JPH0725796B2 - Cholesterol-lowering substances - Google Patents

Description

[Detailed Description of the Invention] "Technical Field" The present invention relates to cholesterol-lowering substances.

"Background Art" Because the prevention and improvement of hypercholesterolemia, a risk factor for arteriosclerosis, through diet is an extremely effective measure, various studies have been conducted on the effects of dietary protein on serum cholesterol levels, and it has become known that plant proteins such as soy protein are more effective at lowering serum cholesterol levels than animal proteins such as casein.

In addition, it has been reported that peptides with specific molecular weights among enzymatic decomposition products of proteins have a cholesterol-lowering effect (Patent Publication No. 2003-2006).
60-11425) is also known.

However, no cholesterol-lowering substance comprising a hydrophobic polymer of an enzymatic decomposition product of a protein as in the present invention is known.

"Disclosure of the Invention" The present inventors have developed a method for preventing and treating hypercholesterolemia through diet.
In researching the effects of dietary protein on serum cholesterol levels with the aim of improving this, we discovered that vegetable proteins such as soybean protein are more effective at lowering serum cholesterol levels than animal proteins such as casein, and that peptides with a specific molecular weight have a cholesterol-lowering effect (JP 60-11425).As we continued to study the effects of these factors on cholesterol metabolism and the mechanism behind the cholesterol-lowering effect, we became convinced that there must be fractions among enzymatic hydrolysates of proteins that have an excellent cholesterol-lowering effect, and we continued our research with the aim of finding such enzymatic hydrolysates of proteins with an excellent cholesterol-lowering effect.

The present inventors have been conducting research in accordance with the above-mentioned objectives, and while studying the cholesterol-lowering effects of various hydrolyzates obtained by enzymatic hydrolysis of proteins, they have discovered that the precipitate fraction (water-insoluble but SDS (Sodium Dodecyl Sulfate)) produced by enzymatic hydrolysis of proteins has the effect of lowering cholesterol.
The present invention was accomplished based on the discovery that benzoyl benzoate (a soluble benzoyl benzoate) has a significant cholesterol-lowering effect.

That is, the present invention relates to a water-insoluble fraction obtained by removing a water-soluble fraction from an enzymatic hydrolysis product of a protein, and the water-insoluble fraction is a water-insoluble fraction obtained by removing a water-soluble fraction from an enzymatic hydrolysis product of a protein.
It is soluble in SDS solution and has a molecular weight of 10
It is a cholesterol-lowering substance with a cholesterol level of 00-150,000.

The insoluble fraction constituting the cholesterol-lowering substance of the present invention is water-insoluble in aqueous systems, usually in the pH range of 2-8.

The water-insoluble fraction constituting the cholesterol-lowering substance of the present invention is soluble in a surfactant solution such as SDS (sodium dodecyl sulfate). In other words, this water-insoluble fraction is a hydrophobic polymer with an excellent hydrophobic structure.

That is, the cholesterol-lowering substance of the present invention contains a water-insoluble fraction (hydrophobic polymer) consisting of a hydrophobic portion that is resistant to enzymatic degradation and/or a hydrophobic portion that is produced during enzymatic degradation of a protein, which remains after removal of the hydrophilic portion by enzymatic degradation of the protein.

The water-insoluble fraction constituting the cholesterol-lowering substance of the present invention has a molecular weight of generally about 1,000 to 150,000 (preferably 2,000 to 50,000) when solubilized in a 1% SDS solution (pH 6.9).
That is, about 1,000 to 150,000 (preferably 2,000
The enzymatic hydrolysis product (up to 100,000) forms a water-insoluble fraction of the enzymatic hydrolysis product of the protein either directly or by forming a polymer,
The cholesterol-lowering substance of the present invention is a hydrophobic polymer having an excellent hydrophobic structure, and the proportion of hydrophobic amino acids (Tyr, Phe, Val, Ile, Leu, and Trp) in the amino acid composition is suitably 30% by weight or more (preferably 35% by weight or more).

