JP2782153B2 - Method for producing angiotensin converting enzyme inhibitory peptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to Ile-Pro-Pro and / or Val-Pro-P which exhibit angiotensin converting enzyme inhibitory activity.
The present invention relates to a method for producing an angiotensin converting enzyme inhibitory peptide containing ro.

[0002]

2. Description of the Related Art Angiotensin converting enzyme (Angiotensi)
n Converting Enzyme (hereinafter referred to as ACE) is mainly present in lung and vascular endothelial cells and is angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His) degraded by renin.
-Leu) to release a dipeptide (His-Leu) from the C-terminus, producing angiotensin II which has a vascular wall smooth muscle contraction effect and a strong blood pressure increasing effect. In addition, this enzyme has a function of decomposing and inactivating bradykinin having a hypotensive effect. As described above, ACE produces a pressurized peptide (acidiotensin II) and, on the other hand, degrades and inactivates a hypotensive peptide (bradykinin), thereby exhibiting an effect of increasing blood pressure. Therefore, various substances have been developed that inhibit the increase in blood pressure by inhibiting this enzyme activity.

[0003] As ACE inhibitors, many natural products and synthetic products have been reported, including snake venom peptides.
Synthetic substances such as captopril (D-2-methyl-3-mercaptopropanoyl-L-proline) have already been put to practical use as oral antihypertensive agents. However, these medicines often have side effects and the like, and always have to pay attention to safety issues. Therefore, antihypertensive drugs with low toxicity and high safety are expected, and ACE inhibitors derived from natural products have been studied in various fields. For example, casein [Susumu MARU-YAMA et al.
al. ABC, 51 (9), 2557-2561 (1987)], fish meat protein [Hiroyuki Uchida, Journal of the Japanese Society of Agricultural Chemistry, 66 (1), 25-29 (1992)]
ACE-inhibiting peptides obtained from enzymatic degradation products such as the above have been reported.

However, A obtained from such natural products
The content of the peptide having CE inhibitory activity is very small,
In order to actually obtain effective data by oral administration, an effect cannot be expected with a normal intake, and it is desired to develop a method for concentrating and purifying an effective peptide having a strong specific activity more easily in a large amount.

Also, Ile-Ala-Ile-Pro-Pro, Ala-Ile-Pro-P
ro or Ile-Pro-Pro has been proposed as a peptide having an antihypertensive effect (JP-A-3-120225).
However, as a method for producing such a peptide having an antihypertensive effect, only a chemical synthesis method has been proposed. In such a chemical synthesis method, it is difficult to industrially produce a desired peptide. There is a disadvantage that there is.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing a large amount of a peptide which is highly safe, has an effective ACE inhibitory activity, and can be used as a pharmaceutical or a functional food. Is to provide.

[0007]

According to the present invention, Ile-Pr
A method for producing an angiotensin-converting enzyme-inhibiting peptide comprising o-Pro and / or Val-Pro-Pro, comprising: lactic acid bacterium or lactic acid bacterium and yeast are mixed with milk protein, corn,
Protein, wheat, wheat protein, soy, defatted soy, soybean
Selected from the group consisting of proteins and mixtures thereof,
A method for producing an angiotensin-converting enzyme-inhibiting peptide, comprising culturing in a medium containing a peptide and / or protein comprising Ile-Pro-Pro and / or Val-Pro-Pro as a constituent, and purifying the culture. You.

Hereinafter, the present invention will be described in more detail.

In the method for producing an ACE-inhibiting peptide of the present invention, lactic acid bacteria or lactic acid bacteria and yeast are first treated with Ile-Pro-Pro
And / or culture in a medium containing Val-Pro-Pro as a component.

