Composition for inhibiting cholesterol absorption, method for producing same, and method for improving cholesterol-absorption-inhibitory action

Abstract

[Problem] To provide a composition that is improved in cholesterol-absorption-inhibitory action of pea protein, and to provide a method for producing said composition. Additionally, to provide a method for improving the cholesterol-absorption-inhibitory action of pea protein. [Solution] The aforementioned problem is solved by a composition for inhibiting cholesterol absorption, the composition containing pea protein having a particle diameter of 180 μm or less as an active ingredient. Preferably, a food, a beverage, a medicine, or a feed contains the composition for inhibiting cholesterol absorption. Also provided is a method for producing a composition for inhibiting cholesterol absorption, the method comprising a step for refining the particle diameter of pea protein. Furthermore provided is a method for improving the cholesterol-absorption-inhibitory action of pea protein by miniaturizing the particle diameter of pea protein.

Description

Cholesterol absorption inhibiting composition, method for producing the same, and method for improving cholesterol absorption inhibiting effect

[0001] The present invention relates to a composition for inhibiting cholesterol absorption containing pea protein, a method for producing the same, and a method for improving the cholesterol absorption inhibitory effect.

[0002] In Japan, arteriosclerotic diseases, such as heart diseases such as myocardial infarction and angina pectoris, and cerebrovascular disorders such as cerebral infarction, account for approximately 23% of deaths, making them one of the leading causes of death among Japanese people. Increased blood cholesterol levels are one of the risk factors for arteriosclerotic diseases, and elevated blood cholesterol levels are thought to predict the future onset and death from heart disease (Non-Patent Document 1). Therefore, lowering blood cholesterol levels can prevent arteriosclerotic diseases and ultimately extend average life expectancy and healthy life expectancy.

[0003] Blood cholesterol levels are controlled by dietary cholesterol absorption and synthesis in the liver. Dietary cholesterol is absorbed by the small intestine. Cholesterol synthesized in the liver is secreted into the small intestine along with bile acids and circulates throughout the body by being reabsorbed (Non-Patent Document 2). Therefore, a material that reduces cholesterol absorption in the small intestine can inhibit both dietary cholesterol absorption and the reabsorption of synthesized cholesterol, thereby effectively suppressing blood cholesterol. In fact, Ezetimibe, which inhibits cholesterol absorption in the intestinal tract, is used as a pharmaceutical to treat high cholesterol (Non-Patent Document 1).

[0004] Pea protein is one of the materials that can reduce blood cholesterol levels. Pea protein has been reported to have cholesterol-lowering properties (Non-Patent Documents 3, 4, and 5). Another patent document suggests that pickled beans, when combined with indigestible polysaccharides, have cholesterol-lowering properties (Patent Document 1). The mechanism of action has been suggested to involve regulation of lipid metabolism-related gene expression, the involvement of intestinal flora, and peptide inhibition of cholesterol transporters (Non-Patent Documents 3, 5, and 6).

[0005] However, the detailed mechanism of action is unknown, and its effects on humans are limited (Non-Patent Document 7). Therefore, there are no foods in Japan that claim to have health benefits and contain pea protein as an ingredient. On the other hand, pea protein has been attracting attention in recent years, mainly in Europe and the United States, as an ingredient in plant-based foods, and demand for it is expected to continue to increase in the future.

[0006] Therefore, enhancing the cholesterol absorption inhibitory effect of pea protein and imparting functionality to it could be a useful technology that could lead to the prevention of arteriosclerotic diseases and the extension of average life expectancy and healthy life expectancy.

[0007] Japanese Patent Application Laid-Open No. 61-132149

[0008] Edited by the Japan Arteriosclerosis Society, Arteriosclerotic Disease Prevention Guidelines 2022 Edition, 2022. Int J Mol Sci., 20(19), 4939.Mol Nutr Food Res., 54(Suppl. S1), S24-30.Br J Nutr., 110(8), 1394-401.iScience., 24(12), 103435.Peptides., 90, 83-89.Br J Nutr., 107(8), 1176-83.

