JP2009215301A - Protease inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cysteine protease inhibitor which excels in safety, can be produced in a large amount at a low cost, and has action to effectively inhibit the activity of cysteine protease. <P>SOLUTION: The cysteine protease inhibitor includes lactoperoxidase present in milk as an active ingredient and a food and drink composition or a feed composition includes the cysteine protease inhibitor. The cysteine protease inhibitor is effective for diseases caused by cysteine protease including osteoporosis, malignant tumor hypercalcemia, a periodontal disease, breast cancer, prostatic cancer, and a bacterium-virus infectious disease. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a cysteine protease inhibitor containing lactoperoxidase as an active ingredient, and prevention of osteoporosis, malignant neoplastic hypercalcemia, periodontal disease, breast cancer, prostate cancer, bacterial / viral infection, etc. -It is a cysteine protease inhibitor that can be used for therapeutic agents, foods and drinks, feeds, and the like.

  Proteases having a thiol group at the active center are collectively called cysteine protease (thiol protease). Cathepsin L, cathepsin B, and cathepsin K are one of typical cysteine proteases together with calcium-dependent neutral protease (CAMP), papain, ficin, bromelain and the like. Substances having an inhibitory action on these cysteine proteases are diseases associated with cysteine proteases such as muscular dystrophy, muscular atrophy, myocardial infarction, stroke, Alzheimer's disease, disturbance of consciousness and movement, head injury As a therapeutic agent for multiple sclerosis, neuropathy of peripheral nerve, cataract, inflammation, allergy, fulminant hepatitis, osteoporosis, hypercalcemia, breast cancer, prostate cancer, benign prostatic hyperplasia, etc. It is expected as a platelet aggregation inhibitor.

  In recent years, the relationship between cathepsin L, cathepsin B, cathepsin K and osteoporosis or malignant neoplastic hypercalcemia has been elucidated by research by Katsunuma et al. Application to medicine as a therapeutic agent for neoplastic hypercalcemia is drawing attention (see, for example, Non-Patent Document 1). In bone tissue, bone formation by osteoblasts (osteoblasts) and bone resorption by osteoclasts are carried out throughout life, and bone weight increases due to bone formation exceeding bone resorption during growth. On the other hand, on the other hand, since bone resorption exceeds bone formation in old age, bone weight is reduced and osteoporosis develops. There are various causes of such osteoporosis, and bone collapse (bone resorption) can be cited as one of the main causes. This is further divided into two causes as follows. That is, one is caused by calcium absorption and deposition failure, and more specifically, calcium supply, transfer, absorption, and deposition. Vitamin D derivatives, female hormones (estrogens), etc. It seems that they are involved.

  The other is to promote the degradation of collagen, which is a bone supporting tissue, and the degradation of bone collagen by cysteine protease groups secreted from lysosomes in osteoclasts, particularly cathepsin L, cathepsin B and cathepsin K. This is the main cause. These cathepsin L, cathepsin B and cathepsin K secreted from lysosomes in osteoclasts promote the degradation of collagen in bone tissue, thereby dissolving old bone and freeing calcium into the blood together with hydroxyproline. It is done. Therefore, by inhibiting the collagen resolution of cathepsin L, cathepsin B, and cathepsin K, it is possible to prevent excessive bone disintegration, and thus, osteoporosis can be treated. As therapeutic agents for these osteoporosis, estrogen, anabolic hormone, calcium agent, vitamin D, calcitonin, bisphosphonate and the like are known. In addition, as for osteoporosis therapeutic agents whose action mechanism is so-called cysteine protease inhibition of cathepsin L inhibition, cathepsin B inhibition, or cathepsin K inhibition, the development of osteoporosis therapeutic agents using several cysteine protease inhibitors is being promoted ( For example, see Patent Document 1 and Patent Document 2), further development of a therapeutic agent for osteoporosis is desired.

On the other hand, hypercalcemia is a metabolic abnormality in which the serum calcium concentration exceeds the normal value, and is often seen in tumor patients. If this is left unattended, it is said that the life of a patient is about 10 days. Most of the causes are tumor bone metastases. When the tumor metastasizes to bone, bone destruction occurs and calcium is released into the blood. This calcium is processed in the kidney, but when the speed of bone destruction exceeds the capacity of the kidney, hypercalcemia develops. As a treatment method, a method of promoting calcium excretion from the kidney by using an infusion solution of physiological saline combined with furosemide, a method of using calcitonin which is a therapeutic agent for osteoporosis, and the like are known. That is, it can be said that an osteoporosis therapeutic agent that suppresses bone resorption is also effective as a therapeutic agent for malignant hypercalcemia.