The lactic acid bacteria used in the culture include:
For example, Lactobacillus helveticus (Lactobacillus
helveticus) and Lactobacillus delbruek (Lactobacillus delbruek)
bulgaricus) and other lactic acid bacteria belonging to the genus Lactobacillus; Lactococcus lactis (Lactococcus lact).
lactic acid bacteria belonging to the genus Lactococcus such as Streptococcus thermophilus
lactic acid bacteria belonging to the genus Streptococcus such as s); lactic acid bacteria belonging to the genus Leuconostoc such as Leuconostoc lactis; Bifidobacterium longum, Bifidobacterium breve Lactic acid bacteria belonging to the genus Bifidobacterium or a mixture thereof can be preferably mentioned. When used, they can be used as a mixture with yeast or the like. At this time, as the yeast, for example, Saccharomyces cerevisiae (Saccharom
yeasts belonging to the genus Saccharomyces such as yces cerevisiae; yeasts belonging to the genus Candida such as Candida utilis; Kluyveromyces marxianus var.
lactis) and yeasts belonging to the genus Kluyveromyces, or mixtures thereof. The yeast inoculation ratio when using the yeast is preferably 1 to 10 yeasts for about 1000 lactic acid bacteria.

[0011] The lactic acid bacteria or the culture medium to the Ile-Pro-Pro and / or Val-Pro-Pro constituents for culturing lactic acid bacteria and yeast, I le-Pro-Pro and / or Val-Pro- Pro
A food material having a peptide and / or protein containing as a constituent component, specifically, whole milk of animal milk, skim milk, milk protein such as casein ; corn, corn protein, wheat, wheat protein, soybean, defatted soybean, soybean Food materials such as proteins, for example, Briggs River Broth (Brig
gs Liver Broth), MRS broth, and other known lactic acid bacteria fermentation media.

The lactic acid bacterium or lactic acid bacterium and yeast are
Conditions for culturing in a medium containing Pro-Pro and / or Val-Pro-Pro as a constituent component vary depending on lactic acid bacteria, the type of yeast or a combination thereof, but preferably the Ile-Pr
o-Pro and / or a food material having a peptide and / or protein containing Val-Pro-Pro as a component is added to a medium as a solution with, for example, water and sterilized, and then lactic acid bacteria or lactic acid bacteria and yeast are added. And culture. At this time, the culture temperature is preferably 25 to 45 ° C., and the culture time is preferably 3 to 72 hours. In order to make lactic acid bacteria act effectively, the pH at the start of the culture is preferably 6 to 72 hours.
It is desirable to adjust to ~ 7. The end point of the culture can be determined, for example, when the number of lactic acid bacteria becomes 10 ⁷ / ml or PH 5.5 or less. The lactic acid bacteria or the inoculated amount of the lactic acid bacteria and yeast when performing the culture is the same as the above-described Il.
For a medium containing e-Pro-Pro and / or Val-Pro-Pro as a component, the number of lactic acid bacteria is about 5 × 10 ^{5 to} 5 × 10 ⁷ cells / ml.
It is preferred that Further, the culturing may be performed in a plurality of times. During the culture, the pH of the medium can be adjusted to 5 to 7 by adding an alkaline agent such as sodium hydroxide.

[0013] In the culture thus cultured,
Most of the peptides and / or proteins containing Ile-Pro-Pro and / or Val-Pro-Pro as components are present after being decomposed into tripeptides that are Ile-Pro-Pro and / or Val-Pro-Pro. However, an oligopeptide of up to about 10 residues including Ile-Pro-Pro and / or Val-Pro-Pro may be present.

Next, in the production method of the present invention, the desired ACE inhibitory peptide can be obtained by purifying the above culture as a culture solution. At this time, the supernatant obtained by centrifuging the culture solution can be subjected to the next purification treatment. Centrifugation of the culture solution is preferably performed by a high-speed centrifuge or the like, preferably at a rotation number of 5,000 to 200.
It can be obtained by centrifugation at 00 rpm for 1 to 10 minutes.

[0015] The above-mentioned purification treatment is carried out by
The E-inhibitory peptide is a treatment to reduce the lactic acidity to such an extent that it can be directly supplied to foods and drinks, etc., wherein the lactic acidity is 1.5 or less, particularly 1.2 to 0 after purification. It is preferable to lower the temperature to about 1. Examples of such purification treatment include, for example, electrodialysis treatment, ion exchange resin treatment, hollow fiber membrane dialysis treatment, reverse osmotic pressure treatment, or treatment with weak hydrophobic chromatography, and then treatment with strong hydrophobic chromatography. A treatment performed by eluting the adsorbed fraction with a polar solvent can be preferably mentioned,
These processes can be arbitrarily selected and combined.

