 79.2 ± 2.0 Polymanuronate group ^{* 2} 54.3 ± 2.4 40.8 ± 1.7 Polyman euronate + polygluronate group ^{* 3} 60.0 ± 2.7 49.2 ± 1.9 Polygluronate Group ^{* 4} 72.1 ± 2.9 51.9 ± 1.9

* 1, * 2, * 3, * 4; See [Table 8]

Serum and Soy Phospholipid Lowering Effects Experimental group Serum Phospholipids (mg / dL) Soy Phospholipids (mg / g) Basic diet 48.9 ± 1.5 10.2 ± 0.8 Control group ^{* 1} 98.8 ± 3.2 24.5 ± 1.5 Polymanuronate group ^{* 2} 63.8 ± 2.6 14.8 ± 0.9 Polyman euronate + polygluronate group ^{* 3} 68.5 ± 2.9 15.7 ± 0.7 Polygluronate Group ^{* 4} 68.0 ± 3.0 18.6 ± 0.7

* 1, * 2, * 3, * 4; See [Table 8]

5. Effects of Low Molecular Weight Polymanuronate on High Density Lipoprotein- and Low Density Lipoprotein-cholesterol

As shown in Table 3 and Table 13, the high density lipoprotein-cholesterol content was the lowest in the cholesterol diet group and the lowest in the low molecular polymanuronate diet group. In addition, the experimental feed group also showed a much higher content than the cholesterol diet group. The hepatic lipids were the lowest in the basic diet group and the low molecular polymanuronate diet group had the highest content. The low-density lipoprotein cholesterol content was the highest in the cholesterol diet group and the lowest in the basic diet group. The experimental diet group showed a significantly lower effect than the cholesterol diet group, which was remarkable in the low molecular polymanuronate diet group. Hepatic lipids also showed a high effect of low-density lipoprotein cholesterol in the experimental diet group, and the results were the same as in serum. And, the effect was found to be the most excellent in the low molecular polymanuronate diet group. The low molecular weight polyman euroneate of the present invention increased the content of high density lipoproteins in serum by 4.6 times and the low density lipoprotein content was reduced by 59%. In the liver, high density lipoprotein increased 1.2 times and low density lipoprotein decreased 74%.

Increased serum and hepatic high density lipoprotein cholesterol Experimental group Serum High Density Lipoprotein (mg / ㎗) Soy High Density Lipoprotein (mg / g) Basic diet 27.8 ± 1.1 3.3 ± 0.1 Control group ^{* 1} 8.6 ± 0.2 5.7 ± 0.3 Polymanuronate group ^{* 2} 39.4 ± 0.9 6.8 ± 0.2 Polyman euronate + polygluronate group ^{* 3} 23.5 ± 0.6 5.4 ± 0.4 Polygluronate Group ^{* 4} 15.2 ± 0.7 4.9 ± 0.3

* 1, * 2, * 3, * 4; See [Table 8]

Serum and liver low density lipoprotein cholesterol lowering effect Experimental group Serum Low Density Lipoprotein (mg / dl) Soy Low Density Lipoprotein (mg / g) Basic diet 7.3 ± 0.3 4.1 ± 0.3 Control group ^{* 1} 275.6 ± 3.4 29.9 ± 0.5 Polymanuronate group ^{* 2} 113.9 ± 1.4 7.9 ± 0.2 Polyman euronate + polygluronate group ^{* 3} 184.0 ± 2.4 14.2 ± 0.3 Polygluronate Group ^{* 4} 203.6 ± 2.9 17.2 ± 0.3

* 1, * 2, * 3, * 4; See [Table 8]

6. Effect of Low Molecular Polymanuronate on Serum GOT and GPT

As a result of the same experiment as in the above 3, as shown in Table 14, the change of GOT and GPT by the low molecular polymanuronate of the present invention has a 38% reduction in GOT and 30% in GPT compared to the control group. Indicated.