For example, soybean protein itself has a superior cholesterol-lowering effect compared to casein, etc., and the water-insoluble fraction (cholesterol-lowering substance) obtained by enzymatic hydrolysis of soybean protein has a molecular weight of about 1,000 to 150,000 (preferably 2,000 to 100,000) when solubilized in a 1% SDS solution (pH 6.9).
The chromatogram pattern by (Performance Liquid Chromatography) is, for example, about 86000, 7400 and 2500.
The main peaks are approximately 86,000, 25,000, 7,600 and 2,900.
It is preferable that the main peak is between about 100,000 and 2,000 molecular weights, which indicates the molecular weight of the protein. In addition, the proportion of hydrophobic amino acids (Tyr, Phe, Val, Ile, Leu, and Trp) in the amino acid composition is 30% by weight.
More than 35% by weight (preferably more than 35% by weight) is suitable.

As explained above, the cholesterol-lowering substance of the present invention is composed of the water-insoluble fraction of the enzymatic hydrolysis product of such a protein having a serum cholesterol-lowering effect, and can be administered as a liquid (suspension) or dry powder. It can also be administered in tablet form using an excipient. If necessary, proteins other than the water-insoluble fraction, carbohydrates, fiber, vitamins, minerals, and other excipients can be freely added. It can also be used as an ingredient in various foods as diet. Since it is derived from natural proteins and does not have side effects from excessive intake, it can be freely administered in an amount according to the serum cholesterol level.

Next, the method for producing the cholesterol-lowering substance of the present invention will be explained.

The cholesterol-lowering substance of the present invention can be produced by enzymatically hydrolyzing a protein in an aqueous system and then fractionating the water-insoluble fraction.

The protein to be used is preferably one having many hydrophobic regions.
Proteins rich in insoluble fractions (hydrophobic fractions) remaining after enzymatic hydrolysis of the hydrophobic regions in an aqueous system, resulting in lower molecular weight fractions and their removal as water-soluble fractions, are preferred. Proteins rich in hydrophobic regions may be of any origin, whether animal (whey proteins such as casein and lactalbumin, egg white, proteins obtained from birds, animals, fish, and shellfish, etc.), vegetable (oilseed proteins such as soybean protein, peanut protein, rapeseed protein, cereal proteins such as gluten and glutenin), or microbial (yeast protein, etc.). However, according to the research of the present inventor, vegetable proteins such as soybean protein are more preferable because they are richer in water-insoluble fractions having a cholesterol-lowering effect than animal proteins such as casein.

The enzymatic hydrolysis treatment may be carried out under any conditions as long as the desired hydrophobic polymer is obtained.
Known enzymes can be used, including animal-derived proteases such as trypsin and pancreatin, plant-derived proteases such as papain, ficin and bromelain, fungal proteases such as Aspergillus, bacterial proteases such as Patylus, and other microbial proteases.

Furthermore, it does not matter whether the protease is acidic, neutral or alkaline.

Either an endo-type protease or an exo-type protease may be used, but it is preferable to use a combination of an endo-type protease and an exo-type protease, since this will result in an enzymatically hydrolyzed insoluble fraction with less bitterness.

The conditions for enzymatic hydrolysis may be any conditions that are suitable for the action of the enzyme (e.g., action temperature, action pH, action E/S ratio, action time, action concentration), and preferably those that result in a larger insoluble fraction upon enzymatic hydrolysis. For example, although the substrate concentration depends on the type of protein and enzyme used, it tends to be preferable for it to be higher than the optimal concentration for hydrolysis, and the temperature, pH, etc. may deviate somewhat from the optimal conditions for hydrolysis. For example, in the case of soybean protein, a concentration of about 3 to 20% is preferred.

Next, the water-insoluble fraction obtained by enzymatic hydrolysis of the protein in an aqueous system can be fractionated. Usually, the water-insoluble fraction that precipitates in a neutral aqueous system can be fractionated, and depending on the enzymatic hydrolysis conditions, the insoluble fraction that forms in an aqueous system with a pH of about 2 to 8 can be fractionated.

The fractionation can be carried out by known fractionation methods for insoluble substances such as centrifugation, filtration, etc. Suitable filtration methods include microfiltration and filter press.

In practical production, the insoluble fraction obtained by these methods is often contaminated with a soluble fraction, but the insoluble fraction referred to in the present invention may contain a soluble fraction to the extent that it is contaminated when the insoluble fraction is collected. If necessary, a purer insoluble fraction can be obtained by purifying the insoluble fraction by repeated washing with water, etc.