The electrodialysis treatment is performed using, for example, a known electrodialysis apparatus having an anion exchange membrane and a cation exchange membrane or an anion exchange membrane in which different types of membranes are combined in an electrodialysis tank. Preferably, the applied voltage per total effective membrane area between the cathode and the anode in the electrodialysis cell is 1 to 10 V / m ² , and the current density (A / cm ² )
It is preferable that the ratio between the specific conductivity (S / cm) and the culture solution be adjusted to about 3. Further, the temperature of the processing solution is 4 to 70.
C., and the treatment time is preferably 10 to 60 minutes.

The ion exchange resin treatment can be carried out, for example, by adding a known ion exchange resin such as a commercially available product to the treatment liquid and stirring for 0.5 to 5 hours. At this time, the temperature of the treatment liquid is preferably 4 to 95 ° C.

The hollow fiber membrane dialysis treatment is performed, for example, using a known hollow fiber type hollow fiber made of a synthetic polymer resin such as a commercially available cellulose acetate, polyethylene alcohol, polysulfone, polyacrylonitrile, or polyimide. It can be performed using a thread membrane dialysis device or the like. At this time, the processing temperature is preferably 4 to 9
The treatment is preferably performed at 5 ° C. for a treatment time of 30 to 600 minutes, and the culture solution to be treated may be either the internal solution side or the external solution side, but is preferably circulated to the external solution side.

In the reverse osmotic pressure treatment, for example, a synthetic polymer membrane of cellulose acetate, polyimide, polyvinyl alcohol, polybenzimidazoline or the like is used as a membrane material, and a known reverse osmosis apparatus is used. Can do it. In this case, the liquid temperature is preferably 4 to 80 ° C., and the processing liquid flow rate is 0.5 to 2.0 m ³ / h. , Operating pressure 5-50k
It is preferably adjusted to g / cm ² .

After the treatment with the weak hydrophobic chromatography and then the strong hydrophobic chromatography,
In the treatment performed by eluting the adsorbed fraction with a polar solvent (hereinafter referred to as treatment step A), the treatment liquid is first treated by weakly hydrophobic chromatography, for example, by a CN (propionitrile) group, a carbon number Silica-based resin having 1 to 3 alkyl groups bonded, more specifically, trade name "Amberlite
XAD-7 "(manufactured by Organo Co., Ltd.) or other resin is treated by a column method or a batch method to adsorb and remove highly hydrophobic peptides and to remove the flow-through fraction containing weakly hydrophobic peptides. Can be recovered. Next, for treatment by strong hydrophobic chromatography, for example, a silica-based resin having a phenyl group and an alkyl group having 8 to 18 carbon atoms bonded thereto, more specifically, a trade name “Amberlite XAD-2”
By treating with a column method or a batch method using a resin such as (manufactured by Organo Corporation), a weakly hydrophobic peptide fraction containing an ACE inhibitory peptide can be adsorbed.

Next, the above-mentioned adsorbed fraction is, for example, methanol,
Polar solvents such as ethanol, 1-propanol, 2-propanol and acetonitrile, particularly preferably 50% by volume
The desired ACE inhibitory peptide can be obtained by elution with a methanol or 50% by volume ethanol solution or the like.

The ACE inhibitory peptide obtained by the production method of the present invention contains Ile-Pro-Pro or Val-Pro-Pro, and the peptide usually contains hydrochloride, sulfate, succinate, citrate And a peptide to which a salt acceptable in the pharmaceutical industry such as tartrate is added.

The ACE inhibitory peptide obtained by the production method of the present invention is usually obtained as a mixture of the above peptides,
Contains other components, but when added to food and drink, etc.
The treatment liquid after the purification treatment can be used as it is. When used as an ACE inhibitor, the peptide mixture can be used as an active ingredient as it is, or the purity can be improved by fractionating and purifying the active peptide by HPLC or the like.

