CN105025914B

CN105025914B - Infection defense agent

Info

Publication number: CN105025914B
Application number: CN201480012689.8A
Authority: CN
Inventors: 浦园浩司; 高桥朋树; 水野友葵; 吉冈孝一郎; 小林敏也; 加藤健
Original assignee: Megmilk Snow Brand Co Ltd
Current assignee: Megmilk Snow Brand Co Ltd
Priority date: 2013-03-08
Filing date: 2014-03-07
Publication date: 2020-02-18
Anticipated expiration: 2034-03-07
Also published as: AU2014226864B2; HK1210942A1; CN105025914A; WO2014136931A1; NZ711985A; AU2014226864A1; JP2014172862A; TWI646969B; JP6346405B2; TW201446257A; MY179921A

Abstract

The infection defense agent contains milk agglutinin and/or decomposition product obtained by treating milk agglutinin with protease as active ingredient. Since the milk of mammals is used as a raw material, the infection defense agent can be produced simply and economically and can be ingested daily and safely.

Description

Infection defense agent

Technical Field

The present invention relates to an infection defense agent (prophylactic and/or therapeutic agent) that exhibits an excellent defense effect against influenza virus, is useful for the prevention or treatment of influenza, and has excellent stability and safety. The present invention also relates to a food or beverage for infection defense, a nutritional composition for infection defense, and a feed for infection defense, each of which contains an infection defense agent.

Background

Annual influenza viruses cause epidemics in humans by aerial infection. Health improvements and medical advances have reduced the threat of influenza epidemics in recent years, but influenza viruses can still cause death. Influenza viruses are classified into three types A, B and C. Among them, influenza a viruses may mutate and cause pandemics. Vaccines are a common technique for preventing influenza virus infection. Unfortunately, vaccines are not necessarily satisfactory for the prevention of infection because infectious influenza viruses that may undergo antigenic shifts or antigenic drift are often not compatible with the antigen of the vaccine. Therefore, vaccination of children is not currently mandatory in japan. Therapeutic agents for influenza such as amantadine, oseltamivir, and zanamivir need to take into account side effects (e.g., hallucinations and sleep disorders) and the emergence of resistant bacteria (resisant bacteria), and thus such agents need to be used with caution. Under these circumstances, there is a need for development of foods, drinks or feeds which can be ingested safely on a daily basis and are expected to be useful for prevention or treatment of influenza.

Conventional food ingredients having an infection defense effect against influenza virus include sphingomyelin, lactoferrin, and kappa-casein glycomacropeptide (kappa-casein) produced by the action of rennet (rennet) or pepsin on kappa-casein. It is also known that exopolysaccharide produced by Lactobacillus bulgaricus (Lactobacillus bulgaricus)1073R-1, which is one of the Lactobacillus contained in fermented milk, acts on the intestinal tract to increase the production amount of IgA and increases NK activity via Peyer's patches, thereby exhibiting infection defense effect against influenza virus.

Milk lectin (lactadherin) is a glycoprotein present in milk fat globule membrane and accounts for 10% of the protein contained in milk fat globule membrane. The molecular weight of lactadherin is 43kDa to 53kDa, the isoelectric point is 7.0, and it includes two epidermal growth factor-like domains. Milk agglutinin contained in cow's milk is also called PAS-VI-VII (PAS6/7), and milk agglutinin contained in rat's milk is also called MFG-E8. Milk agglutinin is thought to play a role in regulating gastrointestinal function in newborns, and is incorporated into infant milk powder for the purpose of preventing newborns from becoming infected with rotavirus. Unfortunately, it has not been known that milk agglutinin and/or its decomposition products have an infection defense effect against influenza virus.

Documents of the prior art

Patent document

Patent document 1: japanese patent application laid-open No. 2008-37788

Non-patent document

Non-patent document 1: bioscience, Biotechnology and Biochemistry,57,1214, 1215,1993

Non-patent document 2: international Immunopharmacology,11,2246-

Disclosure of Invention

Problems to be solved by the invention

An object of the present invention is to provide an infection defense agent that exhibits an infection defense effect against influenza viruses and is useful for the prevention or treatment of influenza. Another object of the present invention is to provide a food or drink for infection prevention, a nutritional composition for infection prevention, and a feed for infection prevention, each containing the infection prevention agent.