The present inventors have already disclosed the following cysteine protease inhibitors that can be used for such purposes.
(1) Cathepsin L-specific inhibitory polypeptide (Patent Document 1)
(2) Thiol protease inhibitor (Patent Document 3)
(3) Valine derivative and its use (Patent Document 4)
(4) Thiol protease inhibitor (Patent Document 5)
(5) FA-70C1 substance (Patent Document 6)
(6) FA-70D substance, its production method and its use (Patent Document 7)
However, further development of a cysteine protease inhibitor that can be used as a safe material without antigenicity has been desired.

  On the other hand, it has been known so far that protease inhibitors exist in breast milk. Known as protease inhibitors contained in breast milk include α1-antichymotrypsin, α1-antitrypsin, inter α2-trypsin inhibitor, α2-antiplasmin, α2-macroglobulin, antithrombin III, Inhibitors such as antileucoprotease are also contained in trace amounts in breast milk (see, for example, Non-Patent Document 2).

Regarding proteins having cysteine protease inhibitory activity (including cystatin) in milk, the following has already been disclosed.
(1) A novel cysteine protease inhibitor having a sugar chain derived from bovine colostrum and having a molecular weight of about 57 kDa (Patent Document 8)
(2) A novel cysteine protease inhibitor having a molecular weight of 16 ± 2 kDa or 13 ± 2 kDa derived from bovine colostrum (Patent Document 9)
(3) A novel protein having a molecular weight of 16 ± 2 kDa or 13 ± 2 kDa derived from human milk and a method for producing the same (Patent Document 10)
(4) Bone resorption inhibitor containing, as an active ingredient, milk-derived basic cystatin and / or milk-derived basic cystatin degradation product prepared from milk (Patent Document 19)
(5) Cystatin C contained in milk basic protein (MBP), and bone resorption inhibitory effect by the cystatin C in vitro (Non-patent Document 10)

Examples of proteins contained in a large amount in mammalian milk include lactoperoxidase, lactoferrin, and casein. Lactoperoxidase is an oxidoreductase contained not only in mammalian milk but also in secretions such as saliva, tears, and airway mucus (for example, Non-Patent Document 3), and industrially is produced on a large scale from milk. It refine | purifies (for example, patent document 11). Lactoperoxidase has been reported to have various biological functions such as antibacterial activity, antiviral activity, antioxidant activity, anticancer activity, and immunomodulatory activity (eg, Non-Patent Documents 4 to 9). In addition, the use of lactoperoxidase, peroxide donors and thiocyanates to produce a medicament for the treatment of Helicobacter pylori infection (for example, Patent Document 12), and pathogen infection prevention and treatment agents added to the mixed feed of cultured aquatic animals (for example, , Patent Document 13), anti-aging agent (for example, Patent Document 14), liver function improving agent (for example, Patent Document 15), peroxidase prevention and treatment application (for example, Patent Document 16), corneal disorder therapeutic agent, etc. A technique related to (for example, Patent Document 17) is disclosed. Furthermore, the present applicant has already disclosed a urease-inactivating composition (Patent Document 18) containing lactoperoxidase, thiocyanic acid, and hydrogen peroxide as active ingredients.

Japanese Patent Laid-Open No. 7-179596 Special table 2002-501502 gazette JP-A-9-212425 JP 2001-139534 A JP 7-242600 A JP 2000-72797 A International Publication No. 97/31122 Pamphlet JP-A-7-2896 Japanese Patent Laid-Open No. 7-126294 Japanese Patent Laid-Open No. 10-80281 Japanese Patent Laid-Open No. 5-41981 JP 2000-509367 Japanese Patent No. 3103615 Japanese Patent No. 3103167 JP 2001-226289 A Japanese National Patent Publication No. 6-501453 Japanese Patent No. 2840795 JP 2002-238554 A Japanese Patent Laid-Open No. 2000-28187

