JP5248755B2 - Angiotensin I-converting enzyme inhibitor and method for producing the same - Google Patents

Description

The present invention relates to an angiotensin I-converting enzyme inhibitor obtained by subjecting cacao nibs or cacao germ, and/or an extract of cacao nibs or cacao germ with water, methanol, ethanol, ethyl acetate, or a mixed solvent thereof, to a depolyphenol treatment using one or more substances selected from the group consisting of polyvinylpyrrolidone, methacrylic acid ester polymers, and polyphenol oxidase, and a method for producing the same.

Hypertension, along with diabetes, hyperlipidemia, and obesity, is a cause of arteriosclerosis. In addition, since it can be accompanied by serious complications, its treatment and prevention have been attracting a great deal of attention.

One of the solutions to hypertension is known to be the inhibition of angiotensin I converting enzyme (hereinafter abbreviated as ACE) in the renin-angiotensin system, which is known as a mechanism for increasing blood pressure. ACE is mainly present in the lungs, vascular endothelial cells, and renal proximal tubules, and is an enzyme that converts the substrate angiotensin I to angiotensin II. This angiotensin II acts as a powerful blood pressure increasing hormone, promoting vasoconstriction by binding to receptors in blood vessels. It is also known that ACE has the activity of breaking down bradykinin in the kinin-kallikrein system (antihypertensive system). Inhibition of ACE activity suppresses the production of angiotensin II and the breakdown of bradykinin, so ACE inhibitors are clinically effective in treating and preventing hypertension.

Recently, it has been found that ingredients derived from foods and natural products have ACE inhibitory activity. Examples include peptides obtained from casein (see, for example, Patent Document 1 and Non-Patent Document 1), sardines (see, for example, Patent Document 2 and Non-Patent Document 2), wakame seaweed (see, for example, Patent Documents 3, 4, 5, 6, and 7), and salmon eggs (see, for example, Patent Document 8), polyphenols such as tea polyphenols (see, for example, Non-Patent Document 3) and fruit polyphenols (see, for example, Patent Document 9), and plant-derived extracts such as eucalyptus extract (see, for example, Patent Document 10) and soybean extract (see, for example, Patent Document 11).

On the other hand, cacao (Theobroma cacao) has also been recognized to have a blood pressure lowering effect. It has been reported that cocoa extracts with increased amounts of cacao polyphenols by reducing the degree of fermentation during the manufacturing process to suppress polyphenol loss have blood pressure lowering activity (see, for example, Patent Documents 12 and 13), and that lignin, a type of polyphenol contained in cacao, lowers the blood pressure of spontaneously hypertensive rats (see, for example, Non-Patent Document 4). It has also been revealed that compositions containing cholesterol lowering agents and cacao polyphenols (see, for example, Patent Document 14) or compositions containing L-arginine and cacao polyphenols (see, for example, Patent Document 15) have therapeutic and preventive effects on heart disease. However, it has not been clarified that components other than polyphenols in cacao exhibit ACE inhibitory activity, or that amino acid compositions derived from cacao exhibit ACE inhibitory activity.

Japanese Unexamined Patent Publication No. 6-128287 Japanese Unexamined Patent Publication No. 7-188282 JP 2002-138100 A JP 2003-246795 A JP 2003-246796 A JP 2003-246797 A JP 2003-252897 A JP 2005-145827 A JP 2002-47196 A Japanese Patent Application Publication No. 11-60498 JP 2005-95156 A JP 2003-204758 A JP 2001-500016 A JP 2003-530410 A JP 2002-505864 A Bioscience, Biotechnology and Biochemistry, 58, 12, 2244-2245, 1994 Journal of the Japanese Society of Nutrition and Food Science, 53, 2, 77-85, 2000 Journal of the Agricultural Chemical Society of Japan, 61, 803-808, 1987 Journal of the Agricultural Chemical Society of Japan, 68, 957-965, 1994

The inventors have subjected cocoa beans and/or cocoa bean extracts to a polyphenol removal process and found that the resulting treated product has high ACE inhibitory activity. The inventors have also found that an amino acid composition derived from cocoa beans and/or cocoa bean extracts in particular has high ACE inhibitory activity. As mentioned above, there have been many reports on the blood pressure lowering effect of cocoa, but only the polyphenols contained in cocoa have been shown to be an effective ingredient, and this is the first time that it has been revealed that components in cocoa other than polyphenols exhibit ACE inhibitory activity.

