Method for producing lactoferrin hydrolysate

Abstract

To efficiently produce a lactoferrin hydrolysate having high content of a peptide composed of Leu-Arg-Ile-Pro-Ser-Lys and exhibiting angiotensin-converting enzyme inhibitory activity.SOLUTION: A method for producing a lactoferrin hydrolysate containing a peptide composed of Leu-Arg-Ile-Pro-Ser-Lys, is characterized by using thermolysin and lysyl endopeptidase for lactoferrin as the raw material.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present technology relates to a method for producing a lactoferrin hydrolysate containing a peptide consisting of Leu-Arg-Ile-Pro-Ser-Lys. [Background technology]

[0002] Limited hydrolysis (fragmentation) using protease enzymes or acids has been used as a means of developing new or improving functional properties of food proteins. It has been shown that peptides obtained in this way are superior in terms of digestion and absorption to free amino acid mixtures in terms of absorption rate and post-absorption amino acid balance (Non-Patent Document 1). However, it has been pointed out that limited hydrolysis (fragmentation) produces bitter peptides with a bitter or acrid taste, resulting in materials with poor taste and poor palatability. Decomposition products with extremely low amino acid liberation rates often have a particularly bitter taste, which can be a hindrance when ingested. This fact has also limited their use. On the other hand, the number of patients with allergies caused by dietary proteins has rapidly increased, and it has become clear that allergies caused by, for example, whey proteins, especially β-lactoglobulin, are occurring frequently, especially among infants (Non-Patent Document 1). This has led to a demand for reducing antigenic substances in foods and substantially eliminating the antigenicity of proteins and peptides. Thus, there is a strong demand for the development of peptide materials that are useful in food processing and that suppress unpleasant tastes such as bitterness and antigenicity.

[0003] Lactoferrin is a non-heme iron-binding glycoprotein found in mammalian milk, saliva, tears, semen, various mucus, etc. It is a multifunctional protein with iron-binding, cell growth-promoting, immunomodulatory, and antibacterial properties. Bovine lactoferrin can be easily obtained in large quantities from raw skim milk or cheese whey, which are routinely handled in dairy factories, and can be used immediately as a commercial product.

[0004] Lactoferrin hydrolysates obtained by hydrolyzing lactoferrin and lactoferrin peptides having specific amino acid sequences in lactoferrin, such as lactoferricin (a registered trademark of the present applicant), are known to have many physiological activities, such as antibacterial activity (Patent Document 1, Non-Patent Document 2), tyrosinase activity (Patent Document 2), apoptosis inducer (Patent Document 3), oral cancer metastasis inhibitor (Patent Document 4), and liver function improver (Patent Document 5).

[0005] Since lactoferrin hydrolysates and lactoferrin peptides are naturally occurring peptides that do not contain chemicals or chemically synthesized amino acid derivatives, they are healthy and safe for humans and animals. Therefore, lactoferrin hydrolysates and lactoferrin peptides are extremely valuable and can be used as safe and effective medicines, foods, feeds, and a wide variety of products, such as eye drops, oral preparations, cosmetics, skin preparations, infant foods, clinical foods, functional foods, and pet products. On the other hand, although various angiotensin converting enzyme inhibitory peptides are known, the angiotensin converting enzyme inhibitory activity of these peptides is still insufficient for their function in foods. There is a need for more efficient isolation of peptides derived from natural products that have higher angiotensin-converting enzyme inhibitory activity and simple structures, and for their application in foods, medicines, etc. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 9-124504 [Patent Document 2] Japanese Patent Application Publication No. 5-320068 [Patent Document 3] Japanese Patent Application Publication No. 10-45618 [Patent Document 4] Japanese Patent Application Publication No. 10-59864 [Patent Document 5] International Publication No. 00 / 06192 Brochure [Non-patent literature]

[0007] [Non-Patent Document 1] Dairy Science and Food Research, Vol. 39, No. A-283, 1990 [Non-patent document 2] Antimicrobial Agents and Chemotherapy, Vol. 41, No. 1, 1997, pp. 54-59 Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present invention is to more efficiently produce a lactoferrin hydrolysate that contains a high content of the peptide consisting of Leu-Arg-Ile-Pro-Ser-Lys and has angiotensin-converting enzyme inhibitory activity. [Means for solving the problem]

[0009] The present invention is a method for producing a lactoferrin hydrolysate containing a peptide consisting of Leu-Arg-Ile-Pro-Ser-Lys, characterized in that lactoferrin is subjected to enzymatic treatment using thermolysin and lysyl endopeptidase.