The insoluble fraction obtained by fractionation may be used as a cholesterol-lowering substance as it is after enzyme inactivation, neutralization if necessary, and sterilization if necessary, or it may be dried in the usual way (using known drying methods such as spray drying, fluidized bed drying, and freeze drying) to form a powder, which can then be used as a cholesterol-lowering substance.

The cholesterol-lowering substance of the present invention contains the insoluble fraction obtained as described above as an active ingredient for lowering cholesterol.

As described above in detail, the present invention has made it possible to develop a cholesterol-lowering substance that is highly effective in lowering serum cholesterol.

The following examples illustrate embodiments of the present invention.

Example 1 Soy protein isolate ("Fujipro NEW-R" manufactured by Fuji Oil Co., Ltd.) 1
00 parts by weight (hereinafter referred to as parts) were made into a 10% aqueous solution of pH 7, and enzymatically hydrolyzed with 1 part each of Protin PN (manufactured by Daiwa Kasei Co., Ltd.: originating from Aspergillus oryzae) and Protin AC (manufactured by Daiwa Kasei Co., Ltd.: originating from Bacillus subtilis) at 50°C for 5 hours. The hydrolyzed solution was centrifuged (5000 rpm x 20 minutes) to obtain a precipitated fraction. Approximately twice the volume of water was added, and the mixture was stirred and washed. The precipitated fraction was centrifuged again in the same manner. The enzyme was inactivated by heating at 80°C for 30 minutes, and the resulting precipitate was freeze-dried to obtain 30 parts of a cholesterol-lowering substance.

Next, the amino acid composition of the raw material soy protein isolate (SPI), cholesterol-lowering substance (HMF), and purified HMF obtained by washing HMF five times with 10 times the amount of water were analyzed using a conventional hydrochloric acid hydrolysis and amino acid analyzer. The results are shown in Table 1.

From the above, it was found that the proportion of hydrophobic amino acids (marked with *) in the amino acid composition was increased, with the composition of hydrophobic amino acids (marked with *) in SPI being 27.6% by weight, compared to 37.5% by weight in HMF and 38.1% by weight in purified HMF.

Next, 5-week-old male Sprague-Dawley rats (body weight 105-108) were fed a high-cholesterol diet containing cholesterol-lowering substances (HMF) (see Table 2) and a high-cholesterol diet containing SPI (see Table 2).
g) (8-9 animals/group) for 24 days, and after 24 days, blood was collected and the animals were dissected to measure serum and liver cholesterol levels.
The results are shown in Table 3.

Table 2 High cholesterol diet Protein 20g Fat 10g Water-soluble vitamin mix 1g Salt mix 4g Choline chloride 0.15g Cellulose powder 2g Vitamin A 400μg Vitamin D 5μg Tocopherol 10mg Cholesterol 0.5g Sucrose Remaining total 100g From the above results, the cholesterol-lowering substance (HMF) showed a very significant serum cholesterol lowering effect.

Experimental Example 1 A cholesterol-lowering substance (HM) obtained in the same manner as in Example 1
F) was dissolved in 1% SDS (sodium dodesyl sulfate) and HP
The molecular weight pattern was examined by LC (TSK gel 3000SW, column 7.5 mm diameter x 60 cm). BSA (Bovine serum albumin) was used as a marker.
lbumen; molecular weight 67,000), soybean trypsin inhibitor (molecular weight 20,000), insulin B chain (molecular weight 3.50
0), pentaglutamic acid (molecular weight 664). The elution solvent was 0.025M phosphate buffer (pH 6.9) containing SDS, and 0.
The fractions were collected at 8 ml/min. Detection was performed using UV at 280 nm and absorbance at OD28
The HPLC chromatogram pattern is shown in Figure 1. From this, it was found that HMF has major peaks at molecular weights of 85,000, 7,400, and 2,500, and also contains a small amount of low-molecular-weight material with a peak at molecular weight of 120, with a molecular weight in the range of approximately 1,000 to 150,000.

Experimental Example 2 The HPLC chromatogram of purified HMF obtained in the same manner as in Example 1 was examined in the same manner as in Experimental Example 1. The results are shown in Figure 2.