In other words, the object of the present invention is to provide a naturally occurring ACE inhibitor that is highly safe and highly active as a food, a method for producing the same, and a food or beverage that contains the same.

In order to solve the above problems, the present inventors subjected cocoa beans and/or cocoa bean extracts to a polyphenol removal treatment and conducted an ACE activity inhibition test. Specifically, the inventors found that the cocoa bean extracts obtained by extracting unextracted cocoa beans and/or cocoa beans with a solvent such as water, methanol, ethanol, acetone, ethyl acetate, or a mixture of these solvents were treated with an adsorption aid such as polyvinylpyrrolidone or with an enzyme treatment to efficiently remove polyphenols, and further found that fractions rich in amino acids were obtained by performing an amino acid composition concentration treatment such as column fractionation, and that these fractions had significant ACE inhibitory activity, thus completing the present invention.

That is, the present invention relates to an angiotensin I-converting enzyme inhibitor obtained by subjecting cacao nibs or cacao germ, and/or an extract of cacao nibs or cacao germ with water, methanol, ethanol, ethyl acetate, or a mixed solvent thereof, to a depolyphenol treatment with one or more substances selected from the group consisting of polyvinylpyrrolidone, methacrylic acid ester polymers, and polyphenol oxidase, and a method for producing the same.

The present invention relates to an angiotensin I converting enzyme inhibitor, which comprises further subjecting the polyphenol-removed product to a column fractionation treatment for concentrating an amino acid composition, and a method for producing the same.

As described above, the angiotensin I-converting enzyme inhibitor of the present invention has the effect of effectively inhibiting ACE, which is involved in increasing blood pressure. Therefore, by adding the angiotensin I-converting enzyme inhibitor of the present invention to various foods and beverages, it can be used to treat or prevent hypertension. Furthermore, the angiotensin I-converting enzyme inhibitor of the present invention has a significantly higher ACE inhibitory effect than sardine peptide, which is considered to be effective in lowering blood pressure.

The angiotensin I converting enzyme inhibitor of the present invention is derived from food (cocoa beans), so there are no safety issues.

In addition, because the angiotensin I-converting enzyme inhibitor of the present invention has been subjected to a polyphenol-removal process, it is expected to have an excellent taste with less of the bitterness characteristic of polyphenols, making it easier to ingest than conventional products.

The product of the present invention can be applied to various foods, beverages, and preparations. In addition, the cacao beans that are the raw material for the product of the present invention have long been used as ingredients for foods, beverages, and natural additives, and there are no safety issues.

The present invention is described in detail below.

The cacao beans used as the raw material for the product of the present invention are usually heat-treated cacao nibs (edible part) or cacao germ (germ part), but the heat treatment method and parts are not limited to these.

There is no particular limitation on the method for obtaining the cocoa bean extract from cocoa beans, but the cocoa bean extract of the present invention can be obtained by adding one or more mixed solvents of water, methanol, ethanol, lower alcohols such as n-propanol, and organic solvents such as ethyl acetate, using a conventional extraction method. However, considering that the product of the present invention is intended for human consumption as a food or beverage or pharmaceutical preparation, it is preferable to use a combination of water and ethanol as the extraction solvent from the standpoint of safety.

The extraction conditions can be any temperature between 20 and 90°C, but 50 to 80°C for 1 to 5 hours is preferred. The resulting extract can be filtered and then concentrated or freeze-dried under reduced pressure for further use.

Examples of processes for removing polyphenols from the above-mentioned cocoa bean extract or the cocoa beans themselves include solvent extraction processes such as water-acetone, adsorption aid treatments such as Sephadex, methacrylic acid ester polymers, and polyvinylpolypyrrolidone, enzyme treatments such as polyphenol oxidase, the ferric chloride method, and combinations of these methods.

Furthermore, as a method for concentrating the amino acid composition, a composition with high amino acid content can be obtained by purifying the treated product using organic solvents, ion exchange column chromatography, etc., such as column fractionation. This composition with high amino acid content is thought to be composed of protein hydrolysates, peptides, and amino acids.

In addition, since the angiotensin I converting enzyme inhibitor of the present invention has excellent aroma and taste and is highly safe, it can be incorporated into, for example, chewing gum, candy, tablets, gummy jelly, chocolate, and other confectioneries, sherbet, beverages, and other foods and beverages, as well as preparations such as tablets and mouthwashes, and can be used on a daily basis.