[0010] That is, the present technology provides the following: [1] A method for producing a lactoferrin hydrolysate containing a peptide consisting of Leu-Arg-Ile-Pro-Ser-Lys, the method comprising enzymatically treating lactoferrin to produce a lactoferrin hydrolysate, the enzymatic treatment using thermolysin and lysyl endopeptidase as hydrolases, and reacting the thermolysin at a concentration of 0.1 to 5% by mass and the lysyl endopeptidase at a concentration of 0.1 to 5% by mass. [2] The manufacturing method described in [1], wherein the content of the peptide consisting of Leu-Arg-Ile-Pro-Ser-Lys per 1 g of the lactoferrin hydrolysate is 1 mg or more. [3] The method for producing a lactoferrin hydrolysate according to [1] or [2], wherein the enzyme treatment is carried out so that the decomposition rate of the lactoferrin hydrolysate is 40% or less. [4] The method for producing a lactoferrin hydrolysate according to any one of [1] to [3], wherein the peptide has angiotensin-converting enzyme inhibitory activity. [Effects of the Invention]

[0011] According to this technology, it is possible to efficiently produce a lactoferrin hydrolysate that is rich in the peptide consisting of Leu-Arg-Ile-Pro-Ser-Lys and has angiotensin-converting enzyme inhibitory activity. The effects described here are not necessarily limited to those described herein, and may be any of the effects described in the present technology. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of MS / MS analysis of the compound obtained in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0013] The manufacturing method of this technology is characterized by using thermolysin and lysyl endopeptidase on the raw material lactoferrin, and is a method for producing a lactoferrin hydrolysate containing a peptide consisting of Leu-Arg-Ile-Pro-Ser-Lys. The peptide consisting of Leu-Arg-Ile-Pro-Ser-Lys has an amino acid sequence (SEQ ID NO: 1) consisting only of Leu-Arg-Ile-Pro-Ser-Lys, and is a peptide excised from lactoferrin.

[0014] Lactoferrin is an iron-binding glycoprotein found in the milk, tears, saliva, blood, etc. of mammals, such as sheep, goats, pigs, mice, buffalo, camels, yaks, horses, donkeys, llamas, cows, and humans. The lactoferrin used in the present invention may be derived from any mammal and is not particularly limited, but in terms of content and availability, lactoferrin derived from milk of, for example, cows, humans, etc. The milk may be any of colostrum, transitional milk, normal milk, and terminal milk. The lactoferrin used in the present invention may be lactoferrin isolated by standard methods (e.g., ion chromatography) from processed milk products such as skim milk and whey; recombinant lactoferrin produced by genetic engineering from microorganisms, animal cells, transgenic animals, or the like; synthetic lactoferrin; or a mixture thereof. Lactoferrin may be either non-glycosylated or glycosylated. Commercially available lactoferrin produced on an industrial scale (e.g., manufactured by Morinaga Milk Industry Co., Ltd.) can be used as such lactoferrin.

[0015] The hydrolase is not particularly limited as long as it can hydrolyze proteins to produce the peptides of the present invention, but when lactoferrin is used as the protein, commercially available enzymes containing thermolysin (Thermoase (Amano Enzyme)) are preferred. Other endopeptidases such as lysyl endopeptidase (Wako Pure Chemical Industries) and pepsin (Amano Enzyme) may be used in combination.

[0016] Thermolysin is a thermostable metalloproteinase produced by fermentation from a bacterial species called Bacillus thermoproteolyticus rokko. It cleaves at the N-terminus of the hydrophobic residues leucine, phenylalanine, valine, isoleucine, alanine, and methionine. Thermolysin can be isolated and / or purified. The CAS number for thermolysin is 9073-78-3. The thermolysin is preferably derived from Bacillus thermoproteolyticus, more preferably from Bacillus thermoproteolyticus rokko. The pH in the enzymatic treatment using thermolysin is preferably 5.5 to 9.5, more preferably 6.5 to 9.0. The temperature in the enzymatic treatment using thermolysin is preferably 10 to 85°C, more preferably 40 to 75°C. The thermolysin preferably specifically cleaves peptide bonds of hydrophobic amino acid residues, more preferably leucine peptide bonds.

