TWI646969B - Use of milk lectin and/or its decomposition products - Google Patents

Abstract

An infection protection agent containing lactadherin and / or a degraded product of lactadherin treated with a proteolytic enzyme as an active ingredient. Infection protection agents are made from mammalian milk as a raw material, can be easily and economically manufactured, and can be safely ingested daily.

Description

Use of lactadherin and / or its decomposed product

The invention relates to an infection protection agent which has excellent protection effect against influenza virus infection, is useful for prevention or treatment of influenza, and has excellent stability and safety. The present invention further relates to a food or drink for infection prevention, a nutritional composition for infection prevention, or a feed for infection prevention containing an infection prevention agent.

Influenza viruses are transmitted to the human body from the air and cause epidemics year after year. In recent years, threats have 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 susceptible to mutations and easily cause a global pandemic called "pandemic." Prevention of influenza virus infection is mainly carried out by vaccination. However, because influenza viruses are prone to mutations such as antigen transfer or antigen drift, the epidemic virus and vaccine antigens are often inconsistent. The current situation is that the preventive effects produced by vaccines may not be satisfied. As a result, child immunizations are still not mandatory in Japan. Furthermore, as a therapeutic agent for the flu, amantadine, oseltamivir, zanamivir, etc. may be mentioned, but the side effects such as hallucinations or sleep disorders or the appearance of drug-resistant bacteria need to be considered. Problems need to be paid full attention to in its use. Based on this situation, a food or feed that can be safely taken in daily life and can be expected to achieve prevention or treatment of influenza is to be developed.

As a food ingredient having a protective effect against influenza virus infection, kappa caseinose, which is a peptide of the genus sphingomyelin, lactoferrin, and kappa casein, is known to act on rennet or gastric protein Macropeptide (κ-casein glyco-macropeoptide). In addition, it is also known that the polysaccharide produced by Lactobacillus bulgaricus 1073R-1, which is a kind of lactic acid bacteria contained in fermented milk and the like, acts on the intestinal tract to increase the production of IgA, and at the same time, makes NK active through Peyer's patches Rises, and has an infection protection effect against the influenza virus.

Lactin 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, its isoelectric point is 7.0, and it has two subdomains of epidermal growth factor. In addition, milk lectin is also called PAS-VI-VII (PAS6 / 7) in cattle, and milk lectin contained in the milk of mice is also expressed as "MFG-E8". In addition, milk lectins are considered to play a role in regulating the digestive tract function of newborns, and are blended in artificial milk powder for infants in order to prevent newborns from being infected by rotavirus. However, it has not been known that lactadherin and / or its decomposed product has an infection protection effect against influenza virus.

[Previous Technical 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

The object of the present invention is to provide an infection protection effect against influenza virus, an infection protection agent for prevention or treatment, and an infection protection food product, an infection protection nutrition composition or infection protection mixed with the infection protection agent. With feed.

As a result of intensive studies conducted by the inventors of the present invention to solve the above-mentioned problems, it was found that lactadherin and / or its decomposed product has an excellent anti-influenza protection effect against influenza virus. That is, the present invention includes the following aspects:

(1) An infection protection agent against influenza virus, which uses lactadherin and / or a degradation product thereof as an active ingredient.

(2) The anti-influenza-protective agent according to (1), wherein the milk lectin and / or its degraded matter are derived from cow milk.

(3) The anti-influenza protective agent against influenza virus according to (1) or (2), wherein the lactohemagglutinin degraded product is obtained by degrading the lactohemagglutinin using a proteolytic enzyme.

(4) The anti-influenza protective agent according to (3), wherein the proteolytic enzyme is at least 1 selected from the group consisting of trypsin, trypsin, chymotrypsin, pepsin, and papain. Species.

(5) A food or drink for infection prevention, a nutritional composition for infection prevention, or a feed for infection prevention, which contains an infection protection agent against influenza virus according to any one of (1) to (4).

(6) An infection prevention method against influenza virus, which comprises ingesting an infection prevention agent according to any one of (1) to (4) by oral ingestion.

(7) A method for using an infection-preventing agent, which comprises applying an infection-preventing agent containing lactadherin and / or its decomposed product as an active ingredient to a patient infected with influenza virus.