Means for solving the problems

The present inventors have conducted extensive studies to solve the above problems, and as a result, have found that milk lectin and/or a decomposition product of milk lectin has an excellent infection defense effect against influenza virus.

The present invention includes the following aspects:

aspect (1) an infection defense agent against influenza virus, which comprises a collectin and/or a decomposition product of the collectin as an active ingredient.

Aspect (2) the infection defense agent against influenza virus according to aspect (1), wherein the milk agglutinin and/or the decomposition product of milk agglutinin is derived from cow's milk.

Aspect (3) the infection defense agent against influenza virus according to aspect (1) or (2), wherein the decomposition product of lactadherin is prepared by decomposing lactadherin with a protease.

Aspect (4) the infection defense agent against an influenza virus according to aspect (3), wherein the protease is at least one selected from the group consisting of trypsin, pancreatin (pancreatin), chymotrypsin, pepsin and papain.

Aspect (5) an infection-preventing food or drink, an infection-preventing nutritional composition, or an infection-preventing feed, which comprises the infection-preventing agent against an influenza virus according to any one of aspects (1) to (4).

Aspect (6) an infection defense method against an influenza virus, which comprises orally ingesting the infection defense agent according to any one of aspects (1) to (4).

Aspect (7) a method for using an infection defense agent comprising a collectin and/or a decomposition product of a collectin as an active ingredient, the method comprising applying the infection defense agent to an influenza virus-infected patient.

ADVANTAGEOUS EFFECTS OF INVENTION

The infection defense agent (prophylactic and/or therapeutic agent) of the present invention exhibits a significant infection defense effect against influenza virus, and is useful for the prevention or treatment of influenza.

Detailed Description

Milk lectin, which is an active ingredient of infection defense agents, can be prepared by desalting (desalting) or concentrating components (a), (B) or (C) using Ultrafiltration (UF) membranes, Microfiltration (MF) membranes, Reverse Osmosis (RO) membranes or ion exchange resins, wherein component (a) is buttermilk, which is an aqueous phase component prepared during the production of butter (butter) from raw milk; the component (B) is a water phase component discharged when the oil phase of high fat milk is changed (phaseinversion) by heating or shearing the high fat cream with fat content of more than 60% obtained by separating the cream with fat content of 40-50% obtained by separating the raw milk by a separator and separating the cream with fat content of more than 60% by a separator; and component (C) is butter serum (butter serum), which is an aqueous phase component separated from the heated and melted butter. Alternatively, milk agglutinin may be prepared by the following procedure: hydrochloric acid is added to the butter whey to a pH of 4.4, calcium chloride is added to the resulting mixture and the mixture is maintained at 50 ℃ for 30 minutes, followed by separation, and the resulting precipitate is dissolved or suspended in water, followed by desalting or concentrating the solution or suspension using Ultrafiltration (UF) membranes, Microfiltration (MF) membranes, Reverse Osmosis (RO) membranes, or ion exchange resins. A collectin decomposition product which is an active ingredient of an infection defense agent can be prepared by decomposing a collectin with a protease. Examples of the protease include commercially available food & industrial protease products such as protease A "Amano" SD (trade name), Thermoase PC10F (trade name) and Protin SD-AY10 (trade name), trypsin, pancreatin, chymotrypsin, pepsin and papain. These proteases may be used in combination. The above-prepared collectin and/or collectin decomposition product can be freeze-dried or spray-dried.

The milk agglutinin and/or breakdown products of milk agglutinin may be prepared from milk of mammals such as human, cow, buffalo, goat or sheep, produced by genetic engineering techniques, or refined from blood or organs of such mammals. The milk agglutinin and/or its decomposition product can be produced at low cost by such a material through a simple process, and the resulting product can be ingested on a daily basis safely. In particular, the milk agglutinin and/or the decomposition product of milk agglutinin is preferably derived from cow's milk. The milk agglutinin and/or breakdown products of milk agglutinin may be commercially available refined reagents.