Nobuhiko Katsunuma, “BIO media”, Vol. 7, No. 6, 1992, p. 73-77 Isao Kiyosawa, “Nutrition of Breast Milk”, Kanehara Publishing, p. 80-81 American Journal of Respiratory and Critical Care Medicine. USA, Vol. 166, 2002, p. S57-S61 Journal of Nutritional Biochemistry, USA, Vol. 11, 2000, p. 94-102 Life Sciences, USA, Vol. 43, 1988, p. 739-745 Life Sciences, USA, 47, 1990, p. 703-709 Journal of Dairy Research, UK, Vol. 63, 1996, p. 257-267 Journal of Dairy Research, UK, Vol. 64, 1997, p. 281-288 Veterinary Immunology and Immunopathology, The Netherlands, Vol. 56, 1997, p. 85-96 Bioscience, Biotechnology, and Biochemistry, Japan, Vol. 66, No. 12, 2002, p. 2531-2536

  The present inventors have tried to identify a protein having an inhibitory effect on cysteine protease contained in milk, and found that lactoperoxidase has an effect of effectively inhibiting the enzyme activity of cysteine protease. In addition, lactoperoxidase is a kind of oxidoreductase, and it has never been reported that it inhibits the enzymatic activity of cysteine protease, among other proteases that are proteolytic enzymes, while originally having the function of an enzyme. Was not.

In Patent Documents 8, 9, and 10, proteins having a cysteine protease inhibitory effect contained in milk have molecular weights of 16 ± 2 kDa, 13 ± 2 kDa, and 57 kDa, respectively, and lactoperoxidase (molecular weight of 80 kDa). It is not described at all.
Furthermore, the protein having a cysteine protease inhibitory effect contained in milk described in Patent Document 19 and Non-Patent Document 10 is cystatin C, which is a protein essentially different from lactoperoxidase, There is no mention of the presence or absence of cysteine protease inhibitory action by oxidase.
Lactoperoxidase, which is an active ingredient of the present invention, effectively inhibits the enzymatic activity of cysteine protease by itself, and is simpler and more effective than conventional proteins having cysteine protease inhibitory activity in milk. Can be easily prepared as a cysteine protease inhibitor.
An object of the present invention is to provide a cysteine protease inhibitor containing lactoperoxidase as an active ingredient, which is a milk protein and a material that can be widely used as food.
Another object of the present invention is to provide a cysteine protease inhibitor having an effect in the prevention and treatment of diseases caused by cysteine protease.

  As a result of earnest search for a cysteine protease inhibitor that can be used as a safe material without antigenicity, the present inventors have found that lactoperoxidase, a milk-derived protein, has cysteine protease inhibitory activity, The present invention has been completed.

The gist of the present invention is as follows (1) to (4).
(1) A cysteine protease inhibitor containing lactoperoxidase as an active ingredient.
(2) The cysteine protease inhibitor according to (1), which has an effect on prevention and / or treatment of a disease caused by cysteine protease.
(3) The cysteine protease inhibitor according to (2) above, wherein the diseases caused by cysteine protease are osteoporosis, malignant neoplastic hypercalcemia, periodontal disease, breast cancer, prostate cancer, and bacterial / viral infection.
(4) A food / beverage composition or a feed composition comprising the cysteine protease inhibitor according to any one of (1) to (3).

The present invention relates to a cysteine protease inhibitor containing lactoperoxidase as an active ingredient, and the effects exhibited by the present invention are as follows.
(1) Since it is a protein contained in milk, it can be produced inexpensively, conveniently and in large quantities, and can be easily prepared as a cysteine protease inhibitor.
(2) It can be used as a prophylactic and / or therapeutic agent for diseases caused by cysteine protease.
(3) It can be taken orally by adding to food and drink or feed.

It is the figure which showed the cysteine protease inhibitory effect (papain residual activity) of the bovine lactoperoxidase with respect to papain.

  Next, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely modified within the scope of the present invention. In the present specification, percentages are expressed by mass unless otherwise specified.

  The cysteine protease inhibitor of the present invention is a cysteine protease inhibitor containing lactoperoxidase as an active ingredient. The lactoperoxidase used in the present invention is not particularly limited, but preferably derived from mammals, and more preferably bovine, human, horse, sheep, goat and the like. As an example of bovine lactoperoxidase, an accession number (hereinafter abbreviated as EMBL-Accession No.): M58150 registered in the nucleotide and amino acid sequence of European Molecular Biology Laboratory (EMBL) Examples thereof include those shown in SEQ ID NO: 1. Examples of human and sheep lactoperoxidase include EMBL-Accession No .: AY324876 (human) and EMBL-Accession No .: AF027970 (sheep), respectively.