In order to use the ACE inhibitory peptide obtained by the production method of the present invention as an ACE inhibitor, it can be used, for example, by adding it to other pharmaceutically acceptable additives or foods and drinks. Can be taken as a solid, liquid or the like by oral administration or the like. The dosage varies depending on the purpose of prevention or treatment, and also varies depending on the course of progression of the symptoms. However, when administered for treatment or prevention in adults, the dose per day is calculated in terms of the amount of the ACE inhibitory peptide. , Preferably in the range of 0.1 mg to 100 mg, preferably 1 to 30 mg.

[0025]

Industrial Applicability According to the method for producing an ACE inhibitory peptide of the present invention, a specific ACE inhibitory peptide exhibiting excellent ACE inhibitory activity can be easily and inexpensively produced by a small amount of oral administration. Extremely effective.

[0026]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

[0027]

Example 1 Preparation of Fermented Milk a 18 g of skim milk powder was dissolved in 200 g of water,
After sterilizing for 1 minute, cool to room temperature and cool to Lactobacillus helveticus JCM-1003 (Lactobacillus helv-eticus J
CM-1003) 2.0 × 10 ⁷ / ml, Saccharomyces
Saccharomyces cerevisiae ATCC-9804
-9804) was inoculated at 2.0 × 10 ⁵ / ml and cultured at 37 ° C. for 24 hours to obtain a primary starter (5 × 10 5 lactic acid bacteria).
⁸ / ml, yeast number 5 × 10 ⁶ / ml). Next, 360 g of skim milk powder was dissolved in 4 kg of water, sterilized at 90 ° C., cooled to room temperature, and inoculated with the primary starter. Further, the cells were cultured at 37 ° C. for 24 hours and used as a secondary starter. Next, 9.0 kg of skim milk powder was dissolved in 100 kg of water, sterilized at 90 ° C., cooled to room temperature, inoculated with the secondary starter, cultured at 37 ° C. for 24 hours, and fermented milk a was about 112 kg. I got

Determination of peptide b contained in fermented milk a 5 ml of the obtained fermented milk a was subjected to 10,000 rpm,
After centrifugation for 10 minutes, the obtained supernatant is referred to as `` Amberlite XAD-
7 "(manufactured by Organo Corporation).
The column was washed with 5 ml of distilled water, combined with the non-adsorbed fraction, and then applied to a column to which two trade names “Sep-pakC18” (manufactured by Waters) were connected, and washed with 5 ml of distilled water. Thereafter, the adsorbed fraction was eluted with 5 ml of methanol, centrifuged under reduced pressure, and lyophilized. The obtained dried product was dissolved in a 0.05 M ammonium acetate buffer (pH 6.89), and then separated by HPLC using a column (trade name “GS320HQ”, manufactured by Asahi Kasei Corporation) equilibrated with the buffer. . This separation is performed at a flow rate of 0.6 ml / min, and an elution time of 14.5.
A peak of 15.5 minutes was collected and subjected to quantitative analysis under the following conditions. The result is shown in FIG. As a standard sample, a fixed amount of chemically synthesized Ile-Pro-Pro and Val
-Pro-Pro was subjected to HPLC under the same conditions to determine the area,
The amount of peptide was calculated from the area ratio with the sample from fermented milk a. Also, from the results of FIG. 1, in the fermented milk a112 kg, 3060 mg of Val-Pro-Pro and 196 mg of Ile-Pro-Pro
It was found that 0 mg was contained.

HPLC conditions Column: Water Bonds, Micro Bonder Sphere 5 μC18 Eluent: A solution: 0.1 wt% TFA aqueous solution B solution: 0.1 wt% TFA in acetonitrile solution Gradient elution (B / (A + B) ) × 100 (%) 0% → 40% (60 min) Flow rate: 1 ml / min, Detection: Preparation of ultraviolet absorbing peptide b at 215 nm Next, in order to purify the peptide contained in the obtained fermented milk a, Was carried out.