The amount of the additive should be at least about 0.001% by weight, preferably at least about 0.01% by weight, in the dried form, of the food, beverage, or preparation.

The cacao beans that are the raw material for the product of the present invention have long been used as a food ingredient and natural additive, and there are absolutely no safety concerns regarding these processed products or the food, beverages, and preparations that contain them.

The present invention will be explained in more detail below with reference to test examples, but these are not intended to limit the scope of the present invention.

Test Example 1

This test was conducted to prepare an extract from cocoa beans.

1) Test samples: Cacao nibs and cacao germ were used.

2) Test method Extracts were prepared as follows.

i) Solvent extraction of cacao nibs 1.5 L of 50% ethanol was added to 200 g of cacao nibs and left to stand at 80° C. for 1 hour to perform extraction. This was filtered, and the filtrate was concentrated under reduced pressure and then freeze-dried. This operation was repeated twice to obtain 25 g of dried extract NA.

ii) Solvent extraction of cacao nibs 1.5 L of water was added to 200 g of cacao nibs, and the mixture was left to stand at 70° C. for 3 hours to perform extraction. The mixture was filtered, and the filtrate was concentrated under reduced pressure and then freeze-dried. This operation was repeated twice to obtain 30 g of the extract dried product NB.

iii) Solvent extraction of cacao nibs 1 L of 100% ethanol was added to 200 g of cacao nibs and left to stand at 60° C. for 1 hour to perform extraction. This was filtered and the filtrate was dried under reduced pressure. This operation was repeated twice to obtain 10 g of dried extract NC.

iv) Solvent extraction of cacao nibs 1 L of 100% methanol was added to 200 g of cacao nibs and left to stand at 60° C. for 2 hours to perform extraction. This was filtered and the filtrate was dried under reduced pressure. This operation was repeated twice to obtain 11 g of dried extract ND.

v) Solvent extraction of cacao nibs 1 L of 100% ethyl acetate was added to 200 g of cacao nibs and left to stand at 70° C. for 1 hour to perform extraction. This was filtered and the filtrate was dried under reduced pressure. This operation was repeated twice to obtain 10 g of dried extract NE.

vi) Solvent extraction of cacao germ 600 mL of 50% ethanol was added to 90 g of cacao germ and left to stand at 50° C. for 3 hours to perform extraction. This was filtered, and the filtrate was concentrated under reduced pressure and then freeze-dried. This procedure was repeated twice to obtain 12 g of dried extract JA.

vii) Solvent extraction of cacao germ 1.2 L of water was added to 180 g of cacao germ, and the mixture was left to stand at 70° C. for 3 hours to carry out extraction. The mixture was filtered, and the filtrate was concentrated under reduced pressure and then freeze-dried. This procedure was repeated twice to obtain 12 g of the dried extract JB.

viii) Solvent extraction of cacao germ 1.5 L of 100% ethanol was added to 200 g of cacao germ, and the mixture was left to stand at 60° C. for 1 hour to carry out extraction. The mixture was filtered, and the filtrate was dried under reduced pressure. This procedure was repeated twice to obtain 10 g of dried extract JC.

ix) Solvent extraction of cacao germ 1.2 L of 100% methanol was added to 180 g of cacao germ, and the mixture was left to stand at 60° C. for 2 hours to carry out extraction. The mixture was filtered, and the filtrate was dried under reduced pressure. This procedure was repeated twice to obtain 12 g of dried extract JD.

x) Solvent extraction of cacao germ 1.5 L of 100% ethyl acetate was added to 200 g of cacao germ, and the mixture was left to stand at 70° C. for 1 hour to carry out extraction. The mixture was filtered, and the filtrate was dried under reduced pressure. This procedure was repeated twice to obtain 12.5 g of dried extract JE.

3) Test results Extracts NA to NE were obtained from the cacao mass by this treatment. Extracts JA to JE were obtained from the cacao germ.

Test Example 2

This test was conducted to prepare a polyphenol-free product from cocoa bean extract.

1) Test Samples The solvent extracts of cacao nibs (NA to NE) and the solvent extracts of cacao germ (JA to JE) prepared in Test Example 1 were used.