  The lactoperoxidase used in the present invention can be obtained from milk such as humans, cows, horses, sheep and goats. For example, as in the method disclosed in Japanese Patent Application Laid-Open No. 5-41981 (Title of Invention: Live Fungus-Containing Liquid Composition), from unheated whey or skim milk, according to a conventional method (for example, ion chromatography) It can be manufactured industrially. In addition, lactoperoxidase produced in microorganisms, animal cells, transgenic animals and the like by genetic engineering techniques can also be suitably used in the present invention. For example, the method of Shin et al. [Biochemical and Biophysical Research Communications, Vol. 271, 2000, p. 831-836] can also be used recombinant lactoperoxidase expressed and purified. In the present invention, commercially available lactoperoxidase can also be used, and the commercially available lactoperoxidase may be a natural product-derived lactoperoxidase (for example, manufactured by Biopol), or genetic recombination. May be lactoperoxidase produced by

The lactoperoxidase that can be used in the present invention includes a modified lactoperoxidase as described above. Specifically, as a variant, one or several amino acids are substituted in the amino acid sequence such as EMBL-Accession No .: M58150 or SEQ ID NO: 1, EMBL-Accession No .: AY324876, EMBL-Accession No .: AF027970, etc. And a protein having a deleted or added sequence and having a cysteine protease inhibitory activity. Here, the term “several” means 2 to 50, preferably 2 to 20, and particularly preferably 2 to 10. In addition, as a variant, amino acid sequences such as EMBL-Accession No .: M58150 or SEQ ID NO: 1, EMBL-Accession No .: AY324876, EMBL-Accession No .: AF027970, etc. are 70% or more, preferably 80% or more, A protein having a homologous sequence of 90% or more, particularly preferably 95% or more, and having cysteine protease inhibitory activity can be exemplified. These lactoperoxidase variants are preferably modified so that the cysteine protease inhibitory activity is retained and the antigenicity to humans is as low as possible. The modified lactoperoxidase as described above expresses a gene obtained by mutating a gene encoding wild-type lactoperoxidase by a PCR method or the like in a known expression system, and measures the cysteine protease inhibitory activity of the obtained protein. Can be obtained. In addition, a microorganism into which a gene encoding wild-type lactoperoxidase has been introduced is subjected to mutation treatment by ultraviolet irradiation or the like, and a modified lactoperoxidase is purified from the obtained microorganism, and its cysteine protease inhibitory activity is measured. Can also be obtained. However, in the present invention, the “variant” does not necessarily have to be produced by modifying the wild type, and may be a naturally occurring mutant. In the present invention, when lactoperoxidase is used by adding it to food and drink or feed, it is possible to use wild-type lactoperoxidase in which amino acids are not substituted, deleted or added as much as possible. preferable.

  Since lactoperoxidase itself has the action of inhibiting the activity of cysteine protease, in the present invention, cysteine protease inhibitor (hereinafter sometimes referred to as “inhibitor of the present invention”). It can be used as an active ingredient. Cysteine protease which lactoperoxidase inhibits is not particularly limited, but preferably cathepsin B, cathepsin L, cathepsin K and papain, more preferably papain. Cysteine protease inhibitory activity can be measured according to a method such as Barrett [Methods in Enzymology, 80, 535-561, 1981]. Moreover, in the test example of this invention mentioned later, it describes about this measuring method.

In the present invention, the “active ingredient” means a component having an action of inhibiting the activity of cysteine protease, and lactoperoxidase does not have to be the main component of the inhibitor of the present invention. For example, in addition to lactoperoxidase, the inhibitor of the present invention may contain components having an action of inhibiting the activity of cysteine protease, casein and lactoferrin which are other useful proteins in milk.
As long as it has cysteine protease inhibitory activity, the active ingredient of the present invention may be used by mixing a lactoperoxidase partial peptide or a lactoperoxidase hydrolysis mixture (peptide mixture) in addition to lactoperoxidase. The active ingredient may be a partial peptide of lactoperoxidase or a hydrolysis mixture (peptide mixture) of lactoperoxidase.