GOT / GPT lowering effect in serum Experimental group GOT (Karmen) GPT (Carmen) Basic diet 23.6 ± 1.7 18.5 ± 1.4 Control group ^{* 1} 45.2 ± 2.3 23.4 ± 2.5 Polymanuronate group ^{* 2} 27.8 ± 2.1 16.3 ± 1.5 Polyman euronate + polygluronate group ^{* 3} 31.9 ± 1.8 18.5 ± 1.8 Polygluronate Group ^{* 4} 33.4 ± 2.0 18.8 ± 1.9

* 1, * 2, * 3, * 4; See [Table 8]

7. Acute Toxicity Test

Eighty-week-old male ICR mice were administered orally at 2 g / kg of low molecular weight polymanuronate. Six hours after dosing was observed every hour, and for two weeks the general state and motility, weight trend, appearance and autonomic symptoms were carefully observed.

Daily observations for 2 weeks after oral administration showed no changes in exercise activity, weight change, convulsions and reflex activity.

In other words, LD50 was over 2000 mg / kg.

Example 4 Heavy Metal Adsorption Capacity of Polymanuronate

Purified algae alginic acid, polymanuronate and polygluronate separated and purified according to the present invention were dissolved in distilled water and the concentration was adjusted to 400 μg / ml. Metal salts were dissolved in distilled water and prepared at 0, 50 or 100 mM concentration. Each of the algae alginic acid, polymanuronate and polygluronate solutions was mixed with each metal cation solution in a volume ratio of 4: 1. The mixture was incubated for 2 hours at room temperature and centrifuged (20 minutes at 1,800 × g). The concentration of polymer in each supernatant was determined by the phenol-sulfuric acid method [Dubois et al., 1956.Anal. Chem., 28, 350-356]. The concentration of precipitated polymer was calculated and used to calculate the relative affinity of the polymer for each heavy metal cation. The results are shown in Table 15.

Precipitation of Polymers by Heavy Metal Ions Heavy metal ion Concentration (nM) ^{* 1} Polymanuronate Polygluronate Alginic acid calcium 8.0 8.5 17.6 cadmium 3.5 3.6 3.6 cobalt 20.2 9.9 11.5 Copper 3.5 4.6 3.2 iron 2.7 3.4 2.7 Mercury 18.0 77.7 100 < magnesium 100 < 100 < 100 < manganese 37.2 90.2 63.5 rubidium 15.5 16.9 24.1 strontium 15.6 16.6 23.1 zinc 15.2 18.3 14.5 lead 5.2 5.5 5.3

*One. The concentration of metal ions needed to precipitate 50% of the polymer from 400 μg / ml (w / v) of each polymer solution (polymanuronate solution, polygluronate solution and alginic acid solution).

As for the binding ability between polymanuronate and heavy metals, it showed excellent binding ability with iron, copper, cadmium, lead and calcium, and others with zinc, strontium, rubidium, mercury and cobalt. However, the binding ability with magnesium was weak.

Polygluronate showed a similar tendency to polymanuronate as a whole, but showed lower binding capacity than polymanuronate in manganese and mercury.

On the other hand, alginic acid showed a lower tendency of binding to metals than polymanuronate and polygluronate as a whole.

In the method for preparing low molecular polymanuronate of the present invention, by using an organic acid in the step of hydrolyzing the polymer alginic acid, there is no problem of device corrosion and the like compared to the method using inorganic acids such as hydrochloric acid or sulfuric acid, and after neutralization treatment There is an advantage that no treatment is required, and the hydrolysis time is significantly shortened and cost is reduced compared to other enzymes and hydrolysis under high temperature and high pressure.

According to the method of the present invention, there is an advantage in that a low molecular weight polyman euronate at a predetermined level having a purity of 90% or more can be obtained.

The low molecular polymanuronate obtained by the method of the present invention is an active ingredient of natural alginic acid, and is a high-purity single substance having high solubility and high solubility without enhancing the cholesterol inhibitory effect and other useful effects of the high molecular alginic acid. , If it is used as a food additive or an adjuvant, even if used in a small amount can achieve the desired effect, such as excellent cholesterol lowering, there is an advantage that it is possible to adjust the content for the desired function control in food.

Claims (2)

Low molecular polymanuronate mixtures with an average molecular weight of 10,000 to 100,000 daltons A low molecular polymanuronate mixture with an average molecular weight of 10,000 to 100,000 Daltons, purely separated by at least 90% purity.