WO2000057898A1

WO2000057898A1 - Compositions for reducing blood cholesterol

Info

Publication number: WO2000057898A1
Application number: PCT/JP2000/001994
Authority: WO
Inventors: Mitsutaka Kohno; Motohiko Hirotsuka; Toshiaki Aoyama; Yukio Hashimoto; Makoto Kito
Original assignee: Fuji Oil Co., Ltd.
Priority date: 1999-03-31
Filing date: 2000-03-30
Publication date: 2000-10-05
Also published as: JP2003088334A; AU3454900A

Abstract

Compositions which contain as the active ingredient 11S globulin fractionated from soybean protein and make it possible to safely and efficiently reduce blood cholesterol level.

Description

Description Composition for reducing blood cholesterol Technical field

The present invention relates to a composition for reducing cholesterol in blood, and more particularly, to a composition for reducing cholesterol in blood, which contains one of the physiologically active components fractionated from soybean protein as an active ingredient. Background art

Soybean protein is not only excellent in nutritional properties among vegetable proteins, but also has various bioactive effects in recent years, and is a food material that has attracted attention as a physiological functioning agent.

The ability of soy protein to lower blood cholesterol is well known.It is said that a component of soy protein specifically binds to bile acids, etc., which cause cholesterol, and is excreted as feces. Have been done. In addition, foods that declare the effect of reducing cholesterol (soy protein foods; “Soy soybeans” manufactured by Fuji Oil Co., Ltd.) have become licensed health foods approved by the Ministry of Health and Welfare.

Physiological functions such as cholesterol-reducing ability are thought to be expressed from a specific fraction of soybean protein as described above, and the cholesterol-reducing ability fraction as a peptide component is identified. Much research has been done on. Takano et al.

(Atherosclerosis, 72, 115, 1988 and J. Nutr., 120, 977, 1990) stated that the blood cholesterol concentration reduction effect is closely related to the digestion process of soy protein, and that soy protein pepsin or In vivo studies with rats have confirmed that the indigestible fraction obtained upon digestion with microbial proteases exhibits a very pronounced cholesterol-lowering effect.

Although it was an in vitro study, Iwami et al. (Journal of the Soy Protein Research Society, 15, 74, 1994) reported that a peptide that binds to bile acid, a precursor of blood cholesterol, is a major component of soy protein. One of the 11S globulin It was suggested that the peptide may be a peptide consisting of 14 amino acids in the acidic subunit that is a component of the above.

Similarly, in a study of the cholesterol-reducing ability fraction in vitro, according to Lovati MR et al. (J. Agric. Food Chera., 46, 2474, 1998), a low-density lipoprotein known as bad cholesterol was identified. Investigation of the effect of lowering human hepatocytes has shown that 7S globulin, a major component of soy protein, has an effect along with 11S globulin.

As described above, at present, no clear conclusion has been found as to where soy protein exists in the blood cholesterol lowering ability of soy protein. Purpose of the invention

An object of the present invention is to specify a blood cholesterol-reducing ability fraction in soybean protein, fractionate the fraction, provide a cholesterol-reducing agent, and provide it as a pharmaceutical. Another object of the present invention is to use cholesterol-reducing foods, foods, nutritional foods, functional foods, foods for specified health uses such as foods and drinks, and can be widely used for cholesterol reduction and prevention of cholesterol accumulation. It is to provide a composition. These and other objects and effects of the present invention will be further clarified by the following description. Summary of the Invention

The present inventors have assiduously studied and found the following.

(1) When fractionating soybean protein components from defatted soybeans by the method of Tan Shibasaki et al., Which can reduce 11 S globulin to 70 ° / ₀ or more without using a reducing agent. It can be fractionated to purity.