[0009] An object of the present invention is to provide a composition having an improved cholesterol absorption inhibitory effect of pea protein, a method for producing the same, and a method for improving the cholesterol absorption inhibitory effect of pea protein.

[0010] As a result of extensive research, the inventors discovered that the cholesterol absorption inhibitory effect of pea protein is negatively correlated with the particle size of the protein particles, and that the cholesterol absorption inhibitory effect can be improved by reducing the particle size, thereby completing the present invention.

[0011] That is, the present invention relates to the following inventions [1] to [6]. [1] A first aspect of the present invention is a composition for inhibiting cholesterol absorption, containing as an active ingredient a pea protein having a particle size of 180 μm or less. [2] A second aspect of the present invention is a food or drink containing the cholesterol absorption inhibitory composition according to [1]. [3] A third aspect of the present invention is a pharmaceutical product containing the cholesterol absorption inhibitory composition according to [1]. [4] A fourth aspect of the present invention is a feed containing the cholesterol absorption inhibitory composition according to [1]. [5] A fifth aspect of the present invention is a method for producing a cholesterol absorption inhibitory composition, characterized by comprising a step of reducing the particle size of pea protein. [6] A sixth aspect of the present invention is a method for improving the cholesterol absorption inhibitory effect of pea protein, characterized by reducing the particle size of pea protein. [7] A seventh aspect of the present invention is a method for using pea protein having a particle size of 180 μm or less, characterized by being used for the purpose of improving the cholesterol absorption inhibitory effect. [8] An eighth aspect of the present invention is a composition comprising a pea protein having a particle size of 180 μm or less for use in the treatment of cholesterol absorption inhibition. [9] A ninth aspect of the present invention is a non-therapeutic use of a pea protein having a particle size of 180 μm or less for improving cholesterol absorption inhibition.

[10] A tenth aspect of the present invention is the use of a pea protein having a particle size of 180 μm or less in the manufacture of a therapeutic drug for cholesterol absorption inhibition.

[0012] The present invention provides a composition for inhibiting cholesterol absorption in which the cholesterol absorption inhibitory effect of pea protein is improved, a method for producing the same, and a method for improving the cholesterol absorption inhibitory effect of pea protein.

[0013] 1 is a graph showing the cholesterol transport inhibitory activity of pea protein and existing pharmaceutical compounds. 2 is a graph showing the correlation between pea protein particle size and cholesterol transport inhibitory activity. 3 is a graph showing that the enhancement of effect by micronizing pea protein particles is observed in pea proteins in general. 4 is a graph showing that the enhancement of effect by micronizing soy protein particles is not observed in soy protein.

[0014] The cholesterol absorption inhibiting composition of the present invention, which has improved cholesterol absorption activity of the pea protein, its production method, and a method for improving the cholesterol absorption inhibiting activity of the pea protein, will be described in detail below. Note that the following descriptions are given for the purpose of illustrating the present invention and are not intended to limit the present invention.

[0015] In the present invention, "pea protein" refers to a protein contained in peas. As long as pea protein is contained, not only extracted or purified pea protein but also whole pea flour and the like may be used. Furthermore, in the present invention, "particle size" refers to the so-called median diameter (center value) based on volume, a value called D50, and refers to the value at which half of all volume-based particles lie above this value and the remaining half lie below this value.

[0016] [Composition for inhibiting cholesterol absorption] The composition for inhibiting cholesterol absorption of the present invention is composed of a pea protein having a particle size of 180 μm or less as an active ingredient. When the particle size is 180 μm or less, the composition has a cholesterol absorption inhibitory effect equivalent to or greater than that of existing intestinal sterol absorption inhibitors (e.g., ezetimibe). The upper limit of the particle size is preferably 100 μm or less, and more preferably 50 μm or less.

