TW201446257A - Infection prevention agent - Google Patents

Abstract

The invention provides an infection prevention agent containing, as an active ingredient, lactadherin and/or a degradation product obtained by treating lactadherin with a protease enzyme. Because the infection prevention agent uses mammalian milk as a raw material, it can be produced easily and economically, and can be safely taken daily.

Description

Infective protectant

The present invention is excellent in the effect of protecting against infection by influenza virus, and is an infection-protecting agent which is useful for prevention or treatment of influenza and which is excellent in stability and safety. Further, the present invention relates to an infectious infection food or drink containing an infection repellent, a nutritional composition for infection protection, or a feed for infection protection.

Influenza viruses are infected from the air to the human body and cause epidemics year after year. In recent years, the threat has been reduced due to improvements in health or advances in medicine, but there are still fatal cases. There are three types of influenza viruses: type A, type B, and type C. Among them, type A viruses are prone to mutations, and are prone to cause a global pandemic called "pandemic." Prevention against influenza virus infection is mainly carried out by vaccination. However, since influenza viruses are prone to mutations such as antigen transfer or antigen migration, the viruses of the epidemic and the antigens of the vaccine are often inconsistent, and the current situation is that the preventive effect produced by the vaccine may not be satisfied. Therefore, in Japan, vaccination against children is still not enforced. Further, as a therapeutic agent for influenza, there may be mentioned amantadine, oseltamivir, and zanamivir, but it is necessary to consider side effects such as hallucinations or sleep disorders or the emergence of drug-resistant bacteria. The problem needs to be fully paid attention to in its use. Based on this situation, it is necessary to develop a food or drink or a feed which can be safely taken in daily life and which is expected to achieve prevention or treatment of influenza. As a food ingredient having an infection-protecting effect against influenza virus, κ-casein which causes chymosin or gastric protein to act on sphingomyelin or lactoferrin or gamma-casein-derived peptide is hitherto known. Κ-casein glyco-macropeoptide. In addition, it is also known that a polysaccharide produced by Lactobacillus bulgaricus 1073R-1, which is a kind of lactic acid bacteria contained in fermented milk, acts on the intestinal tract to increase the yield of IgA, and at the same time, NK activity is achieved via Peyer's patches. It rises and has an infection protection effect against influenza virus.

Milk lectin is one of the glycoproteins that make up the fat globule membrane of milk. In cow's milk, it accounts for about 10% of the protein contained in the fat globule membrane. Its molecular weight is 43kDa~53kDa, isoelectric point is 7.0, and it has two sub-domains of epidermal growth factor. In addition, the milk lectin is also called PAS-VI-VII (PAS6/7) in cattle, and the milk lectin contained in the milk of mice also shows "MFG-E8". Furthermore, lactadherin is thought to be responsible for regulating the functions of the neonatal digestive tract, and is incorporated into artificial milk for infants to prevent neonatal infection with rotavirus. However, the protective effect of lactadherins and/or their decomposition products against influenza virus infection has not been known. [Prior Art Literature] [Patent Literature]

[Patent Document 1] Japanese Patent Laid-Open No. 2008-37788 [Non-patent Literature]

[Non-Patent Document 1] Bioscience, Biotechnology and Biochemistry, 57, 1214-1215, 1993 [Non-Patent Document 2] International Immunopharmacology, 11, 2246-2250, 2011

[Problems to be solved by the invention]

An object of the present invention is to provide an infection-protecting agent for preventing or treating influenza, an infection-preventing agent for prevention or treatment, an infection-protecting food or drink, a protective nutrient composition for infection protection, or infection protection. Use feed. [Means for solving the problem]

As a result of intensive studies to solve the above problems, the inventors of the present invention have found that lactadherin and/or its decomposed products have excellent infection protection effects against influenza virus. That is, the present invention encompasses the following aspects: (1) An infection preventive agent against influenza virus, which comprises lactadherin and/or a decomposition product thereof as an active ingredient. (2) The infection-protecting agent against influenza virus according to (1), wherein the lactadherin and/or its decomposition product is derived from cow's milk. (3) The infection-protecting agent against influenza virus according to (1) or (2), wherein the lactadherin-degrading substance is obtained by decomposing lactadherin using a proteolytic enzyme. (4) The infection-protecting agent against influenza virus according to (3), wherein the proteolytic enzyme is selected from at least 1 selected from the group consisting of trypsin, trypsin, chymotrypsin, pepsin, and papain. Kind. (5) A food or drink for preventing infection, a nutritional composition for infection protection, or a protective food for infection, which comprises the infection preventing agent against influenza virus according to any one of (1) to (4). (6) A method for protecting against infection by influenza virus by orally ingesting the infection-protecting agent according to any one of (1) to (4). (7) A method of using an infection repellent for applying an infection-protecting agent containing lactadherin and/or a decomposition product thereof as an active ingredient to a patient infected with influenza virus. [Effects of the Invention]

The infection-protecting agent of the present invention has a remarkable protective effect against influenza virus infection, and is useful for prevention or treatment of influenza.