The main peaks were molecular weights of 85,000, 7,400, and 2,500, and the molecular weight was 120
The molecular weight of the reduced low molecular weight substance peaking at about 1000 to
It was found to be in the range of 150,000.

Example 2 In the same manner as in Example 1, Sprague Dowry was
Male Igne-Dawley rats (6 rats/group) were fed a high-cholesterol diet (see Table 2) containing 20% of the protein shown in Table 4 as a nitrogen source for 14 days, and serum cholesterol levels were determined using the enzymatic method in the same manner as in Example 1. The results are shown in Table 5.

Mean ± SE, n = 6, significant differences between different symbols (p < 0.
05) From the above results, HMF (No. 1) exhibits an extremely excellent cholesterol-lowering effect, and this cholesterol-lowering effect is
The cholesterol-lowering effect was proportional to the amount of HMF added, and even when HMF was replaced to some extent with casein, an excellent cholesterol-lowering effect was observed.

Example 3 Soy protein isolate ("Fujipro NEW-R" manufactured by Fuji Oil Co., Ltd.) 1
00 parts by weight (the following parts) were made into a 10% aqueous solution of pH 2.0, and 0.5 parts of pepsin (Difco; 1:10000, Eacto) was added, and the mixture was incubated at 37°C.
After enzymatic decomposition for 17 hours, the decomposition solution was centrifuged (5000 rpm x 20
The precipitate was washed by adding about twice the volume of water and stirring, and then centrifuged again in the same manner. The precipitate was then heated at 80°C for 30 minutes.
The enzyme was inactivated by heating for 1 minute, and the mixture was freeze-dried to obtain 30 parts of a cholesterol-lowering substance (referred to as HMP-2).

The HPLC chromatogram pattern measured in the same manner as in Experimental Example 1 is shown in FIG.

The molecular weights ranged from about 1,000 to 150,000, with major peaks at about 86,000, 25,000, 7,600, and 2,900.

The amino acid composition was examined in the same manner as in Example 1, and the results are shown in Table 6.

From the above, as mentioned above, the hydrophobic amino acid composition of SPI is 2
It was found that the proportion of hydrophobic amino acids (marked with *) increased to 35.9 wt% in HMF-2 compared to 7.6 wt% in the previous study.

Example 4 A cholesterol-lowering substance (HMP-
2) In the same manner as in Experimental Example 1, Sprague-Dawley male rats (6 rats/group) were fed a high-cholesterol diet (see Table 2) containing 20% of the nitrogen source for 14 days, and the cholesterol levels in the liver and serum were examined using the enzymatic method in the same manner as in Example 1.

As a control, a similar experiment was carried out using isolated soybean protein ("Fujipro-R" manufactured by Fuji Oil Co., Ltd.). The results are shown in Table 1.
Shown in 7.

As is clear from the above, the cholesterol-lowering substance (HMF-
2) showed a significant cholesterol-lowering effect.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a drawing showing the HPLC chromatogram at OD 280 nm of the cholesterol-lowering substance (HMF) obtained in Example 1.

FIG. 2 is a drawing showing the HPLC chromatogram at OD 280 nm of the cholesterol-lowering substance (purified HMF) obtained in Example 1.

FIG. 3 is a drawing showing the HPLC chromatogram at OD 280 nm of the cholesterol-lowering substance (purified HMF-2) obtained in Example 3.

Claims (4)

[Claims] Claim 1: A water-insoluble fraction obtained by removing a water-soluble fraction from an enzymatic hydrolysis product of a protein, which contains SDS (Sodium dodecyl sulfate).
ester solution, and the molecular weight after solubilization in SDS solution is 1000
A cholesterol-lowering substance showing ~150,000. 2. The cholesterol-lowering substance according to claim 1, wherein the protein is soy protein. Claim 3: 1% SDS (Sodium dodecyl sulfate) in the water-insoluble fraction
3. The cholesterol-lowering substance according to claim 1 or 2, which has a molecular weight of 2,000 to 100,000 after solubilization with the hydroxybenzoate. Claim 4: Hydrophobic amino acids (Tyr, Ph) in the amino acid composition
3. The cholesterol-lowering substance according to claim 1, wherein the proportion of ATP (Aldrich, Val, Ile, Leu and Trp) is 30% by weight or more (preferably 35% by weight or more).