[0017] (Regarding Action and Mechanism) Known methods for inhibiting blood cholesterol absorption include inhibiting cholesterol synthesis in the liver and inhibiting cholesterol absorption in the intestinal tract. The cholesterol absorption inhibitory effect of the pea protein of the present invention is due to the latter inhibitory effect on cholesterol absorption in the intestinal tract, as shown in Test Examples 1 to 3 described below. It has been revealed that the smaller the particle size of pea protein, i.e., the better the cholesterol absorption inhibitory effect, but the exact mechanism of action has not been identified. However, it is speculated that the improved cholesterol absorption inhibitory effect is due to factors such as improved interaction between the micronized pea protein and cholesterol and intestinal cells, and structural changes in the protein due to increased contact with polar solvents caused by increased surface area, resulting in changes in its chemical and physical properties. Furthermore, as shown in Test Example 4, there is no correlation between particle size and cholesterol absorption inhibitory effect in soybean protein, suggesting that the enhanced cholesterol absorption inhibitory effect in the intestinal tract caused by reducing particle size is not a general protein property.

[0018] Furthermore, water-soluble dietary fiber is known to dissolve in water, adsorb bile acids and carbohydrates that promote the absorption of cholesterol and fat in the intestines, and excrete them from the body. However, the majority of dietary fiber contained in peas, which are the raw material for the pea protein used in the present invention, is insoluble dietary fiber, and since pea protein is a protein material, its fiber content is thought to be at most 3% or less. From this, it is speculated that the cholesterol absorption inhibition effect is not due to the effect of the dietary fiber contained in the pea protein of the present invention.

[0019] (Pea Protein) The pea protein used in the present invention can be obtained, for example, by crushing peas, separating the resulting pea powder, extracting it, and spray-drying it. Methods commonly used in the food industry can be used to extract, separate, or concentrate proteins from peas. The pea protein used in the present invention can be purchased as a commercially available powder product and then refined to a desired particle size before use.

[0020] (Method for Adjusting Pea Protein Particle Size) The method for adjusting the particle size of pea protein is not particularly limited, and may include adjusting the spray-drying conditions during powder production, or performing powder production by freeze-drying and adjusting the freeze-drying conditions. Alternatively, the particle size may be adjusted by ultrasonically disrupting a pea protein suspension before powderization or using a homogenizer.

[0021] Furthermore, a method may also be used in which a commercially available pea protein powder is dissolved or dispersed in a liquid, followed by ultrasonic treatment or homogenization. In either case, particle aggregation can be suppressed and the fine particle size can be maintained by adjusting the pea protein concentration, using an emulsifier, stabilizer, or aggregation inhibitor such as starch that is commonly used in the production of foods and pharmaceuticals, adjusting general temperature conditions or pH, or a combination of these methods. When crushing in a liquid, the timing of this is not particularly limited, and particles that have been crushed in advance to adjust their particle size may be used as the raw material, or the particle size of the pea protein can be adjusted by crushing a mix obtained by mixing the pea protein with other raw materials of the product.

[0022] (Method of Measuring Particle Diameter) In the present invention, the particle diameter is measured by measuring powdered pea protein by a laser diffraction light scattering method, and the value (D50) at which the volume-based cumulative frequency percentage of pea protein particles (cumulative frequency percentage in the frequency particle size distribution when particle sizes are plotted from the smallest to the largest) is 50% is defined as the particle diameter (μm).

[0023] (Food and drink) The cholesterol absorption-inhibiting composition of the present invention can be used as a raw material for food and drink as it is, and can be produced by the standard method for each food except for adding the cholesterol absorption-inhibiting composition. Therefore, an effective amount of the cholesterol absorption-inhibiting composition of the present invention can be incorporated into any food and drink, or can be added to the raw materials during the production process of the food and drink. Examples of food and drink include, but are not limited to, dairy products such as cheese, fermented milk, dairy lactic acid bacteria drinks, lactic acid bacteria drinks, butter, margarine, etc., milk drinks, fruit juice drinks, soft drinks, etc., egg products such as jelly, candy, pudding, mayonnaise, confectioneries and breads such as butter cake, various milk powders, infant foods, nutritional compositions, etc.

