WO2014136931A1 - Infection protection agent - Google Patents

Abstract

This infection protection agent contains, as an active component, lactadherin and/or a decomposition product obtained by treating lactadherin with a proteolytic enzyme. Having mammalian milk as the raw ingredient, this infection protection agent can be easily and economically produced and can be safely ingested on a daily basis.

Description

Infectious agent

The present invention relates to an infection protective agent that is excellent in infection protective effect against influenza virus, is useful for prevention and treatment of influenza, and has excellent stability and safety. The present invention further relates to an infection-protecting food or drink, an infection-protecting nutritional composition, or an infection-protecting feed, which further contains an infection-protecting agent.

Influenza viruses infect humans from the air and cause epidemics every year. In recent years, although the threat has been diminished due to improvements in hygiene and advances in medicine, there are cases where deaths still occur. There are three types of influenza viruses, type A, type B, and type C. Of these, type A viruses are prone to mutation and are likely to cause a global pandemic called pandemic. Prevention against influenza virus infection is mainly carried out by vaccination. However, because influenza viruses are prone to mutations such as antigen shift and antigen drift, the prevalent virus and the antigen of the vaccine often do not match, and the preventive effect of the vaccine is always satisfactory. The current situation is not. For this reason, at present, child vaccination is not mandatory in Japan. Influenza drugs include amantadine, oseltamivir, and zanamivir. However, side effects such as hallucinations and sleep disorders and the emergence of resistant bacteria must be taken into account. is there. Under these circumstances, it is desired to develop foods and drinks that can be safely taken on a daily basis and that can be expected to prevent or treat influenza.
So far, κ-casein glycomacropeptide, which is a peptide produced when rennet or pepsin is allowed to act on sphingomyelin, lactoferrin, or κ-casein, is known as a food ingredient having an infection protection effect against influenza virus. In addition, a polysaccharide produced outside Lactobacillus bulgaricus 1073R-1 which is a kind of lactic acid bacteria contained in fermented milk or the like acts on the intestinal tract to increase the production amount of IgA, and through the Peyer's patch It is also known to increase NK activity and have an infection protective effect against influenza virus.

Lactoadherin is one of the glycoproteins that make up milk fat globule membrane, and in milk it accounts for about 10% of the protein contained in fat globule membrane. Its molecular weight is 43 kDa to 53 kDa, its isoelectric point is 7.0, and it has two epidermal growth factor-like domains. In addition, lactadherin is also referred to as PAS-VI-VII (PAS6 / 7) in cattle, and lactadherin contained in mouse milk is also expressed as MFG-E8. Furthermore, lactoadherin is thought to play a role in regulating the gastrointestinal function of newborns, and is blended in infant formula for the purpose of preventing infection of newborns with rotavirus. However, it is not known that lactoadherin and / or its degradation products have an infection-protecting effect against influenza virus.

JP 2008-37788 A

Bioscience, Biotechnology, Biochemistry, 57, 1214-1215, 1993 International Immunopharmacology, 11, 2246-2250, 2011

The present invention has an infection protective effect against influenza virus and is useful for the prevention and treatment of influenza, and a food and drink for infection protection, a nutritional composition for infection protection, or an infection protection compound containing the infection protective agent The challenge is to provide feed.

As a result of diligent studies to solve the above-mentioned problems, the present inventors have found that lactadherin and / or a degradation product thereof have an excellent infection protection effect against influenza virus.
That is, the present invention includes the following modes.
(1) An infection protective agent against influenza virus comprising lactoadherin and / or a degradation product thereof as an active ingredient.
(2) The infection protective agent against influenza virus according to (1), wherein the lactoadherin and / or a degradation product thereof is derived from milk.
(3) The infection protective agent against influenza virus according to (1) or (2), wherein the lactoadherin degradation product is a product obtained by degrading lactoadherin using a proteolytic enzyme.
(4) The infection protective agent against influenza virus according to (3), wherein the proteolytic enzyme is at least one selected from the group consisting of trypsin, pancreatin, chymotrypsin, pepsin, and papain.
(5) A food or drink for infection protection, a nutrition composition for infection protection, or a feed for infection protection containing the infection protective agent against the influenza virus according to any one of (1) to (4).
(6) An infection protection method against influenza virus by orally ingesting the infection protection agent according to any one of (1) to (4).
(7) A method for using an infection protective agent, wherein an infection protective agent comprising lactoadherin and / or a degradation product thereof as an active ingredient is applied to an influenza virus-infected patient.