  The cysteine protease inhibitor of the present invention can be administered orally or parenterally to mammals including humans in combination with lactoperoxidase or a pharmaceutically acceptable pharmaceutical carrier. The dosage unit form of the preparation of the present invention is not particularly limited, and can be appropriately selected according to the purpose of treatment. Specifically, tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, syrups Examples include suppositories, suppositories, injections, ointments, patches, eye drops, nasal drops and the like. Additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, surfactants, solvents for injections, etc. that are commonly used as pharmaceutical carriers for pharmaceutical preparations. Can be used. The amount of lactoperoxidase contained in the preparation of the present invention is not particularly limited and may be appropriately selected. For example, it is 0.0005 to 60% by mass in the preparation, preferably 0.005 to 40% by mass. Is good.

  The administration method of the preparation of the present invention is not particularly limited, and is determined according to various preparation forms, the patient's age, sex, other conditions, the degree of symptoms of the patient and the like. The dosage of the active ingredient in the preparation of the present invention is appropriately selected depending on the usage, patient age, sex, disease severity, other conditions, and the like. Usually, the amount of lactoperoxidase as an active ingredient is 0.1 to 1200 mg / kg / day, preferably 10 to 500 mg / kg / day. Can be administered in divided doses.

The cysteine protease inhibitor of the present invention is a disease involving cysteine protease, such as allergy, muscular dystrophy, atrophy, myocardial infarction, stroke, Alzheimer's disease, multiple sclerosis, cataract, osteoporosis, malignant neoplastic hypercalcemia, Prophylactic / therapeutic agent for benign prostatic hyperplasia, breast cancer, prostate cancer, etc., suppressor of cancer cell proliferation and metastasis, or suppression of growth of bacteria (staphylococcus aureus V8, etc.) and viruses (poliovirus, herpes virus, etc.) Useful as an agent. The cysteine protease inhibitor of the present invention may be used alone, but can also be used in combination with the known prophylactic / therapeutic agent for the disease or the bacterial / viral growth inhibitor. By using in combination, the preventive / therapeutic effect of the disease or the bacterial / virus growth inhibitory effect can be enhanced. The known prophylactic / therapeutic agent for the disease or the bacterial / viral growth inhibitor used in combination may be contained as an active ingredient in the inhibitor of the present invention, or not contained in the inhibitor of the present invention. In addition, they may be combined and commercialized as separate drugs.

  The food-drinks composition of this invention can be manufactured by adding the cysteine protease inhibitor or lactoperoxidase of this invention to the raw material of a foodstuff or a drink, and can be ingested orally. The said raw material can use what is used for the normal drink and foodstuff. The food / beverage product composition of the present invention can be prepared in the same manner as a normal food / beverage product composition except that a cysteine protease inhibitor is added. As a form of the food and beverage composition, beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit juice drinks, lactic acid bacteria drinks (including concentrated concentrates and powders for adjustment of these drinks); ice confectionery such as ice cream, sorbet, shaved ice, etc. Candy, chewing gum, candy, gum, chocolate, tablet confectionery, snack confectionery, biscuits, jelly, jam, cream, baked confectionery, etc .: dairy products such as processed milk, milk beverage, fermented milk, butter; bread; enteral Examples include nutritional foods, liquid foods, milk for childcare, sports drinks; and other functional foods.

  In the food / beverage product composition of the present invention, the amount of lactoperoxidase added is appropriately set depending on the form of the food / beverage product composition. What is necessary is just to add so that it may become ~ 40 mass%.

  As a form which mix | blends and ingests in food-drinks composition, for example, a soft drink, milk drink, etc. which mix | blended lactoperoxidase powder, lactoperoxidase aqueous solution (syrup etc.) etc. as a cysteine protease inhibitor of this invention, or these Concentrates and powders for preparation; dairy products such as processed milk and fermented milk; enteral nutritional foods; functional foods and the like.

  Examples of the shape of the food / beverage product composition include tablet-like supplements. This eliminates the need to control the amount of meal and calorie intake per day in consideration of the intake of other foods, and allows the intake of active ingredients to be accurately grasped.

  The feed composition of the present invention can be produced by adding lactoperoxidase to the feed and can be orally administered to general mammals, livestock, fish farms, and pet animals. Examples of the feed composition include pet food, livestock feed, fish feed, etc., mixed with cereals, potatoes, potatoes, fish meal, bone meal, fats and oils, skimmed milk powder, whey, mineral feed, yeasts, etc. Thus, the feed composition of the present invention can be produced.