[0024] Examples of foods and beverages containing the cholesterol absorption inhibiting composition of the present invention include general foods and beverages, as well as foods for specified health uses, foods with functional claims, nutritional supplements, supplements, etc. Plant-based meat substitutes and plant-based milk substitutes that use finely divided pea protein as an alternative ingredient are examples of foods and beverages that contain pea protein.

[0025] If desired, foods and beverages containing the cholesterol absorption inhibiting composition of the present invention may contain additives and ingredients, such as antioxidants, flavorings, acidulants, colorings, emulsifiers, preservatives, seasonings, sweeteners, spices, pH adjusters, stabilizers, vegetable oils, animal oils, sugars and sugar alcohols, vitamins, organic acids, fruit juice extracts, vegetable extracts, grains, beans, vegetables, meat, seafood, etc., either alone or in combination of two or more. The amounts of these ingredients and additives to be added can be determined appropriately.

[0026] The form of the cholesterol absorption inhibiting food or beverage of the present invention is not particularly limited, and may be, for example, a liquid or fluid form such as a beverage or liquid diet, a jelly, paste, semi-liquid or gel form, or a solid, bar or powder form.

[0027] (Pharmaceuticals) The cholesterol absorption-inhibiting composition of the present invention can be used as a raw material for pharmaceuticals as is, and can be produced by standard methods such as tablets, capsules, powders, syrups, etc., except for the addition of the cholesterol absorption-inhibiting composition. When formulating, commonly used additives such as excipients, binders, disintegrants, and flavoring agents may be appropriately mixed. In other words, the cholesterol absorption-inhibiting composition of the present invention can be in any form as long as the effects of the present invention can be obtained. Examples of dosage forms include solid or powdered preparations such as powders, fine granules, granules, tablets, capsules, and pills, as well as liquid preparations such as suspensions, emulsions, syrups, and extracts.

[0028] There are no particular limitations on the recipient or daily intake of the cholesterol absorption inhibiting composition of the present invention, and for example, when the recipient is a human, the composition can be administered to minors under 18 years of age, adults, elderly people over 65 years of age, etc. The daily intake amount is also not particularly limited, as it differs depending on age, symptoms, body weight, and purpose.

[0029] (Feed) The cholesterol absorption-inhibiting composition of the present invention can be used as a raw material for feed as is, and feed may be produced by a standard method for producing feed except for adding the cholesterol absorption-inhibiting composition. Thus, an effective amount of the cholesterol absorption-inhibiting composition of the present invention may be blended into any feed, similar to foods and beverages, or may be added to raw materials during the feed production process.

[0030] The amount of the pea protein with adjusted particle size to be blended varies depending on the form, dosage form, symptoms, body weight, and use of the subject to be administered, and is not particularly limited, but can be blended at, for example, 0.001 to 100 (w / w)%.

[0031] [Method for producing a composition for inhibiting cholesterol absorption] The composition for inhibiting cholesterol absorption of the present invention can be obtained by reducing the particle size of a pea protein. Specifically, the composition can be obtained by subjecting a granulated powdered pea protein obtained by a known method to a step of reducing the particle size to 180 μm or less (refining step).

[0032] The method for reducing the particle size of pea protein is not particularly limited, and the above-mentioned well-known micronization methods may be used. For example, it is preferable to dissolve or suspend the pea protein in water or any liquid or mixture thereof that is generally used as a raw material for foods, beverages, or pharmaceuticals, and then use ultrasonic disruption or a homogenizer in the liquid. Furthermore, a step of measuring the particle size may be provided after this micronization step. The particle size may be measured using the above-mentioned (method for measuring particle size).