The milk agglutinin and/or breakdown products of milk agglutinin may be used as infection defense agents without any treatment, or may be optionally prepared as powders, granules, tablets, capsules or drinks by conventional processes. The freeze-dried or spray-dried collectin and/or the decomposition product of the collectin may be used as an infection defense agent without any treatment, or may be prepared as any product by a conventional process.

The resulting milk lectin product may be incorporated into nutritional agents, food and drink products (e.g., yogurt, beverages, and wafers), nutritional compositions, or feed.

The food or drink for infection prevention, the nutritional composition for infection prevention, or the feed for infection prevention of the present invention may contain any material commonly contained in any other food or drink or feed, in addition to the milk lectin and/or the decomposition product of milk lectin. Examples of such materials include stabilizers, sugars, lipids, flavors, vitamins, minerals, flavonoids, and polyphenols. The food or drink for infection defense, the nutritional composition for infection prevention or the feed for infection prevention may contain any component that exhibits an infection defense effect in combination with milk lectin and/or a decomposition product of milk lectin as an active ingredient, such as sphingomyelin or lactoferrin.

The food or drink for infection prevention, the nutritional composition for infection prevention or the feed for infection prevention may contain any amount of milk agglutinin and/or a decomposition product of milk agglutinin. In order to achieve an infection prevention effect against influenza viruses, it is preferable to adjust the amount of the collectin and/or the decomposition product of the collectin introduced into the pharmaceutical, food, drink, or feed so that the daily oral dose of the collectin and/or the decomposition product of the collectin per adult is 0.1mg or more.

The infection defense agent can be prepared as any pharmaceutical product by adding an appropriate auxiliary agent to milk agglutinin and/or a decomposition product of milk agglutinin as an active ingredient. The preparation of pharmaceutical products may involve the use of common excipients or diluents, such as fillers, extenders, binders, disintegrants, surfactants or lubricants. Examples of the excipient include sucrose, lactose, starch, crystalline cellulose, mannitol, light anhydrous silicic acid, magnesium aluminate, synthetic aluminum silicate, magnesium aluminate silicate (magnesium aluminate), calcium carbonate, sodium bicarbonate, calcium hydrogen phosphate, and calcium carboxymethylcellulose. These excipients are used either alone or in combination.

While the present invention is described with reference to an embodiment, this embodiment forms a part of the present disclosure and should not be construed as limiting the present invention. Various alternative embodiments, examples, and operating techniques will be apparent to those skilled in the art from this disclosure.

For example, a method of using an infection defense agent is provided, in which an infection defense agent containing lactoagglutinin and/or a decomposition product of lactoagglutinin described in the embodiments as an active ingredient is used for an influenza virus-infected patient. The milk agglutinin and/or the decomposition product of milk agglutinin used in this method may be isolated or purified from the raw materials described in the following examples. The infection defense agent can also be used in non-human mammals including domestic animals such as dogs, monkeys, cats, cows, horses, pigs, chickens, and sheep.

Examples

The present invention is described in detail through examples and test examples, which should not be construed as limiting the present invention.

Example 1

Acetone (100kg) was added to the freeze-dried unsterilized buttermilk powder (10kg) and the mixture was treated with a quark separator to completely remove the precipitate. Acetone was removed from the resulting supernatant with an evaporator, and the residue was dissolved in deionized water. Subsequently, the solution was treated with an ultrafiltration membrane having a cut-off molecular weight (cut-off weight) of 60kDa to recover a filtrate. The filtrate was then treated with an ultrafiltration membrane having a 30kDa molecular weight cut-off to remove impurities, followed by desalting and concentration. Thereafter, the resultant was freeze-dried, thereby preparing a lactadherin powder (example sample 1) (68 g). The lectin powder has a lectin content of 78%. The milk lectin thus prepared can be used as an infection defense agent without any treatment.