2) Test method: 10 g of each extract was dissolved in 500 mL of the same solvent as the extraction solvent, and 50 g of polyvinylpolypyrrolidone (PVPP), a polyphenol adsorbent, was added thereto and stirred for 2 hours to adsorb the polyphenols. The mixture was then filtered with suction, and the filtrate was concentrated under reduced pressure and freeze-dried.

3) Test results Polyphenol-free products (NAP to NEP) of each cacao nib extract (NA to NE) were obtained in amounts of 6.0 g, 5.0 g, 5.0 g, 5.5 g, and 6.0 g, respectively. Polyphenol-free products (JA to JE) of each cacao germ extract (JA to JE) were obtained in amounts of 6.0 g, 5.5 g, 6.0 g, 5.5 g, and 6.0 g, respectively.

Test Example 3

This test was conducted to prepare a polyphenol-free product from cocoa beans.

1) Test samples: Cacao nibs and cacao germ were used.

2) Test Method Extracts were prepared as follows.

i) Polyphenol removal treatment of cacao nibs 1.5L of water was added to 200g of crushed cacao nibs, and 50g of polyvinylpyrrolidone (PPVP) as a polyphenol adsorbent was added and stirred and adsorbed for 24 hours to remove polyphenols. Then, the mixture was left to stand at 80°C for 1 hour to perform extraction. This was filtered, and the filtrate was concentrated under reduced pressure and then freeze-dried. This operation was repeated twice to obtain 2.5g of extracted dried product NP.

ii) Polyphenol removal treatment of cacao germ 1.5 L of 50% acetone was added to 200 g of crushed cacao germ, and 50 g of polyvinylpyrrolidone (PPVP) was added and stirred for 24 hours to remove polyphenols. The mixture was then left to stand at 45°C for 1 hour for extraction. The mixture was filtered, and the filtrate was concentrated under reduced pressure and freeze-dried. This operation was repeated twice to obtain 3 g of extracted dried product JP.

Test Example 4

This test was carried out to concentrate the amino acid composition of the polyphenol-free treated product.

1) Test Samples NAP, NBP, JAP, and JBP prepared in Test Example 2 were used.

2) Test method i) Treatment with cation exchange resin NAP, NBP, JAP, and JBP were fractionated into column adsorbed and non-adsorbed fractions using Dowex 50W x 4 (The Dow Chemical Company) prepared in H ⁺ form. That is, 5 g of the test sample was subjected to Dowex 50W x 4, eluted with 3 L of water to remove the unadsorbed portion, and then eluted with 2.5 L of 2N ammonia water. The obtained eluted fraction was concentrated and then freeze-dried.

ii) Treatment by ion exchange gel filtration NAP, NBP, JAP, and JBP were fractionated into column adsorbed and non-adsorbed fractions using SP Sephadex C-25 (Pharmacia) prepared in H ⁺ form. That is, 5 g of the donated sample was applied to SP Sephadex C-25, eluted with 3 L of water to remove the unadsorbed portion, and then eluted with 2.5 L of 2% NaCl aqueous solution. The fraction was further fractionated and desalted using Dowex 50W x 4 (The Dow Chemical Company). The eluted fraction obtained was concentrated and then freeze-dried.

3) Test results: By treatment with a cation exchange resin, fractions NAPD, NBPD, JAPD, and JBPD were obtained, which are fractions having concentrated amino acid compositions from NAP, NBP, JAP, and JBP. In addition, by treatment with ion exchange gel filtration, fractions NAPS, NBPS, JAPS, and JBPS were obtained, which are fractions having concentrated amino acid compositions from NAP, NBP, JAP, and JBP.

Test Example 5

This test was conducted to measure the ACE inhibitory activity, polyphenol content, and amino acid content of each sample.

1) Test Samples The samples prepared in Test Examples 1 to 4 were used.

2) Test Methods i) Measurement of ACE Inhibitory Activity This was carried out according to the method described in Biochemical Pharmacology 20.1637, 1971.

A test solution was prepared by dissolving the test sample in 10% dimethyl sulfoxide to a final concentration of 500 ppm. In addition, a reaction solution was prepared by dissolving the substrate hippuryl histidiyl leucine in a solution containing 600 mM sodium chloride in 400 mM phosphate buffer (pH 8.5) to prepare a 2 mg/ml reaction solution. 0.1 ml of the reaction solution was added to 0.05 ml of the test solution and shaken at 37°C for 3 minutes, and 0.1 ml of ACE adjusted to 0.1 U/ml was added to this mixed solution and reacted at 37°C for 1 hour. After 1 hour, the reaction was stopped by adding 0.5 ml of 1N hydrochloric acid. In order to obtain hippuric acid produced by this reaction, 1.5 ml of ethyl acetate was added to the reaction solution and extracted. Hippuric acid was quantified by HPLC, and the ACE inhibition rate of the test sample was calculated using the following formula (A = peak area of hippuric acid extracted from the test sample, B = peak area of hippuric acid extracted from 10% dimethyl sulfoxide).