(2) The acidic subunit and the basic subunit, which are components of 7S globulin and 11S globulin purified simultaneously with the above 11S globulin, were fractionated, and casein was used as a control. According to a weekly animal experiment, only 11 S globulin had significant cholesterol hypoactivity in blood. Thus, blood cholesterol-reducing ability exists in 11S globulin, and when it is orally administered after fractionating it into its constituent acidic and basic subunits, the blood cholesterol-reducing ability present there is reduced. The result of reducing the effectiveness to such a degree that it does not show a significant difference is surprising finding. One possible reason for this is that the ability of soy protein to lower blood cholesterol exists in either acidic or basic subunits of 11S globulin, but it can be fractionated into acidic and basic subunits. If it is later provided as a protein source, the blood cholesterol-reducing fraction present there is likely to be rapidly digested in the gastrointestinal tract, reducing its effect.

Also, since the ability to reduce cholesterol in blood is present only in 11S globulin and not in 7S globulin, the protein fraction of that component, that is, the degree of purification from soy protein to 11s globulin, is low. It is expected that the cholesterol reduction effect per protein will be improved due to the improvement.

The present invention has been completed based on these findings, and fractionates 11S globulin, which is a main component of soybean protein, and fractionates it with a protein purity of 70% or more, preferably 80% or more. It is intended to provide a composition for reducing cholesterol in blood, comprising the obtained 11 S globulin as an active ingredient.

Further, the present invention provides a blood cholesterol reduction using soybean protein isolated from soybean containing 11 S globulin of 60% or more, preferably 70% or more of the total protein content in seeds as an effective component by breeding technology. A composition for use is also provided. Detailed description of the invention

In the present specification, 11S globulin generally refers to a globulin that is a generic name of soluble globular proteins and has an ultracentrifugal sedimentation coefficient of molecular weight equivalent to 11S. Globulin has 2S, 7S, 11S, and 15S in molecular weight distribution, among which 7

It is known that S and 11S are contained in large amounts in storage proteins of legumes such as soybeans. In addition, soybean 11S globulin is also called glycinin. The acidic subunit is one of the polypeptides constituting the above-mentioned 11S globulin and is rich in acidic amino acids. The basic subunit is 11S globulin Is one of the other polypeptides that are rich in basic amino acids. The basic subunit and the above-mentioned acidic subunit are linked by one disulfide (SS) bond to form an intermediate subunit. 11S globulin is the assembly of six of these intermediate units.

In the present invention, a fraction having a high content of 11 S globulin fractionated from soybean protein is used as an active ingredient. To fractionate 11S globulin from soybean protein, the Tan 'Shibasaki method (Thahn, VH, and Shibasaki, K., J. Agric. Food Chem., 24, 117, 1976) Of course, other so-called cold-insoluble fractions (CIF) by the so-called cryoprecision (Briggs, DR and Mann, and Cereal Chem, 27, 243, 1950) and those by Wolf et al. WJ and Sly, DA, Cereal Chem, 44, 653, 1967) Fractionation method by addition of 0.1 N calcium chloride, and fractionation method in which an enzyme or enzyme having phytic acid degrading activity acts on soybean protein And any other fractionation method. However, in this case, 11S globulin of sufficient purity that can be used without using a reducing agent can be fractionated, and when used as a cholesterol reducing agent, a broader range of applications can be expected if the reducing agent is not included. .

In the present invention, as another method, defatted soybeans are produced from soybeans containing 60%, preferably 70% or more, of the total protein in the seeds by a breeding technique, and a reducing agent is produced therefrom. Except not to use, the isolated soybean protein containing 11 S globulin having a purity of 70%, preferably 80% or more, is used as an active ingredient by a usual method for producing an isolated soybean protein. In this case as well as above, it is possible to obtain 11S globulin of a purity that can be sufficiently used without using a reducing agent, and even when used as a cholesterol-reducing agent, a wider range of applications can be expected if no reducing agent is contained. .