  In the feed composition of the present invention, the amount of lactoperoxidase added is appropriately set depending on the form of the feed composition, but is 0.0005 to 60% by mass, preferably 0.005 to 40% by mass in normal feed. What is necessary is just to add so that it may become.

Next, the production of bovine lactoperoxidase is shown as a reference example.
[Reference example]
Production of lactoperoxidase 1000 kg of unheated whey was added to 10 liters of CM-Sephadex C-50 resin (
The solution was passed through a column packed with Amersham Biotech. Next, after washing with 100 kg of water, 20 liters of 20 mM sodium phosphate buffer (pH 7.0) containing 0.3 M sodium chloride was passed through to elute lactoperoxidase adsorbed on the resin. The eluate was concentrated and desalted with an ultrafiltration membrane (manufactured by Asahi Kasei Co., Ltd.) having a fractional molecular weight of 10,000. About 1 liter of the obtained concentrated solution was aseptically filtered through a membrane cartridge (manufactured by Nalgen) and freeze-dried to obtain 40 g of powdered sterile lactoperoxidase.

  This lactoperoxidase sample was subjected to electrophoresis using an SDS-polyacrylamide gel having an acrylamide concentration of 10 to 20%, stained with Coomassie Brilliant Blue R250 (Sigma), and an image processing apparatus (Ato Corporation). ), The purity of bovine lactoperoxidase was about 50%.

Next, the present invention will be described in detail with reference to test examples.
[Test Example 1]
This test was conducted to measure the inhibitory effect of lactoperoxidase on cysteine protease.
(1) Test method Commercial bovine lactoperoxidase (manufactured by Sigma) was used as a test sample, and cysteine protease inhibitory activity was measured against papain (manufactured by Wako Pure Chemical Industries, Ltd.), which is a cysteine protease. The inhibitory activity was measured with reference to the method of Barrett et al. [Methods in Enzymology, 80, 535-561, 1981] as follows. That is, bovine lactoperoxidase dissolved in 0.1 M acetate buffer (pH 5.5) at various concentrations and Z-Phe-Arg-MCA (Benzyloxycarbonyl-L-Phenylalanyl-L-Arginine 4-Methyl-Coumaryl as a substrate) -7-Amide: final concentration 20 mM: manufactured by Peptide Laboratories), papain solution (final concentration: 15 units / ml) was added to each sample, mixed, reacted at 37 ° C. for 10 minutes, and digested The fluorescence intensity (excitation wavelength: 370 nm, emission wavelength: 460 nm) of AMC (7-Amino-4-Methyl-Coumarin) released from the substrate that received the light was measured using a fluorescence spectrometer (manufactured by Hitachi, Ltd.).

(2) Test results The test results are shown in Fig. 1. FIG. 1 is a diagram showing the inhibitory effect (papain residual activity) of bovine lactoperoxidase on papain. Papain-based cysteine protease activity is 94% at a bovine lactoperoxidase concentration of 10 ⁻⁶ M, 10 ⁻ It was almost completely inhibited by ⁵ M. Therefore, it was found that bovine lactoperoxidase has cysteine protease inhibitory activity against papain.

  EXAMPLES Next, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.

  Lactose (Megure) 600g, Corn starch (Nisshin Flour Mills) 400g, Crystalline cellulose (Wako Pure Chemical Industries) 400g and Lactoperoxidase 600g are sifted with 50 mesh sieve (Yamato Kagaku). In a polyethylene bag with a thickness of 0.5 mm, invert and mix, and using a fully automatic capsule filling machine (manufactured by Cesele Pedini, press type), the powder is capsuled (manufactured by Elanco Japan, No. 1 gelatin capsule, Op. Yellow No. 6 Body (empty weight 75 mg) was filled with an internal volume of 275 mg to obtain 7000 capsules having cysteine protease inhibitory activity.