[0017] Various proteases can be used in the present invention, but preferred are endopeptidases (bacterial proteases and animal pancreatic proteases), such as lysyl endopeptidase. Protein components in a sample can be fragmented by protease treatment. The action of the lysyl endopeptidase is preferably to specifically cleave peptide bonds of non-terminal lysines. Bacterial protease is preferably added at a ratio of 100 to 5,000 activity units per gram of protein, and animal pancreatic protease is preferably added at a ratio of 3,000 to 8,000 activity units per gram of protein. The lysyl endopeptidase is preferably derived from Achromobacter lyticus. The pH in the enzymatic treatment using the lysyl endopeptidase is preferably 6.5 to 10.0, more preferably 8.0 to 9.5. The temperature in the enzymatic treatment using the lysyl endopeptidase is preferably 10 to 70°C, more preferably 30 to 50°C.

[0018] <Method of producing lactoferrin hydrolysate> The method for producing lactoferrin hydrolysate according to the present technology is not particularly limited in terms of the detailed steps, as long as it uses thermolysin and lysyl endopeptidase on the raw material lactoferrin to obtain a lactoferrin hydrolysate containing a peptide consisting of Leu-Arg-Ile-Pro-Ser-Lys (LRIPSK (SEQ ID NO: 1)). The production method according to the present technology can be freely selected and adopted from various steps used in known methods for producing lactoferrin hydrolysates. An example of the manufacturing method will be specifically described below.

[0019] <Preparation of raw material solution> A lactoferrin-containing raw material is dissolved or dispersed in a solvent such as water to prepare a lactoferrin-containing solution. The lactoferrin used as a raw material is as described above. The lactoferrin is used as a substrate for thermolysin and lysyl endopeptidase. Examples of ingredients other than lactoferrin include pH adjusters, surfactants, etc., and one or more of these may be selected. The solvent is not particularly limited, but it is preferable to use distilled water as the water. The lactoferrin concentration in the lactoferrin-containing solution is not particularly limited, but is preferably in the range of 0.5 to 30% by mass, more preferably 1 to 25% by mass, and even more preferably 2 to 15% by mass, calculated as protein. This concentration range can improve efficiency and operability.

[0020] The lactoferrin-containing solution is preferably sterilized by heating at 70 to 90°C for about 15 seconds to 10 minutes to prevent deterioration due to bacterial contamination.

[0021] Next, it is preferable to add an alkaline or acidic agent to the lactoferrin-containing solution to adjust the pH to or near the optimum pH for the hydrolase used. The pH of the lactoferrin-containing solution is preferably 6.5 to 10.0, more preferably 7.0 to 9.5, and even more preferably 7.5 to 9.0. The alkaline or acidic agent used in the method of the present invention may be any alkaline or acidic agent that is acceptable for food or pharmaceutical use. Specific examples of acidic agents include hydrochloric acid, citric acid, phosphoric acid, acetic acid, etc., and examples of alkaline agents include sodium hydroxide, potassium hydroxide, potassium carbonate, etc. These can be used alone or in combination.

[0022] <Enzyme reaction> Next, a predetermined amount of hydrolase is added to the lactoferrin-containing solution, and the mixture is reacted at a temperature of about 10 to 85°C for 0.1 to 48 hours. The solution to which the enzyme has been added is reacted at an appropriate temperature depending on the type of enzyme to hydrolyze lactoferrin, thereby obtaining a lactoferrin hydrolysate. The hydrolysis reaction time is preferably 0.5 to 48 hours, more preferably 1 to 24 hours, and even more preferably 2 to 12 hours, and the reaction is continued until a desired amount of the peptide of the present invention is produced while monitoring the produced peptide by HPLC or the like.