The obtained fermented milk a 60 liters was
After centrifugation at 00 rpm, ammonium sulfate was added to the supernatant to adjust the pH to 3.5, and about 2 liters of "Amb-erlite XAD-7" (manufactured by Organo) resin which had been adjusted to pH 3.5 in advance was added thereto. After stirring for one minute, the filtrate is collected with a bleaching cloth, and about 2 liters of "Amberlite XAD-2" (manufactured by Organo) resin, which has been adjusted to pH 3.5 in advance, is added to the filtrate, and stirred at room temperature for about 30 minutes. Then, the resin was recovered with a bleaching cloth. After washing the recovered resin with 5 liters of PB,
The active fraction was collected with 5 liters of 100% by volume methanol. The amount of the recovered peptide was 7 g, the activity recovery rate was 72%, and the purity of the recovered peptide fraction was about 26% by HPLC analysis.

[0031]

Example 2 Preparation of Highly Purified Preparation of Peptide b Preparation of Peptide c 7 g of the recovered peptide fraction obtained in the preparation of the peptide b was adjusted to pH 3.5 by further adding an equivalent amount of PB, and then 10 volumes were prepared in advance. "SP-Sephadex column" equilibrated with PB containing 5% methanol (Pharmacia)
The solution was passed through 500 ml (8 cmφ × 10 cm). After washing with 5 liters of PB containing 10% by volume of methanol, 500
An active fraction was obtained with 2 liters of PB containing mM NaCl. The obtained active fraction was subjected to quantification and purity analysis under the same conditions as in the HPLC, and further, ACE inhibitory activity described later was measured. The amount of the recovered peptide was 3.5 g, and Ile-Pro-
Pro and Val-Pro-Pro were contained at 576 mg and 1153 mg, respectively, and the purity was 49%. Active fraction 2 obtained
The liter was concentrated under reduced pressure to about 200 ml using an evaporator, and then freeze-dried under vacuum to obtain 65.0 g of a dry powder.
(Containing 61.5 g of salt and salts). The results of Examples 1 and 2 are shown in Table 1 and FIG. Next, the peptide obtained by HPLC was analyzed according to the following method.

Amino acid composition analysis 5 μg of each of the obtained peptides was dissolved in 6N hydrochloric acid having a constant boiling point and hydrolyzed under vacuum at 110 ° C. for 24 hours. The product name was “Hitachi L-8500 amino acid analyzer” (Hitachi, Ltd.) The composition was analyzed by ninhydrin coloring method according to the formula (Ile: Pro) (molar ratio) of 1.00: 2.0.
8, Val: Pro (molar ratio) was 1.00: 2.02.

Amino acid sequence analysis Trade name “Shimadzu Protein Sequencer PSQ-1”
Type "(manufactured by Shimadzu Corporation) as a result of N-terminal amino acid sequence analysis.
Or Val-Pro-Pro.

Measurement of ACE Inhibitory Activity The method of Cushman and Cheung [DW Cushman and HSCheu
ng, Biochem. Pharma-col., 20 1637 (1971)].

That is, the prepared peptide b was 0.1 M
Borate buffer (containing 0.3 M NaCl, pH 8.
Concentrations of 12.5, 25.0, 50.0, 10 using 3)
After adjusting to 0 and 200 μg / ml, each was put into a test tube (0.08 ml), and 5 m was added thereto with 0.1 M borate buffer (containing 0.3 M NaCl, pH 8.3) as a substrate.
M adjusted to Hypr-histidylleucine (Hip-His-Le
u, Sigma) and 0.22 ml of an aqueous enzyme solution (0.1 u / ml, Sigma) were further added and reacted at 37 ° C. for 30 minutes. Thereafter, 0.25 ml of 1N hydrochloric acid was added to stop the reaction, and then 1.7 m
l of ethyl acetate was added and stirred for 20 seconds. Then 300
After centrifugation at 0 rpm for 10 minutes to collect 1.4 ml of the ethyl acetate layer, the mixture was heated at 120 ° C. for 40 minutes to remove the solvent. After removing the solvent, 1 ml of distilled water was added, and the absorption 228 nm value of the extracted hypoprilic acid was measured, and this was defined as the ACE inhibitory activity. The inhibition rate was calculated from the following equation. As a result, when the inhibitor concentration IC ₅₀ at an inhibition rate of 50% was determined, the obtained peptide b was 20 μM. HPLC
7 μM for Ile-Pro-Pro further fractionated by
-Pro-Pro was 14 μM. The titer of the inhibitor giving the IC ₅₀ value is defined as 1 unit (U).