ACE inhibition rate (%) = (1-A/B) x 100

ii) Measurement of polyphenol content Measurement was performed using the Folin-Ciocalteu method.

0.1 g of the test sample, 35 ml of distilled water, and 5 ml of Folin-Ciocalteu reagent (Sigma) were added to a 50 mL measuring flask, mixed, and allowed to stand for 1 minute. 5 ml of 20% sodium carbonate solution was then added, and the mixture was immediately brought up to 50 ml. After mixing and allowing to stand for 1 hour, the absorbance at a wavelength of 765 nm was measured. (-)-Epicatechin (Sigma) was used as a calibration sample, and the amount of polyphenols in each sample was calculated based on this.

iii) Measurement of Protein Amount The protein amount (amino acid composition amount) was measured using the Kjeldahl method.

1 g of the test sample, 10 ml of sulfuric acid, 8 ml of 30% hydrogen peroxide, and an appropriate amount of decomposition accelerator were added to a Kjeldahl reaction digestion tube, and the protein was digested at 420°C using a digestion heating device (Ticator). 100 ml of pure water was added to this digestion solution, and the nitrogen content was determined using a distillation/titration device (Ticator). The nitrogen content is expressed by the following formula:

Nitrogen (%) = 0.0014 x ( _V1 - _V0 ) x f/S x 100
V ₀ : Volume of 0.1N sulfuric acid standard solution required for titration (mL): Blank V ₁ : Volume of 0.1N sulfuric acid standard solution required for titration (mL): Test sample f: 0.1N sulfuric acid standard solution factor S: Test sample amount (g)

The protein content was calculated using the following formula:

Protein content (%) = Nitrogen content (%)* x 6.25**
*: Value after subtracting caffeine and theobromine nitrogen (%)
**: Protein conversion factor

3) Test results The results are shown in Tables 1 and 2.

As shown in Tables 1 and 2, the angiotensin I converting enzyme inhibitor of the present invention obtained by the above method is mainly composed of a composition consisting of amino acids, and by removing polyphenols, the amount of amino acid composition is increased, resulting in an ACE inhibitor that exhibits significantly higher activity.

Test Example 6

This study was conducted to determine whether the ACE inhibitory activity of the amino acid composition of cocoa bean extract is primarily due to the amino acid composition.

1) Test Samples NAd and NAP were used, which were prepared by diluting with 10% dimethyl sulfoxide so that the polyphenol content of the NA obtained in Test Example 1 was the same as the polyphenol content of the NAP obtained in Test Example 4.

2) Test Method ACE inhibition was measured according to the method described in Biochemical Pharmacology 20.1637, 1971.

A test solution was prepared by dissolving the test sample in 10% dimethyl sulfoxide to a final concentration of 500 ppm. In addition, a reaction solution was prepared by dissolving the substrate hippuryl histidiyl leucine in a solution containing 600 mM sodium chloride in 400 mM phosphate buffer (pH 8.5) to prepare a 2 mg/ml reaction solution. 0.1 ml of the reaction solution was added to 0.05 ml of the test solution and shaken at 37°C for 3 minutes, and 0.1 ml of ACE adjusted to 0.1 U/ml was added to this mixed solution and reacted at 37°C for 1 hour. After 1 hour, the reaction was stopped by adding 0.5 ml of 1N hydrochloric acid. In order to obtain hippuric acid produced by this reaction, 1.5 ml of ethyl acetate was added to the reaction solution and extracted. Hippuric acid was quantified by HPLC, and the ACE inhibition rate of the test sample was calculated using the following formula (A = peak area of hippuric acid extracted from the test sample, B = peak area of hippuric acid extracted from 10% dimethyl sulfoxide).

ACE inhibition rate (%) = (1-A/B) x 100

3) Test results The results are shown in Table 3.

As shown in Table 3, despite the same polyphenol content, the cocoa extract NAP of the present invention exhibited higher ACE inhibitory activity than NAd. From the above, it was revealed that the amino acid composition of the cocoa extract has high ACE inhibitory activity.