Example 2

The lectin powder (500mg) prepared in example 1 was dissolved in purified water (100mL), and the pH of the solution was adjusted to 8 with sodium bicarbonate. Thereafter, trypsin (manufactured by Sigma) (i.e., protease) was added to the solution to make the final lectin concentration 0.01%, and the mixture was treated with the enzyme at 37 ℃ for one hour. The mixture was then heat treated at 85 ℃ for 10 minutes to inactivate the enzyme. The resultant mixture was freeze-dried, thereby preparing a lectin decomposition powder (example sample 2) (463 mg). The collectin decomposition product thus prepared has a molecular weight of 5kDa or less. The collectin decomposition product can be used as an infection defense agent without any treatment.

Example 3

The lectin powder (10g) prepared in example 1 was dissolved in purified water (200mL), and the solution was then maintained at 45 ℃. Protease a "Amano" SD (manufactured by Amano Enzyme inc.) (2g) was added to the solution, and the mixture was treated with the Enzyme for two hours. The mixture was then heat treated at 80 ℃ for 10 minutes to inactivate the enzyme. The resulting mixture was freeze-dried to prepare a collectin decomposition product powder (example sample 3) (8.2 g). The collectin decomposition product thus prepared has a molecular weight of 5kDa or less. The collectin decomposition product can be used as an infection defense agent without any treatment.

[ test example 1]

(confirmation of infection prevention Effect against influenza Virus)

Mice (Balb/c, male, 6 years old) were infected nasally with A/Guinzhou virus (i.e., influenza A virus) or B/Ibaraki virus (i.e., influenza B virus). A solution of example sample 1 (content of lactadherin: 100. mu.g/mL) and a solution of example sample 2 (content of lactadherin-decomposing compound: 100. mu.g/mL) were prepared. Each solution was nasally administered at a dose of 1. mu.L/nasal cavity (dose: 0.1. mu.g), and the effect of preventing infection with influenza virus was measured from the virus titer (virus titer) in the nasal cavity wash. For comparison, a set of mice nasally infected with each influenza virus was provided as a control. Plaque method (plaque method) using MDCK cells was used for the measurement of the preventive effect. Table 1 shows the results.

[ Table 1]

Numerical values: mean ± standard deviation (n ═ 8).

*: significantly lower than the corresponding control (p < 0.05).

Referring to table 1, nasal administration of a lactadherin or a lactadherin decomposing compound showed infection preventing effects against influenza a and B viruses, particularly against influenza a viruses.

[ test example 2]

(confirmation of infection prevention Effect against influenza Virus by oral administration)

With influenza virus PR8(H1N1) (1X 10)³pfu) infected mice (Balb/c, male, 6 years old). Before viral infection, mice were divided into a group to which lactoagglutinin (example sample 1) or a lactoagglutinin degradation product (example sample 3) (dose: 0.1mg/kg body weight) was orally administered, and a group to which lactoagglutinin (example sample 1) or a lactoagglutinin degradation product (example sample 3) (dose: 10mg/kg body weight) was orally administered. The effect of preventing infection with influenza virus was determined from the virus titer in the nasal wash three days after infection with virus.

For comparison, a group of mice orally administered with lactoferrin (10mg/kg body weight) before viral infection was provided as a positive control, and a group of mice orally administered with a solvent (water only) before viral infection was provided as a control. The plaque assay using MDCK cells was used for the measurement of the preventive effect. Table 2 shows the results.

[ Table 2]

Numerical values: mean ± standard deviation (n ═ 10)

*1: significantly lower than the control (p <0.05)

*2:: is significantly lower than 10mg of lactoferrin (p <0.05)

Referring to table 2, oral administration of milk agglutinin or milk agglutinin breakdown significantly reduced intranasal viral titers compared to controls. The preventive effect of milk agglutinin or milk agglutinin decomposition products is remarkably higher than that of lactoferrin administered orally. These results indicate that the collectin or the decomposition product of the collectin exhibits an excellent infection prevention effect against influenza virus. The results also show that this effect is achieved by orally administering the infection defense agent at a dose of 0.1mg/kg mouse body weight or more.