[0023] The enzymatic reaction is preferably carried out so that the content of the peptide consisting of Leu-Arg-Ile-Pro-Ser-Lys per gram of lactoferrin hydrolysate is 1 mg or more. The peptide content in the lactoferrin hydrolysate can be determined by the following method. The hydrolysate is separated by reverse-phase HPLC under the following condition A. A TSQ (manufactured by Thermo Fisher Scientific) can be used as the mass spectrometer.

[0024] [LC-MS Condition A] Column: Inertsil WP300 C18 mm I.D. x 250 mm (GL Sciences, Inc.) Column temperature: 40℃ Measured mass (m / z): 713.4 (MH+) Flow rate: 0.2ml / min Eluent (A): Aqueous solution containing 0.1% TFA Eluent (B): Acetonitrile containing 0.1% TFA

[0025] Analysis is performed using a linear gradient of eluent (A / B) from 98 / 2 to 75 / 25 after 6 minutes and 20 / 80 after 12 minutes, and the peptide in question (eluted at 8.6 minutes) is calculated from the area value using a standard (chemically synthesized product).

[0026] It is preferable that the enzymatic treatment be carried out so that the decomposition rate of the lactoferrin hydrolysate is 40% or less. The decomposition rate of the lactoferrin hydrolysate can be measured by the following calculation method (formol nitrogen amount measurement method).

[0027] The total nitrogen content of the sample is measured by the Kjeldahl method (Food Analysis Methods, edited by the Japan Food Industry Association, p. 102, Korin Co., Ltd., 1984), and the formol nitrogen content of the sample is measured by the formol titration method (Food Engineering Experiment Book, edited by Mitsuda et al., Vol. 1, p. 547, Yokendo, 1970). From these measured values, the decomposition rate can be calculated using the following formula (1). Decomposition rate (%) = (formol nitrogen amount / total nitrogen amount) × 100 (1)

[0028] The enzymatic reaction can be stopped, for example, by inactivating the enzyme in the hydrolyzed solution, which can be carried out by a conventional method, such as heat inactivation. The conditions for the heat inactivation treatment (heating temperature, heating time, etc.) can be appropriately set to ensure sufficient inactivation, taking into account the thermal stability of the enzyme used. In the present technology, as an example, the treatment can be carried out at a temperature range of 80 to 130°C for a holding time of 30 minutes to 2 seconds.

[0029] The enzyme reaction product obtained by the above-mentioned enzyme reaction may be separated and purified by a conventional separation and purification method. For example, this can be achieved by separating the enzyme from the reactants by treatment with an ultrafiltration membrane or the like. When an enzyme designated as a food additive is used, there is no need to isolate and purify the peptide of the present invention from the hydrolyzed liquid, and it can be used as a food ingredient containing ingredients involved in specified health foods or functional ingredients involved in functional food claims. Impurities may be removed from the enzyme-treated product of the present technology, and a desired fraction or desired peptide may be recovered from the enzyme-treated product of the present technology.

[0030] Examples of separation and purification methods include precipitation, membrane separation, HPLC separation and purification, ion exchange chromatography, and crystallization. Examples of separation and purification methods include filtration, microfiltration, membrane separation treatments such as ultrafiltration, resin adsorption separation, column chromatography, HPLC separation and purification, precipitation, and crystallization, and one or a combination of two or more of these can be selected.

[0031] The membrane separation treatment can be carried out using known devices. Examples of such devices include microfiltration modules and ultrafiltration modules. Examples of ultrafiltration modules include those with a molecular weight cutoff of 3,000 to 20,000, preferably 3,000 to 10,000. These filtration modules are commercially available, for example, from Asahi Kasei Corporation under the names of the Microza MF series and Microza UF series. In this case, a solution containing lactoferrin degradation products is obtained as the membrane-permeable fraction after membrane separation treatment. By performing membrane separation treatment, insoluble matter generated during the hydrolysis reaction and / or thermal inactivation of the enzyme present in the hydrolysis-inactivated solution can be removed.