Test Example 7

This test was conducted to compare and examine the ACE inhibitory activity of the angiotensin I converting enzyme inhibitor of the present invention with that of "Marine Peptide" (Nissin Oillio Group, Ltd.), a food for specified health uses that contains sardine peptide, which is effective in lowering blood pressure.

The test samples were "Marine Peptide" (Nisshin Oillio Group, Inc., protein content 44%), a food for specified health uses containing sardine peptides that are effective in lowering blood pressure, and the fraction NAP obtained in Test Example 4 (a polyphenol-removed product of cacao nib extract, protein content 30.6%).

2) Test Method ACE inhibition was measured according to the method described in Biochemical Pharmacology 20.1637, 1971.

At that time, taking into consideration the difference in protein content between the two, the final protein concentrations were made uniform before conducting the test.

That is, a test solution was prepared by dissolving NAP in 10% dimethyl sulfoxide to a final concentration of 500 ppm (marine peptide was 348 ppm). In addition, a reaction solution was prepared by dissolving the substrate hippuryl histidiyl leucine in a solution containing 600 mM sodium chloride in 400 mM phosphate buffer (pH 8.5) at 2 mg/ml. 0.1 ml of the reaction solution was added to 0.05 ml of the test solution and shaken at 37°C for 3 minutes, and 0.1 ml of ACE adjusted to 0.1 U/ml was added to this mixed solution and reacted at 37°C for 1 hour. After 1 hour, the reaction was stopped by adding 0.5 ml of 1N hydrochloric acid. In order to obtain hippuric acid produced by this reaction, 1.5 ml of ethyl acetate was added to the reaction solution and extracted. Hippuric acid was quantified by HPLC, and the ACE inhibition rate of the test sample was calculated using the following formula (A = peak area of hippuric acid extracted from the test sample, B = peak area of hippuric acid extracted from 10% dimethyl sulfoxide).

ACE inhibition rate (%) = (1-A/B) x 100

3) Test results The results are shown in Table 4.

As shown in Table 4, NAP, a polyphenol-depleted cacao nib extract that is an angiotensin I converting enzyme inhibitor of the present invention, showed a significantly higher ACE inhibition rate than marine peptides.

The above studies have demonstrated that the angiotensin I-converting enzyme inhibitor of the present invention exhibits a significantly higher ACE inhibitory effect than sardine peptide, which is considered to be effective in lowering blood pressure.

The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited by these examples.

Tablets were prepared using the NAPS obtained in Test Example 4 according to the following formula:

D-mannitol 44.0 parts Lactose 40.0 parts Crystalline cellulose 10.0 parts Hydroxypropyl cellulose 5.0 parts
Fraction NAPS 1.0 part

Using the NBPS obtained in Test Example 4, lozenges were prepared according to the following formula.

Glucose 72.3 parts Lactose 19.0 parts Gum arabic 6.0 parts Flavoring 1.0 parts Sodium monofluorophosphate 0.7 parts
Fraction NBPS 1.0 part

Chewing gum was prepared using the NBPD obtained in Test Example 4 according to the following recipe.

Gum base 20.0 parts Sugar 55.0 parts Glucose 15.0 parts Starch syrup 9.0 parts Flavoring 0.5 parts
Fraction NBPD 0.5 part

Using the JAPS obtained in Test Example 4, candies were prepared according to the following recipe.

Sugar 50.0 parts Starch syrup 34.0 parts Flavoring 0.5 parts Fragrance JAPS 0.5 parts
Water 15.0 parts

Using the JBPD obtained in Test Example 4, tablets were prepared according to the following recipe.

Sugar 76.4 parts Glucose 19.0 parts Sucrose fatty acid ester 0.2 parts Flavoring 0.2 parts Fraction JBPD 0.1 parts
Water 4.1 parts

Using the JBPS obtained in Test Example 4, gummy jelly was prepared according to the following recipe.

Gelatin 60.0 parts Starch syrup 23.0 parts Sugar 8.5 parts Vegetable oil 4.5 parts Mannitol 2.9 parts Lemon juice 1.0 parts
Fraction JBPS 0.1 part

Chocolate was prepared using the NBPD obtained in Test Example 4 according to the following recipe.