The milk lectin which is an active ingredient of the infection repellent can be separated by, for example, buttery milk (B) which is an aqueous phase component produced by the production of butter from raw milk, and (B) 40% to 50% of the fat component obtained by separating the fat component, which is separated by 60% or more of the high-fat emulsifiable concentrate, which is separated by a separator, and subjected to heat treatment or shear treatment, and the water discharged during the conversion of the high-fat cream phase The phase component, or (C) is a butter whey of an aqueous phase component which is separated by heating the butter, and uses an ultrafiltration (UF) membrane or a microfiltration (MF) membrane, a reverse osmosis membrane (RO) membrane, and an ion exchange resin. Wait for desalting or concentration. Further, the pH can be adjusted to 4.4 by adding hydrochloric acid to the butter whey, and then calcium chloride is added and kept at 50 ° C for 30 minutes to separate the precipitate, which is again dissolved or suspended in water or the like, and ultrafiltration is used ( UF) membrane or microfiltration (MF) membrane, reverse osmosis membrane (RO) membrane, ion exchange resin, etc. are desalted or concentrated. Further, the milk lectin decomposed product which is an active ingredient of the infection repellent can be obtained by decomposing the lactadherin by any proteolytic enzyme. As a proteolytic enzyme, trypsin can be listed in addition to commercially available food and industrial proteases such as Protease A "Amano" SD (trade name), THERMOASE PC10F (trade name), and PROTIN SD-AY10 (trade name). , trypsin, chymotrypsin, pepsin, papain, etc., and may also be used in combination. The milk lectin and/or its decomposition product prepared as described above may also be dried by freeze drying, spray drying or the like.

The milk lectin and/or its decomposition product can be prepared by using milk obtained from mammals such as humans, cows, buffalo, goats, and sheep, those produced by genetic engineering methods, and those purified by blood or internal organs. The reason for this is that it can be manufactured simply and economically, and can be safely taken in daily life. Among them, it is preferably derived from cow's milk. Further, a reagent which is purified and commercially available as a lactadherin and/or a decomposition product thereof may also be used.

The milk lectin and/or its decomposition product can be directly used as an infection repellent, but it can also be used as a powder, granule, lozenge, capsule, beverage, etc. according to the usual method according to the requirements. Further, the lactadherin and/or its decomposed product dried by freeze drying or spray drying may be used as an infection repellent as it is, or may be used in accordance with a usual method. Further, after these preparations are formulated, they may be blended in a nutraceutical, a food or drink, a nutritional composition, or a feed such as a yogurt, a drink, or a muffin.

Infection-protecting foods, nutritional compositions for infection protection, and infection-protective feeds may contain stabilizers or sugars, lipids, flavors, vitamins, minerals, and flavonoids in addition to the lactadherin and/or its decomposition products. Other foods such as polyphenols, raw materials usually contained in feed, etc. Further, in addition to the lactadherin and/or its decomposition product as an active ingredient, it may be used together with a component exhibiting an infection-protecting action, such as sphingomyelin or lactoferrin.

The blending amount of the lacto-agglutinin and/or the decomposition product thereof in the food for infection protection, the nutritional composition for infection protection, and the feed for infection protection is not particularly limited, and the effect of protecting against influenza virus infection is It is preferred to adjust the amount of the pharmaceutical, food or beverage, and feed by daily intake of 0.1 mg or more of lactadherin and/or its decomposition product per adult.

The infection-preventing agent can be formulated into any form by adding an appropriate auxiliary agent to the lactadherin and/or its decomposition product as an active ingredient. At the time of formulation, a diluent or an excipient such as a commonly used filler, extender, binder, disintegrant, surfactant, lubricant, or the like can be used. As the excipient, for example, sucrose, lactose, starch, crystalline cellulose, mannitol, light phthalic anhydride, magnesium aluminate, synthetic aluminum citrate, magnesium aluminum metasilicate, calcium carbonate, sodium hydrogencarbonate, phosphoric acid may be added. One type or a combination of two or more types of calcium hydride and calcium carboxymethyl cellulose.