Lactose (Mirai) 7.5 kg, skim milk powder (Morinaga Milk Industry) 2.5 kg, desalted whey (Domo) 1 kg, casein sodium (New Zealand Daily Board) 0.5 kg, and a small amount Water-soluble vitamins and minerals were dissolved in 50 kg of water, and an aqueous phase was prepared in the tank. Separately, 1.96 kg of vegetable oil (manufactured by Taiyo Oil & Fats Co., Ltd.), 0.04 kg of lecithin (manufactured by Ajinomoto Co., Inc.) and a small amount of fat-soluble vitamins were mixed and dissolved to prepare an oil phase. The oil phase was added to the aqueous phase in the tank, mixed, heated to 70 ° C., and then homogenized with a homogenizer at a pressure of 14.7 MPa. Next, the mixture was sterilized at 90 ° C. for 10 minutes, concentrated, and spray-dried to prepare about 12 kg of intermediate product powder. To 2 kg of this intermediate product powder, 40 g of bovine lactoperoxidase produced by the method of Reference Example was added and mixed uniformly with a mixer to produce about 2 kg of prepared milk powder having cysteine protease inhibitory activity.

  A whey protein enzyme degradation product (manufactured by Morinaga Milk Industry Co., Ltd.) 10.8 kg, dextrin (manufactured by Showa Sangyo Co., Ltd.) 36 kg, and a small amount of water-soluble vitamins and minerals were dissolved in 200 kg of water, and an aqueous phase was prepared in the tank. Separately, soybean salad oil (manufactured by Taiyo Yushi Co., Ltd.) 3 kg, palm oil (manufactured by Taiyo Yushi Co., Ltd.) 8.5 kg, safflower oil (manufactured by Taiyo Yufu Co., Ltd.) 2.5 kg, lecithin (manufactured by Ajinomoto Co., Inc.) 0.2 kg, An oil phase was prepared by mixing and dissolving 0.2 kg of a fatty acid monoglyceride (manufactured by Kao Corporation) and a small amount of a fat-soluble vitamin. The oil phase was added to the aqueous phase in the tank, stirred and mixed, then heated to 70 ° C., and further homogenized with a homogenizer at a pressure of 14.7 MPa. Next, the mixture was sterilized at 90 ° C. for 10 minutes, concentrated and spray-dried to prepare about 59 kg of intermediate product powder. To 50 kg of this intermediate product powder, 6.8 kg of sucrose (manufactured by Hokuren Co., Ltd.), 167 g of amino acid mixed powder (manufactured by Ajinomoto Co., Inc.) and 60 g of lactoperoxidase (manufactured by Biopol) are added and mixed uniformly. About 56 kg of enteral nutrition food powder having inhibitory activity was produced.

  150 g of lactoperoxidase (manufactured by Biopol), 100 g of lactulose powder (manufactured by Morinaga Milk Industry Co., Ltd.), 635 g of malt dextrin (manufactured by Matsutani Chemical Industry Co., Ltd.), 85 g of skim milk powder (manufactured by Morinaga Milk Industry Co., Ltd.), Each powder of 1 g of F eye), 5 g of yogurt flavor (manufactured by Saneigen F.F.I.), and 24 g of glycerin fatty acid ester preparation (manufactured by Riken Vitamin Co., Ltd.) was added and mixed uniformly. (Made by Hata Steel Co., Ltd.), 0.5 g per tablet, continuously tableting the mixed powder at a tableting speed of 12 tablets / min and a pressure of 9.8 kPa to inhibit cysteine protease 1800 tablets (about 900 g) having activity were produced.

  The cysteine protease inhibitor of the present invention contains cysteine protease activity because its active ingredient is contained in a food material such as milk protein, is highly safe for humans, and can be taken orally as a food or beverage composition. It can be used for applications such as the production of novel functional foods, nutritional supplements, foods for specified health use, etc. that have an effect on the prevention and / or treatment of diseases caused by. In addition, since the cysteine protease inhibitor of the present invention can control the activity of proteases contained in foods, it can be used as a food property regulator in the field of food processing.

Claims (4)

A cysteine protease inhibitor containing lactoperoxidase as an active ingredient. The cysteine protease inhibitor according to claim 1, which has an effect on prevention and / or treatment of a disease caused by cysteine protease. The cysteine protease inhibitor according to claim 2, wherein the diseases caused by cysteine protease are osteoporosis, malignant neoplastic hypercalcemia, periodontal disease, breast cancer, prostate cancer, and bacterial / viral infection. The food-drinks composition or feed composition formed by adding the cysteine protease inhibitor as described in any one of Claims 1-3.

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