[0032] The resin adsorption separation method can be carried out by known methods, for example, by packing a column with a resin and passing the deactivated liquid through the column. Examples of resins include trade names such as Diaion (manufactured by Mitsubishi Chemical Corporation), Sepabeads (manufactured by Mitsubishi Chemical Corporation), Amberlite XAD (manufactured by Organo Corporation), and HS (manufactured by Ajinomoto Fine-Techno Co., Ltd.). Resin adsorption separation can be carried out continuously by packing these resins into a column and continuously injecting and eluting the deactivated liquid. Alternatively, it can be carried out batchwise by adding a resin to the hydrolysis-deactivated liquid, allowing it to come into contact with the hydrolysis-deactivated liquid for a certain period of time, and then separating the hydrolysis-deactivated liquid from the resin. Factors that cause turbidity, precipitation, aggregation, browning, etc. during storage (e.g., peptides containing many hydrophobic amino acids) may remain in the hydrolysis-deactivated liquid, and these factors can be removed by resin adsorption separation.

[0033] <Uses of lactoferrin hydrolysate and each separated and purified fraction> The peptide of the present invention has angiotensin converting enzyme inhibitory activity and can therefore be used as an active ingredient of angiotensin converting enzyme inhibitors. The peptide of the present invention has angiotensin converting enzyme inhibitory activity and bradykinin inactivation suppressive activity, and exhibits a blood pressure lowering effect. Therefore, it can be used as a preventive or therapeutic agent for various diseases caused by hypertension, such as cerebral hemorrhage, cerebral infarction, angina pectoris, myocardial infarction, renal failure, etc., specifically as a hypotensive agent.

[0034] Furthermore, angiotensin-converting enzyme inhibitors are known to be effective against essential hypertension of unknown cause, and the peptide of the present invention is also expected to exhibit therapeutic or preventive effects against essential hypertension. In addition, it can also be used as a therapeutic or preventive drug for diseases such as cardiac hypertrophy and angina pectoris, for which angiotensin converting enzyme inhibitors are known to be effective.

[0035] The angiotensin converting enzyme inhibitor of the present invention may be administered either orally or parenterally, but oral administration is preferred. Parenteral administration includes intravenous injection, rectal administration, inhalation, and the like. Dosage forms for oral administration include tablets, capsules, troches, syrups, granules, powders, ointments and the like.

[0036] In preparing the formulation, in addition to the whey protein hydrolysate, ingredients that are normally used in formulations, such as excipients, pH adjusters, colorants, and flavoring agents, can be used. Furthermore, known or future drugs having angiotensin converting enzyme inhibitory activity can also be used in combination.

[0037] Furthermore, the peptide of the present invention can be incorporated as an active ingredient into food products, and processed into foods having angiotensin converting enzyme inhibitory activity as one embodiment of angiotensin converting enzyme inhibitors. Such foods include tablets, liquid food, feed (including for pets), etc., regardless of whether they are in liquid, paste, solid, powder, etc., as well as wheat flour products such as bread, macaroni, spaghetti, noodles, cake mix, fried chicken flour, breadcrumbs, etc.; instant foods such as instant noodles, cup noodles, retort / prepared foods, canned foods, microwave foods, instant soup / stew, instant miso soup / cleaning liquid, canned soup, freeze-dried foods, and other instant foods; processed agricultural products such as canned agricultural products, canned fruit, jams / marmalades, pickles, boiled beans, dried agricultural products, and cereals (processed grain products); Canned seafood, fish ham and sausage, fish paste products, seafood delicacies, tsukudani (simmered fish dishes), etc.; canned livestock products, pastes, livestock ham and sausage, etc.; processed milk products, such as processed milk, milk drinks, yogurt, lactic acid bacteria drinks, cheese, ice cream, infant formula, cream, and other dairy products; butter, margarine, vegetable oil, and other fats and oils; basic seasonings, such as soy sauce, miso, sauces, tomato seasonings, mirin, and vinegar; complex seasonings and foods, such as cooking mixes, curry bases, sauces, dressings, noodle soups, spices, and other complex seasonings; frozen food ingredients frozen foods such as semi-cooked frozen foods and cooked frozen foods; sweets such as candy, caramel, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice snacks, bean snacks, dessert sweets and other sweets; beverages such as carbonated drinks, natural fruit juice, fruit juice drinks, soft drinks with fruit juice, fruit pulp drinks, fruit drinks with fruit pieces, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks and other beverages; other commercially available foods such as baby food, furikake rice seasoning and ochakukenori seaweed.