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

Milk agglutinin, which is an effective component of an infection protection agent, can be used to separate raw milk from (A) butter milk, which is an aqueous phase component produced when butter is produced from raw milk, and (B). The cream with a fat content of 40-50% obtained by the separator is again separated by a separator to obtain more than 60% of the high-fat cream, which is heat-treated or sheared. The water discharged during the high-fat cream phase conversion The phase component, or (C) butter whey which is an aqueous phase component separated when the butter is heated and dissolved, uses an ultrafiltration (UF) membrane or a microfiltration (MF) membrane, a reverse osmosis membrane (RO) membrane, and an ion exchange resin. Obtained by desalting or concentrating. In addition, by adding hydrochloric acid to butter whey to adjust the pH to 4.4, calcium chloride is added, and the precipitate is separated by holding at 50 ° C for 30 minutes, and then dissolved or suspended in water, etc., and then subjected to ultrafiltration ( UF) membrane or microfiltration (MF) membrane, reverse osmosis membrane (RO) membrane, ion exchange resin, etc. are obtained by desalting or concentrating. Furthermore, a milk agglutinin degradation product, which is an effective component of an infection protection agent, can be obtained by decomposing the milk agglutinin with an arbitrary protein-degrading enzyme. As proteolytic enzymes, except Protease A "Amano" SD (quote Product name), THERMOASE PC10F (trade name), PROTIN SD-AY10 (trade name) and other commercially available foods. In addition to industrial protease agents, trypsin, trypsin, chymotrypsin, pepsin, papain, etc. can also be listed, and these various combinations can also be used. The lectin and / or its decomposed product prepared as described above can also be dried by freeze-drying or spray-drying.

Lactin and / or its degraded product can be prepared from milk obtained from mammals such as humans, cattle, buffalo, goats, and sheep, produced by genetic engineering methods, or purified from blood or internal organs. The reason is that it can be manufactured simply and economically, and can be safely ingested in daily life. Among them, those derived from milk are preferred. In addition, a purified and commercially available reagent for lectin and / or its decomposed product may be used.

Lactin and / or its degraded product can be used directly as an infection protection agent, but it can also be formulated into powders, granules, lozenges, capsules, beverages and the like according to common methods according to requirements. In addition, lysin and / or its decomposed product dried by freeze-drying or spray-drying can also be used directly as an infection protection agent, or they can be formulated and used according to a common method.

Furthermore, after these formulations are formulated, they can also be mixed with nutritional supplements, foods such as yogurt, beverages, muffins, nutritional compositions, or feeds.

Food and beverage for infection protection, nutritional composition for infection protection, and feed for infection protection, in addition to the lectin and / or its decomposed product, may also contain stabilizers or sugars, lipids, flavors, vitamins, minerals, and flavonoids , Polyphenols and other foods and drinks, raw materials usually contained in feed, etc. Moreover, in addition to lactadherin and / or a degradation product thereof as an active ingredient, it can also be used together with a component exhibiting an infection-preventing effect, such as sphingomyelin or lactoferrin.

The blending amount of milk lectins and / or their decomposed substances in foods for infection protection, nutritional compositions for infection protection, and feed for infection protection is not particularly limited. In order to exert the effect of preventing infection by influenza viruses, It is better to adjust the blending amount of medicinal products, foods, and feeds in such a way that an adult takes 0.1 mg or more of lactadherin and / or its degraded product per day.

The infection protection agent can be formulated into an arbitrary form by adding an appropriate adjuvant to the lectin and / or its decomposed product as an active ingredient. For formulation, diluents or excipients such as fillers, extenders, binders, disintegrants, surfactants, and lubricants that are commonly used can be used. As an excipient, for example, sucrose, lactose, starch, crystalline cellulose, mannitol, light silicic anhydride, magnesium aluminate, synthetic aluminum silicate, magnesium aluminum metasilicate, calcium carbonate, sodium bicarbonate, and phosphoric acid can be added. One type of calcium hydrogen, calcium carboxymethyl cellulose, or a combination of two or more types.

As described above, the present invention is described in the embodiments, but the discussion forming part of the disclosure should not be construed as limiting the present invention. From this disclosure, those skilled in the art can understand various alternative embodiments, examples, and operating techniques.