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

For example, lactadherin, which is an active ingredient of an infection-protecting agent, is (A) buttermilk, which is an aqueous phase component produced when producing butter from raw milk, and (B) fat content 40 to 40 obtained by separator separation of raw milk. Heat treatment or shearing treatment is performed on 60% or more high fat cream obtained by separating 50% cream with a separator again, and the water phase component discharged when the high fat cream is phase-inverted, or ( C) Detach butaselam, which is an aqueous phase component that separates when butter is heated and dissolved, using an ultrafiltration (UF) membrane, microfiltration (MF) membrane, reverse osmosis membrane (RO) membrane, ion exchange resin, etc. It can be obtained by salting or concentrating. After adjusting the pH to 4.4 by adding hydrochloric acid to Bataselam, adding calcium chloride and holding the precipitate at 50 ° C. for 30 minutes to separate or suspend the precipitate again in water or the like It can be obtained by desalting or concentrating using an ultrafiltration (UF) membrane, microfiltration (MF) membrane, reverse osmosis membrane (RO) membrane, ion exchange resin or the like. Furthermore, a lactoadherin degradation product which is an active ingredient of an infection protective agent can be obtained by degrading the above-mentioned lactoadherin with any proteolytic enzyme. Proteolytic enzymes include protease A “Amano” SD (trade name), Samoase PC10F (trade name), protin SD-AY10 (trade name) and other commercially available food and industrial protease agents, trypsin, pancreatin, Examples include chymotrypsin, pepsin, papain and the like, and a plurality of these may be used in combination. The lactoadherin and / or its degradation product prepared as described above can be dried by freeze drying, spray drying, or the like.

Lactoadherin and / or its degradation products are prepared from milk of mammals such as humans, cows, buffalos, goats, sheep, etc., produced by genetic engineering techniques, purified from blood and organs, etc. Can be used. This is because it can be produced simply and economically and can be safely taken on a daily basis. Of these, those derived from milk are preferred. It is also possible to use a purified and commercially available reagent for lactoadherin and / or its degradation product.

Lactoadherin and / or its degradation product may be used as an infection protection agent as it is, but if necessary, it can be formulated into powders, granules, tablets, capsules, drinks, etc. according to conventional methods. It can also be used. In addition, lactadherin and / or its degradation product dried by freeze-drying or spray-drying can also be used as an infection-protecting agent as it is, and can also be formulated and used according to a conventional method.
Furthermore, after formulating these, it is also possible to mix this with foods and beverages such as nutrients, yogurt, beverages, wafers, nutritional compositions or feeds.

In addition to lactadherin and / or its degradation products, stabilizers, saccharides, lipids, flavors, vitamins, minerals, flavonoids, polyphenols are used as infection protection foods and drinks, infection protection nutrition compositions and infection protection feeds Etc., and other raw materials usually contained in foods and drinks can be contained. Moreover, in addition to the active ingredient lactoadherin and / or its degradation product, it can be used together with an ingredient exhibiting an infection-protecting action, such as sphingomyelin and lactoferrin.

There are no particular restrictions on the amount of lactoadherin and / or its degradation products in food and drink for infection prevention, nutrition composition for infection prevention and feed for infection prevention, but in order to exert an infection protection effect against influenza virus It is preferable to adjust the compounding amount in pharmaceuticals, foods and drinks, and feeds so that 0.1 mg or more of lactadherin and / or its degradation product can be taken orally per adult per day.

The infection preventive agent can be formulated into any form by adding a suitable auxiliary agent to the above-mentioned active ingredient kutoadherin and / or its degradation product. In the formulation, diluents or excipients such as fillers, extenders, binders, disintegrants, surfactants, lubricants and the like that are usually used can be used. Examples of the excipient include sucrose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicic acid, magnesium aluminate, synthetic aluminum silicate, magnesium magnesium metasilicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate One or two or more of carboxymethylcellulose calcium and the like can be added in combination.