Inhibition rate = [(AB) / (AC)] × 100 (%) A: 228 when no sample (peptide b or c) is contained
Absorbance at nm B: 228 when sample (peptide b or c) is added
Absorbance at nm C: Absorbance at 228 nm without addition of enzyme and sample (peptide b or c) Measurement of antihypertensive effect upon oral administration to rats The following method for fermented milk a, peptide b and peptide c obtained below The hypotensive effect was measured according to

26-week-old male spontaneously hypertensive (SHR)
Rats (Charles River Japan, 4 animals per group)
In the animal room at 3 ± 2 ° C. and 55 ± 5% humidity, water and feed (manufactured by CLEA Japan, trade name “CE-2”) were used as test animals, which were acclimated and fed freely. SHR, which had been fasted overnight from the day before the test, was added to saline (2 ml /
(Rat), fermented milk a (2 ml / rat), and peptide b (150 μg / rat) and peptide c (80 μg / rat) dissolved in 2 ml of physiological saline were orally administered by gastric tube. The systolic blood pressure was measured immediately before administration, 4 hours after administration, and 8 hours after administration, respectively. To measure blood pressure,
Non-warmed, non-invasive rat sphygmomanometer (CSI,
It was measured by the tail-cuff method using a trade name “PE-300 type”). Table 2 shows the results of the difference (decrease in blood pressure) between the systolic blood pressure immediately before administration and the systolic blood pressure after 4 hours and 8 hours.

[0038]

[Table 1]

[0039]

[Table 2]

[0040]

Example 3 1.2 liters of the fermented milk a prepared in Example 1 was centrifuged at 10,000 rpm for 5 minutes, and 1 liter of the obtained supernatant was electrodialyzed (trade name "TS-1", Tokuyama Soda Co., Ltd.). (Manufactured by a company). The electrodialysis by the electrodialysis apparatus is performed using an anion membrane (trade name “A
FN-7 ", one sheet effective area = ² dm ² , manufactured by Tokuyama Soda Co., Ltd. and a cationic membrane (trade name“ CM-1 ”, one sheet effective area = ² dm ² , manufactured by Tokuyama Soda Co., Ltd.) 10 Using a pair of ion exchange membranes, deoxidation treatment was performed under the operating conditions of an initial voltage of 19.0 V, a current value of 1.60 A, and a liquid temperature of 15 to 30 ° C., to obtain 980 ml of a treatment liquid. Table 3 shows the results obtained by measuring the lactic acidity, the ACE inhibitory activity, and the like of the treated liquid every 5 minutes and comparing it with the fermented milk a before the treatment. Further, according to the quantification method shown in Example 1, Val-Pr
It was found that 24.1 mg of o-Pro and 16.0 mg of Ile-Pro-Pro were contained.

[0041]

[Table 3]

[0042]

Example 4 18 liters of the fermented milk a prepared in Example 1 was homogenized (trade name "15M-8TA type", AP
V. Gorin Co., Ltd.) at a processing pressure of 160 to 2
Homogenized at 00 Kg / cm ² . Next, 100 ml of the homogenized fermented milk was regenerated with a sodium hydroxide solution according to a conventional method and washed with water, and then washed with water.
RA-93ZU "(manufactured by Rohm and Haas Co., Ltd.) and stirred for about 3 hours, and then the resin was separated to obtain 90 ml of fermented milk. Lactic acidity and ACE inhibitory activity of the fermented milk before and after the treatment were measured and compared. Table 4 shows the results. Further, according to the quantification method shown in Example 1, the treatment solution contained 1.5 mg of Val-Pro-Pro, and Ile-Pro-Pro
Was found to be contained in an amount of 1.0 mg.