[0033] [Method for improving the cholesterol absorption inhibitory effect of pea protein] According to another aspect of the present invention, there is provided a method for improving the cholesterol absorption inhibitory effect of pea protein by reducing the particle size of the pea protein. The method for improving the cholesterol absorption inhibitory effect of the present invention can be carried out according to the description of the cholesterol absorption inhibitory composition of the present invention.

[0034] The present invention will be specifically described in detail below using test examples, although the embodiments of the present invention are not limited thereto.

[0035] Test Example 1: Cholesterol transport inhibitory activity of pea proteins Each pea protein was added to a cholesterol micelle-containing medium at a concentration of 1 mg / mL, and the cholesterol transport inhibitory activity was evaluated. The pea proteins used and their particle sizes are shown in Table 1. The negative control used was sterilized water in the same amount as the sample, and the positive control used was Ezetimibe (Cayman Chemical), a small intestinal cholesterol transporter inhibitor. The methods for measuring particle size, culturing intestinal epithelial cells, and testing cholesterol transport inhibition are described below.

[0036]

[0037] The results are shown in Figure 1. It was revealed that pea protein has cholesterol transport inhibitory activity equal to or greater than that of Ezetimibe, and that the effect tends to be stronger as the particle size becomes smaller (Figure 1). The letters (a, b, c) written above the bar graph in Figure 1 indicate whether there is a significant difference between groups, and groups without the same letter indicate that there is a significant difference between them.

[0038] Test Example 2: Correlation between pea protein particle size and cholesterol transport inhibitory activity Using the sample pea protein a used in Test Example 1, an evaluation was conducted to determine whether a correlation exists between particle size and cholesterol transport inhibitory activity. Pea protein suspensions were subjected to ultrasonic disruption to prepare pea proteins with different particle sizes. These were added to a cholesterol micelle-containing medium at a concentration of 0.5 mg / mL, and the cholesterol transport inhibitory activity was evaluated. The methods for measuring particle size, culturing intestinal epithelial cells, and testing cholesterol transport inhibition are described below.

[0039] The results are shown in Figure 2. It was revealed that there was a positive correlation between the amount of cholesterol transported and the particle size with a correlation coefficient of 0.796, that is, the smaller the particle size, the stronger the cholesterol transport inhibitory activity.

[0040] [Test Example 3] Verification that the enhancement of the cholesterol transport inhibitory effect by micronization is generally observed in pea proteins To investigate whether the enhancement of the cholesterol transport inhibitory effect by micronization of pea protein is a property generally observed in pea protein materials, two more types of pea protein materials were examined. Each material was micronized by ultrasonic disruption, and these were added to a cholesterol micelle-containing medium at a concentration of 1 mg / mL to evaluate whether the cholesterol transport inhibitory activity was enhanced. The methods for measuring particle size, culturing intestinal epithelial cells, and testing cholesterol transport inhibition are described below.

[0041] The results are shown in Figure 3. It was confirmed that the cholesterol transport inhibitory activity of both types of pea protein materials was enhanced by micronization. The letters (a, b, c) written above the bar graph in Figure 3 indicate whether or not there was a significant difference between groups, and groups without the same letter indicated that there was a significant difference between them.

[0042] [Test Example 4] Correlation between particle size of soy protein and cholesterol transport inhibitory activity To confirm whether the enhanced cholesterol transport inhibitory effect due to the reduction in particle size of the protein material is a property unique to pea protein, we evaluated whether a similar effect could be obtained with soy protein. Four types of soy protein were added to a cholesterol micelle-containing medium at a concentration of 1 mg / mL, and the cholesterol transport amount was evaluated. The particle size measurement method, intestinal epithelial cell culture method, and cholesterol transport inhibition test method are described below.