The present invention is described by way of example only, and is not intended to limit the invention. From this disclosure, various alternative embodiments, examples, and operational techniques will be apparent to those of ordinary skill in the art. For example, there is provided a method of using an infection repellent which uses the lactadherin and/or its decomposition product as an active ingredient described in the embodiment as an infectious agent for an influenza virus-infected patient. In this case, as the lactadherin and/or its decomposition product, for example, it can be separated and purified from the raw material as described in the column of the examples described later. In addition, the infectious protective agent can also be applied to mammals other than humans, such as domestic animals such as dogs, monkeys, cats, cows, horses, pigs, chickens, and sheep.

The present invention is illustrated by the following examples and test examples, but these are merely for the purpose of illustration, and the invention is not limited thereto. [Example 1]

After adding 100 kg of acetone to 10 kg of the freeze-dried unsterilized buttermilk powder, the mixture was treated with a Quark separator, whereby the precipitate was completely removed to obtain a supernatant. The acetone was removed from the supernatant by an evaporator and dissolved again in deionized water, and then treated with an ultrafiltration membrane having a molecular weight of 60 kDa, and the permeate was recovered. Next, the permeate was subjected to removal of impurities by an ultrafiltration membrane having a molecular weight of 30 kDa, and subjected to desalting and concentration, followed by lyophilization to obtain 68 g of a milk lectin powder (Example 1). The milk lectin powder contains 78% milk lectin. The lactadherin thus obtained can be directly used as an infection preventive agent. [Example 2]

500 mg of the milk lectin powder prepared in Example 1 was dissolved in 100 ml of purified water, and the pH was adjusted to 8 using baking soda. Then, trypsin (manufactured by SIGMA), which is a proteolytic enzyme, was added to have a final concentration of 0.01%, and the enzyme treatment was carried out at 37 ° C for 1 hour. Subsequently, the enzyme was deactivated by heat treatment at 85 ° C for 10 minutes, and then freeze-dried to obtain 463 mg of a milk lectin decomposed product powder (Example 2). The molecular weight of the obtained lactadherin-degraded product is 5 kDa or less, and can be directly used as an infection preventive agent. [Example 3]

10 g of the milk lectin powder prepared in Example 1 was dissolved in 200 ml of purified water, and then kept at 45 ° C, and 2 g of Protease A "Amano" SD (manufactured by Amano Enzyme Co., Ltd.) was added thereto, and the enzyme treatment was carried out for 2 hours. Subsequently, the enzyme was deactivated by heat treatment at 80 ° C for 10 minutes, and then lyophilized to obtain 8.2 g of a milk lectin decomposed product powder (Example 3). The molecular weight of the obtained lactadherin-degraded product is 5 kDa or less, and can be directly used as an infection preventive agent.

[Test Example 1] (Confirmation of the effect of prevention of infection against influenza virus) A/Guinzhou virus as influenza A virus, B/Ibaraki virus as influenza B virus, nasally infected mice (Balb/c; public; 6 weeks old), a solution in which the lactadherin of Example 1 was dissolved in 100 μg/ml and a lactate agglutinin-decomposed product of Example 2 was added to a solution of 100 μg/ml, respectively, in a solution of 1 μl/intranasal Amount (administration amount: 0.1 μg) for nasal administration, and the infection prevention effect against influenza virus, and the group infected with each influenza virus alone (control group), using the virus in the nasal cavity cleaning solution The price is compared and verified. Further, it was judged that a plaque method using MDCK cells was employed. The results are shown in Table 1.

Values represent mean ± standard deviation (n = 8). * indicates a significantly lower level (p < 0.05) than each control group.

According to the results of Table 1, by administering the lactadherin and its decomposition products intranasally, the infection prevention effect can be confirmed for both type A and type B, and the effect against influenza A virus is remarkable.

[Test Example 2] (Confirmation of infection prevention effect of influenza virus by oral administration) The influenza virus PR8 (H1N1) was infected with a virus amount of 1 × 10 ³ pfu (Balb/c; public; 6 weeks old) . Before the infection, the antibody of Example 1 and Example 3 was administered orally to a group of 0.1 mg of lactadherin or a decomposition product per 1 kg of body weight of the mouse, and 10 mg of lactadherin or its decomposition product was orally administered. In the group, the viral power in the nasal cleansing solution after virus infection for 3 days was used to determine the prevention effect against the influenza virus. Further, as a positive control, a group of 10 mg of lactoferrin was orally administered, and a group of water (control group) which was orally administered as a vehicle was used, and a plaque method using MDCK cells was used. Compare verification. The results are shown in Table 2.

Values represent mean ± standard deviation (n = 10). *1 indicates a significantly lower level (p<0.05) than each control group. *2 indicates a significantly lower level (p<0.05) than 10 mg of lactoferrin.