[0043]

[Table 4]

[0044]

Example 5 500 m of fermented milk homogenized in Example 4
l is a hollow fiber type dialysis machine (trade name "KL-
2-30 ", an effective membrane area of 0.12 m ² , manufactured by Kuraray Co., Ltd.), and a dialysis treatment was carried out by a cross-flow filtration method. In the dialysis treatment, ion-exchanged water (10.5 liters / m ² · h) is fed into the module by a pump, and the fermented milk a (30 liters / m ² · h) is pumped outside the module.
m ² · h) was circulated, and the dialysis treatment was performed for 4 hours by performing external pressure filtration of the fermented milk a. In addition, samples were taken over time, and the lactate acidity, ACE inhibitory activity, etc. were measured and compared with those before treatment. Table 5 shows the results. Furthermore, Val-Pro-Pro was added to the treatment solution for 4 hours by the quantification method described in Example 1.
It was found that 14 mg and 4.10 mg of Ile-Pro-Pro were contained.

[0045]

[Table 5]

[0046]

Example 6 Eighteen liters of the fermented milk homogenized in Example 4 was subjected to electrodialysis using an electrodialysis apparatus (trade name "TS-1", manufactured by Tokuyama Soda Co., Ltd.). Electrodialysis using an electrodialysis device is performed using an anion membrane (trade name “AFN-
7 ", an ion consisting of 10 pairs of one membrane effective area = ² dm ² , manufactured by Tokuyama Soda Co., Ltd. and an anion membrane (trade name" ACS ", one membrane effective area = ² dm ² , manufactured by Tokuyama Soda Co., Ltd.) Using an exchange membrane, an initial voltage of 19.0 V and a current value of 1.
The treatment was performed for 30 minutes under the operating conditions of 76 A and a liquid temperature of 20 to 35 ° C. to obtain 1.0 liter of a treatment liquid. Lactic acidity, ACE inhibitory activity, etc. were measured for the treated liquid every 5 minutes and compared with the fermented milk a before the treatment. Table 6 shows the results. Further, according to the quantification method shown in Example 1, Val-Pro-
It was found that 24.7 mg of Pro and 16.5 mg of Ile-Pro-Pro were contained.

[0047]

[Table 6]

[Brief description of the drawings]

FIG. 1 shows Val- of peptide b prepared in Example 1.
It is a graph which shows each area peak of Pro-Pro and Ile-Pro-Pro.

FIG. 2 shows Val- of peptide c prepared in Example 2.
It is a graph which shows each area peak of Pro-Pro and Ile-Pro-Pro.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1: 865) (72) Inventor Toshiaki Takano 2-4-1 Ebisu Minami, Shibuya-ku, Tokyo Calpis Food Industry Co., Ltd. (56) References JP-A-3-123469 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12P 21/00-21/06 C12P 39/00 BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A method for producing an angiotensin-converting enzyme-inhibiting peptide comprising Ile-Pro-Pro and / or Val-Pro-Pro, comprising: lactic acid bacterium or lactic acid bacterium and yeast ;
Koshi, corn protein, wheat, wheat protein, soybean, dehull
Group consisting of fatty soybeans, soy protein and mixtures thereof
Ri is selected, Ile-Pro-Pro and / or cultured in a medium containing a peptide and / or protein as a constituent component of Val-Pro-Pro, angiotensin converting enzyme inhibitory peptide which is characterized in that purification Manufacturing method. 2. The method for producing an angiotensin-converting enzyme inhibiting peptide according to claim 1, wherein after the culturing, the obtained culture solution is centrifuged, and the supernatant after the centrifugation is purified. 3. The purification treatment is performed by electrodialysis treatment, ion exchange resin treatment, hollow fiber membrane dialysis treatment, reverse osmotic pressure treatment, weak hydrophobic chromatography, and then strong hydrophobic chromatography. And performing the treatment by one or more treatments selected from the group consisting of treatments performed by eluting the adsorbed fraction with a polar solvent.
A method for producing the angiotensin converting enzyme inhibitory peptide according to the above.