The composition of the present invention can be made into a composition for oral administration by using the obtained fraction or isolated soybean protein as an active ingredient. The fraction and the isolated soybean protein used as the active ingredient of the present invention are safe and can be used as food, and the amount and the amount of the compound incorporated in the composition are not particularly limited. It may be added to food as a dietary treatment. In the case of oral administration, the dosage is half or less than 9 g / day for adults, which has been required for isolated soy protein. A sufficient cholesterol reduction effect can be expected with about g.

The composition for oral administration can be in the form of, for example, a powder, a sugar-coated tablet, a granule or the like according to a method known per se, and other excipients and additives can be added as appropriate.

Hereinafter, the present invention will be described in more detail with reference to Production Examples and Examples, but the present invention is not limited to these exemplifications.

Production Example 1

Preparation of 1 1 S globulin

Water was added to the defatted soybeans at a weight ratio of 1:10, and the mixture was stirred for 1 hour while adjusting the pH to 7. ° as needed. The mixture was centrifuged (4,000 rpm, 20 ° C for 10 minutes). . The obtained supernatant was adjusted to pH 6.4, left at 4 ° C overnight, and centrifuged (4,000 rpm, 10 minutes at 4 ° C). The resulting precipitate was collected, hydrated, neutralized to pH 7.0, sterilized, and spray-dried to obtain a desired 11S globulin.

The 11S globulin thus obtained was subjected to SDS-polyacrylamide gel electrophoresis, and then the degree of purity of the stained protein band was determined to be 85.4%. It was confirmed that the purity was sufficiently tolerable. Production Example 2

Preparation of isolated soy protein from soybeans rich in 11S globulin

According to breeding technology, water is added at a weight ratio of 1:10 to defatted soybeans containing 111.2% of the total protein content of 11S globulin in the seeds, and the pH is adjusted to 7.0 as needed. After stirring for 1 hour, the mixture was centrifuged (4,000 rpm, 20 ° C for 10 minutes). The resulting supernatant was adjusted to pH 5.5 and centrifuged (4,000 rpm, 10 minutes at 4 ° C). The resulting precipitate was collected, hydrated, neutralized to pH 7.0, sterilized, and spray-dried to obtain the desired isolated soy protein.

The isolated soybean protein thus obtained was subjected to SDS-polyacrylamide gel electrophoresis. Thereafter, the degree of purity of the stained protein band was determined to be 78.6%, which was sufficient for the following examination. The purity was confirmed.

Production Example 3 Separation and fractionation of each subunit from 11 s globulin

The 1 1 S globulin solution 14. 2% strength obtained by the above was subjected to steam sterilizer blowing water vapor pressure 6 kgf ZCM ^2, after blowing of 2 seconds steam, hot 1 minute at 1 40 ° C Was held.

After the solution has cooled down to about 50 ° C, centrifuge (8,000 rpm,

(At about 20 ° C for 10 minutes) to separate into supernatant and precipitate. The supernatant was freeze-dried as it was, and the precipitate was suspended by adding approximately the same amount of water as the weight of the precipitate, followed by freeze-drying. Each of the thus obtained subunits was subjected to SDS-polyacrylamide gel electrophoresis to examine the pattern. As a result, the purity of the acidic subunit in the supernatant was 93.6%, and no contamination with the basic subunit was observed. The purity of the basic substance in the precipitate fraction was 83.9%, and no acidic substance was detected.

Production Example 4

7 Preparation of S globulin (control)

The supernatant obtained by centrifugation (4,000 rpm, 10 minutes at 4 ° C) was left overnight at 4 ° C in Preparation Example 1 above, and the pH of the supernatant obtained was adjusted to 4.5. Then, centrifugation was performed (4,000 pm, 4 ° C for 10 minutes). The resulting precipitate was recovered, added to water, neutralized to pH 7.0, sterilized, and spray-dried to obtain 7S globulin.

The 7S globulin obtained in this way was subjected to SDS-polyacrylamide gel electrophoresis and found to have a purity of 72.4%, which was sufficient to withstand the following examination.