As described above, the present invention has been described according to the embodiments. However, it should not be understood that the statements forming a part of this disclosure limit the present invention. From this disclosure, various alternative embodiments, examples and operational techniques will be apparent to those skilled in the art.
For example, there is provided a method for using an infection protective agent, which applies the infection protective agent comprising lactadherin and / or a degradation product thereof described in the embodiments as an active ingredient to an influenza virus-infected patient. In this case, as the lactoadherin and / or its degradation product, for example, those isolated and purified from the raw materials as described in the Examples section below can be used. The infection-protecting agent can also be applied to humans and other mammals such as dogs, monkeys, cats, cattle, horses, pigs, chickens, sheep and other livestock.

Hereinafter, the present invention will be described in detail with reference to examples and test examples, but these are merely illustrative, and the present invention is not limited by these.

After adding 100 kg of acetone to 10 kg of lyophilized unsterilized buttermilk powder, the precipitate was completely removed by treatment with a quark separator to obtain a supernatant. Acetone was removed from the supernatant using an evaporator and dissolved again in deionized water, and then treated with an ultrafiltration membrane having a fractional molecular weight of 60 kDa, and the permeate was collected. Next, impurities were removed from this permeate with a ultrafiltration membrane having a fractional molecular weight of 30 kDa, desalted and concentrated, and then freeze-dried to obtain 68 g of lactadherin powder (Example Product 1). The lactadherin powder contained 78% lactadherin. The lactoadherin thus obtained can be used as it is as an infection-protecting agent.

The powder of lactoadherin prepared in Example 1 was dissolved in 100 ml of purified water, and the pH was adjusted to 8 using sodium bicarbonate. Thereafter, trypsin (manufactured by Sigma) as a proteolytic enzyme was added to a final concentration of 0.01%, followed by enzyme treatment at 37 ° C. for 1 hour. And after heat-processing at 85 degreeC for 10 minute (s), the enzyme was deactivated, it lyophilized | freeze-dried and the powder (Example product 2) of the lactoadherin decomposition product was obtained. The molecular weight of the lactoadherin degradation product thus obtained is 5 kDa or less, and can be used as it is as an infection protection agent.

10 g of the lactoadherin powder prepared in Example 1 was dissolved in 200 ml of purified water, and maintained at 45 ° C., and 2 g of protease A “Amano” SD (Amano Enzyme) was added, followed by enzyme treatment for 2 hours. . And after heat-processing at 80 degreeC for 10 minute (s), the enzyme was deactivated, it freeze-dried and 8.2g of powder (Example product 3) of the lactoadherin decomposition product was obtained. The molecular weight of the lactoadherin degradation product thus obtained is 5 kDa or less, and can be used as it is as an infection protection agent.

[Test Example 1]
(Confirmation of infection prevention effect against influenza virus)
A / Guinzhou virus as influenza A virus and B / Ibaraki virus as influenza B virus were intranasally infected in mice (Balb / c, male, 6 weeks old). At the same time, Example product 1 was 100 μg of lactadherin. / Ml and a solution in which the lactoadherin degradation product of Example Product 2 was dissolved at 100 μg / ml, respectively, at a dose of 1 μl / nasal (dose: 0.1 μg). The infection prevention effect against influenza viruses was compared and verified using the group (control) infected with each influenza virus alone and the virus titer in the nasal wash. For the determination, a plaque method using MDCK cells was used. The results are shown in Table 1.

From the results in Table 1, infection prevention effects were confirmed for both types A and B by intranasal administration of lactoadherin and its degradation product, and the effect on influenza A virus was particularly remarkable.

[Test Example 2]
(Confirmation of infection prevention effect of influenza virus by oral administration)
Influenza virus PR8 (H1N1) was infected to mice (Balb / c, male, 6 weeks old) at a viral load of 1 × 10 ³ pfu. Prior to infection, 0.1 mg of lactadherin or its degradation product was orally administered per kg body weight of mouse using Example product 1 and 3 and 10 mg of lactadherin or its degradation product was orally administered. The infection prevention effect against influenza virus was determined by the virus titer in the nasal wash 3 days after the virus infection.
In addition, as a positive target, a group in which 10 mg of lactoferrin was orally administered was set before infection, and a group (control) in which only water as a solvent was orally administered was compared with a plaque method using MDCK cells. The results are shown in Table 2.