[0043] The results are shown in Figure 4. Although soy protein also has cholesterol transport inhibitory activity, no enhancement of the effect was observed by reducing the particle size. Therefore, it was suggested that the enhancement of activity by reducing the particle size is not a universal property of proteins, but is a property specific to pea protein. Furthermore, this result does not deny that protein materials other than pea protein may have similar characteristics, and it may also be observed in protein materials similar to pea protein (such as broad bean protein materials). The letters (a, b, c) written above the bar graph in Figure 4 indicate whether there is a significant difference between groups, and groups without the same letter indicate a significant difference.

[0044] [Measurement of particle size of protein material] Particle size was measured using an MT3000II (Microtrac Bell). After suspending the protein material in sterilized water, the particle was dropped into the device and measured under conditions of a particle refractive index of 1.81 and a solvent refractive index of 1.33. The measurement results were expressed as the volume-based cumulative frequency 50% particle size (D50) or cumulative frequency 90% particle size (D90).

[0045] [Culturing of intestinal epithelial cells] Intestinal epithelial cells (Caco-2) were cultured on a cell culture insert (CORNING) at a density of 1.8 × 10４ The cells were seeded at 1000 x 1000 cells / well. The medium used was Dulbecco's Modified Eagle Medium (DMEM) (Thermo Fisher Scientific) supplemented with 1% MEM-NEAA (Thermo Fisher Scientific) and 1% Penicillin-Streptomycin (Thermo Fisher Scientific), and 10% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific). The medium was changed every 2-3 days, and the cells were used for the test 17 days after seeding.

[0046] Cholesterol transport inhibition test: 10 μM Top Fluor Cholesterol (Avanti Polar Lipids), 4 mM sodium taurocholate (Nacalai Tesque), and 100 μM lecithin (egg yolk) (Nacalai Tesque) were added to phenol red-free culture medium and vigorously mixed to prepare a cholesterol micelle-containing medium. The apical medium of Caco-2 cells cultured on cell culture inserts was replaced with sample-supplemented cholesterol micelle-containing medium, and the basal medium was replaced with phenol red-free culture medium, followed by overnight incubation. After incubation, the basal medium was collected and fluorescence was measured to quantify the Top Fluor Cholesterol concentration, and the cholesterol transport amount of Caco-2 cells was measured.

[0047] According to the present invention, it is possible to provide a pea protein material that enhances the cholesterol absorption inhibitory effect of pea protein or that has an enhanced cholesterol absorption inhibitory effect. In particular, pea protein materials have been attracting attention in recent years as raw materials for plant-based foods, and the present invention may be a useful technology for developing functional foods. As described above, the present invention enhances the cholesterol absorption inhibitory effect of foods containing pea protein, thereby reducing cholesterol levels in consumers, thereby extending healthy lifespan and improving quality of life. Furthermore, by feeding the pea protein to livestock, it is possible to improve the blood lipid profile of the livestock.

Claims

1. A composition for inhibiting cholesterol absorption, comprising as an active ingredient pea protein having a particle size of 180 μm or less.

2. A food or drink containing the cholesterol absorption inhibiting composition according to claim 1.

3. A pharmaceutical comprising the cholesterol absorption inhibiting composition according to claim 1.

4. A feed containing the cholesterol absorption inhibiting composition according to claim 1.

5. A method for producing a composition for inhibiting cholesterol absorption, comprising a step of reducing the particle size of pea protein.

6. A method for improving the cholesterol absorption inhibitory effect of pea protein, which comprises reducing the particle size of pea protein.

7. A method for using pea protein having a particle size of 180 μm or less, characterized in that it is used for the purpose of improving the cholesterol absorption inhibitory effect.

8. A composition comprising pea protein having a particle size of 180 μm or less for use in the treatment of inhibiting cholesterol absorption.

9. Non-therapeutic use of pea protein having a particle size of 180 μm or less for improving cholesterol absorption inhibition.

10. Use of pea protein having a particle size of 180 μm or less in the manufacture of a therapeutic drug for inhibiting cholesterol absorption.

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