Example 1

Test for confirming the effect of reducing cholesterol in blood by rats

Using rats, a test was conducted to confirm the effect of lowering blood cholesterol. Each of the soy protein components obtained in Production Examples 1 to 4 except for Production Example 2 above, and casein (vitamin-free casein, manufactured by Oriental Yeast Co., Ltd.) were used as test feeds, respectively. Contains 20% as dietary protein and 0.5 cholesterol. / ₀ and 0.1% sodium monooleate, then sucrose and corn The starch was used in a mixture ratio of 1: 2.

The specific composition is shown in Table 1. Feed composition of each test group Unit;%

Test group casein 11 g acidic sub basic sub 7 S glo robulin unity brin casein 22.5----

1 1 S globulin-21.1---Acidic subunit--21.6--Basic subunit---20.0

7 S globulin-1-23.2 Sucrose 20.0 20.0 20.0 20.0 20.0 Corn oil 5.0 5.0 5.0 5.0 5.0 Vitamin mix 1.0 1.0 1.0 1.0 1.0 Minerano remix 3.5 3.5 3.5 3.5 3.5 Cellulose powder 5.0 5.0 5.0 5.0 5.0 Choline bitartrate 0.2 0.2 0.2 0.2 0.2 Cholesterol 0.5 0.5 0.5 0.5 0.5 Sodium cholate 0.125 0.125 0.125 0.125 0.125 α-corn starch 42.175 43.575 43.075 44.675 41.475 The test animals were 5 week-old male Wistar rats (rats weighing 90 g to 10 g). Purchased from Japan SLC and preliminarily bred on a commercial solid feed (Oriental “CRF-1”) for 1 week, then divided into 5 groups of 6 animals per group so that the body weight of each test material group becomes uniform. For 3 weeks. Rats were housed individually in individual cages and kept under a 12-hour light cycle (lighting from AM 7:00 to PM 7:00) at a temperature of 23 ± 1 ° C and a humidity of 55 ± 5%. . Free access to water and feed during the breeding period o

The test period was 21 days, and the weight gain during that period was measured, and the results shown in Table 2 were obtained. In addition, no significant difference was observed in the amount of increase in body weight between the tests.

Table 2

Changes in body weight of each test group Unit: g Test group Casein 11 S glo Acid Sulfate Basic sub 7 S Globulin Nitrite Unit At the start of the test 151.0 ± 1.3 151.1 ± 0.8 151.2 ± 1.0 151.2 ± 1.0 151.1 ± 0.9 At the end of the test 243 8 ± 1.6 248.1 ± 4.3 249.8 ± 3.3 250.5 ± 5.2 240.8 ± 5.5 Weight gain 92.7 ± 1.5 97.1 ± 4.198 ・ 7 ± 3.0 99.3 ± 4.8 89.7 ± 6.0 2. After completion of the test meal for 1 day, the animals were fasted for 6 hours from the morning of the 22nd day (AM 8:00), and the laparotomy was performed under Nembutal anesthesia. Plasma was separated from blood by centrifugation (3,000 r.p.m., 10 minutes at 5 ° C), and the total cholesterol concentration was measured. In addition, about the measured value, the significant difference test was performed by the t test between each two groups. The results are shown in Table 3.

Table 3

Changes in blood cholesterol level in each test group Unit: mg dl

As described above, since the blood cholesterol concentration can be efficiently reduced and the safety is extremely high, the composition of the present invention is used as a prophylactic or therapeutic agent for reducing the blood cholesterol concentration. Not only is it useful, but it can also be used as a food or drink for that purpose.

Claims

The scope of the claims

1. A composition for reducing blood cholesterol, comprising as an active ingredient 11S globulin fractionated from soybean protein at a protein purity of 70% or more.

2. A composition for reducing cholesterol in blood, comprising as an active ingredient isolated soybean protein produced from soybeans containing 60% or more of S-globulin in soybean seeds.

3. The composition for reducing blood cholesterol according to claim 1, wherein the composition is prepared without adding a reducing agent in the production process.