Example 4

(preparation of Capsule for prevention of influenza Virus infection)

The raw materials were mixed in the proportions shown in table 3, and the mixture was granulated by a conventional process and then encapsulated, thereby preparing a capsule for preventing influenza virus infection.

[ Table 3]

Example 5

(preparation of tablets for prevention of influenza Virus infection)

The raw materials were mixed in the proportions shown in table 4, and the mixture was formed into a compact (1g) by a conventional process, followed by tableting, thereby preparing an influenza virus infection-preventing tablet.

[ Table 4]

Example 6

(preparation of liquid nutritional composition for prevention of influenza Virus infection)

Example sample 1(50g) was dissolved in deionized water (4,950g) and the solution was heated to 50 ℃. Thereafter, the solution was stirred with a TK homogenizer (TK ROBO MICS, manufactured by PRIMIX Corporation) at 6,000rpm for 30 minutes, thereby preparing a lectin solution having a lectin content of 39g/5 kg. The milk lectin solution (5.0kg) was mixed with casein (5.0kg), soy protein (5.0kg), fish oil (1.0kg), perilla oil (3.0kg), dextrin (17.0kg), mineral mixture (6.0kg), vitamin mixture (1.95kg), emulsifier (2.0kg), stabilizer (4.0kg) and perfume (0.05 kg). The mixture was put into 200-mL sterilization bags (Retart pouches) and sterilized with a sterilization pot (type 1 pressure vessel, model: RCS-4CRTGN, manufactured by Hisaka Works, Ltd., manufactured) at 121 ℃ for 20 minutes, thereby preparing a liquid nutritional composition (50kg) for the prevention of influenza virus infection.

Example 7

(preparation of beverage for prevention of influenza Virus infection)

Skim milk powder (300g) was dissolved in deionized water (409g) and example sample 2(1g) was dissolved in the solution. The solution was heated to 50 ℃ and stirred with an ULTRA-disperser (ULTRA-TURRAX T-25, manufactured by IKA Japan) at 9,500rpm for 30 minutes. The resulting mixture was mixed with maltitol (100g), acidulant (2g), reduced starch syrup (20g), flavor (2g) and deionized water (166 g). Thereafter, the mixture was put into 100-mL glass bottles and sterilized at 95 ℃ for 15 seconds and then sealed, thereby preparing 10 bottles of influenza virus infection-preventing beverages (100mL each).

Example 8

(preparation of feed for preventing influenza Virus infection for dog)

Example sample 1(2kg) was dissolved in deionized water (98kg) and the solution was heated to 50 ℃. Thereafter, the solution was stirred with a TK homogenizer (MARK II 160, manufactured by PRIMIX Corporation) at 3,600rpm for 40 minutes, thereby preparing a lectin solution having a lectin content of 1.56g/100 g. A lectin solution (10kg) was mixed with soybean flour (soy bean lees) (12kg), skimmed milk powder (14kg), soybean oil (4kg), corn oil (2kg), palm oil (23.2kg), corn starch (14kg), wheat flour (flour) (9kg), bran (2kg), a vitamin mixture (5kg), cellulose (2.8kg) and a mineral mixture (2kg), and the mixture was sterilized at 120 ℃ for four minutes, thereby preparing an influenza virus infection-preventing feed (100 kg).

Claims

1. Use of a purified collectin and/or a decomposition product of a collectin in the preparation of an infection defense agent against influenza virus, the infection defense agent comprising a purified collectin and/or a decomposition product of a collectin as an active ingredient, the decomposition product of a collectin being prepared by decomposing a collectin with a protease.

2. Use according to claim 1, wherein the refined collectin and/or breakdown products of collectin are derived from cow's milk.

3. The use according to claim 1 or 2, wherein the protease is at least one selected from the group consisting of trypsin, pancreatin, chymotrypsin, pepsin and papain.

4. Use of the infection defense agent obtained by the use according to any one of claims 1 to 3 for the preparation of a medicament for a method of infection defense against influenza virus, the method comprising orally ingesting the infection defense agent obtained by the use according to any one of claims 1 to 3.