For example, the present invention provides a method for using an infection protection agent using the milk lectin and / or its decomposed product described in the embodiment as an active ingredient in an infection protection agent for a patient infected with influenza virus. In this case, as the lectin and / or its decomposed product, for example, one obtained by separating and purifying the raw material as described in the Example column described later can be used. In addition, the infection protection agent can also be applied to mammals other than humans, such as dogs, monkeys, cats, cattle, horses, pigs, chickens, sheep and other domestic animals.

The following examples and test examples illustrate the present invention in detail, but these are for illustration only, and the present invention is not limited in any way.

[Example 1]

100 kg of acetone was added to 10 kg of freeze-dried non-sterilized buttermilk powder, and then treated with a Quark separator to completely remove the precipitate to obtain a supernatant. After removing acetone from the supernatant with an evaporator and dissolving it again in deionized water, it was treated with an ultrafiltration membrane with a molecular weight of 60 kDa, and the permeate was recovered. Next, the permeate was separated to remove impurities by an ultrafiltration membrane with a molecular weight of 30 kDa, desalted and concentrated, and then freeze-dried to obtain 68 g of a lectin powder (Example Product 1). The powder of this lectin contains 78% of lectin. The thus obtained milk lectin can be directly used as an infection protection agent.

[Example 2]

500 mg of the 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 (produced by SIGMA) as a proteolytic enzyme was added to a final concentration of 0.01%, and an enzyme treatment was performed at 37 ° C for 1 hour. Next, the enzyme was deactivated by performing a heat treatment at 85 ° C for 10 minutes, and then freeze-dried to obtain 463 mg of a powder of lactolysin degradation product (Example Product 2). The molecular weight of the decomposed product obtained in this way is 5 kDa or less, and it can be directly used as an infection protection agent.

[Example 3]

After dissolving 10 g of the lectin powder prepared in Example 1 in 200 ml of purified water, it was maintained at 45 ° C, 2 g of Protease A "Amano" SD (manufactured by Amano Enzyme) was added, and an enzyme treatment was performed for 2 hours. Next, the enzyme was deactivated by heating at 80 ° C for 10 minutes, and then freeze-dried to obtain 8.2 g of a powder of lactolysin degradation product (Example Product 3). The molecular weight of the decomposed product obtained in this way is 5 kDa or less, and it can be directly used as an infection protection agent.

[Test Example 1]

(Confirmation of prevention effect against influenza virus infection)

A / Guinzhou virus as influenza A virus and B / Ibaraki virus as influenza B virus were nasally infected in mice (Balb / c; male; 6 weeks old), and the milk of Example 1 was coagulated The solution was made into a solution of 100 μg / ml and the solution of the lectin degradation product in Example 2 into a solution of 100 μg / ml, and was administered nasally at a dose of 1 μl / intranasal dose (administration amount: 0.1 μg) The prevention effect against influenza virus infection was compared and verified with the nasal infection group (control group) infected with each influenza virus by using the virus power in the nasal cleaning solution. In addition, the plaque method using MDCK cells was used as the determination system. The results are shown in Table 1.

Values represent mean ± standard deviation (n = 8).

※ Indicates significantly lower compared with each control group (p <0.05).

According to the results shown in Table 1, by administering lactadherin and its degradation products to the nasal cavity, infection prevention effects were confirmed for both types A and B, and the effect against influenza A virus was particularly significant.

[Test Example 2]

(Confirmation of infection prevention effect of orally administered influenza virus)

Mice (Balb / c; male; 6 weeks old) were infected with influenza virus PR8 (H1N1) at a viral amount of 1 × 10 ³ pfu. Prior to infection, it was divided into groups of Example 1 and Example 3 which were orally administered with 0.1 mg of lactadherin or its decomposed product per 1 kg of body weight of mice and 10 mg of lactadherin or its decomposed product orally The group used the virus value in the nasal cleaning solution 3 days after the virus infection to determine the prevention effect against the influenza virus infection.

As a positive control, before the infection, a group of 10 mg lactoferrin was orally administered, and a group (control group) of only water as a vehicle was administered orally, and a plaque method using MDCK cells was used. Compare and verify. The results are shown in Table 2.

Values represent mean ± standard deviation (n = 10).

※ 1 means significantly lower than each control group (p <0.05).