According to the results of Table 2, by intranasal administration of lactadherin or its decomposition product, the viral power in the nasal cavity was significantly lower than that of the control group. Moreover, this effect is remarkably improved compared to lactoferrin which is also administered orally. From this result, it is known that lactadherin and its decomposed products have excellent infection protection effects against influenza virus. Further, this effect can be clearly confirmed when the mouse is orally administered with 0.1 mg or more per 1 kg of body weight. [Embodiment 4]

(Preparation of Influenza Virus Infection Prevention Capsule) After mixing the raw materials in the blending amounts shown in Table 3, granulation was carried out by a usual method and filled in a capsule to prepare a capsule for influenza virus infection protection.

[Embodiment 5]

(Preparation of tablet for preventing influenza virus infection) The raw materials were mixed in a blending amount shown in Table 4, and then molded into 1 g according to a usual method, and tableting was carried out to prepare a tablet for infection protection.

[Embodiment 6]

(Preparation of liquid nutrient composition for influenza virus infection prevention) 50 g of Example 1 was dissolved in 4,950 g of deionized water, and heated to 50 ° C, and then TK HOMO MIXER (TK ROBO MICS; special machine chemical industry) was used. The company's system) was stirred and mixed at 6,000 rpm for 30 minutes to obtain a lactadherin solution having a milk agglutinin content of 39 g/5 kg. 5.0 kg of the lactadherin solution, blended with 5.0 kg of casein, 5.0 kg of soy protein, 1.0 kg of fish oil, 3.0 kg of perilla oil, 17.0 kg of dextrin, 6.0 kg of mineral mixture, 1.95 kg of vitamin mixture, 2.0 kg of emulsifier 4.0 kg of stabilizer, 0.05 kg of perfume, and filled into a 200 ml retort pouch, and sterilized at 121 ° C for 20 minutes using a sterilizer (first type pressure vessel; TYPE: RCS-4CRTGN; manufactured by HISAKA) A liquid nutritional composition for preventing infection of 50 kg of influenza virus infection was prepared. [Embodiment 7]

(Preparation of Influenza Virus Infection Prevention Drink) After dissolving 300 g of skim milk powder in 409 g of deionized water, 1 g of Example 2 was dissolved and heated to 50 ° C, and then ULTRA DISPERSER (ULTRA-TURRAX T-25; IKA JAPAN) was used. Company-made), stirred and mixed at 9,500 rpm for 30 minutes. 100 g of maltitol, 2 g of sourdil, 20 g of reducing syrup, 2 g of perfume, and 166 g of deionized water were added, and then filled into a 100 ml glass bottle, and sterilized at 95 ° C for 15 seconds, and then sealed to prepare influenza virus infection prevention. 10 bottles of beverage (content 100 ml). [Embodiment 8]

(Preparation of feed for prevention of influenza virus infection in dogs) 2 kg of the Example 1 was dissolved in 98 kg of deionized water, and heated to 50 ° C, and then TK HOMO MIXER (MARK II 160 type; manufactured by Special Machine Chemical Co., Ltd.) was used. The mixture was stirred and mixed at 3,600 rpm for 40 minutes to obtain a lactadherin solution having a lactadin content of 1.56 g/100 g. 10 kg of the lactadherin solution is blended with 12 kg of soybean meal, 14 kg of skim milk powder, 4 kg of soybean oil, 2 kg of corn oil, 23.2 kg of palm oil, 14 kg of corn starch, 9 kg of wheat flour, 2 kg of rice bran, 5 kg of vitamin mixture, 2.8 kg of cellulose, 2 kg of the mineral mixture was sterilized at 120 ° C for 4 minutes to prepare 100 kg of influenza virus infection prevention feed.

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Claims (7)

An infection preventive agent against influenza virus, which comprises lactadherin and/or a decomposition product thereof as an active ingredient. An infection-protecting agent against influenza virus according to claim 1, wherein the lactadherin and/or its decomposition product is derived from cow's milk. An infection-protecting agent against influenza virus according to claim 1 or 2, wherein the lactadherin-degrading substance is obtained by decomposing a lactadherin using a proteolytic enzyme. An infection-protecting agent against influenza virus according to claim 3, wherein the proteolytic enzyme is selected from the group consisting of trypsin, trypsin, chymotrypsin, pepsin, and papain. 1 species. The invention relates to a food and drink for infection protection, a nutritional composition for infection protection or a feed for infection protection, which comprises the infection protection agent against influenza virus according to any one of claims 1 to 4. A method for protecting against infection by influenza virus by ingesting an infection repellent according to any one of claims 1 to 4 of the patent application. The invention relates to a method for using an infection-protecting agent, which is applied to an influenza virus-infected patient by using an infection-protecting agent containing a lactadin and/or a decomposition product thereof as an active ingredient.