From the results in Table 2, the intranasal virus titer was significantly reduced by oral administration of lactoadherin or its degradation product compared to the control. Moreover, the effect was significantly higher than that of lactoferrin administered orally in the same manner. From these results, it was found that lactadherin and its degradation products have an excellent protective effect against influenza viruses. It was also clarified that this effect was observed when 0.1 mg or more per kg body weight of the mouse was orally administered.

(Preparation of capsules for preventing influenza virus infection)
After mixing the raw materials with the formulation shown in Table 3, the mixture was granulated by a conventional method and filled into capsules to produce capsules for preventing influenza virus infection.

(Preparation of influenza virus infection prevention tablets)
After mixing the raw materials with the formulation shown in Table 4, it was molded into 1 g by a conventional method and tableted to produce influenza virus infection preventing tablets.

(Preparation of liquid nutritional composition for preventing influenza virus infection)
50 g of Example product 1 was dissolved in 4,950 g of deionized water, heated to 50 ° C., and then stirred and mixed at 6,000 rpm for 30 minutes with a TK homomixer (TK ROBO MICS; manufactured by Tokushu Kika Kogyo Co., Ltd.). Thus, a lactoadherin solution having a lactoadherin content of 39 g / 5 kg was obtained. 5.0 kg of this case, 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, emulsifier 2.0kg, 4.0kg stabilizer, 0.05kg fragrance is blended and filled into a 200ml retort pouch, then 121 ° C with a retort sterilizer (Type 1 pressure vessel, TYPE: RCS-4CRTGN, manufactured by Nisaka Seisakusho) And sterilized for 20 minutes to produce 50 kg of a liquid nutritional composition for preventing influenza virus infection.

(Preparation of influenza virus infection prevention beverage)
After dissolving 300 g of skim milk powder in 409 g of deionized water, 1 g of Example products 2 and 1 g were dissolved, heated to 50 ° C., and then heated with Ultra Disperser (ULTRA-TURRAX T-25; manufactured by IKA Japan). The mixture was stirred and mixed at 500 rpm for 30 minutes. After adding maltitol 100g, acidulant 2g, reduced starch syrup 20g, fragrance 2g, deionized water 166g, it is filled into a 100ml glass bottle, sterilized at 95 ° C for 15 seconds, sealed, and 10 drinks for preventing influenza virus infection (100 ml) was prepared.

(Preparation of feed for preventing influenza virus infection for dogs)
2 kg of Example Product 1 was dissolved in 98 kg of deionized water, heated to 50 ° C., and then stirred and mixed at 3,600 rpm for 40 minutes with a TK homomixer (MARK II 160 type; manufactured by Tokushu Kika Kogyo Co., Ltd.). A lactoadherin solution having a lactoadherin content of 1.56 g / 100 g was obtained. 10 kg of this lactoadherin solution, 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 bran, 5 kg of vitamin mixture, 2.8 kg of cellulose, and mineral mixture 2 kg was mixed and sterilized at 120 ° C. for 4 minutes to produce 100 kg of feed for preventing influenza virus infection.

Claims (7)

Infectious agent against influenza virus containing lactoadherin and / or its degradation product as an active ingredient. The infection protective agent against influenza virus according to claim 1, wherein the lactadherin and / or a degradation product thereof is derived from milk. The infection protective agent against influenza virus according to claim 1 or 2, wherein the lactoadherin degradation product is a product obtained by degrading lactoadherin using a proteolytic enzyme. The infection protective agent against influenza virus according to claim 3, wherein the proteolytic enzyme is at least one selected from the group consisting of trypsin, pancreatin, chymotrypsin, pepsin and papain. An infection-protecting food or drink, an infection-protecting nutritional composition, or an infection-preventing feed comprising the infection-protecting agent against the influenza virus according to any one of claims 1 to 4. An infection protection method against influenza virus by orally ingesting the infection protection agent according to any one of claims 1 to 4. A method of using an infection protective agent, wherein an infection protective agent comprising lactadherin and / or a degradation product thereof as an active ingredient is applied to an influenza virus-infected patient.