Powdered sugar 40.8 parts Cocoa bitters 20.0 parts Whole milk powder 20.0 parts Cocoa butter 17.0 parts Mannitol 1.0 parts NBPD 1.0 parts
Fragrance 0.2 parts

Using the JAP obtained in Test Example 2, a sherbet was prepared according to the following recipe.

Orange juice 25.0 parts Sugar 25.0 parts Egg white 10.0 parts Fractionated JAP 2.0 parts
Water 38.0 parts

Biscuits were prepared using the NBP obtained in Test Example 2 according to the following recipe.

Weak flour, grade 1 25.0 parts Medium flour, grade 1 22.0 parts Refined sugar, 5.0 parts Salt, 1.0 parts Glucose, 1.0 parts Palm shortening, 12.0 parts Sodium bicarbonate, 0.2 parts Sodium bisulfite, 0.2 parts Rice flour, 2.0 parts Whole milk powder, 1.0 parts Milk substitute, 0.6 parts NBP, fractionated, 1.0 parts
Water 29.0 parts

Using the JAP obtained in Test Example 2, ice cream was prepared according to the following recipe.

Skim milk powder 50.0 parts Fresh cream 25.0 parts Sugar 10.0 parts Egg yolk 10.0 parts Fraction JAP 1.0 parts Flavoring 0.1 parts
3.9 parts water

A powder formulation was prepared using the JBP obtained in Test Example 2 according to the following recipe.

Corn starch 55.0 parts Carboxymethyl cellulose 40.0 parts
Fraction JBP 5.0 parts

Chewing gum was prepared using the NC obtained in Test Example 1 according to the following recipe.

Gum base 20.0 parts Sugar 55.0 parts Glucose 15.0 parts Starch syrup 9.0 parts Flavoring 0.5 parts
Extract NC 0.5 parts

Using the JAP obtained in Test Example 1, ice cream was prepared according to the following recipe.

Skim milk powder 50.0 parts Fresh cream 25.0 parts Sugar 10.0 parts Egg yolk 10.0 parts Extract Na 1.0 parts Flavoring 0.1 parts
3.9 parts water

A beverage was prepared using the NP obtained in Test Example 3 according to the following recipe.

Orange juice 30.0 parts Isomerized sugar 15.0 parts Citric acid 0.1 parts Vitamin C 0.1 parts Fraction NP 0.1 parts Flavoring 0.1 parts
Water 54.6 parts

Jam was prepared using the JP obtained in Test Example 3 according to the following recipe.

Fruit pulp 4.0 parts Sugar 65.0 parts Clarified juice 25.0 parts Citric acid 0.5 parts JP fraction 2.0 parts
3.5 parts water

Claims (6)

An angiotensin I converting enzyme inhibitor obtained by subjecting cacao nibs and/or an extract of cacao nibs with water, methanol, ethanol, ethyl acetate, or a mixed solvent thereof to one or more polyphenol removal treatments selected from the group consisting of polyvinylpyrrolidone, methacrylic acid ester polymers, and polyphenol oxidase . An angiotensin I converting enzyme inhibitor obtained by subjecting cacao germ and/or an extract of cacao germ with water, methanol, ethanol, ethyl acetate, or a mixed solvent thereof to one or more polyphenol removal treatments selected from the group consisting of polyvinylpyrrolidone, methacrylic acid ester polymers, and polyphenol oxidase . An angiotensin I converting enzyme inhibitor obtained by further subjecting the polyphenol-removed product according to claim 1 or 2 to a column fractionation treatment to concentrate the amino acid composition. A method for producing an angiotensin I converting enzyme inhibitor, comprising subjecting cacao nibs and/or an extract of cacao nibs with water, methanol, ethanol, ethyl acetate, or a mixed solvent thereof to a depolyphenol treatment using one or more enzymes selected from the group consisting of polyvinylpyrrolidone, methacrylic acid ester polymers, and polyphenol oxidase . A method for producing an angiotensin I converting enzyme inhibitor, comprising subjecting cocoa germ and/or an extract of the cocoa germ with water, methanol, ethanol, ethyl acetate, or a mixed solvent thereof to a depolyphenol treatment using one or more substances selected from the group consisting of polyvinylpyrrolidone, methacrylic acid ester polymers, and polyphenol oxidase . The method for producing the angiotensin I converting enzyme inhibitor according to claim 4 or 5, further comprising subjecting the polyphenol-removed product to a column fractionation treatment to concentrate the amino acid composition.