* 2 means significantly lower than 10mg lactoferrin (p <0.05).

According to the results in Table 2, the oral viral strength in the nasal cavity was significantly reduced compared with the control group by administering lactadherin or its decomposed product orally. Moreover, this effect is remarkably improved compared with the same lactoferrin administered orally. From this result, it can be seen that lactadherin and its decomposed product have excellent infection protection effect against influenza virus. This effect was clearly confirmed when mice were orally administered with 0.1 mg or more per 1 kg of body weight.

[Example 4]

(Preparation of capsules for prevention of influenza virus infection)

After mixing the raw materials in the blending amounts shown in Table 3, they were granulated according to the usual method and filled into capsules to prepare capsules for protection against influenza virus infection.

[Example 5]

(Preparation of tablets for prevention of influenza virus infection)

After mixing the raw materials in the blending amounts shown in Table 4, they were formed into 1 g according to a common method, and were tableted to prepare tablets for infection protection.

[Example 6]

(Preparation of liquid nutritional composition for prevention of influenza virus infection)

50 g of Example 1 was dissolved in 4,950 g of deionized water, heated to 50 ° C, and then mixed with TK HOMO MIXER (TK ROBO MICS; manufactured by SEIKI) at 6,000 rpm for 30 minutes to obtain milk. A milk lectin solution with a lectin content of 39g / 5kg. To 5.0 kg of this lectin solution, 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, and 2.0 kg of emulsifier were blended. , 4.0 kg of stabilizer, 0.05 kg of spices, filled into a 200 ml retort bag, and sterilized using a sterilization kettle sterilizer (the first pressure vessel; TYPE: RCS-4CRTGN; manufactured by Nisaka Manufacturing Co., Ltd.) at 121 ° C for 20 minutes 50 kg of liquid nutrition composition for prevention of influenza virus infection.

[Example 7]

(Preparation of beverages for prevention of influenza virus infection)

After 300 g of skim milk powder was dissolved in 409 g of deionized water, 1 g of Example 2 was dissolved and heated to 50 ° C., and then stirred and mixed at 9,500 rpm using ULTRA DISPERSER (ULTRA-TURRAX T-25; manufactured by IKA Japan). minute. After adding 100g of maltitol, 2g of sour sauce, 20g of reducing syrup, 2g of flavor, and 166g of deionized water, fill it into a 100ml glass bottle, sterilize it at 95 ° C for 15 seconds, and seal it to prepare influenza virus infection prevention Use 10 bottles of beverages (content 100ml).

[Example 8]

(Preparation of feed for preventing influenza virus infection in dogs)

2 kg of Example 1 was dissolved in 98 kg of deionized water, heated to 50 ° C, and then TK HOMO MIXER (MARK II 160; manufactured by Special Mechanized Industries) was stirred and mixed at 3,600 rpm for 40 minutes to obtain milk aggregation. A lectin solution of 1.56g / 100g. To 10 kg of this lectin solution, 12 kg of soybean meal, 14 kg of skimmed milk powder, 4 kg of soybean oil, 2 kg of corn oil, 23.2 kg of palm oil, and 14 kg of corn were blended. Starch, 9 kg of wheat flour, 2 kg of rice bran, 5 kg of vitamin mixture, 2.8 kg of cellulose, and 2 kg of mineral mixture were sterilized at 120 ° C. for 4 minutes to prepare 100 kg of feed for preventing influenza virus infection.

Claims (4)

An application of lactadherin and / or its decomposed product is used for manufacturing an anti-influenza protective agent against influenza virus. The anti-influenza protective agent uses refined lectin and / or its decomposed product as active ingredients. Each adult ingests 0.1 mg or more of the milk lectin and / or its decomposed product orally. For example, the use of lactadherin and / or its decomposed product in the scope of patent application item 1, wherein the lactadherin and / or its decomposed product is derived from cow's milk. For example, the use of lactadherin and / or its decomposed product in the scope of the patent application No. 1 or 2, wherein the lactadherin degraded product is obtained by decomposing the lactadherin using a protein degrading enzyme. For example, the use of lactadherin and / or its degraded product in item 3 of the patent application, wherein the proteolytic enzyme is selected from the group consisting of trypsin, trypsin, chymotrypsin, pepsin, and papain At least one of the group.