[0038] In the angiotensin converting enzyme inhibitor of the present invention, the amount of the peptide of the present invention incorporated is preferably at least 0.001% by weight based on the final composition of the angiotensin converting enzyme inhibitor. The dosage of the angiotensin-converting enzyme inhibitor of the present invention varies depending on age, symptoms, etc., but is usually 0.001 to 3000 mg / day, preferably 0.01 to 30 mg / day, and may be administered in one to three divided doses per day.

[0039] Therefore, the enzyme-treated lactoferrin product containing a high content of the peptide Leu-Arg-Ile-Pro-Ser-Lys (LRIPSK (SEQ ID NO: 1)) of the present technology and each separated and purified fraction containing the peptide Leu-Arg-Ile-Pro-Ser-Lys (LRIPSK (SEQ ID NO: 1)) can be used as a food composition for inhibiting angiotensin-converting enzyme, or can be incorporated into these food compositions for use. Furthermore, the enzyme-treated lactoferrin product containing a high content of the peptide Leu-Arg-Ile-Pro-Ser-Lys (LRIPSK (SEQ ID NO: 1)) of the present technology and each separated and purified fraction containing the peptide Leu-Arg-Ile-Pro-Ser-Lys (LRIPSK (SEQ ID NO: 1)) can be used for the prevention, amelioration, or treatment of various diseases (including those at risk) for which efficacy is expected, or can be used as an ingredient for the prevention, etc. of these various diseases, or can be used as an ingredient in medicines or foods for the prevention, etc. of these various diseases, or can be used to manufacture medicines, foods, etc. for these various diseases.

[0040] As such, this technology can be used in a wide range of fields, including pharmaceuticals, foods, food compositions, topical skin preparations, and functional foods. [Example]

[0041] Example 1: Production of peptides by enzymatic hydrolysis of lactoferrin <1> Enzymatic degradation of lactoferrin 250 mg of commercially available lactoferrin (Mirai Co., Ltd.) was added to 5 ml of water and thoroughly dispersed. Sodium hydroxide was added to adjust the pH of the solution to 7.6, completely dissolving the lactoferrin to prepare a lactoferrin aqueous solution with a concentration of approximately 5%. The lactoferrin aqueous solution was sterilized by heating at 80°C for 10 minutes, and the temperature was adjusted to 40°C. 1 AU (amidase unit) of lysyl endopeptidase (Wako Pure Chemical Industries, Ltd.) was added to initiate the hydrolysis reaction. After one hour, 500 activity units (2,500 activity units per gram of protein) of thermoase (Amano Enzyme Inc.) were added, and the temperature was adjusted to 70°C, after which the hydrolysis reaction was continued. Four hours after the addition of thermoase, the hydrolyzate was ultrafiltered using an ultrafiltration membrane (Amicon Co., Ltd.) with a molecular weight cutoff of 3,000. 1 ml of the resulting hydrolyzate filtrate was subjected to the following HPLC.

[0042] <2> Peptide separation by HPLC The casein hydrolysate was separated by reverse phase HPLC under the HPLC conditions shown in HPLC Condition 1 below. [HPLC conditions 1] Column: Cadenza CD-18 10 mm I.D. x 250 mm (Intact) Detection: UV 215nm Flow rate: 3ml / min Eluent A: 2% aqueous acetonitrile containing 0.1% TFA Eluent B: 25% aqueous acetonitrile containing 0.1% TFA

[0043] The hydrolysate was separated using a linear gradient starting from 100% eluent A and then 100% eluent B after 30 minutes. The angiotensin-converting enzyme inhibitory activity of the eluted fractions was measured using the method described below. A peptide with angiotensin-converting enzyme inhibitory activity was eluted at a retention time of 34 minutes. This peptide was further purified by HPLC. The conditions used are shown in HPLC Condition 2 below. [HPLC conditions 2] Column: Cadenza CD-18 10 mm I.D. x 250 mm (Intact) Detection: UV 215nm Flow rate: 3ml / min Eluent A: 2% acetonitrile in water containing 0.2% formic acid Eluent B: 25% aqueous acetonitrile containing 0.2% formic acid

[0044] Under linear gradient conditions, starting from 100% eluent A and then 100% eluent B after 30 minutes, a strong angiotensin-converting enzyme inhibitory activity was observed in the peak at a retention time of 24 minutes. The angiotensin I converting enzyme inhibitory activity of this peak was IC50 [sample concentration (μg / ml) required to inhibit angiotensin I converting enzyme activity by 50%] = 0.15μg / ml. The compound at the above active peak was analyzed by MS / MS using a Shimadzu protein sequencer (PPSQ-23A) and a Thermo Fisher Scientific TSQ mass spectrometer. The molecular weight (M) was 712.4 m / z = 713.4 (MH+) as the parent ion. Daughter ions at m / z = 585.28, 331.11, 270.10, and 253.05 were detected, as shown in Figure 1. These ions were found to be identical to the chemically synthesized peptide Leu-Arg-Ile-Pro-Ser-Lys (LRIPSK). The reaction filtrate (i.e., lactoferrin hydrolysate) contained 4 mg of the hexapeptide Leu-Arg-Ile-Pro-Ser-Lys (LRIPSK) per gram of lactoferrin hydrolysate.

[0045] (Example 2) Angiotensin-converting enzyme inhibitory effect of peptides The hexapeptide Leu-Arg-Ile-Pro-Ser-Lys (LRIPSK) obtained in Example 1 was analyzed according to the method of Kushman et al. [Biochemical Pharmacology, Vol. 20, pp. 1637-1648 (1971)], as described in Japanese Patent No. 5430803. The hexapeptide was dissolved in 0.1 M borate buffer (containing 0.3 M NaCl, pH 8.3) and 0.08 ml was added to a test tube. 0.2 ml of enzyme substrate (hippurylhistidylleucine, Sigma) adjusted to 5 mM with 0.1 M borate buffer (containing 0.3 M NaCl, pH 8.3) was added and incubated at 37°C for 3 minutes. Next, 0.02 ml of rabbit lung angiotensin-converting enzyme (Sigma) adjusted to 0.1 U / ml with distilled water was added, and the mixture was incubated at 37°C for 30 minutes.

[0046] The reaction was terminated by adding 0.25 ml of 1N hydrochloric acid, followed by adding 1.7 ml of ethyl acetate, stirring vigorously for 20 seconds, and centrifuging at 3,000 rpm for 10 minutes. 1.4 ml of the ethyl acetate layer was collected. The resulting ethyl acetate layer was heated to remove the solvent, and then 1.0 ml of distilled water was added. The absorbance of the extracted hippuric acid (absorbance at 228 nm) was measured and used as the enzyme activity.

[0047] The inhibitory activity was calculated using the following formula, and IC50 [sample concentration (μg / ml or μM) required to inhibit angiotensin-converting enzyme activity by 50%] was determined. The results are shown in Table 1. Inhibition rate = (AB) / (AC) × 100% A: Enzyme activity without sample (peptide) (absorbance at 228 nm) B: Enzyme activity when sample is added (absorbance at 228 nm) C: Enzyme activity without enzyme or sample (absorbance at 228 nm)

[0048] [Table 1]

Claims

1. A method for producing a lactoferrin hydrolysate containing a peptide consisting of Leu-Arg-Ile-Pro-Ser-Lys, the method comprising enzymatically treating lactoferrin to produce a lactoferrin hydrolysate, the enzymatic treatment using thermolysin and lysyl endopeptidase as hydrolases, and reacting the thermolysin at a concentration of 0.1 to 5% by mass and the lysyl endopeptidase at a concentration of 0.1 to 5% by mass.

2. 2. The method for producing lactoferrin hydrolysate according to claim 1, wherein the content of the peptide consisting of Leu-Arg-Ile-Pro-Ser-Lys per 1 g of the lactoferrin hydrolysate is 1 mg or more.

3. The method according to claim 1 or 2, wherein the enzyme treatment is carried out so that the decomposition rate of the lactoferrin hydrolysate is 40% or less.

4. The method for producing a lactoferrin hydrolysate according to claim 1 or 2, wherein the peptide has angiotensin converting enzyme inhibitory activity.

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